Assay discrepancies using human coagulation factor VIII chromogenic kits: Results from a plasma-derived factor VIII collaborative study (BSP112).

Chromogenic assay discrepancies were reported at General European Official Medicines Control Laboratories Network (GEON) meetings by laboratories testing FVIII-products. The objectives of the present investigation were to carry out a controlled collaborative study to examine these reports and to delineate the reasons for these discrepancies by assessing affected and unaffected FVIII products. The laboratories followed a strict study protocol, which included assessing their own individual observed factor X (FX) activation times, i.e. the time to reach 50% of maximal FX activation (T1/2), for each chromogenic kit. This measurement was used, in parallel with the kit manufacturers' prescribed FX activation times, to assess the performance of the chromogenic potency assays on FVIII test products. This study confirmed a significant discrepancy between Coatest® and Coamatic® kits and between Siemens and Coamatic® kits when the kit manufacturers' prescribed T1/2 incubation times were followed. Coamatic® kits tended to produce higher potencies than the Coatest® or Siemens kits. Furthermore, FX activation assays revealed marked differences between individual laboratories for all three chromogenic kits in the observed T1/2 incubation times, which also did not correspond to the prescribed T1/2 incubation times. The resulting differences in potency between kits, in some cases, were significantly reduced when using the actual observed T1/2 incubation times instead of the prescribed T1/2 incubation times. The study showed that FVIII potency discrepancies can occur between chromogenic kits. To compensate for this, laboratories should ideally perform FX activation curves for each new chromogenic kit in order to determine the correct observed T1/2 incubation times, which can then be used to determine FVIII potencies in therapeutic concentrates.

Calibration of the Ph. Eur. human coagulation Factor VIII Concentrate BRP batch 6.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for human coagulation Factor VIII (FVIII) Concentrate is used as working standard for potency determination of human coagulation FVIII preparations by chromogenic assay. BRP batch 5 was established in 2015 and its stocks were running low. Therefore, the European Directorate for the Quality of Medicines & HealthCare (EDQM) initiated a project (BSP156) for the calibration of a replacement batch. The potency of BRP batch 6 was assigned during an international collaborative study involving 16 laboratories worldwide, with reference to the WHO 8th International Standard (IS) and BRP batch 5. Participants were instructed to perform 3 independent FVIII potency assays following their own routine validated methods for the chromogenic assay, which is the assay prescribed by the Ph. Eur. As an outcome of the study, Ph. Eur. human coagulation FVIII Concentrate BRP batch 6 was assigned a consensus potency of 9.9 IU/ampoule for the chromogenic assay. The Ph. Eur. BRP batch 6 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable for a reference preparation. The Ph. Eur. BRP batch 6 was adopted at the 167th session of the Ph. Eur. Commission in June 2020 and is available from the EDQM under product code H0920000.

Effect of Withania somnifera (L.) Dunal aqueous root extract on reinstatement using conditioned place preference and brain GABA and dopamine levels in alcohol dependent animals.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal (WS), a known'Rasayana' (rejuvenating agent) as per Ayurveda is prescribed to promote health, to increase longevity and to hasten recovery in disease convalescent stages. WS has demonstrated protective effect on alcohol dependence and withdrawal anxiety in previous experimental studies. AIM OF THE STUDY: To evaluate effect of WS on conditioned place behavioral paradigm (model of relapse) and on GABA and dopamine levels in critical brain areas in alcohol dependent animals. METHODOLOGY: Following Animal Ethics Committee permission, the mice (n = 24) were divided into the following study groups for experiment 1: 1 -distilled water (vehicle control), 2 -WS and 3 -Naltrexone. They were conditioned on conditioned place preference (CPP) using alcohol (2 gm/kg)/saline (1 ml) administered intraperitoneally for 8 days. WS and Naltrexone were administered during the period of extinction (6-8 days). Effect of WS (650 mg/kg) on reinstating behaviour of mice (time spent in alcohol paired compartment) primed with alcohol injection was noted. In experiment 2, effect of WS (450 mg/kg/) on GABA and dopamine levels in the midbrain, striatum and cortex (ng/gm) were measured in alcohol dependent rats (n = 24) following the first phase of standardisation assay (n = 36). The rats were made alcohol dependent for 15 days (intermittent access model) and WS was administered concurrently. GABA and dopamine levels were measured on Day 16. RESULTS: WS group showed decrease in time spent in alcohol paired compartment alike Naltrexone and it differed significantly compared to the distilled water control group (p < 0.05) Alcohol-dependent rats showed significant decrease in GABA and increase in dopamine levels vs distilled water in the midbrain, striatum and cortex. WS and Naltrexone administration showed rise in GABA and fall in dopamine in all the isolated brain parts in the respective groups (p < 0.05 vs alcohol treated group). CONCLUSION: Withania somnifera protected animals from relapse and showed beneficial effects on the brain neurotransmitters involved in alcohol dependence. The study provides substantial evidence for its potential application in alcohol use disorder.

De-colourization of textile effluent using immobilized Geotrichum candidum: an insight into mycoremediation.

Textile effluent is generally complicated to manage because of its extremely noxious and recalcitrant coloured compositions. Mycoremediation is an extensively used strategy for the competent degradation of hazardous pollutants present in textile effluent. Fungus could be immobilized in synthetic or natural matrices. The current study shows the decolourization of the textile effluent by 85·5 and 98·5% within 6 h using suspended and immobilized fungus, Geotrichum candidum with optimized parameters like inoculum size (5%), pH (4·5), and temperature (30°C). To maintain a high biomass of fungal population and enhance the retention of fungal strain in the contaminated sites, the fungi need to be immobilized. Hence, the fungus was immobilized naturally onto the selected inert support that is, coconut fibres by the means of adsorption, where they grew as active films on the fibres after being grown in the culture broth. The optimized process parameters of inoculum size, fibre quantity and agitation speed for immobilized G. candidum were 5%, 2·2 g l-1 of effluent and 100 rev min-1 respectively. High level of laccase (22 and 25 U l-1 in suspended and immobilized fungal cells treatment respectively) was observed during the process of decolourization and it was found that decolourization was directly proportional to the laccase activity. The UV-vis, FTIR, 1 H NMR and GC-MS analyses of treated textile industrial wastewater revealed the degradation of toxic pollutants in the textile effluent and formation of lower molecular weight intermediates. The study revealed a higher efficacy of immobilized G. candidum in comparison to suspended fungal culture, employing ligninolytic enzyme laccase, which catalyzes the degradation/transformation of aromatic dyes in the textile effluent thus decolourizing it.

Elucidation of fungal dye-decolourizing peroxidase (DyP) and ligninolytic enzyme activities in decolourization and mineralization of azo dyes.

AIM: The aim of the study is to investigate the efficiency of Geotrichum candidum in the decolourization and mineralization of synthetic azo dyes. METHODS AND RESULTS: It includes screening of enzymes from G. candidum and its optimization, followed by decolourization and mineralization studies. Decolourization was observed to be maximum in methyl orange (94·6%) followed by Congo red (85%), trypan blue (70·4%) and Eriochrome Black T (55·6%) in 48 h, suggesting the plausible degradation of the azo dyes by G. candidum. The enzyme activity study showed that DyP-type peroxidase has highest activity of 900 mU ml-1 compared to that of laccase (405 mU ml-1 ) and lignin peroxidase (LiP) (324 mU ml-1 ) at optimized pH (6) and temperature (35°C). Moreover, the rate of decolourization was found to be directly proportional to the production of laccase and LiP, unlike DyP-type peroxidase. Furthermore, mineralization study demonstrated reduction in aromatic amines, showing 20% mineralization of methyl orange. CONCLUSION: Geotrichum candidum with its enzyme system is able to efficiently decolourize and mineralize the experimental azo dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficient decolourization and mineralization of azo dyes makes G. candidum a promising alternative in the treatment of textile effluent contaminated with azo dyes.

Over-the-counter medicines: Global perspective and Indian scenario.

Patients often approach a pharmacist instead of visiting a doctor for minor ailments such as cough, cold, allergies, pain, fever, acidity, diarrhea, and skin-related conditions. Purchase of specific medicines over the counter is legally recognized in most countries. 'Over-the-Counter (OTC) Medicines' means drugs which are legally allowed to be sold by pharmacists without need for a prescription. The term does not have a legal definition in India. Technically, drugs are OTC unless they are specifically stated as prescription only drugs. OTC drugs allow faster and cheaper access to healthcare; however, their misuse and adverse health effects cause concerns. This article describes concept of OTC medicines and practices in India against the background of globally prevalent regulations and practices. A recognized category of OTC medicines by law, patient awareness programs, and support of pharmacists and pharmaceutical companies are required to optimize the use of OTC medicines in India.

Co-evolution of spliceosomal disassembly interologs: crowning J-protein component with moonlighting RNA-binding activity.

Spliceosome disassembly is catalyzed by the NineTeen-related (NTR) complex, which is constituted by several proteins, including Cwc23, Ntr1, and Ppr43. Cwc23 is an essential J-protein in Saccharomyces cerevisiae that recruits Ntr1, an NTC-related G-patch protein, to the spliceosome. Ntr1 interacts with Prp43, a DExD/H box RNA helicase protein, which facilitates the disassembly of spliceosomal intermediates. The interaction between Ntr1 and Prp43 is conserved and crucial for the disassembly process. However, the J-protein component of this complex is not studied in other eukaryotes. In silico analysis supported by results of yeast complementation and two-hybrid studies suggests that while Prp43 is highly conserved, both Ntr1 and Cwc23 are co-evolving components of the disassembly triad. The J-domain of Cwc23, which is otherwise dispensable for its function, is highly conserved, whereas the functionally critical C-terminus has significantly diverged in Cwc23 orthologs. Some eukaryotic orthologs of Cwc23 contain a distinct RNA recognition motif at their C-terminus and are able to bind RNA in vitro. Based on the results presented in this study, we propose that RNA-binding activity in some eukaryotic orthologs of Cwc23 might provide additional functional diversity or robustness to the J-protein/Hsp70 machine in spliceosomal remodelling processes.

Endocrine regulation of sperm release.

Spermiation (sperm release) is the culmination of a spermatid's journey in the seminiferous epithelium. After a long association with the Sertoli cell, spermatids have to finally 'let go' of the support from Sertoli cells in order to be transported to the epididymis. Spermiation is a multistep process characterised by removal of excess spermatid cytoplasm, recycling of junctional adhesion molecules by endocytosis, extensive cytoskeletal remodelling and final spermatid disengagement. Successful execution of all these events requires coordinated regulation by endocrine and paracrine factors. This review focuses on the endocrine regulation of spermiation. With the aim of delineating how hormones control the various aspects of spermiation, this review provides an analysis of recent advances in research on the hormonal control of molecules associated with the spermiation machinery. Because spermiation is one of the most sensitive phases of spermatogenesis to variations in hormone levels, understanding their molecular control is imperative to advance our knowledge of the nuances of spermatogenesis and male fertility.

An in vitro experiment to simulate how easy tablets are to swallow.

The compliance of patients to solid oral dosage forms is strongly conditioned by the perceived ease of swallowing, especially in geriatric and pediatric populations. This study proposes a method, based on an in vitro model of the human oropharyngeal cavity, to study quantitatively the oral phase of human swallowing in presence of single or multiple tablets. The dynamics of swallowing was investigated varying the size and shape of model tablets and adjusting the force applied to the mechanical setup to simulate tongue pressure variations among individuals. The evolution of the velocity of the bolus, the oral transit time, and the relative position of the solid oral dosage form within the liquid bolus were measured quantitatively from high speed camera recordings. Whenever the solid dosage forms were big enough to interact with the walls of the in vitro oral cavity, a strong effect of the volume of the medication in respect of its swallowing velocity was observed, with elongated tablets flowing faster than spherical tablets. Conversely, the geometrical properties of the solid oral dosage forms did not significantly affect the bolus dynamics when the cross section of the tablet was lower than 40% of that of the bolus. The oral phase of swallowing multiple tablets was also considered in the study by comparing different sizes while maintaining a constant total mass. The predictive power of different theories was also evaluated against the experimental results, providing a mechanistic interpretation of the dynamics of the in vitro oral phase of swallowing. These findings and this approach could pave the way for a better design of solid oral medications to address the special needs of children or patients with swallowing disorders and could help designing more successful sensory evaluations and clinical studies.

Calibration of the Ph. Eur. human coagulation Factor VIII concentrate BRP batch 5.

The European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) for Factor VIII Concentrate batch 5 was established through a collaborative study involving 14 laboratories organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to be used as working standard for potency determination of human coagulation Factor VIII (FVIII) preparations. The potency of the BRP batch 5 was assigned with reference to the WHO 8th International Standard (IS) for FVIII Concentrate and the BRP batch 4. Participants were instructed to perform 3 independent Factor VIII potency assays following their own routine validated methods by the chromogenic assay as it is the assay prescribed by the European Pharmacopoeia. This publication reports the results obtained during the study. The consensus potency, 9.9 IU/ampoule (n = 14) when assessed against both standards, with inter-laboratory geometric coefficients of variation (GCV) of 3.2 % and 1.9 % against the WHO 8th IS and the BRP batch 4 respectively, was consistent with the expected value. The Ph. Eur. BRP batch 5 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable as a reference preparation. The Ph. Eur. BRP batch 5 was adopted at the 151st session of the European Pharmacopoeia Commission in March 2015 and is available from the EDQM.

Linear dichroism and optical anisotropy of silver nanoprisms in polymer films.

We present optical studies of two different size distributions of silver triangular nanoprisms, one with a dipole resonance at 520 nm and the other with a dipole resonance at 650 nm, placed in different media. Significant wavelength-dependent depolarization of scattered light from the silver nanoprisms suspended in water indicates strong interference of multiple surface plasmon resonant modes in the same particle. We use this depolarization as a probe of light scattering by the nanoprisms in a lipid solution due to the rejection of a polarized background scattering. Also, the silver nanoprisms were embedded in a polyvinyl alcohol polymer matrix and oriented by stretching the polymer/nanoprism nanocomposite films. We observe significantly increased linear dichroism in the region associated with the plasmonic in-plane dipole mode upon stretching. Additionally, there is a weaker linear dichroism in the region associated with out-of-plane modes, which vanish in the extinction spectrum of the stretched nanocomposite film.

Characterization of cytopathogenicity of classical swine fever virus isolate induced by Newcastle disease virus.

Classical swine fever virus (CSFV), the causative agent of classical swine fever, belongs to the family Flaviviridae and genus Pestivirus. Some pestiviruses exhibit cytopathic effect in cell culture but exact phenomenon is unknown. Over expression of NS2-3 gene, presence of defective interfering particle and exaltation of Newcastle disease virus (END) phenomenon could be the reasons of cytopathogenicity. In the present study, a CSFV isolate exhibiting cytopathic effect (CPE) in Madin-Darby Canine Kidney (MDCK) cell line was characterized. To characterize cytopathogenicity of such isolate, END test was carried out. Interference of Newcastle disease virus (NDV) in MDCK adapted CSFV was confirmed by RT-PCR and virus neutralization test. Absence of CPE and NDV specific nucleic acid after neutralization confirmed the induction of CPE by NDV. Further, identity of the CSFV isolate in MDCK cell line by immunoperoxidase test, immunoblotting and RT-PCR post NDV neutralization established the virus replication without CPE (non-cytopathic isolate). Findings suggest that, there could be a chance of mixed infection of both CSFV and NDV in the piglet from which the sample was collected for virus isolation.

Association of Previous Smoking Habit and Perceived Social Discrimination with the Risk of Multi-Drug Resistant Tuberculosis in Central Nepal.

BACKGROUND: Multidrug-resistant tuberculosis (MDR TB) caused by Mycobacterium tuberculosis resistant to both Isoniazid and Rifampicin with or without resistant to other drug, is among the most alarming pandemic problem. The objectives of this study was to assess the risk factors of MDR TB in Central Nepal. METHODS: A matched case control study was conducted among 186 cases of MDR TB and 372 non-MDR TB controls from central region of Nepal. Pretested questionnaires containing socio-economic, cultural & behavioral; environmental, biological and health service factors were used. Variables significant in bivariate analysis were entered in multiple regression models for further analysis. RESULTS: After adjusting for confounders, previous smoking habit (aOR= 4.5,(95%CI(1.24-16.2)) (p=0.04), and perceived social discrimination (aOR=5.83,95%CI (1.77-19.71)) (P=0.021) independently predicted greater MDR TB risk. CONCLUSIONS: Encouraging MDR TB cases for smoking cessation through awareness activities should be a priority. Stigma reduction programs should include the empowerment of patients and communities while promoting TBrelated research for further exploration into the risk factors of TB and associated stigma.

The spatial distribution of actin and mechanical cycle of myosin are different in right and left ventricles of healthy mouse hearts.

The contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. Indeed, differences have been observed in the contractile performance between left and right ventricular myocytes in dilated cardiomyopathy, in congestive heart failure, and in energy usage and speed of contraction at light loads in healthy hearts. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance, or power among the RV and LV in dogs. This report shows that on a mesoscopic scale [when only a few molecules are studied (here three to six molecules of actin) in ex vivo ventricular myofibrils], the two ventricles in rigor differ in the degree of orientational disorder of actin within in filaments and during contraction in the kinetics of the cross-bridge cycle.

Long lived BSA Au clusters as a time gated intensity imaging probe.

The work presented here reports the use of long lifetime (>1 µs) BSA Au clusters as a cellular/tissue, time gated, intensity imaging probe. By collecting the emission signal 50 ns post excitation, one can off-gate the intense auto-fluorescence background, thereby greatly enhancing the clarity/specificity in fluorescence imaging.

Study of photophysical properties of different metal complexes of Alq3.

Different metal complexes of Alq3 (K(+) , Sr(2+) , Ca(2+) and Mg(2+) ) were synthesized by the precipitation method. The characterization of photoluminescence showed that presence of Mg(2+) ion enhances photoluminescence of Alq3 phosphor. The emission spectra are observed at 560 nm when excited at a wavelength of 440 nm. The phosphor is excited at a longer wavelength in the blue region of small energy, so that it could be used as lamp phosphor. It is observed that the prepared phosphor AlMgq5 is more suitable than Alq3. The ionic radii of K(+) , Sr(2+) , Ca(2+) and Mg(2+) ions are in decreasing order. Therefore, the remarkable properties of AlMgq5 could be considered as promising materials as opto- or optoelectronic materials.

Factor VIII Bypassing Activity (FEIBA) assays: standardization and development of the 1st NIBSC Working Standard for FEIBA--results from a collaborative study.

Factor-Eight-Inhibitor-Bypassing-Activity (FEIBA) is a bypassing-agent used to control spontaneous bleeding or cover surgical interventions in Haemophiliacs who develop neutralizing antibodies against FVIII/FIX. The market lot-release of FEIBA is dependent on specific clot-based assays, carried out by both the manufacturer and regulatory authorities, relative to manufacturer's in-house standards, which are produced on a small-scale and are replaced frequently. We sought to standardize the FEIBA assay by developing a FEIBA primary standard which would be internationally available in sufficiently large quantities, with a predicted lifetime of many years. A collaborative study involving the manufacturer and three regulatory authorities, was carried out in which a candidate material, sample B (06/172), was calibrated by assays relative to the manufacturer's in-house FEIBA standards (C and D). All laboratories used their routine validated methods (16 APTT-assays, 8 ACTIN-FS-assays and 27 DAPTTIN-assays). Intra-laboratory geometric coefficients of variation (GCVs) for candidate B ranged from 3% to 29% (GCVs <9% from majority of labs). Assessment of inter-laboratory variability gave overall GCV values of 6.9% and 4.4% relative to standards C and D, respectively, for all methods. There was good agreement in potency estimation between laboratories using each of the three methods, with the overall potencies by the three methods differing by less than 10% of the overall mean, giving an overall combined potency of 28.0 units per ampoule. All participants agreed that candidate B (06/172) be established as the 1st NIBSC Working Standard for FEIBA with an assigned potency of 28.0 units per ampoule, based on combined results for both methods, relative to either standard C or D.

Community attitudes towards leprosy affected persons in Pokhara municipality of western Nepal.

BACKGROUND: Stigma is a social process of interpretation of an attribute. Leprosy has been seen as the epitome of stigmatization. The psychosocial impact a person has to bear in a society after the diagnosis weighs heavier than the physical afflictions it causes, which does not get cured with the mere medical treatment. There are various factors which construct the perception of stigma in both leprosy affected persons and unaffected persons. The main purpose of this study was to determine the level of perceived stigma and the risk factors contributing to it among community people living in ward 15, Pokhara municipality. METHODS: Cross-sectional descriptive study among 281 community people above the age of 18 years was conducted. Two sets of questionnaire form with additional Explanatory Model Interview Catalogue (EMIC) for each individual were used. RESULTS: Among 281 community people, the median score of perceived stigma was 12 while it ranged from 0-30. Ethnic groups, Brahmins, Dalits and minorities had highest perceived stigma score of 15 and above compared to the rest (p=0.001), community people living at the distance more than 2 km had highest perceived stigma score of 15 compared to those living closer to the hospital (p=0.019) and nuclear family had highest perceived stigma score of 15 compared to the joint family (p=0.014). People who lacked information on leprosy had higher score of perceived stigma compared to those who had information on leprosy (p=0.002).Similarly, those who perceived leprosy to be difficult to treat (p<0.001) and a severe disease (p<0.001) had highest score of perceived stigma. CONCLUSIONS: Stigma in leprosy was found highly associated with the lack of information about leprosy and their perception in treatment and disease severity. Stigma reduction strategies should focus on health education, targeting to alleviate their perception about the disease with their active participation.