bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequencing using a two-steps strategy reveals high genetic diversity in the S gene of SARS-CoV-2 after a high transmission period in Tunis, Tunisia
Authors: Fares Wasfi , Ghedira Kais , Gdoura Mariem , Chouikha Anissa , Haddad-Boubaker Sondes , Khedhiri Marwa , Ayouni Kaouthar , Lamari Asma , Touzi Henda , Hammemi Walid , Medeb Zina , Sadraoui Amel , Hogga Nahed , ben Alaya Nissaf , Triki Henda 
Published: June 2021

AbstractRecent efforts have reported numerous variants that influence SARS-CoV-2 viral characteristics including pathogenicity, transmission rate and ability of detection by molecular tests. Whole genome sequencing based on NGS technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low and middle income countries. To decrease sequencing cost, we developed a couple of primers allowing to generate partial sequences in the viral S gene allowing rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred and one nasopharyngeal specimens collected during the decreasing phase of a high transmission COVID-19 wave in T unisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allowed the detection of first introduction in the country of already known VOCs and VOIs as well as others variants that have interesting genomic mutations and need to be kept under surveillance.ImportanceThe method of choice for SARS-CoV-2 variants detection is whole genome sequencing using NGS technologies. Resources for this technology remain limited in many low and middle income countries where it is not possible to perform whole genome sequencing for representative number of SARS-CoV-2 positive cases. In the present work, we developed a novel strategy based on a first partial sanger screening in the S gene including key mutations of the already known VOCs and VOIs for rapid identification of these VOCs and VOIs and helps to better select specimens that need to be sequenced by NGS technologies. The second step consisting in whole genome sequencing allowed to have a holistic view of all variants within the selected viral strains and confirmed the initial classification of the strains based on partial S gene sequencing.
The Foster method: Rapid and non-invasive detection of clinically significant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens
Authors: Mackay John F. , Hewett Rebecca E. , Smith Noa T. , Waters Tammy L. , Scandrett John S. 
Published: June 2021

AbstractClinical signs of American Foulbrood (AFB) can be difficult to diagnose and thus disease is often missed and leads to further spread. Diagnosis is centred on the beekeeper’s skill in recognising clinical symptoms – a highly subjective and time-consuming activity. Previous testing methods have relied on sampling that necessitates dismantling the hive and/or requires multiple visits to retrieve passive samples. The Foster method is a novel environmental DNA sampling method using colony entrance swabs together with a dual-target qPCR forPaenibacillus larvae: the causative bacteria of AFB disease. The quantification data generated can be used to detect hives with clinically significant infections, even before visual symptoms are apparent. Such a sampling method will be applicable to other bee pathogens and incursion pests.ImportanceDiscovery of American foulbrood disease in a honeybee colony typically means the destruction of the bees and hive by burning, in New Zealand and many other countries. This discovery is typically by visual examination of capped brood by the beekeeper - a subjective skill that means the disease is being missed or not recognised. It is a time-consuming and exacting process to inspect hives for AFB. Here we present a novel rapid sampling method that does not require opening/ dismantling the hive, in conjunction with a dual-target quantitative PCR assay for the bacteria responsible,Paenibacillus larvae. Using the resulting quantitative data, hives presenting visual clinical symptoms or likely to soon become clinical, can be determined and the hives dealt with appropriately before further spread occurs. This study provides the basis for a novel way of sampling for honeybee pathogens and pests.
Validation of selective agars for detection and quantification of Escherichia coli resistant to critically important antimicrobials
Authors: Lee Zheng Z. , Abraham Rebecca , O’Dea Mark , Harb Ali , Hunt Kelly , Lee Terence , Abraham Sam , Jordan David 
Published: June 2021

ABSTRACTSuccess in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed more rapidly, affordably and on a larger scale. An approach based on robotics and agars incorporated with antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of designed experiments involving 1,202 plates identified the best candidate-combinations from six commercially available agars and five antimicrobials using 18 Escherichia coli strains as either pure cultures or inoculums within faeces. The effect of various design factors on colony counts were analysed in generalised linear models. Without antimicrobials, Brilliance™E. coli (Brilliance) and CHROMagar™ ECC (CHROMagar) agars yielded 28.9% and 23.5% more colonies than MacConkey agar. The order of superiority of agars remained unchanged when faecal samples with and without spiking of resistant E. coli were inoculated onto agars with or without specific antimicrobials. When incorporating antimicrobials at varying concentrations, it was revealed that ampicillin, tetracycline and ciprofloxacin are suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was only suitable for incorporation at 8 and 16 μg/mL while ceftiofur was only suitable at 1 μg/mL. CHROMagar™ ESBL agar supported growth of a wider diversity of extended-spectrum cephalosporin-resistant E. coli. The findings demonstrate the potential for combining robotics with agars to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance.IMPORTANCEEstablished models of surveillance for AMR in livestock typically have a low sampling intensity which creates a tremendous barrier to understanding the variation of resistance amongst animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process high volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reaction including accurately detecting and quantifying their presence even when present at low concentration. As Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli by assessing the growth performance on agars with antimicrobials. The findings are the first step towards exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock with wider adaptations in food, clinical microbiology and public health.
Differences in IgG antibody responses following BNT162b2 and mRNA-1273 Vaccines
Authors: Montoya José G. , Adams Amy E. , Bonetti Valérie , Deng Sien , Link Nan A. , Pertsch Suzanne , Olson Kjerstie , Li Martina , Dillon Ellis C. , Frosch Dominick L. 
Published: June 2021

ABSTRACTStudies examining antibody responses by vaccine brand are lacking and may be informative for optimizing vaccine selection, dosage, and regimens. The purpose of this study is to assess IgG antibody responses following immunization with BNT162b2 (30 μg S protein) and mRNA-1273 (100 μg S protein) vaccines. A cohort of clinicians at a non-for-profit organization is being assessed clinically and serologically following immunization with BNT162b2 or mRNA-1273. IgG responses were measured at the Remington Laboratory by an IgG against the SARS-CoV-2 spike protein-receptor binding domain. Mixed-effect linear (MEL) regression modeling was used to examine whether the SARS-CoV-2 IgG level differed by vaccine brand, dosage, or days since vaccination. Among 532 SARS-CoV-2 seronegative participants, 530 (99.6%) seroconverted with either vaccine. After adjustments for age and gender MEL regression modeling revealed that the average IgG increased after the second dose compared to the first dose (p<0.001). Overall, titers peaked at week six for both vaccines. Titers were significantly higher for mRNA-1273 vaccine on days 14-20 (p < 0.05), 42-48 (p < 0.01), 70-76 (p < 0.05), 77-83 (p < 0.05), and higher for BNT162b2 vaccine on days 28-34 (p < 0.001). In two participants taking immunosuppressive drugs SARS-CoV-2 IgG remained negative. mRNA-1273 elicited both earlier and higher IgG antibody responses than BNT162b2, possibly due to the higher S-protein delivery. Prospective clinical and serological follow-up of defined cohorts such as this may prove useful in determining antibody protection and whether differences in antibody kinetics between the vaccines have manufacturing relevance and clinical significance.
Stem Cell Microarrays for Assessing Growth Factor Signaling in Engineered Glycan Microenvironments
Authors: Michalak Austen L. , Trieger Greg W. , Trieger Kelsey , Godula Kamil 
Published: June 2021

ABSTRACTExtracellular glycans, such as glycosaminoglycans (GAGs), provide an essential regulatory component during the development and maintenance of tissues. GAGs, which harbor binding sites for a range of growth factors and other morphogens, help establish gradients of these molecules in the extracellular matrix (ECM) and promote the formation of active signaling complexes when presented at the cell surface. As such, GAGs have been pursued as biologically active components for the development of biomaterials for cell-based regenerative therapies. However, their structural complexity and compositional heterogeneity make establishing structure-function relationships for this class of glycans difficult. Here, we describe a stem cell array platform, in which GAG polysaccharides are conjugated to adhesion proteins and introduced into a polyacrylamide hydrogel network to directly measure their contributions to the activation of growth factor signaling pathways in cells. With the recent emergence of powerful synthetic and recombinant technologies to produce well-defined GAG structures, a platform for analyzing both growth factor binding and signaling in response to the presence of these biomolecules will provide a powerful tool for integrating glycans into biomaterials to advance their biological properties and applications.Abstract FigureThe present study describes the integration of glycosaminoglycan-protein conjugates into a hydrogel-supported stem cell microarray platform to analyze the activity of extracellular glycans in growth factor signaling. Such platforms can enable rapid development and optimization of functional glycomaterials for stem cell-based regenerative therapies.
In situ X-ray assisted electron microscopy staining for large biological samples
Authors: Ströh Sebastian , Hammerschmith Eric W. , Tank David W. , Seung H. Sebastian , Wanner Adrian A. 
Published: June 2021

AbstractElectron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient.Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the “black box” of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in reduced osmium solution.X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.
Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia
Authors: Chun Byeong Jae , Aryal Surya , Sun Bin , Bruno Josh , Richards Chris , Bachstetter Adam D. , Kekenes-Huskey Peter M. 
Published: June 2021

1AbstractAdenosine triphosphate (ATP) drives microglia motility and cytokine production by activating P2X- and P2Y- class purinergic receptors with extracellular ATP and its metabolites. Purinergic receptor activation gives rise to diverse intracellular Ca2+signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these diverse waveforms influence microglia function is not well established. We developed a computational model trained with published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+signaling and migration and how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced calcium waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+responses. We, therefore, next evaluated how purinoreceptors and ectonucleotidase work in tandem. Our modeling results indicate that small transients are sufficient to promote motility, while large and sustained transients are needed for cytokine responses. Lastly, we predict how these phenotypical responses vary in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model can be extrapolated to diverse microglia subtypes. These findings provide important insights into how differences in prurinergic receptor expression influence the microglia’s responses to ATP.
Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration
Authors: Choi Jinwook , Jang Yu Jin , Dabrowska Catherine , Iich Elhadi , Evans Kelly V. , Hall Helen , Janes Sam M. , Simons Benjamin D. , Koo Bon-Kyoung , Kim Jonghwan , Lee Joo-Hyeon 
Published: June 2021

AbstractWhile the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Upon damage, IL-1β signalling-dependent modulation of Jag1/2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives reprogramming and acquisition of differentiation plasticity. We identify a core role for the transcription factor Fosl2/Fra2 in secretory cell fate conversion to alveolar type 2 (AT2) cells retaining the distinct genetic and epigenetic signatures of secretory lineages. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate the functional role of a IL-1β-Notch-Fosl2 axis for the fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration.
MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function
Authors: Chambers Ian , Kumar Praveen , Lichtenberg Jens , Wang Pengcheng , Yu Jianshi , Phillips John , Kane Maureen , Bodine David , Hamza Iqbal 
Published: June 2021

AbstractMultidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. InCaenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter asmrp-5mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent inC. elegansraising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.Significance StatementMRPs are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRR5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.
Photoreactive hydrogel stiffness influences volumetric muscle loss repair
Authors: Basurto Ivan M. , Passipieri Juliana A. , Gardner Gregg M. , Smith Kathryn K. , Amacher Austin R. , Hansrisuk Audrey I. , Christ George J. , Caliari Steven R. 
Published: June 2021

AbstractVolumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink via thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase (MMP)-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 ± 0.002, 3.0 ± 0.002, and 10.6 ± 0.006 kPa mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by in situ photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks post-injury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks post-injury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more de novo muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries.
SPOT-Contact-Single: Improving Single-Sequence-Based Prediction of Protein Contact Map using a Transformer Language Model
Authors: Singh Jaspreet , Litfin Thomas , Singh Jaswinder , Paliwal Kuldip , Zhou Yaoqi 
Published: June 2021

AbstractMotivationAccurate prediction of protein contact-map is essential for accurate protein structure and function prediction. As a result, many methods have been developed for protein contact map prediction. However, most methods rely on protein-sequence-evolutionary information, which may not exist for many proteins due to lack of naturally occurring homologous sequences. Moreover, generating evolutionary profiles is computationally intensive. Here, we developed a contact-map predictor utilizing the output of a pre-trained language model ESM-1b as an input along with a large training set and an ensemble of residual neural networks.ResultsWe showed that the proposed method makes a significant improvement over a single-sequence-based predictor SSCpred with 15% improvement in the F1-score for the independent CASP14-FM test set. It also outperforms evolutionary-profile-based methods TrRosetta and SPOT-Contact with 48.7% and 48.5% respective improvement in the F1-score on the proteins without homologs (Neff=1) in the independent SPOT-2018 set. The new method provides a much faster and reasonably accurate alternative to evolution-based methods, useful for large-scale prediction.AvailabilityStand-alone-version of SPOT-Contact-Single is available at https://github.com/jas-preet/SPOT-Contact-Single. Direct prediction can also be made at https://sparks-lab.org/server/spot-contact-single. The datasets used in this research can also be downloaded from the GitHub.Contactjaspreetsingh2@griffithuni.edu.au, k.paliwal@griffith.edu.au, and zhouyq@szbl.ac.cnSupplementary informationSupplementary data are available at Bioinformatics online.