Micelle‐enhanced spectrofluorimetric method for quantification of entacapone in tablets and human plasma

In this paper, a simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of entacapone (ETC). The proposed method is based on forming a highly fluorescent product through the reduction of ETC with Zn/HCl. The produced fluorophore exhibits strong fluorescence at λ_em 345 nm after excitation at λ_ex 240 nm. The use of fluorescence enhancers such as Tween‐80 and carboxy methyl cellulose (CMC) greatly enhanced the fluorescence of the produced fluorophore by 150% and 200%, respectively. Calibration curves showed good linear regression (r2 > 0.9998) within test ranges of 0.05–2.0 and 0.02–1.80 μg mL^?1 with lower detection limits of 1.27 × 10^?2 and 4.8 × 10^?3 μg mL^?1 and lower quantification limits of 4.21 × 10^?2 and 1.61 × 10^?2 μg mL^?1 upon using Tween‐80 and or CMC, respectively. The method was successfully applied to the analysis of ETC in its pharmaceutical formulations (either alone or in presence of other co‐formulated drugs). The results were in good agreement with those obtained using the official method. The methods were further extended to determine the drug in human plasma samples, and to study the pharmacokinetics of ETC. The paper is the first report on the spectrofluorimetric determination of entacapone.     

The outcome of twin pregnancies complicated by single fetal death after 20 weeks of gestation.

A retrospective study involving 972 twin births was conducted to evaluate the maternal and fetal outcomes of twin pregnancies complicated by single fetal death. The incidence of single fetal death in twin pregnancies after 20 weeks was 3.3%. Preterm birth rates for 37 and 32 gestational weeks were 81.3% and 41.6% respectively. The median interval between the diagnosis of fetal death and the delivery was 11 days (range 1-27 days). Eighteen (56%) infants were delivered by cesarean and 14 (43%) vaginally. Twin-twin transfusion syndrome (TTTS) was the cause of single fetal death in 8 of 32 twin pregnancies (25%). Ten of the surviving co-twins were lost in the neonatal period (31.3%) and half of those neonatal deaths were due to TTTS. TTTS is the major contributor for perinatal mortality in same-sex twins complicated by single fetal death. The death of one twin in utero should not be the only indication for preterm delivery, and in case of severe prematurity with a stable intrauterine environment; expectant management may be advisable until fetal lung maturation ensues.     

Selection of evolutionary models for phylogenetic hypothesis testing using parametric methods

Recent molecular studies have incorporated the parametric bootstrap method to test a priori hypotheses when the results of molecular based phylogenies are in conflict with these hypotheses. The parametric bootstrap requires the specification of a particular substitutional model, the parameters of which will be used to generate simulated, replicate DNA sequence data sets. It has been both suggested that, (a) the method appears robust to changes in the model of evolution, and alternatively that, (b) as realistic model of DNA substitution as possible should be used to avoid false rejection of a null hypothesis. Here we empirically evaluate the effect of suboptimal substitution models when testing hypotheses of monophyly with the parametric bootstrap using data sets of mtDNA cytochrome oxidase I and II (COI and COII) sequences for Macaronesian Calathus beetles, and mitochondrial 16S rDNA and nuclear ITS2 sequences for European Timarcha beetles. Whether a particular hypothesis of monophyly is rejected or accepted appears to be highly dependent on whether the nucleotide substitution model being used is optimal. It appears that a parameter rich model is either equally or less likely to reject a hypothesis of monophyly where the optimal model is unknown. A comparison of the performance of the Kishino–Hasegawa (KH) test shows it is not as severely affected by the use of suboptimal models, and overall it appears to be a less conservative method with a higher rate of failure to reject null hypotheses.     

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Stem cell therapy and diabetic erectile dysfunction: A critical review

Erectile dysfunction (ED) has been identified as one of the most frequent chronic complications of diabetes mellitus (DM). The prevalence of ED is estimated to be about 67.4% in all DM cases worldwide. The pathophysiological process leading to ED involves endothelial, neurological, hormonal, and psychological factors. In DM, endothelial and neurological factors play a crucial role. Damages in the blood vessels and erectile tissue due to insulin resistance are the hallmark of ED in DM. The current treatments for ED include phosphodiesterase-5 inhibitors and penile prosthesis surgery. However, these treatments are limited in terms of just relieving the symptoms, but not resolving the cause of the problem. The use of stem cells for treating ED is currently being studied mostly in experimental animals. The stem cells used are derived from adipose tissue, bone, or human urine. Most of the studies observed an improvement in erectile quality in the experimental animals as well as an improvement in erectile tissue. However, research on stem cell therapy for ED in humans remains to be limited. Nevertheless, significant findings from studies using animal models indicate a potential use of stem cells in the treatment of ED.     

An Estimating Equations Approach for Modelling Kappa

Agreement between raters for binary outcome data is typically assessed using the kappa coefficient. There has been considerable recent work extending logistic regression to provide summary estimates of interrater agreement adjusted for covariates predictive of the marginal probability of classification by each rater. We propose an estimating equations approach which can also be used to identify covariates predictive of kappa. Models may include an arbitrary and variable number of raters per subject and yet do not require any stringent parametric assumptions. Examples used to illustrate this procedure include an investigation of factors affecting agreement between primary and proxy respondents from a case‐control study and a study of the effects of gender and zygosity on twin concordance for smoking history.     

Design,synthesis, molecular modeling and biological evaluation of novel Benzoxazole-Benzamide conjugates via a 2-Thioacetamido linker as potential anti-proliferative agents,VEGFR-2 inhibitors and apoptotic inducers

A novel series of 2-thioacetamide linked benzoxazole-benzamide conjugates 1–15 was designed as potential inhibitors of the vascular endothelial growth factor receptor-2 (VEGFR-2). The prepared compounds were evaluated for their potential antitumor activity and their corresponding selective cytotoxicity was estimated using normal human fibroblast (WI-38) cells. Compounds 1, 9–12 and 15 showed good selectivity and displayed excellent cytotoxic activity against both HCT-116 and MCF-7 cancer cell lines compared to sorafenib, used as a reference compound. Furthermore, compounds 1 and 11 showed potent VEGFR-2 inhibitory activity. The cell cycle progression assay showed that 1 and 11 induced cell cycle arrest at G2/M phase, with a concomitant increase in the pre-G1 cell population. Further pharmacological studies showed that 1 and 11 induced apoptosis and inhibited the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins in both cell lines. Therefore, compounds 1 and 11 might serve as promising candidates for future anticancer therapy development.     

Effectiveness of a monthly schedule of follow-up for the treatment of uncomplicated severe acute malnutrition in Sokoto,Nigeria: A cluster randomized crossover trial

BackgroundCommunity-based management of severe acute malnutrition (SAM) involves weekly or biweekly outpatient clinic visits for clinical surveillance and distribution of therapeutic foods. Distance to outpatient clinics and high opportunity costs for caregivers can represent major barriers to access. Reducing the frequency of outpatient visits while providing training to caregivers to recognize clinical danger signs at home between outpatient visits may increase acceptability, coverage, and public health impact of SAM treatment. We investigated the effectiveness of monthly clinic visits compared to the standard weekly follow-up in the outpatient treatment of uncomplicated SAM in northwestern Nigeria.Methods and findingsWe conducted a cluster randomized crossover trial to test the noninferiority of nutritional recovery in children with uncomplicated SAM receiving monthly follow-up compared to the standard weekly schedule. From January 2018 to November 2019, 3,945 children aged 6 to 59 months were enrolled at 10 health centers (5 assigned to monthly follow-up and 5 assigned to weekly follow-up) in Sokoto, Nigeria. In total, 96% of children (n = 1,976 in the monthly follow-up group and 1,802 in the weekly follow-up group) were followed until program discharge, and 91% (n = 1,873 in the monthly follow-up group and 1,721 in the weekly follow-up group) were followed to 3 months postdischarge. The mean age at admission was 15.8 months (standard deviation [SD] 7.1), 2,097/3,945 (53.2%) were girls, and the mean midupper arm circumference (MUAC) at admission was 105.8 mm (SD 6.0). In a modified intention-to-treat analysis, the primary outcome of nutritional recovery, defined as having MUAC ≥125 mm on 2 consecutive visits, was analyzed using generalized linear models, with generalized estimating equations to account for clustering. Nutritional recovery was lower in the monthly follow-up group compared to the weekly group (1,036/1,976, 52.4% versus 1,059/1,802, 58.8%; risk difference: −6.8%), and noninferiority was not demonstrated (lower bound of the confidence interval [CI] was −11.5%, lower than the noninferiority margin of 10%). The proportion of children defaulting was lower in the monthly group than in the weekly group (109/1,976, 5.5% versus 151/1,802, 8.4%, p = 0.03). Three months postdischarge, children in the monthly group were less likely to relapse compared to those in the weekly group (58/976, 5.9% versus 78/1,005, 7.8%, p = 0.03), but cumulative mortality at 3 months postdischarge was higher in the monthly group (159/1,873, 8.5% versus 106/1,721, 6.2%, p < 0.001). Study results may depend on context-specific factors including baseline level of care and the clinical status of children presenting to health centers, and, thus, generalizability of these results may be limited.ConclusionsWhere feasible, a weekly schedule of clinic visits should be preferred to maintain effectiveness of SAM treatment. Where geographic coverage of programs is low or frequent travel to outpatient clinics is difficult or impossible, a monthly schedule of visits may provide an alternative model to deliver treatment to those in need. Modifications to the outpatient follow-up schedule, for example, weekly clinic visits until initial weight gain has been achieved followed by monthly visits, could increase the effectiveness of the model and add flexibility for program delivery.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03140904","term_id":"NCT03140904"}}NCT03140904.

Matt D.T. Hitchings and colleagues investigate the effectiveness of a monthly schedule of follow-up for the management of uncomplicated severe acute malnutrition in children aged 6-59 months in Nigeria.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.