bluuebunny/crossref_metadata_2025 huggingface dataset:
Social isolation is linked to classical risk factors of Alzheimer’s disease-related dementias
Authors: Shafighi Kimia , Villeneuve Sylvia , Rosa-Neto Pedro , Badhwar AmanPreet , Poirier Judes , Sharma Vaibhav , Iturria-Medina Yasser , Silveira Patricia P , Dube Laurette , Glahn David , Bzdok Danilo
Published: September 2021

Alzheimer’s disease and related dementias is a major public health burden – compounding over upcoming years due to longevity. Recently, clinical evidence hinted at the experience of social isolation in expediting dementia onset. In 502,506 UK Biobank participants and 30,097 participants from the Canadian Longitudinal Study of Aging, we revisited traditional risk factors for developing dementia in the context of loneliness and lacking social support. Across these measures of subjective and objective social deprivation, we have identified strong links between individuals’ social capital and various indicators of Alzheimer’s disease and related dementias risk, which replicated across both population cohorts. The quality and quantity of daily social encounters had deep connections with key aetiopathological factors, which represent 1) personal habits and lifestyle factors, 2) physical health, 3) mental health, and 4) societal and external factors. Our population-scale assessment suggest that social lifestyle determinants are linked to most neurodegeneration risk factors, highlighting them promising targets for preventive clinical action.
Optimizing epitope conformational ensembles usingα-synuclein cyclic peptide “glycindel” scaffolds: A customized immunogen method for generating oligomer-selective antibodies for Parkinson’s disease
Authors: Hsueh Shawn C.C. , Aina Adekunle , Roman Andrei Yu. , Cashman Neil R. , Peng Xubiao , Plotkin Steven S.
Published: September 2021

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modelled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines (“glycindels”), to best mimic a misfolding-specific conformation of an epitope ofα-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds ofα-synucleinin silicobased on their ensemble overlap properties with the fibril, oligomer-model, and isolated monomer ensembles. We present experimental data of seeded aggregation that supports nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome, by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap was quantified by the Jensen-Shannon Divergence, and a new measure introduced here—the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble, and which may be a more useful measure in developing immunogens that confer conformational-selectivity to an antibody.

Graphical TOC Entry
Embracing imperfection: machine-assisted invertebrate classification in real-world datasets
Authors: Blair Jarrett , Weiser Michael D. , de Beurs Kirsten , Kaspari Michael , Siler Cameron , Marshall Katie E.
Published: September 2021

Despite growing concerns over the health of global invertebrate diversity, terrestrial invertebrate monitoring efforts remain poorly geographically distributed. Machine-assisted classification has been proposed as a potential solution to quickly gather large amounts of data; however, previous studies have often used unrealistic or idealized datasets to train their models.

In this study, we describe a practical methodology for including machine learning in ecological data acquisition pipelines. Here we train and test machine learning algorithms to classify over 56,000 bulk terrestrial invertebrate specimens from morphometric data and contextual metadata. All vouchered specimens were collected in pitfall traps by the National Ecological Observatory Network (NEON) at 27 locations across the United States in 2016. Specimens were photographed, and morphometric data was extracted as feature vectors using ImageJ. Issues stemming from inconsistent taxonomic label specificity were resolved by making classifications at the lowest identified taxonomic level (LITL). Taxa with too few specimens to be included in the training dataset were classified by the model using zero-shot classification.

When classifying specimens that were known and seen by our models, we reached an accuracy of 72.7% using extreme gradient boosting (XGBoost) at the LITL. Models that were trained without contextual metadata underperformed models with contextual metadata by an average of 7.2%. We also classified invertebrate taxa that were unknown to the model using zero-shot classification, with an accuracy of 39.4%, resulting in an overall accuracy of 71.5% across the entire NEON dataset.

The general methodology outlined here represents a realistic application of machine learning as a tool for ecological studies. Hierarchical and LITL classifications allow for flexible taxonomic specificity at the input and output layers. These methods also help address the ‘long tail’ problem of underrepresented taxa missed by machine learning models. Finally, we encourage researchers to consider more than just morphometric data when training their models, as we have shown that the inclusion of contextual metadata can provide significant improvements to accuracy.
The Effects of Incline Level on Optimized Lower-Limb Exoskeleton Assistance
Authors: Franks Patrick W. , Bryan Gwendolyn M. , Reyes Ricardo , O’Donovan Meghan P. , Gregorczyk Karen N. , Collins Steven H.
Published: September 2021

For exoskeletons to be successful in real-world settings, they will need to be effective across a variety of terrains, including on inclines. While some single-joint exoskeletons have assisted incline walking, recent successes in level-ground assistance suggest that greater improvements may be possible by optimizing assistance of the whole leg. To understand how exoskeleton assistance should change with incline, we used human-in-the-loop optimization to find whole-leg exoskeleton assistance torques that minimized metabolic cost on a range of grades. We optimized assistance for three expert, able-bodied participants on 5 degree, 10 degree and 15 degree inclines using a hip-knee-ankle exoskeleton emulator. For all assisted conditions, the cost of transport was reduced by at least 50% relative to walking in the device with no assistance, a large improvement to walking that is comparable to the benefits of whole-leg assistance on level-ground. This corresponds to large absolute reductions in metabolic cost, with the most strenuous conditions reduced by 4.9 W/kg, more than twice the entire energy cost of level walking. Optimized extension torque magnitudes and exoskeleton power increased with incline, with hip extension, knee extension and ankle plantarflexion often growing as large as allowed by comfort-based limits. Applied powers on steep inclines were double the powers applied during level-ground walking, indicating that larger exoskeleton power may be optimal in scenarios where biological powers and costs are higher. Future exoskeleton devices can be expected to deliver large improvements in walking performance across a range of inclines, if they have sufficient torque and power capabilities.
Individualized Assays of Temporal Coding in the Ascending Human Auditory System
Authors: Borjigin Agudemu , Hustedt-Mai Alexandra R. , Bharadwaj Hari M.
Published: September 2021

Neural phase-locking to temporal fluctuations is a fundamental and unique mechanism by which acoustic information is encoded by the auditory system. The perceptual role of this metabolically expensive mechanism, the neural phase-locking to temporal fine structure (TFS) in particular, is debated. Although hypothesized, it is unclear if auditory perceptual deficits in certain clinical populations are attributable to deficits in TFS coding. Efforts to uncover the role of TFS have been impeded by the fact that there are no established assays for quantifying the fidelity of TFS coding at the individual level. While many candidates have been proposed, for an assay to be useful, it should not only intrinsically depend on TFS coding, but should also have the property that individual differences in the assay reflect TFS coding per se over and beyond other sources of variance. Here, we evaluate a range of behavioral and electroencephalogram (EEG)-based measures as candidate individualized measures of TFS sensitivity. Our comparisons of behavioral and EEG-based metrics suggest that extraneous variables dominate both behavioral scores and EEG amplitude metrics, rendering them ineffective. After adjusting behavioral scores using lapse rates, and extracting latency or percent-growth metrics from EEG, interaural timing sensitivity measures exhibit robust behavior-EEG correlations. Together with the fact that unambiguous theoretical links can be made relating binaural measures and phase-locking to TFS, our results suggest that these “adjusted” binaural assays may be well-suited for quantifying individual TFS processing.
Model-Based Meta-Analysis of Relapsing Mouse Model Studies from the Critical Path to Tuberculosis Drug Regimens Initiative Database
Authors: Berg Alexander , Clary James , Hanna Debra , Nuermberger Eric , Lenaerts Anne , Ammerman Nicole , Ramey Michelle E , Hartley Dan , Hermann David
Published: September 2021

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease-related cause of death worldwide, necessitating the development of new and improved treatment regimens. Non-clinical evaluation of candidate drug combinations via the relapsing mouse model (RMM) is an important step in regimen development, through which candidate regimens that provide the greatest decrease in probability of relapse following treatment in mice may be identified for further development. Although RMM studies are a critical tool to evaluate regimen efficacy, making comprehensive "apples to apples" comparisons of regimen performance in the RMM has been a challenge, in large part due to the need to evaluate and adjust for variability across studies arising from differences in design and execution. To address this knowledge gap, we performed a model-based meta-analysis on data for 17 unique regimens obtained from a total of 1592 mice across 28 RMM studies. Specifically, a mixed-effects logistic regression model was developed that described the treatment duration-dependent probability of relapse for each regimen and identified relevant covariates contributing to inter-study variability. Using the model, covariate-normalized metrics of interest, namely treatment duration required to reach 50% and 10% relapse probability, were derived and used to compare relative regimen performance. Overall, the model-based meta-analysis approach presented herein enables cross-study comparison of efficacy in the RMM, and provides a framework whereby data from emerging studies may be analyzed in the context of historical data to aid in selecting candidate drug combinations for clinical evaluation as TB drug regimens.
Effect of SFX-01 on proliferation of human glioblastoma cell lines
Authors: Leung Euphemia , Wright Edwina , Baguley Bruce C.
Published: September 2021

Glioblastoma (GBM) is the most aggressive and lethal of brain tumours and there is a clear need for novel therapies. SFX-01, a stabilised formu of sulforaphane (SFN) that is complexed with α-cyclodextrin, is being investigated as a potential anticancer drug. And as part of this investigation we have determined its effect on the proliferation of a series of tumour cell lines. Growth-inhibitory concentrations (IC50 values) of SFX-01 and reference SFN were highly correlated but showed a divergence at higher drug concentrations that is likely to reflect drug binding to α-cyclodextrin complexes. Because SFN is redox-sensitive, we also compared the effects of these drugs on four GBM lines that had been derived and cultured under physiological oxygen (5%) conditions. Comparisons were also made using a three-dimensional spheroid model, used to mimic the in vivo state of glioblastomas and to simulate barriers to drug delivery in vivo. SFX-01 and reference SFN and inhibited proliferation of four GBM cell lines in monolayers as well as in spheroids; α-cyclodextrin alone had no significant effect. Our results are consistent with the hypothesis that SFN is liberated from SFX-01 at concentrations sufficient to inhibit cell growth. We envision that SFX-01 will have improved pharmacokinetic properties in vivo, warranting further pre-clinical investigation and clinical development.
The value of genotype-specific reference for transcriptome analyses
Authors: Guo Wenbin , Coulter Max , Waugh Robbie , Zhang Runxuan
Published: September 2021

High quality transcriptome assembly using short reads from RNA-seq data still heavily relies upon reference-based approaches, of which the primary step is to align RNA-seq reads to a single reference genome of haploid sequence. However, it is increasingly apparent that while different genotypes within a species share “core” genes, they also contain variable numbers of “specific” genes that are only present a subset of individuals. Using a common reference may thus lead to a loss of genotype-specific information in the assembled transcript dataset and the generation of erroneous, incomplete or misleading transcriptomics analysis results. With the recent development of pan-genome information in many species, it is important that we understand the limitations of single genotype references for transcriptomics analysis. In this study, we quantitively evaluated the advantages of using genotype-specific reference genomes for transcriptome assembly and analysis using cultivated barley as a model. We mapped barley cultivar Barke RNA-seq reads to the Barke genome and to the cultivar Morex genome (common barley genome reference) to construct a genotype specific Reference Transcript Dataset (sRTD) and a common Reference Transcript Datasets (cRTD), respectively. We compared the two RTDs according to their transcript diversity, transcript sequence and structure similarity and the accuracy they provided for transcript quantification and differential expression analysis. Our evaluation shows that the sRTD has a significantly higher diversity of transcripts and alternative splicing events. Despite using a high-quality reference genome for assembly of the cRTD, we miss ca. 40% transcripts present in the sRTD and cRTD only has ca. 70% true assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression and differential alternative splicing analysis. However, gene level quantification and comparative expression analysis are less affected by the source RTD, which indicates that analysing transcriptomic data at the gene level may be a reasonable compromise when a high-quality genotype-specific reference is not available.
Dinosaur bonebed amber from an original swamp forest soil
Authors: Álvarez-Parra Sergio , la Fuente Ricardo Pérez-de , Peñalver Enrique , Barrón Eduardo , Alcalá Luis , Pérez-Cano Jordi , Martín-Closas Carles , Trabelsi Khaled , Meléndez Nieves , Del Valle Rafael López , Lozano Rafael P , Peris David , Rodrigo Ana , Sarto i Monteys Víctor , Bueno-Cebollada Carlos A , Menor-Salván César , Philippe Marc , Sánchez-García Alba , Peña-Kairath Constanza , Arillo Antonio , Espílez Eduardo , Mampel Luis , Delclòs Xavier
Published: September 2021

Dinosaur bonebeds with amber content, yet scarce, offer a superior wealth and quality of data on ancient terrestrial ecosystems. However, the preserved palaeodiversity and/or taphonomic characteristics of these exceptional localities had hitherto limited their palaeobiological potential. Here we describe the amber from the Lower Cretaceous dinosaur bonebed of Ariño (Teruel, Spain) using a multidisciplinary approach. Amber is found in both a root layer with amber strictly in situ and a litter layer namely composed of aerial pieces unusually rich in bioinclusions, encompassing 11 insect orders, arachnids, and a few plant and vertebrate remains, including a feather. Additional palaeontological data ‒ charophytes, palynomorphs, ostracods‒ are provided. Ariño arguably represents the most prolific and palaeobiologically diverse locality in which fossiliferous amber and a dinosaur bonebed have been found in association, and the only one known where the vast majority of the palaeontological assemblage suffered no or low-grade pre-burial transport. That has enabled unlocking unprecedentedly complete and reliable palaeoecological data out of two complementary windows of preservation from the same site.
PhosPiR: An automated phospho-proteomic pipeline in R
Authors: Hong Ye , Flinkman Dani , Suomi Tomi , Pietilä Sami , James Peter , Coffey Eleanor , Elo Laura L.
Published: September 2021

Large-scale phospho-proteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from this data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from largescale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phospho-site annotation and translation across species, multi-level enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.
Control Cell Migration by Engineering Integrin Ligand Assembly
Authors: Hu Xunwu , Roy Sona Rani , Jin Chengzhi , Li Guanying , Zhang Qizheng , Natsuko Asano , Asahina Shunsuke , Kajiwara Tomoko , Feng Bolu , Aoki Kazuhiro , Takahara Atsushi , Zhang Ye
Published: September 2021

Advances in mechanistic understanding of integrin-mediated adhesion highlight the importance of precise control of ligand presentation in directing cell migration. Top-down nanopatterning limited the spatial presentation to sub-micron. To enhance it to molecular level, we propose a bottom-up nanofabrication strategy. Via self-assembly and co-assembly, precise control of ligand presentation is succeeded by varying the proportions of assembling ligand and nonfunctional peptide. Assembled nanofilaments fulfill multi-functions exerting enhancement to suppression effect on cell migration with tunable amplitudes. Self-assembled nanofilaments possessing super high ligand density selectively suppress cancer cell migration by preventing integrin/actin disassembly at cell rear, which provides new insights to ligand-density-dependent-modulation, revealing valuable inputs to therapeutic innovations in tumor metastasis.

One-Sentence Summary

Engineering integrin ligand assembly from bottom-up offers a generalized tool to selectively control cell migration with tunable amplitudes.
Chronic sodium bromide treatment relieves autistic-like behavioral deficits in three mouse models of autism
Authors: Derieux Cécile , Léauté Audrey , Brugoux Agathe , Jacaz Déborah , Pin Jean-Philippe , Kniazeff Julie , Merrer Julie Le , Becker Jerome AJ
Published: September 2021

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behavior. To date, no pharmacological treatment has been approved that ameliorates social behavior in patients with ASD. Based on the excitation/inhibition imbalance theory of autism, we hypothesized that bromide ions, long used as an antiepileptic medication, could relieve core symptoms of ASD. We evaluated the effects of chronic sodium bromide (NaBr) administration on autistic-like symptoms in three genetic mouse models of autism: Oprm1-/- , Fmr1-/- and Shank3Δex13-16-/- mice. We showed that chronic NaBr treatment relieved autistic-like behaviors in these three models. In Oprm1-/- mice, these beneficial effects were superior to those of chronic bumetanide administration. At transcriptional level, chronic NaBr in Oprm1 null mice was associated with increased expression of genes coding for chloride ions transporters, GABAA receptor subunits, oxytocin and mGlu4 receptor. Lastly, we uncovered synergistic alleviating effects of chronic NaBr and a positive allosteric modulator (PAM) of mGlu4 receptor on autistic-like behavior in Oprm1-/- mice. We evidenced in heterologous cells that bromide ions behave as PAMs of mGlu4, providing a molecular mechanism for such synergy. Our data reveal the therapeutic potential of bromide ions, alone or in combination with a PAM of mGlu4 receptor, for the treatment of ASDs.
Investigation of a derived adverse outcome pathway (AOP) network for endocrine-mediated perturbations
Authors: Ravichandran Janani , Karthikeyan Bagavathy Shanmugam , Samal Areejit
Published: September 2021

An adverse outcome pathway (AOP) is a compact representation of the available mechanistic information on observed adverse effects upon environmental exposure. Sharing of events across individual AOPs has led to the emergence of AOP networks. Since AOP networks are expected to be functional units of toxicity prediction, there is current interest in their development tailored to specific research question or regulatory problem. To this end, we have developed a detailed workflow to construct a comprehensive endocrine-specific AOP (ED-AOP) network. Connectivity analysis of the ED-AOP network comprising 48 AOPs reveals 7 connected components and 12 isolated AOPs. Subsequently, we apply standard network measures to perform an in-depth analysis of the two largest connected components of the ED-AOP network. Notably, the graph-theoretic analyses led to the identification of important events including points of convergence or divergence in the ED-AOP network. Detailed analysis of the largest component in the ED-AOP network gives insights on the systems-level perturbations caused by endocrine disruption, emergent paths, and stressor-event associations. In sum, the derived ED-AOP network can be used to address the current knowledge gaps in the existing regulatory framework and aid in better risk assessment of environmental chemicals.
Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform
Authors: Gruber Franz S. , Johnston Zoe C. , Norcross Neil R. , Georgiou Irene , Wilson Caroline , Read Kevin D. , Gilbert Ian H. , Swedlow Jason R. , de Silva Sarah Martins , Barratt Christopher LR
Published: September 2021

Study question

Can a high-throughput screening platform facilitate male fertility drug discovery?

Summary answer

A high-throughput screening platform identified a large number of compounds that enhanced sperm motility.

What is known already

Several efforts to find small molecules modulating sperm function have been performed but not using high-throughput technology.

Study design, size, duration

Healthy donor semen samples were used and samples were pooled (3-5 donors per pool). Primary screening was performed in singlicate; dose-response screening was performed in duplicate (independent donor pools).

Participants/materials, setting, methods

Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 uM) to identify those compounds with enhancing effects on motility. A total of ∼17,000 compounds from the following libraries: ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box were screened. Dose response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a University setting.

Main results and the role of chance

From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to DMSO treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified in particular PDE10A inhibitors as well as number of compounds not previously identified/known to enhance human sperm motility such as those related to GABA signaling.

Limitations, reasons for caution

Compounds have been tested with prepared donor spermatozoa and only incubated for a short period of time. Therefore, the effect of compounds on whole semen or with longer incubation time may be different. All experiments were performed in vitro.

Wider implications of the findings

This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential inhibitors as promising start-point for a medicinal chemistry programme for potential enhancement of male infertility. Moreover, with disclosure of the results of screening we present a substantial resource to inform further work in the field

Study funding/competing interest(s)

This study was supported by the Bill and Melinda Gates Foundation and Scottish Funding Council and Scottish Universities Life Science Alliance.
MAGI-MS: Multiple seed-centric module discovery
Authors: Chow Julie C. , Zhou Ryan , Hormozdiari Fereydoun
Published: September 2021

Complex disorders manifest by the interaction of multiple genetic and environmental factors. Through the construction of genetic modules that consist of highly co-expressed genes, it is possible to identify genes that participate in common biological pathways relevant to specific phenotypes. We have previously developed tools MAGI and MAGI-S for genetic module discovery by incorporating co-expression and protein-interaction networks. Here we introduce an extension to MAGI-S, denoted as Merging Affected Genes into Integrated Networks - Multiple Seeds (MAGI-MS), that permits the user to further specify a disease pathway of interest by selecting multiple seed genes likely to function in the same molecular mechanism. By providing MAGI-MS with pairs of seed genes involved in processes underlying certain classes of neurodevelopmental disorders, such as epilepsy, we demonstrate that MAGI-MS can reveal modules enriched in genes relevant to chemical synaptic transmission, glutamatergic synapse, and other functions associated with the provided seed genes.

Availability and implementation

MAGI-MS is free and is available at: https://github.com/jchow32/MAGI-MS
The role of antigen recognition in the γδ T cell response at the controlled stage ofM. tuberculosisinfection
Authors: Chowdhury Roshni Roy , Valainis John R. , Kask Oliver , Ohanyan Mane , Sun Meng , Huang Huang , Dubey Megha , von Boehmer Lotta , Sola Elsa , Huang Xianxi , Nguyen Patricia K. , Scriba Thomas J. , Davis Mark M. , Bendall Sean C. , Chien Yueh-hsiu
Published: September 2021

γδT cells contribute to host immune defense uniquely; but how they function in different stages (e.g., acute versus chronic) of a specific infection remains unclear. As the role ofγδT cells in early, activeMycobacterium tuberculosis(Mtb) infection is well documented, we focused on elucidating theγδT cell response in persistent or controlled Mtb infection. Systems analysis of circulatingγδT cells from a South African adolescent cohort identified a distinct population of CD8+γδT cells that expanded in this state. These cells had features indicative of persistent antigenic exposure but were robust cytolytic effectors and cytokine/chemokine producers. While theseγδT cells displayed an attenuated response to TCR-mediated stimulation, they expressed Natural Killer (NK) cell receptors and had robust CD16 (FcγRIIIA)-mediated cytotoxic response, suggesting alternative ways forγδT cells to control this stage of the infection. Despite this NK- like functionality, the CD8+γδT cells consisted of highly expanded clones, which utilized TCRs with different Vγ/δpairs. Theses TCRs could respond to an Mtb-lysate, but not to phosphoantigens, which are components of Mtb-lysate that activateγδT cells in acute Mtb infection, indicating that the CD8+γδT cells were induced in a stage-specific, antigen-driven manner. Indeed, trajectory analysis showed that theseγδT cells arose from naive cells that had traversed distinct differentiation paths in this infection stage. Importantly, increased levels of CD8+γδT cells were also found in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may lead to similarγδT cell responses.
Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity
Authors: Garrido Jose L. , Medina Matias , Bravo Felipe , McGee Sarah , Fuentes Francisco , Calvo Mario , Bowman James W. , Bahl Christopher D. , Barría Maria Inés , Brachman Rebecca A. , Alvarez Raymond A.
Published: September 2021

Despite SARS-CoV-2 being a “novel” coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (FcγR2a & FcγR3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2’FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2’FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.
The genetic basis of tail-loss evolution in humans and apes
Authors: Xia Bo , Zhang Weimin , Wudzinska Aleksandra , Huang Emily , Brosh Ran , Pour Maayan , Miller Alexander , Dasen Jeremy S. , Maurano Matthew T. , Kim Sang Y. , Boeke Jef D. , Yanai Itai
Published: September 2021

The loss of the tail is one of the main anatomical evolutionary changes to have occurred along the lineage leading to humans and to the “anthropomorphous apes”1,2. This morphological reprogramming in the ancestral hominoids has been long considered to have accommodated a characteristic style of locomotion and contributed to the evolution of bipedalism in humans3–5. Yet, the precise genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Primate genome sequencing projects have made possible the identification of causal links between genotypic and phenotypic changes6–8, and enable the search for hominoid-specific genetic elements controlling tail development9. Here, we present evidence that tail-loss evolution was mediated by the insertion of an individual Alu element into the genome of the hominoid ancestor. We demonstrate that this Alu element – inserted into an intron of the TBXT gene (also called T or Brachyury10–12) – pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated a mouse model that mimics the expression of human TBXT products by expressing both full-length and exon-skipped isoforms of the mouse TBXT ortholog. We found that mice with this genotype exhibit the complete absence of a tail or a shortened tail, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype, albeit with incomplete penetrance. We further noted that mice homozygous for the exon-skipped isoforms exhibited embryonic spinal cord malformations, resembling a neural tube defect condition, which affects ∼1/1000 human neonates13. We propose that selection for the loss of the tail along the hominoid lineage was associated with an adaptive cost of potential neural tube defects and that this ancient evolutionary trade-off may thus continue to affect human health today.
The 3D spatial constraint on 6.1 million amino acid sites in the human proteome
Authors: Li Bian , Roden Dan M. , Capra John A.
Published: September 2021

Quantification of the tolerance of protein-coding sites to genetic variation within human populations has become a cornerstone of the prediction of the function of genomic variants. We hypothesize that the constraint on missense variation at individual amino acid sites is largely shaped by direct 3D interactions with neighboring sites. To quantify the constraint on protein-coding genetic variation in 3D spatial neighborhoods, we introduce a new framework called COntact Set MISsense tolerance (or COSMIS) for estimating constraint. Leveraging recent advances in computational structure prediction, large-scale sequencing data from gnomAD, and a mutation-spectrum-aware statistical model, we comprehensively map the landscape of 3D spatial constraint on 6.1 amino acid sites covering >80% (16,533) of human proteins. We show that the human proteome is broadly under 3D spatial constraint and that the level of spatial constraint is strongly associated with disease relevance both at the individual site level and the protein level. We demonstrate that COSMIS performs significantly better at a range of variant interpretation tasks than other population-based constraint metrics while also providing biophysical insight into the potential functional roles of constrained sites. We make our constraint maps freely available and anticipate that the structural landscape of constrained sites identified by COSMIS will facilitate interpretation of protein-coding variation in human evolution and prioritization of sites for mechanistic or functional investigation.
Mechanistic insights into dynamic mutual regulation of USP14 and proteasome
Authors: Zhang Shuwen , Zou Shitao , Yin Deyao , Finley Daniel , Wu Zhaolong , Mao Youdong
Published: September 2021

Proteasomal degradation of ubiquitylated proteins is sophisticatedly regulated at multiple levels1–3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme USP14 that associates reversibly with the proteasome. How USP14 is activated and regulates the proteasome function remains unknown4–7. Here we report cryo-electron microscopy (cryo-EM) structures of human USP14 in complex with the 26S proteasome in nine conformational states at 3.0-3.6 Å resolution, captured during polyubiquitylated protein degradation. Time-resolved cryo-EM analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14 and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, USP14 activation allosterically reprograms conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8–10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11–13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide unprecedented insights into the complete functional cycles of USP14-regulated proteasome and of USP14 activation-deubiquitylation-disassembly and establish mechanistic foundations for USP14-targeted therapeutic discovery.
Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing
Authors: Marquet Mike , Zöllkau Janine , Pastuschek Jana , Viehweger Adrian , Schleußner Ekkehard , Makarewicz Oliwia , Pletz Mathias W. , Ehricht Ralf , Brandt Christian
Published: September 2021

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore’s adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.
Transitioning to confined spaces impacts bacterial swimming and escape response
Authors: Lynch Jonathan B. , James Nicholas , McFall-Ngai Margaret , Ruby Edward G. , Shin Sangwoo , Takagi Daisuke
Published: September 2021

Symbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them towards their eventual destination. However, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacteriumVibrio fischeri,which forms a binary symbiosis with the Hawaiian bobtail squid,Euprymna scolopes, must navigate tight physical confinement, squeezing through a bottleneck constricting to ~2 μm in width on the way to its eventual home. Using microfluidicin vitroexperiments, we discovered thatV. fischericells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period between reversals. Additional experiments in asymmetric capillary tubes confirmed thatV. fischeriquickly escape from tapered ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demonstrate that non-trivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats.

Significance Statement

Symbiotic bacteria that navigate to and through specific host tissues often face tight physical confinement. This work reveals that confinement-associated changes in swimming can dramatically alter taxis, shaping bacterial localization in conjuncture with other motility-directing cues. This work helps explain how bacteria can avoid getting stuck in confined areas while transiting to privileged spaces, adding confinement as an environmental cue that symbiotic bacteria use to shape their motility behavior.
Muscle weakness precedes atrophy during cancer cachexia and is associated with muscle-specific mitochondrial stress
Authors: Delfinis Luca J. , Bellissimo Catherine A. , Gandhi Shivam , DiBenedetto Sara N. , Rosa-Caldwell Megan E. , Rahman Fasih A. , Wiggs Michael P. , Schlattner Uwe , Quadrilatero Joe , Greene Nicholas P. , Perry Christopher G.R.
Published: September 2021

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but distinct responses between muscles and across time remains unclear. We aimed to determine the time-dependent and muscle-specific responses to Colon-26 (C26) cancer-induced cachexia in mice. At 2 weeks post-inoculation, the presence of small tumours did not alter body or muscle mass but decreased force production in the quadriceps and diaphragm. Pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. At 4 weeks, large tumours corresponded to lower body mass, muscle mass, and cross-sectional area of fibers in quadriceps and diaphragm. Force production in quadriceps was unchanged but remained lower in diaphragm vs control. Mitochondrial respiration was increased while H2O2 emission was unchanged in both muscles vs control. Mitochondrial creatine sensitivity was compromised in quadriceps. These findings indicate muscle weakness precedes atrophy in quadriceps and diaphragm but is linked to heterogeneous mitochondrial alterations. Eventual muscle-specific restorations in force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscles to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.
Identification of the safe harbor locus, AAVS1, from porcine genome and site-specific integration of recombinase-mediated cassette exchange system in porcine fibroblasts using CRISPR/Cas9
Authors: Lee Choongil , Yum Soo-Young , Choi Woojae , Kim Seokjoong , Jang Goo , Koo Okjae
Published: September 2021

Gene integration at site-specific loci, such as safe harbor regions for stable expression via transgenesis, is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.
Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11
Authors: Dillard Kaylee E. , Terrace Cynthia , Javanmardi Kamyab , Kim Wantae , Forsberg Kevin J. , Finkelstein Ilya J.
Published: September 2021

Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but are generally specific for a narrow subset of Cas nucleases that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits diverse Cas9 sub-typesin vitroand human cells. Single-molecule fluorescence imaging reveals that AcrIIA11 interferes with the first steps of target search by reducingS. aureusCas9’s diffusion on non-specific DNA. DNA cleavage is inhibited because the AcrIIA11:Cas9 complex is kinetically trapped at PAM-rich decoy sites, preventing Cas9 from reaching its target. This work establishes that DNA trapping can be used to inhibit a broad spectrum of Cas9 orthologsin vitroand during mammalian genome editing.