The following alphabetical list represents papers published in 2020 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Science Citation Index (also known as the Web of Science) Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.

P.S. The journal links only work if you have a license to those respective online journals.

2020 Titles :

Adelmann, C.H., Traunbauer, A.K., Chen, B., Condon, K.J., Chan, S.H., Kunchok, T., Lewis, C.A., and Sabatini, D.M. (2020). MFSD12 mediates the import of cysteine into melanosomes and lysosomes. Nature. Online ahead of print. Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome-the organelle, related to the lysosome, that synthesizes pigment-but have unclear functions(1,2). Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans(3,4). We find that MFSD12 is required to maintain normal levels of cystine-the oxidized dimer of cysteine-in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation(5,6). Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin(7-9). Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes. Full Text

Al-Shekaili, H.H., Petkau, T.L., Pena, I., Lengyell, T.C., Verhoeven-Duif, N.M., Ciapaite, J., Bosma, M., van Faassen, M., Kema, I.P., Horvath, G., et al. (2020). A Novel Mouse Model for Pyridoxine-Dependent Epilepsy Due to Antiquitin Deficiency. Human Molecular Genetics Accepted Manuscript. Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA), and pipecolic acid (PIP) both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.Full Text

Bessho-Uehara, M., Huang, W., Patry, W.L., Browne, W.E., Weng, J.K., and Haddock, S.H.D. (2020). Evidence for de novo Biosynthesis of the Luminous Substrate Coelenterazine in Ctenophores. iScience Vol.23 no.12, 101859 . Coelenterazine is a key substrate involved in marine bioluminescence which is used for light-production by at least nine phyla. Some luminous animals, such as the hydromedusa Aequorea, lack the ability to produce coelenterazine endogenously and instead depend on dietary sources. Little is known about the source organisms or the metabolic process of coelenterazine biosynthesis. Here, we present evidence that ctenophores are both producers and suppliers of coelenterazine in marine ecosystems. Using biochemical assays and mass spectrometry analyses, we detected coelenterazine from cultured ctenophores fed with a non-luminous coelenterazine-free diet. We propose that ctenophores are an emerging model organism to study coelenterazine biosynthesis and the origins of bioluminescence. Full Text

Boija, A., Klein, I.A., and Young, R.A. (2020). Biomolecular condensates and cancer. Cancer Cell. Malignant transformation is characterized by dysregulation of diverse cellular processes that have been the subject of detailed genetic, biochemical, and structural studies, but only recently has evidence emerged that many of these processes occur in the context of biomolecular condensates. Condensates are membrane-less bodies, often formed by liquid-liquid phase separation, that compartmentalize protein and RNA molecules with related functions. New insights from condensate studies portend a profound transformation in our understanding of cellular dysregulation in cancer. Here we summarize key features of biomolecular condensates, note where they have been implicated-or will likely be implicated-in oncogenesis, describe evidence that the pharmacodynamics of cancer therapeutics can be greatly influenced by condensates, and discuss some of the questions that must be addressed to further advance our understanding and treatment of cancer.Full Text

Briskin , D., Wang, P.Y., and Bartel, D.P. (2020). The biochemical basis for the cooperative action of microRNAs. PNAS. In cells, closely spaced microRNA (miRNA) target sites within a messenger RNA (mRNA) can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. Using purified miRNA-Argonaute (AGO2) complexes, synthetic target RNAs, and a purified domain of TNRC6B (GW182 in flies) that is able to simultaneously bind multiple AGO proteins, we examined both the occupancies and binding affinities of miRNA-AGO2 complexes and target RNAs with either one site or two cooperatively spaced sites. On their own, miRNA-AGO2 complexes displayed little if any cooperative binding to dual sites. In contrast, in the presence of the AGO-binding region of TNRC6B, we observed strong cooperative binding to dual sites, with almost no singly bound target RNAs and substantially increased binding affinities and Hill coefficients. Cooperative binding was retained when the two sites were for two different miRNAs or when the two sites were bound to miRNAs loaded into two different AGO paralogs, AGO1 and AGO2. The improved binding affinity was attributable primarily to a reduced rate of dissociation between miRNA-AGO complexes and their dual-site targets. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.Full Text

*Brown, K.M., Sibley, L.D., and Lourido, S. (2020). High-Throughput Measurement of Microneme Secretion in Toxoplasma gondii. Methods in Molecular Biology 2071, 157-169. Micronemes are specialized secretory organelles present in all motile forms of apicomplexan parasites. Microneme vesicles hold adhesins and other proteins that are secreted to facilitate parasite attachment, invasion of host cells, and egress following replication-all processes indispensable for cell-to-cell transmission of these obligate intracellular parasites. Defining the signaling pathways that lead to microneme secretion is an important part of understanding the infectious cycle of apicomplexan parasites. However, the classical method of measuring microneme secretion by immunoblotting for microneme proteins in parasite excreted/secreted antigen (ESA) preparations is low-throughput and only semiquantitative. We recently reported a new luciferase-based method for measuring microneme secretion in a 96-well format with high sensitivity in the model apicomplexan Toxoplasma gondii. Here, we aim to elaborate on this detection method and review current practices for stimulating microneme secretion in vitro. Full Text

Bury, L., Moodie, B., Ly, J., McKay, L.S., Miga, K.H., and Cheeseman, I.M. (2020). Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations. ELife 9:e59770. Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions. Full Text

Cao , Y., Lim, E., Xu, M., Weng, J.K., and Marelli, B. (2020). Precision Delivery of Multiscale Payloads to Tissue-Specific Targets in Plants. Advanced Science (Weinheim).The precise deployment of functional payloads to plant tissues is a new approach to help advance the fundamental understanding of plant biology and accelerate plant engineering. Here, the design of a silk-based biomaterial is reported to fabricate a microneedle-like device, dubbed "phytoinjector," capable of delivering a variety of payloads ranging from small molecules to large proteins into specific loci of various plant tissues. It is shown that phytoinjector can be used to deliver payloads into plant vasculature to study material transport in xylem and phloem and to perform complex biochemical reactions in situ. In another application, it is demonstrated Agrobacterium-mediated gene transfer to shoot apical meristem (SAM) and leaves at various stages of growth. Tuning of the material composition enables the fabrication of another device, dubbed "phytosampler," which is used to precisely sample plant sap. The design of plant-specific biomaterials to fabricate devices for drug delivery in planta opens new avenues to enhance plant resistance to biotic and abiotic stresses, provides new tools for diagnostics, and enables new opportunities in plant engineering.Full Text

Chen, C., and Yamashita, Y.M. (2020). Alstrom syndrome gene is a stem cell-specific regulator of centriole duplication in the Drosophila testis. eLife 9 : e59368. Asymmetrically dividing stem cells often show asymmetric behavior of the mother versus daughter centrosomes, whereby the self-renewing stem cell selectively inherits the mother or daughter centrosome. Although the asymmetric centrosome behavior is widely conserved, its biological significance remains largely unclear. Here we show that Alms1a, a Drosophila homolog of the human ciliopathy gene Alstrom syndrome, is enriched on the mother centrosome in Drosophila male germline stem cells (GSCs). Depletion of alms1a in GSCs, but not in differentiating germ cells, results in rapid loss of centrosomes due to a failure in daughter centriole duplication, suggesting that Alms1a has a stem cell-specific function in centrosome duplication. Alms1a interacts with Sak/Plk4, a critical regulator of centriole duplication, more strongly at the GSC mother centrosome, further supporting Alms1a's unique role in GSCs. Our results begin to reveal the unique regulation of stem cell centrosomes that may contribute to asymmetric stem cell divisions.Full Text

Chivukula , R.R., Maley, J.H., Dudzinski, D.M., Hibbert, K., and Hardin, C.C. (2020). Evidence-Based Management of the Critically Ill Adult With SARS-CoV-2 Infection. Journal of Intensive Care Medicine (Online ahead of print). Human infection by the novel viral pathogen SARS-CoV-2 results in a clinical syndrome termed Coronavirus Disease 2019 (COVID-19). Although the majority of COVID-19 cases are self-limiting, a substantial minority of patients develop disease severe enough to require intensive care. Features of critical illness associated with COVID-19 include hypoxemic respiratory failure, acute respiratory distress syndrome (ARDS), shock, and multiple organ dysfunction syndrome (MODS). In most (but not all) respects critically ill patients with COVID-19 resemble critically ill patients with ARDS due to other causes and are optimally managed with standard, evidence-based critical care protocols. However, there is naturally an intense interest in developing specific therapies for severe COVID-19. Here we synthesize the rapidly expanding literature around the pathophysiology, clinical presentation, and management of COVID-19 with a focus on those points most relevant for intensivists tasked with caring for these patients. We specifically highlight evidence-based approaches that we believe should guide the identification, triage, respiratory support, and general ICU care of critically ill patients infected with SARS-CoV-2. In addition, in light of the pressing need and growing enthusiasm for targeted COVID-19 therapies, we review the biological basis, plausibility, and clinical evidence underlying these novel treatment approaches.

Chivukula , R.R., Montoro, D.T., Leung, H.M., Yang, J., Shamseldin, H.E., Taylor, M.S., Dougherty, G.W., Zariwala, M.A., Carson, J., Daniels, M.L.A., Frenkel, E.M., Sabatini, D.M., et al. (2020). A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance. Nature Medicine [Epub ahead of print]. Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells(1,2). Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies(3). Previous studies have identified mutations that affect ciliary structure and nucleation in PCD(4), but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance. Full Text

Choudhuri, A., Trompouki, E., Abraham, B.J., Colli, L.M., Kock, K.H., Mallard, W., Yang, M.L., Vinjamur, D.S., Ghamari, A., Sporrij, A., Richard A Young, et al. (2020). Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits. Nature Genetics. Online ahead of print. Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-β-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli. Full Text

Cohen , M.A., Zhang, S., Sengupta, S., Ma, H., Bell, G.W., Horton, B., Sharma, B., George, R.E., Spranger, S., and Jaenisch, R. (2020). Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras. Cell Stem Cell [Epub ahead of print]. Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system. Full Text

Daneshvar, K., Ardehali, M.B., Klein, I.A., Hsieh, F.K., Kratkiewicz, A.J., Mahpour, A., Cancelliere, S.O.L., Zhou, C., Cook, B.M., Li, W., Richard A. Young, et al. (2020). lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation. Nature Cell Biology. Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncRNA phase-separated condensates have a broader role as regulators of transcription. Full Text

Dees, C., Pötter, S., Zhang, Y., Bergmann, C., Zhou, X., Luber, M., Wohlfahrt, T., Karouzakis, E., Ramming, A., Gelse, K., Rudolf Jaenisch, et al. (2020). TGF-β-induced epigenetic deregulation of SOCS3 facilitates STAT3 signaling to promote fibrosis. The Journal of Clinical Investigation 130, 2347-2363. Fibroblasts are key effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an "autonomous," self-maintaining profibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, TGF-β induced the expression of DNA methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of STAT3 to promote fibroblast-to-myofibroblast transition, collagen release, and fibrosis in vitro and in vivo. Reestablishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGF-β-dependent fibroblast activation, and ameliorated experimental fibrosis in murine models. These findings identify a pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of targeted therapies in fibrotic diseases. Full Text

*Du, X., Smirnov, A., Pluskal, T., Jia, W., and Sumner, S. (2020). Metabolomics Data Preprocessing Using ADAP and MZmine 2. Methods in Molecular Biology 2104, 25-48.The informatics pipeline for making sense of untargeted LC-MS or GC-MS data starts with preprocessing the raw data. Results from data preprocessing undergo statistical analysis and subsequently mapped to metabolic pathways for placing untargeted metabolomics data in the biological context. ADAP is a suite of computational algorithms that has been developed specifically for preprocessing LC-MS and GC-MS data. It consists of two separate computational workflows that extract compound-relevant information from raw LC-MS and GC-MS data, respectively. Computational steps include construction of extracted ion chromatograms, detection of chromatographic peaks, spectral deconvolution, and alignment. The two workflows have been incorporated into the cross-platform and graphical MZmine 2 framework and ADAP-specific graphical user interfaces have been developed for using ADAP with ease. This chapter summarizes the algorithmic principles underlying key steps in the two workflows and illustrates how to apply ADAP to preprocess LC-MS and GC-MS data.Full Text

Eisen , T.J., Eichhorn, S.W., Subtelny, A.O., Lin, K.S., McGeary, S.E., Gupta, S., and Bartel, D.P. (2020). The Dynamics of Cytoplasmic mRNA Metabolism. Molecular Cell 77, 786-799. For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rate constants, which correspond to cytoplasmic lifetimes. Indeed, with few exceptions, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from either endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rate constants of short-tailed mRNAs vary broadly (1000-fold) and are larger for short-tailed mRNAs that have previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rate constants to impart a similarly large range in stabilities. Full Text

Eisen , T.J., Eichhorn, S.W., Subtelny, A.O., and Bartel, D.P. (2020). MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs. Molecular cell 77, 775-785. MicroRNAs (miRNAs) specify the recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates the acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels, even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs. Full Text

Factor, D.C., Barbeau, A.M., Allan, K.C., Hu, L.R., Madhavan, M., Hoang, A.T., Hazel, K.E.A., Hall, P.A., Nisraiyya, S., Najm, F.J., Olivia Corradin , et al. (2020). Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS. Cell 181, 382-395. Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. Full Text

Fame , R.M., Cortes-Campos, C., and Sive, H.L. (2020). Brain Ventricular System and Cerebrospinal Fluid Development and Function: Light at the End of the Tube: A Primer with Latest Insights. BioEssays 42(3):e1900186.The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field. Full Text

Fang , W., and Bartel, D.P. (2020). MicroRNA Clustering Assists Processing of Suboptimal MicroRNA Hairpins through the Action of the ERH Protein. Molecular Cell 78, 289-302. Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor. Full Text

Fassl , A., Brain, C., Abu-Remaileh, M., Stukan, I., Butter, D., Stepien, P., Feit, A.S., Bergholz, J., Michowski, W., Otto, T., David M. Sabatini, et al. (2020). Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances. Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies. Full Text

Fink, G.R. (2020). A Morgan Legacy. Genetics 216, 611-612. The Thomas Hunt Morgan Medal recognizes lifetime contributions to the field of genetics. The 2020 recipient is Gerald R. Fink of Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research, recognizing the discovery of principles central to genome organization and regulation in eukaryotic cells. Full Text

Gaglia G., Rashid, R., Yapp, C., Joshi, G.N., Li, C.G., Lindquist, S.L., Sarosiek, K.A., Whitesell, L., Sorger, P.K., and Santagata, S. (2020). HSF1 phase transition mediates stress adaptation and cell fate decisions. Nature Cell Biology 22, 151-158.Under proteotoxic stress, some cells survive whereas others die. The mechanisms governing this heterogeneity in cell fate remain unknown. Here we report that condensation and phase transition of heat-shock factor 1 (HSF1), a transcriptional regulator of chaperones(1,2), is integral to cell-fate decisions underlying survival or death. During stress, HSF1 drives chaperone expression but also accumulates separately in nuclear stress bodies called foci(3-6). Foci formation has been regarded as a marker of cells actively upregulating chaperones(3,6-10). Using multiplexed tissue imaging, we observed HSF1 foci in human tumours. Paradoxically, their presence inversely correlated with chaperone expression. By live-cell microscopy and single-cell analysis, we found that foci dissolution rather than formation promoted HSF1 activity and cell survival. During prolonged stress, the biophysical properties of HSF1 foci changed; small, fluid condensates enlarged into indissoluble gel-like arrangements with immobilized HSF1. Chaperone gene induction was reduced in such cells, which were prone to apoptosis. Quantitative analysis suggests that survival under stress results from competition between concurrent but opposing mechanisms. Foci may serve as sensors that tune cytoprotective responses, balancing rapid transient responses and irreversible outcomes. Full Text

Geiler-Samerotte, K.A., Li, S., Lazaris, C., Taylor, A., Ziv, N., Ramjeawan, C., Paaby, A.B., and Siegal, M.L. (2020). Extent and context dependence of pleiotropy revealed by high-throughput single-cell phenotyping.PLoS Biology 18 (8) : e3000836. . Pleiotropy-when a single mutation affects multiple traits-is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context. Full Text

Getz, M.A., Weinberg, D.E., Drinnenberg, I.A., Fink, G.R., and Bartel, D.P. (2020). Xrn1p acts at multiple steps in the budding-yeast RNAi pathway to enhance the efficiency of silencing. Nucleic Acids Research. RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5'-to-3' exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand and degradation of sliced target RNA. Full Text

Godfrey , A.K., Naqvi, S., Chmatal, L., Chick, J.M., Mitchell, R.N., Gygi, S.P., Skaletsky, H., and Page, D.C. (2020). Quantitative analysis of Y-Chromosome gene expression across 36 human tissues. Genome research. Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes. Full Text

Goodheart, J.A., Minsky, G., Brynjegard-Bialik, M.N., Drummond, M.S., Munoz, J.D., Fallon, T.R., Schultz, D.T., Weng, J.K., Torres, E., and Oakley, T.H. (2020). Laboratory culture of the California Sea Firefly Vargula tsujii (Ostracoda: Cypridinidae): Developing a model system for the evolution of marine bioluminescence. Scientific reports 10, 10443. Bioluminescence, or the production of light by living organisms via chemical reaction, is widespread across Metazoa. Laboratory culture of bioluminescent organisms from diverse taxonomic groups is important for determining the biosynthetic pathways of bioluminescent substrates, which may lead to new tools for biotechnology and biomedicine. Some bioluminescent groups may be cultured, including some cnidarians, ctenophores, and brittle stars, but those use luminescent substrates (luciferins) obtained from their diets, and therefore are not informative for determination of the biosynthetic pathways of the luciferins. Other groups, including terrestrial fireflies, do synthesize their own luciferin, but culturing them is difficult and the biosynthetic pathway for firefly luciferin remains unclear. An additional independent origin of endogenous bioluminescence is found within ostracods from the family Cypridinidae, which use their luminescence for defense and, in Caribbean species, for courtship displays. Here, we report the first complete life cycle of a luminous ostracod (Vargula tsujii Kornicker & Baker, 1977, the California Sea Firefly) in the laboratory. We also describe the late-stage embryogenesis of Vargula tsujii and discuss the size classes of instar development. We find embryogenesis in V. tsujii ranges from 25-38 days, and this species appears to have five instar stages, consistent with ontogeny in other cypridinid lineages. We estimate a complete life cycle at 3-4 months. We also present the first complete mitochondrial genome for Vargula tsujii. Bringing a luminous ostracod into laboratory culture sets the stage for many potential avenues of study, including learning the biosynthetic pathway of cypridinid luciferin and genomic manipulation of an autogenic bioluminescent system.Full Text

Graef, J.D., Wu, H., Ng, C., Sun, C., Villegas, V., Qadir, D., Jesseman, K., Warren, S.T., Jaenisch, R., Cacace, A., et al. (2020). Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons. The European Journal of Neuroscience 51, 2143-2157.Fragile X syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5'-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient-derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP-positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP-expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons. Full Text

Guan, Y., Leu, N.A., Ma, J., Chmatal, L., Ruthel, G., Bloom, J.C., Lampson, M.A., Schimenti, J.C., Luo, M., and Wang, P.J. (2020). SKP1 drives the prophase I to metaphase I transition during male meiosis.Science Advances . The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, gammaH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.Full Text

*Gura, M.A., Mikedis, M.M., Seymour, K.A., de Rooij, D.G., Page, D.C., and Freiman, R.N. (2020). Dynamic and regulated TAF gene expression during mouse embryonic germ cell development. PLoS Genetics 16, e1008515 [Epub ahead of print]. Germ cells undergo many developmental transitions before ultimately becoming either eggs or sperm, and during embryonic development these transitions include epigenetic reprogramming, quiescence, and meiosis. To begin understanding the transcriptional regulation underlying these complex processes, we examined the spatial and temporal expression of TAF4b, a variant TFIID subunit required for fertility, during embryonic germ cell development. By analyzing published datasets and using our own experimental system to validate these expression studies, we determined that both Taf4b mRNA and protein are highly germ cell-enriched and that Taf4b mRNA levels dramatically increase from embryonic day 12.5-18.5. Surprisingly, additional mRNAs encoding other TFIID subunits are coordinately upregulated through this time course, including Taf7l and Taf9b. The expression of several of these germ cell-enriched TFIID genes is dependent upon Dazl and/or Stra8, known regulators of germ cell development and meiosis. Together, these data suggest that germ cells employ a highly specialized and dynamic form of TFIID to drive the transcriptional programs that underlie mammalian germ cell development. Full Text

Harding , C.R., Sidik, S.M., Petrova, B., Gnädig, N.F., Okombo, J., Herneisen, A.L., Ward, K.E., Markus, B.M., Boydston, E.A., Fidock, D.A., and Sebastian Lourido. (2020). Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility. Nature Communications 11(1) : 4813. Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA. Full Text

Hariri, L.P., North, C.M., Shih, A.R., Israel, R.A., Maley, J.H., Villalba, J.A., Vinarsky, V., Rubin, J., Okin, D.A., Sclafani, A., Chivukula, R.R., et al. (2020). Lung Histopathology in COVID-19 as Compared to SARS and H1N1 Influenza: A Systematic Review. Chest (Online ahead of print). Patients with severe Coronavirus Disease 2019 (COVID-19) have respiratory failure with hypoxemia and acute bilateral pulmonary infiltrates, consistent with acute respiratory distress syndrome (ARDS). It has been suggested that respiratory failure in COVID-19 represents a novel pathologic entity. RESEARCH QUESTION: How does the lung histopathology described in COVID-19 compare to the lung histopathology described in SARS and H1N1 influenza? STUDY DESIGN: and Methods: We conducted a systematic review to characterize the lung histopathologic features of COVID-19 and compare them against findings of other recent viral pandemics, H1N1 influenza and SARS. We systematically searched MEDLINE and PubMed for studies published up to June 24, 2020 using search terms for COVID-19, H1N1 influenza and SARS with keywords for pathology, biopsy, and autopsy. Using PRISMA-IPD guidelines, our systematic review analysis included 26 articles representing 171 COVID-19 patients; 20 articles representing 287 H1N1 patients; and eight articles representing 64 SARS patients. RESULTS: In COVID-19, acute phase diffuse alveolar damage (DAD) was reported in 88% of patients, which was similar to the proportion of cases with DAD in both H1N1 (90%) and SARS (98%). Pulmonary microthrombi were reported in 57% of COVID-19 and 58% of SARS patients, as compared to 24% of H1N1 influenza patients. INTERPRETATION: DAD, the histologic correlate of ARDS, is the predominant histopathologic pattern identified in lung pathology from patients with COVID-19, H1N1 influenza and SARS. Microthrombi were reported more frequently in both patients with COVID-19 and SARS as compared to H1N1 influenza. Future work is needed to validate this histopathologic finding and, if confirmed, elucidate the mechanistic underpinnings and characterize any associations with clinically important outcomes. Full Text

Helman, A., Cangelosi, A.L., Davis, J.C., Pham, Q., Rothman, A., Faust, A.L., Straubhaar, J.R., Sabatini, D.M., and Melton, D.A. (2020). A Nutrient-Sensing Transition at Birth Triggers Glucose-Responsive Insulin Secretion. Cell metabolism 31 : 1004-1016. A drastic transition at birth, from constant maternal nutrient supply in utero to intermittent postnatal feeding, requires changes in the metabolic system of the neonate. Despite their central role in metabolic homeostasis, little is known about how pancreatic beta cells adjust to the new nutritional challenge. Here, we find that after birth beta cell function shifts from amino acid- to glucose-stimulated insulin secretion in correlation with the change in the nutritional environment. This adaptation is mediated by a transition in nutrient sensitivity of the mTORC1 pathway, which leads to intermittent mTORC1 activity. Disrupting nutrient sensitivity of mTORC1 in mature beta cells reverts insulin secretion to a functionally immature state. Finally, manipulating nutrient sensitivity of mTORC1 in stem cell-derived beta cells in vitro strongly enhances their glucose-responsive insulin secretion. These results reveal a mechanism by which nutrients regulate beta cell function, thereby enabling a metabolic adaptation for the newborn.Full Text

Henninger, J.E., Oksuz, O., Shrinivas, K., Sagi, I., LeRoy, G., Zheng, M.M., Andrews, J.O., Zamudio, A.V., Lazaris, C., Hannett, N.M., Tong Ihn Lee, Richard A Young, , et al. (2020). RNA-Mediated Feedback Control of Transcriptional Condensates. Cell. Online ahead of print. Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process. Full Text

Herbert, Z.T., Thimmapuram, J., Xie, S., Kershner, J.P., Kolling, F.W., Ringelberg, C.S., LeClerc, A., Alekseyev, Y.O., Fan, J., Podnar, J.W., Love, J.A. , et al. (2020). Multisite Evaluation of Next-Generation Methods for Small RNA Quantification. Journal of Biomolecular Techniques : JBTS [Epub ahead of print]. Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest. Full Text

Herneisen , A.L., Sidik, S.M., Markus, B.M., Drewry, D.H., Zuercher, W.J., and Lourido, S. (2020). Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling. ACS Chemical Biology. Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.Full Text

Hogan , R., Flamier, A., Nardini, E., and Bernier, G. (2020). The Role of BMI1 in Late-Onset Sporadic Alzheimer's Disease. Genes. Late-onset sporadic Alzheimer's disease (LOAD) seems to contain a "hidden" component that cannot be explained by classical Mendelian genetics, with advanced aging being the strongest risk factor. More surprisingly, whole genome sequencing analyses of early-onset sporadic Alzheimer's disease cohorts also revealed that most patients do not present classical disease-associated variants or mutations. In this short review, we propose that BMI1 is possibly epigenetically silenced in LOAD. Reduced BMI1 expression is unique to LOAD compared to familial early-onset AD (EOAD) and other related neurodegenerative disorders; moreover, reduced expression of this single gene is sufficient to reproduce most LOAD pathologies in cellular and animal models. We also show the apparent amyloid and Tau-independent nature of this epigenetic alteration of BMI1 expression. Lastly, examples of the mechanisms underlying epigenetic dysregulation of other LOAD-related genes are also illustrated. Full Text

Hsu, J., Huang, H.T., Lee, C.T., Choudhuri, A., Wilson, N.K., Abraham, B.J., Moignard, V., Kucinski, I., Yu, S., Hyde, R.K., Richard A. Young, et al. (2020). CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation. PNAS Online ahead of print. Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation. Full Text

Huang, Q., Cohen, M.A., Alsina, F.C., Devlin, G., Garrett, A., McKey, J., Havlik, P., Rakhilin, N., Wang, E., Xiang, K., Rudolf Jaenisch, et al. (2020). Intravital imaging of mouse embryos. Science 368, 181-186. Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution. Full Text

Hughes, J.F., Skaletsky, H., Pyntikova, T., Koutseva, N., Raudsepp, T., Lin, D.H., Brown, L.G., Bellott, D.W., Cho, T.J., Dugan-Rocha, S., Khan, Z., David C. Page, et al. (2020). Sequence analysis in Bos taurus reveals pervasiveness of X-Y arms races in mammalian lineages. Genome Research. Published in Advance. Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution. Full Text

Jacobowitz J.R., and Weng, J.K. (2020). Exploring Uncharted Territories of Plant Specialized Metabolism in the Postgenomic Era. Annual review of plant biology 71, 631-658. For millennia, humans have used plants for food, raw materials, and medicines, but only within the past two centuries have we begun to connect particular plant metabolites with specific properties and utilities. Since the utility of classical molecular genetics beyond model species is limited, the vast specialized metabolic systems present in the Earth's flora remain largely unstudied. With an explosion in genomics resources and a rapidly expanding toolbox over the past decade, exploration of plant specialized metabolism in nonmodel species is becoming more feasible than ever before. We review the state-of-the-art tools that have enabled this rapid progress. We present recent examples of de novo biosynthetic pathway discovery that employ various innovative approaches. We also draw attention to the higher-order organization of plant specialized metabolism at subcellular, cellular, tissue, interorgan, and interspecies levels, which will have important implications for the future design of comprehensive metabolic engineering strategies. Full Text

Jiang M., Chavarria, T.E., Yuan, B., Lodish, H.F., and Huang, N.J. (2020).Phosphocholine accumulation and PHOSPHO1 depletion promote adipose tissue thermogenesis. PNAS 117, 15055-15065. Phosphocholine phosphatase-1 (PHOSPHO1) is a phosphocholine phosphatase that catalyzes the hydrolysis of phosphocholine (PC) to choline. Here we demonstrate that the PHOSPHO1 transcript is highly enriched in mature brown adipose tissue (BAT) and is further induced by cold and isoproterenol treatments of BAT and primary brown adipocytes. In defining the functional relevance of PHOPSPHO1 in BAT thermogenesis and energy metabolism, we show that PHOSPHO1 knockout mice are cold-tolerant, with higher expression of thermogenic genes in BAT, and are protected from high-fat diet-induced obesity and development of insulin resistance. Treatment of mice with the PHOSPHO1 substrate phosphocholine is sufficient to induce cold tolerance, thermogenic gene expression, and allied metabolic benefits. Our results reveal a role of PHOSPHO1 as a negative regulator of BAT thermogenesis, and inhibition of PHOSPHO1 or enhancement of phosphocholine represent innovative approaches to manage the metabolic syndrome. Full Text

Kanarek, N., Petrova, B., and Sabatini, D.M. (2020). Dietary modifications for enhanced cancer therapy. Nature 579, 507-517. Tumours depend on nutrients supplied by the host for their growth and survival. Modifications to the host's diet can change nutrient availability in the tumour microenvironment, which might represent a promising strategy for inhibiting tumour growth. Dietary modifications can limit tumour-specific nutritional requirements, alter certain nutrients that target the metabolic vulnerabilities of the tumour, or enhance the cytotoxicity of anti-cancer drugs. Recent reports have suggested that modification of several nutrients in the diet can alter the efficacy of cancer therapies, and some of the newest developments in this quickly expanding field are reviewed here. The results discussed indicate that the dietary habits and nutritional state of a patient must be taken into account during cancer research and therapy. Full Text

Kato, Y., Steiner, T.M., Park, H.Y., Hitchcock, R.O., Zaid, A., Hor, J.L., Devi, S., Davey, G.M., Vremec, D., Tullett, K.M., Hidde L Ploegh, et al. (2020). Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination. Journal of Immunology Online ahead of print. Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination. Full Text

Khalil , Jaenisch, R.,and Mooney, D.J. (2020). Engineered tissues and strategies to overcome challenges in drug development. Advanced Drug Delivery Reviews [Epub ahead of print]. Current preclinical studies in drug development utilize high-throughput in vitro screens to identify lead drug candidates, followed by both in vitro and in vivo models to predict lead candidates' pharmacokinetic and pharmacodynamic properties. The goal of these studies is to reduce the number of lead drug candidates down to the most likely to succeed in later human clinical trials. However, only 1 in 10 drug candidates that emerge from preclinical studies will succeed and become an approved therapeutic. Lack of efficacy or undetected toxicity represents roughly 75% of the causes for these failures, despite these parameters being the primary exclusion criteria in preclinical studies. Recently, advances in both biology and engineering have created new tools for constructing new preclinical models. These models can complement those used in current preclinical studies by helping to create more realistic representations of human tissues in vitro and in vivo. In this review, we describe current preclinical models to identify their value and limitations and then discuss select areas of research where improvements in preclinical models are particularly needed to advance drug development. Following this, we discuss design considerations for constructing preclinical models and then highlight recent advances in these efforts. Taken together, we aim to review the advances as of 2020 surrounding the prospect of biological and engineering tools for adding enhanced biological relevance to preclinical studies to aid in the challenges of failed drug candidates and the burden this poses on the drug development enterprise and thus healthcare. Full Text

Kim C.Y., Mitchell, A.J., Glinkerman, C.M., Li, F.S., Pluskal, T., and Weng, J.K. (2020). The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants. Nature communications 11, 1867. Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.Full Text

Klein I.A., Boija, A., Afeyan, L.K., Hawken, S.W., Fan, M., Dall'Agnese, A., Oksuz, O., Henninger, J.E., Shrinivas, K., Sabari, B.R., et al. (2020). Partitioning of cancer therapeutics in nuclear condensates. Science 368, 1386-1392. The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy. Full Text

Kong, H., Reczek, C.R., McElroy, G.S., Steinert, E.M., Wang, T., Sabatini, D.M., and Chandel, N.S. (2020). Metabolic determinants of cellular fitness dependent on mitochondrial reactive oxygen species. Science Advances Vol. 6, no. 45, eabb7272. Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness. Full Text

Kory, N., Uit de Bos, J., van der Rijt, S., Jankovic, N., Güra, M., Arp, N., Pena, I.A., Prakash, G., Chan, S.H., Kunchok, T., Lewis, C.A. , and David M. Sabatini (2020). MCART1/SLC25A51 is required for mitochondrial NAD transport. Science Advances 6(43):eabe5310. The nicotinamide adenine dinucleotide (NAD(+)/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to adenosine triphosphate generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD(+) transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as coessential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD(+) and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD(+) transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells. Full Text

Krishnan, A., Kloehn, J., Lunghi, M., Chiappino-Pepe, A., Waldman, B.S., Nicolas, D., Varesio, E., Hehl, A., Lourido, S., Hatzimanikatis, V., et al. (2020). Functional and Computational Genomics Reveal Unprecedented Flexibility in Stage-Specific Toxoplasma Metabolism. Cell host & Microbe[Epub ahead of print]. To survive and proliferate in diverse host environments with varying nutrient availability, the obligate intracellular parasite Toxoplasma gondii reprograms its metabolism. We have generated and curated a genome-scale metabolic model (iTgo) for the fast-replicating tachyzoite stage, harmonized with experimentally observed phenotypes. To validate the importance of four metabolic pathways predicted by the model, we have performed in-depth in vitro and in vivo phenotyping of mutant parasites including targeted metabolomics and CRISPR-Cas9 fitness screening of all known metabolic genes. This led to unexpected insights into the remarkable flexibility of the parasite, addressing the dependency on biosynthesis or salvage of fatty acids (FAs), purine nucleotides (AMP and GMP), a vitamin (pyridoxal-5P), and a cofactor (heme) in both the acute and latent stages of infection. Taken together, our experimentally validated metabolic network leads to a deeper understanding of the parasite's biology, opening avenues for the development of therapeutic intervention against apicomplexans. Full Text

*Kumari, S., Mak, M., Poh, Y.C., Tohme, M., Watson, N., Melo, M., Janssen, E., Dustin, M., Geha, R., and Irvine, D.J. (2020). Cytoskeletal tension actively sustains the migratory T-cell synaptic contact. The EMBO Journal, e102783.[Epub ahead of print]. When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact. Full Text

Kurley, S.J., Tischler, V., Bierie, B., Novitskiy, S.V., Noske, A., Varga, Z., Zürrer-Härdi, U., Brandt, S., Carnahan, R.H., Cook, R.S., et al. (2020). A Requirement for p120-catenin in the metastasis of invasive ductal breast cancer. Journal of cell science (Online ahead of print) . We have examined the effects of targeted p120 KO in a PyMT mouse model of invasive ductal (mammary) cancer (IDC). Mosaic p120 ablation had little effect on primary tumor growth but caused significant pro-metastatic alterations in the tumor microenvironment leading ultimately to a marked increase in the number and size of pulmonary metastases. Surprisingly, although early effects of p120-ablation included decreased cell-cell adhesion and increased invasiveness, cells lacking p120 were almost entirely unable to colonized distant metastatic sites in vivo. The relevance of this observation to human IDC was established by analysis of a large clinical dataset of 1126 IDCs. As reported by others, p120 downregulation in primary IDC predicted worse overall survival. However, as in the mice, distant metastases were almost invariably p120 positive, even in matched cases where the primary tumors were p120 negative. Collectively, our results demonstrate a strong positive role for p120 (and presumably E-cadherin) during metastatic colonization of distant sites. On the other hand, downregulation of p120 in the primary tumor enhanced metastatic dissemination indirectly via pro-metastatic conditioning of the tumor microenvironment. Full Text

Li , C.H., Coffey, E.L., Dall'Agnese, A., Hannett, N.M., Tang, X., Henninger, J.E., Platt, J.M., Oksuz, O., Zamudio, A.V., Afeyan, L.K., et al. (2020). MeCP2 links heterochromatin condensates and neurodevelopmental disease. Nature. MeCP2 (methyl CpG binding protein 2) is a key component of constitutive heterochromatin, which plays important roles in chromosome maintenance and transcriptional silencing(1-3). Mutations in MeCP2 cause Rett syndrome (RTT)(3-5), a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like symptoms that manifests in girls during early childhood. Heterochromatin, long considered a dense and relatively static structure(1,2), is now understood to exhibit properties consistent with a liquid-like condensate(6,7). Here we report that MeCP2 is a dynamic component of heterochromatin condensates in cells, is stimulated by DNA to form liquid-like condensates, contains multiple domains that contribute to condensate formation, manifests physicochemical properties that selectively concentrate heterochromatin cofactors compared to components of transcriptionally active condensates, and when altered by RTT-causing mutations is disrupted in its ability to form condensates. We propose that MeCP2 enhances heterochromatin/euchromatin separation through its condensate partitioning properties and that condensate disruption may be a common consequence of mutations found in patients with RTT. Full Text

Li, M.Y., Naik, T.S., Siu, L.Y.L., Acuto, O., Spooner, E., Wang, P., Yang, X., Lin, Y., Bruzzone, R., Ashour, J., et al. (2020). Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles. Nature Communications 11(1):5189. Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn(-/-) cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations - one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism. Full Text

Li , X., Wang, J., Wang, L., Feng, G., Li, G., Yu, M., Li, Y., Liu, C., Yuan, X., Zang, G., et al. (2020). Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging. PNAS [Epub ahead of print]. Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.Full Text

Li, Y., Ivica, N.A., Dong, T., Papageorgiou, D.P., He, Y., Brown, D.R., Kleyman, M., Hu, G., Chen, W.W., Sullivan, L.B., et al. (2020). MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme. Nature Communications 11 : 4837. ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.Full Text

Lin, B., Luo, J., and Lehmann, R. (2020). Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo.Nature Communications 11 : 4477. Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Here, using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), we show that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. We demonstrate that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease. Full Text

Liston S.D., Whitesell, L., McLellan, C.A., Mazitschek, R., Petraitis, V., Petraitiene, R., Kavaliauskas, P., Walsh, T.J., and Cowen, L.E. (2020). Antifungal activity of gepinacin-scaffold GPI anchor biosynthesis inhibitors with improved metabolic stability. Antimicrobial Agents and Chemotherapy. The GPI-anchor biosynthesis inhibitor gepinacin demonstrates broad-spectrum antifungal activity and negligible mammalian toxicity in culture but is metabolically labile. The stability and bioactivity of 39 analogs were tested in vitro, to identify LCUT-08, a stabilized lead with increased potency and promising single-dose pharmacokinetics. Unfortunately, no antifungal activity was seen at the maximum dosing achievable in a neutropenic rabbit model. Nevertheless, structure-activity relationships identified here suggest strategies to further improve compound potency, solubility, and stability. Full Text

*Liu , G.Y., and Sabatini, D.M. (2020). mTOR at the nexus of nutrition, growth, ageing and disease. Nature Reviews Molecular Cell Biology[Epub ahead of print].The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention. Full Text

Lodish , H.F. (2020). Over 60 Years of Experimental Hematology (without a License). Experimental Hematology. Available online.. I am deeply honored to receive the International Society for Experimental Hematology (ISEH) 2020 Donald Metcalf Lecture Award. Although I am not a physician and have had no formal training in hematology, I have had the privilege of working with some of the top hematologists in the world, beginning in 1970 when Dr. David Nathan was a sabbatical visitor in my laboratory and introduced me to hematological diseases.  And I take this award to be given not just to me but to an exceptional group of MD and PhD trainees and visitors in my laboratory who have cloned and characterized many proteins and RNAs important for red cell development and function. Many of these projects involved taking exceptionally large risks in developing and employing novel experimental technologies. Unsurprisingly, all of these trainees have gone on to become leaders in hematology and, more broadly, in molecular cell biology and molecular medicine. To illustrate some of the challenges we have faced and the technologies we had to develop, I have chosen several of our multiyear projects to describe in some detail: elucidating the regulation of translation of α- and β-globin mRNAs and the defect in beta thalassemia in the 1970s; cloning the Epo receptor and several red cell membrane proteins in the 1980s and 1990s; and more recently, determining the function of many microRNAs and long noncoding RNAs in red cell development. I summarize how we are currently utilizing single-cell transcriptomics (scRNAseq) to understand how dividing transit-amplifying burst-forming unit erythroid progenitors balances the need for more progenitor cells with the need for terminally differentiated erythroid cells, and to identify drugs potentially useful in treating Epo-resistant anemias such as Diamond Blackfan anemia. I hope that the lessons I learned in managing these diverse fellows and projects, initially without having grants to support them, will be helpful to others who would like to undertake ambitious and important lines of research in hematology.

Lokapally , A., Neuhaus, H., Herfurth, J., and Hollemann, T. (2020). Interplay of TRIM2 E3 Ubiquitin Ligase and ALIX/ESCRT Complex: Control of Developmental Plasticity During Early Neurogenesis. Cells. Tripartite motif 2 (TRIM2) drives neurite outgrowth and polarization, is involved in axon specification, and confers neuroprotective functions during rapid ischemia. The mechanisms controlling neuronal cell fate determination and differentiation are fundamental for neural development. Here, we show that in Xenopus, trim2 knockdown affects primary neurogenesis and neural progenitor cell survival. Embryos also suffer from severe craniofacial malformation, a reduction in brain volume, and the loss of motor sensory function. Using a high-throughput LC-MS/MS approach with GST-Trim2 as bait, we pulled down ALG-2 interacting protein X (Alix) from Xenopus embryonic lysates. We demonstrate that the expression of trim2/TRIM2 and alix/ALIX overlap during larval development and on a cellular level in cell culture. Interestingly, trim2 morphants showed a clustering and apoptosis of neural progenitors, which are phenotypic hallmarks that are also observed in Alix KO mice. Therefore, we propose that the interaction of Alix and Trim2 plays a key role in the determination and differentiation of neural progenitors via the modulation of cell proliferation/apoptosis during neurogenesis. Full Text

Luengo, A., Li, Z., Gui, D.Y., Sullivan, L.B., Zagorulya, M., Do, B.T., Ferreira, R., Naamati, A., Ali, A., Lewis, C.A., et al. (2020). Increased demand for NAD(+) relative to ATP drives aerobic glycolysis. Molecular Cell. Online ahead of Print. Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD(+)/NADH ratio. This change in NAD(+)/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD(+) regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD(+)/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD(+) to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD(+) regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD(+) is in excess of the demand for ATP. Full Text

Ma, H., Jeppesen, J.F., and Jaenisch, R. (2020). Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive β Cell Destruction. Cell Reports Medicine 1(6):100097. Autoimmune destruction of pancreatic β cells underlies type 1 diabetes (T1D). To understand T cell-mediated immune effects on human pancreatic β cells, we combine β cell-specific expression of a model antigen, CD19, and anti-CD19 chimeric antigen receptor T (CAR-T) cells. Coculturing CD19-expressing β-like cells and CD19 CAR-T cells results in T cell-mediated β-like cell death with release of activated T cell cytokines. Transcriptome analysis of β-like cells and human islets treated with conditioned medium of the immune reaction identifies upregulation of immune reaction genes and the pyroptosis mediator GSDMD as well as its activator CASP4. Caspase-4-mediated cleaved GSDMD is detected in β-like cells under inflammation and endoplasmic reticulum (ER) stress conditions. Among immune-regulatory genes, PDL1 is one of the most upregulated, and PDL1 overexpression partially protects human β-like cells transplanted into mice. This experimental platform identifies potential mechanisms of β cell destruction and may allow testing of therapeutic strategies.Full Text

Mace, K., Krakowiak, J., El-Samad, H., and Pincus, D. (2020). Multi-kinase control of environmental stress responsive transcription. PloS One 15, e0230246. Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling. Full Text

Majumder , S., and Jain, A. (2020) Osmotic Stress Triggers Phase Separation Molecular Cell 79 (6): 876-877. In this issue of Molecular Cell, Jalihal et al. (2020) show that cell volume changes upon osmotic stress result in rapid and reversible condensation of numerous multivalent proteins.Full Text

Marescal, O., and Cheeseman, I.M. (2020). Cellular Mechanisms and Regulation of Quiescence. Developmental Cell 55(3):259-271. Quiescence is a state of reversible proliferative arrest in which cells are not actively dividing and yet retain the capacity to reenter the cell cycle upon receiving an appropriate stimulus. Quiescent cells are remarkably diverse-they reside in different locations throughout the body, serve distinct roles, and are activated by a variety of signals. Despite this diversity, all quiescent cells must be able to persist in a nondividing state without compromising their proliferative potential, which requires changes to core cellular programs. How drastically different cell types are able to implement extensive changes to their gene-expression programs, metabolism, and cellular structures to induce a common cellular state is a fascinating question in cell and developmental biology. In this review, we explore the diversity of quiescent cells and highlight the unifying characteristics that define the quiescent state.Full Text

Markus, B.M., Waldman, B.S., Lorenzi, H.A., and Lourido, S. (2020). High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii. Frontiers in Cellular and Infection Microbiology 10:617998. Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5'-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5' termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5' leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite. Full Text

Matsumoto, T., Harima, S., Weng, J.K., and Nihei, K. (2020). Systematic approach to the chemical synthesis of arabidopyrones, the unique alpha-pyrones of Arabidopsis metabolites. Synthetic Communications (Published online). Total synthesis of two arabidopyrones,iso-arabidopyl alcohol (1) andiso-arabidopic acid (2) isolated fromArabidopsis thalianawas achieved for the first time using Claisen condensation and Wittig reaction as the key steps. In addition, arabidopic acid (4) was synthesized from the methyl ester of arabidopyl alcohol (3). Thus, chemical synthesis of the unique natural alpha-pyrones1-4was accomplished with a short synthetic route by a systematic approach from readily available substances.Full Text

Melnik, A., Cappelletti, V., Vaggi, F., Piazza, I., Tognetti, M., Schwarz, C., Cereghetti, G., Ahmed, M.A., Soste, M., Matlack, K., et al. (2020). Comparative analysis of the intracellular responses to disease-related aggregation-prone proteins. Journal of proteomics 225, 103862. Aggregation-prone proteins (APPs) have been implicated in numerous human diseases but the underlying mechanisms are incompletely understood. Here we comparatively analysed cellular responses to different APPs. Our study is based on a systematic proteomic and phosphoproteomic analysis of a set of yeast proteotoxicity models expressing different human disease-related APPs, which accumulate intracellular APP inclusions and exhibit impaired growth. Clustering and functional enrichment analyses of quantitative proteome-level data reveal that the cellular response to APP expression, including the chaperone response, is specific to the APP, and largely differs from the response to a more generalized proteotoxic insult such as heat shock. We further observe an intriguing association between the subcellular location of inclusions and the location of the cellular response, and provide a rich dataset for future mechanistic studies. Our data suggest that care should be taken when designing research models to study intracellular aggregation, since the cellular response depends markedly on the specific APP and the location of inclusions. Further, therapeutic approaches aimed at boosting protein quality control in protein aggregation diseases should be tailored to the subcellular location affected by inclusion formation. SIGNIFICANCE: We have examined the global cellular response, in terms of protein abundance and phosphorylation changes, to the expression of five human neurodegeneration-associated, aggregation-prone proteins (APPs) in a set of isogenic yeast models. Our results show that the cellular response to each APP is unique to that protein, is different from the response to thermal stress, and is associated with processes at the subcellular location of APP inclusion formation. These results further our understanding of how cells, in a model organism, respond to expression of APPs implicated in neurodegenerative diseases like Parkinson's, Alzheimer's, and ALS. They have implications for mechanisms of toxicity as well as of protective responses in the cell. The specificity of the response to each APP means that research models of these diseases should be tailored to the APP in question. The subcellular localization of the response suggest that therapeutic interventions should also be targeted within the cell. Full Text

Michowski, W., Chick, J.M., Chu, C., Kolodziejczyk, A., Wang, Y., Suski, J.M., Abraham, B., Anders, L., Day, D., Dunkl, L.M., Richard A Young, et al. (2020). Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells. Molecular Cell 78, 459-476. The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs. Full Text

Mikedis, M.M., Fan, Y., Nicholls, P.K., Endo, T., Jackson, E.K., Cobb, S.A., de Rooij, D.G., and Page, D.C. (2020). DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors. eLife 9:e56523. Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3' UTRs of ~2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3' UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia. Full Text

Miller-Vedam, L.E., Bräuning, B., Popova, K.D., Schirle Oakdale, N.T., Bonnar, J.L., Prabu, J.R., Boydston, E.A., Sevillano, N., Shurtleff, M.J., Stroud, R.M., et al. (2020). Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients. eLife 9:e62611. Online ahead of print. Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes. Full Text

Moyer, T.J., Kato, Y., Abraham, W., Chang, J.Y.H., Kulp, D.W., Watson, N., Turner, H.L., Menis, S., Abbott, R.K., Bhiman, J.N., et al. (2020). Engineered immunogen binding to alum adjuvant enhances humoral immunity. Nature Medicine [Epub ahead of print]. Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant. Full Text

Murai , N., Mitalipova, M., and Jaenisch, R. (2020). Functional analysis of CX3CR1 in human induced pluripotent stem (iPS) cell-derived microglia-like cells. The European Journal of Neuroscience. Microglia are the primary immune cells of the central nervous system and crucial to proper development and maintenance of the brain. Microglia have been recognized to be associated with neurodegenerative diseases and neuroinflammatory disorders. CX3C chemokine receptor 1 (CX3CR1), which is specifically expressed in microglia, regulates microglia homeostatic functions such as microglial activation and is downregulated in aged brain and disease associated microglia in rodents, yet its role in human microglia is not fully understood. In this study, we investigated the function of CX3CR1 in human microglia using human induced pluripotent stem (iPS) cell-derived microglia-like cells. Human iPS cell-derived microglia-like cells expressed microglial markers, and showed an activated state and phagocytic activity. Using CRISPR/Cas9 genome editing we deleted CX3CR1 in human iPS cells and found an increased inflammatory responses and phagocytic activity in mutant as compared to wild-type microglia-like cells. In addition, the CX3C chemokine ligand 1 (CX3CL1, a ligand for CX3CR1) significantly decreased the upregulation of IL-6 by lipopolysaccharide stimulation in human iPS cell-derived microglia-like cells. These results suggest that CX3CR1 in human microglia may contribute to microglial homeostasis by regulating inflammatory response and phagocytosis. Full Text

Narayan , P., Sienski, G., Bonner, J.M., Lin, Y.T., Seo, J., Baru, V., Haque, A., Milo, B., Akay, L.A., Graziosi, A., Susan Lindquist, et al. (2020). PICALM Rescues Endocytic Defects Caused by the Alzheimer's Disease Risk Factor APOE4. Cell Reports 33 (1): 108224. The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including late-onset Alzheimer's disease (AD). We investigate the cellular consequences of APOE4 in human iPSC-derived astrocytes, observing an endocytic defect in APOE4 astrocytes compared with their isogenic APOE3 counterparts. Given the evolutionarily conserved nature of endocytosis, we built a yeast model to identify genetic modifiers of the endocytic defect associated with APOE4. In yeast, only the expression of APOE4 results in dose-dependent defects in both endocytosis and growth. We discover that increasing expression of the early endocytic adaptor protein Yap1802p, a homolog of the human AD risk factor PICALM, rescues the APOE4-induced endocytic defect. In iPSC-derived human astrocytes, increasing expression of PICALM similarly reverses endocytic disruptions. Our work identifies a functional interaction between two AD genetic risk factors-APOE4 and PICALM-centered on the conserved biological process of endocytosis. Full Text

Navarro, A.P., and Cheeseman, I.M. (2020). Chromosome Segregation: Evolving a Plastic Chromosome-Microtubule Interface. Current Biology : CB 30(4):R174-R17.Despite a conserved requirement in mediating chromosome segregation, kinetochores display remarkable plasticity in their structure and composition. New work in holocentric insect species highlights the molecular rewiring that occurs when key structural components of the kinetochore are lost and centromere structure is changed. Full Text

Ngo, B., Kim, E., Osorio-Vasquez, V., Doll, S., Bustraan, S., Liang, R.J., Luengo, A., Davidson, S.M., Ali, A., Ferraro, G.B., David M. Sabatini, et al. (2020). Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition. Cancer Discovery 10, 1352-1373. A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis. Full Text

Nothias, L.F., Petras, D., Schmid, R., Dührkop, K., Rainer, J., Sarvepalli, A., Protsyuk, I., Ernst, M., Tsugawa, H., Fleischauer, M., Tomáš Pluskal, et al. (2020). Feature-based molecular networking in the GNPS analysis environment. Nature Methods 17, 905-908. Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry. Full Text

Ognjenovic, N.B., Bagheri, M., Mohamed, G.A., Xu, K., Chen, Y., Mohamed Saleem, M.A., Brown, M.S., Nagaraj, S.H., Muller, K.E., Gerber, S.A., et al. (2020). Limiting Self-Renewal of the Basal Compartment by PKA Activation Induces Differentiation and Alters the Evolution of Mammary Tumors. Developmental Cell (In Press, Corrected Proof). Differentiation therapy utilizes our understanding of the hierarchy of cellular systems to pharmacologically induce a shift toward terminal commitment. While this approach has been a paradigm in treating certain hematological malignancies, efforts to translate this success to solid tumors have met with limited success. Mammary-specific activation of PKA in mouse models leads to aberrant differentiation and diminished self-renewing potential of the basal compartment, which harbors mammary repopulating cells. PKA activation results in tumors that are more benign, exhibiting reduced metastatic propensity, loss of tumor-initiating potential, and increased sensitivity to chemotherapy. Analysis of tumor histopathology revealed features of overt differentiation with papillary characteristics. Longitudinal single-cell profiling at the hyperplasia and tumor stages uncovered an altered path of tumor evolution whereby PKA curtails the emergence of aggressive subpopulations. Acting through the repression of SOX4, PKA activation promotes tumor differentiation and represents a possible adjuvant to chemotherapy for certain breast cancers. Full Text

Orozco , J.M., Krawczyk, P.A., Scaria, S.M., Cangelosi, A.L., Chan, S.H., Kunchok, T., Lewis, C.A., and Sabatini, D.M. (2020). Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nature Metabolism. The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1. Full Text

Perdomo-Pantoja, A., Holmes, C., Cottrill, E., Rindone, A.N., Ishida, W., Taylor, M., Tomberlin, C., Lo, S.L., Grayson, W.L., and Witham, T.F. (2020). Comparison of Freshly Isolated Adipose Tissue-derived Stromal Vascular Fraction and Bone Marrow Cells in a Posterolateral Lumbar Spinal Fusion Model. Spine. Published Ahead-of-Print. STUDY DESIGN: Rat posterolateral lumbar fusion model. OBJECTIVE: To compare the efficacy of freshly isolated adipose tissue-derived stromal vascular fraction (A-SVF) and bone marrow cells (BMC) cells in achieving spinal fusion in a rat model. SUMMARY OF BACKGROUND DATA: Adipose tissue-derived stromal cells (ASCs) offer advantages as a clinical cell source compared to bone marrow-derived stromal cells (BMSCs), including larger available tissue volumes and reduced donor site morbidity. While pre-clinical studies have shown that ex vivo expanded ASCs can be successfully used in spinal fusion, the use of A-SVF cells better allows for clinical translation. METHODS: A-SVF cells were isolated from the inguinal fat pads, while BMC were isolated from the long bones of syngeneic 6-8-week-old Lewis rats and combined with Vitoss (Stryker) bone graft substitute for subsequent transplantation. Posterolateral spinal fusion surgery at L4-L5 was performed on 36 female Lewis rats divided into 3 experimental groups: [1] Vitoss bone graft substitute only (VO group); [2] Vitoss + 2.5x10 A-SVF cells/side; and, [3] Vitoss + 2.5x10 BMC/side. Fusion was assessed eight weeks post-surgery via manual palpation, micro-computed tomography (μCT) imaging, and histology. RESULTS: μCT imaging analyses revealed that fusion volumes and μCT fusion scores in the A-SVF group were significantly higher than in the VO group; however, they were not significantly different between the A-SVF group and the BMC group. The average manual palpation score was highest in the A-SVF group compared with the BMC and VO groups. Fusion masses arising from cell-seeded implants yielded better bone quality than non-seeded bone graft substitute. CONCLUSIONS: In a rat model, A-SVF cells yielded a comparable fusion mass volume and radiographic rate of fusion to BMC when combined with a clinical-grade bone graft substitute. These results suggest the feasibility of using freshly isolated A-SVF cells in spinal fusion procedures. Full Text

Perea-Resa, C., Bury, L., Cheeseman, I.M., and Blower, M.D. (2020). Cohesin Removal Reprograms Gene Expression upon Mitotic Entry. Molecular Cell [Epub ahead of print]. As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1. Full Text

Picard , C.L., and Gehring, M. (2020). Identification and Comparison of Imprinted Genes Across Plant Species. Methods in Molecular Biology 2093, 173-201.Genomic imprinting is a phenomenon that occurs in flowering plants and mammals, whereby a gene is expressed in a parent-of-origin-specific manner. Although imprinting has now been examined genome-wide in a number of species using RNA-seq, the analyses used to assess imprinting vary between studies, making consistent comparisons between species difficult. Here we present a simple, easy-to-use bioinformatic pipeline for imprinting analyses suitable for any tissue, including plant endosperm. All relevant scripts can be downloaded. As an illustrative example, we reanalyze published data from A. thaliana and Z. mays endosperm using the pipeline and then demonstrate how to use the results to assess the conservation of imprinting between these species. We also introduce the Plant Imprinting Database, a repository for published imprinting datasets in plants that can be used to view, compare, and download data. Full Text

Pluskal, T., Hoffmann, N., Du, X., and Weng, J.-K. (2020). Mass Spectrometry Development Kit (MSDK): a Java Library for Mass Spectrometry Data Processing. Processing Metabolomics and Proteomics Data with Open Software: A Practical Guide , pp. 399-405.

Povilus , R.A., DaCosta, J.M., Grassa, C., Satyaki, P.R.V., Moeglein, M., Jaenisch, J., Xi, Z., Mathews, S., Gehring, M., Davis, C.C., et al. (2020). Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses. PNAS 117(15): 8649-8656. For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms approximately 140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution. Full Text

Qiu, Q., Hu, P., Qiu, X., Govek, K.W., Cámara, P.G., and Wu, H. (2020). Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq. Nature Methods Online ahead of print. Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.Full Text

Ray G.J., Boydston, E.A., Shortt, E., Wyant, G.A., Lourido, S., Chen, W.W., and Sabatini, D.M. (2020). A PEROXO-Tag Enables Rapid Isolation of Peroxisomes from Human Cells. iScience 23, 101109. Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes. Full Text

Rubinfien, J., Atabay, K.D., Nichols, N.M., Tanner, N.A., Pezza, J.A., Gray, M.M., Wagner, B.M., Poppin, J.N., Aken, J.T., Gleason, E.J., et al. (2020). Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP). FASEB BioAdvances 2, 160-165. Human spaceflight endeavors present an opportunity to expand our presence beyond Earth. To this end, it is crucial to understand and diagnose effects of long-term space travel on the human body. Developing tools for targeted, on-site detection of specific DNA sequences will allow us to establish research and diagnostics platforms that will benefit space programs. We describe a simple DNA diagnostic method that utilizes colorimetric loop-mediated isothermal amplification (LAMP) to enable detection of a repetitive telomeric DNA sequence in as little as 30 minutes. A proof of concept assay for this method was carried out using existing hardware on the International Space Station and the results were read instantly by an astronaut through a simple color change of the reaction mixture. LAMP offers a novel platform for on-orbit DNA-based diagnostics that can be deployed on the International Space Station and to the broader benefit of space programs. Full Text

Sabari , B.R., Dall'Agnese, A., and Young, R.A. (2020). Biomolecular Condensates in the Nucleus.Trends in biochemical sciences . Nuclear processes such as DNA replication, transcription, and RNA processing each depend on the concerted action of many different protein and RNA molecules. How biomolecules with shared functions find their way to specific locations has been assumed to occur largely by diffusion-mediated collisions. Recent studies have shown that many nuclear processes occur within condensates that compartmentalize and concentrate the protein and RNA molecules required for each process, typically at specific genomic loci. These condensates have common features and emergent properties that provide the cell with regulatory capabilities beyond canonical molecular regulatory mechanisms. We describe here the shared features of nuclear condensates, the components that produce locus-specific condensates, elements of specificity, and the emerging understanding of mechanisms regulating these compartments. Full Text

Santoriello, C., Sporrij, A., Yang, S., Flynn, R.A., Henriques, T., Dorjsuren, B., Custo Greig, E., McCall, W., Stanhope, M.E., Fazio, M., Brian J Abraham , et al. (2020). RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature Cell Biology 22, 372-379. The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo(1). To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln(z24)) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. Full Text

Schlissel , G., and Li, P. (2020). Synthetic Developmental Biology: Understanding Through Reconstitution. Annual Review of Cell and Developmental Biology 36, 339-357. Reconstitution is an experimental strategy that seeks to recapitulate biological events outside their natural contexts using a reduced set of components. Classically, biochemical reconstitution has been extensively applied to identify the minimal set of molecules sufficient for recreating the basic chemistry of life. By analogy, reconstitution approaches to developmental biology recapitulate aspects of developmental events outside an embryo, with the goal of revealing the basic genetic circuits or physical cues sufficient for recreating developmental decisions. The rapidly growing repertoire of genetic, molecular, microscopic, and bioengineering tools is expanding the complexity and precision of reconstitution experiments. We review the emerging field of synthetic developmental biology, with a focus on the ways in which reconstitution strategies and new biological tools have enhanced our modern understanding of fundamental questions in developmental biology.Full Text

Scimone M.L., Atabay, K.D., Fincher, C.T., Bonneau, A.R., Li, D.J., and Reddien, P.W., (2020). Muscle and neuronal guidepost-like cells facilitate planarian visual system regeneration. Science. Neuronal circuits damaged or lost after injury can be regenerated in some adult organisms, but the mechanisms enabling this process are largely unknown. We used the planarian Schmidtea mediterranea to study visual system regeneration after injury. We identify a rare population of muscle cells tightly associated with photoreceptor axons at stereotyped positions in both uninjured and regenerating animals. Together with a neuronal population, these cells promote de novo assembly of the visual system in diverse injury and eye transplantation contexts. These muscle guidepost-like cells are specified independently of eyes, and their position is defined by an extrinsic array of positional information cues. These findings provide a mechanism, involving adult formation of guidepost-like cells typically observed in embryos, for axon pattern restoration in regeneration. Full Text

Seebacher, F., Zeigerer, A., Kory, N., and Krahmer, N. (2020). Hepatic lipid droplet homeostasis and fatty liver disease. Seminars in Cell & Developmental Biology . In cells, lipids are stored in lipid droplets, dynamic organelles that adapt their size, abundance, lipid and protein composition and organelle interactions to metabolic changes. Lipid droplet accumulation in the liver is the hallmark of non-alcoholic fatty liver disease (NAFLD). Due to the prevalence of obesity, the strongest risk factor for steatosis, NAFLD and its associated complications are currently affecting more than 1 billion people worldwide. Here, we review how triglyceride and phospholipid homeostasis are regulated in hepatocytes and how imbalances between lipid storage, degradation and lipoprotein secretion lead to NAFLD. We discuss how organelle interactions are altered in NAFLD and provide insights how NAFLD progression is associated with changes in hepatocellular signaling and organ-crosstalk. Finally, we highlight unsolved questions in hepatic LD and lipoprotein biology and give an outlook on therapeutic options counteracting hepatic lipid accumulation.

Seiglie, J., Platt, J., Cromer, S.J., Bunda, B., Foulkes, A.S., Bassett, I.V., Hsu, J., Meigs, J.B., Leong, A., Putman, M.S., et al. (2020). Diabetes as a Risk Factor for Poor Early Outcomes in Patients Hospitalized With COVID-19. Diabetes care. Early edition. Diabetes and obesity are highly prevalent among hospitalized patients with coronavirus disease 2019 (COVID-19), but little is known about their contributions to early COVID-19 outcomes. We tested the hypothesis that diabetes is a risk factor for poor early outcomes, after adjustment for obesity, among a cohort of patients hospitalized with COVID-19. RESEARCH DESIGN AND METHODS: We used data from the Massachusetts General Hospital (MGH) COVID-19 Data Registry of patients hospitalized with COVID-19 between 11 March 2020 and 30 April 2020. Primary outcomes were admission to the intensive care unit (ICU), need for mechanical ventilation, and death within 14 days of presentation to care. Logistic regression models were adjusted for demographic characteristics, obesity, and relevant comorbidities. RESULTS: Among 450 patients, 178 (39.6%) had diabetes-mostly type 2 diabetes. Among patients with diabetes versus patients without diabetes, a higher proportion was admitted to the ICU (42.1% vs. 29.8%, respectively, P = 0.007), required mechanical ventilation (37.1% vs. 23.2%, P = 0.001), and died (15.9% vs. 7.9%, P = 0.009). In multivariable logistic regression models, diabetes was associated with greater odds of ICU admission (odds ratio 1.59 [95% CI 1.01-2.52]), mechanical ventilation (1.97 [1.21-3.20]), and death (2.02 [1.01-4.03]) at 14 days. Obesity was associated with greater odds of ICU admission (2.16 [1.20-3.88]) and mechanical ventilation (2.13 [1.14-4.00]) but not with death. CONCLUSIONS: Among hospitalized patients with COVID-19, diabetes was associated with poor early outcomes, after adjustment for obesity. These findings can help inform patient-centered care decision making for people with diabetes at risk for COVID-19. Abstract

Seranova, E., Palhegyi, A.M., Verma, S., Dimova, S., Lasry, R., Naama, M., Sun, C., Barrett, T., Rosenstock, T.R., Kumar, D., Cohen, M.A., et al. (2020). Human induced pluripotent stem cell models of neurodegenerative disorders for studying the biomedical implications of autophagy. Journal of Molecular Biology [Epub ahead of print]. Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers as well as the nature of the autophagy defects may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell-types such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modelling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation, and outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models. Full Text

Shi, C.Y., Kingston, E.R., Kleaveland, B., Lin, D.H., Stubna, M.W., and Bartel, D.P. (2020). The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation. Science. Online ahead of print. MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread post-transcriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. Here we found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings suggested and supported a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin-proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals. Full Text

*Shortt E., and Lourido, S., (2020). Plate-Based Quantification of Stimulated Toxoplasma Egress. Methods in Molecular Biology 2071, 171-186. Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipulated genetically or pharmacologically. Here we describe two methods of assessing stimulated parasite egress from host cells by measuring the permeabilization of host-cell membranes that occurs during this process. The first method measures the release of lactate dehydrogenase (LDH) from host cells, which is quantified in a colorimetric assay that detects LDH by the enzymatic generation of red formazan. The second method measures entry of the cell-impermeant 4',6-diamidino-2-phenylindole (DAPI) DNA dye, which stains host-cell nuclei (HCN) as parasites egress. Both described methods complement, with higher throughput, video-microscopy approaches that are well suited to examine the dissociation of parasite vacuoles that follows host-cell permeabilization. Full Text

Sivakumaren, S.C., Shim, H., Zhang, T., Ferguson, F.M., Lundquist, M.R., Browne, C.M., Seo, H.S., Paddock, M.N., Manz, T.D., Jiang, B., Kwiatkowski, N.P., et al. (2020). Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors. Cell Chemical Biology [Epub ahead of print]. The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kalpha/beta/gamma. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders. Full Text

Surface, L.E., Burrow, D.T., Li, J., Park, J., Kumar, S., Lyu, C., Song, N., Yu, Z., Rajagopal, A., Bae, Y., David M. Sabatini, et al. (2020). ATRAID regulates the action of nitrogen-containing bisphosphonates on bone. Science Translational Medicine. Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.Full Text

Tao T., Shi, H., Mariani, L., Abraham, B.J., Durbin, A.D., Zimmerman, M.W., Powers, J.T., Missios, P., Ross, K.N., Perez-Atayde, A.R., Richard A Young, et al. (2020). LIN28B regulates transcription and potentiates MYCN-induced neuroblastoma through binding to ZNF143 at target gene promotors. PNAS. LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7-independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis. Full Text

Tomasello D.L., and Sive, H. (2020). Noninvasive Multielectrode Array for Brain and Spinal Cord Local Field Potential Recordings from Live Zebrafish Larvae. Zebrafish (Online ahead of print). Zebrafish are an important and expanding experimental system for brain research. We describe a noninvasive electrophysiology technique that can be used in living larvae to measure spontaneous activity in the brain and spinal cord simultaneously. This easy-to-use method uses a commercially available multielectrode array to detect local field potential parameters, and allows for relative coordinated (network) measurements of activity. We demonstrate sensitivity of this system by measuring activity in larvae treated with the antiepileptic drug valproic acid. Valproic acid decreased larval movement and startle response, and decreased spontaneous brain activity. Spinal cord activity did not change after treatment, suggesting valproic acid primarily affects brain function. The observed differences in brain activity, but not spinal cord activity, after valproic acid treatment indicates that brain activity differences are not a secondary effect of decreased startle response and movement. We provide a step-by-step protocol for experiments presented that a novice could easily follow. This electrophysiological method will be useful to the zebrafish neuroscience community. Full Text

Tomezsko , P.J., Corbin, V.D.A., Gupta, P., Swaminathan, H., Glasgow, M., Persad, S., Edwards, M.D., McIntosh, L., Papenfuss, A.T., Emery, A.,Tammy C. T. Lan, and Silvi Rouskin (2020). Determination of RNA structural diversity and its role in HIV-1 RNA splicing. Nature 582, 438-442. Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors(1,2). Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements(3). Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages(4), our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized(5) alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis(6-8) that heterogeneity in RNA conformation regulates splice-site use and viral gene expression. Full Text

Tomezsko , P., Swaminathan, H., and Rouskin, S. (2020). Viral RNA structure analysis using DMS-MaPseq. Methods RNA structure is critically important to RNA viruses in every part of the replication cycle. RNA structure is also utilized by DNA viruses in order to regulate gene expression and interact with host factors. Advances in next-generation sequencing have greatly enhanced the utility of chemical probing in order to analyze RNA structure. This review will cover some recent viral RNA structural studies using chemical probing and next-generation sequencing as well as the advantages of dimethyl sulfate (DMS)-mutational profiling and sequencing (MaPseq). DMS-MaPseq is a robust assay that can easily modify RNA in vitro, in cell and in virion. A detailed protocol for whole-genome DMS-MaPseq from cells transfected with HIV-1 and the structure of TAR as determined by DMS-MaPseq is presented. DMS-MaPseq has the ability to answer a variety of integral questions about viral RNA, including how they change in different environments and when interacting with different host factors. Full Text

Torrens-Spence , M.P., Chiang, Y.C., Smith, T., Vicent, M.A., Wang, Y., and Weng, J.K. (2020). Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins. PNAS 117, 10806-10817. Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations. Full Text

Tsvetkov , P., Eisen, T.J., Heinrich, S.U., Brune, Z., Hallacli, E., Newby, G.A., Kayatekin, C., Pincus, D., and Lindquist, S. (2020). Persistent Activation of mRNA Translation by Transient Hsp90 Inhibition. Cell Reports 32 (6): 108001. The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a role promoting the evolution of new heritable traits. To better understand how Hsp90 can affect mRNA translation, we screen more than 1,600 factors involved in mRNA regulation for physical interactions with Hsp90 in human cells. The mRNA binding protein CPEB2 strongly binds Hsp90 via its prion domain. In a yeast model, transient inhibition of Hsp90 results in persistent activation of a CPEB translation reporter even in the absence of exogenous CPEB that persists for 30 generations after the inhibitor is removed. Ribosomal profiling reveals that some endogenous yeast mRNAs, including HAC1, show a persistent change in translation efficiency following transient Hsp90 inhibition. Thus, transient loss of Hsp90 function can promote a nongenetic inheritance of a translational state affecting specific mRNAs, introducing a mechanism by which Hsp90 can promote phenotypic variation. Full Text

*Waldman , B.S., Schwarz, D., Wadsworth, M.H., 2nd, Saeij, J.P., Shalek, A.K., and Lourido, S. (2020). Identification of a Master Regulator of Differentiation in Toxoplasma. Cell [Epub ahead of print]. Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections. Full Text

Wang, H.Y., Wu, S., Capecchi, M.R., and Jaenisch, R. (2020). A brief review of genome editing technology for generating animal models. Frontiers of Agricultural Science and Engineering 7, 123-128.The recent development of genome editing technologies has given researchers unprecedented power to alter DNA sequences at chosen genomic loci, thereby generating various genetically edited animal models. This mini-review briefly summarizes the development of major genome editing tools, focusing on the application of these tools to generate animal models in multiple species.Full Text

Wang, Y., Sangaré, L.O., Paredes-Santos, T.C., Hassan, M.A., Krishnamurthy, S., Furuta, A.M., Markus, B.M., Lourido, S., and Saeij, J.P.J. (2020). Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages. Nature Communications 11, 5258. Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages. Full Text

Weng J.K. (2020). Plant Solutions for the COVID-19 Pandemic and Beyond: Historical Reflections and Future Perspectives. Molecular Plant 13, 803-807. Here, I reflect on the COVID-19 pandemic from the perspective of plant science. First, plants have served as the main source of medicine for humans since the beginning of our species. Some of the earliest modern medicines are indeed plant natural products for treating infectious diseases. Plants have a lot to offer for treating COVID-19 and other infectious diseases, but it will require interdisciplinary research efforts to fully realize this potential. Second, the countermeasures that were quickly deployed against COVID-19 this time, including disease detection and potential treatments, are resulted from previous science and technology development in broad disciplines. This strongly advocates for not just maintaining but significantly increasing societal funding into basic sciences, including plant science, in order to better prepare us for future pandemics and other societal challenges. Last but not least, the global COVID-19 crisis has exposed several weaknesses of human nature, and in many ways echoes other looming crises, such as climate change and food insecurity. Plant science could contribute to the solutions of these problems, but such effort needs to be integrated into a global grand strategy yet to be established. Full Text

Williams, B.P., and Gehring, M. (2020). Principles of Epigenetic Homeostasis Shared Between Flowering Plants and Mammals. Trends in Genetics : TIG. In diverse eukaryotes, epigenetic information such as DNA methylation is stably propagated over many cell divisions and generations, and can remain the same over thousands or millions of years. However, this stability is the product of dynamic processes that add and remove DNA methylation by specialized enzymatic pathways. The activities of these dynamic pathways must therefore be finely orchestrated in order to ensure that the DNA methylation landscape is maintained with high fidelity - a concept we term epigenetic homeostasis. In this review, we summarize recent insights into epigenetic homeostasis mechanisms in flowering plants and mammals, highlighting analogous mechanisms that have independently evolved to achieve the same goal of stabilizing the epigenetic landscape.Full Text

Wilson, M.M., Robert A. Weiberg, Lees, J.A., and Guen, V.J. (2020). Emerging Mechanisms by which EMT Programs Control Stemness. Trends in cancer. Tissue regeneration relies on adult stem cells (SCs) that possess the ability to self-renew and produce differentiating progeny. In an analogous manner, the development of certain cancers depends on a subset of tumor cells, called cancer stem cells (CSCs), with SC-like properties. In addition to being responsible for tumorigenesis, CSCs exhibit elevated resistance to therapy and thus drive tumor relapse post-treatment. The epithelial-mesenchymal transition (EMT) programs promote SC and CSC stemness in many epithelial tissues. Here, we provide an overview of the mechanisms underlying the relationship between stemness and EMT programs, which may represent therapeutic vulnerabilities for the treatment of cancers. Full Text

Yang C., Zhang, W., Dong, X., Fu, C., Yuan, J., Xu, M. , Liang, Z., Qiu, C., and Xu, C. (2020). A natural product solution to aging and aging-associated diseases. Pharmacology & Therapeutics: 107673. Online ahead of print. Aging is a natural biological progress accompanied by the gradual decline in physiological functions, manifested by its close association with an increased incidence of human diseases and higher vulnerability to death. Those diseases include neurological disorders, cardiovascular diseases, diabetes, and cancer, many of which are currently without effective cures. Even though aging is inevitable, there are still interventions that can be developed to prevent/delay the onset and progression of those aging-associated diseases and extend healthspan and/or lifespan. Here, we reviewed decades of research that reveals the molecular pathways underlying aging and forms the biochemical basis for anti-aging drug development. Importantly, due to the vast chemical space of natural products and the rich history of herb medicines in treating human diseases documented in different cultures, natural products have played essential roles in aging research. Using several of the most promising natural products and their derivatives as examples, we discussed how natural products serve as an inspiration resource that helped the identification of key components/pathways underlying aging, their mechanisms of action inside the cell, and the functional scaffolds or targeting mechanisms that can be learned from natural products for drug engineering and optimization. We argue that natural products might eventually provide a solution to aging and aging-associated diseases. Full Text

Yang J., Antin, P., Berx, G., Blanpain, C., Brabletz, T., Bronner, M., Campbell, K., Cano, A., Casanova, J., Christofori, G., Robert A. Weiberg, EMT International Association (TEMTIA), et al. (2020). Guidelines and definitions for research on epithelial-mesenchymal transition. Nature Reviews Molecular Cell Biology 21, 341-352. Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT. Full Text

Zeming, K.K., Sato, Y., Yin, L., Huang, N.J., Wong, L.H., Loo, H.L., Lim, Y.B., Lim, C.T., Chen, J., Preiser, P.R., et al. (2020). Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture. Lab on a Chip. Online ahead of print. In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost. DFF sorting of reticulocytes from whole erythroid culture showed a 2.4-fold increase in cell recovery compared to FACS albeit with a lower purity; DLD sorting showed comparable cell recovery and purity with FACS using an inverse-L pillar structure to emphasize size and deformability sorting of reticulocytes. The viability and functional assurance of purified reticulocytes showed conserved cell deformability and supported the propagation of malaria parasites. Collectively, our study on label-free RBCs isolation represents a significant technical advancement towards developing in vitro generated viable human RBCs, opening opportunities for close-loop cell manufacturing, downstream therapeutic and research purposes. Full Text

Zou, Y., Henry, W.S., Ricq, E.L., Graham, E.T., Phadnis, V.V., Maretich, P., Paradkar, S., Boehnke, N., Deik, A.A., Reinhardt, F., Joshua Fairman , Heather R Keys, Robert A Weinberg, et al. (2020). Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature Online ahead of print. Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers(1). The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions(2-5). However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis. Full Text