Effects of an interactive handgrip game on surgical patients requiring intensive care: An assessor-blinded randomized controlled trial.

OBJECTIVE: To explore the effect of an interactive handgrip game on psychological distress and handgrip strength among critically ill surgical patients. DESIGN: A randomised controlled trial. SETTING: A surgical intensive care unit. INTERVENTION: Participants were enrolled in the program within 48 hours of admission to the intensive care unit. Patients in the intervention group played a 20-minute interactive handgrip game twice daily for a maximum of three days in the intensive care unit in addition to routinely passive physical rehabilitation. Patients in the routine care group had a daily target of 20 min of passive physical rehabilitation as needed. MEASUREMENT: The primary outcomes included depression, anxiety, and stress measured using the shortened version of the Depression Anxiety Stress Score scale. The secondary outcomes were perceived sleep evaluated using the Richards-Campbell Sleep Questionnaire, delirium assessed using the Intensive Care Delirium Screening Checklist, and handgrip strength measured using handgrip dynamometry within a handgrip device. RESULTS: Two hundred and twenty-seven patients were eligible and 70 patients were recruited in the intervention (n = 35) and routine care groups (n = 35). The patients in the intervention group had lower scores (median = 6.0, 4.0, and 12.0) for depression, anxiety, and stress compared with those in the routine care group (12.0, 12.0, and 20.0; all p < 0.05). The interactive handgrip game did not significantly improve sleep quality and prevent the occurrence of delirium (both p > 0.05). The patients who received the interactive handgrip game intervention exhibited significantly enhanced handgrip strength in both hands over time (both p < 0.001). CONCLUSION: An interactive handgrip game may benefit the psychological well-being and handgrip strength of critically ill patients. IMPLICATIONS FOR CLINICAL PRACTICE: Interactive handgrip games is effective active exercise which should be integrated into routine nursing practice.

Dynamic prevalence of sleep disturbance among critically ill patients in intensive care units and after hospitalisation: A systematic review and meta-analysis.

BACKGROUND: Sleep disturbance is a common complaint among critically ill patients in intensive care units and after hospitalisation. However, the prevalence of sleep disturbance among critically ill patients varies widely. OBJECTIVE: To estimate the prevalence of sleep disturbance among critically ill patients in the intensive care unit and after hospitalisation. METHODS: Electronic databases were searched from their inception until 15 August 2022. Only observational studies with cross-sectional, prospective, and retrospective designs investigating sleep disturbance prevalence among critically ill adults (aged ≥ 18 years) during intensive care unit stay and after hospitalisation were included. RESULTS: We found 13 studies investigating sleep disturbance prevalence in intensive care units and 14 investigating sleep disturbance prevalence after hospitalisation, with 1,228 and 3,065 participants, respectively. The prevalence of sleep disturbance during an ICU stay was 66 %, and at two, three, six and ≥ 12 months after hospitalisation was 64 %, 49 %, 40 %, and 28 %, respectively. Studies using the Richards-Campbell Sleep Questionnaire detected a higher prevalence of sleep disturbance among patients in intensive care units than non-intensive care unit specific questionnaires; studies reported comparable sleep disturbance prevalence during intensive care stays for patients with and without mechanical ventilation. CONCLUSION: Sleep disturbance is prevalent in critically ill patients admitted to an intensive care unit and persists for up to one year after hospitalisation, with prevalence ranging from 28 % to 66 %. The study results highlight the importance of implementing effective interventions as early as possible to improve intensive care unit sleep quality.

Association between hypomagnesemia and mortality among dialysis patients: a systematic review and meta-analysis.

Background: Malnutrition-inflammation-atherosclerosis (MIA) syndrome is caused by the inflammatory cytokines in end stage renal disease (ESRD) patients, and MIA complex-related factors may be associated with hypomagnesemia and mortality. However, the association between serum magnesium level and mortality for dialysis patients is still not clear. Additionally, no meta-analysis has investigated the impact of serum magnesium on peritoneal dialysis and hemodialysis, separately. Methods: We searched published studies in PubMed, Embase, Cochrane, Collaboration Central Register of Controlled Clinical Trials, and Cochrane Systematic Reviews through April 2022. Studies associated with serum magnesium and all-cause mortality or cardiovascular (CV) mortality in ESRD on kidney replacement therapy (KRT) patients were included. A hazard ratio (HR) with 95% confidence intervals (CI) was used to report the outcomes. Results: Twenty-one studies involving 55,232 patients were included. Overall, there was a significant association between hypomagnesemia and all-cause mortality for dialysis patients (HR: 1.67, 95% CI [1.412-2.00], p < 0.001; certainty of evidence: moderate) using a mixed unadjusted and adjusted HR for analysis. There was also a significantly increased risk of CV mortality for individuals with hypomagnesemia compared with the non-hypomagnesemia group (HR 1.56, 95% CI [1.08-2.25], p < 0.001; certainty of evidence: moderate). In addition, a subgroup analysis demonstrated that hypomagnesemia was associated with a high risk of both all-cause mortality and CV mortality (all-cause mortality, HR:1.80, 95% CI [1.48-2.19]; CV mortality, HR:1.84, 95% CI [1.10-3.07]) in hemodialysis (HD) patients, but not in participants receiving peritoneal dialysis (PD; all-cause mortality, HR:1.26, 95% CI [0.84-1.91]; CV mortality, HR:0.66, 95% CI [0.22-2.00]). The systematic review protocol was prespecified and registered in PROSPERO [CRD42021256187]. Conclusions: Hypomagnesemia may be a significant risk factor for all-cause mortality and CV mortality in KRT patients, especially in those receiving hemodialysis. However, because of the limited certainty of evidence, more studies are required to investigate this association.

A novel flavivirus entry inhibitor, BP34610, discovered through high-throughput screening with dengue reporter viruses.

Dengue virus (DENV) is a global health problem that affects approximately 3.9 billion people worldwide. Since safety concerns were raised for the only licensed vaccine, Dengvaxia, and since the present treatment is only supportive care, the development of more effective therapeutic anti-DENV agents is urgently needed. In this report, we identified a potential small-molecule inhibitor, BP34610, via cell-based high-throughput screening (HTS) of 12,000 compounds using DENV-2 reporter viruses. BP34610 reduced the virus yields of type 2 DENV-infected cells with a 50% effective concentration (EC50) and selectivity index value of 0.48 ±â€¯0.06 µM and 197, respectively. Without detectable cytotoxicity, the compound inhibited not only all four serotypes of DENV but also Japanese encephalitis virus (JEV). Time-of-addition experiments suggested that BP34610 may act at an early stage of DENV virus infection. Sequencing analyses of several individual clones derived from BP34610-resistant viruses revealed a consensus amino acid substitution (S397P) in the N-terminal stem region of the E protein. Introduction of S397P into the DENV reporter viruses conferred an over 14.8-fold EC90 shift for BP34610. Importantly, the combination of BP34610 with a viral replication inhibitor, ribavirin, displayed synergistic enhancement of anti-DENV-2 activity. Our results identify an effective small-molecule inhibitor, BP34610, which likely targets the DENV E protein. BP34610 could be developed as an anti-flavivirus agent in the future.

A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication.

Host cells infected with dengue virus (DENV) often trigger endoplasmic reticulum (ER) stress, a key process that allows viral reproduction, without killing the host cells until the late stage of the virus life-cycle. However, little is known regarding which DENV viral proteins interact with the ER machinery to support viral replication. In this study, we identified and characterized a novel host factor, stress-associated ER protein 1 (SERP1), which interacts with the DENV type 2 (DENV-2) NS4B protein by several assays, for example, yeast two-hybrid, subcellular localization, NanoBiT complementation, and co-immunoprecipitation. A drastic increase (34.5-fold) in the SERP1 gene expression was observed in the DENV-2-infected or replicon-transfected Huh7.5 cells. The SERP1 overexpression inhibited viral yields (37-fold) in the DENV-2-infected Huh7.5 cells. In contrast, shRNAi-knockdown and the knockout of SERP1 increased the viral yields (3.4- and 16-fold, respectively) in DENV-2-infected HEK-293 and Huh7.5 cells, respectively. DENV-2 viral RNA replication was severely reduced in stable SERP1-expressing Huh7.5 cells transfected with DENV-2 replicon plasmids. The overexpression of DENV-2 NS4B alleviated the inhibitory effect of SERP1 on DENV-2 RNA replication. Taking these results together, we hypothesized that SERP1 may serve as an antiviral player during ER stress to restrict DENV-2 infection. Our studies revealed novel anti-DENV drug targets that may facilitate anti-DENV drug discovery.

A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses.

A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.

Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 µM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus.

Dengue virus (DENV) is a public health threat to approximately 40% of the global population. At present, neither licensed vaccines nor effective therapies exist, and the mechanism of viral RNA replication is not well understood. Here, we report the development of efficient Renilla luciferase reporter-based DENV replicons that contain the full-length capsid sequence for transient and stable DENV RNA replication. A comparison of the transient and stable expression of this RNA-launched replicon to replicons containing various deletions revealed dengue replicon containing entire mature capsid RNA element has higher replicon activity. An efficient DNA-launched DENV replicon, pCMV-DV2Rep, containing a full-length capsid sequence, was created and successfully applied to evaluate the potency of known DENV inhibitors. Stable cell lines harboring the DENV replicon were easily established by transfecting pCMV-DV2Rep into BHK21 cells. Steady and high replicon reporter signals were observed in the stable DENV replicon cells, even after 30 passages. The stable DENV replicon cells were successfully used to determine the potency of known DENV inhibitors. A high-throughput screening assay based on stable DENV replicon cells was evaluated and shown to have an excellent Z' factor of 0.74. Altogether, the development of our efficient DENV replicon system will facilitate the study of virus replication and the discovery of antiviral compounds.

Resistance studies of a dithiazol analogue, DBPR110, as a potential hepatitis C virus NS5A inhibitor in replicon systems.

Hepatitis C virus (HCV), a member of the Flaviviridae family, affects approximately 3% of the world's population and is becoming the leading cause of liver disease in the world. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In our previous study, we identified a potential HCV NS5A inhibitor, BP008. After further systemic optimization, we discovered a more potent HCV inhibitor, DBPR110. DBPR110 reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and a selective index value of 3.9 ± 0.9 pM and >12,800,000, respectively. DBPR110 reduced HCV2a replicon activity with an EC(50) and a selective index value of 228.8 ± 98.4 pM and >173,130, respectively. Sequencing analyses of several individual clones derived from the DBPR110-resistant RNAs purified from cells harboring genotype 1b and 2a HCV replicons revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. P58L/T and Y93H/N in genotype 1b and T24A, P58L, and Y93H in the genotype 2a replicon were the key substitutions for resistance selection. In the 1b replicon, V153M, M202L, and M265V play a compensatory role in replication and drug resistance. Moreover, DBPR110 displayed synergistic effects with alpha interferon (IFN-α), an NS3 protease inhibitor, and an NS5B polymerase inhibitor. In summary, our results present an effective small-molecule inhibitor, DBPR110, that potentially targets HCV NS5A. DBPR110 could be part of a more effective therapeutic strategy for HCV in the future.

Resistance analysis and characterization of a thiazole analogue, BP008, as a potent hepatitis C virus NS5A inhibitor.

Hepatitis C virus (HCV) is a global health problem, affecting approximately 3% of the world's population. The standard treatment for HCV infection is often poorly tolerated and ineffective. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In this report, BP008, a potent small-molecule inhibitor of HCV replication, was developed from a class of compounds with thiazol core structures by means of utilizing a cell-based HCV replicon system. The compound reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and selective index value of 4.1 ± 0.7 nM and >12,195, respectively. Sequencing analyses of several individual clones derived from BP008-resistant RNAs purified from cells harboring HCV1b replicon revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. Q24L, P58S, and Y93H are the key substitutions for resistance selection; F149L and V153M play the compensatory role in the replication and drug resistance processes. Moreover, BP008 displayed synergistic effects with alpha interferon (IFN-α), NS3 protease inhibitor, and NS5B polymerase inhibitor, as well as good oral bioavailability in SD rats and favorable exposure in rat liver. In summary, our results pointed to an effective small-molecule inhibitor, BP008, that potentially targets HCV NS5A. BP008 can be considered a part of a more effective therapeutic strategy for HCV in the future.

Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes.

Reverse genetics is a powerful tool to study single-stranded RNA viruses. Despite tremendous efforts having been made to improve the methodology for constructing flavivirus cDNAs, the cause of toxicity of flavivirus cDNAs in bacteria remains unknown. Here we performed mutational analysis studies to identify Escherichia coli promoter (ECP) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (DENV2) and Japanese encephalitis virus (JEV) genomes. Eight and four active ECPs were demonstrated within nt 1 to 3000 of the DENV2 and JEV genomes, respectively, using fusion constructs containing DENV2 or JEV segments and empty vector reporter gene Renilla luciferase. Full-length DENV2 and JEV cDNAs were obtained by inserting mutations reducing their ECP activity in bacteria without altering amino acid sequences. A severe cytopathic effect occurred when BHK21 cells were transfected with in vitro-transcribed RNAs from either a DENV2 cDNA clone with multiple silent mutations within the prM-E-NS1 region of dengue genome or a JEV cDNA clone with an A-to-C mutation at nt 90 of the JEV genome. The virions derived from the DENV2 or JEV cDNA clone exhibited infectivities similar to those of their parental viruses in C6/36 and BHK21 cells. A cis-acting element essential for virus replication was revealed by introducing silent mutations into the central portion (nt 160 to 243) of the core gene of DENV2 infectious cDNA or a subgenomic DENV2 replicon clone. This novel strategy of constructing DENV2 and JEV infectious clones could be applied to other flaviviruses or pathogenic RNA viruses to facilitate research in virology, viral pathogenesis, and vaccine development.

Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay.

Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC(50)) of 15.43 ± 2.12 µM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC(50)) of 0.17 ± 0.01 µM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.