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As a member of the integrin family, integrin α3β1 (ITGA3) has been linked to intercellular communication and serves an important role in the signaling among cells and the extracellular matrix. MicroRNA (miR)‑199a‑5p has been demonstrated to be related to the pathogenesis and progression of multiple malignant diseases. However, the biological functions of miR‑199a‑5p and ITGA3 in colorectal cancer (CRC) have rarely been reported. The aim of the present study was to explore the roles of miR‑199a‑5p and ITGA3 in CRC. Immunohistochemistry staining and western blotting were applied to detect the protein expression of ITGA3 in CRC tissues and cells. Reverse transcription‑quantitative PCR was performed to investigate the expression of miR‑199a‑5p and ITGA3 mRNA. HCT‑116 cells were transfected with miR‑199a‑5p mimics, mimics control, short hairpin RNA targeting ITGA3, or pcDNA‑ITGA3 for the functional experiments. Dual luciferase reporter assay was applied to confirm whether miR‑199a‑5p targeted the 3' untranslated region (3'UTR) of ITGA3. The MTT, Transwell and wound healing assays were used to evaluate the proliferation, invasion and migration of CRC cells. Immunofluorescence assay was used to monitor the epithelial‑mesenchymal transition (EMT) biomarker expression. The results demonstrated downregulation of miR‑199a‑5p and upregulation of ITGA3 in CRC tissues and cell lines. miR‑199a‑5p mimics and knockdown of ITGA3 suppressed the proliferation, invasion and migration of CRC cells. Bioinformatics analysis and luciferase reporter assay indicated that miR‑199a‑5p targeted the 3'UTR of the ITGA3 transcript, and overexpression of ITGA3 reversed the tumor‑suppressive effects of miR‑199a‑5p elevation. In addition, the immunofluorescence assay suggested that miR‑199a‑5p mimics suppressed the EMT of CRC cells, whereas the overexpression of ITGA3 restored this effect. In conclusion, miR‑199a‑5p may act as a tumor suppressor by targeting and negatively regulating ITGA3 in CRC.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

Background Thyroid cancer (TC) is the most common endocrine malignancy worldwide. The incidence of TC is high and increasing worldwide due to continuous improvements in diagnostic technology. Therefore, identifying accurate prognostic predictions to stratify TC patients is important. Methods Raw data were downloaded from the TCGA database, and pairwise comparisons were applied to identify differentially expressed immune-related lncRNA (DEirlncRNA) pairs. Then, we used univariate Cox regression analysis and a modified Lasso algorithm on these pairs to construct a risk assessment model for TC. We further used qRT‒PCR analysis to validate the expression levels of irlncRNAs in the model. Next, TC patients were assigned to high- and low-risk groups based on the optimal cutoff score of the model for the 1-year ROC curve. We evaluated the signature in terms of prognostic independence, predictive value, immune cell infiltration, immune status, ICI-related molecules, and small-molecule inhibitor efficacy. Results We identified 14 DEirlncRNA pairs as the novel predictive signature. In addition, the qRT‒PCR results were consistent with the bioinformatics results obtained from the TCGA dataset. The high-risk group had a significantly poorer prognosis than the low-risk group. Cox regression analysis revealed that this immune-related signature could predict prognosis independently and reliably for TC. With the CIBERSORT algorithm, we found an association between the signature and immune cell infiltration. Additionally, immune status was significantly higher in low-risk groups. Several immune checkpoint inhibitor (ICI)-related molecules, such as PD-1 and PD-L1, showed a negative correlation with the high-risk group. We further discovered that our new signature was correlated with the clinical response to small-molecule inhibitors, such as sunitinib. Conclusions We have constructed a prognostic immune-related lncRNA signature that can predict TC patient survival without considering the technical bias of different platforms, and this signature also sheds light on TC’s overall prognosis and novel clinical treatments, such as ICB therapy and small molecular inhibitors.

Background Human DNA methylation is a common epigenetic regulatory mechanism, and it plays a critical role in various diseases. However, the potential role of DNA methylation in Ewing sarcoma (ES) is not clear. This study aimed to explore the regulatory roles of DNA methylation in ES. Methods The microarray data of gene expression and methylation were downloaded from the Gene Expression Omnibus (GEO) database, and analyzed via GEO2R. Venn analysis was then applied to identify aberrantly methylated-differentially expressed genes (DEGs). Subsequently, function and pathway enrichment analysis was conducted, a protein-protein interaction (PPI) network was constructed, and hub genes were determined. Besides, a connectivity map (CMap) analysis was performed to screen bioactive compounds for ES treatment. Results A total of 135 hypomethylated high expression genes and 523 hypermethylated low expression genes were identified. The hypomethylated high expression genes were enriched in signal transduction and the apoptosis process. Meanwhile, hypermethylated low expression genes were related to DNA replication and transcription regulation. The PPI network analysis indicated C3, TF, and TCEB1 might serve as diagnostic and therapeutic targets of ES. Furthermore, CMap analysis revealed 6 chemicals as potential options for ES treatment. Conclusions The introduction of DNA methylation characteristics over DEGs is helpful to understand the pathogenesis of ES. The identified hub aberrantly methylated DEGs and chemicals might provide some novel insights on ES treatment.

Background. Intervertebral disc degeneration (IDD) disease is a global challenge because of its predominant pathogenic factor in triggering low back pain, whereas cartilaginous endplate degeneration (CEPD) is the main cause of IDD. Accumulating evidence have indicated that the differentially expressed microRNAs (DEMs) and differentially expressed genes (DEGs) have been determined to be involved in multiple biological processes to mediate CEPD progression. However, the differentially expressed circular RNAs (DECs) and their potential biofunctions in CEPD have not been identified. Methods. GSE153761 dataset was analyzed using R software to predict DECs, DEMs, and DEGs. Pathway enrichment analysis of DEGs and host genes of DECs and protein-protein interaction network of DEGs were conducted to explore their potential biofunctions. Furthermore, we explore the potential relationship between DEGs and DECs. Results. There were 74 DECs, 17 DEMs, and 68 DEGs upregulated whereas 50 DECs, 16 DEMs, and 67 DEGs downregulated in CEPD group. Pathway analysis unveiled that these RNAs might regulate CEPD via mediating inflammatory response, ECM metabolism, chondrocytes apoptosis, and chondrocytes growth. A total of 17 overlapping genes were predicted between the host genes of DEGs and DECs, such as SDC1 and MAOA. Moreover, 6 upregulated DECs, of which hsa_circ_0052830 was the most upregulated circRNA in CEPD, were derived from the host genes SDC1, whereas 8 downregulated DECs were derived from the host genes MAOA. Conclusion. This will provide novel clues for future experimental studies to elucidate the pathomechanism of CEPD and therapeutic targets for CEPD-related diseases.

Objective. To study the pharmacological mechanisms of Siwu decoction (SWD) on primary dysmenorrhea (PDM) and verify with molecular docking. Methods. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was utilized to acquire the active compounds and their corresponding target genes. The GeneCards database was utilized in the search for target genes that were associated with PDM. The intersection genes from the active target genes of SWD and those associated with PDM represented the active target genes of SWD that act on PDM. The Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were both carried out by RGUI 3.6.1 and Cytoscape 3.6.0 software. Cytoscape was also utilized for creating a compound-target network, and a protein-protein interaction (PPI) network was created through the STRING database. Molecular docking simulations of the macromolecular protein target receptors and their corresponding compounds were performed using AutoDockTool 1.5.6 and AutoDock Vina software. Results. We identified 14 active compounds as well as 97 active target genes of SWD by using the TCMSP. We compared the 97 active target genes of SWD to the 299 target genes related to PDM, and 23 active target genes for SWD that act on PDM which correlated with 11 active compounds were detected. The compound-target network as well as the PPI network were created, in addition to selecting the most essential compounds and their targets in order to create a key compound-target network. The most essential compounds were kaempferol, beta-sitosterol, stigmasterol, and myricanone. The key targets were AKT1, PTGS2, ESR1, AHR, CASP3, and PGR. Lastly, molecular docking was used to confirm binding of the target with its corresponding compound. Conclusion. The pharmacological mechanisms of SWD that act on PDM were investigated, and the active compounds in the SWD for treating PDM were further verified.

Background. The competing endogenous RNA- (ceRNA-) mediated regulatory mechanisms are known to play a pivotal role in intervertebral disc degeneration (IDD). Our research intended to establish a ceRNA regulatory network related to IDD through bioinformatics analyses. Methods. The expression profiles of circRNA, miRNA, and mRNA were obtained from the public Gene Expression Omnibus (GEO) datasets. Then, we use sequence-based bioinformatics methods to select differentially expressed mRNAs (DEmRNAs), microRNAs (DEmiRNAs), or circRNAs (DEcircRNAs) related to IDD. We used ChEA3 to verify the targets of transcription factors (TFs). Then, we used DAVID to annotate the DEmRNAs. Finally, we constructed a potentially circRNA-miRNA-mRNA network related to IDD by predicting in the database (ENCORI, TargetScan, miRecords, miRmap, and circBank). Results. We identified 31 common DEmRNAs by Venn analysis, of which MMP2 was regarded as the key hub genes. Simultaneously, miR-423-5p and miR-185-5p were predicted as the upstream molecules of MMP2. Furthermore, a total of six DEcircRNAs were predicted as the upstream circRNAs of miR-423-5p and miR-185-5p. Then, a potential circRNA-miRNA-mRNA network related to IDD was constructed by bioinformatics analysis. Conclusion. A comprehensive ceRNA regulatory network was constructed, which was found to be significant in IDD progression.

BACKGROUND Little is known about the relationship between the site of infection, type of pathogen, and the occurrence of sepsis-associated liver dysfunction (SALD). This population study aimed to identify the sites and types of infection in SALD patients. MATERIAL AND METHODS We conducted a retrospective observational study using the Medical Information Mart for Intensive Care III. Patients with sepsis were divided into a SALD group and a control group. We evaluated the effect of the location of culture-positive specimens and the distribution of pathogens on the occurrence of SALD and then compared the clinical outcomes. RESULTS A total of 14 596 admissions were included, and the incidence of SALD was 11.96%. Positive bile culture (odds ratio [OR] 7.450, P

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9–11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Potato is the 4th largest staple food in the world currently. As a high biomass crop, potato harbors excellent potential to produce energy-rich compounds such as triacylglycerol as a valuable co-product. We have previously reported that transgenic potato tubers overexpressing WRINKLED1, DIACYLGLYCEROL ACYLTRANSFERASE 1, and OLEOSIN genes produced considerable levels of triacylglycerol. In this study, the same genetic engineering strategy was employed on potato leaves. The overexpression of Arabidopsis thaliana WRINKED1 under the transcriptional control of a senescence-inducible promoter together with Arabidopsis thaliana DIACYLGLYCEROL ACYLTRANSFERASE 1 and Sesamum indicum OLEOSIN driven by the Cauliflower Mosaic Virus 35S promoter and small subunit of Rubisco promoter respectively, resulted in an approximately 30- fold enhancement of triacylglycerols in the senescent transgenic potato leaves compared to the wild type. The increase of triacylglycerol in the transgenic potato leaves was accompanied by perturbations of carbohydrate accumulation, apparent in a reduction in starch content and increased total soluble sugars, as well as changes of polar membrane lipids at different developmental stages. Microscopic and biochemical analysis further indicated that triacylglycerols and lipid droplets could not be produced in chloroplasts, despite the increase and enlargement of plastoglobuli at the senescent stage. Possibly enhanced accumulation of fatty acid phytyl esters in the plastoglobuli were reflected in transgenic potato leaves relative to wild type. It is likely that the plastoglobuli may have hijacked some of the carbon as the result of WRINKED1 expression, which could be a potential factor restricting the effective accumulation of triacylglycerols in potato leaves. Increased lipid production was also observed in potato tubers, which may have affected the tuberization to a certain extent. The expression of transgenes in potato leaf not only altered the carbon partitioning in the photosynthetic source tissue, but also the underground sink organs which highly relies on the leaves in development and energy deposition.

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

Emergence of the third-generation cephalosporin-resistant hypervirulent Klebsiella pneumoniae due to the acquisition of a self-transferable blaDHA-1-carrying plasmid by an ST23 strain

Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco (Nicotiana tabacum) leaf and potato (Solanum tuberosum) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways.

Background The aim of this study was to analyze the clinical testing data of syphilis suspected children, to give more comprehensive detection information and offer experimental basis for the clinical diagnosis of syphilis. Methods From April 2010 to December 2012, 141 suspected syphilis children, 0-3 years old in XuZhou Children's Hospital were selected and divided into two groups: infants group (0-1 years old, 119 cases) and children group (1-3 years old, 22 cases). Blood samples were collected from these children and following experimental detection methods were used: the rapid plasma reagin (RPR) test, the colloidal gold test (SYP), the enzyme-linked immuno-sorbent assay (ELISA) and the Treponema pallidum particle agglutination (TPPA) test. The relevant experimental data were analyzed by SPSS 13.0 software. Results The positive rate of ELISA was the highest, RPR was the lowest; the positive rate of SYP and TPPA were higher than RPR, the positive rate of SYP and TPPA were lower than ELISA, and the differences were statistically significant. Among the 86 false positives, the rate for ELISA was the highest, and no TPPA false positive was found. False positive were higher in the children group than the infant group. Conclusions High false positive rate of ELISA could be caused by hemolysis. RPR had low sensitivity in suspected syphilis neonates, and SYP was suitable for emergency treatment. TPPA was fit for the diagnosis of syphilis. Thus a combination of all these methods would be the best choice to cure syphilis infection in children. Final diagnosis can only be confirmed after periodically reexamining samples of suspected syphilis children.

The physical and chemical characteristics of soil and water sources affect desert plants’ growth, which is essential for the ecological protection in arid areas. The typical patch patterns of Haloxylon ammodendron in the oasis-desert ecotone in the southern margin of the Manas River Basin consists of bare patches (BP) and vegetation patches (VP). The water sources of H. ammodendron were studied using stable isotope technology, and the soil physical and chemical properties were monitored and analyzed. The results showed that the soil moisture presented a reversed “S” type curve, and the total salt content of the soil presented an “S” type curve. A “wet island” and “cold island” were formed in the low salt area with H. ammodendron at the center. NaCl was most abundant in the BP soil, and the milligram equivalent of Cl− was 80–90%, while CaSO4 was most abundant in the VP soil, in which the milligram equivalent of SO42− was 80–100%. Before the rain, H. ammodendron mainly relied on the soil water from a deeper layer (≥60 cm) to maintain its growth. However, after the rain, H. ammodendron mainly relied on shallow soil water (&lt, 60 cm) to maintain its growth.

Objective: To explore the mechanism of Kangguan decoction in the treatment of coronavirus disease 2019 (COVID-19) and then perform preliminary verification. Methods: The effective compounds and target genes of Kangguan decoction were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. COVID-19 related target genes were searched in the GeneCards database. The active target genes of Kangguan decoction acting on COVID-19 were identified to perform GO function enrichment and KEGG pathway enrichment analysis. The compound-target network and protein-protein interaction were constructed;Molecular docking simulations of macromolecular protein target receptors and their corresponding compounds were performed. The clinical data of COVID-19 patients were retrieved from their electronic medical records of Nantong Third People's Hospital. Results: We screened out 137 effective compounds and 274 effective target genes of Kangguan decoction from TCMSP. The active target genes of Kangguan decoction were compared with the COVID-19 related target genes, and 63 active target genes for Kangguan decoction acting on COVID-19 were identified. GO function enrichment and KEGG pathway enrichment analysis were performed. The compound-target network and PPI network were constructed and the key compounds and key targets were selected to construct a key compound-target network. Finally, the binding of the target and its corresponding components was verified by molecular docking and two clinical cases with obvious clinical efficacy after Kangguan decoction application were demonstrated. Conclusion: The pharmacological mechanism of Kangguan decoction acting on COVID-19 has been explored, and the active compounds and targets of Kangguan decoction acting on COVID-19 and clinical efficacy for Kangguan decoction treating COVID-19 patients have been preliminarily verified.

The objective of the present study was to evaluate the combined application of measuring cystatin C (Cys-C) and hemoglobin A1c (HbA1c) levels for early renal injury in pediatric patients with type 2 diabetes. A total of 130 children with type 2 diabetes admitted to our hospital from May 2013 to July 2015 were selected. Patients were divided according to whether there was complication of renal injury. In group A (n=65), the patients had renal injury and in group B (n=65), the patients did not have renal injury. The levels of Cys-C and HbA1c in the two groups were examined. The results showed that the levels of Cys-C and HbA1c of patients in group A were significantly higher than those in group B (P

MicroRNAs are important regulators of the pathogenesis of B-cell acute lymphoblastic leukaemia (B-ALL). In this study, we identified miR-3173 and its predicted target gene PTK2 were correspondingly differentially expressed in B-ALL patients. In B-ALL cell lines, CCK-8 proliferation assay revealed that miR-3173 could inhibit the cell proliferation. Moreover, transwell assay revealed that miR-3173 could also inhibit cell migration and invasion in B-ALL cell lines. Luciferase assays revealed that miR-3173 directly bound to the 3'untranslated region of PTK2, and western blotting showed that miR-3173 suppressed the expression of PTK2 at the protein level. Generally, this study indicates that miR-3173 negatively regulates PTK2 and inhibits proliferation and invasion of B-ALL cell lines. Thus, miR-3173 may represent a potential therapeutic molecule for B-ALL intervention.

Neurogenic locus notch homolog protein (Notch) signaling mediates intracellular communication and may regulate cell fate decisions, including cell proliferation, differentiation, and apoptosis. Mesenchymal stem cells (MSCs) possess immunomodulatory properties and the potential for use in stem cell replacement treatments. The aim of the present study was to evaluate the therapeutic effects of human placenta‑deviated MSCs (hPMSCs) in asthma and to investigate the mechanisms of Notch signaling mediated by transplanted MSCs. A Sprague‑Dawley rat ovalbumin (OVA)‑sensitized acute asthma model was established and challenged. MSCs derived from human placenta (hPMSCs) were transplanted into the asthmatic rats. Transplantation resulted in reduced Notch‑1, Notch‑2 and jagged‑1, and increased Notch‑3, Notch‑4 and delta‑like ligand (delta)‑4 expression in lung, blood, and lymph samples. Notch‑1, Notch‑2, and jagged‑1 expression in OVA‑treated rats was significantly decreased compared with controls and hPMSC‑treated rats; however, Notch‑3, Notch‑4 and delta‑4 expression was significantly increased. Serum interferon‑γ significantly increased after hPMSCs transplantation, whereas interleukin‑4 and immunoglobulin E decreased. In OVA‑treated rats, Notch‑1, Notch‑2 and jagged‑1 levels were increased in the lymph compared with the blood, although Notch‑4 and delta‑4 levels were decreased. Peribronchial infiltration of cells and goblet cell hyperplasia were markedly decreased in the OVA + hPMSCs group compared with those in the OVA‑treated and control groups. Alterations in Notch signaling pathway expression were accompanied by decreased inflammatory cell infiltration, goblet cell hyperplasia and mucus production in lung tissues. The results of the present study are consistent with hPMSC suppression of asthma symptoms and inflammation by regulating the Notch signaling pathway in the rat asthma model.

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

Background: The role of post-translational modification of regulatory β-subunits in the control of large conductance potassium (BK) channels is largely unknown. Results: β4-subunit palmitoylation controls surface trafficking of BK channel α-subunit splice variants. Conclusion: Palmitoylation of β4 masks an α-subunit trafficking motif to control surface delivery. Significance: Palmitoylation of regulatory subunits provides a dynamic mechanism to control surface trafficking of specific BK channel variants., Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels.

The host acquired immune response, especially the humoral immunity, plays key roles in preventing bacterial pneumonia in the lung. Our previous research demonstrated that interleukin 17-producing γδ T cells (IL17-γδ T cells) have a protective effect on the early innate immune response during acute pulmonary Pseudomonas aeruginosa infection. However, whether IL17-γδ T cells also play a role in humoral immunity is unknown. In this study, an acute pulmonary P. aeruginosa infection model was established in wild-type and γδ TCR−/− C57BL/6 mice. The expression of IL-17 on γδ T cells isolated from infected lung tissues increased rapidly and peaked at day 7 after acute infection with P. aeruginosa. Compared with wild-type infected mice, the levels of total immunoglobulins including IgA, IgG, and IgM in the serum and BALF were significantly decreased in γδ TCR−/− mice, with the exception of IgM in the BALF. Moreover, CD69 expression in B cells from the lungs and spleen and the level of BAFF in the plasma were also decreased in γδ TCR−/− mice. IL17-γδ T cell transfusion significantly improved the production of immunoglobulins, B cell activation and BAFF levels in γδ TCR−/− mice compared with γδ TCR−/− mice without transfusion; this effect was blocked when cells were pretreated with an IL-17 antibody. Together, these data demonstrate that IL17-γδ T cells are involved in CD19+ B cell activation and the production of immunoglobulins during acute pulmonary P. aeruginosa infection. Thus, we conclude that IL17-γδ T cells may facilitate the elimination of bacteria and improve survival through not only innate immunity but also humoral immunity.

Background: Enzymes controlling (de)palmitoylation of ion channels are poorly defined. Results: Palmitoylation of BK channels by zDHHC22 and zDHHC23 and depalmitoylation by LYPLA1 and LYPLAL1 controls BK channel cell surface expression. Conclusion: Acyl protein transferases and thioesterases display substrate specificity and control BK channel surface expression. Significance: Understanding how channels are (de)palmitoylated is essential for defining the role of palmitoylation in ion channel physiology., Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0–S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ∼30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium-and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by similar to 55% in the C53:54:56 A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Palmitoylation is emerging as an important and dynamic regulator of ion channel function; however, the specificity with which the large family of acyl palmitoyltransferases ( zinc finger Asp-His-His-Cystype-containing acyl palmitoyltransferase (DHHCs)) control channel palmitoylation is poorly understood. We have previously demonstrated that the alternatively spliced stress-regulated exon (STREX) variant of the intracellular C-terminal domain of the large conductance calcium- and voltage- activated potassium (BK) channels is palmitoylated and targets the STREX domain to the plasma membrane. Using a combined imaging, biochemical, and functional approach coupled with loss-of-function ( small interfering RNA knockdown of endogenous DHHCs) and gain-of-function ( overexpression of recombinant DHHCs) assays, we demonstrate that multiple DHHCs control palmitoylation of the C terminus of STREX channels, the association of the STREX domain with the plasma membrane, and functional channel regulation. Cysteine residues 12 and 13 within the STREX insert were the only endogenously palmitoylated residues in the entire C terminus of the STREX channel. Palmitoylation of this dicysteine motif was controlled by DHHCs 3, 5, 7, 9, and 17, although DHHC17 showed the greatest specificity for this site upon overexpression of the cognate DHHC. DHHCs that palmitoylated the channel also co-assembled with the channel in co-immunoprecipitation experiments, and knockdown of any of these DHHCs blocked regulation of the channel by protein kinase A-dependent phosphorylation. Taken together our data reveal that a subset of DHHCs controls STREX palmitoylation and function and suggest that DHHC17 may preferentially target cysteine-rich domains. Finally, our approach may prove useful in elucidating the specificity of DHHC palmitoylation of intracellular domains of other ion channels and transmembrane proteins.

Large conductance calcium- and voltage-activated potassium (BK) channels assemble as macromolecular signaling complexes and are potently regulated by reversible protein phosphorylation. However, although numerous studies have revealed regulation of BK channels through changes in direct phosphorylation of the pore-forming alpha-subunits the functional role of changes in phosphorylation of defined adapter/signaling proteins within the complex on channel function are essentially not known. Here, we demonstrate that mammalian BK channels are potently regulated by endogenous protein-tyrosine kinase and protein-tyrosine phosphatase activity closely associated with the channel. BK channel regulation was not dependent upon direct phosphorylation of the BK alpha-subunit, rather channel function was controlled by the tyrosine phosphorylation status of the adapter protein cortactin that assembles directly with the BK channel. Our data thus reveal a novel mode for BK channel regulation by reversible tyrosine phosphorylation and strongly support the hypothesis that phosphorylation-dependent regulation of accessory proteins within the BK channel signaling complex represents an important target for control of BK channel function.

Homophilic ligation of CD31, a member of the Ig superfamily of adhesion receptors, promotes macrophage clearance of apoptotic leukocytes by a mechanism hitherto not described. In studying CD31-dependent regulation of β1-integrin binding of fibronectin-coated Latex™ beads, we discovered a role for the voltage-gated potassium channel ether-à-go-go-related gene (ERG) as a downstream effector of CD31 signaling. ERG was identified by tandem mass spectrometry as a 140-kDa protein, which was selectively modified with biotin following the targeted delivery of a biotin-transfer reagent to CD31 using Fab fragments of an anti-CD31 mAb. Similar results were obtained with macrophages but not K562 cells, expressing a truncated cytoplasmic tail of CD31, which failed to regulate bead binding. Colocalization of CD31 with ERG was confirmed by immunofluorescence for K562 cells and macrophages. We now demonstrate that the resting membrane potential of macrophages is depolarized on contact with apoptotic cells and that CD31 inhibits the ERG current, which would otherwise function to repolarize. Sustained depolarization favored the firm binding of phagocytic targets, a prerequisite for efficient engulfment. Our results identify ERG as a downstream effector of CD31 in the regulation of integrin-dependent binding of apoptotic cells by macrophages.

Large conductance voltage- and calcium-activated potassium (BK Ca ) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming α-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of α-subunits, it is not known whether phosphorylation of all or single α-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the α-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four α-subunits is required for channel activation. In contrast, inhibition of BK Ca channel activity requires phosphorylation of only a single α-subunit at a splice insert (STREX)-specific PKA consensus site (S4 STREX ). Thus, distinct modes of BK Ca channel regulation by PKA phosphorylation exist: an “all-or-nothing” rule for activation and a “single-subunit” rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BK Ca channels by reversible protein phosphorylation.

Large conductance, calcium- and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to associate with the channel and regulate its activity. A synthetic polypeptide encompassing the central d position leucine residues in LZ1 blocks the regulation of recombinant mouse BK channels by endogenous PKA in HEK293 cells. In contrast, neither an alanine-substituted LZ1 peptide nor a peptide corresponding to another, more C-terminal putative leucine zipper, LZ2, had any effect on regulation of the channels by endogenous PKA. Mutagenesis of the central two LZ1 d position leucines to alanine in the BK channel also eliminated regulation by endogenous PKA in HEK293 cells without altering the channel sensitivity to activation by voltage or by exogenous purified PKA. Inclusion of the STREX splice insert in the BK channel protein, which switches channel regulation by PKA from stimulation to inhibition, did not alter the requirement for an intact LZ1. Although PKA does not bind directly to the channel protein in vitro, mutation of LZ1 abolished co-immunoprecipitation of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in the BK channel protein may provide anchoring for other regulatory enzyme complexes.

Background: Gene expression analysis is powerful for investigating the underlying mechanisms of Duchenne muscular dystrophy (DMD). Previous studies mainly neglected co-expression or transcription factor (TF) information. Here we integrated TF information into differential co-expression analysis (DCEA) to explore new understandings of DMD pathogenesis. Methods: Using two microarray datasets from Gene Expression Omnibus (GEO) database, we firstly detected differentially expressed genes (DEGs) and pathways enriched with DEGs. Secondly, we constructed differentially regulated networks to integrate the TF-to-target information and the differential co-expression genes. Results: A total of 454 DEGs were detected and both KEGG pathway and ingenuity pathway analysis revealed that pathways enriched with aberrantly regulated genes are mostly involved in the immune response processes. DCEA results generated 610 pairs of DEGs regulated by at least one common TF, including 78 pairs of co-expressed DEGs. A network was constructed to illustrate their relationships and a subnetwork for DMD related molecules was constructed to show genes and TFs that may play important roles in the secondary changes of DMD. Among the DEGs which shared TFs with DMD, six genes were co-expressed with DMD, including ATP1A2, C1QB, MYOF, SAT1, TRIP10 ,a ndIFI6. Conclusion: Our results may provide a new understanding of DMD and contribute potential targets for future therapeutic tests. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/ 13000_2014_210

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.

Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming alpha-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to beta-subunits in vivo. Functional expression of the STREX-1 containing alpha-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of alpha-subunits expressing the mouse STREX-1 exon.

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 μm dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel α-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5′-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nm of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel alpha subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Endothelial hyperpermeability is a significant problem in many cardiovascular disorders, including vascular inflammation, ischemia-reperfusion injury, thrombosis and atherosclerosis. BuShen-KangShuai (BS-KS) tablet, traditional Chinese medicines, has been used for many years in China to treat

Large conductance calcium- and voltage- gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C-terminus of the pore-forming α-subunits. However, how PKA phosphorylation of the α-subunit effects changes in channel activity are unknown. The STREX variant of BK channels is inhibited by PKA as a result of phosphorylation of a serine residue within the evolutionary conserved STREX insert. As this inhibition is dependent upon phosphorylation of only a single α-subunit in the channel tetramer we hypothesised that phosphorylation results in major conformational rearrangements of the C-terminus. Using a combined imaging, biochemical and electrophysiological strategy we have defined the mechanism of PKA-inhibition of BK channels. We demonstrate that the cytosolic C-terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionary conserved cysteine residues. PKA-phosphorylation of STREX dissociates the C-terminus from the plasma membrane resulting in channel inhibition. Abolition of channel palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl-transferases prevents PKA-mediated inhibition. Thus PKA inhibition of BK channels is conditional upon the palmitoylation status of the channel. Palmitoylation and phosphorylation are both dynamically regulated thus cross-talk between these two major post-translational signalling cascades provides a novel mechanism for conditional regulation of BK channels. Interplay of these distinct signalling cascades has important implications for the dynamic regulation of BK channels and the control of physiological homeostasis.

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming α-subunits. However, the molecular mechanism(s) by which phosphorylation of the α-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single α-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

Calcium-activated potassium (BK) channels play a central role in regulating multiple physiological processes, from the control of blood flow to neuronal excitability. Coordinated regulation of BK channel activity by changes in actin cytoskeleton dynamics has been implicated in several of these processes and related disease states such as epilepsy and stroke. However, how BK channels interact with the actin cytoskeleton is essentially unknown. Here we demonstrate noncanonical Src homology domain 3 (SH3) binding site motifs in the intracellular C terminus of the BK channel pore-forming alpha-subunit that are conserved from fish to humans. These noncanonical motifs target multiple SH3 domain cellular signaling proteins to BK channels, including the SH3 adapter protein cortactin (EMS1). We demonstrate that cortactin provides a molecular bridge between BK channels and the cortical actin cytoskeleton in cells. Disruption of the SH3-mediated interaction prevents the regulation of BK channel activity controlled by changes in actin cytoskeletal dynamics. Targeting of cortactin to BK channels via a novel, noncanonical SH3 domain binding motif has important implications for the coordination of BK channel function in normal physiology and disease.

The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

1. Large-conductance Ca(2+)- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO alpha-subunit variants expressed in human embryonic kidney (HEK 293) cells. 2. STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nM) for 2 h. 3. The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nM okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI-2. 4. ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4(STREX)A) were also activated by PKA. Importantly, DEX blocked PKA activation of S4(STREX)A channels in a PP2A-dependent manner. 5. Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets.

The regulation of large conductance calcium- and voltage-activated potassium (BK) currents by activation of the protein kinase C (PKC) and glucocorticoid signalling pathways was investigated in AtT20 D16:16 clonal mouse anterior pituitary corticotroph cells. Maximal activation of PKC using the phorbol esters, 4β-phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13 dibutyrate (PDBu) and 12-deoxyphorbol 13-phenylacetate (dPPA) elicited a rapid, and sustained, inhibition of the outward steady-state voltage- and calcium- dependent potassium current predominantly carried through BK channels. The effect of PMA was blocked by the PKC inhibitors bisindolylmaleimide I (BIS; 100 nM) and chelerythrine chloride (CHE; 25 μM) and was not mimicked by the inactive phorbol ester analogue 4α-PMA. PMA had no significant effect on the 1 mM tetraethylammonium (TEA)-insensitive outward current or pharmacologically isolated, high voltage-activated calcium current. PMA had no significant effect on steady-state outward current in cells pre-treated for 2 h with 1 μM of the glucocorticoid agonist dexamethasone. Dexamethasone had no significant effect on steady-state outward current amplitude or sensitivity to 1 mM TEA and did not block PMA-induced translocation of the phorbol ester-sensitive PKC isoforms, PKCα and PKCe, to membrane fractions. Taken together these data suggest that in AtT20 D16:16 corticotroph cells BK channels are important targets for PKC action and that glucocorticoids inhibit PKC signalling downstream of PKC activation. In many neuroendocrine cells of the anterior pituitary gland, activation of the protein kinase C (PKC) intracellular signalling pathway leads to sustained cellular excitability and neurosecretion although the cellular mechanisms and targets for PKC are poorly understood (Ozawa & Sand, 1986; Mason et al. 1988). In anterior pituitary corticotrophs PKC mediates the sustained phase of adrenocorticotrophin (ACTH) secretion stimulated by activation of the phospholipase C pathway by the hypothalamic secretagogue vasopressin (Carvallo & Aguilera, 1989; Oki et al. 1990). Vasopressin elicits a biphasic elevation of intracellular free calcium (Corcuff et al. 1993; Tse & Lee, 1998) and during the sustained phase of calcium influx stimulates PKC translocation and enhances PKC activity at the plasma membrane, an effect that is mimicked by the cell-permeant PKC-activating phorbol esters (Carvallo & Aguilera, 1989). In AtT20 mouse corticotroph cells phorbol-ester-mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage-dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al. 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC-induced calcium influx results, at least in part, from inhibition of TEA-sensitive potassium conductances. This inhibition results in membrane depolarization and subsequent, indirect, enhancement of voltage-gated calcium influx (Reisine & Guild, 1987; Reisine, 1989). However, ionic conductances regulated by PKC activation in corticotrophs have not been identified. In rat GH4C1 pituitary cells activation of PKC results in inhibition of the TEA-sensitive large conductance calcium- and voltage-activated potassium (BK) channels (Shipston & Armstrong, 1996), which act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997). Furthermore, BK channels are an important target for cellular regulation by two distinct, physiologically relevant, intracellular signalling pathways in AtT20 corticotrophs. Activation of the cAMP-dependent protein kinase pathway results in inhibition of BK channels leading to a robust secretory response. Protein kinase A (PKA)-mediated inhibition of BK channel function is blocked by activation of a protein-synthesis-dependent signalling cascade activated by glucocorticoid hormones. The cross-talk between these two pathways at the level of BK channels is an important determinant of the secretory response in this system (Shipston et al. 1996; Lim et al. 1998; Tian et al. 1998). As TEA-sensitive BK channels act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997) and PKC-mediated calcium influx is dependent upon inhibition of a TEA-sensitive conductance in AtT20 cells (Reisine & Guild, 1987; Reisine, 1989), we have addressed whether BK channels are an important cellular target for PKC action in this system. Furthermore, as glucocorticoids block PKC-stimulated ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995) and antagonize PKA-mediated inhibition of BK channels in this system (Shipston et al. 1996; Tian et al. 1998) we have addressed the question as to whether glucocorticoids also block PKC-mediated regulation of BK channels in AtT20 D16:16 corticotroph cells.

Adrenal glucocorticoid hormones rapidly exert powerful effects on neurons, immune and neuroendocrine cells through induction of de novo protein synthesis. In this study, we investigated, using mouse clonal anterior pituitary AtT20 D16:16 corticotrophs, whether (i) glucocorticoids rapidly inhibit glucose transport and (ii) whether this inhibition of glucose transport is directly correlated with early inhibition of ACTH secretion. Glucose uptake in AtT20 D16:16 cells was Na+-independent because the Na+-independent glucose transport inhibitor phloretin (100 microM) completely inhibited specific 14C-deoxygluose (DoG) uptake and replacement of extracellular Na+ with N-methyl D-glucamine+ had no effect. Furthermore, the Na+-independent glucose transporters, GLUTs 1 and 3 were expressed in AtT20 D16:16 cells. The synthetic type II glucocorticoid receptor agonist dexamethasone, rapidly, within 2 h, inhibited DoG uptake into AtT20 D16:16 cells through a mechanism that was dependent on de novo mRNA synthesis. Glucocorticoid inhibition of glucose transport was not correlated with early inhibition of ACTH secretion because removal of glucose from the external medium had no effect on CRF-stimulated ACTH secretion or the efficacy of early glucocorticoid inhibition of ACTH release. Although the Na+-independent glucose transport inhibitor phloretin significantly inhibited CRF-stimulated ACTH release, this effect of phloretin was a result of its potent activation of large conductance calcium-activated potassium (BK) channels. These data suggest that different molecular pathways and/or glucocorticoid induced proteins underlie the mechanism(s) of early glucocorticoid inhibition of glucose uptake and ACTH release, respectively.

Background Although Klebsiella pneumoniae bloodstream infections (KP-BSIs) have recently attracted attention due to an alarming raise in morbidity and mortality, there have been few reports on the epidemiology of KP-BSIs in mainland China. We sought to describe the epidemiological, microbiological, and clinical characteristics of KP-BSIs, focusing on the risk factors of carbapenem resistance and patient mortality. Methods A retrospective analysis of WHONET data of KP-BSI patients admitted to a teaching hospital in Shanghai, China, between January 1, 2011 and December 31, 2015 was performed, and the annual percentage of patients with carbapenem-resistant K. pneumoniae (CRKP) was determined. Risk factors related to the carbapenem resistance and patient mortality were analyzed using binary logistic regression model. The genetic relatedness of CRKP strains isolated from intensive care unit (ICU) patients was determined by pulsed-field gel electrophoresis (PFGE). Results A total of 293 incidences of KP-BSIs were identified in a 5-year period, 22.18% of these (65/293) were CRKP strains, and the proportion of CRKP-BSI in ICU was 59.62% (31/52), equaling the levels observed in the epidemic regions. A number of KP-BSIs (114), obtained from January 1, 2014, to December 31, 2015, were further investigated. Skin and soft tissue infection source (odds ratio [OR] 26.63, 95% confidence interval [CI] 4.8–146.8) and ICU-acquired infection (OR 5.82, 95% CI 2.0–17.2) was shown to be powerful risk factors leading to the development of CRKP-BSI. The crude 28-day mortality rates of KP-BSI and CRKP-BSI patients were 22.8% and 33.3%, respectively. Lung as the probable source of infection (OR 4.23, 95% CI 1.0–17.3), and high Sequential Organ Failure Assessment (SOFA) score (OR 1.40, 95% CI 1.2–1.6) were strong prognostic factors determining crude 28-day KP-BSI mortality rates. PFGE analysis demonstrated that 10/11 random CRKP isolates in ICU belonged to the same clonal type. Conclusions During the study period, we observed a significant increase in the occurrence of CRKP infections among the identified KP-BSIs in our hospital and especially in ICU, and we demonstrated that carbapenem resistance is associated with the increased mortality of KP-BSI patients.