Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

Human Hemorrhagic Pulmonary Leptospirosis: Pathological Findings and Pathophysiological Correlations

Background

Leptospirosis is a re-emerging zoonosis with protean clinical manifestations. Recently, the importance of pulmonary hemorrhage as a lethal complication of this disease has been recognized. In the present study, five human necropsies of leptospirosis (Weil‘s syndrome) with extensive pulmonary manifestations were analysed, and the antibodies expressed in blood vessels and cells involved in ion and water transport were used, seeking to better understand the pathophysiology of the lung injury associated with this disease.

Principal Findings

Prominent vascular damage was present in the lung microcirculation, with decreased CD34 and preserved aquaporin 1 expression. At the periphery and even inside the extensive areas of edema and intraalveolar hemorrhage, enlarged, apparently hypertrophic type I pneumocytes (PI) were detected and interpreted as a non-specific attempt of clearence of the intraalveolar fluid, in which ionic transport, particularly of sodium, plays a predominant role, as suggested by the apparently increased ENaC and aquaporin 5 expression. Connexin 43 was present in most pneumocytes, and in the cytoplasm of the more preserved endothelial cells. The number of type II pneumocytes (PII) was slightly decreased when compared to normal lungs and those of patients with septicemia from other causes, a fact that may contribute to the progressively low PI count, resulting in deficient restoration after damage to the alveolar epithelial integrity and, consequently, a poor outcome of the pulmonary edema and hemorrhage.

Conclusions

Pathogenesis of lung injury in human leptospirosis was discussed, and the possibility of primary non-inflammatory vascular damage was considered, so far of undefinite etiopathogenesis, as the initial pathological manifestation of the disease.     

Antibody Levels to Persistent Pathogens and Incident Stroke in Mexican Americans

Background

Persistent pathogens have been proposed as risk factors for stroke; however, the evidence remains inconclusive. Mexican Americans have an increased risk of stroke especially at younger ages, as well as a higher prevalence of infections caused by several persistent pathogens.

Methodology/Principal

Findings Using data from the Sacramento Area Latino Study on Aging (n = 1621), the authors used discrete-time regression to examine associations between stroke risk and (1) immunoglobulin G antibody levels to Helicobacter pylori (H. pylori), Cytomegalovirus, Varicella Zoster Virus, Toxoplasma gondii and Herpes simplex virus 1, and (2) concurrent exposure to several pathogens (pathogen burden), defined as: (a) summed sero-positivity, (b) number of pathogens eliciting high antibody levels, and (c) average antibody level. Models were adjusted for socio-demographics and stroke risk factors. Antibody levels to H. pylori predicted incident stroke in fully adjusted models (Odds Ratio: 1.58; 95% Confidence Interval: 1.09, 2.28). No significant associations were found between stroke risk and antibody levels to the other four pathogens. No associations were found for pathogen burden and incident stroke in fully adjusted models.

Conclusions/Significance

Our results suggest that exposure to H. pylori may be a stroke risk factor in Mexican Americans and may contribute to ethnic differences in stroke risk given the increased prevalence of exposure to H. pylori in this population. Future studies are needed to confirm this association.     

Genetic variants of microRNA-146a gene: an indicator of systemic lupus erythematosus susceptibility,lupus nephritis,and disease activity

Genetic variations of microRNA encoding genes influence various sorts of diseases by modifying the expression or activity of microRNAs. MicroRNA 146a is an epigenetic regulator of immune response through controlling the type I interferon (IFN) and nuclear factor kappa B (NF-κB) pathways. Genetic variations of microRNA 146a impact the susceptibility to systemic lupus erythematosus (SLE) and its clinical presentations. This study aimed to investigate the polymorphisms of microRNA-146a gene (rs2431697 and rs57095329) in patients with SLE and its association with disease activity. Sixty-five patients with SLE and 40 apparently healthy controls were enrolled in this study. Patients were subjected to history taking, clinical examination, and disease activity evaluation by SLEDAI score. The microRNA-146a variants were determined by allele discrimination real-time PCR method in all participants. We found a statistically significant association between rs2431697 T allele and SLE (P-value?<?0.05), but there was no significant association between rs57095329 and SLE. The T/T genotype of microRNA-146a rs2431697 was associated with lupus nephritis, higher disease activity, and autoantibodies production. The microRNA-146a rs2431697 T allele could be a potential risk factor that contributes to SLE susceptibility, development of lupus nephritis, and disease activity.

     

Detection of two CSN1S1 variants in Egyptian buffalo

The present study investigates CSN1S1 casein gene polymorphism in Egyptian buffalo. CSN1S1 was analyzed in 17 unrelated Egyptian lactating buffalo. The amplified segment includes the last 43 amino acids of Exon 17 and part of Intron 17. In the present study we report for the first time the presence of 2 variants 178^Ser (TCA) and 178^Leu (TTA) in Egyptian buffalo CSN1S1 gene. The genotypic frequencies in the investigated Egyptian buffalo sample were 0.47, 0.058 and 0.47 for homozygous 178^Ser, for homozygous 178^Leu and heterozygous 178^Leu/Ser, respectively. The 178^Ser and 178^Leu variant frequencies are 0.64 and 0.36, respectively which indicates the superiority of variant 178^Ser in Egyptian buffalo. The allelic frequency in Egyptian buffalo is not much different from the corresponding allelic frequency in Italian buffalo (0.69 and 0.31 for 178^Ser and 178^Leu, respectively) as reported by Chianese et al. [3]. This is not surprising since they both belong to Mediterranean type.     

Nutrigerontology: a key for achieving successful ageing and longevity

During the last two centuries the average lifespan has increased at a rate of approximately 3 months/year in both sexes, hence oldest old people are becoming the population with the fastest growth in Western World. Although the average life expectancy is increasing dramatically, the healthy lifespan is not going at the same pace. This underscores the importance of studies on the prevention of age-related diseases, in order to satisfactorily decrease the medical, economic and social problems associated to advancing age, related to an increased number of individuals not autonomous and affected by invalidating pathologies. In particular, data from experimental studies in model organisms have consistently shown that nutrient signalling pathways are involved in longevity, affecting the prevalence of age-related loss of function, including age-related diseases. Accordingly, nutrigerontology is defined as the scientific discipline that studies the impact of nutrients, foods, macronutrient ratios, and diets on lifespan, ageing process, and age-related diseases. To discuss the potential relevance of this new science in the attainment of successful ageing and longevity, three original studies performed in Sicily with local foods and two reviews have been assembled in this series. Data clearly demonstrate the positive effects of nutraceuticals, functional foods and Mediterranean Diet on several biological parameters. In fact, they could represent a prevention for many age-related diseases, and, although not a solution for this social plague, at least a remedy to alleviate it. Thus, the possibility to create a dietary pattern, based on the combined strategy of the use of both nutraceuticals and functional foods should permit to create a new therapeutic strategy, based not only on a specific bioactive molecule or on a specific food but on a integrated approach that, starting from the local dietary habits, can be led to a “nutrafunctional diet” applicable worldwide.     

Optimizing the spectrofluorimetric determination of cefdinir through a Taguchi experimental design approach

The aim of this work is to optimize a spectrofluorimetric method for the determination of cefdinir (CFN) using the Taguchi method. The proposed method is based on the oxidative coupling reaction of CFN and cerium(IV) sulfate. The quenching effect of CFN on the fluorescence of the produced cerous ions is measured at an emission wavelength (λ_em) of 358 nm after excitation (λ_ex) at 301 nm. The Taguchi orthogonal array L₉ (3⁴) was designed to determine the optimum reaction conditions. The results were analyzed using the signal‐to‐noise (S/N) ratio and analysis of variance (ANOVA). The optimal experimental conditions obtained from this study were 1 mL of 0.2% MBTH, 0.4 mL of 0.25% Ce(IV), a reaction time of 10 min and methanol as the diluting solvent. The calibration plot displayed a good linear relationship over a range of 0.5–10.0 µg/mL. The proposed method was successfully applied to the determination of CFN in bulk powder and pharmaceutical dosage forms. The results are in good agreement with those obtained using the comparison method. Finally, the Taguchi method provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.     

Orthophthaldehyde as a fluorescent probe for the feasible and sensitive assay of heptaminol: application to dosage forms and real human plasma

The antihypotensive drug heptaminol was determined using a spectrofluorimetric method and ortho-phthaladehyde as a fluorescence probe. The drug was mixed with the reagent in the presence of 2-mercaptoethanol and the reaction was carried out in slightly alkaline aqueous solution containing 0.1 M sodium hydroxide. The resulting product exhibited high fluorescence activity that was measured at 451 nm after excitation at 334 nm. The linearity range of the method was 5–100 ng ml⁻¹ with a lower detection limit of 1.8 ng ml⁻¹. The procedure was evaluated according to the International Council of Harmonization guidelines. The proposed method was applied to analyze the drug in pharmaceutical tablets and oral drops. In addition, the present study represents the first spectrofluorimetric method for the determination of the cited drug in real human plasma. The method provided high recovery percentages without any interference from coexisting pharmaceutical excipients or the components of human plasma.     

Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).