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.

Avian Influenza A Virus Polymerase Can Utilize Human ANP32 Proteins To Support cRNA but Not vRNA Synthesis.

Host restriction limits the emergence of novel pandemic strains from the influenza A virus avian reservoir. For efficient replication in mammalian cells, the avian influenza RNA-dependent RNA polymerase must adapt to use human orthologues of the host factor ANP32, which lack a 33-amino-acid insertion relative to avian ANP32A. Here, we find that influenza polymerase requires ANP32 proteins to support both steps of genome replication: cRNA and vRNA synthesis. However, avian strains are only restricted in vRNA synthesis in human cells. Therefore, avian influenza polymerase can use human ANP32 orthologues to support cRNA synthesis, without acquiring mammalian adaptations. This implies a fundamental difference in the mechanism by which ANP32 proteins support cRNA versus vRNA synthesis. IMPORTANCE To infect humans and cause a pandemic, avian influenza must first adapt to use human versions of the proteins the virus hijacks for replication, instead of the avian orthologues found in bird cells. One critical host protein is ANP32. Understanding the details of how host proteins such as ANP32 support viral activity may allow the design of new antiviral strategies that disrupt these interactions. Here, we use cells that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps of influenza genome replication. Unexpectedly, however, we found that avian influenza can use human ANP32 proteins for the first step of replication, to copy a complementary strand, without adaptation but can only utilize avian ANP32 for the second step of replication that generates new genomes. This suggests ANP32 may have a distinct role in supporting the second step of replication, and it is this activity that is specifically blocked when avian influenza infects human cells.

Innovating Student Registered Nurse Anesthetist Clinical Learning Through United States Air Force Pilot Training Practices: A Review of the Literature.

The clinical learning environment is essential for student registered nurse anesthetists (SRNAs) to develop intricate clinical knowledge and acquire proficiency in technical skills required for anesthetic care. The perioperative experience of an SRNA can differ greatly based on the program, preceptor, hospital rotation, or geographic location. This literature review synthesizes the historical and current state of certified registered nurse anesthetist (CRNA)/SRNA preceptorship in the clinical setting. Themes analyzed include the current CRNA/SRNA learning and teaching environment, student perceptions of effective CRNA preceptors, evaluation tools and feedback practices, and overall CRNA preceptor preparedness as well as the availability and effectiveness of preceptor training programs. We compare their findings to best practices seen in the United States Air Force (USAF) pilot training program because of its similar "high stakes" learning environment and utilization of a preceptor teaching model. Actionable recommendations, based on CRNA preceptorship data, preceptorship theory, and the USAF pilot training model are presented in the effort to improve preceptor transfer of knowledge and SRNA clinical competence.

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.

Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics.

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.

CRNA Engagement During the COVID-19 Crisis: Optimization of Resource Management, Organizational Climate, and Contributions to Care.

COVID-19 has a strong hold on New York City, and it has similar effects on other areas of the country. As COVID-19 strains healthcare systems and the certified registered nurse anesthetists (CRNAs) who work within them, optimization of CRNA organizational climate promotes transformation of care delivery and may positively impact provider and patient outcomes. This article describes one healthcare system's newly refined processes for managing the surge of COVID-19 patients. It also describes the novel contributions of CRNAs to all aspects of care provision from supply and resource management to management of patients in critical care environments, to refinement of intubation and airway management for this patient population. Lessons learned during this pandemic from two facilities within this healthcare system are described. When a healthcare system's culture respects and encourages collaboration and innovation, dynamic changes can be implemented effectively during times of crises. Also, critically helpful is when CRNA organizational climate promotes equal partnerships with physicians, and administration values the CRNA contributions to care. In this case, collaboration of all stakeholders promoted best practices and improved care provision. Lessons learned may be applied to facilities where the COVID-19 surge is occurring.

Fluorescence-Based Measurements of Membrane-Bound Angiotensin Converting Enzyme 2 Activity Using Xenopus Laevis Oocytes.

Functional investigations of enzymes involving cellular expression systems are important for pharmacological studies. The precise control of expression is challenging in transiently transfected mammalian cell lines. Here, we explored the ability of Xenopus laevis oocytes to express a membrane-bound enzyme for functional characterization using standard 96-well plates and a fluorescence-based plate reader assay. We microinjected oocytes with cRNA encoding the angiotensin converting enzyme 2 (ACE2) and measured the enzymatic activity in single oocytes using a commercial fluorescence-based assay. The injected oocytes showed up to a 50-fold increase in fluorescence compared to uninjected oocytes. This fluorescence intensity was dose-dependent on the amount of ACE2 cRNA. These results suggest that Xenopus oocytes can be used for the functional evaluation of membrane-bound enzymes, decreasing the experimental workload.

Minimum Atropine Dosing in Pediatric Patients: Does CRNA Practice Reflect Current Recommendations?

The purpose of this study was to evaluate certified registered nurse anesthetist (CRNA) current pediatric atropine dosing practices. Emphasis was placed on rationale for dosing and knowledge regarding current literature and guidelines. An electronic survey was deployed by the American Association of Nurse Anesthetists (AANA)'s survey department to a total of 2,905 CRNAs who are current AANA members. The survey was completed by 98 CRNAs, of which 67 selected that they do not administer anesthesia to pediatric patients weighing less than 5 kg and were excluded from further survey participation. The responses from the remaining 31 CRNAs were utilized for data analysis (n = 31). Approximately two thirds of participants (64.5%) were unaware of available guidelines pertaining to pediatric dosing of atropine within the last 5 years. A statistically significant difference existed when analyzing whether awareness of guidelines was associated with knowledge of the correct American Heart Association recommended pediatric atropine dose. Providers who were aware of guidelines reported the correct dose 100% of the time, whereas those unaware of guidelines reported the correct dose only 65% of the time (P = .03). Variability in clinical practices and sources guiding practice should be addressed to avoid potential overdosing in the vulnerable neonatal population.

Operating room relay strategy for turnover time improvement: a quality improvement project.

INTRODUCTION: Operating room (OR) management plays a pivotal role in the healthcare system due to the high cash flow it yields. Enhancing communication in the OR, which is the common root problem for delays, might improve OR efficiency and revenues for healthcare. This study aims to evaluate the impact of an OR relay strategy on turnover time (TOT). METHODS: A quality improvement project was conducted. In the intervention group, a certified registered nurse anaesthetist (CRNA) remained outside of the OR, coordinating the steps to get the next patient ready. This CRNA communicated with the anaesthesia providers within the OR via a Microsoft Team chat. The TOT for the control group was recorded from the electronic anaesthesia record system. RESULTS/DATA ANALYSIS: Analysis of 636 turnovers was performed with non-parametric tests. The OR relay strategy decreased TOT for most ORs, with statistically significant results for three of the ORs and the overall ORs system. A decreased in variability between TOTs was evidenced for the overall OR and the majority of the ORs evaluated individually. CONCLUSION: The OR relay strategy has a positive impact on TOT.

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.

Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal-to-zygotic transition in mouse early embryos.

OBJECTIVES: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains. MATERIALS AND METHODS: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. RESULTS: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce+/+ 80.1% vs. Prkce-/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. CONCLUSIONS: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes.

Prescription Drug Insurance and Cost-Related Medication Nonadherence Among Lesbian, Gay, and Bisexual Individuals in Canada.

Purpose: This study estimates the frequency of uninsurance for prescription drugs and cost-related medication nonadherence (CRNA) among lesbian, gay, and bisexual (LGB) persons in Canada, compared with the heterosexual population. Methods: Logistic regression was used to quantify associations between sexual orientation, insurance status, and CRNA within the national probability-based Canadian Community Health Survey, 2015-2016. This sample included 98,413 individuals aged 15-80 years, including 2803 LGB individuals. Results: From our sample of Canadians, 22.2% of LGB respondents reported being uninsured for prescription drugs, compared with 20.0% of heterosexual persons (unadjusted odds ratio [UOR] 1.14, 95% confidence interval [CI] 0.97-1.35). LGB individuals had more than twice the odds of reporting CRNA compared with heterosexual individuals (UOR 2.48, 95% CI 1.99-3.10). This disparity was most pronounced among bisexual respondents, who had over three times the odds of reporting CRNA in comparison to heterosexual respondents (UOR 3.45, 95% CI 2.65-4.51). The odds ratio (OR) for CRNA comparing bisexual with heterosexual individuals remained statistically significant after adjustment for race/ethnicity, gender/sex, and age (OR 2.67, 95% CI 1.97-3.61) and was further attenuated with adjustment for partnership status, employment status, income, educational attainment, prescription drug insurance status, general health status, and immigration status (OR 2.09, 95% CI 1.51-2.89). Conclusion: LGB Canadians reported more CRNA but comparable prescription drug insurance frequencies to heterosexual persons. Factors pertaining to medication access (e.g., income, partnership status) and health needs appear to be the most important contributors to disparities.

Cost-related nonadherence can be explained by a general nonadherence framework.

BACKGROUND: A conceptual framework has been developed specifically for cost-related nonadherence (CRNA) that differs from models proposed for general medication nonadherence. OBJECTIVE: This study aimed to demonstrate that CRNA studies are best explained by a conceptual framework developed for general medication nonadherence. METHODS: A systematic literature review was conducted using MEDLINE via PubMed, CINAHL, ScienceDirect, and Google Scholar databases from 2008 to 2020. Articles were considered for inclusion if they were research studies, used a self-reported measure for CRNA, and provided self-reported data on factors associated with CRNA. RESULTS: A total of 58 studies were identified and included in the review. Factors related to financial pressures were consistently associated with CRNA corresponding to conceptual frameworks for both CRNA and general medication nonadherence. However, noneconomic factors, classified as moderators in the CRNA framework (i.e., patient factors, disease factors, clinician factors), consistently demonstrated independent effects, often with similar strength of association compared with economic factors. Overall, the pattern of risk factors identified in CRNA studies was consistent with general nonadherence except for indicators of poor health. Poor health was often associated with an increased risk of CRNA, whereas the inverse association is generally observed in general nonadherence studies (i.e., nonadherence higher in primary prevention vs. secondary prevention). However, the apparent disagreement was likely caused by the general population studied rather than a unique causal pathway for CRNA. CONCLUSION: Financial difficulties are extremely common among people who take prescription medications. However, current evidence is insufficient to support a conceptual framework that differs from general medication nonadherence.

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.

The Opioid Crisis and the CRNA: Providing Emergency Care for Patients With Opioid Use Disorder.

Certified Registered Nurse Anesthetists (CRNAs) care for patients with opioid use disorder frequently. Goals are to support recovery, prevent relapse, and effectively and safely treat perioperative pain. During emergencies, care may be urgent to prevent patient harm, potentially interfering with helpful interventions. This article discusses care principles that CRNAs should follow to assure that the anesthetic care goals are achieved during emergent care of patients with opioid use disorder.

Synthesis of 2'-O-alkylcarbamoylethyl-modified oligonucleotides with enhanced nuclease resistance that form isostable duplexes with complementary RNA.

To expand the variety of 2'-O-modified oligonucleotides, we synthesized 2'-O-carbamoylethyl-modified oligonucleotides bearing ethyl, n-propyl, n-butyl, n-pentyl, and n-octyl groups on their nitrogen atoms. The corresponding nucleosides were synthesized using 2'-O-benzyloxycarbonylethylthymidine, which was easily converted into the carboxylic acid through hydrogeneration; subsequent condensation with the appropriate amine gave the desired nucleoside. We evaluated the effect of the 2'-O-alkylcarbamoylethyl modifications on duplex stability by analyzing melting temperature, which revealed the formation of isostable duplexes. In addition, we also revealed that these modifications, especially octylcarbamoylethyl, endowed these oligonucleotides with resistance toward a 3'-exonuclease. These results highlight the usefulness of the 2'-O-alkylcarbamoylethyl modification for various biological applications.

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.

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.