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Vaginal malodour is a sign of dysbiosis. The biogenic amines (BAs) cadaverine, putrescine and tyramine are known to be causative compounds. Recent reports suggest these compounds produced by pathogens might have a role beyond causing malodour; namely inhibiting the growth of lactobacilli bacteria that are crucial in the maintenance of vaginal homeostasis. The aim of this study was to identify whether certain lactobacilli strains could reduce BAs and to evaluate how Lactobacillus species were affected by these compounds. Using LC–MS and HPLC-UV, five Lactobacillus crispatus strains were identified as being capable of significantly reducing BAs from the media under in vitro conditions. Through 16S rRNA gene sequencing of vaginal swabs exposed to Bas, cadaverine was found to reduce the relative abundance of lactobacilli. When L. crispatus was exposed to media supplemented with BAs with an HCl adjusted lower pH, its growth was enhanced, demonstrating the relevance of the maintenance of an acidic vaginal environment. If strains are to be developed for probiotic application to alleviate bacterial vaginosis and other conditions affecting large numbers of women worldwide, their ability to adapt to Bas and regulate pH should be part of the experimentation.

Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of 21OH deficiency.

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2α/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and - independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa &beta, (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis. publishedVersion

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6&prime, modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636&ndash, 655 nm. Among them, the combination of PxRE-R215K mutant with 6&prime, (1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

Using a data driven analysis of a high-content screen, we have uncovered new regulators of epithelial-to-mesenchymal transition (EMT) induced cell migration. Our results suggest that increased expression of miR614 can alter cell intrinsic gene expression to enhance single cell and collective migration in multiple contexts. Interestingly, miR614 specifically increased the expression of the EMT transcription factor Slug while not altering existing epithelial character or inducing other canonical EMT regulatory factors. Analysis of two different cell lines identified a set of genes whose expression is altered by the miR614 through direct and indirect mechanisms. Prioritization driven by functional testing of 25 of the miR614 suppressed genes uncovered the mitochondrial small GTPase Miro1 and the transmembrane protein TAPT1 as miR614 suppressed genes that inhibit migration. Notably, the suppression of either Miro1 or TAPT1 was sufficient to increase Slug expression and the rate of cell migration. Importantly, reduced TAPT1 expression correlated with an increased risk of relapse in breast cancer patients. Together, our results reveal how increased miR614 expression and the suppression of TAPT1 and Miro1 modulate the EMT state and migratory properties of breast cancer cells.

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

Galectins are a family of animal lectins with high affinity for &beta, galactosides. Galectins are able to bind to bacteria, and a few mammalian galectins are known to kill the bound bacteria. In fish, no galectins with direct bactericidal effect have been reported. In the present study, we identified and characterized a tandem repeat galectin-8 from tongue sole Cynoglossus semilaevis (designated CsGal-8). CsGal-8 possesses conserved carbohydrate recognition domains (CRDs), as well as the conserved HXNPR and WGXEE motifs that are critical for carbohydrate binding. CsGal-8 was constitutively expressed in nine tissues of tongue sole and up-regulated in kidney, spleen, and blood by bacterial challenge. When expressed in HeLa cells, CsGal-8 protein was detected both in the cytoplasm and in the micro-vesicles secreted from the cells. Recombinant CsGal-8 (rCsGal-8) bound to lactose and other carbohydrates in a dose dependent manner. rCsGal-8 bound to a wide range of gram-positive and gram-negative bacteria and was co-localized with the bound bacteria in animal cells. Lactose, fructose, galactose, and trehalose effectively blocked the interactions between rCsGal-8 and different bacteria. Furthermore, rCsGal-8 exerted potent bactericidal activity against some gram-negative bacterial pathogens by directly damaging the membrane and structure of the pathogens. Taken together, these results indicate that CsGal-8 likely plays an important role in the immune defense against some bacterial pathogens by direct bacterial interaction and killing.

The early onset and rapid progression of cutaneous squamous cell carcinoma (cSCC) leads to high mortality rates in individuals with recessive dystrophic epidermolysis bullosa (RDEB). Currently, the molecular mechanisms underlying cSCC development in RDEB are not well understood and there are limited therapeutic options. RDEB-cSCC arises through the accumulation of genetic mutations, however, previous work analyzing gene expression profiles have not been able to explain its aggressive nature. Therefore, we generated a model to study RDEB-cSCC development using cellular reprograming and re-differentiation technology. We compared RDEB-cSCC to cSCC that were first reprogrammed into induced pluripotent stem cells (RDEB-cSCC-iPSC) and then differentiated back to keratinocytes (RDEB-cSCC-iKC). The RDEB-cSCC-iKC cell population had reduced proliferative capacities in vitro and in vivo, suggesting that reprogramming and re-differentiation leads to functional changes. Finally, we performed RNA-seq analysis for RDEB-cSCC, RDEB-cSCC-iPSC, and RDEB-cSCC-iKC and identified different gene expression signatures between these cell populations. Taken together, this cell culture model offers a valuable tool to study cSCC and provides a novel way to identify potential therapeutic targets for RDEB-cSCC.

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist beta-alanine, we observed a release of IL-6. beta-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the G alpha q/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-kappa B) and stimulation of IL-6 release. This IL-6 release could be blocked by a G alpha q inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for beta-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

Systemic Acquired Resistance (SAR) improves immunity of plant systemic tissue after local exposure to a pathogen. Guard cells that form stomatal pores on leaf surfaces recognize bacterial pathogens via pattern recognition receptors, such as Flagellin Sensitive 2 (FLS2). However, how SAR affects stomatal immunity is not known. In this study, we aim to reveal molecular mechanisms underlying the guard cell response to SAR using multi-omics of proteins, metabolites and lipids. Arabidopsis plants previously exposed to pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when they are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreased number of bacteria in leaves. Multi-omics has revealed molecular components of SAR response specific to guard cells functions, including potential roles of reactive oxygen species (ROS) and fatty acid signaling. Our results show an increase in palmitic acid and its derivative in the primed guard cells. Palmitic acid may play a role as an activator of FLS2, which initiates stomatal immune response. Improved understanding of how SAR signals affect stomatal immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5&prime, non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135&ndash, 555) and RVFV non-coding RNAs, IGR and 5&rsquo, NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135&ndash, 555, IGR, and 5&rsquo, NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5&rsquo, NCR with a dissociation constant of 5.77 &plusmn, 0.15 &micro, M and 9.85 &plusmn, 0.11 &micro, M, respectively. As DDX17135&ndash, 555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135&ndash, 555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135&ndash, 555 interacting with and unwinding RVFV non-coding regions.

Obesity, a characteristic of metabolic syndrome, is also associated with chronic inflammation and the development of autoimmune diseases. However, the relationship between obesity and autoimmune diseases remains to be investigated in depth. Here, we compared hepatic gene expression profiles among high-fat diet (HFD) mice using the primary biliary cholangitis (PBC) mouse model based on the chronic expression of interferon gamma (IFN&gamma, ) (ARE-Del-/- mice). The top differentially expressed genes affected by upstream transcriptional regulators IFN&gamma, LPS, and TNF&alpha, displayed an overlap in HFD and ARE-Del-/- mice, indicating that obesity-induced liver inflammation may be dependent on signaling via IFN&gamma, The top pathways altered in HFD mice were mostly involved in the innate immune responses, which overlapped with ARE-Del-/- mice. In contrast, T cell-mediated signaling pathways were exclusively altered in ARE-Del-/- mice. We further evaluated the therapeutic effect of luteolin, known as anti-inflammatory flavonoid, in HFD and ARE-Del-/- mice. Luteolin strongly suppressed the MHC I and II antigen presentation pathways, which were highly activated in both HFD and ARE-Del-/- mice. Conversely, luteolin increased metabolic processes of fatty acid oxidation and oxidative phosphorylation in the liver, which were suppressed in ARE-Del-/- mice. Luteolin also strongly induced PPAR signaling, which was downregulated in HFD and ARE-Del-/- mice. Using human GWAS data, we characterized the genetic interaction between significant obesity-related genes and IFN&gamma, signaling and demonstrated that IFN&gamma, is crucial for obesity-mediated inflammatory responses. Collectively, this study improves our mechanistic understanding of the relationship between obesity and autoimmune diseases. Furthermore, it provides new methodological insights into how immune network-based analyses effectively integrate RNA-seq and microarray data.

This article belongs to the Special Issue Crop Genome Editing., The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family., This research was funded by the Spanish Ministry of Science and Innovation (Project PID2019-110847RB-I00), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía (Project P20_01005), and the European Regional Development Fund (FEDER). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

IgA nephropathy (IgAN) has been considered to have a relationship with infection in the tonsil, because IgAN patients often manifest macro hematuria just after tonsillitis. In terms of oral-area infection, the red complex of periodontal bacteria (Porphyromonas gingivalis (P. gingivalis), Treponema denticol (T. denticola) and Tannerella forsythia (T. forsythia)) is important, but the relationship between these bacteria and IgAN remains unknown. In this study, the prevalence of the red complex of periodontal bacteria in tonsil was compared between IgAN and tonsillitis patients. The pathogenicity of IgAN induced by P. gingivalis was confirmed by the mice model treated with this bacterium. The prevalence of P. gingivalis and T. forsythia in IgAN patients was significantly higher than that in tonsillitis patients (p < 0.001 and p < 0.05, respectively). A total of 92% of tonsillitis patients were free from red complex bacteria, while only 48% of IgAN patients had any of these bacteria. Nasal administration of P. gingivalis in mice caused mesangial proliferation (p < 0.05 at days 28a nd 42; p < 0.01 at days 14 and 56) and IgA deposition (p < 0.001 at day 42 and 56 after administration). Scanning-electron-microscopic observation revealed that a high-density Electron-Dense Deposit was widely distributed in the mesangial region in the mice kidneys treated with P. gingivalis. These findings suggest that P. gingivalis is involved in the pathogenesis of IgAN.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower &Delta, F/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4&ndash, 6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a &Delta, F/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar &Delta, F/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower &Delta, F/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a &Delta, F/F dynamic range of 9.4 and a contrast of 2.3&ndash, 3.5, respectively.

The iron-sulfur subunit (SDH2) of succinate dehydrogenase plays a key role in electron transport in plant mitochondria. However, it is yet unknown whether SDH2 genes are involved in leaf senescence and yield formation. In this study, we isolated a late premature senescence mutant, lps1, in rice (Oryza sativa). The mutant leaves exhibited brown spots at late tillering stage and wilted at the late grain-filling stage and mature stage. In its premature senescence leaves, photosynthetic pigment contents and net photosynthetic rate were reduced, chloroplasts and mitochondria were degraded. Meanwhile, lps1 displayed small panicles, low seed-setting rate and dramatically reduced grain yield. Gene cloning and complementation analysis suggested that the causal gene for the mutant phenotype was OsSDH2-1 (LOC_Os08g02640), in which single nucleotide mutation resulted in an amino acid substitution in the encoded protein. OsSDH2-1 gene was expressed in all organs tested, with higher expression in leaves, root tips, ovary and anthers. OsSDH2-1 protein was targeted to mitochondria. Furthermore, reactive oxygen species (ROS), mainly H2O2, was excessively accumulated in leaves and young panicles of lps1, which could cause premature leaf senescence and affect panicle development and pollen function. Taken together, OsSDH2-1 plays a crucial role in leaf senescence and yield formation in rice.

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed systematic analysis of CSE gene family in poplar and investigated the possible roles of CSEs and CSE-like genes in Populus. We performed a genome-wide analysis of the CSE family, including functional and phylogenetic analyses of CSE and CSE-like genes using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12 might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of Populus tomentosa (Popto) CSE1, -2 and -12 showed that PoptoCSE1 and -2 kept CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression pattern and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides systematic understanding of this novel gene family and suggests the CSE function in monolignol biosynthesis in Populus.

Synovial mesenchymal stem cell (SMSC) is the promising cell source of cartilage regeneration but has several issues to overcome such as limited cell proliferation and heterogeneity of cartilage regeneration ability. Previous reports demonstrated that basic fibroblast growth factor (bFGF) can promote proliferation and cartilage differentiation potential of MSCs in vitro, although no reports show its beneficial effect in vivo. The purpose of this study is to investigate the promoting effect of bFGF on cartilage regeneration using human SMSC in vivo. SMSCs were cultured with or without bFGF in a growth medium, and 2 &times, 105 cells were aggregated to form a synovial pellet. Synovial pellets were implanted into osteochondral defects induced in the femoral trochlea of severe combined immunodeficient mice, and histological evaluation was performed after eight weeks. The presence of implanted SMSCs was confirmed by the observation of human vimentin immunostaining-positive cells. Interestingly, broad lacunae structures and cartilage substrate stained by Safranin-O were observed only in the bFGF (+) group. The bFGF (+) group had significantly higher O&rsquo, Driscoll scores in the cartilage repair than the bFGF (&minus, ) group. The addition of bFGF to SMSC growth culture may be a useful treatment option to promote cartilage regeneration in vivo.

In recent years, it has been realized that the tau protein is a key player in multiple neurodegenerative diseases. Positron emission tomography (PET) radiotracers that bind to tau filaments in Alzheimer&rsquo, s disease (AD) are in common use, but PET tracers binding to tau filaments of rarer, age-related dementias, such as Pick&rsquo, s disease, have not been widely explored. To design disease-specific and tau-selective PET tracers, it is important to determine where and how PET tracers bind to tau filaments. In this paper, we present the first molecular modelling study on PET probe binding to the structured core of tau filaments from a patient with Pick&rsquo, s disease (TauPiD). We have used docking, molecular dynamics simulations, binding-affinity and tunnel calculations to explore TauPiD binding sites, binding modes, and binding energies of PET probes (AV-1451, MK-6240, PBB3, PM-PBB3, THK-5351 and PiB) with TauPiD. The probes bind to TauPiD at multiple surface binding sites as well as in a cavity binding site. The probes show unique surface binding patterns, and, out of them all, PM-PBB3 proves to bind the strongest. The findings suggest that our computational workflow of structural and dynamic details of the tau filaments has potential for the rational design of TauPiD specific PET tracers.

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human–Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding protein-5 (IGFBP-5) is a conserved member of the IGFBP family of proteins that is overexpressed in SSc and IPF lung tissues. In this study, we investigated the functional role of IGFBP-5 in the development of fibrosis in vivo using a transgenic model. We generated transgenic mice ubiquitously expressing human IGFBP-5 using CRISPR/Cas9 knock-in. Our data show that the heterozygous and homozygous mice are viable and express human IGFBP-5 (hIGFBP-5). Transgenic mice had increased expression of extracellular matrix (ECM) genes, especially Col3a1, Fn, and Lox in lung and skin tissues of mice expressing higher transgene levels. Histologic analysis of the skin tissues showed increased dermal thickness, and the lung histology showed subtle changes in the heterozygous and homozygous mice as compared with the wild-type mice. These changes were more pronounced in animals expressing higher levels of hIGFBP-5. Bleomycin increased ECM gene expression in wild-type mice and accentuated an increase in ECM gene expression in transgenic mice, suggesting that transgene expression exacerbated bleomycin-induced pulmonary fibrosis. Primary lung fibroblasts cultured from lung tissues of homozygous transgenic mice showed significant increases in ECM gene expression and protein levels, further supporting the observation that IGFBP-5 resulted in a fibrotic phenotype in fibroblasts. In summary, transgenic mice expressing human IGFBP-5 could serve as a useful animal model for examining the function of IGFBP-5 in vivo.

Pseudomonas putida&rsquo, s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs&rsquo, metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22&ndash, 35 years and 74&ndash, 94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.

The gastrointestinal tract (GIT) plays a key role in regulating nutrient metabolism and appetite responses. This study aimed to identify changes in the GIT that are important in the development of diet related obesity and diabetes. GIT samples were obtained from C57BL/6J male mice chronically fed a control diet or a high sucrose diet (HSD) and analysed for changes in gene, protein and metabolite levels. In HSD mice, GIT expression levels of fat oxidation genes were reduced, and increased de novo lipogenesis was evident in ileum. Gene expression levels of the putative sugar sensor, slc5a4a and slc5a4b, and fat sensor, cd36, were downregulated in the small intestines of HSD mice. In HSD mice, there was also evidence of bacterial overgrowth and a lipopolysaccharide activated inflammatory pathway involving inducible nitric oxide synthase (iNOS). In Caco-2 cells, sucrose significantly increased the expression levels of the nos2, iNOS and nitric oxide (NO) gas levels. In conclusion, sucrose fed induced obesity/diabetes is associated with changes in GI macronutrient sensing, appetite regulation and nutrient metabolism and intestinal microflora. These may be important drivers, and thus therapeutic targets, of diet-related metabolic disease.

Despite advances in the diagnosis and treatment of the central nervous system malignancy glioma, overall survival remains poor. Cytoskeleton-associated protein 2-like (CKAP2L), which plays key roles in neural progenitor cell division, has also been linked to poor prognosis in lung cancer. In the present study, we investigated the role of CKAP2L in glioma. From bioinformatics analyses of datasets from The Cancer Gene Atlas and the Chinese Glioma Genome Atlas, we found that CKAP2L expression correlates with tumor grade and overall survival. Gene set enrichment analysis (GSEA) showed that MITOTIC_SPINDLE, G2M_CHECKPOINT, and E2F_TARGETS are crucially enriched phenotypes associated with high CKAP2L expression. Using U87MG, U118MG, and LNZ308 human glioma cells, we confirmed that CKAP2L knockdown with siCKAP2L inhibits glioma cell proliferation, migration, invasion, and epithelial-mesenchymal transition. Interestingly, CKAP2L knockdown also induced cell cycle arrest at G2/M phase, which is consistent with the GSEA finding. Finally, we observed that CKAP2L knockdown led to significant increases in miR-4496. Treating cells with exogenous miR-4496 mimicked the effect of CKAP2L knockdown, and the effects of CKAP2L knockdown could be suppressed by miR-4496 inhibition. These findings suggest that CKAP2L is a vital regulator of miR-4496 activity and that CKAP2L is a potentially useful prognostic marker in glioma.

There is increasing recognition that environmental nano-biological interactions in model species, and the resulting effects on progeny, are of paramount importance for nanomaterial (NM) risk assessment. In this work, Daphnia magna F0 mothers were exposed to a range of silver and titanium dioxide NMs. The key biological life history traits (survival, growth and reproduction) of the F1 intergenerations, at the first (F1B1), third (F1B3) and fifth (F1B5) broods, were investigated. Furthermore, the F1 germlines of each of the three broods were investigated over 3 more generations (up to 25 days each) in continuous or removed-from NM exposure, to identify how the length of maternal exposure affects the resulting clonal broods. Our results show how daphnids respond to NM-induced stress, and how the maternal effects show trade-offs between growth, reproduction and survivorship. The F1B1 (and following germline) had the shortest F0 maternal exposure times to the NMs, and thus were the most sensitive showing reduced size and reproductive output. The F1B3 generation had a sub-chronic maternal exposure, whereas the F1B5 generation suffered chronic maternal exposure where (in most cases) the most compensatory adaptive effects were displayed in response to the prolonged NM exposure, including enhanced neonate output and reduced gene expression. Transgenerational responses of multiple germlines showed a direct link with maternal exposure time to &lsquo, sub-lethal&rsquo, effect concentrations of NMs (identified from standard OECDs acute toxicity tests which chronically presented as lethal) including increased survival and production of males in the F1B3 and G1B5 germlines. This information may help to fine-tune environmental risk assessments of NMs and prediction of their impacts on environmental ecology.

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.

Rapeseed (Brassica napus) is one of the major important oil crops worldwide and is largely cultivated in the Qinghai-Tibetan plateau (QTP), where long and strong solar-radiation is well-known. However, the molecular mechanisms underlying rapeseed’s response to light stress are largely unknown. In the present study, the color of rapeseed seedlings changed from green to purple under high light (HL) stress conditions. Therefore, changes in anthocyanin metabolism and the transcriptome of rapeseed seedlings cultured under normal light (NL) and HL conditions were analyzed to dissect how rapeseed responds to HL at the molecular level. Results indicated that the contents of anthocyanins, especially glucosides of cyanidin, delphinidin, and petunidin, which were determined by liquid chromatography-mass spectrometry (LC-MS), increased by 9.6-, 4.2-, and 59.7-fold in rapeseed seedlings exposed to HL conditions, respectively. Next, RNA-sequencing analysis identified 7390 differentially expressed genes (DEGs), which included 4393 up-regulated and 2997 down-regulated genes. Among the up-regulated genes, many genes related to the anthocyanin-biosynthetic pathway were enriched. For example, genes encoding dihydroflavonol reductase (BnDFR) and anthocyanin synthase (BnANS) were especially induced by HL conditions, which was also confirmed by RT-qPCR analysis. In addition, two PRODUCTION OF ANTHOCYANIN PIGMENTATION 2 (BnPAP2) and GLABRA3 (BnGL3) genes encoding MYB-type and bHLH-type transcription factors, respectively, whose expression was also up-regulated by HL stress, were found to be associated with the changes in anthocyanin biosynthesis. Many genes involved in the jasmonic acid (JA)-biosynthetic pathway were also up-regulated under HL conditions. This finding, which is in agreement with the well-known positive regulatory role of JA in anthocyanin biosynthesis, suggests that the JA may also play a key role in the responses of rapeseed seedlings to HL. Collectively, these data indicate that anthocyanin biosynthesis-related and JA biosynthesis-related pathways mediate HL responses in rapeseed. These findings collectively provide mechanistic insights into the mechanisms involved in the response of rapeseed to HL stress, and the identified key genes may potentially be used to improve HL tolerance of rapeseed cultivars through genetic engineering or breeding strategies.

Fetal undernutrition programs cardiometabolic diseases, with higher susceptibility in males. The mechanisms implicated are not fully understood and may be related to sex differences in placental adaptation. To evaluate this hypothesis, we investigated placental oxidative balance, vascularization, glucocorticoid barrier, and fetal growth in rats exposed to 50% global nutrient restriction from gestation day 11 (MUN, n = 8) and controls (n = 8). At gestation day 20 (G20), we analyzed maternal, placental, and fetal weights, oxidative damage, antioxidants, corticosterone, and PlGF (placental growth factor, spectrophotometry), and VEGF (vascular endothelial growth factor), 11&beta, HSD2, p22phox, XO, SOD1, SOD2, SOD3, catalase, and UCP2 expression (Western blot). Compared with controls, MUN dams exhibited lower weight and plasma proteins and higher corticosterone and catalase without oxidative damage. Control male fetuses were larger than female fetuses. MUN males had higher plasma corticosterone and were smaller than control males, but had similar weight than MUN females. MUN male placenta showed higher XO and lower 11&beta, HSD2, VEGF, SOD2, catalase, UCP2, and feto-placental ratio than controls. MUN females had similar feto-placental ratio and plasma corticosterone than controls. Female placenta expressed lower XO, 11&beta, HSD2, and SOD3, similar VEGF, SOD1, SOD2, and UCP2, and higher catalase than controls, being 11&beta, HSD2 and VEGF higher compared to MUN males. Male placenta has worse adaptation to undernutrition with lower efficiency, associated with oxidative disbalance and reduced vascularization and glucocorticoid barrier. Glucocorticoids and low nutrients may both contribute to programming in MUN males.

The Hsd11b2 enzyme converts cortisol into its inactive form, cortisone and regulates cortisol levels, in particular in response to stress. Taking advantage of CRISPR/Cas9 technology, we generated a hsd11b2 zebrafish mutant line to evaluate the involvement of this gene in stress response regulation. The absence of a functional Hsd11b2 affects survival of zebrafish, although homozygous hsd11b2−/− mutants can reach adulthood. Reproductive capability of hsd11b2−/− homozygous adult males is almost completely abrogated, while that of females is reduced. Interestingly, basal cortisol levels and glucocorticoid-dependent transcriptional activities are not affected by the mutation. In agreement with basal cortisol results, we also demonstrated that basal response to light (LMR-L/D) or mechanical (VSRA) stimuli is not significantly different in wild-type (hsd11b2+/+) compared to mutant larvae. However, after exposure to an acute stressor, the cortisol temporal patterns of synthesis and release are prolonged in both 5 days post fertilization larvae and one-year-old adult hsd11b2−/− zebrafish compared to wild-type siblings, showing at the same time, at 5 dpf, a higher magnitude in the stress response at 10 min post stress. All in all, this new zebrafish model represents a good tool for studying response to different stressors and to identify mechanisms that are induced by cortisol during stress response.

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T1 and T2 progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.

The LOR (LURP-one related) family genes encode proteins containing a conserved LOR domain. Several members of the LOR family genes are required for defense against Hyaloperonospora parasitica (Hpa) in Arabidopsis. However, there are few reports of LOR genes in response to abiotic stresses in plants. In this study, a genome-wide survey and expression levels in response to abiotic stresses of 36 LOR genes from Glycine max were conducted. The results indicated that the GmLOR gene family was divided into eight subgroups, distributed on 14 chromosomes. A majority of members contained three extremely conservative motifs. There were four pairs of tandem duplicated GmLORs and nineteen pairs of segmental duplicated genes identified, which led to the expansion of the number of GmLOR genes. The expansion patterns of the GmLOR family were mainly segmental duplication. A heatmap of soybean LOR family genes showed that 36 GmLOR genes exhibited various expression patterns in different tissues. The cis-acting elements in promoter regions of GmLORs include abiotic stress-responsive elements, such as dehydration-responsive elements and drought-inducible elements. Real-time quantitative PCR was used to detect the expression level of GmLOR genes, and most of them were expressed in the leaf or root except that GmLOR6 was induced by osmotic and salt stresses. Moreover, GmLOR4/10/14/19 were significantly upregulated after PEG and salt treatments, indicating important roles in the improvement of plant tolerance to abiotic stress. Overall, our study provides a foundation for future investigations of GmLOR gene functions in soybean.

Sporadic occurrence of inherited eye disorders has been reported in cattle but so far pathogenic variants were found only for rare forms of cataract but not for retinopathies. The aim of this study was to characterize the phenotype and the genetic aetiology of a recessive form of congenital day-blindness observed in several cases of purebred Original Braunvieh cattle. Electroretinography in an affected calf revealed absent cone-mediated function, whereas the rods continue to function normally. Brain areas involved in vision were morphologically normal. When targeting cones by immunofluorescence, a decrease in cone number and an accumulation of beta subunits of cone cyclic-nucleotide gated channel (CNGB3) in the outer plexiform layer of affected animals was obvious. Achromatopsia is a monogenic Mendelian disease characterized by the loss of cone photoreceptor function resulting in day-blindness, total color-blindness, and decreased central visual acuity. After SNP genotyping and subsequent homozygosity mapping with twelve affected cattle, we performed whole-genome sequencing and variant calling of three cases. We identified a single missense variant in the bovine CNGB3 gene situated in a ~2.5 Mb homozygous genome region on chromosome 14 shared between all cases. All affected cattle were homozygous carriers of the p.Asp251Asn mutation that was predicted to be deleterious, affecting an evolutionary conserved residue. In conclusion, we have evidence for the occurrence of a breed-specific novel CNGB3-related form of recessively inherited achromatopsia in Original Braunvieh cattle which we have designated OH1 showing an allele frequency of the deleterious allele of ~8%. The identification of carriers will enable selection against this inherited disorder. The studied cattle might serve as an animal model to further elucidate the function of CNGB3 in mammals.

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.

Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID50/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values &lt, 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

To investigate the antiviral effect of lithium chloride (LiCl) on the replication of Marek’s disease virus (MDV) in chicken embryonic fibroblast (CEF) cells, real-time PCR, Western blotting, plaque counting, and indirect immunofluorescence experiments were performed at different time points of LiCl treated CEF cells with virus infection. The results demonstrated that LiCl could affect multiple steps of virus replication and inhibit viral gene expression and protein synthesis in a dose- and time-dependent manner. Moreover, LiCl could directly affect viral infectivity as well. In addition, LiCl significantly affected the gene expression of IFN-β related genes in virus-infected cells. These results indicate that LiCl significantly inhibits MDV replication and proliferation in CEF cells and it has the potential to be used as an antiviral agent against MDV.

We aim to compare the relative heritability contributed by variants of behavior-related environmental phenotypes and elucidate the role of these factors in the conundrum of “missing heritability” of type 2 diabetes. Methods: We used Linkage-Disequilibrium Adjusted Kinships (LDAK) and LDAK-Thin models to calculate the relative heritability of each variant and compare the relative heritability for each phenotype. Biological analysis was carried out for the phenotype whose variants made a significant contribution. Potential hub genes were prioritized based on topological parameters of the protein-protein interaction network. We included 16 behavior-related phenotypes and 2607 valid variants. In the LDAK model, we found the variants of alcohol consumption and caffeine intake were identified as contributing higher relative heritability than that of the random variants. Compared with the relative expected heritability contributed by the variants associated with type 2 diabetes, the relative expected heritability contributed by the variants associated with these two phenotypes was higher. In the LDAK-Thin model, the relative heritability of variants of 11 phenotypes was statistically higher than random variants. Biological function analysis showed the same distributions among type 2 diabetes and alcohol consumption. We eventually screened out 31 hub genes interacting intensively, four of which were validated and showed the upregulated expression pattern in blood samples seen in type 2 diabetes cases. Conclusion: We found that alcohol consumption contributed higher relative heritability. Hub genes may influence the onset of type 2 diabetes by a mediating effect or a pleiotropic effect. Our results provide new insight to reveal the role of behavior-related factors in the conundrum of “missing heritability” of type 2 diabetes.

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Gibberellins (GAs) are diterpenoid phytohormones regulating various aspects of plant growth and development, such as internode elongation and seed germination. Although the GA biosynthesis pathways have been identified, the transcriptional regulatory network of GA homeostasis still remains elusive. Here, we report the functional characterization of a GA-inducible OsABF1 in GA biosynthesis underpinning plant height and seed germination. Overexpression of OsABF1 produced a typical GA-deficient phenotype with semi-dwarf and retarded seed germination. Meanwhile, the phenotypes could be rescued by exogenous GA3, suggesting that OsABF1 is a key regulator of GA homeostasis. OsABF1 could directly suppress the transcription of green revolution gene SD1, thus reducing the endogenous GA level in rice. Moreover, OsABF1 interacts with and transcriptionally antagonizes to the polycomb repression complex component OsEMF2b, whose mutant showed as similar but more severe phenotype to OsABF1 overexpression lines. It is suggested that OsABF1 recruits RRC2-mediated H3K27me3 deposition on the SD1 promoter, thus epigenetically silencing SD1 to maintain the GA homeostasis for growth and seed germination. These findings shed new insight into the functions of OsABF1 and regulatory mechanism underlying GA homeostasis in rice.

Salicylic acid (SA) and jasmonic acid (JA) are essential plant immune hormones, which could induce plant resistance to multiple pathogens. However, whether common components are employed by both SA and JA to induce defense is largely unknown. In this study, we found that the enhanced disease susceptibility 8 (EDS8) mutant was compromised in plant defenses to hemibiotrophic pathogen Pseudomonas syringae pv. maculicola ES4326 and necrotrophic pathogen Botrytis cinerea, and was deficient in plant responses to both SA and JA. The EDS8 was identified to be THO1, which encodes a subunit of the THO/TREX complex, by using mapping-by-sequencing. To check whether the EDS8 itself or the THO/TREX complex mediates SA and JA signaling, the mutant of another subunit of the THO/TREX complex, THO3, was tested. THO3 mutation reduced both SA and JA induced defenses, indicating that the THO/TREX complex is critical for plant responses to these two hormones. We further proved that the THO/TREX interacting protein SERRATE, a factor regulating alternative splicing (AS), was involved in plant responses to SA and JA. Thus, the AS events in the eds8 mutant after SA or JA treatment were determined, and we found that the SA and JA induced different alternative splicing events were majorly modulated by EDS8. In summary, our study proves that the THO/TREX complex active in AS is involved in both SA and JA induced plant defenses.

It is established that high rates of morbidity and mortality caused by fungal infections are related to the current limited number of antifungal drugs and the toxicity of these agents. Imidazolium salts as azole derivatives can be successfully used in the treatment of fungal infections in humans. Steroid-functionalized imidazolium salts were synthesized using a new, more efficient method. As a result, 20 salts were obtained with high yields, 12 of which were synthesized and characterized for the first time. They were derivatives of lithocholic acid and 3-oxo-23,24-dinorchol-4-ene-22-al and were fully characterized by 1H and 13C nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and high resolution mass spectrometry (HRMS). Due to the excellent activity against bacteria and Candida albicans, new research was extended to include tests on five species of pathogenic fungi and molds: Aspergillus niger ATCC 16888, Aspergillus fumigatus ATCC 204305, Trichophyton mentagrophytes ATCC 9533, Cryptococcus neoformans ATCC 14116, and Microsporum canis ATCC 11621. The results showed that the new salts are almost universal antifungal agents and have a broad spectrum of activity against other human pathogens. To initially assess the safety of the synthesized salts, hemocompatibility with host cells and cytotoxicity were also examined. No toxicity was observed at the concentration at which the compounds were active against pathogens.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa.&nbsp, P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice.However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Chemoattraction is a normal and essential process, but it can also be involved in tumorigenesis. This phenomenon plays a key role in glioblastoma (GBM). The GBM tumor cells are extremely difficult to eradicate, due to their strong capacity to migrate into the brain parenchyma. Consequently, a complete resection of the tumor is rarely a possibility, and recurrence is inevitable. To overcome this problem, we proposed to exploit this behavior by using three chemoattractants: CXCL10, CCL2 and CCL11, released by a biodegradable hydrogel (GlioGel) to produce a migration of tumor cells toward a therapeutic trap. To investigate this hypothesis, the agarose drop assay was used to test the chemoattraction capacity of these three chemokines on murine F98 and human U87MG cell lines. We then studied the potency of this approach in vivo in the well-established syngeneic F98-Fischer glioma-bearing rat model using GlioGel containing different mixtures of the chemoattractants. In vitro assays resulted in an invasive cell rate 2-fold higher when chemokines were present in the environment. In vivo experiments demonstrated the capacity of these specific chemoattractants to strongly attract neoplastic glioblastoma cells. The use of this strong locomotion ability to our end is a promising avenue in the establishment of a new therapeutic approach in the treatment of primary brain tumors.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p &lt, 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.