Binding Protein Research Articles

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation

AbstractThe C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Association of urinary EGF, FABP3, and VCAM1 levels with the progression of early diabetic kidney disease

Introduction Diabetic kidney disease (DKD) is a common cause of chronic kidney disease with around 25-40% of patients with diabetes being affected. The course of DKD is variable and estimated glomerular filtration rate (eGFR) and albuminuria, the currently used clinical markers, are not able to accurately predict the individual disease trajectory, in particular in early stages of the disease. The aim of this study was to assess the association of urine levels of selected protein biomarkers with the progression of DKD at an early stage of disease. Methods We measured 22 protein biomarkers using the Mesoscale Discovery platform in 461 urine samples of the PROVALID cohort, an observational study of patients with type 2 diabetes mellitus followed at the primary health care level for a minimum of four years. Odds ratios (OR) were estimated for the effect of marker values above median on fast progression using unadjusted and adjusted logistic regression models. RNA expression at the single cell level in kidney biopsy samples obtained from a cohort of young persons with type 2 diabetes mellitus was in addition determined for markers showing significant associations with disease progression. Results Increased urinary levels of epidermal growth factor (EGF) were linked to lower odds of fast progression (defined as annual eGFR decline greater than 2.58 ml/min per 1.73 m2) with an odds ratio (OR) of 0.60 (95% CI 0.46, 0.78). The association with outcome was even stronger when adjusting for a set of 14 baseline clinical parameters including age, biological sex, eGFR, body mass index, albuminuria, and HbA1c. Elevated urinary levels of fatty acid binding protein 3 (FABP3) and vascular cell adhesion molecule 1 (VCAM1) were each significantly associated with fast progression with an OR of 1.44 (95% CI 1.11, 1.87) and an OR of 1.41 (95% CI 1.08, 1.83), respectively. Enriched expression of EGF and FABP3 was observed in distal convoluted tubular cells and VCAM1 in parietal epithelial cells at single cell level from biopsies of patients with early DKD. Conclusion In summary we show that lower urinary levels of EGF and higher urinary levels of FABP3 and VCAM1 are significantly associated with DKD progression in early-stage disease.

Mesenchymal stem cell-derived exosomes carrying miR-486-5p inhibit glycolysis and cell stemness in colorectal cancer by targeting NEK2.

Colorectal cancer (CRC) is a major global concern. Mesenchymal stem cell-derived exosomes (MSC-EXOs) have demonstrated efficacy as a therapeutic approach for colorectal cancer. However, the precise mechanism by which MSC-EXOs treat colorectal cancer remains unclear. Human umbilical cord (hUC)-MSC-EXOs were isolated and identified. Cell Counting Kit-8 (CCK-8), Transwell, and colony formation assays were used to assess the activity of CRC cells. Glucose consumption, lactic acid production, and extracellular acidification rate (ECAR) were measured to assess glycolytic activity. Cell stemness was assessed using a sphere-formation assay. Furthermore, MSC-exosomal microRNAs (miRNAs) in CRC tissues were analyzed using the EVmiRNA database, and aberrantly expressed miRNAs in CRC cells were obtained from the Gene Expression Omnibus (GEO) database. The binding relationship between miR-486-5p and the never in mitosis gene A-related kinase 2 (NEK2) was predicted using the Starbase database and validated through RNA binding protein immunoprecipitation (RIP) and dual luciferase assays. These results showed that hUC-MSC-EXOs inhibited the proliferation and metastasis of CRC cells. Moreover, glycolysis and stemness abilities of CRC cells also decreased after treatment with hUC-MSC-EXOs. miR-486-5p was found to be enriched in hUC-MSC-EXOs and significantly downregulated in CRC cells. miR-486-5p directly bound to NEK2. Overexpression of NEK2 reversed the inhibitory effect of miR-486-5p on CRC cell glycolysis and stemness. Our study highlights that hUC-MSC-EXO miR-486-5p inhibits glycolysis and cell stemness in CRC by targeting NEK2. This finding offers compelling evidence supporting the potential application of hUC-MSC-EXOs in the treatment of CRC.

M6A-methylase METTL3 promotes retinal angiogenesis through modulation of metabolic reprogramming in RPE cells.

Retinal neovascularization (RNV) disease is one of the leading causes of blindness, yet the molecular underpinnings of this condition are not well understood. To delve into the critical aspects of cell-mediated angiogenesis, we analyzed our previously published single-cell data. Our analysis revealed that retinal pigment epithelium (RPE) cells serve a crucial promotional function in angiogenesis. RPE cells were regulated by N6-methyladenosine (m6A). Next, we detected several critical m6A methylase in hypoxic ARPE-19 cells and in oxygen-induced retinopathy (OIR) mice, our results revealed a significant decrease in the level of methyltransferase like 3 (METTL3). METTL3 specific inhibitor STM2457 intravitreal injection or METTL3 conditional knockout mice both showed a significantly reduced neovascularization area of retina. Additionally, the angiogenesis-related abilities of human retinal endothelial cells (HRECs) were diminished after co-cultured with ARPE-19 treated with STM2457 or sh-METTL3 in vitro. Furthermore, through the integration of Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing, we discovered that the metabolic enzyme quinolinate phosphoribosyltransferase (QPRT) was directly modified by METTL3 and recognized by the YTH N6-methyladenosine RNA binding protein C1 (YTHDC1). Moreover, after over-expressing QPRT, the angiogenic abilities of HRECs were improved through the phosphorylated phosphatidylinositol-3-kinase (p-PI3K)/ phosphorylated threonine kinase (p-AKT) pathway. Collectively, our study provided a novel therapeutic target for retinal angiogenesis.

SEC-SAXS/MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded-Over Conformations.

Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a "moonlighting" role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis-competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N-terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC-MALS confirms that it is monomeric at high concentrations, but without any apparent inter-molecular self-association. SEC-SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best-fit models have the N-terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent "folded over" conformations. We hypothesize the best-fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed-folded proteins.

Expression and Purification of pAG-MNase for CUT&RUN.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a chromatin profiling strategy that releases specific DNA-protein complexes from in situ chromatin to the supernatant. CUT&RUN employs the hybrid protein pAG-MNase, composed of IgG binding proteins A and G, followed by micrococcal nuclease. Here, I describe the expression and purification of recombinant pAG-MNase as well as the assessment of its enzymatic activity. Purified pAG-MNase is hyperactive and enables large-scale analyses.

Cognotoxemia: endotoxemia and gender predict changes in working memory performance in healthy adults

IntroductionExamining the contribution of peripheral systems to cognitive function under healthy circumstances may improve our understanding of the systems that confer risk or resilience in diseased states. Endotoxemia—a pro-inflammatory response to the translocation of bacteria that reside in the gut on other sources (e.g., respiratory tract; infection) into the blood—was hypothesized to relate to worsened cognitive functioning. Gender was explored as a moderator.MethodsA sample of 162 healthy adults (25–65 years old) provided plasma, from which a measure of endotoxemia was determined [i.e., the ratio of lipopolysaccharide binding protein (LBP) to soluble cluster of differentiation 14 receptors (sCD14)]. Participants performed an array of laboratory and ambulatory cognitive tasks at three timepoints, each separated by 9 months. Two sets of multilevel models were used: Prospective models, linking endotoxemia at baseline with changes in cognition across time, and coupling models, which examine correlations of endotoxemia with cognition across time.ResultsA prospective model indicated lower levels of endotoxemia at baseline predicted improvements in working memory across the three timepoints; higher levels were associated with no change in cognitive performance. Gender was not found to modulate this finding. Interestingly, a coupling analysis of endotoxemia and gender across time showed that in men, those with higher endotoxemia performed better at the working memory task overall; in women, working memory performance was similar regardless of endotoxemia level.ConclusionThis work provides initial evidence that endotoxemia may be associated with a dampening of improvement in working memory, improvement consistent with practice effects, which should be expected in a sample of healthy, relatively young adults. The findings also provide preliminary evidence that, at least for men, higher degrees of endotoxemia are not inherently negative, and may link with short term positive outcomes for working memory.

Borg5/Cdc42EP1 restricts contractility and motility in epithelial MDCK cells.

The Borg/Cdc42EP family comprises septin binding proteins, which are known to promote septin-dependent stress fibers and acto-myosin contractility. We show here that epithelial Borg5/Cdc42ep1 instead limits contractility, cell-cell adhesion tension and motility as is required for the acquisition of columnar, isotropic cell morphology in mature MDCK monolayers. Borg5 depletion inhibited the development of the lateral F-actin cortex, stimulated microtubule-dependent leading-edge lamellae as well as radial stress fibers and, independently of the basal F-actin phenotype, caused anisotropy of apical surfaces within compacted monolayers. We determined that Borg5 limits septin-colocalization with microtubules, and that like Septin 2, Borg5 interacts with the rod-domain of Myosin- IIA. The interaction of Myosin-IIA with Borg5 was reduced in the presence of septins. Because septins also mediate myosin activation, we propose that Borg5 limits contractility in MDCK cells in part by counteracting septin-associated myosin activity.

An Integrated TCN-CrossMHA Model for Predicting circRNA-RBP Binding Sites.

Circular RNA (circRNA) has the capacity to bind with RNA binding protein (RBP), thereby exerting a substantial impact on diseases. Predicting binding sites aids in comprehending the interaction mechanism, thereby offering insights for disease treatment strategies. Here, we propose a novel approach based on temporal convolutional network (TCN) and cross multi-head attention mechanism to predict circRNA-RBP binding sites (circTCA). First, we employ two distinct encoding methodologies to obtain two raw matrices of circRNA sequences. Then, two parallel TCN blocks extract shallow and abstract features of the two matrices separately. The fusion of the two is achieved through cross multi-head attention mechanism and after this, global expectation pooling assigns weights to the concatenated feature. Finally, the task of classifying the input sequence is entrusted to a fully connected (FC) layer. We compare circTCA with other five methods and conduct ablation experiments to demonstrate its effectiveness. We also conduct feature visualization and assess the motifs extracted by circTCA with existing motifs. All in all, circTCA is effective for binding sites prediction of circRNA and RBP.

Ranking Antibody Binding Epitopes and Proteins Across Samples from Whole Proteome Tiled Linear Peptides.

Ultradense peptide binding arrays that can probe millions of linear peptides comprising the entire proteomes of human or mouse, or hundreds of thousands of microbes, are powerful tools for studying the antibody repertoire in serum samples to understand adaptive immune responses. There are few tools for exploring high-dimensional, significant and reproducible antibody targets for ultradense peptide binding arrays at the linear peptide, epitope (grouping of adjacent peptides), and protein level across multiple samples/subjects (i.e. epitope spread or immunogenic regions of proteins) for understanding the heterogeneity of immune responses. We developed HERON (Hierarchical antibody binding Epitopes and pROteins from liNear peptides), an R package, which identifies immunogenic epitopes, using meta-analyses and spatial clustering techniques to explore antibody targets at various resolution and confidence levels, that can be found consistently across a specified number of samples through the entire proteome to study antibody responses for diagnostics or treatment. Our approach estimates significance values at the linear peptide (probe), epitope, and protein level to identify top candidates for validation. We test the performance of predictions on all three levels using correlation between technical replicates and comparison of epitope calls on two datasets, which shows HERON's competitiveness in estimating false discovery rates and finding general and sample-level regions of interest for antibody binding. The HERON R package is available at Bioconductor https://bioconductor.org/packages/release/bioc/html/HERON.html. Supplementary data are available at Bioinformatics online.

Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein.

Autophagy is a highly conserved degradative process that has been linked to various functions, including defending host cells against pathogens. Although the involvement of autophagy in porcine circovirus 2 (PCV2) infection has become apparent, it remains unclear whether selective autophagy plays a critical role in PCV2 restriction. Here we show that retinol-binding protein 4 (RBP4), an adipokine for retinol carrier, initiates the autophagic degradation of PCV2 ORF1 protein. PCV2 infection increases RBP4 protein levels through MAPK-eIF4E axis in living cells. Ectopic expression of RBP4 or recombinant RBP4 treatment promotes the degradation of ORF1 protein. Mechanistically, RBP4 activates TRAF6 to induce K63-linked ubiquitination of ORF1, leading to SQSTM1/p62-mediated selective autophagy for degradation. Consequently, RBP4 deficiency increases viral loads and exacerbates the pathogenicity of PCV2 in vivo. Collectively, these results identify RBP4 as a key host restriction factor of PCV2 and reveal a previously undescribed antiviral mechanism against PCV2 in infected cells.

Rapid and Specific Detection of Bacillus cereus Using Phage Protein-Based Lateral Flow Assays.

Rapid and precise diagnostic techniques are essential for identifying foodborne pathogens, including Bacillus cereus (B. cereus), which poses significant challenges to food safety. Traditional detection methods are limited by long incubation times and high costs. In this context, gold nanoparticle (AuNP)-based lateral flow assays (LFAs) are emerging as valuable tools for rapid screening. However, the use of antibodies in LFAs faces challenges, including complex production processes, ethical concerns, or variability. Here, we address these challenges by proposing an innovative approach using bacteriophage-derived proteins for pathogen detection on LFAs. We used the engineered endolysin cell-wall-binding domain (CBD) and distal tail proteins (Dit) from bacteriophages that specifically target B. cereus. The protein-binding properties, essential for the formation of efficient capture and detection biointerfaces in LFAs, were extensively characterized from the microstructural to the LFA device level. Machine-learning models leverage knowledge of the protein sequence to predict advantageous protein orientations on the nitrocellulose membrane and AuNPs. The study of the biointerface binding quantified the degree of attachment of AuNPs to bacteria, providing, for the first time, a microscopic model of the number of AuNPs binding to bacteria. It highlighted the binding of up to one hundred 40 nm AuNPs per bacterium in conditions mimicking LFAs. Eventually, phage proteins were demonstrated as efficient bioreceptors in a straightforward LFA prototype combining the two proteins, providing a rapid colorimetric response within 15 min upon the detection of 105 B. cereus cells. Recombinantly produced phage binding proteins present an opportunity to generate a customizable library of proteins with precise binding capabilities, offering a cost-effective and ethical alternative to antibodies. This study enhances our understanding of phage protein biointerfaces, laying the groundwork for their utilization as efficient bioreceptors in LFAs and rapid point-of-care diagnostic assays, thus potentially strengthening public health measures.

Cellular NONO protein binds to the flavivirus replication complex and promotes positive-strand RNA synthesis.

A cellular protein, non-POU-domain-containing octamer binding protein (NONO), bound to the replication complex of Japanese encephalitis virus (JEV) by directly interacting with the viral 3' UTR RNA and NS3 protein. These interactions were also identified in West Nile virus (WNV) and Zika virus (ZIKV). The infection of JEV or the expression of JEV NS3 protein in cells could induce relocation of NONO protein from the nucleus to the cytoplasm. In JEV-infected cells, the NS3, NS5, and viral RNA could be concurrently detected in the immunoprecipitation by the NONO-specific antibody, suggesting that NONO could integrate into the replication complex of JEV. Further results of co-immunoprecipitation assays showed that NONO protein interacted with NS3 helicase domains 1 and 2 by its two RNA recognize motifs (RRMs). The knockdown and knockout of NONO in cells could significantly reduce the replication of JEV and ZIKV but had no effect on the replication of vesicular stomatitis virus (VSV). The effect of NONO protein on JEV proliferation occurred during the replication stage, rather than the attachment and entry stages. The level of viral positive-strand RNA in NONO knockout cells was significantly reduced than that in wild-type cells at 12-48 h post-JEV infection. However, the level of negative-strand virus RNA had no difference between NONO knockout and wild-type cells at 12-24 h post-infection. In summary, our study identified a cellular protein that bound to the replication complex of flavivirus and facilitated the synthesis of positive-strand RNA.IMPORTANCEOver half of the world's population is at risk of flaviviruses infection, posing a serious global health concern. To date, there are no antiviral drugs or treatments for the severe symptoms caused by the infection of flaviviruses. Some cellular proteins could participate in the replication of virus, and these cellular proteins were also ideal targets in antiviral strategy. Here, we identified cellular NONO protein was recruited by flavivirus NS3 protein to the cytoplasm, serving as a "scaffold" for viral replication complex. Our findings also revealed that NONO protein was critical for flavivirus positive-strand RNA synthesis. Specific areas where NONO interacted with flavivirus NS3 proteins and viral UTRs have also been identified. These results propose a new mechanism for cellular protein to participate in flavivirus replication and also raise a new potential anti-flavivirus strategy.

Pulmonary-delivered Anticalin Jagged-1 antagonists reduce experimental airway mucus hyperproduction and obstruction.

Mucus hypersecretion and mucus obstruction are pathogenic features in many chronic lung diseases directly linked to disease severity, exacerbation, progression, and mortality. The Jagged-1/Notch pathway is a promising therapeutic target that regulates secretory and ciliated cell trans-differentiation in the lung. However, the Notch pathway is also required in various other organs. Hence, pulmonary delivery of therapeutic agents is a promising approach to target this pathway while minimizing systemic exposure. Using Anticalin® technology, Jagged-1 Anticalin binding proteins were generated and engineered to potent and selective inhalable Jagged-1 antagonists. Their therapeutic potential to reduce airway mucus hyperproduction and obstruction was investigated ex vivo and in vivo. In primary airway cell cultures grown at air-liquid interface and stimulated with inflammatory cytokines, Jagged-1 Anticalin binding proteins reduced both mucin gene expression and mucous cell metaplasia. In vivo, prophylactic and therapeutic treatment with a pulmonary-delivered Jagged-1 Anticalin binding protein reduced mucous cell metaplasia, epithelial thickening and airway mucus hyperproduction in IL-13 and house dust mite allergen-challenged mice, respectively. Further, in a transgenic mouse model with pathophysiologic features of cystic fibrosis and COPD, pulmonary-delivered Jagged-1 Anticalin binding protein reduced hallmarks of airway mucus obstruction. In all in vivo models a reduction of mucous cells with a concomitant increase of ciliated cells was observed. Collectively, these findings support Jagged-1 antagonists' therapeutic potential for patients with muco-obstructive lung diseases, and the feasibility of targeting the Jagged-1/Notch pathway by inhalation.

TYROBP promotes the spread of pancreatic cancer by causing M2 TAM polarization.

M2-polarized tumor-associated macrophages (M2 TAMs) are known to promote cancer progression, and exosomes are crucial mediators of communication within the tumor microenvironment (TME). However, the specific role of exosomes derived from M2 TAMs in pancreatic cancer (PC) progression remains poorly understood. Tyrosine kinase binding protein (TYROBP, also known as DAP12 for DNAX activating protein-12) is a transmembrane signal transduction polypeptide that interacts with immune cell receptors, influencing cellular functions via signal transduction pathways. TYROBP is prominently found in M2 TAMs exosomes, facilitating its transfer to PC cells and suggesting a potential role in PC pathogenesis. This study initially confirmed the presence of TYROBP in M2 TAMs exosomes and its transfer to PC cells via exosomes. The impact of TYROBP on PC proliferation, apoptosis, migration, and invasion was investigated. Special attention was given to TYROBP's influence on PC metastasis and its underlying mechanisms, focusing particularly on the CD44/AKT/ERK signaling pathway. TYROBP expression in PC cells did not significantly affect tumor cell proliferation or apoptosis but demonstrated a notable inhibitory effect on migration and invasion, which was mediated through the CD44/AKT/ERK pathway. Both in vivo and in vitro experiments consistently showed that TYROBP enhanced PC metastasis. This study elucidates that TYROBP plays a direct role in promoting PC metastasis through its association with M2 TAMs polarization. Therefore, TYROBP represents a potential novel therapeutic target for interventions aimed at combatting PC progression.

Curcumin derivative C210 induces Epstein–Barr virus lytic cycle and inhibits virion production by disrupting Hsp90 function

Lytic induction therapy was devised to selectively combat malignancies associated with Epstein–Barr virus (EBV) by triggering viral reactivation from latency. At present, the major challenges of lytic induction therapy are to maximize reactivating efficiencies and meanwhile minimize infectious virion production. C210, a novel curcumin derivative with potent Hsp90 inhibitory activity, was explored for EBV-reactivating and virion-producing effects in EBV-positive nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) cell lines. And the molecular mechanisms underlying these effects were determined. Follow C210 treatment, EBV lytic RNAs and proteins were upregulated, but infectious virions were not produced. Knockdown of heat shock protein 90 (Hsp90) induced expression of lytic RNAs and proteins, and diminished C210-driven EBV lytic induction. Pretreatment with an X box binding protein 1 (XBP1) inhibitor reduced C210-induced EBV lytic RNA. Furthermore, we demonstrated that C210 inhibited the binding of Hsp90 with its clients, signal transducer and activator of transcription 3 (STAT3) and xeroderma pigmentosum group B-complementing protein (XPB), which subsequently promoted their proteasomal degradation. Degradation of STAT3 by C210 enhanced the EBV-reactivating and anticancer capacity of suberoylanilide hydroxamic acid (SAHA). Depletion of XPB blocked SAHA-induced expression of late viral genes and production of infectious virions. These results elucidate a novel Hsp90 inhibitor targeting EBV lytic phase and extend the research on lytic induction strategy, which may offer reference value in the treatment of EBV-positive malignancies.

Essential Oils of The Leaves of Syzygium hemilamprum (F. Muell.) Craven & Biffin.: Chemical Analysis, Antimicrobial, Mosquito Larvicidal, Molecular Docking, and ADMET Studies.

The chemical composition of the essential oil and n-hexane extract from Syzygium hemilamprum leaves was first performed. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the essential oil was predominantly composed of monoterpene hydrocarbons (71.5 %) and oxygenated derivatives (20.2 %), with β-pinene (31.5 %), limonene (19.4 %), α-pinene (12.3 %), and α-terpineol (7.4 %) being the principal constituents. The n-hexane extract contained monoterpene hydrocarbons (42.2 %) and non-terpenic compounds (34.0 %), with β-pinene (32.8 %) and n-hexadecane (10.2 %) as the major components. Antimicrobial and mosquito larvicidal assays demonstrated that both samples exhibited antimicrobial activity against Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Saccharomyces cerevisiae (yeast), with a minimum inhibitory concentration (MIC) of 128 μg/mL for all tested organisms. Both samples also showed significant mosquito larvicidal activity against Aedes aegypti and Culex quinquefasciatus, with LC50 and LC90 values below 20 μg/mL at 24 and 48 hours post-treatment. Molecular docking studies suggested that limonene and α-terpineol could serve as potent inhibitors of mosquito odorant binding proteins. Additionally, an in silico analysis was performed to evaluate the physicochemical and ADMET (absorption, distribution, metabolism, and toxicity) properties of the major constituents of the essential oil.

Dengzhan Shengmai Capsule Ameliorates Cognitive Impairment via Inhibiting ER stress in APP/PS1 Mice

Ethnopharmacological relevanceAlzheimer's disease (AD) is a common type of neurodegenerative disease with the β-amyloid plaques (Aβ) deposition. Previously, Dengzhan Shengmai capsule (DZSM) has been shown to reduce the pathology associated with AD, but the underlying mechanism is unclear. Aim of studyThis study investigated the potential mechanisms of DZSM against AD. Materials and methodsThe six-month-old wild-type male mice and APP/PS1 double transgenic male mice were administered 0.9 % saline or DZSM for 8 weeks by gavage. Open field test, new object recognition test, and Morris Water maze test were used to assess spatial learning and memory. Aβ plaques in brains were visualized using ThT staining. Nissl staining, TUNEL staining, and western blot analyses were used to detect the neuronal function and apoptosis level. The superoxide dismutase (SOD), glutathione peroxidase assay kit (GSH-Px), and malondialdehyde (MDA) kits were performed to assess oxidative stress levels. Then, immunofluorescence and western blot analysis were applied to evaluate ER stress pathway protein levels. Finally, HT22 cells were treated by Aβ1-42 with or without DZSM medicated serum. Cell viability was assessed using the CCK-8 assay, and western blot analysis was applied to evaluate ER stress pathway protein levels. ResultsOpen filed test, new object recognition test and Morris Water maze test showed that DZSM restored cognitive disorders in APP/PS1 mice. Immunohistochemistry and Thioflavin T staining results indicated that DZSM reduced Aβ plaques in the brain. Deeper and denser Nissl bodies were found in APP/PS1 mice after DZSM administration. Besides, APP/PS1 mice treated with DZSM showed a lower level of TUNEL and Bax/Bcl-2 ratio. DZSM improved the acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity while reducing acetylcholinesterase (AChE) and malondialdehyde (MDA) activity. In addition, the levels of ER stress pathway containing Phospho-PKR-like ER kinase (P-PERK), phosphorylate eukaryotic initiation factor 2 (P-eIF2α), activating transcription factor 4 (ATF4), glutamine-rich protein 1 (QRICH1), phosphorylate inositol-requiring protein 1α (P-IRE1α), the spliced form of X-box binding protein 1 (XBP1s), activating transcription factor-6 (ATF6) and C/EBP homologous binding protein (CHOP) were decreased by DZSM. CCK-8 results indicated that DZSM medicated serum played cytoprotective effects on Aβ1-42-induced HT22 cells. Western blot results suggested DZSM possibly inhibited ER stress pathways in Aβ1-42-induced HT22 cells. ConclusionThe potential protective mechanism of DZSM on cognitive impairment in AD might be related to ER stress pathways.

MiR‑155 promotes an inflammatory response in HaCaT cells via the IRF2BP2/KLF2/NF‑κB pathway in psoriasis.

Psoriasis is a chronic inflammatory skin condition with numerous causes, including genetic, immunological and infectious factors. The course of psoriasis is long and recurrence is common; pathogenesis is not completely understood. However, there is an association between advancement of psoriasis and aberrant microRNA (miR or miRNA)‑155 expression. Through bioinformatics, the present study aimed to analyze the differentially expressed genes and miRNAs in psoriasis and its biological mechanism and function psoriatic inflammation. First of all, differentially expressed genes (DEGs) and miRNAs (DEMs) in patients with psoriasis were identified using GEO2R interactive web application. A psoriasis inflammatory model was established using lipopolysaccharide (LPS)‑treated HaCaT keratinocytes, which were transfected with miR‑155 mimic or inhibitor. Cell Counting Kit‑8 was used for the assessment of cell viability and proliferation, and changes in the cell cycle were examined using flow cytometry. ELISA and reverse transcription‑quantitative PCR (RT‑qPCR) were used to detect the expression levels of the inflammatory factors IL‑1β and IL‑6. The dual‑luciferase reporter assay was used to verify the targeting association between miR‑155‑5p and IFN regulatory factor 2 binding protein 2 (IRF2BP2). To verify the targeting association of miR‑155 and the IRF2BP2/kruppel‑like factor 2 (KLF2)/NF‑κB signaling pathway, expression levels of IRF2BP2, KLF2 and p65 were identified by RT‑qPCR and western blotting. IRF2BP2 levels were also confirmed by immunofluorescence, in conjunction with bioinformatics database analysis. Overexpression of miR‑155 inhibited proliferation of HaCaT cells and increased the number of cells in S phase and decreasing number of cells in G1 and G2 phase. In the LPS‑induced inflammatory state, miR‑155 overexpression heightened the inflammatory response of HaCaT cells while inhibition of miR‑155 lessened it. Suppression of inflammatory cytokine expression by miR‑155‑5p inhibitor was reversed by knockdown of IRF2BP2. miR‑155 was shown to interact with IRF2BP2 to negatively regulate its expression, leading to decreased KLF2 expression and increased p65 expression and secretion of inflammatory factors, intensifying the inflammatory response of HaCaT cells. Therefore, miR‑155 may contribute to development of psoriasis by inducing tissue and cell damage by increasing the inflammatory response of HaCaT cells via the IRF2BP2/KLF2/NF‑κB pathway. In conclusion, the results of the present study offer novel perspectives on the role of miR‑155 in the onset and progression of psoriasis.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco)

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2,346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequence alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. Anyway, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.