bluuebunny/crossref_metadata_2025 huggingface dataset:
Do molecular fingerprints identify diverse active drugs in large-scale virtual screening? (no)
Authors: Venkatraman Vishwesh , Gaiser Jeremiah , Demekas Daphne , Roy Amitava , Xiong Rui , Wheeler Travis J.
Published: September 2022

Computational approaches for small-molecule drug discovery now regularly scale to consideration of libraries containing billions of candidate small molecules. One promising approach to increased speed in evaluating billion-molecule libraries is to develop succinct representations of each molecule that enable rapid identification of molecules with similar properties. Molecular fingerprints are thought to provide a mechanism for producing such representations. Here, we explore the utility of commonly-used fingerprints in the context of predicting similar molecular activity. We show that fingerprint similarity provides little discriminative power between active and inactive molecules for a target protein based on a known active – while they may sometimes provide some enrichment for active molecules in a drug screen, a screened dataset will still be dominated by inactive molecules. We also demonstrate that high-similarity actives appear to share a scaffold with the query active, meaning that they could more easily be identified by structural enumeration. Furthermore, even when limited to only active molecules, fingerprint similarity values do not correlate with compound potency. In sum, these results highlight the need for a new wave of molecular representations that will improve the capacity to detect biologically active molecules based on similarity to other such molecules.
The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change
Authors: Rehm Heidi L , Alaimo Joseph T , Aradhya Swaroop , Bayrak-Toydemir Pinar , Best Hunter , Brandon Rhonda , Buchan Jillian G , Chao Elizabeth C. , Chen Elaine , Clifford Jacob , Cohen Ana S A , Conlin Laura K , Das Soma , Davis Kyle W , Gaudio Daniela del , Viso Florencia Del , DiVincenzo Christina , Eisenberg Marcia , Guidugli Lucia , Hammer Monia B , Harrison Steven M , Hatchell Kathryn E , Dyer Lindsay Havens , Hoang Lily U , Holt James M. , Jobanputra Vaidehi , Karbassi Izabela D , Kearney Hutton M , Kelly Melissa A. , Kelly Jacob M. , Kluge Michelle L , Komala Timothy , Kruszka Paul , Lau Lynette , Lebo Matthew S. , Marshall Christian R , McKnight Dianalee , McWalter Kirsty , Meng Yan , Nagan Narasimhan , Neckelmann Christian S , Neerman Nir , Niu Zhiyv , Paolillo Vitoria K , Paolucci Sarah A , Perry Denise , Pesaran Tina , Radtke Kelly , Rasmussen Kristen J , Retterer Kyle , Saunders Carol J , Spiteri Elizabeth , Stanley Christine , Szuto Anna , Taft Ryan J , Thiffault Isabelle , Thomas Brittany C , Thomas-Wilson Amanda , Thorpe Erin , Tidwell Timothy J , Towne Meghan C , Zouk Hana
Published: September 2022

PURPOSE

Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact.

METHODS

Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 - 2021.

RESULTS

We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared to MGPs (32.6%; p<0.0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; p<0.001) whereas the use of GS compared to ES had no impact (22.2% vs 22.6%; p=ns).

CONCLUSION

The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources towards important VUS follow-up.
Allelic Transcription Factor binding shape transcriptional kinetics observed with scRNA-seq in human cell lines
Authors: Jin Bowen , Feng Hao , Bush William S.
Published: September 2022

Gene expression from bulk RNA-seq studies is an average measurement between two chromosomes and across cell populations. Both allelic and cell-to-cell heterogeneity in gene expression results from promoter bursting patterns that repeatedly alternate between an activated and inactivated state. Increased cell-to-cell heterogeneity in gene expression has been associated with aging and stem cell pluripotency. However, studies of bursting kinetics and their molecular mechanism are relatively limited in human cells compared to other species due to laborious single-molecule experiments. Here, we systematically investigate the regulatory effect of genetic variants and transcription factor (TF) binding on transcriptional kinetics at the single chromosome level with GM12878. We found that the transcription initiation rate and burst frequency correlate most with eQTL effect sizes among transcriptional kinetics, which suggests that eQTLs affect average gene expression mainly through altering burst kinetics. We further found that ∼90% of the variance of burst frequency can be explained by TF occupancy in phase with the core promoter. We identified and replicated several examples where eQTL or GWAS catalog loci perturb TF binding affinity and are consequently associated with the change of burst kinetics.
Conserved allosteric inhibition mechanism in SLC1 transporters
Authors: Dong Yang , Wang Jiali , Garibsingh Rachel-Ann , Hutchinson Keino , Shi Yueyue , Eisenberg Gilad , Yu Xiaozhen , Schlessinger Avner , Grewer Christof
Published: September 2022

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined using cryo-EM, in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki= 9.3 μM), much closer to the EAAT1 value of 0.6 μM. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.
FDA-approved drug screening identified micafungin as an antiviral agent against bat-borne emerging zoonotic Pteropine orthoreovirus
Authors: Katta Tetsufumi , Sato Ayato , Kadofusa Naoya , Ishibashi Tomoki , Shimoda Hiroshi , Iida Atsuo , Hondo Eiichi
Published: September 2022

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, severe acute respiratory syndrome (SARS) coronavirus, and SARS-CoV-2. Pteropine orthoreovirus (PRV), which spillover event occurred from fruit bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is a critical tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV.

Highlights

A library of 2,943 FDA-approved drugs was screened to find potential antiviral drugs of Pteropine orthoreovirus.

Six hit compounds dramatically inhibited viral replication in vitro.

Micafungin possessed antiviral activity to multiple strains of PRV.
Noncanonical inflammasome assembly requires caspase-11 catalytic activity and intra-molecular autoprocessing
Authors: Akuma Daniel C. , Grubaugh Daniel , Odunze Chukwuma E. , Shin Sunny , Taabazuing Cornelius Y. , Brodsky Igor E.
Published: September 2022

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammsasomes. Inflammasomes are large oligomeric complexes that recruit caspases to undergo proximity-induced autoprocessing, leading to formation of the enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high molecular weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Using fluorescently labeled Casp11, we found that LPS transfection of macrophages induced Casp11 speck formation, providing direct evidence that Casp11 forms LPS-induced specks in macrophages. Unexpectedly, we found that catalytic activity was required for Casp11 to form LPS-induced specks in macrophages. Importantly, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and Casp11 processing via an exogenous protease was sufficient to induce Casp11 speck formation. These data reveal a previously-undescribed role for Casp11 catalytic activity and self-cleavage in non-canonical inflammasome assembly and indicate that Casp11 catalytic activity and autoprocessing occur upstream of, and mediate, Casp11 oligomerization. Our data provide new insight into the molecular requirements for assembly of Casp11 noncanonical inflammasome complexes.
PepGM: A probabilistic graphical model for taxonomic inference of viral proteome samples with associated confidence scores
Authors: Holstein Tanja , Kistner Franziska , Martens Lennart , Muth Thilo
Published: September 2022

Motivation

Inferring taxonomy in mass spectrometry-based shotgun proteomics is a complex task. In multi-species or viral samples of unknown taxonomic origin, the presence of proteins and corresponding taxa must be inferred from a list of identified peptides which is often complicated by protein homology: many proteins do not only share peptides within a taxon but also between taxa. However, correct taxonomic identification is crucial when identifying different viral strains with high sequence homology – considering, e.g., the different epidemiological characteristics of the various strains of SARS-CoV-2. Additionally, many viruses mutate frequently, further complicating the correct assignment of virus proteomic samples.

Results

We present PepGM, a probabilistic graphical for the taxonomic assignment of virus proteomic samples with strain-level resolution and associated confidence scores. PepGM combines the results of a standard proteomic database search algorithm with belief propagation to calculate the marginal distributions, and thus confidence score, for potential taxonomic assignments. We demonstrate the performance of PepGM using several publicly available virus proteomic datasets, showing its strain-level resolution performance. In two out of eight cases, the taxonomic assignments were only correct on species level, which PepGM clearly indicates by lower confidence scores.

Availability and Implementation

PepGM is written in Python and embedded into a Snakemake workflow. Its is available at https://github.com/BAMeScience/PepGM
Bioefficacy of hydroxytyrosol-rich food supplements on preventing lipid peroxidation in healthy men
Authors: Bender Cecilia , Candi Ilaria , Rogel Eva
Published: September 2022

In the present study we report the efficacy of food matrices derived from olives in preventing oxidation of low-density lipoprotein cholesterol (LDL) and lipid peroxidation. To this end, 12 healthy volunteers were divided into 3 groups and cross-received a single dose of olive phenolics, mainly hydroxytyrosol (HT), provided as a liquid dietary supplement (30.6 or 61.5 mg HT), or as fortified extra virgin olive oil (12.19 mg HT + tyrosol). Blood and urine samples were collected at baseline and up to 12 hours after ingestion. Plasma oxidized LDL levels were measured by ELISA using a monoclonal antibody, while F2-isoprostanes were quantified in urine by UHPLC-MS/MS. Despite the great variability between individuals, a tendency to reduce lipoxidation reactions has been observed after olive phenolics intake in both, blood and urine. In addition, the subgroup of individuals with the highest baseline lipoxidation level showed a decrease in F2-isoprostanes (p < 0.05) after taking the food supplements, as well as a marked decrease in oxidized LDL levels (p < 0.01) after intake of the food supplement with the lowest HT dose. These promising results suggest that HT supplementation could be a useful aid in preventing lipoxidation. Additionally, people with a redox imbalance could benefit even more from supplementing with bioavailable HT.
Transpiration and water use efficiency of sorghum canopies have a large genetic variability and are positively related under naturally high evaporative demand
Authors: Pilloni Raphaël , Aparna Kakkera , Ghazzal Zineb El , Kar Soumyashree , Kumar A Ashok , Hajjarpoor Amir , Affortit Pablo , Ribière William , Kholova Jana , Tardieu François , Vadez Vincent
Published: September 2022

Indoor experiments with individual plants often show that transpiration rate is restricted under high vapor pressure deficit (VPD), resulting in a plateau of transpiration that increases water use efficiency (WUE) of some genotypes. We tested this hypothesis outdoors during dry or rainy seasons of India and Senegal, based on the response of the transpiration of canopy-grown sorghum plants to the reference evapotranspiration that takes both light and VPD into account. This response showed no plateau at high evaporative demand in 47 genotypes, but a large genetic variability was observed for the slope of the relationship over the whole range of evaporative demand. Unexpectedly, this slope was genetically correlated with WUE in two experiments with high evaporative demand: genotypes that most transpired had the highest WUE. Conversely, a negative correlation was observed under low evaporative demand. Genotypes with high WUE and response to evaporative demand were also those allowing maximum light penetration into the canopy. We suggest that this caused the observed high WUE of these genotypes because leaves within the canopy had sufficient light for photosynthesis whereas we observed a lower VPD in the canopy than in open air when leaf area index reached 2.5-3, thereby decreasing transpiration.

Highlights

The transpiration response to evaporative demand was genetically variable and correlated to WUE: genotypes that most transpired had highest light penetration towards leaves subjected to lower VPD than in air.
Mortality without springing a leak: Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold
Authors: El-Saadi Mahmoud I. , Brzezinski Kaylen , Hinz Aaron , Phillips Laura , Wong Alex , Gerber Lucie , Overgaard Johannes , MacMillan Heath A.
Published: September 2022

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown if the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.
Lifetime development changes in rats tracked by urinary proteome
Authors: Pan Xuanzhen , Liu Yongtao , Bao Yijin , Gao Youhe
Published: September 2022

The existing researches mainly focus on the embryonic stage and a short time after that. However, there was little research about the whole life of an individual from childhood to aging and death. For the first time, we used non-invasive urinary proteome technology to track the changes of several important development timepoints in a batch of rats, covering the 10 timepoints from childhood, adolescence, young, middle adulthood, and to old age death. As previous studies on puberty found, sexual or reproductive maturation, mature spermatozoa in seminiferous tubules(first seen), gonadal hormones, decline of estradiol(E), brain growth and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, neuron development.

Like previous studies found in young adulthood, musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential protein enrichment pathway also included skeletal system development, bone regeneration, system development, immune system process, myeloid leukocyte differentiation, growth, and developmental growth. However, at all timepoints in the whole life, there were still many biological pathways of the differential urinary protein enrichment involving multiple organs, tissues, systems, etc., which had not been mentioned by existing studies. This study provided comprehensive and detailed changes in rats’ lifetime development through urinary proteome, supplementing the blank of development research. But also provided a new idea for monitoring the human health change condition and the possibly occurring aging diseases by using the urinary proteome.

As far as we know, this is the first time that the urinary proteome was used to track the changes of several important developmental timepoints in the lifetime of rats.
Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II
Authors: Rengachari Srinivasan , Schilbach Sandra , Kaliyappan Thangavelu , Gouge Jerome , Zumer Kristina , Schwarz Juliane , Urlaub Henning , Dienemann Christian , Vannini Alessandro , Cramer Patrick
Published: September 2022

RNA Polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes such as small nuclear RNA (snRNA) genes is not understood at the structural level. Here we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol II preinitiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE) located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc called wing-1 and wing-2 bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.
Comparing multiscale, presence-only habitat suitability models created with structured survey data and community science data for a rare warbler species at the southern range margin
Authors: Whitenack Lauren E. , Taylor Sara Snell , Tomcho Aimee , Hurlbert Allen H.
Published: September 2022

Golden-winged Warblers (Vermivora chrysoptera, Parulidae) are declining migrant songbirds that breed in the Great Lakes and Appalachian regions of North America. Within their breeding range, Golden-winged Warblers are found in early successional habitats adjacent to mature hardwood forest, and previous work has found that Golden-winged Warbler habitat preferences are scale-dependent. Golden-winged Warbler Working Group management recommendations were written to apply to large regions of the breeding range, but there may be localized differences in both habitat availability and preferences. Rapid declines at the southernmost extent of their breeding range in Western North Carolina and Georgia necessitate investigation into landscape characteristics governing distribution in this subregion. We describe patterns of Golden-winged Warbler presence in Western North Carolina by examining habitat variables at multiple spatial scales using data from standardized Audubon North Carolina (NC) surveys and unstructured community scientist checklists submitted to eBird. We compared model performance and predictions between Audubon NC and eBird models and found that Golden-winged Warbler presence is associated with sites which, at a local scale (150m), have more young forest, less mature forest, and more road cover, and at a landscape scale (2.5km), have less road cover. eBird and Audubon models had similar parameter estimates for all of the land cover variables and similar overall performance. Based on parameter estimates, road density at the landscape scale (2.5km) is the primary variable driving the difference between Golden-winged Warbler breeding sites and random background sites in Western North Carolina. Our results show that eBird data can produce species distribution modeling results that are similar to results obtained from more standardized survey methods.
Toroidal displacement of Klebsiella pneumoniae by Pseudomonas aeruginosa is a unique mechanism to avoid competition for iron
Authors: Pradhan Diana , Tanwar Ajay , Parthasarathy Srividhya , Singh Varsha
Published: September 2022

Competition for resources is one of the major drivers for evolution and retention of new traits in microbial communities. Quorum-dependent traits of opportunistic human pathogen Pseudomonas aeruginosa allow it to survive and thrive in nature. Here, we report a unique surfactant-driven pushing mechanism that P. aeruginosa employs specifically against Klebsiella pneumoniae. The pushing is accomplished in a manner that is dependent on nutrient limitation and quorum sensing. We find that P. aeruginosa employs neither proteases nor toxic secondary metabolites against K. pneumoniae. Rhamnolipid biosurfactant appears to be the only factor required to displace Klebsiella effectively. Both rhamnolipid production and the pushing ability of P. aeruginosa are suppressed by iron supplementation. We show that both these bacteria produce several siderophores in minimal medium and rapidly deplete iron. Under these conditions, P. aeruginosa pushes Klebsiella away from the substratum using rhamnolipid, reducing the competition for iron. Our study describes a unique quorum and iron-responsive mechanism in P. aeruginosa to support its own growth during resource competition.
Determining distinct roles of IL-1α through generation of an IL-1α knockout mouse with no defect in IL-1β expression
Authors: Malireddi R.K. Subbarao , Bynigeri Ratnakar , Kancharana Balabhaskararao , Sharma Bhesh Raj , Burton Amanda R. , Pelletier Stephane , Kanneganti Thirumala-Devi
Published: September 2022

Interleukin 1α (IL-1α) and IL-1β are the founding members of the IL-1 cytokine family, and these innate immune inflammatory mediators are critically important in health and disease. Early studies on these molecules suggested that their expression was interdependent, with an initial genetic model of IL-1α depletion, the IL-1α KO mouse (Il1a-KOline1), showing reduced IL-1β expression. However, studies using this line in models of infection and inflammation resulted in contrasting observations. To overcome the limitations of this genetic model, we have generated and characterized a new line of IL-1α KO mice (Il1a-KOline2) using CRISPR-Cas9 technology. In contrast to cells from theIl1a-KOline1, where IL-1β expression was drastically reduced, bone marrow-derived macrophages (BMDMs) fromIl1a-KOline2mice showed normal induction and activation of IL-1β. Additionally,Il1a-KOline2BMDMs showed normal inflammasome activation and IL-1β expression in response to multiple innate immune triggers, including both pathogen-associated molecular patterns and pathogens. Moreover, usingIl1a-KOline2cells, we confirmed that IL-1α, independent of IL-1β, is critical for the expression of the neutrophil chemoattractant KC/CXCL1. Overall, we report the generation of a new line of IL-1α KO mice and confirm functions for IL-1α independent of IL-1β. Future studies on the unique functions of IL-1α and IL-1β using these mice will be critical to identify new roles for these molecules in health and disease and develop therapeutic strategies.
In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury
Authors: Giannakopoulos Stefanos , Strange Daniel P , Jiyarom Boonyanudh , Abdelaal Omar , Bradshaw Aaron W , Nerurkar Vivek R , Ward Monika A , Bakse Jackson , Yap Jonathan , Vanapruks Selena , Boisvert William , Tallquist Michelle D , Shikuma Cecilia , Sadri-Ardekani Hooman , Clapp Philip , Murphy Sean , Verma Saguna
Published: September 2022

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.
OCA-B/Pou2af1 is sufficient to promote CD4+T cell memory and prospectively identifies memory precursors
Authors: Sun Wenxiang , Hughes Erik P. , Kim Heejoo , Perovanovic Jelena , Charley Krystal R. , Perkins Bryant , Du Junhong , Ibarra Andrea , Syage Amber R. , Hale J. Scott , Williams Matthew A. , Tantin Dean
Published: September 2022

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable anti-tumor immune therapies. Here we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response short lived effector T cell populations are expanded but show increasedGadd45bandSocs2expression, while memory precursor effector cells show increased expression ofBcl2,Il7randTcf7on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+TCMcells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

Significance

CD4+T cell memory is incompletely defined and memory progenitors difficult to identify. Here, we show that expression of the OCA-B transcription coactivator in CD4+T cells is necessary and sufficient to drive productive memory. Using a novel mCherry reporter mouse line, we show that OCA-B expression enriches for responding effector cells with elevated memory potential. The results show that OCA-B expression in T cells is sufficient to promote CD4+memory formation and marks memory precursor cells.
Specific Codons Control Cellular Resources and Fitness
Authors: Love Aaron M. , Nair Nikhil U.
Published: September 2022

As cellular engineering progresses from simply overexpressing proteins to imparting complex metabolic and regulatory phenotypes through multigene expression, judicious appropriation of cellular resources is essential. Since there is degeneracy in codons and their use is biased, codons may control cellular resources at a translational level. We investigate how partitioning tRNA resources by incorporating dissimilar codon usage can drastically alter interdependence of expression level and burden on the host. By isolating the effect of individual codons’ use during elongation, while eliminating confounding factors like mRNA structure, GC content, transcript level, and translation initiation rates, we show that codon choice cantrans-regulate fitness of the host and expression of other heterologous genes. We correlate specific codon usage patterns with host fitness, and derive a coding scheme for multi-gene expression called the Codon Health Index (CHI, χ). This empirically derived coding scheme (χ) enables the design of multi-gene expression systems that avoid catastrophic cellular burden and is robust across multiple growth conditions.
Integration of Gene Expression and DNA Methylation Data Across Different Experiments
Authors: Itai Yonatan , Rappoport Nimrod , Shamir Ron
Published: September 2022

Integrative analysis of multi-omic datasets has proven to be extremely valuable in cancer research and precision medicine. However, obtaining multimodal data from the same samples is often difficult. Integrating multiple datasets of different omics remains a challenge, with only a few available algorithms developed to solve it.

Here, we present INTEND (IntegratioN of Transcriptomic and EpigeNomic Data), a novel algorithm for integrating gene expression and DNA methylation datasets covering disjoint sets of samples. To enable integration, INTEND learns a predictive model between the two omics by training on multi-omic data measured on the same set of samples. In comprehensive testing on eleven TCGA cancer datasets spanning 4329 patients, INTEND achieves significantly superior results compared to four state-of-the-art integration algorithms. We also demonstrate INTEND’s ability to uncover connections between DNA methylation and the regulation of gene expression in the joint analysis of two lung adenocarcinoma single-omic datasets from different sources. INTEND’s data-driven approach makes it a valuable multi-omic data integration tool.

The code for INTEND is available at https://github.com/Shamir-Lab/INTEND.
Evolution of antibody immunity following Omicron BA.1 breakthrough infection
Authors: Kaku Chengzi I. , Starr Tyler N. , Zhou Panpan , Dugan Haley L. , Khalifé Paul , Song Ge , Champney Elizabeth R. , Mielcarz Daniel W. , Geoghegan James C. , Burton Dennis R. , Andrabi Raiees , Bloom Jesse D. , Walker Laura M.
Published: September 2022

Understanding the evolution of antibody immunity following heterologous SAR-CoV-2 breakthrough infection will inform the development of next-generation vaccines. Here, we tracked SARS-CoV-2 receptor binding domain (RBD)-specific antibody responses up to six months following Omicron BA.1 breakthrough infection in mRNA-vaccinated individuals. Cross-reactive serum neutralizing antibody and memory B cell (MBC) responses declined by two- to four-fold through the study period. Breakthrough infection elicited minimal de novo Omicron-specific B cell responses but drove affinity maturation of pre-existing cross-reactive MBCs toward BA.1. Public clones dominated the neutralizing antibody response at both early and late time points, and their escape mutation profiles predicted newly emergent Omicron sublineages. The results demonstrate that heterologous SARS-CoV-2 variant exposure drives the evolution of B cell memory and suggest that convergent neutralizing antibody responses continue to shape viral evolution.
Periplasmic coiled coil formed by assembly platform proteins PulL and PulM is critical for function of the Klebsiella type II secretion system
Authors: Li Yuanyuan , Santos-Moreno Javier , Francetic Olivera
Published: September 2022

Bacteria use type II secretion systems (T2SS) to secrete to their surface folded proteins that confer diverse functions, from nutrient acquisition to virulence. In the Klebsiella species, T2SS-mediated secretion of pullulanase (PulA) requires assembly of a dynamic filament called pseudopilus. The inner membrane assembly platform (AP) complex is essential for PulA secretion and pseudopilus assembly. The AP components PulL and PulM form an inner membrane complex interacting through their C-terminal globular domains and transmembrane segments. Here we investigated the roles of periplasmic helices and cytoplasmic domains of PulL and PulM in their assembly. We found that PulL and PulM variants lacking periplasmic helices were defective for interactions in the bacterial two-hybrid (BACTH) assay. Their function in PulA secretion and assembly of PulG subunits into pseudopilus filaments were strongly reduced. In addition, deleting the cytoplasmic peptide of PulM in variant PulMΔN nearly abolished interaction with PulG in the BACTH assay, without affecting the interaction with PulL. Nevertheless, PulL was degraded in the presence of the PulMΔN variant, suggesting that PulM N-terminal peptide interacts with PulL in the cytoplasm and plays a stabilizing role. We discuss the implication of these results for the mechanism of T2S and type IV pilus assembly.
A hierarchy of cell death pathways confers layered resistance to shigellosis in mice
Authors: Roncaioli Justin L , Babirye Janet Peace , Chavez Roberto A , Liu Fitty L , Turcotte Elizabeth A , Lee Angus Y , Lesser Cammie F , Vance Russell E
Published: September 2022

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP–NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell, Roncaioli et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP–NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides protection from Shigella colonization of the intestinal epithelium, but only in the absence of both NAIP– NLRC4 and Caspase-11. The combined genetic removal of Caspases-1,-11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.
Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome
Authors: Tsu Brian V. , Agarwal Rimjhim , Gokhale Nandan S. , Kulsuptrakul Jessie , Ryan Andrew P. , Castro Lennice K. , Beierschmitt Christopher M. , Turcotte Elizabeth A. , Fay Elizabeth J. , Vance Russell E. , Hyde Jennifer L. , Savan Ram , Mitchell Patrick S. , Daugherty Matthew D.
Published: September 2022

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8’s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.
Dynamic refinement of behavioural restructure mediates dopamine-dependent credit assignment
Authors: Tang Jonathan C.Y. , Paixao Vitor , Carvalho Filipe , Silva Artur , Klaus Andreas , Alves da Silva Joaquim , Costa Rui M.
Published: September 2022

Animals exhibit a diverse behavioral repertoire when exploring new environments and can learn which actions or action sequences produce positive outcomes. Dopamine release upon encountering reward is critical for reinforcing reward-producing actions1–3. However, it has been challenging to understand how credit is assigned to the exact action that produced dopamine release during continuous behavior. We investigated this problem with a novel self-stimulation paradigm in which specific spontaneous movements triggered optogenetic stimulation of dopaminergic neurons. Dopamine self-stimulation rapidly and dynamically changes the structure of the entire behavioral repertoire. Initial stimulations reinforced not only the stimulation-producing target action, but also actions similar to target and actions that occurred a few seconds before stimulation. Repeated pairings led to gradual refinement of the behavioral repertoire to home in on the target. Reinforcement of action sequences revealed further temporal dependencies of refinement. Action pairs spontaneously separated by long time intervals promoted a stepwise credit assignment, with early refinement of actions most proximal to stimulation and subsequent refinement of more distal actions. Thus, a retrospective reinforcement mechanism promotes not only reinforcement, but gradual refinement of the entire behavioral repertoire to assign credit to specific actions and action sequences that lead to dopamine release.
The relationship between PD-L1 and quiescence in melanocyte stem cell aging
Authors: Palmer Joseph W. , Villavicencio Kyrene M. , Idris Misgana , Weddle Dominique , Filipp Fabian V. ,   NISC Comparative Sequencing Program, Pavan William J. , Harris Melissa L.
Published: September 2022

A central aspect of life-long stem cell function in slow cycling stem cells is the proper regulation of cellular quiescence. How the quiescent state is achieved, whether all quiescent cells are equivalent, and if the quiescent stem cell pool changes with age are all questions that remain unanswered. Using quiescent melanocyte stem cells (qMcSC) as a model, we found that stem cell quiescence is neither a singular nor static process and can be heterogeneous. As one example of this heterogeneity, we show that a portion of qMcSCs expresses the immune checkpoint protein PD-L1 at the cell membrane (PD-L1mem+), PD-L1mem+ qMcSCs are better retained with age, and that the aged quiescent McSC pool is transcriptomically more deeply quiescent. Collectively these findings demonstrate that PD-L1 expression is a physiological attribute of quiescence in McSCs and PD-L1mem+ quiescent stem cells may be good targets for reactivation in the context of aging.
Triple COVID-19 vaccination induces humoral and cellular immunity to SARS-CoV-2 with cross-recognition of the Omicron variant and IgA secretion
Authors: Ruhl Louisa , Kühne Jenny F. , Beushausen Kerstin , Keil Jana , Christoph Stella , Sauer Jasper , Falk Christine S.
Published: September 2022

COVID-19 vaccination is the leading strategy to prevent severe courses after SARS-CoV-2 infection. In our study, we analyzed humoral and cellular immune responses in detail to three consecutive homologous or heterologous COVID-19 vaccinations. All individuals (n=20) responded to vaccination with increasing S1- /RBD-/S2-specific IgG levels, whereas specific plasma IgA displayed individual variability. The third dose increased antibody inhibitory capacity (AIC) against immune-escape variants Beta and Omicron independently from age. The mRNA-primed vaccination induced IgG and IgA immunity more efficiently, whereas vector-primed individuals displayed higher levels of memory T and B cells. Vaccinees showed a SARS-CoV-2-specific T cell responses, which were further improved and specified after Omicron breakthrough infections in parallel to appearance of new variant-specific antibodies. In conclusion, the third vaccination was essential to increase IgG levels, mandatory to boost AIC against immune-escape variants and induced SARS-CoV-2-specific T cells. Breakthrough infection with Omicron generates additional spike specificities covering all known variants.
Effects of hydrogen and chicken waste blends in the internal combustion engines for superior engine performance and emission characteristics assisted with graphite oxide
Authors: R. Dinesh , Retnam Stanly Jones , M. Dev Anand , J. Edwin Raja Dhas
Published: September 2022

Purpose

The demand for energy is increasing massively due to urbanization and industrialization. Due to the massive usage of diesel engines in the transportation sector, global warming is increasing rapidly. The purpose of this paper is to use hydrogen as the potential alternative for diesel engine.

Design/methodology/approach

A series of tests conducted in the twin cylinder four stroke diesel engine at various engine speeds. In addition to the hydrogen, the ultrasonication is applied to add the nanoparticles to the neat diesel. The role of nanoparticles on engine performance is effective owing to its physicochemical properties. Here, neat diesel mixed 30% of biodiesel along with the hydrogen at the concentration of 10%, 20% and 30% and 50 ppm of graphite oxide to form the blends DNH10, DNH20 and DNH30.

Findings

Inclusion of both hydrogen and nanoparticles increases the brake power and brake thermal efficiency (BTE) of the engine with relatively less fuel consumption. Compared to all blends, the maximum BTE of 33.3% has been reported by 30% hydrogen-based fuel. On the contrary, the production of the pollutants also reduces as the hydrogen concentration increases.

Originality/value

Majority of the pollutants such as carbon monoxide, carbon dioxide and hydrocarbon were dropped massively compared to diesel. On the contrary, there is no reduction in nitrogen of oxides (NOx). Highest production of NOx was witnessed for 30% hydrogen fuel due to the premixed combustion phase and cylinder temperatures.
Factors affecting unitized curtain wall system adoption for Hong Kong's high-rise residential buildings: a multi-stakeholder perspective
Authors: Kwok Tai Wai , Chang Siwei , Li Heng
Published: September 2022

Purpose

The unitized curtain wall system (UCWS), one of the prefabricated technologies, is increasingly attracting attention in the Hong Kong construction industry. However, this innovative technology still lacks on-site implementation in high-rise residential buildings. To promote its development, this study aims at identifying the influential factors of UCWS adoption in Hong Kong's high-rise residential buildings from a multi-stakeholder perspective.

Design/methodology/approach

Factors were first selected through an in-depth literature review and a semi-structured interview. Then the factors were validated through a questionnaire survey using Cronbach's Alpha Reliability Test. Next, the factors were ranked regarding their importance using mean-score ranking and standard deviation. Meanwhile, different stakeholders were clustered using an experimental factor analysis (EFA) model to find the shared preferences (namely common factors).

Findings

The result shows that reduction of construction time (B1) and insufficient site storage area (C1) are the most important factors. The six stakeholder groups were clustered into two segments. B1 and improved quality control are the shared interests. While C1 and the need of specification change are the common concerns.

Originality/value

There are two major breakthroughs in this study. First is the novelty of research objects. UCWS, particularly its application preference in high-rise residential buildings, has rarely been studied, yet it is urgently required. Second is the novel research perspective. The influential factors were studied from a multi-stakeholder perspective. Not only the significant factors for six specific stakeholders but also the shared preference for stakeholder groups was identified. The findings contribute to promoting UCWS more targeted, efficient and comprehensive, as well as demonstrating the collaborative possibilities of multi-stakeholders.