bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Iron Homeostasis Through Parkin-mediated Lactoferrin Ubiquitylation
Authors: Gholkar Ankur A. , Schmollinger Stefan , Velasquez Erick F. , Lo Yu-Chen , Cohn Whitaker , Capri Joseph , Dharmarajan Harish , Deardorff William J. , Gao Lucy W. , Abdusamad Mai , Whitelegge Julian P. , Torres Jorge Z.
Published: June 2020

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson’s disease. Lactotransferrin is a member of the transferrin iron binding proteins that regulate iron homeostasis and increased levels of Lactotransferrin and its receptor have been observed in neurodegenerative disorders like Parkinson’s disease. Here, we report that Parkin binds to Lactotransferrin and ubiquitylates it to regulate iron homeostasis.
Imaging fascicular organization of peripheral nerves with fast neural Electrical Impedance Tomography (EIT)
Authors: Ravagli Enrico , Mastitskaya Svetlana , Thompson Nicole , Iacoviello Francesco , Shearing Paul R , Perkins Justin , Gourine Alexander V , Aristovich Kirill , Holder David
Published: June 2020

Imaging of the compound action potential (CAP) in fascicles in peripheral nerves could help avoid side effects in neuromodulation by selective stimulation of identified fascicles. Existing methods have low resolution, limited imaging depth, or are invasive. We propose fast neural electrical impedance tomography (EIT), which allows fascicular CAP imaging with a high resolution of ∼200 μm, <1 ms. This uses a non-penetrating flexible cuff electrode array with 14 circumferential electrodes. This has been validated in rat sciatic nerve by comparison to micro-computed tomography (microCT) and histology with fluorescent dextran tracers (n=5). With EIT, there were reproducible localized changes in tissue impedance in response to stimulation of individual fascicles (tibial, peroneal and sural). The reconstructed EIT images corresponded to microCT scans and neural tracer histology, with significant separation between the fascicles (p<0.01), and no significant difference between techniques. The standard deviation from the mean fascicle position for EIT was 86 μm (6% of nerve diameter). This suggests fast neural EIT can reliably image the functional fascicular anatomy of the nerves and so aid selective neuromodulation.
Clathrin Senses Membrane Curvature
Authors: Zeno Wade F. , Hochfelder Jacob B. , Thatte Ajay S. , Wang Liping , Gadok Avinash K. , Hayden Carl C. , Lafer Eileen M. , Stachowiak Jeanne C.
Published: June 2020

The ability of proteins to sense membrane curvature is essential to diverse membrane remodeling processes including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to assemble together at curved membrane sites, leading to local recruitment of the clathrin coat. Because clathrin does not bind to the membrane directly, it has remained unclear whether clathrin plays an active role in sensing curvature or is passively recruited by its adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable to previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly, rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Further, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by two to ten-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface, compared to a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the critical drivers of membrane curvature sensing.
Distribution of droplets and immune responses after aerosol and intra-nasal delivery of influenza virus to the respiratory tract of pigs
Authors: Martini Veronica , Hinchcliffe Michael , Blackshaw Elaine , Joyce Mary , McNee Adam , Beverley Peter , Townsend Alain , MacLoughlin Ronan , Tchilian Elma
Published: June 2020

Recent evidence indicates that local immune responses and tissue resident memory T cells (TRM) are critical for protection against respiratory infections but there is little information on the contributions of upper and lower respiratory tract (URT and LRT) immunity. To provide a rational basis for designing methods for optimal delivery of vaccines to the respiratory tract in a large animal model, we investigated the distribution of droplets generated by a mucosal atomization device (MAD) and two vibrating mesh nebulizers (VMNs) and the immune responses induced by delivery of influenza virus by MAD in pigs. We showed that droplets containing the drug albuterol, a radiolabel (99mTc-DTPA) or a model influenza virus vaccine (S-FLU) have similar aerosol characteristics.99mTc-DTPA scintigraphy showed that VMNs deliver droplets with uniform distribution throughout the lungs as well as the URT. Surprisingly MAD administration (1ml/nostril) also delivered a high proportion of the dose to the lungs, albeit concentrated in a small area. After MAD administration of influenza virus, antigen specific T cells were found at high frequency in nasal turbinates, trachea, broncho-alveolar lavage, lungs, tracheobronchial nodes and blood. We conclude that the pig is useful for investigating optimal targeting of vaccines to the respiratory tract.
Multimodalin vivorecording using transparent graphene microelectrodes illuminates spatiotemporal seizure dynamics at the microscale
Authors: Driscoll Nicolette , Rosch Richard , Murphy Brendan B. , Ashourvan Arian , Vishnubhotla Ramya , Dickens Olivia O. , Johnson A.T. Charlie , Davis Kathryn A. , Litt Brian , Bassett Danielle S. , Takano Hajime , Vitale Flavia
Published: June 2020

Neurological disorders such as epilepsy arise from disrupted brain networks. Our capacity to treat these disorders is limited by our inability to map these networks at sufficient temporal and spatial scales to target interventions. Current best techniques either sample broad areas at low temporal resolution (e.g. calcium imaging) or record from discrete regions at high temporal resolution (e.g. electrophysiology). This limitation hampers our ability to understand and intervene in aberrations of network dynamics. Here we present a technique to map the onset and spatiotemporal spread of acute epileptic seizuresin vivoby simultaneously recording high bandwidth microelectrocorticography and calcium fluorescence using transparent graphene microelectrode arrays. We integrate dynamic data features from both modalities using non-negative matrix factorization to identify sequential spatiotemporal patterns of seizure onset and evolution, revealing how the temporal progression of ictal electrophysiology is linked to the spatial evolution of the recruited seizure core. This integrated analysis of multimodal data reveals otherwise hidden state transitions in the spatial and temporal progression of acute seizures. The techniques demonstrated here may enable future targeted therapeutic interventions and novel spatially embedded models of local circuit dynamics during seizure onset and evolution.
Neural divergence and convergence for attention to and detection of interoceptive and somatosensory stimuli
Authors: Herman Aleksandra M. , Palmer Clare E. , Azevedo Ruben T. , Tsakiris Manos
Published: June 2020

Body awareness is constructed by signals originating from within and outside the body. How do these apparently divergent signals converge? We developed a signal detection task to study the neural convergence and divergence of interoceptive and somatosensory signals. Participants focused on either cardiac or tactile events and reported their presence or absence. Beyond some evidence of divergence, we observed a robust overlap in the pattern of activation evoked across both conditions in frontal areas including the insular cortex, as well as parietal and occipital areas, and for both attention and detection of these signals. Psycho-physiological interaction analysis revealed that right insular cortex connectivity was modulated by the conscious detection of cardiac compared to somatosensory sensations, with greater connectivity to occipito-parietal regions when attending to cardiac signals. Our findings speak in favour of the inherent convergence of bodily-related signals and move beyond the apparent antagonism between exteroception and interoception.
Regulatory plasticity within a complex cytokine-sensing mammary enhancer during lactation
Authors: Lee Hye Kyung , Liu Chengyu , Hennighausen Lothar
Published: June 2020

Enhancers are transcription factor platforms that synergize with promoters to activate gene expression up to several-thousand-fold. While genome-wide structural studies are used to predict enhancers, thein vivosignificance is less clear. Specifically, the biological importance of individual transcription factors within enhancer complexes remains to be understood. Here we investigate the structural and biological importance of individual transcription factor binding sites and redundancy among transcription components within a complex enhancerin vivo. TheCsn1s2bgene is expressed exclusively in mammary tissue and activated several thousand-fold during pregnancy and lactation. Using ChIP-seq we identified a complex lactation-specific candidate enhancer that binds multiple transcription factors and coincides with activating histone marks. Using experimental mouse genetics, we determined that deletion of canonical binding motifs for the transcription factors NFIB and STAT5, individually and combined, had a limited biological impact. Loss of these sites led to a shift of transcription factor binding to juxtaposed sites, suggesting exceptional plasticity that does not require direct protein-DNA interactions. Additional deletions revealed the critical importance of a non-canonical STAT5 binding site for enhancer activity. Our data also suggest that enhancer RNAs are not required for the activity of this specific enhancer. While ChIP-seq experiments predicted an additional candidate intronic enhancer, its deletion did not adversely affect gene expression, emphasizing the limited biological information provided by structural data. Our study provides comprehensive insight into the anatomy and biology of a composite mammary enhancer that activates its target gene several hundred-fold during lactation.
Differentiation of ciliated human midbrain-derived LUHMES neurons
Authors: Lauter Gilbert , Coschiera Andrea , Yoshihara Masahito , Sugiaman-Trapman Debora , Ezer Sini , Sethurathinam Shalini , Katayama Shintaro , Kere Juha , Swoboda Peter
Published: June 2020

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing.

LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain.

In LUHMES, neuronal cilia biology can be investigated along a complete timeline: from proliferation through differentiation to mature neurons.

Summary Statement

With LUHMES, a ciliated human neuronal cell model, the underlying “neurobiology” of cilia and ciliopathies can be investigated along a complete time line: from proliferation through differentiation to mature neurons.
Divergent behavioral consequences of manipulations enhancing pyramidal neuron excitability in the prelimbic cortex
Authors: Rose Timothy R. , de Velasco Ezequiel Marron Fernandez , Vo Baovi N. , Tipps Megan E. , Wickman Kevin
Published: June 2020

Background

Drug-induced neuroadaptations in the prefrontal cortex are thought to underlie impaired executive functions that reinforce addictive behaviors. Repeated cocaine exposure increased layer 5/6 pyramidal neuron excitability in the mouse prelimbic cortex (PL), an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+(GIRK/Kir3) channel activity. GIRK channel suppression in the PL of drug-naïve mice enhanced the motor-stimulatory effect of cocaine. The impact of cocaine on PL GABA neurons, key pyramidal neuron regulators, and the behavioral relevance of increased PL pyramidal neuron excitability, remain unclear.

Methods

The effect of repeated cocaine on mouse layer 5/6 PL GABA neurons was assessed using slice electrophysiology. Adaptations enhancing PL pyramidal neuron excitability were modeled in drug-naïve mice using persistent viral Cre ablation and acute chemogenetic approaches. The impact of these manipulations on PL-dependent behavior was assessed in motor activity and trace fear conditioning tests.

Results

Repeated cocaine treatment did not impact GIRK channel activity in, or excitability of, layer 5/6 PL GABA neurons. GIRK channel ablation in PL pyramidal neurons enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or fear learning. In contrast, direct or indirect chemogenetic activation of PL pyramidal neurons increased baseline and cocaine-induced motor activity and disrupted fear learning. These effects were mirrored by chemogenetic activation of PL pyramidal neurons projecting to the ventral tegmental area.

Conclusions

Manipulations enhancing the excitability of PL pyramidal neurons, including those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure.
Influence of the large–Z effect during contact between butterfly sister species
Authors: Nelson Erik D. , Cong Qian , Grishin Nick V.
Published: June 2020

Comparisons of genomes from recently diverged butterfly populations along a suture zone in central Texas have revealed high levels of divergence on the Z chromosome relative to autosomes, as measured by fixation index,Fst. The pattern of divergence appears to result from accumulation of incompatible alleles, obstructing introgression on the Z chromosome in hybrids. However, it is unknown whether this mechanism is sufficient to explain the data. Here, we simulate the effects of hybrid incompatibility on interbreeding butterfly populations using a model in which populations accumulate cross–incompatible alleles in allopatry prior to contact. We compute statistics for introgression and population divergence during contact between model butterfly populations and compare them to statistics obtained for 15 pairs of butterfly species interbreeding along the Texas suture zone. For populations that have evolved sufficiently in allopatry, the model exhibits high levels of divergence on the Z chromosome relative to autosomes in populations inter-breeding on time scales comparable to periods of interglacial contact between butterfly populations in central Texas. Levels of divergence on the Z chromosome increase when interacting groups of genes are closely linked, consistent with interacting clusters of functionally related genes in butterfly genomes. Results for various periods in allopatry are in qualitative agreement with the pattern of data for butterflies, supporting a picture of speciation in which populations are subjected to cycles of divergence in glacial isolation, and partial fusion during interglacial contact.
Analysis of socioeconomic disadvantage and pace of aging measured in saliva DNA methylation of children and adolescents
Authors: Raffington Laurel , Belsky Daniel W. , Malanchini Margherita , Tucker-Drob Elliot M. , Harden K. Paige
Published: June 2020

Children who grow up in socioeconomically disadvantaged families face increased burden of disease and disability as they mature into adulthood. One hypothesized mechanism for this increased burden is that early-life disadvantage and its associated psychological stress accelerate biological processes of aging, increasing vulnerability to subsequent disease. In order to evaluate this hypothesis and the potential impact of preventive interventions, measures to quantify the early acceleration of biological aging in childhood are needed. Here, we evaluated a novel DNA-methylation measure of the pace of aging, DunedinPoAm, and compared DunedinPoAm results with results for several published epigenetic clocks. Data on saliva DNA-methylation and socioeconomic circumstances were collected fromN= 600 children and adolescents aged 8- to 18-years-old (48% female) participating in the Texas Twin Project. Participants living in more disadvantaged families and neighborhoods exhibited faster pace of aging (r= 0.18,p= 0.001 for both). Latinx-identifying children exhibited faster DunedinPoAm compared to both White- and Latinx-White-identifying children, consistent with higher levels of disadvantage in this group. Children with more advanced pubertal development and those with had higher body-mass index also exhibited faster DunedinPoAm, but these covariates did not account for the observed socioeconomic gradient in methylation pace of aging. In contrast to findings for DunedinPoAm, we did not detect associations of socioeconomic disadvantage with five published epigenetic clocks. Findings suggest that DNA-methylation pace-of-aging measures may prove more sensitive to health damaging effects of adversity, particularly when measurements are taken early in the life course, before substantial aging has occurred.
T cell receptor is required for differentiation but not maintenance of intestinal intraepithelial lymphocytes
Authors: Bilate Angelina M. , London Mariya , Castro Tiago B. R. , Mesin Luka , Kongthong Suppawat , Harnagel Audrey , Victora Gabriel D. , Mucida Daniel
Published: June 2020

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties. Migrating peripheral CD4+T cells, including regulatory (Treg) and conventional T cells (Tconv), acquire an IEL (CD4-IEL) program upon arrival at the epithelium. However, the specific role of the T cell receptor (TCR) in this process remains unclear. Single-cell TCR repertoire and transcriptomic analysis of intraepithelial CD4+T cells revealed different extents of clonal expansion and TCR overlap between cell states; fully differentiated CD4-IELs from Tregs or Tconvs were the least diverse. Conditional deletion of TCR on differentiating CD4+T cells or of MHCII on intestinal epithelial cells prevented CD4-IEL differentiation. However, TCR ablation on developed CD4-IELs did not affect their accumulation. These results indicate that local recognition of a limited set of antigens is an essential signal for the differentiation and adaptation of T cells to the epithelium.
Linking T cell receptor sequence to transcriptional profiles with clonotype neighbor graph analysis (CoNGA)
Authors: Schattgen Stefan A. , Guion Kate , Crawford Jeremy Chase , Souquette Aisha , Barrio Alvaro Martinez , Stubbington Michael J.T. , Thomas Paul G. , Bradley Philip
Published: June 2020

Multi-modal single-cell technologies capable of simultaneously assaying gene expression and surface phenotype across large numbers of immune cells have described extensive heterogeneity within these complex populations, in healthy and diseased states. In the case of T cells, these technologies have made it possible to profile clonotype, defined by T cell receptor (TCR) sequence, and phenotype, as reflected in gene expression (GEX) profile, surface protein expression, and peptide:MHC (pMHC) binding, across large and diverse cell populations. These rich, high-dimensional datasets have the potential to reveal new relationships between TCR sequence and T cell phenotype that go beyond identification of features shared by clonally related cells. In order to uncover these connections in an unbiased way, we developed a graph-theoretic approach---clonotype neighbor-graph analysis or “CoNGA”---that identifies correlations between GEX profile and TCR sequence through statistical analysis of a pair of T cell similarity graphs, one in which cells are linked based on gene expression similarity and another in which cells are linked by similarity of TCR sequence. Applying CoNGA across diverse human and mouse T cell datasets uncovered known and novel associations between TCR sequence features and cellular phenotype including the classical invariant T cell subsets; a novel defined population of human blood CD8+ T cells expressing the transcription factorsHOBITandHELIOS, NK-associated receptors, and a biased TCR repertoire, representing a potential previously undescribed lineage of “natural lymphocytes”; a striking association between usage of a specific V-beta gene segment and expression of theEPHB6gene that is conserved between mouse and human; and TCR sequence determinants of differentiation in developing thymocytes. As the size and scale of single-cell datasets continue to grow, we expect that CoNGA will prove to be a useful tool for deconvolving complex relationships between TCR sequence and cellular state in single-cell applications.
Molecular characterization ofFasciola giganticain Punjab, Pakistan to infer the dispersal route among the neighbouring countries of the Indian subcontinent
Authors: Ur Rehman Zia , Tashibu Atsushi , Tashiro Michiyo , Rashid Imran , Ali Qasim , Zahid Osama , Ashraf Kamran , Shehzad Wasim , Chaudhry Umer , Ichikawa-Seki Madoka
Published: June 2020

Fasciola giganticais considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in millions of dollars production losses to the livestock industry. To understand the dispersal origin and the spread patterns ofF. giganticais important for preventing the disease. A total of 53Fasciolaflukes collected from buffalo and goat in the Punjab province of Pakistan, were identified asF. giganticabased on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference betweenF. giganticafrom buffalo and goats in Pakistan was indicated by the genetic analysis of two distinct mitochondrial markers [NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1)]. Phylogenetic analysis of the seventeennad1haplotypes ofF. giganticafrom Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes were clustered in haplogroup A.Fasciola giganticawith the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, or possibly come from neighbouring Middle East countries. Our study provides a proof of concept for a method that could be used to investigate the epidemiology ofF. giganticaregarding the development of sustainable parasite control strategies.
Metabolome shift associated with thermal stress in coral holobionts
Authors: Williams Amanda , Chiles Eric N. , Conetta Dennis , Pathmanathan Jananan S. , Cleves Phillip A. , Putnam Hollie M. , Su Xiaoyang , Bhattacharya Debashish
Published: June 2020

Coral reef systems are under global threat due to warming and acidifying oceans1. Understanding the response of the coral holobiont to environmental change is crucial to aid conservation efforts. The most pressing problem is “coral bleaching”, usually precipitated by prolonged thermal stress that disrupts the algal symbiosis sustaining the holobiont2,3. We used metabolomics to understand how the coral holobiont metabolome responds to heat stress with the goal of identifying diagnostic markers prior to bleaching onset. We studied the heat tolerantMontipora capitataand heat sensitivePocillopora acutacoral species from the Hawaiian reef system in Kāne’ohe Bay, O’ahu. Untargeted LC-MS analysis uncovered both known and novel metabolites that accumulate during heat stress. Among those showing the highest differential accumulation were a variety of co-regulated dipeptides present in both species. The structures of four of these compounds were determined (Arginine-Glutamine, Lysine-Glutamine, Arginine-Valine, and Arginine-Alanine). These dipeptides also showed differential accumulation in symbiotic and aposymbiotic (alga free) individuals of the sea anemone modelAiptasia4, suggesting their animal provenance and algal symbiont related function. Our results identify a suite of metabolites associated with thermal stress that can be used to diagnose coral health in wild samples.
Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program
Authors: London Mariya , Bilate Angelina M. , Castro Tiago B. R. , Mucida Daniel
Published: June 2020

Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria (LP) and intraepithelial (IE) compartments, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we addressed the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+T cells from mLN, LP and IE segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile. Treg fate–mapping coupled with RNA– and ATAC–sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4 lineage–defining transcription factor ThPOK results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Thus, coordinated replacement of circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting.
Distinct genetic pathways define pre-leukemic and compensatory clonal hematopoiesis in Shwachman-Diamond syndrome
Authors: Kennedy Alyssa L. , Myers Kasiani C. , Bowman James , Gibson Christopher J. , Camarda Nicholas D. , Furutani Elissa , Muscato Gwen M. , Klein Robert H. , Ballotti Kaitlyn , Liu Shanshan , Harris Chad E. , Galvin Ashley , Malsch Maggie , Dale David , Gansner John M. , Nakano Taizo A. , Bertuch Alison , Vlachos Adrianna , Lipton Jeffrey M. , Castillo Paul , Connelly James , Churpek Jane , Edward John R. , Hijiya Nobuko , Ho Richard H. , Hofmann Inga , Huang James N. , Keel Siobán , Lamble Adam , Lau Bonnie W. , Norkin Maxim , Stieglitz Elliot , Stock Wendy , Walkovich Kelly , Boettcher Steffen , Brendel Christian , Fleming Mark D. , Davies Stella M. , Weller Edie A. , Bahl Christopher , Carter Scott L. , Shimamura Akiko , Lindsley R. Coleman
Published: June 2020

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with predisposition to developing leukemia. We found that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations inEIF6orTP53.EIF6mutations cause functional compensation for the germline deficiency by alleviating the SDS ribosome joining defect, improving translation, and reducing p53 activation.TP53mutations decrease checkpoint activation without affecting ribosome assembly. We link development of leukemia with acquisition of biallelicTP53alterations. Our results define distinct pathways of clonal selection driven by germline fitness constraint and provide a mechanistic framework for clinical surveillance.
The conserved ASCL1/MASH-1 ortholog HLH-3 specifies sex-specific ventral cord motor neuron fate inC. elegans
Authors: Perez Lillian M. , Alfonso Aixa
Published: June 2020

Neural specification can be regulated by one or many transcription factors. Here we identify a novel role for one conserved proneural factor, the bHLH protein HLH-3, implicated in the specification of sex-specific ventral cord motor neurons inC. elegans. In the process of characterizing the role ofhlh-3in neural specification, we document that differentiation of the ventral cord type C neurons, VCs, within their motor neuron class, is dynamic in time and space. Expression of VC class-specific and subclass-specific identity genes is distinct through development and dependent on where they are along the A-P axis (and their position in proximity to the vulva). Our characterization of the expression of VC class and VC subclass-specific differentiation markers in the absence ofhlh-3function reveals that VC fate specification, differentiation, and morphology requireshlh-3function. Finally, we conclude thathlh-3cell-autonomously specifies VC cell fate.
Foot shock facilitates reward seeking in an experience-dependent manner
Authors: Strickland JA , Dileo AD , Moaddab M , Ray MH , Walker RA , Wright KW , McDannald MA
Published: June 2020

Animals organize reward seeking around aversive events. An abundance of research shows that foot shock, as well as a shock-associated cue, can elicit freezing and suppress reward seeking. Yet, there is evidence that experience can flip the effect of foot shock to facilitate reward seeking. Here we examine cue suppression, foot shock suppression and foot shock facilitation of reward seeking in a single behavioural setting. Male Long Evans rats received fear discrimination consisting of danger, uncertainty and safety cues. Discrimination took place over a baseline of rewarded nose poking. With limited experience, all cues and foot shock strongly suppressed reward seeking. With continued experience, suppression became specific to shock-associated cues and foot shock facilitated reward seeking. Our results provide a means of assessing positive properties of foot shock, and may provide insight into maladaptive behavior around aversive events.
GO-CRISPR: a highly controlled workflow to discover gene essentiality in loss-of-function screens
Authors: Perampalam Pirunthan , McDonald James I. , Dick Frederick A.
Published: June 2020

Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describeGuide-Only controlCRISPR(GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package,Toolset for theRankedAnalysis of GO-CRISPRScreens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios.
A pan-cancer analysis of CpG Island gene regulation reveals extensive plasticity within Polycomb targets
Authors: Zheng Yueyuan , Huang Guowei , Silva Tiago C. , Yang Qian , Koeffler H Phillip , Berman Benjamin P. , Lin De-Chen
Published: June 2020

CpG Island promoter genes make up more than half of human genes, and a subset regulated by Polycomb-Repressive Complex 2 (PRC2+-CGI) become DNA methylated and silenced in cancer. Here, we perform a systematic regulatory analysis of CGI genes across TCGA cancer types, finding that PRC2+-CGI genes are frequently prone to transcriptional upregulation as well. These upregulated PRC2+-CGI genes control important pathways such as Epithelial-Mesenchymal Transition and TNFα-associated inflammatory response, and have the greatest cancer-type specificity among any class of CGI genes. Using publicly available chromatin datasets and genetic perturbations, we show that transcription factor binding sites (TFBSs) within distal enhancers underlie transcriptional activation of PRC2+-CGI genes, in contrast to PRC2-free CGI genes which are predominantly regulated by promoter TFBSs. Surprisingly, a large subset of PRC2+-CGI genes that are upregulated in one cancer type are also hypermethylated/silenced in at least one other cancer type, highlighting the high degree of regulatory plasticity likely derived from their complex regulatory patterns during normal development.
Loss of Bone Morphogenetic Protein-binding Endothelial Regulator Causes Insulin Resistance
Authors: Mao Hua , Li Luge , Fan Qiying , Angelini Aude , Saha Pradip K. , Wu Huaizhu , Ballantyne Christie M. , Hartig Sean M. , Xie Liang , Pi Xinchun
Published: June 2020

Accumulating evidence suggests chronic inflammation of metabolic tissues plays a causal role in obesity-induced insulin resistance. Yet, how specific endothelial factors exert impacts in metabolic tissues remains undefined. Bone morphogenetic protein (BMP)–binding endothelial regulator (BMPER) adapts endothelial cells to inflammatory stress in diverse organ microenvironments. Here we demonstrate BMPER is a driver of insulin sensitivity. Inducible knockout (iKO) of BMPER causes hyperinsulinemia, glucose intolerance and insulin resistance without increasing inflammation in metabolic tissues. Interestingly, BMPER can directly activate insulin signaling, which requires its internalization and interaction with Niemann-Pick C1 (NPC1), an integral membrane protein that transports intracellular cholesterol. These results suggest the endocrine function of the vascular endothelium maintains glucose homeostasis. Of potential clinical significance, the delivery of BMPER recombinant protein or its overexpression significantly alleviates insulin resistance and hyperglycemia in high-fat diet (HFD)-fed mice and Leprdb/db(db/db)diabetic mice. We conclude that BMPER exhibits therapeutic potential for the treatment of diabetes.
Donor-derived mitochondrial DNA variant peptides elicit allo-specific immune response in transplant patients
Authors: Marishta Argit , Yang Yanqin , Hu Xiaomeng , Jang Moon Kyoo , Cuttin Karen , Jackson Annette M. , Luikart Helen , Deuse Tobias , Khush Kiran K. , Schrepfer Sonja , Agbor-Enoh Sean , Valantine Hannah
Published: June 2020

In stem cell transplant, mitochondrial DNA (mtDNA) nonsynonymous single nucleotide variants (SNVs) between donor and recipient (D-R) trigger alloimmune responses and transplant rejection. Whether similar alloimmune responses occurs in solid-organ transplantation remains unknown, particularly with the presence of human leukocyte antigen mismatches. This study characterized mtDNA SNVs between D-R of 163 human lung transplant pairs, and then, post-transplantation, assessed alloimmune responses against donor-derived mitochondrial peptides using ELISpot to measure interferon gamma (IFNγ) release from recipient’s monocytes. We identified a median of 6 nonsynonymous mtDNA SNVs (Interquartile Range = 4 – 9) per D-R pair. SNVs were predominantly located atMT-CYB, MT-ATP6, andMT-ND3genes. The number of SNVs was higher in D-R race non-concordant pairs than in race-concordant pairs. Donor-derived mitochondrial peptides triggered a 19.8-fold higher IFNγ release compared to recipient-derived peptide. These findings were validated in heart transplantation and show that donor-derived mitochondrial peptides trigger allo-specific immune responses after transplantation.
Multivariate analysis reveals a generalizable human electrophysiological signature of working memory load
Authors: Adam Kirsten C.S. , Vogel Edward K. , Awh Edward
Published: June 2020

Working memory (WM) is an online memory system that is critical for holding information in a rapidly accessible state during ongoing cognitive processing. Thus, there is strong value in methods that provide a temporally-resolved index of WM load. While univariate EEG signals have been identified that vary with WM load, recent advances in multivariate analytic approaches suggest that there may be rich sources of information that do not generate reliable univariate signatures. Here, using data from 4 published studies (n= 286 and >250,000 trials), we demonstrate that multivariate analysis of EEG voltage topography provides a sensitive index of the number of items stored in WM that generalizes to novel human observers. Moreover, multivariate load detection (“mvLoad”) can provide robust information at the single-trial level, exceeding the sensitivity of extant univariate approaches. We show that this method tracks WM load in a manner that is (1) independent of the spatial position of the memoranda, (2) precise enough to differentiate item-by-item increments in the number of stored items, (3) generalizable across distinct tasks and stimulus displays and (4) correlated with individual differences in WM behavior. Thus, this approach provides a powerful complement to univariate analytic approaches, enabling temporally-resolved tracking of online memory storage in humans.
Molecular characterization of mosquitoes (Diptera: Culicidae) from the tropical rainforest of Sierra Nevada de Santa Marta, Colombia
Authors: Muñoz-Gamba Andrew , Laiton-Donato Katherine , Perdomo-Balaguera Erick , Usme-Ciro José , Parra-Henao Gabriel
Published: June 2020

BACKGROUND

The Sierra Nevada de Santa Marta rainforest has diverse fauna due to its position in northern Colombia, a Caribbean region with predominantly tropical, dry, and rainforest ecosystems in which there is a high diversity of mosquito species that may act as arbovirus vectors.

OBJECTIVES

The present study reports the molecular characterization of select mosquito species in this rainforest.

METHODS

Manual capture methods were used to collect mosquitoes, and the specimens were identified via classical taxonomy. TheCOImarker was used for species confirmation, and phylogenetic analysis was performed, using the neighbor-joining method, with the Kimura-2-Parameters model.

FINDINGS

Aedes serratus,Psorophora ferox,Johnbelkinia ulopus,Sabethes cyaneus,Wyeomyia aporonoma,Wyeomyia pseudopecten,Wyeomyia ulocomaandWyeomyia luteoventraliswere identified and intra-species variation >2% for most species.

MAIN CONCLUSIONS

We report the first records on the genetic variability of mosquitoes in this area and phylogenetic reconstructions allowed for identification at the species level, and the corroboration by means of classical taxonomy suggested complementarity of both methods, which may be employed when morphological or molecular data are poor or not available. The genetic and morphological characterization of jungle mosquito populations will help to understand their biology.
Ventral pallidum neurons signal relative threat
Authors: Moaddab Mahsa , Ray Madelyn H. , McDannald Michael A.
Published: June 2020

Ventral pallidum (VP) neurons scale firing increases to reward value and decrease firing to aversive cues. Anatomical connectivity suggests a critical role for the VP in threat-related behavior. Here we tested whether firing decreases in VP neurons conform to relative threat by recording single units while male rats discriminated cues predicting unique foot shock probabilities. Rats behavior and VP single unit firing discriminated danger, uncertainty and safety cues. We found that two VP populations (Low firing and Intermediate firing) signaled relative threat, proportionally decreased firing according shock probability: danger < uncertainty < safety. Low firing neurons showed reward firing increases, consistent with a general signal for relative value. Intermediate firing neurons were unresponsive to reward, revealing a specific signal for relative threat. The results suggest an integral role for the VP in threat-related behavior.
The specificity of sperm-mediated paternal effects in threespined sticklebacks
Authors: Chen Eunice , Zielinski Christian , Deno Jack , Singh Raiza , Bell Alison M , Hellmann Jennifer K
Published: June 2020

Parental effects can help offspring cope with challenging environments, but whether these effects are unique to specific environmental conditions is largely unknown. Parental effects may evolve via a core pathway that generally prepares offspring for risky environments or could be stimuli-specific, with offspring developing phenotypes that are tailored to specific environmental challenges. We exposed threespined sticklebacks (Gasterosteus aculeatus) fathers to a potentially threatening stimulus (net) versus native predator (sculpin). Offspring of sculpin-exposed fathers were more responsive (greater change in activity) to a simulated predator attack, while offspring of net-exposed fathers were less responsive (lower plasma cortisol and fewer antipredator behaviors). To evaluate offspring response to native and non-native stimuli, we sequentially exposed offspring of net-exposed, sculpin-exposed or control fathers to a net, native sculpin model, or non-native trout model. Paternal treatment did not influence offspring response to stimuli; instead, offspring were more responsive to the native sculpin predator compared to nets or non-native trout predator. Collectively, we demonstrate that sperm-mediated paternal effects in response to different, potentially stressful stimuli result in distinct offspring phenotypes. This specificity may be key to understanding the evolution of adaptive parental effects and how parents prime offspring for encountering both evolved and novel environmental stimuli.
GermlineAPOBEC3Bdeletion in Asian women increases somatic hypermutation in breast cancer that is associated with Her2 subtype,PIK3CAmutations, immune activation, and increased survival
Authors: Pan Jia-Wern , Ahmad Zabidi Muhammad Mamduh , Chong Boon-Keat , Meng Mei-Yee , Ng Pei-Sze , Hasan Siti Norhidayu , Sandey Bethan , Bahnu Saira , Rajadurai Pathmanathan , Yip Cheng-Har , Rueda Oscar M. , Caldas Carlos , Chin Suet-Feung , Teo Soo-Hwang
Published: June 2020

A 30-kb deletion that eliminates the coding region ofAPOBEC3B(A3B) is >5 times more common in women of Asian compared to European descent. This polymorphism creates a chimera with theAPOBEC3A(A3A) coding region andA3B3’UTR, and is associated with an increased risk for breast cancer in Asian women. Here, we explored the relationship between theA3Bdeletion polymorphism with tumour characteristics in Asian women. Using whole exome and whole transcriptome sequencing data of 527 breast tumours, we report that germlineA3Bdeletion polymorphism leads to expression of theA3A-Bhybrid isoform and increased APOBEC-associated somatic hypermutation. Hypermutated tumours, regardless ofA3Bgermline status, were associated with the Her2 molecular subtype andPIK3CAmutations. Compared to non-hypermutated tumours, hypermutated tumours also had higher neoantigen burden, tumour heterogeneity and immune activation. Taken together, our results suggest that the germlineA3Bdeletion polymorphism, via theA3A-Bhybrid isoform, contributes to APOBEC-mutagenesis in a significant proportion of Asian breast cancers. In addition, APOBEC somatic hypermutation, regardless ofA3Bbackground, may be an important clinical biomarker for Asian breast cancers.

Graphical Abstract
Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis
Authors: Wu Sunny Z. , Roden Daniel L. , Al-Eryani Ghamdan , Bartonicek Nenad , Harvey Kate , Cazet Aurélie S. , Chan Chia-Ling , Junankar Simon , Hui Mun N. , Millar Ewan A. , Beretov Julia , Horvath Lisa , Joshua Anthony M. , Stricker Phillip , Wilmott James S. , Quek Camelia , Long Georgina V. , Scolyer Richard A. , Yeung Bertrand Z. , Segara Davendra , Mak Cindy , Warrier Sanjay , Powell Joseph E. , O’Toole Sandra , Lim Elgene , Swarbrick Alexander
Published: June 2020

Background

High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms.

Results

We show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of ∼120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing.

Conclusions

Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.
Characterization of the substitution hotspots in SARS-CoV-2 genome using BioAider and detection of a SR-rich region in N protein providing further evidence of its animal origin
Authors: Zhou Zhi-Jian , Qiu Ye , Ge Xing-Yi
Published: June 2020

The novel human coronavirus (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic worldwide. The increasing sequencing data have shown abundant single nucleotide variations in SARS-CoV-2 genome. However, it is difficult to quickly analyze genomic variation and screen key mutations of SARS-CoV-2. In this study, we developed a visual program, named BioAider, for quick and convenient sequence annotation and mutation analysis on multiple genome-sequencing data. Using BioAider, we conducted a comprehensive genome variation analysis on 3,240 sequences of SARS-CoV-2 genome. Herein, we detected 14 substitution hotspots within SARS-CoV-2 genome, including 10 non-synonymous and 4 synonymous ones. Among these hotspots, NSP13-Y541C was predicted to be a crucial substitution which might affect the unwinding activity of NSP13, a key protein for viral replication. Besides, we also found 3 groups of potentially linked substitution hotspots which were worth further study. In particular, we discovered a SR-rich region (aa 184-204) on the N protein of SARS-CoV-2 distinct from SARS-CoV, indicating more complex replication mechanism and unique N-M interaction of SARS-CoV-2. Interestingly, the quantity of SRXX repeat fragments in the SR-rich region well reflected the evolutionary relationship among SARS-CoV-2 and SARS-CoV-2 related animal coronaviruses, providing further evidence of its animal origin. Overall, we developed an efficient tool for rapid identification of mutations, identified substitution hotspots in SARS-CoV-2 genomes, and detected a distinctive polymorphism SR-rich region in N protein. This tool and the detected hotspots could facilitate the viral genomic study and may contribute for screening antiviral target sites.