bluuebunny/crossref_metadata_2025 huggingface dataset:
BMP-AKT-GSK3β Signaling Restores Hair Follicle Stem Cells Decrease Associated with Loss of Sfrp1
Authors: Sunkara Raghava R , Mehta Darshan , Sarate Rahul M , Waghmare Sanjeev K
Published: June 2022

Wnt signaling plays a pivotal role in regulating activation, proliferation, stem cell renewal, and differentiation of hair follicle stem cells (HFSCs). Secreted frizzled-related protein 1 (Sfrp1), a Wnt antagonist is upregulated in the HFSCs; however, its role in the HFSCs regulation is still obscure. Here, we show that Sfrp1 loss showed a depletion of HFSCs, enhanced HFSC proliferation, and faster hair follicle cycle at PD21-PD28; HFSC markers, such as Lgr5 and Axin2, were decreased in both the Sfrp1+/− and Sfrp1−/− HFSCs. In addition, the second hair follicle cycle was also faster compared with WT. Importantly, Sfrp1−/− showed a restoration of HFSC by second telogen (PD49), whereas Sfrp1+/− did not show restoration with still having a decreased HFSC. In fact, restoration of HFSCs was due to a pronounced downregulation of β-catenin activity mediated through a cross-talk of BMP-AKT-GSK3β signaling in Sfrp1−/− compared with Sfrp1+/−, where downregulation was less pronounced. In cultured keratinocytes, Sfrp1 loss resulted in enhanced proliferation and clonogenicity, which were reversed by treating with either BMPR1A or GSK3β inhibitor thereby confirming BMP-AKT-GSK3β signaling involved in β-catenin regulation in both the Sfrp1+/− and Sfrp1−/− mice. Our study reveals a novel function of Sfrp1 by unraveling an in vivo molecular mechanism that regulates the HFSCs pool mediated through a hitherto unknown cross-talk of BMP-AKT-GSK3β signaling that maintains stem cell pool balance, which in turn maintains skin tissue homeostasis.
Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium
Authors: Rusyn Ivan , Sakolish Courtney , Kato Yuki , Stephan Clifford , Vergara Leoncio , Hewitt Philip , Bhaskaran Vasanthi , Davis Myrtle , Hardwick Rhiannon N , Ferguson Stephen S , Stanko Jason P , Bajaj Piyush , Adkins Karissa , Sipes Nisha S , Hunter E Sidney , Baltazar Maria T , Carmichael Paul L , Sadh Kritika , Becker Richard A
Published: June 2022

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.
A review of passenger ride comfort in railway: assessment and improvement method
Authors: Peng Yong , Zhou Jiahao , Fan Chaojie , Wu Zhifa , Zhou Wenjun , Sun Dayan , Lin Yating , Xu Diya , Xu Qian
Published: June 2022

Passenger ride comfort has become a focus of attention in rail transportation equipment design, manufacture and later operation to meet people's demand for travel quality. However, comfort is a very subjective concept, which is difficult to quantify and evaluate directly, and can be affected by various factors, leading to the corresponding technologies for ride comfort improvement becoming diverse. In this paper, recent research on the assessment method and improvement measures of railway passenger ride comfort is reviewed. The main types of ride comfort are summarized first according to the sources of discomfort, including static comfort, vibration comfort, noise comfort, aural pressure comfort, thermal comfort and visual comfort. The current assessment methods of ride comfort are introduced from the aspects of environmental parameters and human parameters based on the nature of evaluation indicators. Finally, the improvement technologies for each type of ride comfort are presented.
13Posthumanisms
Authors: Nayar Pramod K
Published: June 2022

Posthumanism as a school of thought and a theme in literary and cultural texts strives to keep pace with developments such as terraforming or genetic engineering, but also engages philosophically and politically with those developments, in the form of enunciating questions about human identity, nonhuman agency, and ethics. In more recent times, posthuman thought has moved into areas as diverse as climate change, the Anthropocene, legal identity, and pedagogy. It also continues, as this chapter’s review of books, book chapters, and journal articles published in the year 2021 shows, to be interested in legal subjectivity, ideas of personhood, human genealogy, and their cultural expressions in the form of pop culture and literary texts. The chapter is divided into seven sections: 1. Surveying Posthumanism; 2. Posthumanisms and the Anthropocene; 3. Posthuman Performance; 4. Transhumanism; 5. Posthumanism, Law, and Ethics; 6. Posthumanism and the Pandemic; 7. Conclusion.
The Impact of Occupational Stigma on Gig Workers' Workplace Well-being
Authors: Liu Beini , Guo Yuehua , Fu Yaoyao
Published: June 2022

Objective

In this article, the influence of occupational stigma on workplace well-being of platform-based food-delivery workers is examined. The mediation effect of work-contingent self-esteem and the moderating effect of job control are also assessed.

Methods

Questionnaire data from 362 platform-based food-delivery workers were gathered at three time points, and multiple regression analyses were used to test each hypothesis.

Results

Occupational stigma reduces workplace well-being of platform-based food-delivery workers, and this relationship is mediated by work-contingent self-esteem. Job control exerts a negative moderating effect on the relationship between occupational stigma and platform-based food-delivery workers' work-contingent self-esteem.

Conclusions

Strategies for reducing occupational stigma should be prioritized to increase workplace well-being in gig workers. Giving them more control over their work will likely alleviate the detrimental effect of occupational stigma on work-contingent self-esteem.
The phers R package: using phenotype risk scores based on electronic health records to study Mendelian disease and rare genetic variants
Authors: Aref Layla , Bastarache Lisa , Hughey Jacob J.
Published: June 2022

Electronic health record (EHR) data linked to DNA biobanks are a valuable resource for understanding the phenotypic effects of human genetic variation. We previously developed the phenotype risk score (PheRS) as an approach to quantify the extent to which a patient’s clinical features resemble a given Mendelian disease. Using PheRS, we have uncovered novel associations between Mendelian diseaselike phenotypes and rare genetic variants, and identified patients who may have undiagnosed Mendelian disease. Although the PheRS approach is conceptually simple, it involves multiple mapping steps and was previously only available as custom scripts, limiting the approach’s usability. Thus, we developed the phers R package, a complete and user-friendly set of functions and maps for performing a PheRS-based analysis on linked clinical and genetic data. The package includes up-to-date maps between EHR-based phenotypes (i.e., ICD codes and phecodes), human phenotype ontology (HPO) terms, and Mendelian diseases. Starting with occurrences of ICD codes, the package enables the user to calculate phenotype risk scores, validate the scores using case-control analyses, and perform genetic association analyses. By increasing PheRS’s transparency and usability, the phers R package will help improve our understanding of the relationships between rare genetic variants and clinically meaningful human phenotypes.

Availability

The phers R package is free and open-source, and available on CRAN and at https://phers.hugheylab.org.

Contact

jakejhughey@gmail.com

Supplementary information

Supplementary data are available at Bioinformatics online.
MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer
Authors: Soto-Feliciano Yadira M. , Zhu Changyu , Morris John P. , Huang Chun-Hao , Koche Richard P. , Ho Yu-jui , Banito Ana , Chen Chun-Wei , Shroff Aditya , Tian Sha , Livshits Geulah , Chen Chi-Chao , Fennell Myles , Armstrong Scott A. , Allis C. David , Tschaharganeh Darjus F. , Lowe Scott W.
Published: June 2022

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3/KMT2C) histone methyltransferase occur in a range of solid tumors and heterozygous deletions encompassing MLL3 occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Mll3 cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded genes, Ink4a and Arf. Conversely, elevated MLL3 expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Mll3 restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in MLL3 and CDKN2A display mutual exclusivity in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.
Exploring the alternative conformation of a known protein structure based on contact map prediction
Authors: Li Jiaxuan , Wang Lei , Zhu Zefeng , Song Chen
Published: June 2022

The rapid development of deep learning-based methods has considerably advanced the field of protein structure prediction. The accuracy of predicting the 3D structures of simple proteins is comparable to that of experimentally determined structures, providing broad possibilities for structure-based biological studies. Another critical question is whether and how multistate structures can be predicted from a given protein sequence. In this study, analysis of multiple two-state proteins demonstrated that deep learning-based contact map predictions contain structural information on both states, which suggests that it is probably appropriate to change the target of deep learningbased protein structure prediction from one specific structure to multiple likely structures. Furthermore, by combining deep learning- and physics-based computational methods, we developed a protocol for exploring alternative conformations from a known structure of a given protein, by which we successfully approached the holo-state conformation of a leucine-binding protein from its apo-state structure.
Evolution of dispersal and the maintenance of fragmented metapopulations
Authors: Finand Basile , Monnin Thibaud , Loeuille Nicolas
Published: June 2022

Because it affects dispersal risk and modifies competition levels, habitat fragmentation directly constrains dispersal evolution. When dispersal is traded-off against competitive ability, increased fragmentation is often expected to select higher dispersal. Such evolutionary effects could favor the maintenance of the metapopulation by fostering spatial rescue effects. Using an evolutionary model, we first investigate how dispersal evolves in a metapopulation when fragmentation and aggregation of this fragmentation are fixed. Our results suggest that high fragmentation indeed selects for dispersal increase, but this effect is largely reduced in aggregated landscapes, to the point of being nonexistent at the highest aggregation levels. Contrasted dispersal strategies coexist at high fragmentation levels and with no or low aggregation. We then simulate time-varying fragmentation scenarios to investigate the conditions under which evolutionary rescue of the metapopulation happens. Faster evolution of dispersal favors the persistence of the metapopulation, but this effect is very reduced in aggregated landscapes. Overall, our results highlight how the speed of evolution of dispersal and the structuration of the fragmentation will largely constrain metapopulation survival in changing environments.
Subtype-specific single β1 integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling
Authors: Jo Myung Hyun , Li Jing , Jaumouillé Valentin , Hao Yuxin , Coppola Jessica , Yan Jiabin , Waterman Clare M. , Springer Timothy A. , Ha Taekjip
Published: June 2022

Although integrins are known to be mechano-sensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4β1 and RGD-binding integrins (αVβ1 and α5β1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4β1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVβ1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.
Spatial structure alters the site frequency spectrum produced by hitchhiking
Authors: Min Jiseon , Gupta Misha , Desai Michael M. , Weissman Daniel B.
Published: June 2022

The reduction of genetic diversity due to genetic hitchhiking is widely used to find past selective sweeps from sequencing data, but very little is known about how spatial structure affects hitchhiking. We use mathematical modeling and simulations to find the unfolded site frequency spectrum (SFS) left by hitchhiking in the genomic region of a sweep in a population occupying a one-dimensional range. For such populations, sweeps spread as Fisher waves, rather than logistically. We find that this leaves a characteristic three-part SFS at loci very close to the swept locus. Very low frequencies are dominated by recent mutations that occurred after the sweep and are unaffected by hitchhiking. At moderately low frequencies, there is a transition zone primarily composed of alleles that briefly “surfed” on the wave of the sweep before falling out of the wavefront, leaving a spectrum close to that expected in well-mixed populations. However, for moderate-to-high frequencies, there is a distinctive scaling regime of the SFS produced by alleles that drifted to fixation in the wavefront and then were carried throughout the population. For loci slightly farther away from the swept locus on the genome, recombination is much more effective at restoring diversity in one-dimensional populations than it is in well-mixed ones. We find that these signatures of space can be strong even in apparently well-mixed populations with negligible spatial genetic differentiation, suggesting that spatial structure may frequently distort the signatures of hitchhiking in natural populations.
Shared functional specialization in transformer-based language models and the human brain
Authors: Kumar Sreejan , Sumers Theodore R. , Yamakoshi Takateru , Goldstein Ariel , Hasson Uri , Norman Kenneth A. , Griffiths Thomas L. , Hawkins Robert D. , Nastase Samuel A.
Published: June 2022

Humans use complex linguistic structures to transmit ideas to one another. The brain is thought to deploy specialized computations to process these structures. Recently, a new class of artificial neural networks based on the Transformer architecture has revolutionized the field of language modeling, attracting attention from neuroscientists seeking to understand the neurobiology of languagein silico. Transformers integrate information across words via multiple layers of structured circuit computations, forming increasingly contextualized representations of linguistic content. Prior work has focused on the internal representations (the “embeddings”) generated by these circuits. In this paper, we instead analyze the circuit computations directly: we deconstruct these computations into functionally-specialized “transformations” to provide a complementary window onto linguistic computations in the human brain. Using functional MRI data acquired while participants listened to naturalistic spoken stories, we first verify that the transformations account for considerable variance in brain activity across the cortical language network. We then demonstrate that the emergent syntactic computations performed by individual, functionally-specialized “attention heads” differentially predict brain activity in specific cortical regions. These heads fall along gradients corresponding to different layers, contextual distances, and syntactic dependencies in a low-dimensional cortical space. Our findings indicate that large language models and the cortical language network may converge on similar trends of functional specialization for processing natural language.
CaMKIIα promoter-controlled circuit manipulations target both pyramidal cells and inhibitory interneurons in cortical networks
Authors: Veres Judit M. , Andrasi Tibor , Nagy-Pal Petra , Hajos Norbert
Published: June 2022

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vector (AAV) carrying the CaMKIIα-Channelrhodopsin 2-mCherry construct. We show that the reporter protein, mCherry can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY)-containing GABAergic cells, which altogether cover around 50% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the detectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.
Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants
Authors: Yoshida Masahide , Saito Tomoko , Takayanagi Yuki , Totsuka Yoshikazu , Onaka Tatsushi
Published: June 2022

The CRISPR-Cas9 method for generation of knock-in mutations in a rodent embryo yields many F0 generation candidates that may have the designed mutations. Genetic mutations must be confirmed using genomic DNA with a mixture of designed and unexpected mutations. In our study for establishing Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopy at a target allele locus and mosaic genotype of two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism or discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In F1 and F2 generations, droplet digital PCR analysis contributed to establishment of the strain, although a multicopy was falsely detected as one copy by the analysis in the F0 generation. We emphasize that the combination of these five methods allowed us to select promising F0 generations and establish the designed strain and that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.
Rapid presynaptic maturation in naturally regenerating axons
Authors: Browne Lorcan P. , Crespo Andres , Grubb Matthew S.
Published: June 2022

Successful neuronal regeneration requires the re-establishment of synaptic connectivity. Crucial to this process is the reconstitution of presynaptic machinery responsible for controlling neurotransmitter release. In the mammalian adult CNS post-injury regeneration is usually only possible after extensive experimental intervention, and it is unknown how presynaptic function is re-established, let alone how it might be optimised to promote functional recovery. Here we addressed these questions by studying presynaptic maturation during a regenerative process that occurs entirely naturally. After toxin-induced injury, olfactory sensory neurons in the adult mouse olfactory epithelium can regenerate fully, sending axons to the brain to re-establish synaptic contact with postsynaptic partners in the olfactory bulb. Using electrophysiological recordings in acute slices, we found that after initial re-contact, functional connectivity in this system was rapidly established. Moreover, re-connecting presynaptic terminals had almost mature functional properties, including high release probability and a strong capacity for presynaptic inhibition. Release probability then matured quickly, rendering re-established terminals functionally indistinguishable from controls just one week after initial contact. These data show that successful synaptic regeneration in the adult mammalian brain is not quite a ‘plug-and-play’ process; instead, almost-mature presynaptic terminals undergo a rapid phase of functional maturation to re-integrate into established target networks.
Loss of the protein disrupted in schizophrenia 1 (DISC1) impairs cardiac resistance against ischemia
Authors: Marwarha Gurdeep , Pinho Maria J. , Scrimgeour Nathan R , Slagsvold Katrine Hordnes , Wahba Alexander , Røsbjørgen Ragnhild E , Enguita Francisco J. , Yang Kun , Ishizuka Koko , Casas Carlos Andrés Acosta , Pettersen Kristine , Bjørkøy Geir , Sawa Akira , Høydal Morten A
Published: June 2022

Treatable targets that hinder heart failure development following myocardial infarction remain limited. Through an unbiased transcriptional regulation study for ischemic heart disease, we identified the protein disrupted in schizophrenia 1 (DISC1), which has been almost solely characterized in the brain. Here, we show that loss of DISC1 is a major driver of heart disease and ischemic damage. Silencing of DISC1 sensitizes human cardiomyocyte cell lines to hypoxia, whereas DISC1 overexpression is cardioprotective. Mechanistically, we provide evidence that reduced DISC1 protein levels interrupt multiple signaling processes vital for cardiomyocyte survival whereas overexpression of DISC1 enhances pro survival signalling. The present study has implications for understanding scenarios where cardiomyocytes are unable to increase the levels of DISC1 during ischemia, e.g., in individuals with DISC1 genetic defects. Indeed, patients with severe mental illness display 20 years earlier mortality, with IHD as the greatest cause of death. The present study can thus shed light on this long-term enigma in epidemiology. Furthermore, the promising data showing activation of pro survival signalling processes with DISC1 overexpression opens new directions towards potential treatment strategies of patients suffering from ischemic heart disease.
Genome-wide subcellular protein localisation in the flagellate parasite Trypanosoma brucei
Authors: Billington Karen , Halliday Clare , Madden Ross , Dyer Philip , Carrington Mark , Vaughan Sue , Hertz-Fowler Christiane , Dean Samuel , Sunter Jack Daniel , Wheeler Richard John , Gull Keith
Published: June 2022

Trypanosoma brucei is a prototypical trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is hindered since, as with most eukaryotic microbes, ∼50% of the proteins encoded in the genome have completely unknown function. Using fluorescence microscopy and cell lines expressing endogenously tagged proteins we mapped the subcellular localisation of 89% of the proteome, giving clues to function, defining the lineage-specific organelle adaptations for obligate parasitism and mapping the ultra-conserved cellular architecture of eukaryotes. This includes the single flagellum, vital for morphogenesis and pathology: the first comprehensive cartographic analysis of the flagellum in any organism. To demonstrate the power of this resource, we identify novel specialisation of organelle molecular composition through the cell cycle and in specialised subdomains. This is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.
Heritability and host genomic determinants of switchgrass root-associated microbiota in field sites spanning its natural range
Authors: Edwards Joseph A , Saran Usha Bishnoi , Bonnette Jason , MacQueen Alice , Yin Jun , Nguyen Tu uyen , Schmutz Jeremy , Grimwood Jane , Pennacchio Len A. , Daum Chris , del Rio Tijana Glavina , Fritschi Felix B. , Lowry David B. , Juenger Thomas E.
Published: June 2022

A fundamental goal in plant microbiome research is to determine the relative impacts of host and environmental effects on root microbiota composition, particularly how host genotype impacts bacterial community composition. Most studies characterizing the effect of plant genotype on root microbiota undersample host genetic diversity and grow plants outside of their native ranges, making the associations between host and microbes difficult to interpret. Here we characterized the root microbiota of a large population of switchgrass, a North American native C4 bioenergy crop, in three field locations spanning its native range. Our data, composed of >2000 samples, suggest field location is the primary determinant of microbiome composition; however, substantial heritable variation is widespread across bacterial taxa, especially those in the Sphingomonadaceae family. Despite diverse compositions, we find that relatively few highly prevalent bacterial taxa make up the majority of the switchgrass root microbiota, a large fraction of which is shared across sites. Local genotypes preferentially recruit / filter for local microbes, supporting the idea of affinity between local plants and their microbiota. Using genome-wide association, we identified loci impacting the abundance of >400 microbial strains and found an enrichment of genes involved in immune responses, signaling pathways, and secondary metabolism. We found loci associated with over half of the core microbiota (i.e. microbes in >80% of samples) regardless of field location. Finally, we show a genetic relationship between a basal plant immunity pathway and relative abundances of root microbiota. This study brings us closer to harnessing and manipulating beneficial microbial associations via host genetics.
IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies
Authors: Grzelak Ludivine , Roesch Ferdinand , Vaysse Amaury , Biton Anne , Porrot Françoise , Commère Pierre-Henri , Planchais Cyril , Mouquet Hugo , Vignuzzi Marco , Bruel Timothée , Schwartz Olivier
Published: June 2022

Anti-CD20 monoclonal antibodies such as Rituximab, Ofatumumab and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc-dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome-wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti-CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement-dependent-cytotoxicity (CDC). As expected, the screen identifiedMS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B-cell lines.IRF8knockout also decreased the efficacy of antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti-CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting-capacity of anti-CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20-targeting immunotherapies.
Structure of a HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Å Resolution: Routes to Inhibitor Optimization
Authors: Eilers Grant , Gupta Kushol , Allen Audrey , Montermoso Saira , Murali Hemma , Sharp Robert , Hwang Young , Bushman Frederic D. , Van Duyne Gregory
Published: June 2022

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy -terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 Å X-ray crystal structure of the minimal ternary complex between CCD, CTD, and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of π-mediated interactions that suggest specific avenues for future ALLINI development and optimization.
Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalisation
Authors: Urriola-Muñoz Paulina , Pattison Luke A. , Smith Ewan St. John.
Published: June 2022

The naked mole-rat (NMR,Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts (MPSF) ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+-dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted wound closure in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.
G-Trap Assay I: Small GTPases as sensitive immune response biomarkers for active bacteremia
Authors: Clark Ross , Bondu Virginie , Simons Peter , Shevy Laura , Young Stephen , Castillo Eliseo F. , Kanagy Nancy L. , Wandinger-Ness Angela , Howdieshell Thomas , Buranda Tione
Published: June 2022

The annual toll of sepsis is a 33% mortality rate for hospitalized patients with a cost of greater than 60 billion dollars in the U.S. There is a correlation between sepsis mortality rates and time to treatment with broad-spectrum antibiotics. Consequently, antibiotics are prescribed to nearly every patient suspected of bacteremia. However, once determined that broad-spectrum antibiotics are not required, it is unclear how to optimize the de-escalation of the antibiotics. There is an urgent need for methods to distinguish bacteremia from sterile inflammation and to assess antibiotic efficacy. Rho (Rac1 and RhoA) and Ras (Rap1) family GTPases are dynamic nodes of signaling convergence used by immune-activated leukocytes migrating to sites of infection. This study targeted the onset of GTPase activation as a biomarker of infection-induced immune activation in trauma patients. GTP binding assays were performed using a novel GTPase effector trap flow cytometry assay (G-Trap). Here, we demonstrate increased GTP binding to small GTPases (Rac1, Rap1, and RhoA) of resting cells serially exposed to plasma samples from bacteremic trauma patients. Responses to the serial samples showed that GTPase activation was influenced by the concentration of circulating pro- and anti-inflammatory mediators, in tandem with synergy or antagonism from the cytokines and the antibiotic treatment.
G-Trap Assay II: Characterization of blood Leukocyte Functionality differentiates immune activation and immune suppression in bacteremia patient samples
Authors: Simons Peter , Shevy Laura , Bondu Virginie , Wandinger-Ness Angela , Young Stephen , Buranda Tione
Published: June 2022

Sepsis is a severe organ dysfunction syndrome caused by a dysregulation of the immune system’s response to infection. Unfortunately, most infection-causing pathogens aren’t routinely detectable in real-time to enable targeted and lifesaving treatment. Thus, clinicians frequently have limited data on which to base treatment decisions. A complete blood count with differential is available within 24 h, and positive culture is only available in ~30% of cases. Furthermore, a blood culture, the traditional gold standard for accurate diagnosis of bacteremia, may take up to five days for results, long after a clinical decision for sepsis management is required. Circulating leukocytes can sense chemotactic signals released by bloodborne pathogens or focal infections not in the bloodstream. Our earlier study showed that pathogen and host immune factors released in the bloodstream stimulated GTP binding of Ras homology (Rho) GTPases (guanosine triphosphatase) such as Rac1 in quiescent endothelial and human leukocytes after exposure to blood plasma from infected patients.[1] In this study, we measured Rac1•GTP as a biomarker of immune functionality of peripheral blood monocytes and polymorphonuclear cells extracted from blood samples drawn for diagnostic use in blood culture assays; from 120 non-infected control patients and serial blood test samples from 28 patients with a confirmed diagnosis of bloodstream infection. 18 cases presented with Rac1•GTP elevation of ≥3 fold above that of control samples. Ten patients with normal or below-normal GTPase activity, accompanied by neutrophilia or pancytopenia. We used Principal Component Analysis to differentiate the 2D spatial distribution of infected patients and negative controls. Measuring differential leukocyte functionality in infected and control patients’ blood samples with the G-Trap assay may provide an innovative process for a real-time distinction between infection and non-infectious etiologies.
A single cell spatial temporal atlas of skeletal muscle reveals cellular neighborhoods that orchestrate regeneration and become disrupted in aging
Authors: Wang Yu Xin , Holbrook Colin A. , Hamilton James N. , Garoussian Jasmin , Afshar Mohsen , Su Shiqi , Schürch Christian M. , Lee Michael Y. , Goltsev Yury , Kundaje Anshul , Nolan Garry P. , Blau Helen M.
Published: June 2022

Our mobility requires muscle regeneration throughout life. Yet our knowledge of the interplay of cell types required to rebuild injured muscle is lacking, because most single cell assays require tissue dissociation. Here we use multiplexed spatial proteomics and neural network analyses to resolve a single cell spatiotemporal atlas of 34 cell types during muscle regeneration and aging. This atlas maps interactions of immune, fibrogenic, vascular, nerve, and myogenic cells at sites of injury in relation to tissue architecture and extracellular matrix. Spatial pseudotime mapping reveals sequential cellular neighborhoods that mediate repair and a nodal role for immune cells. We confirm this role by macrophage depletion, which triggers formation of aberrant neighborhoods that obstruct repair. In aging, immune dysregulation is chronic, cellular neighborhoods are disrupted, and an autoimmune response is evident at sites of denervation. Our findings highlight the spatial cellular ecosystem that orchestrates muscle regeneration, and is altered in aging.

Highlights

Single cell resolution spatial atlas resolves a cellular ecosystem of 34 cell types in multicellular neighborhoods that mediate efficient skeletal muscle repair

Highly multiplexed spatial proteomics, neural network and machine learning uncovers temporal dynamics in the spatial crosstalk between immune, fibrogenic, vascular, nerve, and muscle stem cells and myofibers during regeneration

Spatial pseudotime mapping reveals coherent formation of multicellular neighborhoods during efficacious repair and the nodal role of immune cells in coordinating muscle repair

In aged muscle, cellular neighborhoods are disrupted by a chronically inflamed state and autoimmunity
Whole blood transcriptome profiles of trypanotolerant and trypanosusceptible cattle highlight a differential modulation of metabolism and immune response during infection byTrypanosoma congolense
Authors: Peylhard Moana , Berthier David , Dayo Guiguigbaza-Kossigan , Chantal Isabelle , Sylla Souleymane , Nidelet Sabine , Dubois Emeric , Martin Guillaume , Sempéré Guilhem , Flori Laurence , Thévenon Sophie
Published: June 2022

Animal African trypanosomosis, caused by blood protozoan parasites transmitted mainly by tsetse flies, represents a major constraint for millions of cattle in sub-Saharan Africa. Exposed cattle include trypanosusceptible indicine breeds, severely affected by the disease, and West African taurine breeds called trypanotolerant owing to their ability to control parasite development, survive and grow in enzootic areas. Until now the genetic basis of trypanotolerance remains unclear. Here, to improve knowledge of the biological processes involved in trypanotolerance versus trypanosusceptibility, we identified bovine genes differentially expressed in five West African cattle breeds during an experimental infection byTrypanosoma congolenseand their biological functions. To this end, whole blood genome-wide transcriptome of three trypanotolerant taurine breeds (N’Dama, Lagune and Baoulé), one susceptible zebu (Zebu Fulani) and one African taurine x zebu admixed breed (Borgou) were profiled by RNA sequencing at four time points, one before and three during infection. As expected, infection had a major impact on cattle blood transcriptome regardless of the breed. The functional analysis of differentially expressed genes over time in each breed confirmed an early activation of the innate immune response, followed by an activation of the humoral response and an inhibition of T cell functions at the chronic stage of infection. More importantly, we highlighted overlooked features, such as a strong disturbance in host metabolism and cellular energy production that differentiates trypanotolerant and trypanosusceptible breeds. N’Dama breed showed the earliest regulation of immune response, associated with a strong activation of cellular energy production, also observed in Lagune, and to a lesser extent in Baoulé. Susceptible Zebu Fulani breed differed from other breeds by the strongest modification in lipid metabolism regulation. Overall, this study provides a better understanding of the biological mechanisms at work during infection, especially concerning the interplay between immunity and metabolism that seems differentially regulated depending on the cattle breeds.
Requirement for specific bacterial genome maintenance pathways in repair of C8-linked pyrrolobenzodiazepine (PBD) bi-aryl monomer-mediated DNA damage
Authors: Joseph Asha Mary , Nahar Kazi , Daw Saheli , Hasan Md. Mahbub , Lo Rebecca , Le Tung B. K. , Rahman Khondaker Miraz , Badrinarayanan Anjana
Published: June 2022

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and antibacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.
Structural bases for blockade and activation of BK channels by Ba2+ ions
Authors: Srivastava Shubhra , Miranda Pablo , de Val Natalia , Giraldez Teresa , Zhu Jianghai , Cachau Raul , Holmgren Miguel
Published: June 2022

Ba2+ increases open probability and blocks the permeation pathway of BK channels. Here we discuss Cryo-EM structures of BK channels with Ba2+. They reveal that Ba2+ occupies site S3 in the selectivity filter, leading to blockade. Ba2+ are also detected at all high affinity Ca2+ binding sites of the gating ring. However, the architectures of this region in these structures represent intermediate transitions between the extreme close and open configurations.
SARS-CoV-2 minor variant genomes at the start of the pandemic contained markers of VoCs
Authors: Dong Xiaofeng , Hiscox Julian A.
Published: June 2022

SARS-CoV-2 emerged through limited zoonotic spillovers and was predicted to have constrained sequence diversity. The dominant consensus and minor variant genomes were determined from the earliest samples associated with the Huanan market and the start of the pandemic. The sequence data confirmed that the dominant consensus genomes shared very close homology. However, there were minor variant genomes present in each sample, which encompassed synonymous and non-synonymous changes. Fusion sequences characteristic of defective RNAs were identified that could be linked between patients. Several substitutions (but not deletions) associated with much later variants of concern (VoCs) were already present as minor variant genomes. This suggests it may be possible to predict futures variants at the start of a pandemic by examining where variability in sequence occurs.
The sets of kinesins and dynein transporting endocytic cargoes determine the effect of tau on their motility
Authors: Beaudet Daniel , Berger Christopher L. , Hendricks Adam G.
Published: June 2022

The misregulation of tau, a neuronal microtubule-associated protein, is linked to defective axonal transport and neurodegenerative disease. We reconstituted the motility of isolated phagosomes along microtubules to ask how the sets of motors transporting a cargo determine its motility and response to tau. Using quantitative photobleaching, we find that early phagosomes (EPs) and late phagosomes (LPs) are associated with different sets of kinesin-1, -2, -3, and dynein. While EPs exhibit unidirectional retrograde transport, LPs move bidirectionally. Previously, we found that tau biases LP transport towards the microtubule minus-end. Here, we find that tau strongly inhibits long-range retrograde EP motility. Tau impedes the forces generated by multi-dynein teams and accelerates dynein unbinding under load. Thus, specific cargoes differentially respond to tau, where dynein-complexes on EPs are more sensitive to tau than those on LPs.
NISNet3D: Three-Dimensional Nuclear Synthesis and Instance Segmentation for Fluorescence Microscopy Images
Authors: Wu Liming , Chen Alain , Salama Paul , Dunn Kenneth , Delp Edward
Published: June 2022

The primary step in tissue cytometry is the automated distinction of individual cells (segmentation). Since cell borders are seldom labeled, researchers generally segment cells by their nuclei. While effective tools have been developed for segmenting nuclei in two dimensions, segmentation of nuclei in three-dimensional volumes remains a challenging task for which few tools have been developed. The lack of effective methods for three-dimensional segmentation represents a bottleneck in the realization of the potential of tissue cytometry, particularly as methods of tissue clearing present researchers with the opportunity to characterize entire organs. Methods based upon deep-learning have shown enormous promise, but their implementation is hampered by the need for large amounts of manually annotated training data. In this paper we describe 3D Nuclei Instance Segmentation Network (NISNet3D), a deep learning-based approach in which training is accomplished using synthetic data, profoundly reducing the effort required for network training. We compare results obtained from NISNet3D with results obtained from eight existing techniques.