Manisha Pathak

The merits of continuous processing over batch processing are well known in the manufacturing industry. Continuous operation results in shorter process times due to omission of hold steps, higher productivity due to reduced shutdown costs, and lowers labor requirement. Over the past decade, there has been an increasing interest in continuous processing within the bioprocessing community, specifically those involved in production of biotherapeutics. Continuous operations in upstream processing (perfusion) have been performed for decades. However, recent development of continuous downstream operations has led the industry to envisage an integrated bioprocessing platform for efficient production. The regulators, key players in the biotherapeutic industry, have also expressed their interest and willingness in this migration from the traditional batch processing. This paper aims to review major developments in continuous bioprocessing in the past decade. A discussion of pros and cons of the different proposed approaches has also been presented.

The intestinal epithelial lining is a very dynamic interface, where multiple interactions occur with the external world. The intestinal epithelial barrier is continuously exposed to a huge load of commensal microorganisms, food-borne antigens, as well as invading enteropathogens. Intestinal epithelial cells (IECs) and underlying immune cells are the main players in maintaining the delicate balance between gut tolerance and inflammation. IECs deferentially express the variety of chemokines and chemokine receptors, and these receptor-ligand interactions not only mediate the infiltration and activation of immune cells but also switch on the survival cascades in IECs. In this review, we discussed how chemokine-chemokine receptor-induced interactions play a central role to coordinate the interplay between IECs and gut immune cells to maintain homeostasis or elicit gut inflammation. Furthermore, we discussed how chemokines and chemokine receptors were used as a target for developing new drugs and therapies to control gut inflammation and autoimmunity.

Abstract The efficiency of the protein refolding process lies in identification of the optimal conditions. However, a number of challenges need to be overcome to achieve this. This review first describes the protein refolding process that is utilized presently for production of protein therapeutics. Next, it discusses the various shortcomings that exist with respect to the present approach. The focus of the paper is on presentation of the significant advancements that have been made in the past decade in the various aspects of protein folding, including use of bioinformatics, mechanistic modeling, analytical monitoring, process optimization, use of additives, high throughput development, on‐column refolding, Quality by Design ( QbD ), Process Analytical Technology ( PAT ), and process intensification. Finally, an approach is proposed that incorporates the best practices that have been identified in the various areas. The paper is expected to be of interest to those in academia and industry working in the area of protein refolding. © 2013 Society of Chemical Industry.

A novel coiled flow inverter (CFI) based plug flow reactor has been developed for continuous refolding of granulocyte colony stimulating factor (GCSF), a biotech therapeutic product. Solubilized inclusion bodies containing the denatured and reduced forms of GCSF were continuously diluted with the refolding buffer using an inline mixing unit. This was followed by protein refolding into a CFI based tubular reactor in which a helical coil was bent at equidistant right angles to cause flow inversion at each bend. This configuration effectively provided substantial cross sectional mixing while maintaining a favourable distribution of residence time. Design of experiments (DOE) based studies was performed to optimize the refolding protocol with respect to redox conditions, pH and dilution ratio. The performance of the continuous refolding protocol has been compared with an optimized batch refolding protocol. It has been demonstrated that enhanced mixing in CFI allows for operation at higher protein concentrations (0.38 mg/ml as compared to 0.19 mg/ml in batch) and results in comparable purity (84% vs. 83% in batch), thereby resulting in a nearly 15 times increase in productivity. This will result in a significant reduction of costs related to downstream purification as well as no need for the large tank that is otherwise required for dilution based batch refolding. The proposed configuration is likely to perform favourably in other biotech unit operations that require mixing and/or sharp residence time distribution such as precipitation.

Biotech unit operations are often characterized by a large number of inputs (operational parameters) and outputs (performance parameters) along with complex correlations among them. A typical biotech process starts with the vial of the cell bank, ends with the final product, and has anywhere from 15 to 30 such unit operations in series. Besides the above‐mentioned operational parameters, raw material attributes can also impact process performance and product quality as well as interact among each other. Multivariate data analysis (MVDA) offers an effective approach to gather process understanding from such complex datasets. Review of literature suggests that the use of MVDA is rapidly increasing, fuelled by the gradual acceptance of quality by design (QbD) and process analytical technology (PAT) among the regulators and the biotech industry. Implementation of QbD and PAT requires enhanced process and product understanding. In this article, we first discuss the most critical issues that a practitioner needs to be aware of while performing MVDA of bioprocessing data. Next, we present a step by step procedure for performing such analysis. Industrial case studies are used to elucidate the various underlying concepts. With the increasing usage of MVDA, we hope that this article would be a useful resource for present and future practitioners of MVDA. © 2014 American Institute of Chemical Engineers Biotechnol. Prog ., 30:967–973, 2014

This paper aims to provide a thorough understanding of how fouling of Protein A resin takes place. Binding and mass transport properties of widely used agarose-based Protein A resin, MabSelect SuRe™, have been examined to understand the mechanism of resin fouling. There could be various factors that impact resin fouling. These include product/impurity build-up due to components in the feed material and ligand degradation due to the use of harsh buffers. To unravel their contributions, cycling studies were performed with and without product loading. The results presented in this paper provide a lucid understanding of the causative factors that limit Protein A chromatographic resin lifetime. The capacity fall for protein A resin at the end of 100th cycle due to use of feed material was found to be five times greater than that without using feed material. Compared to the fresh resin, the cycled resin samples shows 24% reduction in particle porosity and 51% reduction in pore mass transfer coefficient. Transmission electron microscopy (TEM) was used to qualitatively monitor accumulation of foulants on the cycled resin. Fouled resin sample contained a dense residue in the interior and exterior of resin particle both as a film at the bead surface and as granules. The surface activation energy increased five times in the case of fouled resin sample. The major event in fouling was identified as the non-specific adsorption of the feed material components on resin, signaling that pore diffusion is the rate limiting step. It is anticipated that these findings will assist in development of a more robust and economical downstream manufacturing process for monoclonal antibody purification.

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.

Chemokine receptor CCR9 is a G protein-coupled receptor and express on several types of immune cells, including dendritic cells, CD4+ T cells, and B cells. CCR9 drives the migration of immune cells to gradients of its cognate ligand CCL25. The chemokine CCL25 is mostly produced by gut and thymic epithelial cells. Gut- and thymic-homing dendritic cells (DCs) are known to express CCR9, and these cells predominantly localized in the gut lining and thymus. CCR9+ DCs are implicated in regulating inflammation, food allergy, alloimmunity, and autoimmunity. Differential interaction of CCR9+ DCs with lymphoid and myeloid cells in the thymus, secondary lymphoid tissues, and mucosal sites offer crucial insights to immune regulation. In this review, we reviewed the phenotypes, distributions, and interactions of CCR9+ DCs with other immune cells, elucidating their functions and role in inflammation and autoimmunity.

Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC–MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC–MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

The trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.In the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.The present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

Abstract BACKGROUND Assessment of process comparability is often required across different phases of manufacturing (Phase I vs Phase II vs Phase III vs Commercial) as well as other key activities during product commercialization (process scale‐up, technology transfer, process improvement). In this work, chemometrics was applied to compare two versions of a biotech process and identify process steps where significant differences exist as well as the parameters that cause these differences. RESULTS The dataset used in this analysis consists of data from 229 manufacturing batches. Partial least squares has been used for modeling the data. Scatter plots and variable importance plots have been used for evaluating comparability. Process parameters identified as significant by using chemometrics have been compared against those identified from process characterization using traditional lab scale experimental studies. The comparison has been followed by discussion on the pros and cons of the two approaches. To our knowledge this is the first time that such a comparison has been published for biotech processing. CONCLUSIONS The study further demonstrates the usefulness of chemometrics in defining process comparability and in gathering process understanding from analysis of manufacturing data to supplement traditional lab‐scale experimentation. © 2014 Society of Chemical Industry

Biotherapeutics have become the focus of the pharmaceutical industry due to their proven effectiveness in managing complex diseases. Downstream processes of these molecules consist of several orthogonal, high resolution unit operations designed so as to be able to separate variants having very similar physicochemical properties. Typical process development involves optimization of the individual unit operations based on Quality by Design principles in order to define the design space within which the process can deliver product that meets the predefined specifications. However, limited efforts are dedicated to understanding the interactions between the unit operations. This paper aims to showcase the importance of understanding these interactions and thereby arrive at operating conditions that are optimal for the overall process. It is demonstrated that these are not necessarily same as those obtained from optimization of the individual unit operations. Purification of Granulocyte Colony Stimulating Factor (G-CSF), a biotherapeutic expressed in E. coli., has been used as a case study. It is evident that the suggested approach results in not only higher yield (91.5 vs. 86.4) but also improved product quality (% RP-HPLC purity of 98.3 vs. 97.5) and process robustness. We think that this paper is very relevant to the present times when the biotech industry is in the midst of implementing Quality by Design towards process development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:355-362, 2016.

Anemia is a very common condition in pregnancy. It represents one of the most common risk factors for maternal as well as fetal complications. Its early identification, treatment and prevention is necessary to avoid these complications. The objective of this study was to evaluate prevalence of anemia in pregnant women attending outpatient department. In this single center retrospective clinic-based study carried pregnant women attending for their regular ante-natal checkup were included. Demographic details, hemoglobin level and prior obstetric related history was noted. Prevalence of anemia was estimated based on the hemoglobin level. The severity of anemia was categorized as mild (Hb level 10 g/dL to 10.9 g/dL), moderate (Hb level 7 g/dL to 9.99 g/dL), severe (Hb level <7 g/dL).Corelates of anemia were examined based on the demographic parameters. The study included 199 pregnant females with mean (SD) age was 29.6 (4.1) years. Mean (SD) hemoglobin of the study population was 10.6 (1.4) gm. A total of 174 (87.44%) females were house-makers and 165 (82.9%) were from the urban area. Anemia was observed in 76 (38.2%) pregnant females. Mild, moderate and severe anemia was observed in 32 (42.11%), 43 (56.58%) and 1 (1.3%) female respectively. Out of 76 pregnant females with anemia, 66 (86.8%) were housemakers. No significant difference was observed in the mean age (p=0.34) and body weight (p=0.69) of pregnant females with or without anemia. There was no significant difference in the anemia prevalence in rural versus urban pregnant females (p=0.33). Prevalence of anemia in pregnant women was 38.2%. Moderate anemia was more common than mild and severe anemia. There was no significant difference in the mean age or residence of pregnant females with anemia versus without anemia.

This paper presents a quality by design (QbD) based development of a novel native PAGE (N‐PAGE) method as a low‐cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N‐PAGE relevant to those academicians and other small companies involved in early‐stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N‐PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD‐based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N‐PAGE has been in use for protein analysis for several decades, use of N‐PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.

Vaccination is devised/formulated to stimulate specific and prolonged immune responses for long-term protection against infection or disease. A vaccine component, namely adjuvant, enhances antigen recognition by the host immune system and thereby stimulates its cellular and adaptive responses. Especially synthetic Toll-like receptor (TLR) agonists having self-assembling properties are considered as good candidates for adjuvant development. Here, a human TLR4-derived 20-residue peptide (TR-433), present in the dimerization interface of the TLR4–myeloid differentiation protein-2 (MD2) complex, displayed self-assembly and adopted a nanostructure. Both <i>in vitro</i> studies and <i>in vivo</i> experiments in mice indicated that TR-433 is nontoxic. TR-433 induced pro-inflammatory responses in THP-1 monocytes and HEK293T cells that were transiently transfected with TLR4/CD14/MD2 and also in BALB/c mice. In light of the self-assembly and pro-inflammatory properties of TR-433, we immunized with a mixture of TR-433 and either ovalbumin or filarial antigen trehalose-6-phosphate phosphatase (TPP). A significant amount of IgG titers was produced, suggesting adjuvanting capability of TR-433 that was comparable with that of Freund's complete adjuvant (FCA) and appreciably higher than that of alum. We found that TR-433 preferentially activates type 1 helper T cell (T_h1) response rather than type 2 helper T cell (T_h2) response. To our knowledge, this is the first report on the identification of a short TLR4-derived peptide that possesses both self-assembling and pro-inflammatory properties and has significant efficacy as an adjuvant, capable of activating cellular responses in mice. These results indicate that TR-433 possesses significant potential for development as a new adjuvant in therapeutic application.

Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process as per the expectations laid out in the Quality by Design paradigm.

The phosphoglycerate mutase (PGM) enzyme catalyzes the interconversion of 2- and 3-phosphoglycerate in the glycolytic /gluconeogenic pathways that are present in the majority of cellular organisms. They can be classified as cofactor-dependent PGM (dPGM) or cofactor-independent PGM (iPGM). Vertebrates, yeasts, and many bacteria have only dPGM, while higher plants, nematodes, archaea, and many other bacteria have only iPGM. A small number of bacteria, including Escherichia coli and certain archaea and protozoa, contain both forms. The silencing of ipgm in Caenorhabditis elegans (C. elegans) has demonstrated the importance of this enzyme in parasite viability and, therefore, its potential as an anthelmintic drug target. In this study, the role of the Brugia malayi (B. malayi) ipgm in parasite viability, microfilaria release, embryogenesis, and in vivo development of infective larvae post-gene silencing was explored by applying ribonucleic acid (RNA) interference studies.The in vitro ipgm gene silencing by small interfering RNA (siRNA) leads to severe phenotypic deformities in the intrauterine developmental stages of female worms with a drastic reduction (~90%) in the motility of adult parasites and a significantly reduced (80%) release of microfilariae (mf) by female worms in vitro. Almost half of the in vitro-treated infective L3 displayed sluggish movement. The in vivo survival and development of siRNA-treated infective larvae (L3) was investigated in the peritoneal cavity of jirds where a ~45% reduction in adult worm establishment was observed.The findings clearly suggest that iPGM is essential for both larval and adult stages of B. malayi parasite and that it plays a pivotal role in female worm embryogenesis. The results thus validate the Bm-iPGM as a putative anti-filarial drug target.

Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose‐based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC‐MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding. © 2018 American Institute of Chemical Engineers Biotechnol. Prog. , 34:412–419, 2018

Protein A chromatography is quite commonly used for capture of monoclonal antibodies from the clarified cell culture broths. Protein A resins are expensive and economic feasibility demands that the resin be reused for 50–300 cycles. Resin reuse is, however, accompanied by resin fouling, impacting both the binding and mass transfer characteristics of the resin. In the present study, we attempt to model the variations in binding and mass transfer characteristics of a commercially available Protein A resin, mAbSelect SuRe™, as a function of resin’s reuse. Simplified linear driving force modeling and kinetic modeling of Protein A chromatography step elution cycling data has been successfully used to predict resin performance up to 100 cycles based on fouling data up to 50 cycles. Fouling factor for Protein A resin has been empirically modeled as a function of binding and mass transfer characteristics of resin and the resin’s reuse using a combination of Buckingham’s π theorem and statistical analysis. The proposed empirical model enables reliable prediction of performance of Protein A resin as well as offers an improved understanding of the underlying mechanism behind the decline in resin performance during fouling.

The chemokine receptor CCR9 and its only known ligand CCL25 play an important role in gut inflammation and autoimmune colitis. The function of CCR9-CCL25 in the migration of immune cells is well characterized. However, its role in the immune cell differentiation is mostly not known. Using dextran sodium sulfate (DSS)-induced gut inflammation model, we showed that CCR9+ dendritic cells (DCs) specifically CD11b- CD103+ DCs were significantly increased in the gut-associated lymphoid tissues (GALT) compared to control mice. These CCR9+ DCs express lower MHC II and CD86 molecules and had regulatory surface markers (FasL and latency-associated peptide, LAP) in the GALT. In the presence of CCL25, CCR9+ DCs promoted in vitro differentiation of Foxp3+ regulatory CD4+ T cells (Tregs). CCL25-induced differentiation of Tregs was due to intrinsic signaling in the DCs but not through CD4+ T cells, which was driven by the production of thymic stromal lymphopoietin (TSLP) and not IL-10. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the OVA-specific gut-allergic response. Our results suggest CCR9+ DCs have a regulatory function and may provide a new cellular therapeutic strategy to control gut inflammation and allergic immune reaction.

A real time monitoring of fouling in liquid chromatography has been presented. The versatility of the approach has been proven by successful implementation in three case studies with an error <1%. The first application demonstrates the monitoring of protein A ligand density and foulant concentration for assessing performance of protein A chromatography resin during purification of monoclonal antibodies. The observations have been supported from LC-MS/MS studies that were independently performed. The second application involves monitoring of foulant deposition during multimode cation exchange chromatography based purification of human serum albumin. Finally, in the third application, monitoring of foulants during multimodal hydrophobic interaction chromatography of recombinant human granulocyte colony stimulating factor is demonstrated. In all three cases, it is observed that the fluorescence intensity consistently increases with resin reuse as more foulants are deposited over time. The proposed approach can be readily used for real time monitoring of fouling and process control.

Moxidectin is a macrocyclic lactone belonging to milbemycin family closely related to ivermectin and is currently progressing towards Phase III clinical trial against human infection with the filaria Onchocerca volvulus (Leuckart, 1894). There is a single report on the microfilaricidal and embryostatic activity of moxidectin in case of the human lymphatic filarial parasite Brugia malayi (Brug, 1927) in Mastomys coucha (Smith) but without any adulticidal action. In the present study, the in vitro and in vivo antifilarial efficacy of moxidectin was evaluated on, B. malayi. In vitro moxidectin showed 100% reduction in adult female worm motility at 0.6 μM concentration within 7 days with 68% inhibition in the reduction of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) (which is used to detect viability of worms). A 50% inhibitory concentration (IC50) of moxidectin for adult female parasite was 0.242 μM, for male worm 0.186 μM and for microfilaria IC50 was 0.813 μM. In adult B. malayi-transplanted primary screening model (Meriones unguiculatus Milne-Edwards), moxidectin at a single optimal dose of 20 mg/kg by oral and subcutaneous route was found effective on both adult parasites and microfilariae. In secondary screening (M coucha, subcutaneously inoculated with infective larvae), moxidectin at the same dose by subcutaneous route brought about death of 49% of adult worms besides causing sterilisation in 54% of the recovered live female worms. The treated animals exhibited a continuous and sustained reduction in peripheral blood microfilaraemia throughout the observation period of 90 days. The mechanism of action of moxidectin is suggested to be similar to avermectins. The in silico studies were also designed to explore the interaction of moxidectin with glutamate-gated chloride channels of B. malayi. The docking results revealed a close interaction of moxidectin with various GluCl ligand sites of B. malayi.

Lymphatic filariasis leads to profound impairment of parasite-specific T helper type 1 (Th1) and Th2 immune responses and significantly increases the expression of regulatory networks and regulatory effectors like transforming growth factor-β, CD25, cytotoxic T-lymphocyte antigen 4, glucocorticoid-induced tumour necrosis factor receptor (GITR) and regulatory T (Treg) cells, which together play an important role in immunosuppression. While Treg cells suppress the activity of effector cells, monocyte dysfunction, characterized by an alternatively activated immunoregulatory phenotype, is one hypothesis that explains the lack of an antigen-specific T-cell response in infected individuals. In the present study, we administered neutralizing antibodies against the Treg cell-associated markers CD25 and GITR and observed its effects on filaria-induced immunosuppression. Our results show that administration of anti-CD25 and anti-GITR in infected animals not only arrested the accumulation of Treg cells and reduced arginase activity, but also led to an increase in the percentages of Th17 cells in the secondary lymphoid organs of mice. Elevated levels of interferon-γ and decreased levels of interleukin-10 were also noted in the culture supernatants of mouse splenocytes that were treated with neutralizing antibodies. Furthermore, treatment with neutralizing antibodies enhanced the expression of inducible nitric oxide synthase on host macrophages and CD40 on host dendritic cells with concomitant decreased expression of alternative activation markers Arg1, Ym1 and Fizz1, which together lead to reduced parasite burden in treated animals. In summary, administration of neutralizing antibodies helps in breaking the regulatory network in mice and limits parasite-induced immunosuppression at the earliest host-parasite interface.

Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor β (TGF-β) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-β levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-β secretion.

Annona squamosa (AS) has traditionally been used as ethnomedicine. We have earlier extracted and fractionated the twigs of AS based upon its bioactivity and observed its immune potentiating activity that was localized in its three fractions. Present communication deals with the phytochemical analysis and pharmacological investigation of the most active chloroform fraction that led to isolation and identification of a number of compounds whose structures were elucidated using 1D and 2D NMR spectroscopic analysis. Amongst the twelve pure compounds isolated, five compounds Lanuginosine (1), (+)-O-methylarmepavine (2), (+)-anomuricine (3), Isocorydine (4), and N-methyl-6, 7-dimethoxyisoquinolone (5) were evaluated in vivo for their immune modifier activities in BALB/c mice after oral administration at three log doses of 0.3, 1.0 and 3.0mg/kg for 14 consecutive days. Of these, three compounds (1, 2 and 5) showed dose dependent immune stimulating activity. However, the uppermost activity was noted in the compound N-methyl-6, 7-dimethoxyisoquinolone at the 3.0mg/kg oral dose. The activity was assessed in the form of increased splenic T and B cellular proliferation, up-regulated CD4+, CD8+ and CD19+ cell population and accentuation in the peritoneal macrophage function. The compound possibly acted modifying the expression of Th1- and Th2- cytokines via stimulation of pro-inflammatory Th1 cytokines IL-2 and IFN-γ. These results warrant the use of the above compounds as an efficient immune-stimulant or immune-adjuvant against diseases with immune suppression. The analogs of the compound may further be chemically synthesized to achieve desired immune modifying activity.

We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing high degree of protection against filarial larval invasion.

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.

Abstract BACKGROUND Protein A chromatography fouling is accompanied by two major events, one is the loss of protein A ligands and other is fouling due to non‐specific, irreversible interactions of foulants with resin particles. This paper presents implementation of process analytical technology based control for fouling of protein A chromatography resin using a novel, fluorescence based approach. This approach enables direct, in situ measurement of protein A ligand density as well as monitoring of resin fouling during resin reuse. RESULTS A novel, fluorescence based process analytical technology (PAT) tool has been designed and used for screening a variety of cleaning protocols. A two‐step cleaning protocol was created using this methodology. The above mentioned fluorescence based approach was successfully used to monitor fouling and to take a decision on when to initiate cleaning. This resulted in effective maintenance of dynamic binding capacity and step yield at 50 cycles (DBC: 97% of the original value vs 65% with conventional protocols and yield: 93% of the original value vs 73% with conventional protocols) and at 200 cycles (&gt; 90% of the original value). CONCLUSIONS The proposed fluorescence based approach has been effectively used for monitoring and control of resin fouling upon reuse, thereby resulting in a substantial increase in resin lifetime. © 2017 Society of Chemical Industry

Variations due to geographical location and dioecious nature have shown implications in the chemical and pharmacological properties of medicinal plants and their herbal products. Tinospora cordifolia is one of the most important dioecious plant distributed throughout India and very widely used in many herbal products and formulations. In this study a method combining direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight (TOF) mass spectrometer (MS) along with multivariate analysis was developed and applied for metabolic fingerprinting and screening of the major phytochemicals in this plant. Using this approach phytodiversity in plants due to gender and geographical distribution were studied in T. cordifolia stem cuttings without any processing. An aqueous/ethanolic stem extracts of male and female T. cordifolia were also evaluated for immunomodulatory activity in inbred strain of age and sex matched BALB/c mice. A characteristic nine and sixteen marker peaks were respectively, identified as gender and geographical markers for T. cordifolia stem. It also discriminates the herbal and polyherbal formulations of T. cordifolia stem using principal component analysis. Female plant stem extract caused a significant up regulation in the pro-inflammatory and anti-inflammatory cytokines and activated the peritoneal exudate cells leading to significant release in reactive oxygen species and enhanced the in vitro lymphocyte proliferation than male stem extract. This finding underscore the importance of gender in all dioecious medicinal plants where only vegetative parts are used as a source of drug as the pharmacological activity may vary depending on the sex of the plant used.

Rapid quantitation of product titer is a critical input for control of any bioprocess. This measurement, however, is marred by the myriad components that are present in the fermentation broth, often requiring extensive sample pretreatment before analysis. Spectroscopy techniques such as fluorescence spectroscopy are widely recognized as potential monitoring tools. Here, we investigate the possibility of using fluorescence of the culture supernatant as a potential at-line monitoring tool to measure the concentration of a recombinant therapeutic protein expressed in a Pichia pastoris fed-batch fermentation. We propose an integrated method wherein both the target protein and total protein concentrations are predicted using intrinsic riboflavin fluorescence and extrinsic fluorescence, respectively. The root mean square error for estimating the concentrations of the target protein (using riboflavin fluorescence) and total protein (using extrinsic fluorescence) have been estimated to be <0.1 and <0.2, respectively. The proposed approach has been validated for two different biotherapeutic products, human serum albumin and granulocyte colony stimulating factor, that were expressed using Mut+ and Muts strains of P. pastoris, respectively. The proposed approach is rapid (1 min analysis time, 10 min total with at line sampling) and thus could be a significant enabler for process analytical technology implementation in Pichia fermentation.

Imidazolin-2-ones(13-18) on photooxygenation in the presence of methylene blue yielded the corresponding diacylureas as the only products isolated at room temperature. The rate of photooxygenation followed the order 16>17>18>13>14>15. The reaction was also studied at pH 4.4, 6.0 and 9.2 as well as in solvents of varying dielectric constants to explore the nature of the intermediates. It appears that the reaction involves the formation zwitterionic perepoxides leading to dioxetanes which decompose to yield diacylureas.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4-6 months) and adult (~ 2 years) bucks during non-breeding (July-August; n = 4 each) and breeding (October-November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and β1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs.The online version contains supplementary material available at 10.1007/s10616-021-00515-x.

The composition of follicular fluid (FF) varies with the cyclical hormonal changes and developmental stage of follicle. During superovulation programme, the large number of unovulated follicles is a major constraint and affects adversely the embryo recovery. An attempt was made to study the hormonal and biochemical profiles of unovulated follicles in goat superovulated with either Synthetic FSH (133 mg Folltropin) or 1000 IU PMSG (Folligon). The study revealed that Progesterone and testosterone concentrations were significantly (P<0.05) higher in FF of unovulated follicles of suparovulated goats as compared to control. The total and free cholesterol levels were significantly (P<0.05) lower in FF of treated animals then control one. The Alkaline Phosphosphatase activity was recorded lower in treated group while Acid Phosphosphatase activity was observed significantly (P<0.05) high in FF of treated animals compared to control. Iron and zinc concentration were also recorded significantly (P<0.05) higher in FF of superovulated animal compared to control. The total protein concentration was observed high in FF of treated animals than control but the differences were statistically non significant. The concentration of estradiol 17 s, Lactate dehydrogenase and Copper did not show much variation between treatment and control group.

Ratio of low density to high density lipoprotein concentration is critical for normal functioning of human body. Deviation in this ratio has been linked to various diseases, many of which are fatal if not diagnosed at early stages. For example, cardiovascular diseases (CVD) have been linked to the level of low density lipoprotein (LDL). Henceforth, detection of the lipoprotein subtractions is crucial for health of an individual. To date, methods like ultracentrifugation, nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) and gradient gel electrophoresis (GGE) have been used for separation and identification of lipoprotein types and subtypes. However, these methods are expensive, time consuming and require specialized equipments and expertise. This paper aims to propose a low-cost, high-throughput native polyacrylamide gel electrophoresis (N-PAGE) based protocol for analysis of lipoproteins. Quality by Design (QbD) based approach has been utilized. The initial screening of parameters was followed by a fractional factorial design to optimize the protocol. The lipoprotein subtractions obtained by the optimized protocol were compared with the commercially available and commonly used Lipoprint(®) Lipoprotein Subfractions Testing System from Quantimetrix. The proposed method gave comparable results to those obtained with the commercial system. The proposed method is capable of analysis of up to forty different samples in two hours at a cost of approximately 2$/sample. This is an order of magnitude better than the present cost of 265$/sample when using the commercial system. We think that the proposed method would be of particular interest to the developing and under-developed economies of the world, where this cost differential would be deemed quite significant and would make testing affordable to the majority of the population.

In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature.We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques.We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ.Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response.This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Abstract Protein A resins are often reused for multiple cycles to improve process economy during mAb purification. Significant reduction in binding capacity and product recovery are typically observed due to the presence of unwanted materials (foulants) deposited on the resin upon reuse. In this paper, we have used a wide spectrum of qualitative and quantitative analytical tools (particle size analysis, HPLC, fluorescence, SEM, MS, and FTIR) to compare the strengths and shortcomings of different analytical tools in terms of their capability to detect the fouling of the resin and relate it to chromatographic cycle performance. While each tool offers an insight into this complex phenomena, fluorescence is the only one that can be used for real‐time monitoring of resin fouling. A correlation could be established between fluorescence intensity and the process performance attributes (like yield or binding capacity) impacted upon resin reuse. This demonstration of the application of fluorescence for real‐time monitoring correlated empirically with process performance attributes and the results support its use as a PAT tool as part of a process control strategy. While the focus of this paper is on fouling of protein A chromatography resin, the approach and strategy are pertinent to other modes of chromatography as well.

Partogram is an effective method of identifying various abnormalities early and reducing undue labour prolongation.It is also a powerful research tool.The study was performed prospectively over a period of 2 years in the Department of Obstetrics and Gynaecology, S.N.Medical College, Agra.Women with term singleton pregnancies with vertex presentation with no major disproportion at the onset were identified.Partogram was maintained.Active management of labour was done.A total of 569 women fulfilling the selection criteria were considered for study.Out of 569 women, 44 were dropped due to early fetal distress or early decision for cesarean section.Of the remaining 525 women, 429 showed normal labour pattern and 96 showed abnormal patterns.The incidence of protracted active phase was 12.3% in nulliparas and 6% in multiparas.In abnormal labour, cesarean rate was 62.1% among nulliparas and 54.6% among multiparas.Use of WHO composite partogram with a 4-hour action line produced lower incidence of prolonged labours and lower requirement for augmentation of labours (rates being similar to those in normal labours).

Drug manufacture is among the most significant businesses on the planet. For long, this business has been a key productive member of society, and it will remain to be for several years to come. It's difficult to fathom living without pharmaceuticals that treat ailments and enable people live longer, better lives. Drug industry must improve their manufacturing techniques in order to guarantee that operation is both efficient and robust. Artificial intelligence may assist by offering a third-party view on how the medication manufacturing process should be run and recommending improvements in equipment for optimal efficiency. It's vital to get through the jargon and cacophony as we approach closer to a future where AI/ML is more integrated into R&D. When forming judgments regarding data, it's also critical to remember that the scientific process isn't defunct. This will aid in distinguishing hope from hype and lead to more informed decisions on the best use of AI/ML in drug research. However, there are many challenges and hurdles to be handled for the successful AI/ML implementation in pharmaceutical industry.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4–5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (β1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and β-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Infertility is a medical disorder caused by abnormal sperm production, stress, and trauma. Lifestyle factors are part of the main causes of male infertility, which includes obesity, tobacco, and alcohol abuse. Nutritional intake also affects the levels of male infertility positively or negatively. Oligospermia characterized by low sperm count is generally seen in male infertility. Nowadays, endogenous and exogeneous antioxidants have been found to be effective for the treatment of male infertility. Several other factors such as long noncoding RNAs, ADAM, epidermal growth factor receptor, and calcineurin also modulate the production and maturation of the sperm. Various natural products and medicinal plants have been well reported for the treatment of male infertility. Apart from it, pharmaceutical medications such as antidepressants, calcium channel blockers, α-adrenergic blockers, antiepilepsy, and antiretrovirals have been implicated in aggravating male factor infertility. In this chapter, we explored the male infertility causes, targets, treatments, and available medications.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Background: Routine HIV counseling and testing done as a mandatory part of antenatal care in India has lead all pregnant women comes under the prevention of mother to child transmission of HIV(PMTCT) program. Despite such strategies, the effective execution and uptake of these programs remains a major obstacle. It is thus, important to understand experiences of pregnant women undergoing HIV testing to detect the flaws on the part of the provider and the benefiter and eliminate them to strengthen the PMTCT services. Aim: Westudied the acceptability of HIV voluntary counseling and testing (VCT) in antenatal women attending a tertiary health centre of north India. The impact of sociodemographic factors on HIV prevalence and uptake of PMTCT was also studied and the possible reasons for dropouts were determined . Methods: Firstly we performed pretest counseling and sociodemographic data and blood samples collected from the consenting antenatal pregnant women were also taken. Samples were tested for HIV antibodies as per WHO guidelines. Data was analysed and presented as mean, percentages and tables. Results:  Of 30150 pregnant women counseled, 23464 (77.82%) underwent testing.136 / 23464 women tested seropositive. The prevalence of HIV in antenatal women was found to be 0.58%. Majority of these women were young and belonged to the age group 20-24 years (0.23%).22% refused testing, the reasons for which were tried to b sought. Strong associations were found between the HIV seroreactive status and marital status, low education status, low social class, high parity and unemployment. Conclusion:  To eliminate pediatric transmission of HIV and to create more awareness regarding HIV infection and parent to child transmission, there is a need to make VCT and PMTCT programs more acceptable to the population. The observations found in the study were consistent with the national projections.

ABSTRACT Clostridium sordellii, an anaerobic pathogen, is ubiquitously distributed in the environment and causes fatal necrotizing infections in approximately 70% of all reported cases. Characteristic clinical features include absence of fever and rash, dramatic leukemoid reaction (LR), capillary leak and fluid sequestration with hemoconcentration, refractory tachycardia and hypotension, and marked edema of infected tissues without gas production or extensive myonecrosis. C. sordellii has rarely been identified in the genital tract, other Clostridium species colonize the vagina in 4 to 18% of healthy women and commonly are associated with postpartum endometritis and septic abortion. Pregnancy, childbirth, or abortion may predispose a some women to acquire C. sordellii in the vaginal tract. Dilatation of the cervix may lead to ascending infection of necrotic decidual tissue. The acidic pH of the vaginal tract may enhance the cytopathic effects of C. sordellii lethal toxin C. sordellii infections pose difficult clinical challenges and are usually fatal. How to cite this article Agrawal PK, Garg R, Singh R, Pathak A, Pathak M. Clostridium sordellii Infection of Female Genital Tract: A Rare but Fulminating Reaction. J South Asian Feder Obst Gynae 2015;7(3):197-198.

Unwanted process shutdown is an important and challenging problem in petrochemical Industry. Incipient fault detection and diagnosis of a fault while the system is still operating not only avoids abnormal event progression, but also reduce productivity loss and hazards. In this paper, simulated version of Tennessee Eastman (TE) process, which is replica of real time process, is studied. There are 21 types of identified faults out of which two faults, ‘reactor cooling water valve sticking’, and ‘condenser cooling water valve sticking’, have been simulated using MATLAB. Dynamics of important process variables under faulty operation, obtained using simulated data, are presented and analyzed. A brief discussion on the need for a multivariate dimensionality reduction technique is also presented in this paper.

The effects of synthetic FSH preparation (Folltropin-V) and PMSG (Folligon) were studied on superovulatory response, ovarian biometry and transferable embryo recovery in goats during winter (December to March) and rainy (June to October) season of the year. Goats were superovulated using 133 mg Folltropin-V or 1000 IU Folligon along with a control group during each breeding season. The overall increase in the dimensions of superovulated goat ovaries was 1.5 times compared to the control group. The results indicated that irrespective of the season, the overall response in terms of number of ovulation was significantly (P<0.05) higher (12.83± 2.58) in Folltropin-V treated animals than Folligon treated (08.91± 1.90). The number of unovulated follicles was significantly (P<0.05) lower during rainy (2.08 ± 0.86) compared to winter (5.17 ± 1.55) season irrespective of the hormone used for superovulation. The number of total and transferable embryos recovered was significantly (P<0.05) higher in Folltropin-V treated animals (10.33± 0.74 and 5.91 ± 2.14) than the Folligon (7.50± 0.57 and 3.66 ± 1.11) treated animals.

Implementation of chemometrics, design of experiments and neural network analysis for prior process knowledge assessment ( <scp>PPKA</scp> ), failure modes and effect analysis ( <scp>FMEA</scp> ), scale‐down model development ( <scp>SDM</scp> ) and process characterization for a chromatographic purification of Teriparatide

Implementation of chemometrics, design of experiments and neural network analysis for prior process knowledge assessment ( <scp>PPKA</scp> ), failure modes and effect analysis ( <scp>FMEA</scp> ), scale‐down model development ( <scp>SDM</scp> ) and process characterization for a chromatographic purification of Teriparatide

The objective of the present study was to establish a workable approach for the production of germ cell (GC)-depleted recipient goat model using intra-testicular busulfan treatment and transplantation of cultured and enriched caprine-male GC (cmGCs) into the homologous recipients under ultrasonography (USG) guidance. The evaluation of post-transplantation colonization of donor cmGCs and restoration of the normal architecture of seminiferous tubules (ST) was performed. For this, the cmGCs of pre-pubertal male goats were isolated and enriched by differential platting for culture until the third passage. Thereafter, cells were harvested and further enriched by magnetic-activated cell sorting using rabbit-anti-CD90 antibody. After confirmation of metabolic viability (MTT-assay) and cluster-forming ability (crystal violet staining) of CD90+ cmGCs, the cells were labeled with a lipophilic red-fluorescent dye (PKH26) before transplanted into the recipient male goats by injection directly into the mediastinum testes under USG guidance. The colonization and repopulation of transplanted CD90+ cmGCs into the recipient ST was observed up to 8 weeks post-transplantation. The PKH26-labeled donor cell-derived colonies were identified in enzymatically digested ST and cryosections of recipient testes. Moreover, histochemical analyses revealed the restoration of the normal architecture of ST of recipient testis after GC transplantation. Therefore, the results suggest that the reproductive competence of infertile animals can be restored through mGC therapy and thus the methodology presented herein could be useful to obtain donor mGCs-derived functional male gametes in the recipient animal testis.

The present study evaluates and compares the clinical effects of systemic metronidazole and ornidazole in sites with or without scaling and root planing (SRP) in generalized chronic periodontitis patients in terms of gingival scores (GS), Probing depth (PD) and Bleeding on probing (BOP). A total of 40 patients suffering from chronic periodontitis (18-42 years) were selected & randomly divided equally into three groups on the basis of the treatment plan. The clinical parameters were assessed at baseline i.e. day 0, day 7 and day 14 post-treatment. Clinical parameters gingival inflammation, pocket depth and bleeding on probing over a period of 14 days Ornidazole + SRP proved to be a better mode of treatment. Significant improvement was noted in all the five treatment modalities in treating chronic generalized periodontitis.

Introduction: Preeclampsia is a multisystem complex disorder with an increased uterine arterial resistance due to deficiency of e-NOS, resulting in vasoconstriction.L-arginine is the substrate of nitric oxide (NO) and therefore we aimed to study the effect of its supplementation in preeclampsia patients. Materials and Methods:The study was conducted in SN Medical College, Agra and included a total of 120 women out of which 60 were included in the study group and 60 in the placebo group, from 20 weeks of gestation onwards, complicated by Pregnancy induced hypertension and Doppler parameters were done serially at 4-6 weeks interval after supplementation of L-Arginine to see for the improvement.Results: It was found that supplementation with Arginine significantly improved the fetal outcome in patients with Pre-eclampsia.Conclusion: L-Arginine proved to be an efficient intervention to improve neonatal outcomes in patients with hypertensive disorders of pregnancy.

Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.

The present study was planned to develop the best fecal extraction method for progesterone metabolite assay which is based on the concentration of fecal progesterone metabolite (FPM) (5α-pregnan-3α-ol-20-one), obtained in a dried fecal sample of buffaloes.Four clinically healthy female buffaloes of the same age group 4 to 7 years were used for the collection of fecal samples maintained under isomanagerial conditions with an intensive system at the LPM Section of the IVRI institute.The extraction of FPM (5αpregnan-3α-ol-20-one) was done by various methods using organic solvents like methanol, diethyl ether, ethanol, and 90% methanol and found that extraction with 90% methanol the concentration of FPM (5α-pregnan-3α-ol-20-one) (231.98ng/g), is comparatively higher than other methods and best for a dried fecal sample of buffaloes.In conclusion, the above results suggest that FPM (5α-pregnan-3α-ol-20-one) value using 90% methanol as a solvent in the dried fecal sample assay having high value as compared to other solvents extraction methods like methanol, absolute ethanol, and diethyl ether, and we reported best for a dried fecal sample of buffaloes and preserved for further estimation.

Abstract Chemokine receptor CCR9 is known to play an important role in the migration of immune cells to the gut. Intestinal epithelial cells under gut inflammation or in inflammatory bowel disease (IBD) produce several folds higher CCL25, the only known ligand for CCR9, and drive the recruitment of CCR9+ immune cells. Due to its gut tropism activity, CCR9 and CCL25 are suggested as a potential therapeutic target. Still, many of the clinical trials targeting CCR9/CCL25 did not give a positive outcome; instead, they increase the severity of the disease. The non-chemotactic function of CCR9 in the gut inflammation and immunity is not known. Using dextran sodium sulfate (DSS)-induced gut inflammation model in C57BL/6 mice, we showed that CCR9+ DCs, specifically CD11b−CD103+ DCs recruited in high frequency to the gut and gut-associated lymphoid tissues (GALT) in DSS treated mice as compared to control group. These CCR9+ DCs showed lower MHC II and CD86 molecules and had higher regulatory surface markers (FasL and Latency-associated peptide, LAP) in the GALT. Further, we demonstrate that thymic stromal lymphopoietin (TSLP) produced by CCR9+ DCs but not IL-10 or TGF-β, promotes the differentiation of Foxp3+ Treg. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the ovalbumin-specific gut allergic immune response. Together, these results suggest that CCR9+ DCs have a regulatory function and can be explored as an adoptive cellular therapy to control the gut inflammation and allergic immune response.

The aim of experiment was to study the culture characteristics of spermatogonial stem cells (SSCs) obtained from pre-and post-pubertal goat testes. Six animals each of two age groups (3-6 months and 1.0-1.5 years) were included in the study. The testes were collected from slaughtered animals and after washing, morpho-biometric characters such as weight, length, mid circumference and volume of both the left and right testes were recorded. The isolated SSCs were enriched by double filtration through 80 and 60 μm nylon mesh filters. The filtrate was cultured overnight with SSC medium Dulbecco's modified eagle's medium / HamF-12 medium. The colonies were also characterized by alkaline phosphatase activity and demonstration of specific markers octamer binding transcription factor and protein gene product.All the morpho-biometrical parameters of left and right testes, except density, were significantly (P<0.01) different between the groups. The total number of SSCs isolated from pre-pubertal testes (6.85×106 cells/ml) was significantly (P<0.05) higher than post-pubertal testes (2.40×106 cells/ml). The colonies of SSCs inpre-pubertal culture appeared within seven days of culture whereas in post-pubertal SSCs, colonies appeared after 14 days of culture. The results demonstrated that the SSCs in prepubertal goat testes were higher in number, grew faster and formed colonies at least one week earlier compared to the SSCs of post-pubertal testes. Thus, the testes of pre-pubertal goats were a better source to study the physiological and regulatory mechanisms of SSCs.

Abstract The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O 2 ) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O 2 ). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O 2 ) and normoxic (21% O 2 ) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced ( p &lt; 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O 2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly ( p &lt; 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

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Abstract Ausgehend von S‐2‐Methyl‐2‐butanol (I) werden gemäß dem angegebenen Formelschema unter Erhalt der optischen Aktivität die Harnstoffe (V) synthetisiert.

Abstract The imidazolones (IIIa)‐(IIIc) are prepared by reaction of the components (I) and (II); the imidazolones (IIId) are obtained by appropriate alkylation of (IIIa).

Imidazolin-2-ones (1) when subjected to dye sensitized photooxygenation in the presence of various solvents like chloroform, benzene, methanol, water either done or in mixture of any two as well as at different pH's (4.4, 7.0 and 9.2) yielded diacylureas as the major reaction products. The reaction was carried out in the presence of cationic (hexadecyltrimethyl ammonium bromide, CTAB); anionic (sodium dodecyl sulphate, SDS) and neutral (ethoxylated nonylphenol, ES) surfactants in aqueous methanol to explore the nature of intermediates. It was obseeved that CTAB enhances the yield of the product while SDS and ES exhibit no effect. The yield of the product was found to be maximum at 2 × 10 -3 M concentration of CTAB. A plausible mechanism of photooxygenation of imidazolin-2-ones involving zwitterionic perepoxides as a prelude to dioxetane formation has been suggested on the basis of observed surfactant, salt and solvent effects.