The SkillsCenter: Creating scalable research opportunities for STEM students.

Undergraduate students generally need laboratory skills and experience to be accepted into a position within an academic lab or a company. However, those settings are traditionally where students would develop that necessary expertise. We developed a laboratory course paradigm to equip students with the skills they need to access future opportunities.

Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Trans-ethnic and Ancestry-Specific Blood-Cell Genetics in 746,667 Individuals from 5 Global Populations.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10-9, including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Long-term culture-expanded alveolar macrophages restore their full epigenetic identity after transfer in vivo.

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.

Natural innate and adaptive immunity to cancer.

The immune system can identify and destroy nascent tumor cells in a process termed cancer immunosurveillance, which functions as an important defense against cancer. Recently, data obtained from numerous investigations in mouse models of cancer and in humans with cancer offer compelling evidence that particular innate and adaptive immune cell types, effector molecules, and pathways can sometimes collectively function as extrinsic tumor-suppressor mechanisms. However, the immune system can also promote tumor progression. Together, the dual host-protective and tumor-promoting actions of immunity are referred to as cancer immunoediting. In this review, we discuss the current experimental and human clinical data supporting a cancer immunoediting process that provide the fundamental basis for further study of immunity to cancer and for the rational design of immunotherapies against cancer.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Circadian Rhythms and Astrocytes: The Good, the Bad, and the Ugly.

This review explores the interface between circadian timekeeping and the regulation of brain function by astrocytes. Although astrocytes regulate neuronal activity across many time domains, their cell-autonomous circadian clocks exert a particular role in controlling longer-term oscillations of brain function: the maintenance of sleep states and the circadian ordering of sleep and wakefulness. This is most evident in the central circadian pacemaker, the suprachiasmatic nucleus, where the molecular clock of astrocytes suffices to drive daily cycles of neuronal activity and behavior. In Alzheimer's disease, sleep impairments accompany cognitive decline. In mouse models of the disease, circadian disturbances accelerate astroglial activation and other brain pathologies, suggesting that daily functions in astrocytes protect neuronal homeostasis. In brain cancer, treatment in the morning has been associated with prolonged survival, and gliomas have daily rhythms in gene expression and drug sensitivity. Thus, circadian time is fast becoming critical to elucidating reciprocal astrocytic-neuronal interactions in health and disease.

Publisher Correction: c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

In the version of this article initially published, the Supplementary Data file was an incorrect version. The correct version is now provided. The error has been corrected in the HTML and PDF version of the article.

Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche.

Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.

UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome.

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Clearing Your Mind: Mechanisms of Debris Clearance After Cell Death During Neural Development.

Neurodevelopment and efferocytosis have fascinated scientists for decades. How an organism builds a nervous system that is precisely tuned for efficient behaviors and survival and how it simultaneously manages constant somatic cell turnover are complex questions that have resulted in distinct fields of study. Although neurodevelopment requires the overproduction of cells that are subsequently pruned back, very few studies marry these fields to elucidate the cellular and molecular mechanisms that drive nervous system development through the lens of cell clearance. In this review, we discuss these fields to highlight exciting areas of future synergy. We first review neurodevelopment from the perspective of overproduction and subsequent refinement and then discuss who clears this developmental debris and the mechanisms that control these events. We then end with how a more deliberate merger ofneurodevelopment and efferocytosis could reframe our understanding of homeostasis and disease and discuss areas of future study.

c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

The transcription factor c-Maf induces the anti-inflammatory cytokine IL-10 in CD4+ T cells in vitro. However, the global effects of c-Maf on diverse immune responses in vivo are unknown. Here we found that c-Maf regulated IL-10 production in CD4+ T cells in disease models involving the TH1 subset of helper T cells (malaria), TH2 cells (allergy) and TH17 cells (autoimmunity) in vivo. Although mice with c-Maf deficiency targeted to T cells showed greater pathology in TH1 and TH2 responses, TH17 cell-mediated pathology was reduced in this context, with an accompanying decrease in TH17 cells and increase in Foxp3+ regulatory T cells. Bivariate genomic footprinting elucidated the c-Maf transcription-factor network, including enhanced activity of NFAT; this led to the identification and validation of c-Maf as a negative regulator of IL-2. The decreased expression of the gene encoding the transcription factor RORγt (Rorc) that resulted from c-Maf deficiency was dependent on IL-2, which explained the in vivo observations. Thus, c-Maf is a positive and negative regulator of the expression of cytokine-encoding genes, with context-specific effects that allow each immune response to occur in a controlled yet effective manner.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

A CTCF Code for 3D Genome Architecture.

The architectural protein CTCF plays a complex role in decoding the functional output of the genome. Guo et al. now show that the orientation of a CTCF site restricts its choice of interacting partner, thus creating a code that predicts the three-dimensional organization of the genome. We propose a DNA extrusion model to account for orientation-specific loop formation.

A Corticostriatal Path Targeting Striosomes Controls Decision-Making under Conflict.

A striking neurochemical form of compartmentalization has been found in the striatum of humans and other species, dividing it into striosomes and matrix. The function of this organization has been unclear, but the anatomical connections of striosomes indicate their relation to emotion-related brain regions, including the medial prefrontal cortex. We capitalized on this fact by combining pathway-specific optogenetics and electrophysiology in behaving rats to search for selective functions of striosomes. We demonstrate that a medial prefronto-striosomal circuit is selectively active in and causally necessary for cost-benefit decision-making under approach-avoidance conflict conditions known to evoke anxiety in humans. We show that this circuit has unique dynamic properties likely reflecting striatal interneuron function. These findings demonstrate that cognitive and emotion-related functions are, like sensory-motor processing, subject to encoding within compartmentally organized representations in the forebrain and suggest that striosome-targeting corticostriatal circuits can underlie neural processing of decisions fundamental for survival.

Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos.

Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C. elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.