Effectiveness of Narciclasine in Suppressing the Inflammatory Response in Sepsis: Molecular Docking and In Silico Studies.

Narciclasine is an alkaloid belonging to the Amaryllidaceae family which has been reported to have many beneficial properties. Especially its anticancer properties have been widely reported. Here, we have focused on its potential use in suppressing the inflammatory response in sepsis using in silico methods. Lipopolysaccharide (LPS) is an endotoxin which is present in the outer membrane of gram-negative bacteria and is a crucial player in the pathogenesis of gram-negative sepsis. Activation of toll-like receptor 4 (TLR4) signaling by LPS is an important event in the pathogenesis of gram-negative sepsis. This initiates a downstream signaling pathway comprising of several adaptor proteins such as toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase (IRAK)-1, IRAK-4, interferon regulatory factor 3 (IRF-3), tumor necrosis factor receptor-associated factor 6 (TRAF-6) leading to nuclear factor kappa B (NF-κß) activation resulting in elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. S100 calcium binding proteins A8/A9 (S100A8/A9) have been found to be an agonist of TLR4, and it amplifies the inflammatory response in sepsis. Molecular docking studies of narciclasine with target proteins associated with the LPS-TLR4 pathway showed that it has good binding affinity and stable interactions with the targets studied. Molecular dynamics (MD) simulation studies over 100 ns showed that most of the ligand-target complexes were stable. The structures of all the targets except TRAF-6 were retrieved from the Protein Data Bank (PDB) database. Homology modeling was done to predict the 3-dimensional structure of TRAF-6. MD simulation of narciclasine-TRAF-6 complex showed that the structure is stable. Metapocket was used for active site prediction in the target proteins. Toxicity analysis by admetSAR revealed that narciclasine was readily biodegradable and exhibited minimum toxicity. These results indicate that narciclasine has effective anti-inflammatory properties which could be useful in suppressing the inflammatory response in sepsis.

Neonatal Sepsis-Newer Insights.

Sepsis is one of the leading causes of mortality and morbidity among neonates worldwide and especially affects the preterm neonates in resource-restricted settings. The infection may be acquired in utero, from the mother's genital tract or postnatally from the community or hospital environment and personnel. Factors including the time of exposure, size of the inoculum, immunity in the host, and virulence of the infectious agent affect the severity and course of illness. Culture-independent diagnostics, sepsis prediction scores, antibiotic stewardship, and preventive strategies including hand hygiene are ongoing efforts for reducing the neonatal sepsis burden.

Coronavirus Disease 2019 Infection among Children: Pathogenesis, Treatment, and Outcome.

Coronavirus disease 2019 (COVID-19) is a contagious disease that may lead to respiratory distress syndrome and even death. Neonates and children are most vulnerable population to COVID-19 infection; however, the infection is usually milder and has a better prognosis in pediatric patients compared with adults. It remains unclear why pediatric population is less symptomatic than adults. Children frequently experience respiratory infections and their immune system is in developing stage. However, large proportion of the asymptomatic pediatric population may contribute to transmission. This review explored several aspects of COVID-19 infection such as its epidemiology, its molecular pathogenesis with respect to angiotensin-converting enzyme 2 receptor and inflammatory mediators, intrauterine vertical transmission, imaging findings, and complications like cytokine release syndrome (multisystem inflammatory syndrome in children). We also looked at prognostic factors and treatment modalities like corticosteroids, RNA replicate inhibitors, protease inhibitors, Bruton tyrosine kinase inhibitor, that is, acalabrutinib and convalescent plasma therapy. Since there is no strong evidence for the intrauterine transmission, early isolation should be performed to protect a neonate from a COVID-19 infected mother. Development of vaccine and an effective antiviral drug are the need of the hour.

Role of Zinc in Neonatal Sepsis.

Sepsis emerges as a complex clinical syndrome with activation of an innate host response to infections. Despite advancement in therapeutic approaches, infants with sepsis remain hospitalized for longer durations and it remains to be a major health problem in today's world. Zinc as a trace element, has the potential to improve the host's defence mechanism against various pathogenic diseases. During sepsis, a redistribution of zinc from serum into the liver has been observed and earlier studies imply a correlation between serum zinc levels and the outcome of sepsis. Zinc also appears to have a potential to be used as a biomarker of sepsis outcome. There are only few reports available to show the efficacy of zinc supplements in the management of neonatal sepsis.

Are Global DNA methylation and telomere length useful biomarkers for identifying intrauterine growth restricted neonates?

Background: Intrauterine growth restriction (IUGR) is manifested by decreased growth rate of fetus than its normal genetic growth potential. Global DNA methylation is a crucial investigation for identification of epigenetic changes. Epigenetic change regulates Gene transcription, maintenance of genomic stability, and telomere length.Objectives: To investigate whether the global DNA methylation and telomere length are useful for identifying intrauterine growth restriction.Methods: This cohort study was conducted in the Neonatology Department of JIPMER during the period of November 2016 to December 2017. Forty (40) IUGR and forty (40) AGA neonates were recruited. Umbilical cord blood samples were collected at birth. DNA has been separated from the blood samples and using 5-mC DNA ELISA method, the percentage of genomic DNA methylated in these neonates was established. Telomere length (T/S ratio) was measured by using quantitative real time PCR. Data were expressed as a mean ± standard deviation.Results: Genomic DNA methylation varied significantly between IUGR and AGA neonates (IUGR: 3.12 ± 1.24; AGA: 4.40 ± 2.03; p: < .01). There was significant DNA hypo methylation in IUGR neonates. However, telomere length (T/S ratio) was (IUGR: 1.25 ± 0.13; AGA: 1.26 ± 0.22; p: 0.228 (NS)) similar in both groups.Conclusion: Although there is no significant difference in telomere length between IUGR and AGA neonates, global DNA methylation of 3.45 would identify IUGR with a sensitivity and specificity of 69 and 65% respectively.

Effect of zinc supplementation on relative expression of immune response genes in neonates with sepsis: A preliminary study.

Background & objectives: Zinc alters gene expression mainly by binding to a site on the transcription factor. Genome-wide expression studies have shown early repression of genes related to zinc and immunity in adult patients with sepsis. The present study was conducted to evaluate the role of zinc supplementation on relative expression of immune response genes in neonatal sepsis. Methods: In the present study, a sample of convenience of 22 neonates each was selected from the zinc supplemented and control groups using random numbers for expression of immune-related genes by zinc supplementation. These neonates with sepsis were earlier randomized into two groups: with and without zinc supplementation in addition to standard antibiotics and supportive care. Relative expression of immune response genes were analyzed for 22 neonates in each group using quantitative real-time PCR for calprotectin (S100A8/A9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), toll-like receptor-4 (TLR-4), cluster of differentiation 14 (CD14) and lipopolysaccharide-binding protein (LBP) genes. Results: An increase in serum zinc levels was observed in zinc-supplemented group compared to controls. S100A8 gene showed downregulation by three-fold (P <0.001) and S100A9 gene showed upregulation by two-fold (P <0.05) in zinc group compared to controls. CD14 gene showed upregulation by one-fold in zinc-supplemented group compared to controls (P <0.05). No significant fold changes were observed with respect to TNF-α, IL-6, LBP and TLR-4 genes between the two groups. Interpretation & conclusions: The results of our preliminary study showed that the zinc supplementation might modulates the relative expression of immune-related genes involved in sepsis pathway among neonates. However, studies with larger sample size are needed to be done to provide a better picture on the outcome by gene expression in neonatal sepsis by zinc supplementation.

Narciclasine improves outcome in sepsis among neonatal rats via inhibition of calprotectin and alleviating inflammatory responses.

Sepsis is associated with exacerbated inflammatory response which subsequently results in multiple organ dysfunction. Sepsis accounts for high mortality and morbidity among newborns worldwide. Narciclasine is a plant alkaloid which has shown to possess anti-inflammatory properties. In this study we investigated the effect and mechanism of action of narciclasine in neonatal sepsis rat models. The excessive release of S100A8/A9 or calprotectin in neonatal sepsis could be detrimental as it could exacerbate the inflammatory responses. We found that narciclasine significantly reduced the plasma levels of S100A8/A9 and also suppressed its expression in the liver and lung. The systemic and local bacterial load was also reduced in the narciclasine treated rats. The systemic and local production of pro-inflammatory cytokines in plasma and organs (liver and lungs) was significantly reduced in the narciclasine treated rats. The histopathological studies showed that narciclasine prevents the organ damage associated with sepsis and improved the survival of neonatal rats. Sepsis increased the phosphorylated NF-κß p65 protein expression in the liver. Narciclasine suppressed the phosphorylation of NF-κß p65 and the degradation of NF-κß inhibitory protein alpha. It could also suppress the expression of adaptor proteins of the toll like receptor signaling pathway viz., myeloid differentiation factor 88 (MyD88), Interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6). These results suggest that narciclasine protects against sepsis in neonatal rats through the inhibition of calprotectin, pro-inflammatory cytokines and suppression of NF-κß signaling pathway.

Detection of Blastocystis in clinical stool specimens using three different methods and morphological examination in Jones' medium.

BACKGROUND: Blastocystis was identified almost a century ago, yet its biology and pathogenicity status in humans is obscure. Studies on Blastocystis in India are scanty and are mostly microscopy based. This study compared three detection modalities to determine their efficiency in the identification of Blastocystis in human feces. MATERIALS AND METHODS: A total of 279 stool samples were screened using microscopy, culture (Jones' medium), and polymerase chain reaction (PCR)-based methods. Among the three, PCR is considered the gold standard test for detection of Blastocystis, as it helps to authenticate the sensitivity, specificity, and kappa agreement obtained by the other two tests. The morphological features of Blastocystis were recorded at 24, 48, and 72 h. After positive morphological identification, ten samples were cultured on Löwenstein-Jensen (LJ) medium and Locke's egg slant medium. RESULTS: The sensitivity and specificity determined on the basis of microscopy were 36.2% and 99.4%, respectively. On the other hand, Jones' medium showed 67.6% sensitivity and 100% specificity. Further, we documented various morphological and reproductive features of Blastocystis using various staining techniques on cultures positive in Jones' medium. In addition, we also found that LJ medium was not equally efficacious as Jones' medium in assisting the growth of Blastocystis. CONCLUSIONS: Although molecular diagnosis is a necessary tool for understanding the true epidemiology of Blastocystis, in laboratories devoid of molecular detection facilities, stool microscopy in conjunction with stool culture on Jones' medium could serve as the best alternative tool for the detection of Blastocystis.

Short Term Oral Zinc Supplementation among Babies with Neonatal Sepsis for Reducing Mortality and Improving Outcome - A Double-Blind Randomized Controlled Trial.

OBJECTIVE: To evaluate the efficacy of short term zinc supplementation on the mortality rate and neurodevelopment outcome in neonates with sepsis at 12 mo corrected age. METHODS: The clinical trial was undertaken in the neonatal intensive care unit of JIPMER during the time period from September 2013 through December 2016. Neonates with clinical manifestations of sepsis who exhibited two positive screening tests (microESR, C- reactive protein, band cell count) were included and randomized into no zinc and zinc group. The intervention was zinc sulfate monohydrate given at a dose of 3 mg/kg twice a day orally for 10 d along with standard antibiotics. The no zinc group was on antibiotic treatment. Blood samples from both groups were collected at baseline and after day 10. Babies were carefully discharged from the hospital. The babies were followed up till 12 mo corrected age using DASII (Development Assessment Scale for Indian Infants). RESULTS: At the time of enrolment, patient characteristics were similar in both the groups. The mortality rate was significantly higher in no zinc compared to zinc group (5 vs. 13; P = 0.04). Although motor development quotient was similar, mental development quotient was significantly better among babies who received zinc supplementation. CONCLUSIONS: Short term zinc supplementation of newborns with sepsis reduces mortality and improves mental development quotient at 12 mo of age.

Predicting Severity of Acute Kidney Injury in Term Neonates with Perinatal Asphyxia Using Urinary Neutrophil Gelatinase Associated Lipocalin.

OBJECTIVE: To evaluate the utility of urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) as a biomarker for predicting Acute Kidney Injury (AKI) and its severity among neonates with perinatal asphyxia. METHODS: This descriptive study included 120 term neonates with perinatal asphyxia. Renal parameters of neonates were monitored and AKI was ascertained as per Acute Kidney Injury Network criteria. Urinary NGAL was estimated and correlated with severity of AKI. RESULTS: Among the 120 neonates with perinatal asphyxia, 55(46 %) had AKI. The median urinary NGAL level was 165 ng/ml (88.8-245.8) in neonates with AKI compared to 58.97(42.8-74.7) in those without AKI. The median NGAL was 134.45(112.2-162.5), 301.2(255.5-361.2), 416.2(412.2-465.5) in AKI stages 1, 2 and 3 respectively. An NGAL cut off value of 86.82 ng/ml had 87 % sensitivity and 87.7 % specificity in predicting AKI. CONCLUSIONS: Urinary NGAL is a useful biomarker for predicting AKI and its severity among neonates with perinatal asphyxia.

Effect of Zinc Supplementation on Early Outcome of Neonatal Sepsis--A Randomized Controlled Trial.

OBJECTIVE: To find the effect of zinc supplementation on the outcome of neonatal sepsis at one month of age. METHODS: This randomized controlled trial was conducted in a tertiary care neonatal unit, enrolling neonates with clinical features of sepsis and positive blood culture or positive sepsis screening tests. The treatment group received 3 mg/kg/twice a day of zinc sulfate monohydrate orally for 10 d along with standard antibiotic therapy. The control group received standard antibiotic treatment without zinc. Samples were collected from both the groups before and after the treatment. Babies were monitored till discharge and followed up as out-patients till one month of age. RESULTS: Demographic characteristics were similar between the cases and controls. After 10 d of treatment, the mean serum zinc level between the two groups was 737.09 ± 219.97 vs. 801.26 ± 405.56, (p = 0.20). Outcome measures like days of hospital stay (15 vs. 15; p = 0.69) and mortality rate (4.5% vs. 13.6%; p = 0.27) were not found to be significantly different between the groups. At one month of age, more number of control neonates had abnormal neurological findings as compared to the zinc supplemented group [(P = 0.02); RR (95%CI) = 0.28 (0.11-0.73)]. CONCLUSIONS: Zinc supplementation in neonates with sepsis improves the neurological status at one month of age although the mortality reduction was not statistically significant.