bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of m6A modifiers and RNA modifications in uterine fibroids
Authors: George Jitu W. , Cancino Rosa A. , Miller Jennifer L. Griffin , Qiu Fang , Lin Qishan , Rowley M Jordan , Chennathukuzhi Varghese M. , Davis John S. 
Published: August 2023

AbstractUterine leiomyoma or fibroids are the most common prevalent noncancerous tumors of the uterine muscle layer. Common symptoms associated with fibroids include pelvic pain, heavy menstrual bleeding, anemia, and pelvic pressure. These tumors are a leading cause of gynecological care but lack long-term therapy as the origin and development of fibroids are not well understood. Several next-generation sequencing technologies have been performed to identify the underlying genetic and epigenetic basis of fibroids. However, there remains a systemic gap in our understanding of molecular and biological process that define uterine fibroids. Recent epitranscriptomics studies have unraveled RNA modifications that are associated with all forms of RNA and are thought to influence both normal physiological functions and the progression of diseases. We quantified RNA expression profiles by analyzing publicly available RNA-seq data for 15 known epigenetic mediators to identify their expression profile in uterine fibroids compared to myometrium. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key m6A modifiers in fibroids and their matched myometrium. In concordance with our RNA expression profiles, no significant differences were observed in these proteins in uterine fibroids compared to myometrium. To determine abundance of RNA modifications, mRNA and small RNA from fibroids and matched myometrium were analyzed by UHPLC MS/MS. In addition to the prevalent N6-methyladenosine (m6A), we identified 11 other known modifiers but did not identify any aberrant expression in fibroids. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic sub-type. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and varied patient cohort.
Declining old pole physiology gradually enhances gene expression asymmetry in bacteria
Authors: Proenca Audrey M. , Tuğrul Murat , Nath Arpita , Steiner Ulrich K. 
Published: August 2023

AbstractGene expression is a heterogeneous process at the single-cell level. This heterogeneity is often coupled to individual growth rates, which are also highly stochastic, leading to the emergence of multiple physiological states within bacterial populations. Although recent advances have shown that cellular aging acts as a deterministic driver of growth asymmetry, the relationship between aging and gene expression heterogeneity remains elusive. Here we show that old poles undergo a progressive decline in gene expression as mother cells age, contributing to enhance phenotypic heterogeneity in bacterial populations. We quantified the activity of promoters with distinct activity profiles: a constitutive promoter, whose expression positively correlates with growth, and the promoter of RpoS, the general stress response sigma factor, for which growth and expression are mutually inhibitory. We demonstrate that mother cells have lower gene expression for both promoters. This asymmetry could not be explained by metabolic rate differences, but rather by the increasing intracellular heterogeneity of mother cells. As a mother ages, the declining activity of its old pole produces intracellular gradients in gene expression. This intracellular asymmetry manifests in the next generation as mother-daughter asymmetry, thus representing a source of phenotypic heterogeneity for the population. Our results show that bacterial asymmetry is built into the declining physiology of mother cells across generations, illustrating the deterministic nature of aging in bacterial systems. These findings provide further evidence for cellular aging as a mechanism to enhance the variance of metabolic states found in bacterial populations, with possible consequences for stress response and survival.
Force-induced site-specific enzymatic cleavage probes reveal that serial mechanical engagement boosts T cell activation
Authors: Rogers Jhordan , Ma Rong , Hu Yuesong , Salaita Khalid 
Published: August 2023

ABSTRACTThe surface of T cells is studded with T cell receptors (TCRs) that are used to scan target cells to identify peptide-major histo-compatibility complexes (pMHCs) signatures of viral infection or cancerous mutation. It is now established that the TCR-pMHC complex is highly transient and experiences mechanical forces that augment the fidelity of T cell activation. An important question in this area pertains to the role of force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions withF>7.1 piconewtons (pN) between TCRs and pMHCs. Force lifetimes (τF) are tunable from tens of min down to 1.9 min. T cells challenged with FUSE probes presenting cognate antigens with τFof 1.9 min demonstrated dampened markers of early activation, thus demonstrating that repeated mechanical sampling boosts TCR activation. Repeated mechanical samplingF>7.1 pN was found to be particularly critical at lower pMHC antigen densities, wherein the T cell activation declined by 23% with τFof 1.9 min. FUSE probes withF>17.0 pN response showed weaker influence on T cell triggering further showing that TCR-pMHC withF>17.0 pN are less frequent compared toF>7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement in antigen recognition.
High-efficiency transformation and expression of genomic libraries in yeast
Authors: Loock Mira , Antunes Luiza Berenguer , Heslop Rhiannon T , De Lauri Antonio Alfonso , Lira Andressa Brito , Cestari Igor 
Published: August 2023

AbstractSaccharomyces cerevisiaeis a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield about 106cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100x coverage of diverse genome libraries with small amounts of genomic libraries (0.1µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries’ size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method’s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.
Impact of Donor Individuality, Temporal Variation, and Culture Medium Type on Microbiota Composition and Metabolic Activity in Human Fecal Batch Culture
Authors: Liu Zhuang , Gerritsen Jacoline , Smidt Hauke , Zoetendal Erwin G. 
Published: August 2023

ABSTRACTFecal batch culture (FBC) studies often rely on a single fecal sample collection and the use of one type of medium for cultivation, bringing challenges to the interpretation of results and the comparison between studies. This study investigated the impact of donor individuality, temporal variation and culture medium type on microbiota composition and metabolic activity in an FBC setting with the fiber polydextrose (PDX) as carbon and energy source. FBCs were inoculated with fecal microbiota from three healthy donors sampled at three different days (day 1, 2 and 30), using either basal or rich culture medium with PDX as carbon source. Microbiota composition and metabolic activity were determined after 0, 6, 12, and 24 h of incubation. Microbiota composition variation explained by donor individuality dropped from 51% to 16% during incubation, while that explained by medium and PDX supplementation increased from 0% to 17% and 20%, respectively. Independent of the medium, the generaErysipelotrichaceaeUCG-003,BlautiaandFusicatenibacterwere stimulated by PDX supplementation. In basal mediumBacteroidesandAnaerostipesgrew better, whereasBifidobacterium,FaecalibacteriumandMegasphaeragrew better in rich medium. Metabolite variation was explained up to 50% by PDX supplementation during incubations, with butyrate being produced at the highest concentrations among all metabolites. Temporal variation explained less than 3% of the variation in both microbiota and metabolite composition. In conclusion, in this study donor individuality had the most profound impact on microbiota succession while medium and PDX supplementation had larger impacts on metabolic activity in FBCs.IMPORTANCEFBCs or otherin vitromodels are often chosen to assist in obtaining mechanistic insights complementingin vivomicrobiome observations by mimicking the colonic fermentation. However, FBCs are prone to a variety of factors such as the individuality of feces donor, temporal variation in microbiota composition within the individual, and cultivation medium. The importance of our study is in reinforcing that both donor individuality and medium type have major impacts on PDX degradation, whilst the impact of temporal variation is limited. Of interest is that bifidobacterial growth was more stimulated in rich medium with PDX as carbon source, whereas growth of members of theBacteroideteswere more stimulated in basal medium with PDX as carbon source. We recommend that variations in medium and donor samples should be considered when planning and interpretingin vitroincubation studies.
Single-cell transcriptome landscape of developing fetal gonads defines somatic cell lineage specification in humans
Authors: Lardenois A. , Suglia A. , Moore CL. , Evrard B. , Noël L. , Rivaud P. , Besson A. , Toupin M. , Léonard S. , Lesné L. , Coiffec I. , Nef S. , Lavoué V. , Collin O. , Chédotal A. , Mazaud-Guittot S. , Chalmel F. , Rolland AD. 
Published: August 2023

AbstractGonad development is an exciting model to study cell fate commitment. However, the specification and differentiation of somatic cell lineages within the testis and the ovary are incompletely characterized, especially in humans. In fact, a better understanding of sex determination first requires the identification of all the cell types involved and of their dynamic expression programs. Here we present a comprehensive analysis of approximately 128,000 single cells collected from 33 fetal testes and ovaries between 5 and 12 postconceptional weeks. In particular, a focused analysis of somatic cells allowed us to identify a common population of bipotential progenitors derived from the coelomic epithelium of both male and female gonads and capable of committing to either a steroidogenic or a supporting fate. Moreover, we have shown that early supporting cells, prior to further differentiation into Sertoli or granulosa cells, also give rise to the rete testis/ovarii lineage. Finally, we found that the ovary retains the capacity to feed the supporting cell pool for an extended period of time, directly from the surface epithelial cells and, bypassing the bipotential progenitor step. Altogether, our results provide an unprecedented revisiting of the human gonadal sex determination process.
Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine
Authors: Jain Shweta , Yee Andrew G. , Maas James , Gierok Sarah , Xu Hongfei , Stansil Jasmine , Eriksen Jacob , Nelson Alexandra , Silm Katlin , Ford Christopher P. , Edwards Robert H. 
Published: August 2023

AbstractThe burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.Significance statementReinforcement learning requires the phasic dopamine produced by burst firing but synaptic vesicle depletion limits the ability to convey information at high firing rates. We now find that AP-3 has two independent roles in dopamine release. First, AP-3 confers the axonal polarity of dopamine release by targeting vesicular monoamine transporter 2 (VMAT2) to the axon. Second, AP-3 acting locally at the nerve terminal produces synaptic vesicles that respond specifically to high frequency stimulation. Consistent with this, loss of AP-3 impairs reinforcement learning and this reflects the defect in release at high frequency, not the reduction in axonal dopamine.
A Novel Burn / Synovectomy Mouse Model for Temporomandibular Joint Osteoarthritis
Authors: Hsu Ginny Ching-Yun 
Published: August 2023

AbstractTemporomandibular Disorders (TMDs) is present in 33% of the U.S. population. Currently available animal models do not faithfully simulate the native disease progression of TMJ OA. The initiation of TMJ OA requires both local trauma and systemic inflammation. In this study, we present a novel mouse model which reproduces these two conditions. This is achieved by a procedure involving both synovectomy (local trauma) and a distant burn injury (systemic inflammation). Its efficacy at inducing TMJ OA was assessed with histomorphology and radiographic evaluation at 1,3, and 9 weeks after the procedure. We found that burn/synovectomy mice demonstrated significantly more degenerative hard and soft tissue changes in TMJ than uninjured control mice or synovotomy mice. The observed histology in burn/synoectomy mice mimicked native TMJ OA disease progression in a faithful manner. This animal model is invaluable in future research of the mechanism and risk factors of TMJ OA.
The actin binding protein profilin 1 is critical for mitochondria function
Authors: Read Tracy-Ann , Cisterna Bruno A. , Skruber Kristen , Ahmadieh Samah , Lindamood Halli L. , Vitriol Josefine A. , Shi Yang , Lefebvre Austin E.Y.T. , Black Joseph B. , Butler Mitchell T. , Bear James E. , Cherezova Alena , Ilatovskaya Daria V. , Weintraub Neil L. , Vitriol Eric A. 
Published: August 2023

AbstractProfilin 1 (PFN1) is an actin binding protein that is vital for the polymerization of monomeric actin into filaments. Here we screened knockout cells for novel functions of PFN1 and discovered that mitophagy, a type of selective autophagy that removes defective or damaged mitochondria from the cell, was significantly upregulated in the absence of PFN1. Despite successful autophagosome formation and fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells still accumulate damaged, dysfunctional mitochondria. Subsequent imaging and functional assays showed that loss of PFN1 significantly affects mitochondria morphology, dynamics, and respiration. Further experiments revealed that PFN1 is located to the mitochondria matrix and is likely regulating mitochondria function from within rather than through polymerizing actin at the mitochondria surface. Finally, PFN1 mutants associated with amyotrophic lateral sclerosis (ALS) fail to rescue PFN1 knockout mitochondrial phenotypes and form aggregates within mitochondria, further perturbing them. Together, these results suggest a novel function for PFN1 in regulating mitochondria and identify a potential pathogenic mechanism of ALS-linked PFN1 variants.
Molecular basis of Gabija anti-phage supramolecular assemblies
Authors: Yang Xiao-Yuan , Shen Zhangfei , Lin Qingpeng , Fu Tian-Min 
Published: August 2023

SummaryAs one of the most prevalent anti-phage defense systems in prokaryotes, Gabija consists of a Gabija protein A (GajA) and a Gabija protein B (GajB). The assembly and function of the Gabija system remain unclear. Here we present cryo-EM structures of GajA and the GajAB complex, revealing tetrameric and octameric assemblies, respectively. In the center of the complex, GajA assembles into a symmetric tetramer, which recruits two sets of GajB dimer at opposite sides of the complex, resulting in a 4:4 GajAB supramolecular complex for anti-phage defense. Further biochemical analysis showed that GajA alone is sufficient to cut double-stranded DNA and plasmid DNA, which can be inhibited by ATP. Unexpectedly, the GajAB displays enhanced activity for plasmid DNA, suggesting a role of substrate selection by GajB. Together, our study defines a framework for understanding anti-phage immune defense by the GajAB complex.
Tissue-specific increases in hypoxia-inducible factor 1 alpha protein are independent of mRNA levels during acute hypoxic exposure of the Gulf killifish,Fundulus grandis
Authors: Murphy Taylor E. , Harris Jasmine C. , Rees Bernard B. 
Published: August 2023

AbstractThe hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses to low oxygen in animals. It has been extensively studied in mammals, where its tissue levels are regulated by stabilization of the alpha subunit (HIF1α) when oxygen levels decrease. Consistent with this, the initial characterization of HIF1α in fish cells in culture indicated that protein abundance increased during hypoxia even when transcription was blocked. Subsequent studies, however, have reported an increase in HIF1α mRNA levels during hypoxia in certain tissues of selected species, raising the question whether hypoxic exposure brings about coordinated changes in HIF1α mRNA and protein in tissues when measured in the same fish. We have directly addressed this question by determining levels of HIF1α protein and mRNA in the tissues of Gulf killifish,Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2l−1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1α mRNA levels were unaffected by hypoxia in any tissue. Moreover, levels of HIF1α protein and mRNA in the same tissues were not correlated with one another, during either normoxia or hypoxia. These results suggest that, during the initial response to low oxygen, HIF1α protein levels increase as the result of post-translational protein stabilization, rather than new transcription, as predicted from studies in mammalian and fish cells in culture.Summary StatementParallel measurements of protein and mRNA of the hypoxia inducible factor support post-translational protein stabilization rather than new transcription in the initial response of fish to low oxygen
Non-homogenous axonal bouton distribution in whole-brain single cell neuronal networks
Authors: Qian Penghao , Manubens-Gil Linus , Jiang Shengdian , Peng Hanchuan 
Published: August 2023

SummaryWe examined the distribution of pre-synaptic contacts in axons of mouse neurons and constructed whole-brain single-cell neuronal networks using an extensive dataset of 1891 fully reconstructed neurons. We found that bouton locations were not homogeneous throughout the axon and also among brain regions. As our algorithm was able to generate whole-brain single-cell connectivity matrices from full morphology reconstruction datasets, we further found that non-homogeneous bouton locations have a significant impact on network wiring, including degree distribution, triad census and community structure. By perturbing neuronal morphology, we further explored the link between anatomical details and network topology. In our in silico exploration, we found that dendritic and axonal tree span would have the greatest impact on network wiring, followed by synaptic contact deletion. Our results suggest that neuroanatomical details must be carefully addressed in studies of whole brain networks at the single cell level.
Being present: a longitudinal study on the role of mindfulness on engagement and burnout in teachers
Authors: Gabel-Shemueli Rachel , Tzafrir Shay , Rodriguez Perez Berlan , Bahamonde Canepa Danae 
Published: August 2023

PurposeThe purpose of this two-wave longitudinal study was to examine the role of mindfulness as mediator between social support and engagement and as a moderator in the relationship between work overload and burnout, both cross-sectionally and after one year.Design/methodology/approachThe sample consisted of 243 Peruvian teachers. Structural equation modeling and SPSS Process Macro were used to test the hypothesis.FindingsThe results show that mindfulness mediated the relationship between social support and engagement at both times but doesn't moderate the relationship between work overload and burnout at either time.Originality/valueThis study highlights the role of mindfulness as a valuable personal resource in the workplace, as it facilitates a higher functioning at work while identifying relevant organizational characteristics that have an influence in its use.