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Background: Fetal sex and placental development impact pregnancy outcomes and fetal-maternal health, but the critical timepoint of placenta establishment in first trimester is understudied in human pregnancies., Methods: Pregnant subjects were recruited in late first trimester (weeks 10-14) at time of chorionic villus sampling, a prenatal diagnostic test. Leftover placenta tissue was collected and stored until birth outcomes were known, then DNA and RNA were isolated from singleton, normal karyotype pregnancies resulting in live births. DNA methylation was measured with the Illumina Infinium MethylationEPIC BeadChip array (n = 56). Differential methylation analysis compared 25 females versus 31 males using a generalized linear model on 743,461 autosomal probes. Gene expression sex differences were analyzed with RNA-sequencing (n = 74). An integrated analysis was performed using linear regression to correlate gene expression and DNA methylation in 51 overlapping placentas., Results: Methylation analysis identified 151 differentially methylated probes (DMPs) significant at false discovery rate < 0.05, including 89 (59%) hypermethylated in females. Probe cg17612569 (GABPA, ATP5J) was the most significant CpG site, hypermethylated in males. There were 11 differentially methylated regions affected by fetal sex, with transcription factors ZNF300 and ZNF311 most significantly hypermethylated in males and females, respectively. RNA-sequencing identified 152 genes significantly sexually dimorphic at false discovery rate < 0.05. The 151 DMPs were associated with 18 genes with gene downregulation (P < 0.05) in the direction of hypermethylation, including 2 genes significant at false discovery rate < 0.05 (ZNF300 and CUB and Sushi multiple domains 1, CSMD1). Both genes, as well as Family With Sequence Similarity 228 Member A (FAM228A), showed significant correlation between DNA methylation and sexually dimorphic gene expression, though FAM228A DNA methylation was less sexually dimorphic. Comparison with other sex differences studies found that cg17612569 is male-hypermethylated across gestation in placenta and in human blood up to adulthood., Conclusions: Overall, sex dimorphic differential methylation with associated differential gene expression in the first trimester placenta is small, but there remain significant genes that may be regulated through methylation leading to differences in the first trimester placenta., (© 2024. The Author(s).)

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development., Competing Interests: Competing interests statement:E.T.W. is a co-founder and consultant to Kate Therapeutics. K.K.P. and E.T.W. are inventors on a patent related to this work.

Introduction: Fetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes., Objectives: We utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation., Study Design: We analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences., Results: Placenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3., Discussion: This is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes., Competing Interests: Declaration of competing interest The authors report no conflict of interest, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Backgroud: The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated., Results: The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus., Conclusion: The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex., (© 2024. The Author(s).)

Enterovirus A71 (EV-A71) infections are a major Asia-Pacific health issue. However, this infection can cause serious and potentially fatal neurological issues. We attempt to explain EV-A71's molecular virology, epidemiology, and recombination events in this review. The clinical and neurological signs of EV-A71 infections are well documented. The review discusses EV-A71 central nervous system infections' causes, diagnostic criteria, treatment choices, and prognosis. Some consequences are aseptic meningitis, acute flaccid paralysis, and acute transverse myelitis. These problems' pathophysiology and EV-A71's central nervous system molecular processes are examined in the review. EV-A71 infections must be diagnosed accurately for therapy. No particular antiviral medications exist for EV-A71 infections, thus supportive care is the main treatment. The study emphasises addressing symptoms including temperature, dehydration, and pain to ease suffering. EV-A71 CNS infections have different prognoses depending on severity. The review discusses long-term effects and neurological sequelae of EV-A71 infections. In conclusion, Asia-Pacific public health is threatened by EV-A71 infections. This review helps prevent, diagnose, and treat EV-A71 infections by addressing the mechanisms, diagnostic criteria, treatment choices, and prognosis. This study fully examines the challenges and considerations of managing and treating EV-A71 infections. It also recommends future research and development to generate effective viral infection treatments., (© 2024 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Because analytic technologies improve, increasing amounts of data on methylation differences between assisted reproductive technology (ART) and unassisted conceptions are available. However, various studies use different tissue types and different populations in their analyses, making data comparison and integration difficult., Objective: To compare and integrate data on genome-wide analyses of methylation differences due to ART, allowing exposure of overarching themes., Evidence Review: All studies undertaking genome-wide analysis of human methylation differences due to ART or infertility in any tissue type across the lifespan were assessed for inclusion., Findings: Seventeen studies were identified that met the inclusion criteria. One study assessed trophectoderm biopsies, 2 first-trimester placenta, 1 first-trimester fetal tissue, 2 term placenta, 7 cord blood, 3 newborn dried blood spots, 1 childhood buccal smears, 1 childhood peripheral blood, and 2 adult peripheral blood. Eleven studies compared tissues from in vitro fertilization (IVF) conceptions with those of unassisted conceptions, 4 compared intracytoplasmic sperm injection with unassisted conceptions, 4 compared non-IVF fertility treatment (NIFT) with unassisted conceptions, 4 compared NIFT with IVF, and 5 compared an infertile population (conceiving via various methods) with an unassisted presumably fertile population. In studies assessing placental tissue, 1 gene with potential methylation changes due to IVF when compared with unassisted conceptions was identified by 2 studies. In blood, 11 potential genes with methylation changes due to IVF compared with unassisted conceptions were identified by 2 studies, 1 of which was identified by 3 studies. Three potentially affected genes were identified by 2 studies involving blood between intracytoplasmic sperm injection and unassisted populations. There were no overlapping genes identified in any tissue type between NIFT and unassisted populations, between NIFT and IVF, or the infertility combined population when compared with the unassisted fertile population., Conclusions: Comparing studies is challenging due to differing variables between analyses. However, even in similar tissue types and populations, overlapping methylation changes are limited, suggesting that differences due to ART are minimal., Relevance: Information from this systematic review is significant for providers and patients who provide and use ART to understand methylation risks that may be associated with the technology., Competing Interests: Declaration of interests A.M.S. has nothing to disclose. T.L.G. has nothing to disclose. A.E.D. has nothing to disclose. R.A.H. has nothing to disclose. P.M.H. has nothing to disclose. M.H.K. has nothing to disclose. B.J.S. has nothing to disclose. A.S. has nothing to disclose. E.T.W. has nothing to disclose. J. L. C. is a scientific advisor for BINTO. J. W. III is a member of The Advisory Board for Natera. M. D. P. is a Ferring Pharmaceuticals consultant, serves at the Society for the Study of Reproduction Development Committee, previously served as a Team Lead on the Endocrine Society Annual Meeting Steering Committee, and previously received a speaker honorarium from Natera., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Arrow bamboo ( Fargesia nitida ) is a pioneer plant in secondary forest succession in the Sichuan Province mountains. To comprehensively investigate the microbial communities and their functional variations in different rhizocompartments (root endosphere, rhizosphere, and root zone) of arrow bamboo ( Fargesia nitida ), a high-throughput metagenomic study was conducted in the present study. The results showed that the abundances of the dominant bacterial phyla Proteobacteria and Actinobacteria in the bamboo root endosphere were significantly lower than those in the rhizosphere and root zones. In contrast, the dominant fungal phyla, Ascomycota and Basidiomycota, showed the opposite tendency. Lower microbial diversity, different taxonomic composition and functional profiles, and a greater abundance of genes involved in nitrogen fixation ( nifB ), cellulose degradation (beta-glucosidase), and cellobiose transport (cellulose 1, 4-beta-cellobiosidase) were found in the bamboo root endosphere than in the other rhizocompartments. Greater soil total carbon, total nitrogen, NH 4 + -N, microbial biomass carbon, and greater activities of invertase and urease were found in the bamboo root zone than in the adjacent soil (spruce root zone). In contrast, the soil microbial community and functional profiles were similar. At the phylum level, invertase was significantly related to 31 microbial taxa, and the effect of NH 4 + -N on the microbial community composition was greater than that of NO 3 - -N. The soil physicochemical properties and enzyme activities were significantly correlated with microbial function. These results indicate that the root endosphere microbiomes of arrow bamboo were strongly selected by the host plant, which caused changes in the soil nutrient properties in the subalpine coniferous forest., Competing Interests: The authors declare there are no competing interests., (©2023 Zhang et al.)

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Four Gram-positive, aerobic, catalase- and oxidase-negative, rod-shaped, motile endophytic bacterial strains, designated NM3R9 T , NE1TT3, NE2TL11 and NE2HP2 T , were isolated from the inner tissues (leaf and stem) of Sphaeralcea angustifolia and roots of Prosopis laevigata . They were characterized using a polyphasic approach, which revealed that they represent two novel Microbacterium species. Phylogenetic analysis based on 16S rRNA gene sequencing showed that the species closest to NE2HP2 T was Microbacterium arborescens DSM 20754 T (99.6 %) and that closest to NM3R9 T , NE2TL11 and NE2TT3 was Microbacterium oleivorans NBRC 103075 T (97.4 %). The whole-genome average nucleotide identity value between strain NM3R9 T and Microbacterium imperiale DSM 20530 T was 90.91 %, and that between strain NE2HP2 T and M. arborecens DSM 20754 T was 91.03 %. Digital DNA-DNA hybridization showed values of less than 70 % with the type strains of related species. The polar lipids present in both strains included diphosphatidylglycerol, phosphatidylglycerol, glycolipids and unidentified lipids, whereas the major fatty acids included anteiso-C 15 : 0 , anteiso-C 17 : 0 , iso-C 16 : 0 and C 16 : 0 . Whole-cell sugars included mannose, rhamnose and galactose. Strains NM3R9 T and NE2HP2 T showed physiological characteristics different from those present in closely related Microbacterium species. According to the taxonomic analysis, both strains belong to two novel species. The name Microbacterium plantarum sp. nov. is proposed for strain NE2HP2 T (=LMG 30875 T =CCBAU 101117 T ) and Microbacterium thalli sp. nov. for strains NM3R9 T (=LMG 30873 T =CCBAU 101116 T ), NE1TT3 (=CCBAU 101114) and NE2TL11 (=CCBAU 101115).

Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered a MBNL1 and RBFOX1 inducible HEK-293 cell line. Modest induction of exogenous RBFOX1 in this cell line modulated MBNL1-dependent AS outcomes in 3 skipped exon events, despite significant levels of endogenous RBFOX1 and RBFOX2. Due to background RBFOX levels, we conducted a focused analysis of dose-dependent MBNL1 skipped exon AS outcomes and generated transcriptome wide dose-response curves. Analysis of this data demonstrates that MBNL1-regulated exclusion events may require higher concentrations of MBNL1 protein to properly regulate AS outcomes compared to inclusion events and that multiple arrangements of YGCY motifs can produce similar splicing outcomes. These results suggest that rather than a simple relationship between the organization of RBP binding sites and a specific splicing outcome, that complex interaction networks govern both AS inclusion and exclusion events across a RBP gradient., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The common bean ( Phaseolus vulgaris L.) is an important crop in the world that forms root nodules with diverse rhizobia. Aiming to learn the rhizobial communities associated with the common bean in the black soil of Northeast China, 79 rhizobia were isolated from root nodules of two host varieties (Cuican and Jiadouwang) grown in two sites of blackland and were characterized by comparative sequence analyses of 16S rRNA, recA, atpD, nodC , and nifH genes, and whole genome. As a result, Rhizobium indigoferae, R. anhuiense , and R. croatiense as minor groups and three dominant novel Rhizobium species were identified based on their average nucleotide identity and DNA-DNA hybridization values to the type strains of relative species. This community composition of rhizobia associated with the common bean in the tested black soils was unique. Despite their different species affiliations, all of them were identified into the symbiovar phaseoli according to the phylogenies of symbiotic genes, nodC and nifH . While the phylogenetic discrepancies found in nodC, nifH evidenced that the evolutions of nodulation ( nod ) and nitrogen fixation ( nif ) genes were partially independent. In addition, only one dominant rhizobial species was shared by the two common bean varieties grown in the two soil samples, implying that both the plant variety and the soil characteristics affected the compatibility between rhizobia and their hosts. These findings further enlarged the spectrum of common bean-nodulating rhizobia and added more information about the interactions among the soil factors, rhizobial species, and host plants in the symbiosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Liu, Hu, Wang, Gu and Wang.)

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG) exp , harbored in the 3' untranslated region (3' UTR) of dystrophia myotonica protein kinase ( DMPK ) mRNA and drives disease through a gain-of-function mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG) exp in vitro , affording perimidin-2-amine diazirine ( 1 ) that bound to r(CUG) exp . The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1 . Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG) exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites., Competing Interests: The authors declare the following competing financial interest(s): M.D.D. is a founder of Expansion Therapeutics., (© 2023 The Authors. Published by American Chemical Society.)

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs., (© 2023. The Author(s).)

Symbiotic nitrogen fixation between legumes and rhizobia makes a great contribution to the terrestrial ecosystem. The successful symbiosis between the partners mainly depends on the nod and nif genes in rhizobia, while the specific symbiosis is mainly determined by the structure of Nod factors and the corresponding secretion systems (type III secretion system; T3SS), etc. These symbiosis genes are usually located on symbiotic plasmids or a chromosomal symbiotic island, both could be transferred interspecies. In our previous studies, Sesbania cannabina -nodulating rhizobia across the world were classified into 16 species of four genera and all the strains, especially those of Rhizobium spp., harboured extraordinarily highly conserved symbiosis genes, suggesting that horizontal transfer of symbiosis genes might have happened among them. In order to learn the genomic basis of diversification of rhizobia under the selection of host specificity, we performed this study to compare the complete genome sequences of four Rhizobium strains associated with S. cannabina , YTUBH007, YTUZZ027, YTUHZ044 and YTUHZ045. Their complete genomes were sequenced and assembled at the replicon level. Each strain represents a different species according to the average nucleotide identity (ANI) values calculated using the whole-genome sequences; furthermore, except for YTUBH007, which was classified as Rhizobium binae , the remaining three strains were identified as new candidate species. A single symbiotic plasmid sized 345-402 kb containing complete nod , nif , fix , T3SS and conjugal transfer genes was detected in each strain. The high ANI and amino acid identity (AAI) values, as well as the close phylogenetic relationships among the entire symbiotic plasmid sequences, indicate that they have the same origin and the entire plasmid has been transferred among different Rhizobium species. These results indicate that S. cannabina stringently selects a certain symbiosis gene background of the rhizobia for nodulation, which might have forced the symbiosis genes to transfer from some introduced rhizobia to the related native or local-condition-adapted bacteria. The existence of almost complete conjugal transfer related elements, but not the gene virD , indicated that the self-transfer of the symbiotic plasmid in these rhizobial strains may be realized via a virD -independent pathway or through another unidentified gene. This study provides insight for the better understanding of high-frequency symbiotic plasmid transfer, host-specific nodulation and the host shift for rhizobia.

A major issue in the clinical management of epilepsy is the unpredictability of seizures. Yet, traditional approaches to seizure forecasting and risk assessment in epilepsy rely heavily on raw seizure frequencies, which are a stochastic measurement of seizure risk. We consider a Bayesian non-homogeneous hidden Markov model for unsupervised clustering of zero-inflated seizure count data. The proposed model allows for a probabilistic estimate of the sequence of seizure risk states at the individual level. It also offers significant improvement over prior approaches by incorporating a variable selection prior for the identification of clinical covariates that drive seizure risk changes and accommodating highly granular data. For inference, we implement an efficient sampler that employs stochastic search and data augmentation techniques. We evaluate model performance on simulated seizure count data. We then demonstrate the clinical utility of the proposed model by analyzing daily seizure count data from 133 patients with Dravet syndrome collected through the Seizure Tracker ™ system, a patient-reported electronic seizure diary. We report on the dynamics of seizure risk cycling, including validation of several known pharmacologic relationships. We also uncover novel findings characterizing the presence and volatility of risk states in Dravet syndrome, which may directly inform counseling to reduce the unpredictability of seizures for patients with this devastating cause of epilepsy.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that impacts women worldwide. There are several racial and ethnic differences in PCOS phenotypes and in PCOS- associated metabolic dysfunction. In this review, we summarize the current literature on disparities in the diagnosis and outcomes associated with PCOS in the United States. Future studies are needed to address gaps in knowledge for racial and ethnic-specific differences in PCOS, and include a large number of non-White and/or Hispanic participants in PCOS studies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Ongoing research is needed to determine geo-epidemiologic differences of polycystic ovary syndrome (PCOS)., Objective: Determine hormonal and metabolic parameters of women with PCOS in 2 environments., Methods: Prospective cohort study., Setting: Tertiary-care based specialty clinics in Alabama and California., Patients or Other Participants: A total of 1610 women with PCOS by National Institutes of Health Criteria from 1987 to 2010., Interventions: Interview, physical examination, laboratory studies., Main Outcomes Measures: Demographic data, menstrual cycle history, and hormonal and metabolic parameters were collected. Hirsutism was defined as modified Ferriman-Gallwey scores ≥4. Androgen values greater than laboratory reference ranges or >95th percentile of all values were considered elevated (hyperandrogenemia). Metabolic parameters included body mass index (BMI), waist-hip-ratio (WHR), glucose tolerance test, and homeostatic model assessment for insulin resistance (HOMA-IR) scores., Results: Alabama women with PCOS were younger with a higher BMI. After adjustment for age and BMI, Alabama women with PCOS were more likely hirsute (adjusted odds ratio [aOR], 1.8; 95% CI, 1.4-2.4; P < 0.001), with elevated HOMA-IR scores (adjusted beta coefficient 3.6; 95% CI, 1.61-5.5; P < 0.001). California women with PCOS were more likely to have hyperandrogenemia (free testosterone aOR, 0.14; 95% CI, 0.11-0.18; P < 0.001; total testosterone aOR, 0.41; 95% CI, 0.33-0.51). Results were similar when stratified by White race. In Black women with PCOS, BMI and WHR did not differ between locations, yet differences in androgen profiles and metabolic dysfunction remained., Conclusion: Alabama women with PCOS, regardless of Black or White race, were more likely hirsute with metabolic dysfunction, whereas California women with PCOS were more likely to demonstrate hyperandrogenemia, highlighting potential environmental impacts on PCOS., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bradyrhizobium guangxiense CCBAU53363 efficiently nodulates peanut but exhibits incompatible interaction with mung bean. By comparing the common nod region with those of other peanut bradyrhizobia efficiently nodulating these two hosts, distinctive characteristics with a single nodD isoform ( nodD1 ) and a truncated nolA were identified. However, the regulatory roles of NodD1 and NolA and their coordination in legume-bradyrhizobial interactions remain largely unknown in terms of explaining the contrasting symbiotic compatibility. Here, we report that nolA was important for CCBAU53363 symbiosis with peanut but restricted nodulation on mung bean, while nodD1 was dispensable for CCBAU53363 symbiosis with peanut but essential for nodulation on mung bean. Moreover, nolA exerted a cumulative contribution with nodD1 to efficient symbiosis with peanut. Additionally, mutants lacking nolA delayed nodulation on peanut, and both nolA and nodD1 were required for competitive nodule colonization. It is noteworth that most of the nodulation genes and type III secretion system (T3SS)-related genes were significantly downregulated in a strain 53Δ nodD1nolA mutant compared to wild-type strain CCBAU53363, and the downregulated nodulation genes also had a greater impact than T3SS-related genes on the symbiotic defect of 53Δ nodD1nolA on peanut, which was supported by a more severe symbiotic defect induced by 53Δ nodC than that with the 53Δ nodD1nopP , 53Δ nodD1rhcJ , and 53Δ nodD1ttsI mutants. NolA did not regulate nod gene expression but did regulate the T3SS effector gene nopP in an indirect way. Meanwhile, nolA , nodW , and some T3SS-related genes besides nopP were also demonstrated as new "repressors" that seriously impaired CCBAU53363 symbiosis with mung bean. Taken together, the roles and essentiality of nolA and nodD1 in modulating symbiotic compatibility are sophisticated and host dependent. IMPORTANCE The main findings of this study were that we clarified that the roles and essentiality of nodD1 and nolA are host dependent. Importantly, for the first time, NolA was found to positively regulate T3SS effector gene nopP to mediate incompatibility on mung bean. Additionally, NolA does not regulate nod genes, which are activated by NodD1. nolA exerts a cumulative effect with nodD1 on CCBAU53363 symbiosis with peanut. These findings shed new light on our understanding of coordinated regulation of NodD1 and NolA in peanut bradyrhizobia with different hosts.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N 2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-β-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens , while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N 2 -fixing efficiency of the peanut nodules is three times higher than that of S. flavescens . By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein., Competing Interests: Declaration of interests E.T.W. is a cofounder of and consultant to Kate Therapeutics. A.J. is a member of the scientific advisory board of Molecular Cell. P.K.T. served as a consultant to Denali Therapeutics and holds a shared patent with Ionis Pharmaceuticals. Y.N. discloses the patent PCT/JP2017/23,162 related to this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To determine whether deoxyribonucleic acid (DNA) methylation alterations exist in the first-trimester human placenta between conceptions using fertility treatments and those that do not and, if so, whether they are the result of underlying infertility or fertility treatments. We also assessed whether significant alterations led to changes in gene expression., Design: We compared DNA methylation of the first-trimester placenta from singleton pregnancies that resulted in live births from unassisted, in vitro fertilization (IVF), and non-IVF fertility treatment (NIFT) conceptions using the Infinium MethylationEPIC BeadChip array. Significant CpG sites were compared with corresponding ribonucleic acid sequencing analysis in similar cohorts to determine whether methylation alterations lead to differences in gene expression., Setting: Academic medical center., Patient(s): A total of 138 singleton pregnancies undergoing chorionic villus sampling resulting in a live birth were recruited for methylation analysis (56 unassisted, 38 NIFT, and 44 IVF conceptions). Ribonucleic acid-sequencing data consisted of 141 subjects (74 unassisted, 33 NIFT, and 34 IVF conceptions) of which 116 overlapped with the methylation cohort., Intervention(s): In vitro fertilization-conceived pregnancy or pregnancy conceived via NIFT, such as ovulation induction and intrauterine insemination., Main Outcome Measure(s): Significant methylation changes at CpG sites after adjustment for multiple comparisons. The secondary outcome was gene expression changes of significant CpG sites., Result(s): Of the 741,145 probes analyzed in the placenta, few were significant at Bonferroni <0.05: 185 CpG sites (0.025%) significant in pregnancies conceived with the fertility treatments (NIFT + IVF) vs. unassisted conceptions; 28 in NIFT vs. unassisted; 195 in IVF vs. unassisted; and only 13 (0.0018%) in IVF vs. NIFT conceptions. Of all significant CpG sites combined, 10% (35) were located in genes with suggestive gene expression changes, but none were significant after adjustment for multiple comparisons (ribonucleic acid sequencing false discovery rate <0.05). None of the 13 differentially methylated probes in the IVF vs. NIFT placenta were located in genes with suggestive IVF vs. NIFT gene expression differences., Conclusion(s): Underlying infertility is the most significant contributor to the minimal differences in first-trimester placental methylation, and not the specific fertility treatment used, such as IVF., (Copyright © 2022. Published by Elsevier Inc.)

Most cells in the body are mononuclear whereas skeletal muscle fibres are uniquely multinuclear. The nuclei of muscle fibres (myonuclei) are usually situated peripherally which complicates the equitable distribution of gene products. Myonuclear abundance can also change under conditions such as hypertrophy and atrophy. Specialised zones in muscle fibres have different functions and thus distinct synthetic demands from myonuclei. The complex structure and regulatory requirements of multinuclear muscle cells understandably led to the hypothesis that myonuclei govern defined 'domains' to maintain homeostasis and facilitate adaptation. The purpose of this review is to provide historical context for the myonuclear domain and evaluate its veracity with respect to mRNA and protein distribution resulting from myonuclear transcription. We synthesise insights from past and current in vitro and in vivo genetically modified models for studying the myonuclear domain under dynamic conditions. We also cover the most contemporary knowledge on mRNA and protein transport in muscle cells. Insights from emerging technologies such as single myonuclear RNA-sequencing further inform our discussion of the myonuclear domain. We broadly conclude: (1) the myonuclear domain can be flexible during muscle fibre growth and atrophy, (2) the mechanisms and role of myonuclear loss and motility deserve further consideration, (3) mRNA in muscle is actively transported via microtubules and locally restricted, but proteins may travel far from a myonucleus of origin and (4) myonuclear transcriptional specialisation extends beyond the classic neuromuscular and myotendinous populations. A deeper understanding of the myonuclear domain in muscle may promote effective therapies for ageing and disease., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.)

Type I peanut bradyrhizobial strains can establish efficient symbiosis in contrast to symbiotic incompatibility induced by type II strains with mung bean. The notable distinction in the two kinds of key symbiosis-related regulators nolA and nodD close to the nodABCSUIJ operon region between these two types of peanut bradyrhizobia was found. Therefore, we determined whether NolA and NodD proteins regulate the symbiotic adaptations of type I strains to different hosts. We found that NodD1-NolA synergistically regulated the symbiosis between the type I strain Bradyrhizobium zhanjiangense CCBAU51778 and mung bean, and NodD1-NodD2 jointly regulated nodulation ability. In contrast, NodD1-NolA coordinately regulated nodulation ability in the CCBAU51778-peanut symbiosis. Meanwhile, NodD1 and NolA collectively contributes to competitive nodule colonization of CCBAU51778 on both hosts. The Fucosylated Nod factors and intact type 3 secretion system (T3SS), rather than extra nodD2 and full-length nolA, were critical for effective symbiosis with mung bean. Unexpectedly, T3SS-related genes were activated by NodD2 but not NodD1. Compared to NodD1 and NodD2, NolA predominantly inhibits exopolysaccharide production by promoting exoR expression. Importantly, this is the first report that NolA regulates rhizobial T3SS-related genes. The coordinated regulation and integration of different gene networks to fine-tune the expression of symbiosis-related genes and other accessory genes by NodD1-NolA might be required for CCBAU51778 to efficiently nodulate peanut. This study shed new light on our understanding of the regulatory roles of NolA and NodD proteins in symbiotic adaptation, highlighting the sophisticated gene networks dominated by NodD1-NolA., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Objective: Epilepsy monitoring unit (EMU) admissions are critical for presurgical evaluation of drug-resistant epilepsy but may be nondiagnostic if an insufficient number of seizures are recorded. Seizure forecasting algorithms have shown promise for estimating the likelihood of seizures as a binary event in individual patients, but methods to predict how many seizures will occur remain elusive. Such methods could increase the diagnostic yield of EMU admissions and help patients mitigate seizure-related morbidity. Here, we evaluated the performance of a state-space method that uses prior seizure count data to predict future counts., Methods: A Bayesian negative-binomial dynamic linear model (DLM) was developed to forecast daily electrographic seizure counts in 19 patients implanted with a responsive neurostimulation (RNS) device. Holdout validation was used to evaluate performance in predicting the number of electrographic seizures for forecast horizons ranging 1-7 days ahead., Results: One-day-ahead prediction of the number of electrographic seizures using a negative-binomial DLM resulted in improvement over chance in 73.1% of time segments compared to a random chance forecaster and remained >50% for forecast horizons of up to 7 days. Superior performance (mean error = .99) was obtained in predicting the number of electrographic seizures in the next day compared to three traditional methods for count forecasting (integer-valued generalized autoregressive conditional heteroskedasticity model or INGARCH, 1.10; Croston, 1.06; generalized linear autoregressive moving average model or GLARMA, 2.00). Number of electrographic seizures in the preceding day and laterality of electrographic pattern detections had highest predictive value, with greater number of electrographic seizures and RNS magnet swipes in the preceding day associated with a higher number of electrographic seizures the next day., Significance: This study demonstrates that DLMs can predict the number of electrographic seizures a patient will experience days in advance with above chance accuracy. This study represents an important step toward the translation of seizure forecasting methods into the optimization of EMU admissions., (© 2022 International League Against Epilepsy.)

Epilepsy is a disorder characterized by paroxysmal transitions between multistable states. Dynamical systems have been useful for modeling the paroxysmal nature of seizures. At the same time, intracranial electroencephalography (EEG) recordings have recently discovered that an electrographic measure of epileptogenicity, interictal epileptiform activity, exhibits cycling patterns ranging from ultradian to multidien rhythmicity, with seizures phase-locked to specific phases of these latent cycles. However, many mechanistic questions about seizure cycles remain unanswered. Here, we provide a principled approach to recast the modeling of seizure chronotypes within a statistical dynamical systems framework by developing a Bayesian switching linear dynamical system (SLDS) with variable selection to estimate latent seizure cycles. We propose a Markov chain Monte Carlo algorithm that employs particle Gibbs with ancestral sampling to estimate latent cycles in epilepsy and apply unsupervised learning on spectral features of latent cycles to uncover clusters in cycling tendency. We analyze the largest database of patient-reported seizures in the world to comprehensively characterize multidien cycling patterns among 1,012 people with epilepsy, spanning from infancy to older adulthood. Our work advances knowledge of cycling in epilepsy by investigating how multidien seizure cycles vary in people with epilepsy, while demonstrating an application of an SLDS to frame seizure cycling within a nonlinear dynamical systems framework. It also lays the groundwork for future studies to pursue data-driven hypothesis generation regarding the mechanistic drivers of seizure cycles.

Considerable evidence from mouse models and human postmortem brain suggests loss of Muscleblind-like protein 2 (MBNL2) function in brain is a major driver of CNS symptoms in Myotonic dystrophy type 1 (DM1). Increased hypersomnia, fatigue, and surgical complications associated with general anesthesia suggest possible sensitivity to GABAergic inhibition in DM1. To test the hypothesis that MBNL2 depletion leads to behavioral sensitivity to GABA A receptor (GABA A -R) modulation, Mbnl2 knock-out (KO) and wild-type (WT) littermates were treated with the anesthetic sevoflurane, the benzodiazepine diazepam, the imidazopyridine zolpidem, and the benzodiazepine rescue agent, flumazenil (Ro 15-1788), and assessed for various behavioral metrics. Mbnl2 KO mice exhibited delayed recovery following sevoflurane, delayed emergence and recovery from zolpidem, and enhanced sleep time at baseline that was modulated by flumazenil. A significantly higher proportion of Mbnl2 KO mice also loss their righting reflex [loss of righting reflex (LORR)] from a standard diazepam dose. We further examined whether MBNL2 depletion affects total GABA A -R mRNA subunit levels and validated RNA-sequencing data of mis-spliced Gabrg2 , whose isoform ratios are known to regulate GABA sensitivity and associated behaviors. While no other GABA A -R subunit mRNA levels tested were altered in Mbnl2 KO mouse prefrontal cortex, Gabrg2S/L mRNA ratio levels were significantly altered. Taken together, our findings indicate that loss of MBNL2 function affects GABAergic function in a mouse model of myotonic dystrophy (DM1)., (Copyright © 2022 Edokpolor et al.)

Objective: To determine whether ovarian volume (OV) alone is an independent marker for metabolic dysfunction in women with suspected androgen excess., Design: Retrospective cohort study., Setting: Tertiary academic reproductive endocrinology clinic., Patients: Women aged ≥21 years recruited/referred for symptoms related to androgen excess., Interventions: Transvaginal ovarian ultrasound, physical and medical evaluation, 2-hour 75-g oral glucose tolerance test (oGTT), and blood sampling., Main Outcome Measures: Prevalence of hyperandrogenism and metabolic dysfunction., Results: This study included 666 women, of whom 412 (61.9%) and 254 had OVs of >10 and ≤10 mL, respectively. An OV of >10 mL was associated with a higher prevalence of hirsutism (65.1% vs. 51.5%) than an OV of ≤10 mL. Polycystic ovary syndrome by the National Institutes of Health 1990 criteria was found in 67.3% and 51.4% of women with OVs of >10 and ≤10 mL, respectively. Metabolic parameters, including body mass index, waist circumference, and 1-hour insulin levels during the oGTT (odds ratio, 1.98; 95% confidence interval, 1.18-3.31), were significantly higher in women with an OV of >10 mL than in those with an OV of ≤10 mL. An OV of ≤10 mL had a 76.3% negative predictive value for hyperinsulinemia at 1 hour., Conclusions: In women with suspected androgen excess, an OV of >10 mL in at least 1 ovary is not associated with metabolic syndrome but is associated with younger age; an increased body mass index and waist circumference; a higher prevalence of hirsutism, oligoovulation, and polycystic ovary syndrome; and a higher 60-minute insulin level during the oGTT. Overall, an increased OV appears to be a good marker for hyperinsulinemia and hyperandrogenism in women suspected of having an androgen excess disorder., (© 2022 The Authors.)

Myotonic dystrophy (DM) is caused by expansions of C(C)TG repeats in the non-coding regions of the DMPK and CNBP genes, and DM patients often suffer from sudden cardiac death due to lethal conduction block or arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP, Elav-like family member 1 (CELF1). Hypothesis solely targeting MBNL1 or CELF1 pathways that could address all the consequences of repeat expansion in heart remained inconclusive, particularly when the direct cause of mortality and results of transcriptome analyses remained undetermined in Mbnl compound knockout (KO) mice with cardiac phenotypes. Here, we develop Myh6-Cre double KO (DKO) (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice to eliminate Mbnl1/2 in cardiomyocytes and observe spontaneous lethal cardiac events under no anesthesia. RNA sequencing recapitulates DM heart spliceopathy and shows gene expression changes that were previously undescribed in DM heart studies. Notably, immunoblotting reveals a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Our findings demonstrate that complete ablation of MBNL1/2 in cardiomyocytes is essential for generating sudden death due to lethal cardiac rhythms and reveal potential mechanisms for DM heart pathogenesis., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMS Bmal1 -/- ) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMS Bmal1 -/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20 , a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies., Competing Interests: LR, XZ, CD, JM, DH, CW, NW, HO, PD, SL, MP, EW, KE No competing interests declared, (© 2022, Riley et al.)

CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUG n RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies., Competing Interests: Declaration of interests E.T.W. has consulted for Expansion Therapeutics, Entrada Therapeutics, Design Therapeutics, Triplet Therapeutics, and Faze Medicines. E.T.W. is a co-founder of, shareholder in, and adviser to Kate Therapeutics. Financial support for research has been provided to the lab of E.T.W. at the University of Florida by Expansion Therapeutics, Entrada Therapeutics, and Kate Therapeutics. E.T.W. and C.P.K. are inventors on a patent application filed by the University of Florida related to this work., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Enterovirus A71 (EV A71) is one of the most important enteroviruses related to morbidity and mortality in children worldwide. This study aimed to analyse the secular trend of EV A71 in Taiwan from 1998 to 2020 and to evaluate the effectiveness of infection control measures., Methods: We collected the epidemiological data of EV A71 from disease surveillance systems in Taiwan. We analysed the association between the secular trend of EV A71 and preventive measures such as hand washing, case isolation, and suspension of classes., Results: The incidence of enterovirus infections with severe complications (EVSC) decreased from 16.25 per 100,000 children under six in 1998 to less than 9.73 per 100,000 children under six after 2012 (P = 0.0022). The mortality rate also decreased significantly, from 3.52 per 100,000 children under six in 1998 to 0 per 100,000 children under six in 2020 (P < 0.0001). The numbers of EVSC and fatalities were significantly higher in the years when EV A71 accounted for more than 10% of the annual predominant serotypes (p < 0.05). After the implementation of many non-pharmaceutical interventions in 2012, the incidence of EVSC and mortality rate decreased significantly (p < 0.001)., Conclusions: After implementing active enterovirus surveillance and preventive measures, we found that the incidence of EVSC and fatalities due to EV A71 in Taiwan decreased significantly from 1998 to 2020. Continuous surveillance and strengthened infection control policies are still needed in the future., (© 2022. The Author(s).)

Root-rot disease has lead to serious reduction in yields and jeopardized the survival of the economically and ecologically important Zanthoxylum bungeanum trees cultured in Sichuan Province. In order to investigate the interaction between the microbiome and the root-rot disease, a metagenomic analysis was performed to characterize the microbial communities and functions in Z. bungeanum root endosphere, rhizosphere and bulk soil with/without root-rot disease. Soil physicochemical properties, microbial population size and enzyme activities were also analyzed for finding their interactions with the root-rot disease. As results, lower total nitrogen (TN) and available phosphorus (AP) contents but higher pH in rhizosphere and bulk soil, as well as lower substrate-induced respiration (SIR) and higher protease activity in bulk soil of diseased trees were found, in comparison with that of healthy trees. Microbial diversity and community composition were changed by root-rot disease in the endosphere, but not in rhizosphere and bulk soils. The endophytic microbiome of diseased trees presented higher Proteobacteria abundance and lower abundances of Bacteroidetes, Firmicutes and dominant fungal phyla. The relative abundances of nitrogen cycle- and carbon cycle-related genes in endophytic microbiomes were different between the diseased and healthy trees. Based on ANOSIM and PCoA, functional profiles (KEGG and CAZy) of microbiomes in rhizosphere and bulk soil shifted significantly between the diseased and healthy trees. In addition, soil pH, TN, AP, SIR, invertase and protease were estimated as the main factors influencing the shifts of taxonomic and functional groups in microbiomes of rhizosphere and bulk soil. Conclusively, the imbalance of root and soil microbial function groups might lead to shifts in the root endosphere-rhizosphere microenvironment, which in turn resulted in Z. bungeanum root-rot., Competing Interests: The authors declare there are no competing interests., (©2022 Liao et al.)

New sequences of nanocomposites including numerous maleic acid-grafted poly(butylene adipate-co-terephthalate) (g-PBAT) and cellulose nanocrystals (CNC) were efficaciously fabricated via transesterification and polycondensation processes with the covalent bonds between the polymer and reinforcing fillers. The grafting interaction of maleic acid onto PBAT was successfully demonstrated using Fourier transform infrared (FTIR) and 13 C-nuclear magnetic resonance (NMR) spectra. The morphology of g-PBAT/CNC nanocomposites was investigated by wide-angle X-ray diffraction and transmission electron microscopy. Both results indicate that the CNC was randomly dispersed into the g-PBAT polymer matrix. The storage modulus at -80 and 25 °C was significantly enhanced with the incorporation of CNC into g-PBAT matrix. The crystallization rate of g-PBAT/CNC nanocomposites increased as the loading of CNC increased. With the incorporation of 3 wt% CNC, the half-time for crystallization of the g-PBAT/CNC composite decreased about 50~80% as compared with the same isothermal crystallization of pure polymer matrix. All water vapor permeation (WVP) values of all g-PBAT/CNC nanocomposites decreased as the loading of CNC increased. The decrease in WVP may be attributed to the addition of stiff CNC, causing the increase on the permeation route in the water molecules in the g-PBAT polymer matrix.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR < 0.05) miRNAs were identified in the first and 4 in the third trimester, all upregulated in females, including miR-361-5p, significant in both trimesters. Sex-specific analyses across gestation identified 677 differentially expressed (DE) miRNAs at FDR < 0.05 and baseMean>10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In patients with myotonic dystrophy type 1 (DM1), dysregulation of RNA-binding proteins like MBNL and CELF1 leads to alternative splicing of exons and is thought to induce a return to fetal splicing patterns in adult tissues, including the central nervous system (CNS). To comprehensively evaluate this, we created an atlas of developmentally regulated splicing patterns in the frontal cortex of healthy individuals and DM1 patients, by combining RNA-seq data from BrainSpan, GTEx and DM1 patients. Thirty-four splice events displayed an inclusion pattern in DM1 patients that is typical for the fetal situation in healthy individuals. The regulation of DM1-relevant splicing patterns could partly be explained by changes in mRNA expression of the splice regulators MBNL1 , MBNL2 and CELF1 . On the contrary, interindividual differences in splicing patterns between healthy adults could not be explained by differential expression of these splice regulators. Our findings lend transcriptome-wide evidence to the previously noted shift to fetal splicing patterns in the adult DM1 brain as a consequence of an imbalance in antagonistic MBNL and CELF1 activities. Our atlas serves as a solid foundation for further study and understanding of the cognitive phenotype in patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Environmental pollution as a result of heavy metals (HMs) is a worldwide problem and the implementation of eco-friendly remediation technologies is thus required. Metallophores, low molecular weight compounds, could have important biotechnological applications in the fields of agriculture, medicine, and bioremediation. This study aimed to isolate HM-resistant bacteria from soils and sediments of the Lerma-Chapala Basin and evaluated their abilities to produce metallophores and to promote plant growth. Bacteria from the Lerma-Chapala Basin produced metallophores for all the tested metal ions, presented a greater production of As 3+ metallophores, and showed high HM resistance especially to Zn 2+ , As 5+ , and Ni 2+ . A total of 320 bacteria were isolated with 170 strains showing siderophores synthesis. Members of the Delftia and Pseudomonas genera showed above 92 percent siderophore units (psu) during siderophores production and hydroxamate proved to be the most common functional group among the analyzed siderophores. Our results provided evidence that Lerma-Chapala Basin bacteria and their metallophores could potentially be employed in bioremediation processes or may even have potential for applications in other biotechnological fields., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Novel and biodegradable acrylic acid-grafted poly(1,4-butylene adipate-co-terephthalate)/organically modified layered double hydroxide (g-PBAT/m-LDH) nanocomposites were synthesized through the polycondensation and transesterification process, with the covalent linkages between the polymer and the inorganic materials. X-ray diffraction and transmission electron microscopy were used to characterize the structure and morphology of the g-PBAT/m-LDH nanocomposites. The experimental results show that the m-LDH was exfoliated and widely distributed in the g-PBAT matrix. The addition of m-LDH into the g-PBAT extensively improved the storage modulus at -90 °C, when compared to that of the pure g-PBAT matrix. The effects of the minor comonomer of the butylene terephthalate (BT) unit and the addition of m-LDH on the crystallization behavior, and the polymorphic crystals of the g-PBAT at numerous crystallization temperatures, were examined, using a differential scanning calorimeter (DSC). The data indicate that the minor comonomer of the BT unit into g-PBAT can significantly change the starting formation temperatures of the α-form and ꞵ-form crystals, while a change in the starting formation temperatures of the α-form and ꞵ-form crystals using the addition of m-LDH into g-PBAT is not evident.

Chamaecrista mimosoides is an annual herb legume widely distributed in tropical and subtropical Asia and Africa. It may have primitive and independently-evolved root nodule types but its rhizobia have not been systematically studied. Therefore, in order to learn the diversity and species affinity of its rhizobia, root nodules were sampled from C. mimosoides plants growing in seven geographical sites along the coast line of Shandong Peninsula, China. A total of 422 rhizobial isolates were obtained from nodules, and they were classified into 28 recA haplotypes. By using multilocus sequence analysis of the concatenated housekeeping genes dnaK, glnII, gyrB, recA and rpoB, the representative strains for these haplotypes were designated as eight defined and five candidate novel genospecies in the genus Bradyrhizobium. Bradyrhizobium elkanii and Bradyrhizobium ferriligni were predominant and universally distributed. The symbiotic genes nodC and nifH of the representative strains showed very similar topology in their phylogenetic trees indicating their co-evolution history. All the representative strains formed effective root nodules in nodulation tests. The correlation between genospecies and soil characteristics analyzed by CANOCO software indicated that available potassium (AK), organic carbon (OC) and available nitrogen (AN) in the soil samples were the main factors affecting the distribution of the symbionts involved in this current study. The study is the first systematic survey of Chamaecrista mimosoides-nodulating rhizobia, and it showed that Chamaecrista spp. were nodulated by bradyrhizobia in natural environments. In addition, the host spectrum of the corresponding rhizobial species was extended, and the study provided novel information on the biodiversity and biogeography of rhizobia., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Aims: To investigate the effects of three symbiotic Bradyrhizobium strains on peanut growth and on rhizobacterial communities in flowering and harvest stages in an organic farm, also to evaluate the role of plant development in influencing peanut rhizobacterial microbiota and correlations among the inoculants, rhizobacterial communities and plant growth., Methods and Results: Peanut seeds were inoculated with three individual Bradyrhizobium strains, plant growth performance was measured in two developmental stages and rhizobacterial communities were analysed by Illumina sequencing of rpoB gene amplicons from peanut rhizosphere. The three bradyrhizobial inoculants significantly increased the nodule numbers and aboveground fresh weight of peanut plants regardless of the different growth stages, and the pod yields were increased to some extent and significantly positively correlated with Bradyrhizobium abundances in rhizosphere. Principal coordinate analysis indicated that the rhizobacterial communities were strongly influenced by the inoculation and peanut developmental stages. The bradyrhizobia inoculation increased relative abundances of potentially beneficial bacteria in peanut rhizosphere, and also altered rhizobacterial co-occurrence association networks and important network hub taxa. Similarly, plant development also significantly influenced the structure, composition and co-occurrence association networks of rhizobacterial communities., Conclusions: Bradyrhizobial inoculants increased peanut growth and yields, they and plant development affected the assembly of peanut rhizobacterial communities., Significance and Impact of the Study: Rhizobial inoculants improved the host plant performance that might also be associated with the dynamic changes in rhizobacterial community except enhancing the biological nitrogen fixation and helps to profoundly understand the mechanism how rhizobia inoculants improve plant growth and yields., (© 2021 The Society for Applied Microbiology.)