Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

α-Ketoglutarate downregulates thiosulphate metabolism to enhance antibiotic killing.

Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.

Comparative analysis of pregnancy outcomes in preimplantation genetic testing for aneuploidy and conventional in vitro fertilization and embryo transfer: a stratified examination on the basis of the quantity of oocytes and blastocysts from a multicenter randomized controlled trial.

OBJECTIVE: To investigate variations in pregnancy outcomes between preimplantation genetic testing for aneuploidy (PGT-A) and conventional in vitro fertilization and embryo transfer (IVF-ET) treatment across distinct groups categorized by oocyte and blastocyst counts. Because the live birth rate (LBR) of assisted reproductive technology treatment is influenced by the number of oocytes and blastocysts retrieved. Our previous study indicated comparable cumulative LBRs (CLBRs) between conventional IVF-ET and PGT-A. DESIGN: A post hoc exploratory secondary analysis of data from a multicenter randomized controlled trial compared the CLBRs between conventional IVF-ET and PGT-A. SETTING: Academic fertility centers. SUBJECTS: A total of 1,212 infertile women with a good prognosis for a live birth after PGT-A or conventional IVF-ET were included. INTERVENTION: Women underwent PGT-A or conventional IVF-ET. MAIN OUTCOME MEASURE(S): Cumulative LBR, cumulative clinical pregnancy loss (CPL) rate, and good birth outcome. RESULT(S): In the study, all participants were divided into 4 groups on the basis of quartiles of the number of oocytes retrieved, or blastocysts. There was an interaction between whether to perform PGT-A and the oocyte numbers category on cumulative CPL and biochemical pregnancy loss. Chi-square analysis revealed that the PGT-A group showed a lower cumulative frequency of CPL compared with the IVF-ET group (PGT-A vs. IVF-ET: 5.9% vs. 13.7%; relative risk = 0.430; 95% confidence interval, 0.243-0.763) when the number of oocytes retrieved was <15. Although there was no interaction on CLBR when the retrieved oocyte count ranged from 19-23 (19≤ oocytes <23) the PGT-A group exhibited a lower CLBR than the conventional IVF-ET group (PGT-A vs IVF-ET: 75.6% vs 87.1%; relative risk = 0.868; 95% confidence interval, 0.774-0.973), and the average body weight of newborns from the PGT-A group was approximately 142 g lower than that of the conventional IVF-ET group (PGT-A vs. IVF-ET: 3,334 ± 479 g vs. 3,476 ± 473 g). However, no statistically significant difference in the CLBR was observed between the PGT-A and IVF-ET groups in the other oocyte or blastocyst groups. CONCLUSION: When the number of retrieved eggs was <15, the PGT-A group exhibited a lower cumulative CPL rate but no higher CLBR than the conventional IVF-ET group. CLINICAL TRIAL REGISTRATION NUMBER: NCT03118141.

Hepcidin deficiency impairs hippocampal neurogenesis and mediates brain atrophy and memory decline in mice.

BACKGROUND: Hepcidin is the master regulator of iron homeostasis. Hepcidin downregulation has been demonstrated in the brains of Alzheimer's disease (AD) patients. However, the mechanism underlying the role of hepcidin downregulation in cognitive impairment has not been elucidated. METHODS: In the present study, we generated GFAP-Cre-mediated hepcidin conditional knockout mice (HampGFAP cKO) to explore the effect of hepcidin deficiency on hippocampal structure and neurocognition. RESULTS: We found that the HampGFAP cKO mice developed AD-like brain atrophy and memory deficits. In particular, the weight of the hippocampus and the number of granule neurons in the dentate gyrus were significantly reduced. Further investigation demonstrated that the morphological change in the hippocampus of HampGFAP cKO mice was attributed to impaired neurogenesis caused by decreased proliferation of neural stem cells. Regarding the molecular mechanism, increased iron content after depletion of hepcidin followed by an elevated level of the inflammatory factor tumor necrosis factor-α accounted for the impairment of hippocampal neurogenesis in HampGFAP cKO mice. These observations were further verified in GFAP promoter-driven hepcidin knockdown mice and in Nestin-Cre-mediated hepcidin conditional knockout mice. CONCLUSIONS: The present findings demonstrated a critical role for hepcidin in hippocampal neurogenesis and validated the importance of iron and associated inflammatory cytokines as key modulators of neurodevelopment, providing insights into the potential pathogenesis of cognitive dysfunction and related treatments.

A novel survival prediction signature outperforms PAM50 and artificial intelligence-based feature-selection methods.

The robustness of a breast cancer gene signature, the super-proliferation set (SPS), is initially tested and investigated on breast cancer cell lines from the Cancer Cell Line Encyclopaedia (CCLE). Previously, SPS was derived via a meta-analysis of 47 independent breast cancer gene signatures, benchmarked on survival information from clinical data in the NKI dataset. Here, relying on the stability of cell line data and associative prior knowledge, we first demonstrate through Principal Component Analysis (PCA) that SPS prioritizes survival information over secondary subtype information, surpassing both PAM50 and Boruta, an artificial intelligence-based feature-selection algorithm, in this regard. We can also extract higher resolution 'progression' information using SPS, dividing survival outcomes into several clinically relevant stages ('good', 'intermediate', and 'bad) based on different quadrants of the PCA scatterplot. Furthermore, by transferring these 'progression' annotations onto independent clinical datasets, we demonstrate the generalisability of our method on actual patient data. Finally, via the characteristic genetic profiles of each quadrant/stage, we identified efficacious drugs using their gene reversal scores that can shift signatures across quadrants/stages, in a process known as gene signature reversal. This confirms the power of meta-analytical approaches for gene signature inference in breast cancer, as well as the clinical benefit in translating these inferences onto real-world patient data for more targeted therapies.

miR-219-5p attenuates cisplatin resistance of ovarian cancer by inactivating Wnt/ß-catenin signaling and autophagy via targeting HMGA2.

Our previous study confirmed that miR-219-5p inhibits the progression of ovarian cancer (OC) by targeting high mobility group AT-hook 2 (HMGA2), while the role of miR-219-5p on the chemoresistance of OC is unclear. HMGA2 and miR-219-5p expression in OC tumors and various types of OC cells were determined by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The miRNA profiles in A2780 and cisplatin-resistant A2780 cells were investigated via bulk miRNA sequencing, and the interactions of miR-219-5p and HMGA2 were determined by luciferase reporter activity assay. Cell function was verified through Cell Counting Kit-8, invasion assay, wound-healing, and TUNEL assays. HMGA2 level is highly expressed in cisplatin-resistant OC cell lines compared to normal OC cells, while the expression trend of miR-219-5p is the opposite. In addition, we found that miR-219-5p is one of the miRNAs that have the most significant reduction in levels in the cisplatin-resistant A2780/DDP cell line compared to A2780 cells. Then, we reveal that miR-219-5p directly targets HMGA2 in cisplatin-resistant OC cells, and upregulation of miR-219-5p significantly reduces the resistance of OC cells to cisplatin both in vitro and in vivo. Finally, our results suggest that Wnt/ß-catenin signaling and autophagy pathway is involved in the role of miR-219-5p/HMGA2 on resistance of OC cells to cisplatin via gain-of-function experiments. Collectively, the present study shows that miR-219-5p decreases the resistance of OC cells to cisplatin via Wnt/ß-catenin signaling and autophagy by regulating HMGA2, which provides a feasible solution for the resistance of OC to chemotherapy.

Icotinib derivatives as tyrosine kinase inhibitors with anti-esophageal squamous carcinoma activity.

Previous report showed that a variety of icotinib derivatives bearing different 1,2,3-triazole moieties, which could be readily prepared via copper (I)-catalyzed cycloaddition (CuAAC) reaction between icotinib and different azides, exhibited interesting activity against different lung cancer cell lines such as H460, H1975, H1299, A549 or PC-9. To further expand the application scope of the compounds and to validate the function of triazole groups in drug design, the anti-cancer activity of these compounds against esophageal squamous carcinoma (ESCC) cells was tested herein. Preliminary MTT experiments suggested that these compounds were active against different ESCC cell lines such as KYSE70, KYSE410, or KYSE450 as well as their drug-resistant ones. Especially, compound 3l showed interesting anticancer activity against these cell lines. The mode of action was studied via molecular docking, SPR experiments and other biochemical studies, and 3l exhibited higher binding potential to wild-type EGFR than icotinib did. In vivo anticancer study showed that 3l could inhibit tumor growth of cell-line-derived xenografts in ESCC. Study also suggested that 3l was a potent inhibitor for EGFR-TK pathway. Combining these results, 3l represents a promising lead compound for the design of anti-cancer drugs against ESCC.

Morphology-Controlled Synthesis of V1.11S2 for Electrocatalytic Hydrogen Evolution Reaction in Acid Media.

High-performance low-cost catalysts are in high demand for the hydrogen evolution reaction (HER). In the present study, we reported that V1.11S2 materials with flower-like, flake-like, and porous morphologies were successfully synthesized by hydrothermal synthesis and subsequent calcination. The effects of morphology on hydrogen evolution performance were studied. Results show that flower-like V1.11S2 exhibits the best electrocatalytic activity for HER, achieving both high activity and preferable stability in 0.5 M H2SO4 solution. The main reason can be ascribed to the abundance of catalytically active sites and low charge transfer resistance.

Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2.

Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term 'programmable antivirals', with implications for antiviral prophylaxis and post-exposure therapy.

Cadmium induced BEAS-2B cells apoptosis and mitochondria damage via MAPK signaling pathway.

Cadmium, a heavy metal pollutant in industrial production, is found in air, water and soil, which is harmful to human health and can lead to diseases, such as asthma, lung cancer, and emphysema. In this study, the toxicity of cadmium on human bronchial epithelial cells (BEAS-2B) was investigated. Cell viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, apoptosis and the related signaling pathways were detected with MTT assay, Rhodamine staining, DCFH-DA staining, Hoechst33258 staining and Western blot methods respectively. The results showed that the cell viability decreased, the mitochondrial membrane potential declined, ROS was accumulated and apoptotic rate raised in BEAS-2B cells. Meanwhile, the expression of B-cell lymphoma-2 (Bcl-2) was downregulated, while the expression of Bcl-2-associated X protein (Bax) and the cleaved caspase-3 was upregulated, which indicated mitochondria-mediated intrinsic apoptosis pathway was activated. Furthermore, the phosphorylation of JNK, ERK and p38 was enhanced respectively, which manifested that MAPK signaling pathways were activated. Therefore, it could be concluded that cadmium could increase intracellular ROS, result in cellular oxidative stress, activate JNK, ERK and p38 MAPK pathways and ultimately lead to apoptosis of BEAS-2B cells by activating mitochondria-mediated intrinsic apoptosis pathway. This study provided useful information to elucidate the toxicity of cadmium and revealed the possible mechanism for the occurrence of lung disease induced by cadmium.

The Identification of Anthocyanins from Padus racemosa and Its Protective Effects on H2 O2 -Induced INS-1 Cells Damage and STZ-Induced Diabetes Mice.

Oxidative damage in cells induced by reactive oxygen species (ROS) is a main factor in diabetes mellitus diseases progression. The composition of anthocyanins from Padus racemosa (APR) and the protective effects of APR on H2 O2 -induced rat insulinoma (INS-1) cells damage and streptozocin (STZ)-induced diabetes mice were investigated in this study. The main components of APR were cyanidin-cyanidin glucosyl-rutinoside, cyanidin-cyanidin xylosyl-rutinoside, cyanidin-xylosyl-glucoside and cyanidin-rutinoside, which were determined by liquid chromatography-mass spectrometry (LC/MS). APR could scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical and superoxide radical in vitro. ROS level was decreased and the cell viability was increased in INS-1 cells after treated with APR. Cell apoptosis induced by H2 O2 in INS-1 cells was decreased after incubation with APR. APR could decrease the phosphorylation of p38 and the nuclear translocation of p65, which indicated that APR could inhibit the activation of p38 Mitogen-activated protein kinase (MAPK) and Nuclear factor kappa B (NF-κB) cell signaling pathways. Meanwhile, APR could effectively reduce the blood glucose and blood lipid in STZ-induced diabetic mice. These results suggested that APR might be a potential agent for diabetes mellitus diseases treatment.

Repeated radon exposure induced lung injury and epithelial-mesenchymal transition through the PI3K/AKT/mTOR pathway in human bronchial epithelial cells and mice.

Radon exposure is the most frequent cause of lung cancer in non-smokers. The high linear energy transfer alpha-particles from radon decay cause the accumulation of multiple genetic changes and lead to cancer development. Epithelial-mesenchymal transition (EMT) plays an important role in oncogenesis. However, the mechanisms underlying chronic radon exposure-induced EMT attributed to carcinogenesis are not understood. This study aimed to explore the EMT and potential molecular mechanisms induced by repeated radon exposure. The EMT model of 16HBE and BEAS-2B cells was established with radon exposure (20000 Bq/m3, 20 min each time every 3 days). We found repeated radon exposure facilitated epithelial cell migration, proliferation, reduced cell adhesion and ability to undergo EMT through a decrease in epithelial markers and an increase in mesenchymal markers. Radon regulated the expression of matrix metalloproteinase 2 (MMP2) and tissue inhibitors of metalloproteinase 2 (TIMP2) to disrupt the balance of MMP2/TIMP2. In vivo, BALB/c mice were exposed to 105 Bq/m3 radon gas for cumulative doses of 60 and 120 Working Level Months (WLM). Radon inhalation caused lung damage and fibrosis in mice, which was aggravated with the increase of exposure dose. EMT-like transformation also occurred in lung tissues of radon-exposure mice. Moreover, radon radiation increased p-PI3K, p-AKT and p-mTOR in cells and mice. Radon reduced the GSK-3ß level and elevated the active ß-catenin in 16HBE cells. The m-TOR and AKT inhibitors attenuated radon exposure-induced EMT by regulation related biomarkers. These data demonstrated that radon exposure induced EMT through the PI3K/AKT/mTOR pathway in epithelial cells and lung tissue.

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

A study on the protective effects of taxifolin on human umbilical vein endothelial cells and THP-1 cells damaged by hexavalent chromium: a probable mechanism for preventing cardiovascular disease induced by heavy metals.

Hexavalent chromium [Cr(vi)] which is a kind of heavy metal with strong oxidizing ability can induce cardiovascular disease (CVD), while taxifolin can protect cells and organisms against suffering from oxidative stress. In this study, the inhibitory effects of taxifolin against Cr(vi)-induced cell damage in human umbilical vein endothelial cells (HUVECs) and THP-1 cells were investigated. Cr(vi) could increase the phosphorylation of p38 and JNK, regulate the expression of Bax and Bcl-2 in both cell lines. Meanwhile, the Cr(vi) stimulation led to an increase of the expression of ICAM-1 and VCAM-1, and upregulated the adhesion of THP-1 cells to HUVECs. Furthermore, Cr(vi) could induce the activation of the nuclear factor kappa B (NF-κB) signaling pathway, the accumulation of p65 in the nucleus, and the increase in the phosphorylation of IκB and the expression of cleaved caspase-1 and IL-1ß in THP-1 cells. However, taxifolin could reverse the effects by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathways, regulating the expression of apoptosis-related proteins, and alleviating the adhesion of THP-1 cells to HUVECs. Our findings demonstrated that taxifolin was a potential agent to prevent endothelial dysfunction, monocyte inflammation and cell adhesion induced by Cr(vi).

Resveratrol protects human bronchial epithelial cells against nickel-induced toxicity via suppressing p38 MAPK, NF-κB signaling, and NLRP3 inflammasome activation.

Nickel is a common environmental pollutant that can impair the lung, but the underlying mechanisms have not yet been fully elucidated. Furthermore, natural products are generally used to inhibit cell damage induced by heavy metal. Resveratrol possesses wide biological activities, including anti-inflammation and antioxidative stress. This study was conducted to explore the toxicity of nickel on human bronchial epithelial (BEAS-2B) cells and evaluate the protective effect of resveratrol. The results showed that nickel could induce cell apoptosis, increase oxidative stress, and promote the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-8, C-reaction protein. Western blot analysis showed that nickel activated p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B, and nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 pathways, while resveratrol could reverse these effects. Our results suggested that resveratrol could protect BEAS-2B cells from nickel-induced cytotoxicity. Therefore, resveratrol is a potential chemopreventive agent against nickel-induced lung disease.

Protective Effects of Anthocyanins from Coreopsis tinctoria against Oxidative Stress Induced by Hydrogen Peroxide in MIN6 Cells.

Anthocyanins (AC) from Coreopsis tinctoria possesses strong antioxidant properties, while the effects of AC on cells damage induced by reactive oxygen species (ROS) in diabetes mellitus diseases progression have not been reported. The present study was carried out to evaluate the protective property of AC against cellular oxidative stress with an experimental model, H2 O2 -exposed MIN6 cells. AC could reverse the decrease of cell viability induced by H2 O2 and efficiently suppressed cellular ROS production and cell apoptosis. In addition, Real-time PCR and Western blot analyses indicated that AC could protect MIN6 cells against oxidative injury through increasing the translocation of Nrf2 into nuclear, decreasing the phosphorylation level of p38 and up-regulating the protein expression of antioxidant enzyme (SOD1, SOD2 and CAT). Thus, this study provides evidence to support the beneficial effect of AC in inhibiting MIN6 cells from H2 O2 -induced oxidative injury.

Co-expression of multiple heavy metal transporters changes the translocation, accumulation, and potential oxidative stress of Cd and Zn in rice (Oryza sativa).

The OsHMA2, OsLCT1 and OsZIP3 transporters were all involved in zinc (Zn) and cadmium (Cd) transport. So far, only a few researches studied on the co-regulation effect of three transporters related to Zn and Cd transport. The present study showed that rice co-expressing OsLCT1-OsHMA2-OsZIP3 (LHZ) had longer roots and shoots than wild-type (WT) rice after Zn and Cd treatments. The chlorophyll content was significantly higher, and the proline, malondialdehyde and H2O2 contents were significantly lower in co-transgenic lines than in WT under Cd and Zn stress. LHZ in the seedlings of transgenic rice decreased the root-to-shoot translocation of Cd after Cd and Zn treatments. At the filling stage, LHZ line reduced Cd accumulation in grain after Cd treatment. Moreover, LHZ line increased the translocation of Zn to grain and reduced the accumulation of Cd after Zn treatment. These results suggested that LHZ co-expression could effectively decrease the translocation and accumulation of Cd to grains, alleviated the oxidative stress of Cd and Zn, and finally enhanced the quality and safety of rice grains.

Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer.

Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2ß and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2ß-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.

Toxic effects of Cr(VI) on the bovine hemoglobin and human vascular endothelial cells: Molecular interaction and cell damage.

Hexavalent chromium [Cr(VI)] is the main harmful component in the atmosphere released by chemical industry. The study was conducted to assess Cr(VI) inducing cardiovascular diseases (CVDs) in vitro by investigating the effects of Cr(VI) on bovine hemoglobin (BHb) and human umbilical vein endothelial cells (HUVECs). Multi-spectroscopic techniques and molecular docking method were used to determine the interaction of Cr(VI) and BHb. Fluorescence spectra results showed that the quenching constant (Ksv) decreased with temperature raise, indicating that Cr(VI) quenches BHb fluorescence through static quenching mechanism. The number of binding sites was 1.14 (310 K), enthalpy and entropy changes revealed the interaction of Cr(VI) and BHb was driven by hydrogen bonds. The results of synchronous fluorescence and circular dichroism (CD) spectra suggested that Cr(VI) could change BHb conformation and influence the microenvironment of Trp and Tyr residues. Moreover, in order to study Cr(VI) induced HUVECs damage, inflammatory factors were detected at the mRNA level, JNK and p38 MAPK pathways were analyzed. The results shown that Cr(VI) could induce mRNA expression of NLRP3, ICAM-1, VCAM-1, TNF-α and IL-1ß, and increased intracellular ROS. Furthermore, Cr(VI) could induce oxidative stress in HUVECs, and then activate JNK and p38 MAPK pathways, ultimately lead to apoptosis of HUVECs by activating mitochondrial apoptosis pathways. These results suggested that Cr(VI) might bring about CVDs by both changing the BHb conformation and inducing HUVECs damage.