Potential Binding Sites Research Articles

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

Cardiomyocyte sarcolemma modelling and lipid-protein interactions.

The cardiomyocyte sarcolemma is a crucial cellular organelle in heart function. The vital components of the sarcolemma are membrane proteins that generate and control the cardiac action potential, responsible for cardiomyocyte contractibility and heart rhythm. Their malfunction can be caused by congenital mutations and side effects of antiarrhythmic therapies and can lead to life-threatening conditions related to heart failure. Other main components are represented by several lipid types, which not only provide the matrix for membrane proteins but also play active roles in regulating protein function. Shifts in membrane physiology through changes in the lipidomic profile or lipid-protein interactions can also cause cardiac disorders. Our goal is to characterize specific lipid-protein interactions in the sarcolemma by studying lipid redistribution around proteins and identifying lipid binding sites that can potentially regulate protein function. We combined lipidomic, cryo-electron microscopy and X-ray crystallography studies to build computational models of the cardiomyocyte sarcolemma. The membrane models consist of 16 lipid species, which differ in headgroup type, levels of acyl tail unsaturation and length. We embedded a selected membrane protein (cardiac ion channel, transporter or receptor) in a lipid bilayer and performed molecular dynamics simulations with the Martini coarse-grained models. We simulated each system with a corresponding membrane protein for 120 μs and analyzed lipid-protein interactions in terms of lipid redistribution around the proteins via computing lipid depletion-enrichment index, lipid density, and predicting lipid binding sites.Our results reproduce experimental findings of the interactions between lipids and the Kir2.2 potassium channel. We propose potential lipid binding sites for other proteins, highlighting similarities and differences in lipid redistribution and interaction with the cardiomyocyte proteins. Such findings unveil new possible lipid-protein interactions in the cardiomyocyte sarcolemma and provide an understanding of their relevance in heart function.

Characterization of binding interaction between S100A1 and titin's UN2A region.

Titin, the giant sarcomere protein, consists of multiple domains and regions of repeated structural elements that are essential for sarcomeric integrity of the myofibril. The N2A region within titin has been identified as a signaling hub in skeletal muscle that is responsible for various mechanics and interactions in the muscle. The N2A region is located at the junction between the proximal tandem Ig region and the PEVK region and is comprised of four Ig domains and a unique 117 amino acid insertion sequence (UN2A). The insertion sequence resides between the Ig80 and Ig81 domains, and exists in skeletal muscle, as well as the N2AB isoform of cardiac muscle. An analysis of the insertion region sequence revealed a potential binding site for the Ca2+-binding protein S100A1. A combination of circular dichroism and FRET experiments demonstrated the conformational change in UN2A that is induced by S100A1, and can be affected differently due to pH changes. Surface plasmon resonance was used to study the binding kinetics and affinities of this interaction at different pH and pCa values. S100A1's binding to UN2A is calcium-dependent, and the strength of this interaction is affected by pH. The S100A1/UN2A binding interaction has been shown to perturb the structure of UN2A potentially at its stable helical core. The results will provide insight on the nature of this interaction and how it may have a functional role in the muscle.

Oscillatory effects of ketamine using focused ultrasound sensitive liposomes

The blood brain barrier (BBB) poses a significant challenge in the treatment of neurological disorders by preventing the diffusion of medications into the brain. Focused ultrasound (FUS) enables noninvasive modulation of the central nervous system by targeting millimeter-sized regions of the cortical and sub-cortical brain. We have synthesized ultrasound-sensitive nanoparticles (NPs) to encapsulate small, hydrophobic neuromodulators. Recent data has shown that our NPs deliver pharmacological drugs across the BBB when paired with FUS to the desired region of the brain with targeted accuracy. This method enables us to study the impact of small hydrophobic anesthetics across different regions of the brain. The aim of the study is to determine the electrophysiological effects of FUS neuromodulation in an awake small animal model. Specifically, we aim to understand the oscillatory effects of localized ketamine activity on the thalamocortical system. We hypothesized that localized delivery of ketamine in the central thalamus (CL) will induce slow-delta and gamma oscillations comparable to the recorded frequencies obtained from systemic delivery of ketamine. Using in-house electrocorticogram (ECoG) hardware, we recorded signals from the prefrontal cortex, CL, and visual cortex in awake-restraint rats. Preliminary results suggest that FUS-only neuromodulation of the brain induced undetectable oscillatory differences from baseline signals. We then investigated the electrophysiologic signatures of localized ketamine activity. Ketamine-loaded NPs were administered intravenously to rats via bolus injection and FUS was applied to the desired brain region for site-specific drug uncaging. Initial evidence indicated localized delivery of ketamine induced similar slow-delta and gamma oscillations in a smaller timeframe than those recorded after systemic ketamine administration. These results provide insight into the impacts of pharmacological modulation in relation to brain activity. Future directions include analyzing the thalamocortical system as a potential binding site for ketamine to induce anesthetic-specific oscillations.

Predicting plant-specific small molecule binding variability across species.

Agricultural plants must resist myriad stresses such as heat, disease, drought, and herbivory. Plant biochemists are highly incentivized to discover chemical methods of improvement that will mitigate these stresses. Finding novel methods of improving plant function and resistance is difficult and time consuming, however, by taking advantage of millions of years of evolutionary adaptation, we may be able to accelerate research. Here, we use Alphafold, NAMD, Deepsurf, and Autodock Vina, to construct a computational pipeline that that uses amino acid sequences and ligand structures to rapidly screen large panels of small compounds such as isoprene, ethylene, and auxin, for potential binding sites as well as binding affinity on plant proteins. From these binding affinities, we can determine likely targets of small compounds, and whether compounds currently in heavy usage may have off-target effects that have not previously been investigated.

The structural features of the ligand-free moaA riboswitch and its ion-dependent folding

Riboswitches are structural elements of mRNA involved in the regulation of gene expression by responding to specific cellular metabolites. To fulfil their regulatory function, riboswitches prefold into an active state, the so-called binding competent form, that guarantees metabolite binding and allows a consecutive refolding of the RNA. Here, we describe the folding pathway to the binding competent form as well as the ligand free structure of the moaA riboswitch of E. coli. This RNA proposedly responds to the molybdenum cofactor (Moco), a highly oxygen-sensitive metabolite, essential in the carbon and sulfur cycles of eukaryotes. K+- and Mg2+-dependent footprinting assays and spectroscopic investigations show a high degree of structure formation of this RNA already at very low ion-concentrations. Mg2+ facilitates additionally a general compaction of the riboswitch towards its proposed active structure. We show that this fold agrees with the earlier suggested secondary structure which included also a long-range tetraloop/tetraloop-receptor like interaction. Metal ion cleavage assays revealed specific Mg2+-binding pockets within the moaA riboswitch. These Mg2+ binding pockets are good indicators for the potential Moco binding site, since in riboswitches, Mg2+ was shown to be necessary to bind phosphate-carrying metabolites. The importance of the phosphate and of other functional groups of Moco is highlighted by binding assays with tetrahydrobiopterin, the reduced and oxygen-sensitive core moiety of Moco. We demonstrate that the general molecular shape of pterin by its own is insufficient for the recognition by the riboswitch.

Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Molecular dynamics simulations suggest Thiosemicarbazones can bind p53 cancer mutant R175H

Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC – p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi‑sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.

Benchmark single-step ethylene purification from ternary mixtures by a customized fluorinated anion-embedded MOF

Ethylene (C2H4) purification from multi-component mixtures by physical adsorption is a great challenge in the chemical industry. Herein, we report a GeF62- anion embedded MOF (ZNU-6) with customized pore structure and pore chemistry for benchmark one-step C2H4 recovery from C2H2 and CO2. ZNU-6 exhibits significantly high C2H2 (1.53 mmol/g) and CO2 (1.46 mmol/g) capacity at 0.01 bar. Record high C2H4 productivity is achieved from C2H2/CO2/C2H4 mixtures in a single adsorption process under various conditions. The separation performance is retained over multiple cycles and under humid conditions. The potential gas binding sites are investigated by density functional theory (DFT) calculations, which suggest that C2H2 and CO2 are preferably adsorbed in the interlaced narrow channel with high aff0inity. In-situ single crystal structures with the dose of C2H2, CO2 or C2H4 further reveal the realistic host-guest interactions. Notably, rare C2H2 clusters are formed in the narrow channel while two distinct CO2 adsorption locations are observed in the narrow channel and the large cavity with a ratio of 1:2, which accurately account for the distinct adsorption heat curves.

Artemisia argyi essential oil exerts herbicidal activity by inhibiting photosynthesis and causing oxidative damage

Aromatic plant extracts, especially essential oils (EOs), can be used as alternatives to synthetic herbicides due to their effectiveness and easy degradation in environment. Rich in essential oils, Artemisia argyi is a plant that is known for both its medicinal and allelopathic properties. In this study, the chemical composition of A. argyi essential oil (AEO) was examined. Gas chromatographymass spectrometry (GCMS) revealed that relatively high amounts of monoterpenes, including eucalyptol (26.08%), thujone (10.84%), β-caryophyllene (7.46%), borneol (5.09%), and camphor (4.03%), were detected in AEO. Furthermore, AEO was found to inhibit seed germination of Echinochloa crus-galli, Setaria viridis, Portulaca oleracea, and Amaranthus retroflexus as well as kill seedlings of these plants in petri dish experiments. The IC50 values of E. crus-galli, S. viridis, P. oleracea and A. retroflexus were 5.51, 1.02, 25.41, and 0.61 μL/dish, respectively. We demonstrated that A. argyi essential oil aqueous emulsion (AEOAE) exhibited broad-spectrum herbicidal activity and significant short-term contact killing effects on weed plants in both pot and field experiments. S. viridis was taken as a model weed and various physiological indicators were assessed. The results showed that AEOAE induced excessive ROS production in the tested samples, producing an imbalance of the antioxidant system and damage to the integrity of cell membranes, which manifests as increased MDA content and electrolyte leakage. Molecular docking experiments indicated all of above components as potential binding sites with 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzymes, which regulate the biosynthesis of plant tocopherols and plastid quinones to ultimately generate carotenoids. Additionally, AEOAE treatment led to the decrease of HPPD, carotenoid and chlorophyll content in target weeds, revealing that AEOAE may act as an HPPD inhibitor to impede photosynthesis in weed species. Hence, AEO has the potential to be utilized as a source for bio-herbicides that could be adapted for industrial-level production in the future, but its long-term effects should be studied further.

Evolutionary Landscape of Tea Circular RNAs and Its Contribution to Chilling Tolerance of Tea Plant.

Chilling stress threatens the yield and distribution pattern of global crops, including the tea plant (Camellia sinensis), one of the most important cash crops around the world. Circular RNA (circRNA) plays roles in regulating plant growth and biotic/abiotic stress responses. Understanding the evolutionary characteristics of circRNA and its feedbacks to chilling stress in the tea plant will help to elucidate the vital roles of circRNAs. In the current report, we systematically identified 2702 high-confidence circRNAs under chilling stress in the tea plant, and interestingly found that the generation of tea plant circRNAs was associated with the length of their flanking introns. Repetitive sequences annotation and DNA methylation analysis revealed that the longer flanking introns of circRNAs present more repetitive sequences and higher methylation levels, which suggested that repeat-elements-mediated DNA methylation might promote the circRNAs biogenesis in the tea plant. We further detected 250 differentially expressed circRNAs under chilling stress, which were functionally enriched in GO terms related to cold/stress responses. Constructing a circRNA-miRNA-mRNA interaction network discovered 139 differentially expressed circRNAs harboring potential miRNA binding sites, which further identified 14 circRNAs that might contribute to tea plant chilling responses. We further characterized a key circRNA, CSS-circFAB1, which was significantly induced under chilling stress. FISH and silencing experiments revealed that CSS-circFAB1 was potentially involved in chilling tolerance of the tea plant. Our study emphasizes the importance of circRNA and its preliminary role against low-temperature stress, providing new insights for tea plant cold tolerance breeding.

Xanthatin and 8-epi-xanthatin as new potential colchicine binding site inhibitors: a computational study.

Phytocompounds xanthatin and 8-epi-xanthatin, obtained from Xanthium chinese Mill, showed antitumoral activity in vitro related to the microtubules destabilizing properties of these phytocompounds. Five binding sites for microtubule destabilizing agents have been characterized on tubulin by high-resolution X-ray crystallography: vinca domain, colchicine, pironetin, maytansine site, and more recently, the seventh site. This work aims to develop a comprehensive computational strategy to understand and eventually predict the interaction between xanthatin and 8-epi-xanthatin with the destabilizing-antimitotic binding domain of the tubulin heterodimer. In addition, we propose a putative binding site for these phytocompounds into the microtubule destabilizing binding sites on the tubulin heterodimer. Xanthanolides showed higher stability in the colchicine and pironetin binding sites, whit a greater affinity for the former. In addition, we found that xanthanolides and non-classical colchicine binding site inhibitors share a high structural similarity. The 3D structures for xanthatin and 8-epi-xanthatin were obtained using DFT with the hybrid functional B3LYP and the base 6-31G (d,p), implemented in Gaussian 09. The 3D coordinates for tubulin proteins were downloaded from PDB. The complexes tubulin-xanthanolides were predicted using a Monte-Carlo iterated search combined with the BFGS gradient-based optimizer implemented in the AutoDock Vina. The xanthanolides-tubulin complexes were energy minimized by molecular dynamics simulations at vacuum, and their stabilities were evaluated by solvated molecular dynamics simulations during 100ns. All molecular dynamics simulations were performed using the conjugate gradient method implemented in NAMD2 and CHARMM36 forcefield.

Ruthenium(II) Polypyridyl Complexes as FRET Donors: Structure- and Sequence-Selective DNA-Binding and Anticancer Properties.

Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)2(5,5'dmb)]2+ and [Ru(PIP)2(5,5'dmb)]2+ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5'dmb)]2+ acts to block DNA replication fork progression.

Whole-Transcriptome Sequencing Combined with High-Dimensional Proteomic Technologies Reveals the Potential Value of miR-135b-5p as a Biomarker for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a disease with great heterogeneity and a high mortality rate. It is crucial to identify reliable biomarkers for diagnosis, prognosis, and treatment to improve clinical outcomes in patients with HCC. Alpha-fetoprotein (AFP) is not only a widely used biomarker in clinical practice but also plays a complicated role in HCC, and it has recently been considered to be related to immunotherapy. MicroRNAs (miRNAs) are regarded as key regulators and promising biomarkers of HCC. We investigated the role of an AFP-related miRNA, miR-135b-5p, in HCC progression. Identification of miR-135b-5p was performed based on a cohort of 65 HCC cases and the liver hepatocellular carcinoma cohort of The Cancer Genome Atlas (Asian people only). A combination of whole-transcriptome sequencing and high-dimensional proteomic technologies was used to study the role of miR-135b-5p in HCC. Upregulation of miR-135b-5p was detected in patients with HCC with high serum AFP levels (AFP > 400 ng/ml). Elevated miR-135b-5p expression was associated with adverse prognosis. We also identified the relevance between high miR-135b-5p expression and tumor-related pathological characteristics, such as Edmondson grade and vascular invasion. We revealed tyrosine kinase nonreceptor 1 as a potential target of miR-135b-5p. Additionally, the transcriptional start site of miR-135b-5p had potential binding sites for SRY-box transcription factor 9, and the stemness properties of tumor cells were more remarkable in HCC with the upregulation of miR-135b-5p. The molecular characterization of the miR-135b-5p-high group was similar to that of the HCC subclasses containing moderately and poorly differentiated tumors. Finally, gene signatures associated with improved clinical outcomes in immune checkpoint inhibitor therapy were upregulated in the miR-135b-5p-high group. miR-135b-5p could be a biomarker for predicting the prognosis and antiprogrammed cell death protein 1 monotherapy response in HCC.

The genetic polymorphisms at the promoter region of HLA-DQB1 gene, creating responsive elements for NF1/CTF and converting the TFII-D binding site to GR-alpha.

Human leukocyte antigen-DQB1 (HLA-DQB1, OMIM: 604305) is the human major histocompatibility complex (MHC) system. HLA genes are classified into three classes (I, II, and III). The HLA-DQB1 belongs to class II, is mainly involved in the actions of the human immune system and plays a fundamental role in donor-recipient matching in transplantation and can be associated with most autoimmune diseases. In this study, the potential influence(s) of the G-71C (rs71542466) and T-80C (rs9274529) genetic polymorphisms were investigated. These polymorphisms, located in the HLA-DQB1 promoter region, have a significant frequency in the world population. The online software ALGGEN-PROMO.v8.3 was used in this work. The results indicate that the C allele at the -71 position actually creates a new potential binding site for NF1/CTF and the C allele at the -80 position changes the TFII-D binding site into a GR-alpha response element. The NF1/CTF plays the role of activator and the GR-alpha is the inhibitor; thus, according to the roles of these transcription factors, it is suggested that the above-mentioned polymorphisms alter the expression levels of HLA-DQB1. Therefore, this genetic variation is associated with autoimmune diseases; however, this cannot be generalized because this is the first report and more studies are needed in the future.

Testosterone promotes human foreskin fibroblast growth through miR-143-3p targeting IGFBP-3

Testosterone is an important male hormone, which could improve the maintenance and recovery of gonadal function in males as well as the repair of human hypospadias and cell fibrosis. Our study focused on investigating the regulatory effect of testosterone in human foreskin fibroblasts (HFF-1) and regulatory mechanisms involved. In this study, HFF-1 cells were treated with testosterone, and cell viability and migration were assessed by cell counting kit-8 (CCK8) and Transwell assays. The expression levels of androgen receptor (AR), miR-143-3p and insulin-like growth factor binding protein-3 (IGFBP-3) were measured by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. In addition, a potential binding site for miR-143-3p on IGFBP-3 was predicted and its direct binding was further confirmed by a dual luciferase reporter assay. These results showed that testosterone increased the viability and migration of HFF-1 cells. Testosterone could down-regulate miR-143-3p and up-regulate IGFBP-3 and AR. Overexpression of miR-143-3p hindered HFF-1 cell viability and negatively regulated IGFBP-3, whereas inhibition of IGFBP-3 impeded cell viability and migration. Furthermore, miR-143-3p was found to directly bind to IGFBP-3. Overexpression of IGFBP-3 countered the regulation of HFF-1 cells by miR-143-3p mimics. In conclusion, this study showed that testosterone promoted the proliferation and migration of HFF-1 cells and AR signaling, at least via the miR-143-3p/IGFBP-3 axis. This discovery presents a novel insight for testosterone application in male disorders like hypospadias.

Effect and mechanism of microRNA-515-5p in proliferation and apoptosis of trophoblast cells in preeclampsia via manipulating histone deacetylase 2.

Preeclampsia (PE)refers to a pregnancy-specific disease that begins with the placenta. Differentially expressed microRNAs (miRs) are a feature of PE. This study tried to elicit the functional mechanism of miR-515-5p in trophoblast cell behaviors in PE. First, HTR-8/SVneo cells were transfected with miR-515-5p mimic or miR-515-5p inhibitor. Then, relative expression levels of miR-515-5p and histone deacetylase 2 (HDAC2) in HTR-8/SVneo cells were determined by reverse transcription-quantitative polymerase chain reaction. The potential binding site of miR-515-5p and HDAC2 was predicted on Targetscan and their binding relationship was verified via dual-luciferase assay. Proliferation, apoptosis, invasion, and migration of HTR-8/SVneo cells were assessed via cell counting kit-8, flow cytometry, Transwell, and wound healing assays, respectively. Protein levels of Cleaved caspase-3, Bcl-2, and Bax were determined via Western blot. Overexpressed miR-515-5p impeded proliferation and stimulated apoptosis of HTR-8/SVneo cells, and decreased levels of Cleaved caspase-3 and Bax and elevated Bcl-2, whilst opposite results were observed after miR-515-5p inhibition. miR-515-5p targeted HDAC2. Knockdown of HDAC2 annulled the promotional action of miR-515-5p inhibition on proliferative, invasive, and migratory abilities and its antiapoptotic action on HTR-8/SVneo cells. In brief, miR-515-5p affected the proliferation, apoptosis, invasion, and migration of HTR-8/SVneo cells by targeting HDAC2.

Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa

Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis.

Bioinformatics analysis and the association of polymorphisms within the caprine GDF9 gene promoter with economically useful traits in Damani goats

The blood sample from 60 Damani does were collected and genomic DNA was extracted, and DNA integrity were investigated. A 447 bp promoter fragment of the GDF9 gene was amplified and Sanger sequenced for the identification of GDF9 gene polymorphism. Three novel SNPs were identified at positions g. 97(T > A), g. 142 (G > G) and g. 313(C > T) in the promoter region of the caprine GDF9 gene which significantly (P < 0.05) influenced litter size, body measurement, and milk production traits in Damani goats. The genotype CT of SNP1 significantly (P < 0.05) improved litter size, genotype GG of SNP2 significantly (P < 0.05) enhanced milk production, while the genotypes CC of SNP3 significant (P < 0.05) increased body measurement traits in Damani goats. Moreover, in SNP1 loss of 3 transcription factors (TF) binding sites occurred, SNP2 caused loss of two TFs binding sites, and SNP3 caused loss of a single TF binding site. Similarly, SNP1 and SNP2 caused the gain of three new potential TF binding sites, and SNP3 caused gain of two new TF binding sites. It is concluded that caprine GDF9 gene could be used as a candidate gene for litter size, milk production and body measurement traits in Damani goats through marker-assisted selection for future breeding program.

SOX9 and HMGB3 co-operatively transactivate NANOG and promote prostate cancer progression.

The homeodomain-containing transcription factor NANOG is overexpressed in prostate adenocarcinoma (PCa) and predicts poor prognosis. The SOX family transcription factor SOX9, as well as the transcription co-activator HMGB3 of the HMGB family, are also overexpressed and may play pivotal roles in PCa. However, it is unknown whether SOX9 and HMGB3 interact with each other, or if they regulate NANOG gene transcription. We identified potential SOX9 responsive elements in NANOG promoter, and investigated if SOX9 regulated NANOG transcription in co-operation with HMGB3 by experimental analysis of potential SOX9 binding sites in NANOG promoter, reporter gene transcription assays with or without interference or artificial overexpression of SOX9 and/or HMGB3, and protein-binding assays of SOX9-HMGB3 interaction. Clinicopathologic and prognostic significance of SOX9-HMGB3 overexpression in PCa was analyzed. SOX9 activated NANOG gene transcription by preferentially binding to a highly conserved consensus cis-regulatory element (-573 to -568) in NANOG promoter, and promoted the expression of NANOG downstream oncogenic genes. Importantly, HMGB3 functioned as a partner of SOX9 to co-operatively enhance transactivation of NANOG by interacting with SOX9, predominantly via the HMG Box A domain of HMGB3. Overexpression of SOX9 and/or HMGB3 enhanced PCa cell survival and cell migration and were significantly associated with PCa progression. Notably, Cox proportional regression analysis showed that co-overexpression of both SOX9 and HMGB3 was an independent unfavorable prognosticator for both CRPC-free survival (relative risk [RR] = 3.779，95% confidence interval [CI]: 1.159-12.322, p = 0.028) and overall survival (RR = 3.615，95% CI: 1.101-11.876, p = 0.034). These findings showed a novel SOX9/HMGB3/NANOG regulatory mechanism, deregulation of which played important roles in PCa progression.