Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust <it>in vitro </it>RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.</p> <p>Results</p> <p>To characterize the biochemical properties of JEV RdRp, we expressed in <it>Escherichia coli </it>and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U₈₁, at the two nucleotides upstream of the 3'-end of the template.</p> <p>Conclusion</p> <p>As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the <it>in vitro </it>reconstitution of viral RNA replicase complex, we for the first time established an <it>in vitro </it>JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.</p

Systemic EBV+ T-cell lymphoma in elderly patients: comparison with children and young adult patients

Fulminant Epstein–Barr virus (EBV+) T-cell lymphoma in immunocompetent elderly patients is rare and its character has not been well defined. This study analyzed the clinicopathological features of five elderly patients (group A: 50–84 years) and compared them with those of eight children and young adult patients with systemic T-cell lymphomas (group B: 10–34 years). Group A more commonly presented with generalized lymphadenopathy (n = 3) than did group B (n = 1). Chronic active EBV infection (n = 3) and hydroa vacciniforme-like eruptions (n = 1) were seen in group B, while group A showed no evidence of chronic EBV infection, but did show chronic hepatitis B or C virus infections (n = 3). The histological and immunophenotypical findings were similar. All patients died within 1 to 14 months of diagnosis. These findings suggest that EBV+ T-cell lymphoma in elderly patients is a unique disease with an underlying derangement of T-cell immunity and failure to eradicate infected virus. Additional factors related to senility may play a role in the disruption of homeostasis between the virus and the host’s immune system

Possible association of norepinephrine transporter -3081(A/T) polymorphism with methylphenidate response in attention deficit hyperactivity disorder

Background Attention-deficit/hyperactivity disorder (ADHD) is a heritable disorder characterized by symptoms of inattention and/or hyperactivity/impulsivity. Methylphenidate (MPH) has been shown to block the norepinephrine transporter (NET), and genetic investigations have demonstrated that the norepinephrine transporter gene (SLC6A2) is associated with ADHD. The aims of this study were to examine the association of the SLC6A2 -3081(A/T) and G1287A polymorphisms with MPH response in ADHD. Methods This study enrolled 112 children and adolescents with ADHD. A response criterion was defined based on the Clinical Global Impression-Improvement (CGI-I) score, and the ADHD Rating Scale-IV (ARS) score was also assessed at baseline and 8 weeks after MPH treatment. Results We found that the subjects who had the T allele as one of the alleles (A/T or T/T genotypes) at the -3081(A/T) polymorphism showed a better response to MPH treatment than those with the A/A genotype as measured by the CGI-I. We also found a trend towards a difference in the change of the total ARS scores and hyperactivity/impulsivity subscores between subjects with and without the T allele. No significant association was found between the genotypes of the SLC6A2 G1287A polymorphism and response to ADHD treatment. Conclusion Our findings provide evidence for the involvement of the -3081(A/T) polymorphism of SLC6A2 in the modulation of the effectiveness of MPH treatment in ADHD

Prognostic factors for aorta remodeling after thoracic endovascular aortic repair of complicated chronic DeBakey IIIb aneurysms

ObjectivesThe use of thoracic endovascular aortic repair (TEVAR) for chronic DeBakey III type b (CDIIIb) aneurysms is controversial. We analyzed the potential prognostic factors affecting aorta remodeling after this procedure.MethodsA total of 20 patients with CDIIIb aneurysms underwent TEVAR, with full coverage of reentry tears at the descending thoracic aorta. The potential factors affecting false lumen (FL) remodeling were analyzed, including reentry tears (communicating channels visible on the computed tomography angiogram), large intimal tears below the stent graft (≥2 consecutive axial cuts on the computed tomography angiogram), visceral branches arising from the FL, and intercostal arteries (ICAs) arising from the FL.ResultsAll the patients had uneventful in-hospital courses; 2 patients (10%) required reintervention during the follow-up period. Thirteen patients (65%) had complete thrombosis of the FL at stent graft segment. Compared with the complete thrombosis group, the partial thrombosis group had more reentry tears (1.8 vs 2.3, P = .48), large intimal tears (0.8 vs 1.7, P < .05), visceral branches arising from the FL (1.2 vs 2.3, P < .05), and ICAs arising from the FL (3.8 vs 5.1, P = .35). Reentry tears, visceral branches, and ICAs from the FL were significant negative prognostic factors for FL shrinkage (P < .05).ConclusionsAlthough reentry tears above the celiac trunk were fully covered, the visceral branches and ICAs from the FL and all communicating channels below the celiac trunk kept the FL pressurized and were unfavorable prognostic factors for aorta remodeling after TEVAR for CDIIIb aneurysms

Associations between maternal stress during pregnancy and offspring internalizing and externalizing problems in childhood

BACKGROUND: Maternal psychological health during pregnancy has been associated with offspring psychopathology. However, it is uncertain whether these associations are mediated by the postpartum depression and related child-rearing factors. Therefore, we examined the associations between prenatal and postnatal factors and internalizing and externalizing behavioral problems in childhood, focusing on maternal psychological health in school-aged children in Korea. FINDINGS: The current study included 1,003 children (580 boys, 423 girls, mean age 9.05 ± 0.70 years, age range 8–11 years) recruited from schools in five Korean cities. Children’s internalizing and externalizing problems were assessed by the Child Behavior Checklist (CBCL). The parents of the children completed structured questionnaires on perinatal factors. Among 1,003 children, 44 had internalizing problems (IP) and 30 had externalizing problems (EP). When comparing children with IP (n = 44) and without IP (n = 959), severe maternal stress during pregnancy (OR3.36, 95% CI 1.80-6.25) and postpartum depression (OR3.19, 95% CI 1.36-7.53) showed a significant association with the IP. When comparing children with EP (n = 30) and without EP (n = 973), low family income (OR2.19, 95% CI 1.05-4.56), unwanted pregnancy (OR2.76, 95% CI 1.28-5.95) and severe maternal stress during pregnancy (OR2.69, 95% CI 1.29-5.61) with the EP. Only maternal stress during pregnancy was significantly associated with the IP after controlling for postpartum depression and with the EP after controlling for family income and unwanted pregnancy. CONCLUSION: This study suggests the importance of maternal psychological health during perinatal period on children’s mental health. Further prospective studies in a larger sample are required to confirm our findings

Prevention of hypoglycemia-induced neuronal death by minocycline

Diabetic patients who attempt strict management of blood glucose levels frequently experience hypoglycemia. Severe and prolonged hypoglycemia causes neuronal death and cognitive impairment. There is no effective tool for prevention of these unwanted clinical sequelae. Minocycline, a second-generation tetracycline derivative, has been recognized as an anti-inflammatory and neuroprotective agent in several animal models such as stroke and traumatic brain injury. In the present study, we tested whether minocycline also has protective effects on hypoglycemia-induced neuronal death and cognitive impairment. To test our hypothesis we used an animal model of insulin-induced acute hypoglycemia. Minocycline was injected intraperitoneally at 6 hours after hypoglycemia/glucose reperfusion and injected once per day for the following 1 week. Histological evaluation for neuronal death and microglial activation was performed from 1 day to 1 week after hypoglycemia. Cognitive evaluation was conducted 6 weeks after hypoglycemia. Microglial activation began to be evident in the hippocampal area at 1 day after hypoglycemia and persisted for 1 week. Minocycline injection significantly reduced hypoglycemia-induced microglial activation and myeloperoxidase (MPO) immunoreactivity. Neuronal death was significantly reduced by minocycline treatment when evaluated at 1 week after hypoglycemia. Hypoglycemia-induced cognitive impairment is also significantly prevented by the same minocycline regimen when subjects were evaluated at 6 weeks after hypoglycemia. Therefore, these results suggest that delayed treatment (6 hours post-insult) with minocycline protects against microglial activation, neuronal death and cognitive impairment caused by severe hypoglycemia. The present study suggests that minocycline has therapeutic potential to prevent hypoglycemia-induced brain injury in diabetic patients

Association between maternal coronavirus disease 2019 and transient tachypnea of the newborn: a single-center study

Background Limited clinical reports have investigated the effects of maternal coronavirus disease 2019 (COVID-19) on fetuses and neonates. Purpose This retrospective study aimed to assess the impact of maternal COVID-19 on neonates during the perinatal period, including neonatal clinical outcomes, versus the outcomes of neonates of mothers without COVID-19. Methods Neonates born to COVID-19-infected mothers at the National Health Insurance Service Ilsan Hospital between February 2021 and March 2022 were included. Those with gestational age (GA) ≥35+0 weeks who were born within 2 weeks of the maternal infection were matched 1:2 with a control group based on GA. The main outcomes were respiratory diseases, including transient tachypnea of the newborn (TTN), respiratory distress syndrome, meconium aspiration syndrome, the need for respiratory support, and length of hospital stay. Uni- and multivariate logistic regression analyses were performed and adjusted for relevant covariates, including maternal age, obstetric complications (hypertension and gestational diabetes), delivery mode, birth weight, sex, and small-for-gestational-age status. Results The case group comprised 103 neonates (mean GA, 38.5±1.3 weeks; mean birth weight, 3,121±397 g), while the control group included 206 neonates (mean GA, 38.4±1.2 weeks; mean birth weight, 3088±428 g). In the case and control groups, the proportion of cesarean sections was 91% and 40%, respectively, while the proportion of male infants was 56% and 47%, respectively. After adjusting for covariates, the case group had a higher risk of TTN (adjusted odd ratio [AOR], 3.69; 95% confidence interval [CI], 1.69–8.07), noninvasive respiratory ventilator use (AOR, 2.28; 95% CI, 1.05–4.97), and oxygen support (AOR, 4.83; 95% CI, 1.46–15.95). Conclusion Newborns born to COVID-19-infected mothers are at increased risk of TTN and may require respiratory support. Close monitoring of respiratory symptoms is crucial in neonates

Antimicrobial peptide from Bacillus subtilis CSB138: characterization, killing kinetics, and synergistic potency

We studied the prospect of synergy between the antimicrobial peptide p138c and non-peptide antibiotics for increasing the potency and bacterial killing kinetics of these agents. The production of p138c was maximized in the late exponential growth phase of Bacillus subtilis CSB138. Purification of p138c resulted in a total of 4800 arbitrary units (AU) with 19.15-fold and 3.2% recovery. Peptide p138c was thermo-tolerant up to 50 &deg;C and stable at pH 5.8 to 11. The biochemical nature of p138c was determined by a bioassay, similar to tricine-SDS-PAGE, indicating inhibition at 3 kDa. The amino acid sequence of p138c was Gly-Leu-Glu-Glu-Thr-Val-Tyr-Ile-Tyr-Gly-Ala-Asn-Met-X-Ser. Potency and killing kinetics against vancomycin-resistant Staphylococcus aureus improved considerably when p138c was synergized with oxacillin, ampicillin, and penicillin G. The minimal inhibitory concentration (MIC) of p138c showed a 4-, 8-, and 16-fold improvement when p138c was combined with oxacillin, ampicillin, and penicillin G, respectively. The fractional inhibitory concentration index for the combination of p138c and oxacillin, ampicillin, and penicillin G was 0.3125, 0.25, and 0.09, respectively. Synergy with non-peptide antibiotics resulted in enhanced killing kinetics of p138c. Hence, the synergy between antimicrobial peptide and non-peptide antibiotics may enhance the potency and bacterial killing kinetics, providing more potent and rapidly acting agents for therapeutic use. [Int Microbiol 20(1):43-53 (2017)]Keywords: Bacillus subtilis &middot; antimicrobial peptides &middot; killing kinetic