Improving Certified Registered Nurse Anesthetists' Adherence to a Standardized Intraoperative Lung Protective Ventilation Protocol.

PURPOSE: The use of lung protective ventilation (LPV) during general anesthesia is an effective strategy among certified registered nurse anesthetists (CRNAs) to reduce and prevent the incidence of postoperative pulmonary complications. The purpose of this project was to implement a LPV protocol, assess CRNA provider adherence, and investigate differences in ventilation parameters and postoperative oxygen requirements. DESIGN: This quality improvement project was conducted using a pre- and postimplementation design. METHODS: Sixty patients undergoing robotic laparoscopic abdominal surgery and 35 CRNAs at a community hospital participated. An evidence-based intraoperative LPV protocol was developed, CRNA education was provided, and the protocol was implemented. Pre- and postimplementation, CRNA knowledge, and confidence were assessed. Ventilation data were collected at 1-minute intervals intraoperatively and oxygen requirements were recorded in the postanesthesia care unit (PACU). FINDINGS: Use of intraoperative LPV strategies increased 2.4%. Overall CRNA knowledge (P = .588), confidence (P = .031), and practice (P < .001) improved from pre- to postimplementation. Driving pressures decreased from pre- to postimplementation (P < .001). Supplemental oxygen use on admission to the PACU decreased from 93.3% to 70.0%. CONCLUSIONS: Educational interventions and implementation of a standardized protocol can improve the use of intraoperative LPV strategies and patient outcomes.

Cost-related prescription non-adherence and patient-reported outcomes in systemic lupus erythematosus: The Michigan Lupus Epidemiology & Surveillance program.

OBJECTIVES: Medication access and adherence play key roles in determining patient outcomes. We investigated whether cost-related non-adherence (CRNA) to prescription medications was associated with worse patient-reported outcomes in a population-based systemic lupus erythematosus (SLE) cohort. METHODS: Sociodemographic and prescription data were collected by structured interviews in 2014-2015 from patients meeting SLE criteria in the established Michigan Lupus Epidemiology & Surveillance (MILES) Cohort. We examined the associations between CRNA and potential confounders such as sociodemographics and health insurance coverage, and outcome measures of SLE activity and damage using multivariable linear regression. RESULTS: 462 SLE participants completed the study visit: 430 (93.1%) female, 208 (45%) Black, and mean age 53.3 years. 100 (21.6%) participants with SLE reported CRNA in the preceding 12 months. After adjusting for covariates, CRNA was associated with both higher levels of current SLE disease activity [SLAQ: ß coeff 2.7 (95% CI 1.3, 4.1), p < 0.001] and damage [LDIQ ß coeff 1.4 (95% CI 0.5, 2.4), p = 0.003]. Race, health insurance status, and fulfilling Fibromyalgia (FM) Survey Criteria were independently associated with both higher (worse) SLAQ and LDIQ scores; female sex was further associated with higher SLAQ scores. CONCLUSION: Patients with SLE who reported CRNA in the previous 12 months had significantly worse self-reported current disease activity and damage scores compared to those not reporting CRNA. Raising awareness and addressing barriers or concerns related to financial implications and accessibility issues in care plans may help to improve these outcomes.

Effect of temperature and IRF-9 gene-knockout on dynamics of vRNA, cRNA, and mRNA of viral hemorrhagic septicemia virus (VHSV).

The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12-36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3' end of cRNA might be lower than that to the 3' end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication.

Boron Clusters as Enhancers of RNase H Activity in the Smart Strategy of Gene Silencing by Antisense Oligonucleotides.

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with "two faces", namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3'-Iron-1,2,1',2'-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a "smart" strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.

Cystathionine ß-synthase is required for oocyte quality by ensuring proper meiotic spindle assembly.

OBJECTIVES: Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine ß-synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation. MATERIALS AND METHODS: Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study. RESULTS: We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore-microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co-localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of α-tubulin. CONCLUSIONS: These results reveal that CBS is required for the acetylation of α-tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.

Established Protocols for cRNA Expression and Voltage-Clamp Characterization of the P2X7 Receptor in Xenopus laevis Oocytes.

P2X7 receptors (P2X7Rs) are fast ATP4--gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP4- binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results.

The actions of neonicotinoid insecticides on nicotinic acetylcholine subunits Ldα1 and Ldα8 of Leptinotarsa decemlineata and assembled receptors.

The insect nicotinic acetylcholine receptor (nAChR) is a pentameric channel protein and also a target for neonicotinoids. There are few reported studies on the molecular interactions of Leptinotarsa decemlineata nAChRs with neonicotinoids. In this study, we analyzed the response of acetylcholine and neonicotinoids (thiamethoxam [TMX], imidacloprid [IMI], and clothianidin [CLO]) on hybrid receptors constructed by nAChR α1 and α8 subunits of L. decemlineata (Ldα1 and Ldα8) co-expressed with rat ß2 subunit (rß2) at different capped RNA (cRNA) ratios in Xenopus oocytes. In addition, we evaluated the expression changes of Ldα1 and Ldα8 after median lethal dose of TMX treatment for 72 h by quantitative polymerase chain reaction (qPCR). The resulting functional nAChRs Ldα1/rß2 and Ldα1/Ldα8/rß2 showed different pharmacological characteristics. The neonicotinoids tested showed lower agonist affinity on Ldα1/Ldα8/rß2 compared to Ldα1/rß2 at same ratios of subunit cRNAs. The sensitivities of neonicotinoids tested for Ldα1/rß2 and Ldα1/Ldα8/rß2 at cRNA ratios of 5:1, 1:1 and 5:5:1, 1:1:1, respectively, were lower than those for nAChRs at ratios of 1:5 and 1:1:5, respectively, whereas the values of maximum response (Imax ) varied. For Ldα1/Ldα8/rß2, a reduction of Lda8 cRNA resulted in increased sensitivity to IMI and decreased sensitivity to TMX. The expression of Ldα1 and Ldα8 significantly decreased in adults by 82.12% and 47.02%, respectively, while Ldα8 was significantly upregulated by 2.44 times in 4th instar larvae after exposure to TMX. We infer that Ldα1 and Ldα8 together play an important role in the sensitivity of L. decemlineata to neonicotinoids.

CircMMP11 overexpression predicts the poor survival of non-small cell lung cancer and downregulates miR-143 through methylation to suppress cell proliferation.

BACKGROUND: CircMMP11 is a characterized circRNA with oncogenic function in breast cancer. In this study, we explored the involvement of circMMP11 in non-small cell lung cancer (NSCLC). METHODS: Paired cancer and non-cancer tissues were collected from 66 NSCLC patients, and the expression of circMMP11 and miR-143 in these tissues were detected using RT-qPCRs. Overexpression levels of circMMP11 and miR-143 were performed by transfection, and their crosstalk was analyzed by RT-qPCRs. The effect of circMMP11 overexpression on miR-143 methylation was analyzed by methylation-specific PCR. CCK-8 assay was performed to analyze the roles of miR-143 and circMMP11 in regulating NSCLC cell proliferation. RESULTS: We found that circMMP11 was overexpressed in NSCLC and predicted patients' poor survival. Moreover, a close correlation between circMMP11 and miR143 was observed. In NSCLC cells, circMMP11 overexpression reduced miR-143 expression and increased miR-143 methylation. CCK-8 assay analysis showed that miR-143 reversed the enhancing effects of circMMP11 overexpression on cell proliferation. CONCLUSIONS: CircMMP11 is overexpressed in NSCLC and predicts poor survival. In addition, circMMP11 may downregulate miR-143 through methylation to suppress cell proliferation.

Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus.

Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5' and 3' UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1-4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14th nucleotide uridine (U14') of the 5' UTR and the 13th nucleotide adenosine (A13) of the 3' UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14' and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13' and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity.

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

The identification of induction chemo-sensitivity genes of laryngeal squamous cell carcinoma and their clinical utilization.

PURPOSE: To identify potential molecular markers for induction chemotherapy of Laryngeal squamous cell carcinoma (LSCC). METHODS: Differently expressed genes between chemo-sensitive group (seven cases) and chemo-insensitive (five cases) group after induction chemotherapy by TPF were identified by microarrays. Bayes network and Random forest analyses were employed to identify core genes for induction chemotherapy. The diagnostic value of these core genes was also evaluated by ROC analysis. RESULTS: Six genes (SPP1, FOLR3, KYNU, LOC653219, ADH7 and XAGE1A) are highly expressed, while seven gene (CADM1, NDUFA4L2, CCND2, RARRES3, ERAP2, LYD6 and CNTNAP2) present significantly low expression. Among these genes, genes CADM1, FOLR3, KYNU, and CNTNAP2 are core candidates for LSCC chemo-sensitivity. And that the low expression of CADM1 may result in chemo-sensitivity, which leads to high expression of gene FOLR3 and KYNU, and low expression of gene CNTNAP2. Besides, ROC analysis shows that these four genes exhibit effective diagnostic value for induction chemo-sensitivity. CONCLUSIONS: CADM1 may be a potential molecular marker for LSCC induction chemotherapy, while CADM1, FOLR3, KYNU, and CNTNAP2 may provide essential guidance for LSCC diagnosis and follow-up treatment strategies.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Subclassification and Detection of New Markers for the Discrimination of Primary Liver Tumors by Gene Expression Analysis Using Oligonucleotide Arrays.

BACKGROUND/AIMS: The failure to correctly differentiate between intrahepatic cholangiocarcinoma (CC) and hepatocellular carcinoma (HCC) is a significant clinical problem, particularly in terms of the different treatment goals for both cancers. In this study a specific gene expression profile to discriminate these two subgroups of liver cancer was established and potential diagnostic markers for clinical use were analyzed. METHODS: To evaluate the gene expression profiles of HCC and intrahepatic CC, Oligonucleotide arrays (AffymetrixU133A) were used. Overexpressed genes were checked for their potential use as new markers for discrimination and their expression values were validated by reverse transcription polymerase chain reaction and immunohistochemistry analyses. RESULTS: 695 genes/expressed sequence tags (ESTs) in HCC (245 up-/450 down-regulated) and 552 genes/ESTs in CC (221 up-/331 down-regulated) were significantly dysregulated (p＜0.05, fold change >2, ≥70%). Using a supervised learning method, and one-way analysis of variance a specific 270-gene expression profile that enabled rapid, reproducible differentiation between both tumors and nonmalignant liver tissues was established. A panel of 12 genes (e.g., HSP90ß, ERG1, GPC3, TKT, ACLY, and NME1 for HCC; SPT2, T4S3, CNX43, TTD1, HBD01 for CC) were detected and partly described for the first time as potential discrimination markers. CONCLUSIONS: A specific gene expression profile for discrimination of primary liver cancer was identified and potential marker genes with feasible clinical impact were described.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

The zinc spark is an inorganic signature of human egg activation.

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent "zinc spark." The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

SGK3 Sensitivity of Voltage Gated K+ Channel Kv1.5 (KCNA5).

BACKGROUND: The serum & glucocorticoid inducible kinase isoform SGK3 is a powerful regulator of several transporters, ion channels and the Na+/K+ ATPase. Targets of SGK3 include the ubiquitin ligase Nedd4-2, which is in turn a known regulator of the voltage gated K+ channel Kv1.5 (KCNA5). The present study thus explored whether SGK3 modifies the activity of the voltage gated K+ channel KCNA5, which participates in the regulation of diverse functions including atrial cardiac action potential, activity of vascular smooth muscle cells, insulin release and tumour cell proliferation. METHODS: cRNA encoding KCNA5 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild-type SGK3, constitutively active S419DSGK3, inactive K191NSGK3 and/or wild type Nedd4-2. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp. RESULTS: Voltage gated current in KCNA5 expressing Xenopus oocytes was significantly enhanced by wild-type SGK3 and S419DSGK3, but not by K191NSGK3. SGK3 was effective in the presence of ouabain (1 mM) and thus did not require Na+/K+ ATPase activity. Coexpression of Nedd4-2 decreased the voltage gated current in KCNA5 expressing Xenopus oocytes, an effect largely reversed by additional coexpression of SGK3. CONCLUSION: SGK3 is a positive regulator of KCNA5, which is at least partially effective by abrogating the effect of Nedd4-2.

pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA.

Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed.

An Immediate Innate Immune Response Occurred In the Early Stage of E. granulosus Eggs Infection in Sheep: Evidence from Microarray Analysis.

BACKGROUND: Cystic Echinococcosis(CE), caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), is a chronic parasitic zoonosis, with highly susceptible infection in sheep. However, the comprehensive molecular mechanisms that underlie the process of E. granulosus infection in the early stage remain largely unknown. The objective of this present study was to gain a cluster of genes expression profiles in the intestine tissue of sheep infected with CE. METHODS: Nine healthy sheep were divided into infection group and healthy controls, with six infected perorally 5000 E. granulosus eggs suspended in 1000 µl physiological saline and three controls perorally injected 1000 µl physiological saline. All animals were sacrificed at 4 hours post-infection, respectively. The intestine tissue was removed and the RNA was extracted. In the infection group, the biology replicates were designed to make sure the accuracy of the data. The ovine microarrays were used to analyze changes of gene expression in the intestine tissue between CE infected sheep and healthy controls. Real-time PCR was used to assess reliability of the microarray data. RESULTS: By biology repeats, a total of 195 differentially expressed genes were identified between infected group and controls at 4 hours post-infection, with 105 genes related to immune responses, while 90 genes associated with functions including energy metabolism, fat soluble transport, etc. Among the 105 immunity genes, 72 genes showed up-regulated expression levels while 33 showed down-regulation levels. Function analysis showed that most of up-regulated genes were related to innate immune responses, such as mast cell, NK cell, cytokines, chemokines and complement. In addition, Real-time PCR analysis of a random selection of nine genes confirmed the reliability of the microarray data. CONCLUSION: To our knowledge, this is the first report describing gene expression profiles in the intestine tissue of CE infection sheep. These results suggested that the innate immune system was activated to elicit immediate defense in the intestine tissue where E. granulosus invaded in at 4 hour-post infection. Furthermore, future interest will also focus on unraveling similar events, especially for the function of adaptive immunity, but at late stage infection.