bluuebunny/crossref_metadata_2025 huggingface dataset:
The vacuolar iron transporter mediates iron detoxification inToxoplasma gondii
Authors: Aghabi Dana , Sloan Megan , Dou Zhicheng , Guerra Alfredo J. , Harding Clare R.
Published: September 2021

Iron is essential to living cells, acting as a cofactor in a number of essential enzymes in metabolism; however, iron requires proper storage or it can be dangerous to the cell. In both yeast and plants, iron is stored in a vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including inToxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT inT. gondii. We show that deletion of VIT causes a slight growth defectin vitro, however leads to hypersensitivity in the presence of excess iron, confirming its essential role in iron detoxification in the parasite. In the absence of VIT, parasites contain less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by environmental iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that theT. gondiiresponds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in pathogenesis in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification inT. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.
In vitroFunctional Analysis of pgRNA Sites Regulating Assembly of Hepatitis B Virus
Authors: Patel Nikesh , Clark Sam , Weiß Eva U. , Mata Carlos P. , Bohon Jen , Farquhar Erik R. , Maskell Daniel P. , Ranson Neil A. , Twarock Reidun , Stockley Peter G.
Published: September 2021

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficientin vitroassay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of theT=4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry-relaxation of this reconstruction reveals that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase bindsin vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcriptsin vitro, and therefore may play similar rolesin vivo,in concert with other molecular factors.
Land Use Cover changes in the western escarpment of Rift Valley in the Gamo Zone, Southern Ethiopia
Authors: Dingamo Temesgen , Takele Serekebirhan , Demissew Sebsebe , Woldu Zerihun
Published: September 2021

LULC changes are caused by natural and human alterations of the landscape that could largely affect forest biodiversity and the environment. The aim of the study was to analyzed LULC change dynamics in the western escarpment of the rift valley of the Gamo Zone, Southern Ethiopia. Digital satellite images downloaded from USGS were analyzed using ERDAS Imagine (14) and Arc GIS 10.2 software and supervised image classification was used to generate LULC classification, accuracy assessment and Normalized Difference Vegetation Index (NDVI). Drivers of LULC change were identified and analyzed. Four land classes were identified such as forest, farmland, settlement and water-wetland. Settlement and farmlands have increased by 7.83% and 5.88%, respectively. On the other hand, both forest and water bodies and wetland decreased by aerial coverage of 11.03% and 2.68%, respectively. The overall accuracy of the study area was 92.86%, 94.22% and 94.3% with a kappa value of 0.902, 0.92 and 0.922, respectively. NDVI values ranged between -0.42 to 0.73. Agricultural expansion (31.4%), expansion of settlement (25.7%) and Fuelwood collection and Charcoal production (22.9%) were the main driving forces that jeopardize forest biodiversity of the study area. Integrated land use and policy to protect biodiversity loss, forest degradation and climate changes are deemed necessary.
Experimental characterization and automatic identification of stridulatory sounds inside wood
Authors: Bedoya Carol L. , Nelson Ximena J. , Brockerhoff Eckehard G. , Pawson Stephen , Hayes Michael
Published: September 2021

The propagation of animal vocalizations in water and in air is a well-studied phenomenon, but sound produced by bark and wood boring insects, which feed and reproduce inside trees, is poorly understood. Often being confined to the dark and chemically-saturated habitat of wood, many bark- and woodborers have developed stridulatory mechanisms to communicate acoustically. Despite their ecological and economic importance and the unusual medium used for acoustic communication, very little is known about sound production in these insects, or their acoustic interactions inside trees. Here, we use bark beetles (Scolytinae) as a model system to study the effects of wooden tissue on the propagation of insect stridulations and propose algorithms for their automatic identification. We characterize distance-dependence of the spectral parameters of stridulatory sounds, propose data-based models for the power decay of the stridulations in both outer and inner bark, provide optimal spectral ranges for stridulation detectability, and develop automatic methods for their detection and identification. We also discuss the acoustic discernibility of species cohabitating the same log. The species tested can be acoustically identified with 99% of accuracy at distances up to 20 cm and detected to the greatest extent in the 2-6 kHz frequency band. Phloem was a better medium for sound transmission than bark.
Effects of age and surface instability on the control of the center of mass
Authors: van den Bogaart Maud , Bruijn Sjoerd M. , Spildooren Joke , van Dieën Jaap H. , Meyns Pieter
Published: September 2021

During standing, posture can be controlled by accelerating the Center of Mass (CoM) through shifting the center of pressure (CoP) within the base of support by applying ankle moments (“CoP mechanism”), or through the “counter-rotation mechanism”, i.e., changing the angular momentum of segments around the CoM to change the direction of the ground reaction force. Postural control develops over the lifespan; at both the beginning and the end of the lifespan adequate postural control appears more challenging. In this study, we aimed to assess mediolateral balance performance and the related use of the postural control mechanisms in children, older adults and young adults when standing on different (unstable) surfaces. Sixteen pre-pubertal children (6-9y), 17 young adults (18-24y) and eight older adults (65-80y) performed bipedal upright standing trials of 16 seconds on a rigid surface and on three balance boards that could freely move in the frontal plane, varying in height (15-19 cm) of the surface of the board above the point of contact with the floor. Full body kinematics (16 segments, 48 markers, using SIMI 3D-motion analysis system (GmbH) and DeepLabCut and Anipose) were retrieved. Performance related outcome measures, i.e., the number of trials with balance loss and the Root Mean Square (RMS) of the time series of the CoM acceleration, the contributions of the CoP mechanism and the counter-rotation mechanism to the CoM acceleration in the frontal plane and selected kinematic measures, i.e. the orientation of the board and the head and the Mean Power Frequency (MPF) of the balance board orientation and the CoM acceleration were determined. Balance loss only occurred when standing on the highest balance board, twice in one older adult once in one young adult. In children and older adults, the RMS of the CoM accelerations were larger, corresponding to poorer balance performance. Across age groups and conditions, the contribution of the CoP mechanism to the total CoM acceleration was much larger than that of the counter-rotation mechanisms, ranging from 94%-113% vs 23%-38% (with totals higher than 100% indicating opposite effects of both mechanisms). Deviations in head orientation were small compared to deviations in balance board orientation. We hypothesize that the CoP mechanism is dominant, since the counter-rotation mechanism would conflict with stabilizing the orientation of the head in space.
Disruption of fish gut microbiota composition and holobiont’s metabolome by cyanobacterial blooms
Authors: Gallet Alison , Halary Sébastien , Duval Charlotte , Huet Hélène , Duperron Sébastien , Marie Benjamin
Published: September 2021

Background

Cyanobacterial blooms are one of the most common stress encountered by metazoans living in freshwater lentic systems such as lakes and ponds. Blooms reportedly impair fish health, notably through oxygen depletion and production of bioactive compounds including cyanotoxins. However, in the times of the “microbiome revolution”, it is surprising that so little is still known regarding the influence of blooms on fish microbiota. In this study, an experimental approach is used to demonstrate that blooms affect fish microbiome composition and functions, as well as the metabolome of holobionts. To this end, the model teleost Oryzias latipes is exposed to simulated Microcystis aeruginosa blooms of various intensities in a microcosm setting, and the response of bacterial gut communities is evaluated in terms of composition, metagenome-encoded functions and metabolome profiling.

Results

The gut bacterial community of O. latipes exhibits marked responses to the presence of M. aeruginosa blooms in a dose-dependent manner. Notably, abundant gut-associated Firmicutes almost disappear, while potential opportunists increase. The holobiont’s gut metabolome displays major changes, while functions encoded in the metagenome of bacterial partners are more marginally affected. Bacterial communities tend to return to original composition after the end of the bloom suggesting post-bloom resilience, and remain sensitive in case of a second bloom, reflecting a highly reactive gut community.

Conclusion

In the context of increasingly frequent and intense blooms worldwide, results point to the relevance of accounting for short- and long-term microbiome-related effects in fish ecology, with potential outcomes relevant to conservation biology as well as aquaculture.
No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults
Authors: Ercanoglu Meryem Seda , Gieselmann Lutz , Dähling Sabrina , Poopalasingam Nareshkumar , Detmer Susanne , Koch Manuel , Korenkov Michael , Halwe Sandro , Klüver Michael , Di Cristanziano Veronica , Janicki Hanna , Schlotz Maike , Worczinski Johanna , Gathof Birgit , Gruell Henning , Zehner Matthias , Becker Stephan , Vanshylla Kanika , Kreer Christoph , Klein Florian
Published: September 2021

Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.
High-Dimensional Gene Expression and Morphology Profiles of Cells across 28,000 Genetic and Chemical Perturbations
Authors: Haghighi Marzieh , Caicedo Juan , Cimini Beth A. , Carpenter Anne E. , Singh Shantanu
Published: September 2021

Cells can be perturbed by various chemical and genetic treatments and the impact on the cells’ gene expression (transcription, i.e. mRNA levels) and morphology (in an image-based assay) can be measured. The patterns observed in this high-dimensional profile data can power a dozen applications in drug discovery and basic biology research, but both types of profiles are rarely available for large-scale experiments. Here, we provide a collection of four datasets with both gene expression and morphological profile data useful for developing and testing multi-modal methodologies. Roughly a thousand features are measured for each of the two data types, across more than 28,000 thousand chemical and genetic perturbations. We define biological problems that use the shared and complementary information in these two data modalities, provide baseline analysis and evaluation metrics for multi-omic applications, and make the data resource publicly available (http://broad.io/rosetta).
Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques
Authors: Speranza Emily , Purushotham Jyothi N. , Port Julia R. , Schwarz Benjamin , Flagg Meaghan , Williamson Brandi N. , Feldmann Friederike , Singh Manmeet , Pérez-Pérez Lizzette , Sturdevant Gail L. , Roberts Lydia M. , Carmody Aaron , Schulz Jonathan E. , Doremalen Neeltje van , Okumura Atsushi , Lovaglio Jamie , Hanley Patrick W. , Shaia Carl , Germain Ronald N. , Best Sonja M. , Munster Vincent J. , Bosio Catharine M. , de Wit Emmie
Published: September 2021

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.
Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche
Authors: Anllo Lauren , DiNardo Stephen
Published: September 2021

Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis we recently showed that pro niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. We now find that Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which we find is also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.
SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning
Authors: Gamart Julie , Barozzi Iros , Laurent Frédéric , Reinhardt Robert , Martins Laurène Ramos , Oberholzer Thomas , Visel Axel , Zeller Rolf , Zuniga Aimée
Published: September 2021

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-taggedSmad4allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lackingSmad4in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced inSmad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared betweenSmad4- andShh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.
Classification of antimicrobial mechanism of action using bacterial time-lapse imaging
Authors: Ouyang Xudong , Hoeksma Jelmer , Siersma Tjalling K. , Hamoen Leendert W. , Hertog Jeroen den
Published: September 2021

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.
Targeted isolation of panels of diverse human protective broadly neutralizing antibodies against SARS-like viruses
Authors: He Wan-ting , Musharrafieh Rami , Song Ge , Dueker Katharina , Tse Longping V. , Martinez David R. , Schäfer Alexandra , Callaghan Sean , Yong Peter , Beutler Nathan , Torres Jonathan L. , Volk Reid M. , Zhou Panpan , Yuan Meng , Liu Hejuni , Anzanello Fabio , Capozzola Tazio , Parren Mara , Garcia Elijah , Rawlings Stephen A. , Smith Davey M. , Wilson Ian A. , Safonova Yana , Ward Andrew B. , Rogers Thomas F. , Baric Ralph S. , Gralinski Lisa E. , Burton Dennis R. , Andrabi Raiees
Published: September 2021

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1–7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10–13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14–18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.
CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen
Authors: Huang Jun , Rowe David , Zhang Wei , Suelter Tyler , Valent Barbara , Cook David E.
Published: September 2021

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.
Combined free energy calculation and machine learning methods for understanding ligand unbinding kinetics
Authors: Badaoui Magd , Buigues Pedro J , Berta Dénes , Mandana Gaurav M. , Gu Hankang , Földes Tamás , Dickson Callum J , Hornak Viktor , Kato Mitsunori , Molteni Carla , Parsons Simon , Rosta Edina
Published: September 2021

The determination of drug residence times, which define the time an inhibitor is in complex with its target, is a fundamental part of the drug discovery process. Synthesis and experimental measurements of kinetic rate constants are, however, expensive, and time-consuming. In this work, we aimed to obtain drug residence times computationally. Furthermore, we propose a novel algorithm to identify molecular design objectives based on ligand unbinding kinetics. We designed an enhanced sampling technique to accurately predict the free energy profiles of the ligand unbinding process, focusing on the free energy barrier for unbinding. Our method first identifies unbinding paths determining a corresponding set of internal coordinates (IC) that form contacts between the protein and the ligand, it then iteratively updates these interactions during a series of biased molecular-dynamics (MD) simulations to reveal the ICs that are important for the whole of the unbinding process. Subsequently, we performed finite temperature string simulations to obtain the free energy barrier for unbinding using the set of ICs as a complex reaction coordinate. Importantly, we also aimed to enable further design of drugs focusing on improved residence times. To this end, we developed a supervised machine learning (ML) approach with inputs from unbiased “downhill” trajectories initiated near the transition state (TS) ensemble of the string unbinding path. We demonstrate that our ML method can identify key ligand-protein interactions driving the system through the TS. Some of the most important drugs for cancer treatment are kinase inhibitors. One of these kinase targets is Cyclin Dependent Kinase 2 (CDK2), an appealing target for anticancer drug development. Here, we tested our method using two different CDK2 inhibitors for potential further development of these compounds. We compared the free energy barriers obtained from our calculations with those observed in available experimental data. We highlighted important interactions at the distal ends of the ligands that can be targeted for improved residence times. Our method provides a new tool to determine unbinding rates, and to identify key structural features of the inhibitors that can be used as starting points for novel design strategies in drug discovery.
Homocysteine-induced endoplasmic reticulum stress activates FGF21 via CREBH, resulting in browning and atrophy of white adipose tissue in Bhmt knockout mice
Authors: Warrier Manya , Paules Evan M. , Friday Walter B. , Bramlett Frances , Kim Hyunbae , Zhang Kezhong , Trujillo-Gonzalez Isis
Published: September 2021

Betaine-homocysteine methyltransferase (BHMT) catalyzes the transfer of methyl-groups from betaine to homocysteine (Hcy) producing methionine and dimethylgycine. In this work, we characterize Bhmt wildtype (WT) and knockout (KO) mice that were fully backcrossed to a C57Bl6/J background. Consistent with our previous findings, Bhmt KO mice had decreased body weight, fat mass and adipose tissue weight compared to WT. Histological analyses and gene expression profiling indicate that adipose browning was activated in KO mice and contributed to the adipose atrophy observed. BHMT is not expressed in adipose tissue but is abundant in liver, thus, a signal must be originating from the liver that modulates adipose tissue. We found that, in Bhmt KO mice, homocysteine-induced endoplasmic reticulum (ER) stress, with activation of hepatic transcription factor cyclin AMP response element binding protein (CREBH), mediated an increase in hepatic and plasma concentrations of fibroblast growth factor 21 (FGF21), which is known to induce adipose browning. CREBH binds to the promoter regions of FGF21 to activate its expression. Taken together, our data indicate that deletion of a single gene in one-carbon metabolism modifies adipose biology and energy metabolism. It would be interesting to determine whether people with functional polymorphisms in BHMT exhibit a similar adipose atrophy phenotype.
Digital Spatial Profiling of Collapsing Glomerulopathy
Authors: Smith Kelly D. , Henriksen Kammi , Nicosia Roberto F. , Alpers Charles E. , Akilesh Shreeram
Published: September 2021

Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV and SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli from HIV and SARS-CoV-2 infected patients with biopsy confirmed collapsing glomerulopathy. The increased resolution of DSP uncovered heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Therefore, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.
Interplay between mitochondria and reactive oxygen and nitrogen species in metabolic adaptation to hypoxia in facioscapulohumeral muscular dystrophy: potential therapeutic targets
Authors: Heher P , Ganassi M , Weidinger A , Engquist EN , Pruller J , Nguyen TH , Tassin A , Declèves AE , Mamchaoui K , Grillari J , Kozlov AV , Zammit PS
Published: September 2021

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking.

Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO· bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

Highlights

Transcriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathways

Disturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophy

DUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex I

FSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubes

Hypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidants

Graphical abstract
Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities
Authors: Ghaly Timothy M. , Penesyan Anahit , Pritchard Alexander , Qi Qin , Rajabal Vaheesan , Tetu Sasha G. , Gillings Michael R.
Published: September 2021

Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.
MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING
Authors: Wallis Tristan P. , Jiang Anmin , Hou Huiyi , Gormal Rachel S. , Durisic Nela , Balistreri Giuseppe , Joensuu Merja , Meunier Frédéric A.
Published: September 2021

Single-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in “hotspots” on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of individual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.
A thermodynamic model for interpreting tryptophan excitation-energy-dependent fluorescence spectra provides insight into protein conformational sampling and stability
Authors: Kwok A , Camacho IS , Winter S , Knight M , Meade RM , Van der Kamp MW , Turner A , O’Hara J , Mason JM , Jones AR , Arcus VL , Pudney CR
Published: September 2021

It is now over thirty years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. Whilst there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the protein REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides to multi-tryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibody in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating bimolecular simulation with experimental data to yield novel insights.
A thermostable oral SARS-CoV-2 vaccine induces mucosal and protective immunity
Authors: Bellier Bertrand , Saura Alicia , Luján Lucas A. , Molina Cecilia R. , Lujan Hugo D. , Klatzmann David
Published: September 2021

An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (e-VLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made e-VLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated e-VLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered e-VLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines.
Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity inC. elegansoocyte meiosis
Authors: Cavin-Meza Gabriel , Kwan Michelle M. , Wignall Sarah M.
Published: September 2021

While centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system inC. elegansto remove factors from pre-formed oocyte spindles within minutes and assess effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes toC. elegansmeiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.
Targeting Stem-loop 1 of the SARS-CoV-2 5’UTR to suppress viral translation and Nsp1 evasion
Authors: Vora Setu M. , Fontana Pietro , Leger Valerie , Zhang Ying , Fu Tian-Min , Lieberman Judy , Gehrke Lee , Shi Ming , Wang Longfei , Wu Hao
Published: September 2021

SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5’ UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5’ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.
The impact of firm innovativeness on consumer trust in the sharing economy: a moderated mediation model
Authors: Geng Ruoshi , Sun Ruijie , Li Jie , Guo Fan , Wang Wangshuai , Sun Gong
Published: September 2021

Purpose

This paper examined the relationship between firm innovativeness and consumer trust in the sharing economy. In addition, the authors examine the mediating effect of organizational legitimacy and the moderating effect of social worth.

Design/methodology/approach

To examine the hypotheses, the authors collected data from 276 users of a sharing platform (Didi) in China to conduct empirical research. The “lavaan” packages in R and SPSS were used to analyze the data and test the proposed hypotheses.

Findings

The results reveal that sharing platforms' innovativeness is positively related to consumer trust, and this relationship is mediated by organizational legitimacy. Furthermore, sharing platforms' social worth moderates the relationship between firm innovativeness and organizational legitimacy as well as the indirect effect of firm innovativeness on consumer trust via organizational legitimacy.

Practical implications

This article proposes strategies that enable sharing platforms to increase consumer trust, which can also better promote the development of the sharing economy.

Originality/value

This paper contributes to the literature by focusing on the social attributes of the sharing economy. By building a more detailed model of consumer trust, this paper adds to the knowledge on the influencing mechanism of consumer trust in the sharing economy.
Factors affecting the diffusion of traceability practices in an imported fresh produce supply chain in China
Authors: Yi Ying , Bremer Phil , Mather Damien , Mirosa Miranda
Published: September 2021

Purpose

The purpose of this paper is to facilitate the successful adoption of traceability technologies, such as blockchain, into food supply chains and facilitate the understanding of the barriers and enablers to their uptake by channel members' needs so that appropriate enabling strategy can be put in place.

Design/methodology/approach

In this paper, an integrated framework involving five components was used as the methodology: (1) diffusion of innovation theory, (2) the innovation concept, (3) the business structure-conduct-performance paradigm, (4) legitimacy and (5) trust was developed and assessed for validity through interviewing 21 channel members, including distributors, wholesalers, Internet retailers and traditional retailers associated with a global fresh produce company's supply chain in China.

Findings

Barriers negatively framing channel members' attitudes and decisions included a perceived lack of need owing to fresh produce having a short shelf life and being of low value and risk. However, the importance of traceability and the need for effective food recalls were not always understood among channel members, and distributed trust innovations were also suppressed by their lack of compatibility with the Chinese hierarchical culture.

Originality/value

To date, channel members' perception of innovations in food supply chains has not been considered in light of the components proposed in the integrated framework. The adapted framework used in this study ensured a comprehensive assessment of channel members' attitude and motivations toward traceability practices.