Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion.

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

Progress report on new medications for seizures and epilepsy: A summary of the 17th Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XVII). I. Drugs in preclinical and early clinical development.

For >30 years, the Eilat Conference on New Antiepileptic Drugs and Devices has provided a forum for the discussion of advances in the development of new therapies for seizures and epilepsy. The EILAT XVII conference took place in Madrid, Spain, on May 5-8, 2024. Participants included basic scientists and clinical investigators from industry and academia, other health care professionals, and representatives from lay organizations. We summarize in this article information on treatments in preclinical and in early clinical development discussed at the conference. These include AMT-260, a gene therapy designed to downregulate the expression of Glu2K subunits of kainate receptors, in development for the treatment of drug-resistant seizures associated with mesial temporal sclerosis; BHV-7000, a selective activator of heteromeric Kv7.2/7.3 potassium channels, in development for the treatment of focal epilepsy; ETX101, a recombinant adeno-associated virus serotype 9 designed to increase NaV1.1 channel density in inhibitory γ-aminobutyric acidergic (GABAergic) neurons, in development for the treatment of SCN1A-positive Dravet syndrome; GAO-3-02, a compound structurally related to synaptamide, which exerts antiseizure activity at least in part through an action on cannabinoid type 2 receptors; LRP-661, a structural analogue of cannabidiol, in development for the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex; OV329, a selective inactivator of GABA aminotransferase, in development for the treatment of drug-resistant seizures; PRAX-628, a functionally selective potent sodium channel modulator with preference for the hyperexcitable state of sodium channels, in development for the treatment of focal seizures; RAP-219, a selective negative allosteric modulator of transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory protein γ-8, in development for the treatment of focal seizures; and rozanolixizumab, a humanized anti-neonatal Fc receptor monoclonal antibody, in development for the treatment of LGI1 autoimmune encephalitis. Treatments in more advanced development are summarized in Part II of this report.

GRminusRD: A Sensitive Assay to Detect Activation Processes at the Plasma Membrane in Living Cells.

Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.

Evaluation of Bio-Rejuvenator and Compaction Conditions on Stiffness Modulus and Indirect Tensile Strength of Recycled Hot Mix Asphalt.

This study focuses on the investigation of the effect of a reclaimed asphalt material (RAP) and a bio-rejuvenator (mix of vegetable oils) on the stiffness modulus and indirect tensile strength (ITS) values of eight bituminous mixtures produced by using three types of compaction, with different RAP amounts (25% and 50%) and rejuvenator (0%, 0.20%, 0.40% and 0.60% by mass of RAP). A conventional hot mix asphalt was considered as the reference mix. All tests were performed on cylindrical samples produced using: Marshall compaction with 50 blows/side, cored cylindrical specimens from slabs compacted using a roller compactor (39 passes), and, respectively, gyratory compaction on 80 gyrations. Stiffness modulus and ITS values showed strong linear variation with the increase in rejuvenator content, independently of test temperature and type of compaction. The rejuvenating effect of the bio-rejuvenator was observed to counterbalance the impact of RAP. The results at 20 °C for gyratory specimens for the mix with 50% RAP and 0.40% bio-rejuvenator were comparable/closer (under 5% relative difference) to those obtained for the reference mix. A strong correlation between stiffness modulus values of mixes and penetration values of the corresponding binder blends was obtained (R2≥0.977).

ZO-1 and IL-1RAP Phosphorylation: Potential Role in Mediated Brain-Gut Axis Dysregulation in Irritable Bowel Syndrome-like Stressed Mice.

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1ß and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1ß showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

Effect of zearalenone on the jejunum of weaned gilts through the Epac1/Rap1/JNK pathway.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by Fusarium strains that is harmful to the intestinal health of animals and is widely present in contaminated crops. The objective of this study was to investigate the potential therapeutic target of ZEN-induced jejunal damage in weaned gilts. Sixteen weaned gilts either received a basal diet or a basal diet supplemented with 3.0 mg/kg ZEN in a 32-day experiment. The results showed that ZEN at the concentration of 3.0 mg/kg diet activated the inflammatory response and caused oxidative stress of gilts (P < 0.05). ZEN exposure resulted in the up-regulation (P < 0.05) of the Exchange protein directly activated by the cAMP 1/Ras-related protein1/c-Jun N-terminal kinase (Epac1/Rap1/JNK signaling pathway in the jejunum of gilts in vivo and in the intestinal porcine epithelial cells in vitro. The cell viability, EdU-positive cells, and the mRNA expression of B-cell lymphoma-2 (Bcl-2) were decreased, whereas the reactive oxygen species production and the mRNA expressions of Bcl-2-associated X (Bax) and Cysteine-aspartic acid protease 3 (Caspase3) were increased (P < 0.05) by ZEN. However, ZEN increased the mRNA expression of Bcl-2 and decreased the mRNA expressions of Bax and caspase3 (P < 0.05) after the Epac1 was blocked. These results collectively indicated that 3.0 mg ZEN /kg diet induced jejunal damage via the Epac1/Rap1/JNK signaling pathway.

Evaluating the Activity of Ras/Rap GTPases in Dictyostelium.

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.

Cepharanthine inhibits migration, invasion, and EMT of bladder cancer cells by activating the Rap1 signaling pathway in vitro.

BACKGROUND: Cepharanthine, a bioactive constituent of Stephania japonica (Thunb.) Miers, is known for its potent anti-tumor properties. Nevertheless, the precise impact of this substance on bladder cancer remains poorly comprehended. The aim of this study was to demonstrate the effect and mechanism of cepharanthine on the metastasis of human bladder cancer cells. METHODS: The application of network pharmacology was utilized to ascertain the possible targets and signaling pathways of cepharanthine in the treatment of bladder cancer. The antiproliferative effects of cepharanthine were evaluated using Cell Counting Kit-8 and colony formation assays. The migration and invasion capabilities were assessed using Transwell assays and wound healing experiments. Proteins related to the Rap1 signaling pathway, cellular migration, cellular invasion, and Epithelial-Mesenchymal Transition (EMT) were quantified by western blotting. RESULTS: Through database screening, 313 cepharanthine-acting targets, 277 candidate disease targets in bladder cancer, 22 intersecting targets, and 12 core targets were confirmed. The involvement of the Rap1 signaling system was revealed by the Kyoto Encyclopedia of Genes and Genomes' pathway enrichment study. Cepharanthine was shown to decrease bladder cancer cell proliferation, migration, and invasion in vitro. Cepharanthine activated the Rap1 signaling pathway by upregulating Epac1 and downregulating E-cadherin and C3G protein expression, leading to increased expression of Rap1 GTP protein and decreased expression of protein kinase D1 and integrin α5. Rap1 signalling pathway activation resulted in the downregulation of migration and invasion-related proteins, matrix metallopeptidase MMP2, MMP9, as well as EMT-related proteins, N-cadherin and Snail, without affecting vimentin expression. CONCLUSION: Cepharanthine inhibits migration, invasion, and EMT of bladder cancer cells by activating the Rap1 signalling pathway. The results offer helpful insights regarding the possible therapeutic use of cepharanthine for treating bladder cancer.

Exosomal circPVT1 promotes angiogenesis in laryngeal cancer by activating the Rap1b-VEGFR2 signaling pathway.

Laryngeal cancer (LC) is the second most common head and neck cancer and has a decreasing 5-year survival rate worldwide. Circular RNAs regulate cancer development in diverse ways based on their distinct biogenesis mechanisms and expansive regulatory roles. However, currently, there is little research on how exosomal circular RNAs are involved in the development of laryngeal cancer. Here, we demonstrated that circPVT1, a circular RNA derived from the well-studied long noncoding RNA PVT1, is correlated with disease progression in LC and promotes angiogenesis both in vivo and in vitro. Mechanistically, circPVT1 is loaded into LC cell-secreted exosomes and taken up by vascular epithelium cells. By sponging miR-30c-5p, exosomal circPVT1 promotes Rap1b expression, which dramatically enhances VEGFR2 and PI3K/AKT pathway activation, ultimately resulting in the induction of angiogenesis. Furthermore, our xenograft models demonstrated that the combination of shRNA-circPVT1 and cetuximab showed high efficacy in inhibiting tumor growth and angiogenesis. Collectively, these findings uncover a novel mechanism of exosomal circular RNA-mediated angiogenesis modulation and provide a preclinical rationale for testing this analogous combination in patients with LC.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Preparation and Performance Evaluation of Castor Oil-Based Asphalt Regeneration Agent.

Regeneration agents play a critical role in modifying the mechanical properties and durability of RAP asphalt mixtures. This paper aimed to develop a castor oil-based asphalt regeneration agent. The effects of this regeneration agent on the pavement performance of laboratory-aged asphalt and an RAP asphalt mixture were comparatively studied by a series of laboratory tests. For the developed castor oil-based asphalt regeneration agent, the weight ratio of the castor oil to dibutyl phthalate was determined as 1:4. Moreover, the regeneration effectiveness of the castor oil-based regeneration agent was tested on three laboratory-aged asphalt binders and an RAP asphalt binder; the penetration, softening point and ductility of the RAP asphalt binder recovered to 83 dmm, 50.3 °C, and more than 100 cm, respectively. The optimum content of the regeneration agent was 5% by the weight of the aged asphalt binder. Furthermore, the castor oil-based regeneration agent could effectively restore the pavement performance of an RAP asphalt mixture. In this study, the RAP percentage can reach up to 60% by the weight of the HMA mixture using the castor oil-based asphalt regeneration agent according to the Chinese specification.

Performance of High-Dose Reclaimed Asphalt Mixtures (RAPs) in Hot In-Place Recycling Based on Balanced Design.

This study endeavors to employ a balanced design methodology, aiming to equilibrate the resistance to rutting and cracking exhibited by hot in-place recycling asphalt mixtures containing a high dose of reclaimed asphalt pavement (RAP). The primary goal is to ascertain the optimal amount of new binder necessary for practical engineering applications, ensuring a balanced rutting and crack resistance performance of recycled asphalt mixtures. The investigation mainly employed wheel-tracking tests and semi-circular bending tests to assess the rutting and cracking performance of recycled asphalt mixtures with a different dose of RAP (in China, it is common to use RAP with 80% and 90% content as additives for preparing hot in-place recycling asphalt mixtures), and varying quantities of new binders (10%, 20%, and 30% of the binder content in the total RAP added). The results indicated that the addition of new binder reduced the resistance to rutting of the recycling asphalt mixtures but improved their resistance to cracking. Furthermore, for the recycling asphalt mixture with 80% RAP content aged for 5 days, the optimal new binder content is 1.52%, while the mixture with 90% RAP content requires 1.23% of new binder. After 10 days of aging, the optimal new binder content for the recycling asphalt mixture with 80% RAP content is 1.55%, while the mixture with 90% RAP content requires 1.28% of new binder.

TSPAN6 reinforces the malignant progression of glioblastoma via interacting with CDK5RAP3 and regulating STAT3 signaling pathway.

Glioblastoma is the prevailing and highly malignant form of primary brain neoplasm with poor prognosis. Exosomes derived from glioblastoma cells act a vital role in malignant progression via regulating tumor microenvironment (TME), exosomal tetraspanin protein family members (TSPANs) are important actors of cell communication in TME. Among all the TSPANs, TSPAN6 exhibited predominantly higher expression levels in comparison to normal tissues. Meanwhile, glioblastoma patients with high level of TSPAN6 had shorter overall survival compared with low level of TSPAN6. Furthermore, TSPAN6 promoted the malignant progression of glioblastoma via promoting the proliferation and metastatic potential of glioblastoma cells. More interestingly, TSPAN6 overexpression in glioblastoma cells promoted the migration of vascular endothelial cell, and exosome secretion inhibitor reversed the migrative ability of vascular endothelial cells enhanced by TSPAN6 overexpressing glioblastoma cells, indicating that TSPAN6 might reinforce angiogenesis via exosomes in TME. Mechanistically, TSPAN6 enhanced the malignant progression of glioblastoma by interacting with CDK5RAP3 and regulating STAT3 signaling pathway. In addition, TSPAN6 overexpression in glioblastoma cells enhanced angiogenesis via regulating TME and STAT3 signaling pathway. Collectively, TSPAN6 has the potential to serve as both a therapeutic target and a prognostic biomarker for the treatment of glioblastoma.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Continuous monitoring methods of cerebral compliance and compensatory reserve: a scoping review of human literature.

Objective.Continuous monitoring of cerebrospinal compliance (CC)/cerebrospinal compensatory reserve (CCR) is crucial for timely interventions and preventing more substantial deterioration in the context of acute neural injury, as it enables the early detection of abnormalities in intracranial pressure (ICP). However, to date, the literature on continuous CC/CCR monitoring is scattered and occasionally challenging to consolidate.Approach.We subsequently conducted a systematic scoping review of the human literature to highlight the available continuous CC/CCR monitoring methods.Main results.This systematic review incorporated a total number of 76 studies, covering diverse patient types and focusing on three primary continuous CC or CCR monitoring metrics and methods-Moving Pearson's correlation between ICP pulse amplitude waveform and ICP, referred to as RAP, the Spiegelberg Compliance Monitor, changes in cerebral blood flow velocity with respect to the alternation of ICP measured through transcranial doppler (TCD), changes in centroid metric, high frequency centroid (HFC) or higher harmonics centroid (HHC), and the P2/P1 ratio which are the distinct peaks of ICP pulse wave. The majority of the studies in this review encompassed RAP metric analysis (n= 43), followed by Spiegelberg Compliance Monitor (n= 11), TCD studies (n= 9), studies on the HFC/HHC (n= 5), and studies on the P2/P1 ratio studies (n= 6). These studies predominantly involved acute traumatic neural injury (i.e. Traumatic Brain Injury) patients and those with hydrocephalus. RAP is the most extensively studied of the five focused methods and exhibits diverse applications. However, most papers lack clarification on its clinical applicability, a circumstance that is similarly observed for the other methods.Significance.Future directions involve exploring RAP patterns and identifying characteristics and artifacts, investigating neuroimaging correlations with continuous CC/CCR and integrating machine learning, holding promise for simplifying CC/CCR determination. These approaches should aim to enhance the precision and accuracy of the metric, making it applicable in clinical practice.

F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

BACKGROUND: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. METHODS: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. RESULTS: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. CONCLUSIONS: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

General regulatory factors exert differential effects on nucleosome sliding activity of the ISW1a complex.

BACKGROUND: Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. RESULTS: Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. CONCLUSIONS: Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.

EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.

Glucotoxicity is mediated by cytoplasmic distribution of RAP1 in pancreatic ß-cells.

Diabetes mellitus (DM) is a group of chronic metabolic disorders characterized by persistent hyperglycemia. In our study, we analyzed the level and location of RAP1 changes in the development of ß-cell dysfunction induced by glucotoxicity. We employed three pancreatic ß-cell lines, namely INS-1, 1.2B4, and NIT-1, as well as a streptozotocin-induced diabetes rat model. We demonstrate that after high glucose treatment, RAP1 is increased, probably through induction by AKT, allowing RAP1 to shuttle from the nucleus to the cytoplasm and activate NF-κB signaling. Furthermore, non-enzymatic post-translational modifications of RAP1, such as advanced glycation end products and carbonylation may affect the function of RAP1, such as activation of the NF-κB signaling. Taken together, we showed that RAP1 is a new player in the mechanism of glucotoxicity in pancreatic ß-cells.