A rapid and simple sterility test method based on solid culture medium containing blood.

Current sterility test performed for most biological products takes 14 days. We evaluated solid medium, containing 5% blood for use in the membrane filtration (MF) and direct inoculation (DI) sterility test. Representative microorganisms prepared in a sample matrix at approximately 0.1, 1, 10 and 100 colony forming units were tested for growth by compendial MF sterility test using fluid thioglycolate medium and tryptic soy broth and also on the Schaedler blood agar (SBA). Sterility test performed on SBA was significantly more sensitive and faster in detecting various microorganisms than the compendial method, particularly for sample matrix containing 0.01% thimerosal (p < 0.05). SBA detected all microorganisms within 7 days. To implement solid medium in the DI sterility test, multiple BA plates were inoculated with the sample. All representative microorganisms were detected within 5 days. The sterility test using solid medium required 3 different incubation conditions, 30-35 °C aerobically and anaerobically to detect bacteria, and at 20-25 °C aerobically to detect mold and yeast. To eliminate aerobic incubation of solid medium at 20-25 °C, we evaluated representative species of mold and yeast for their growth at 30-35 °C and 20-25 °C in the sterility test performed on solid medium. Penicillium chrysogenum could not be detected at 30-35 °C consistently within 7 days. Sterility test performed on solid medium without any additional technology could be completed in 7 days, as compared to the 14 days required for the current compendial method.

Predicting the potential suitable habitats of genus Nymphaea in India using MaxEnt modeling.

Modeling and mapping the distribution of suitable habitats of aquatic plants are critical for assessing the impact of factors like changing climate on species habitat range shifts, declines, and expansions. Nymphaea is an aquatic perennial herb considered valuable because of its ornamental, economic, medicinal, and ecological importance. In India, the geographical distribution of Nymphaea is diverse, and the suitable habitats of individual species are vulnerable to the changing climate and global warming effects. Despite its increased vulnerability, only a few limited conservation efforts in aquatic environments are being made to date. In several places, the distribution of Nymphaea has been impacted by both anthropogenic and climate-related disturbances. A comprehensive strategy will be needed to meet the socio-ecological challenge of Nymphaea conservation. In this study, we employed maximum entropy (MaxEnt) method to assess how climate change affects the distribution of Nymphaea suitable habitat. The occurrence records of Nymphaea were collected from primary surveys, Global Biodiversity Information Facility (GBIF), and published works. Bioclimatic variables obtained from the Coupled Model Intercomparison Project (CMIP6) were employed as predictor variables in distribution modeling. The projections were made using three SSPs (stringent mitigation scenarios) for the future period of 2050. Our results showed shifts in the suitability ranges of Nymphaea under different projection scenarios. The study provides information about the distribution of suitable habitats for Nymphaea in India, which may be helpful for ongoing efforts to conserve and manage the aquatic plants, particularly in areas that are losing suitable climate conditions.

Aberrant promoter hypermethylation regulates thrombomodulin in high altitude induced deep vein thrombosis.

BACKGROUND: DNA methylation regulates gene expression by inhibiting transcription factor binding to promoter and regulatory regions. Acute hypoxia during altitude exposure is associated with decreased natural anticoagulants and morbid thrombotic events. Thrombomodulin (TM) is a high affinity thrombin binding receptor protein, vital for vascular homeostasis. The purpose of this study is to determine gene expression regulation via methylation of TM gene in high altitude hypoxia induced deep vein thrombosis (DVT) patients. MATERIALS AND RESULTS: Percent 5-methyl cytosine analysis showed increased methylation in high altitude DVT patients (HAP) as compared to high altitude control (HAC) and seal level control (Control) subjects, while TM protein and mRNA levels were decreased in high altitude DVT patients as compared to other two groups. Bisulfite sequencing analysis indicated increased methylation in TM promoter in high altitude DVT patients compared to high altitude controls. Flow cytometry analysis showed decreased TM expression in hypoxia induced primary human umbilical vein endothelial cells (HUVECs). Treatment with specific DNA methyltransferase (DNMT) inhibitor-decitabine during hypoxia, restored TM expression. in vitro global methylation assay showed increased methylation in hypoxia group. Specific concentration of decitabine in hypoxia decreased global methylation showing a direct correlation between DNMTs and methylation. Selective dose of decitabine restored TM levels in HUVECs. DNMT1 and DNMT3B proteins showed to mediate the overall expression of TM. CONCLUSION: TM emerged as a potential candidate for methylation in high altitude DVT patients, regulated by hypoxia-induced epigenetic mechanism. Hypoxia culminates in methylation of DNA sequences in the promoter region of TM gene and increased the expression of DNMT1 and DNMT3B per se in primary HUVECs. Critical DNA methylation events were found to be compromised in high altitude DVT patients.

Contrasting Reproductive Strategies of Two Nymphaea Species Affect Existing Natural Genetic Diversity as Assessed by Microsatellite Markers: Implications for Conservation and Wetlands Restoration.

Nymphaea, commonly known as water lily, is the largest and most widely distributed genus in the order Nymphaeales. The importance of Nymphaea in wetland ecosystems and their increased vulnerability make them a great choice for conservation and management. In this work, we studied genetic diversity in a collection of 90 N. micrantha and 92 N. nouchali individuals from six different states of India, i.e., Assam, Manipur, Meghalaya, Maharashtra, Goa, and Kerala, using simple sequence repeat (SSR) markers developed by low throughput Illumina sequencing (10X coverage of genome) of N. micrantha. Nymphaea nouchali is native to India, whereas N. micrantha is suggested to be introduced to the country for its aesthetic and cultural values. The study revealed extensive polymorphism in N. nouchali, while in N. micrantha, no apparent genetic divergence was detected prompting us to investigate the reason(s) by studying the reproductive biology of the two species. The study revealed that N. micrantha predominantly reproduces asexually which has impacted the genetic diversity of the species to a great extent. This observation is of immense importance for a successful re-establishment of Nymphaea species during restoration programs of wetlands. The information generated on reproductive behaviors and their association with genotypic richness can help in strategizing genetic resource conservation, especially for species with limited distribution. The study has also generated 22,268 non-redundant microsatellite loci, out of which, 143 microsatellites were tested for polymorphism and polymorphic markers were tested for transferability in five other Nymphaea species, providing genomic resources for further studies on this important genus.

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71>) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex(®) Detection System [RMDS]), and 2) by CO(2) monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iAST and iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1CFU than the compendial methods (p<0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p<0.05). RMDS detected all microorganisms significantly faster than the compendial method (p<0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p<0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.

Arabinogalactan protein from Arachis hypogaea: role as carrier in drug-formulations.

Arabinogalactan protein (AGP) a highly water-soluble glyco-conjugate from groundnut (Arachis hypogaea L.) seedling was isolated and purified by precipitation with beta-glucosyl Yariv reagent. Quantification of AGP was done by gel diffusion assay. Purified AGP was conjugated to amphotericin-B (AmB) by Schiff base reaction at pH 11.0, with aim to prepare a water-injectable lesser toxic AGP-AmB conjugate without affecting AmB antifungal potential. The AGP-AmB conjugate antifungal activity was assayed by serial broth dilution and disc method against several Candida albicans clinical isolates. Both AmB and AGP-AmB showed similar MICs and MFCs activities, indicating that AGP do not reduced the antifungal activity of AmB. However, the in vitro and in vivo toxicity assays revealed that AGP-AmB conjugate was lesser toxic than AmB, as high MTD (45 mg/kg body weight) was observed. It is suggested that AGP could be a potent carrier in AmB formulation, which may result in effective treatment of fungal infections.

Reproduction in male rats is vulnerable to treatment with the flavonoid-rich seed extracts of Vitex negundo.

A partially purified flavonoid-rich extract was prepared from the seed of Vitex negundo. The effect of this extract on the reproductive system of male rats was investigated at four different concentrations. All the major accessory sex organs shed weight when the preparation was administered at doses of >or=15 mg/rat/day after 15 days of treatment. The drop in weight was also reflected in disturbed tissue biochemistry. Secretory products such as citric acid in the prostate, fructose in seminal vesicles and epididymal alpha-glucosidase activity, indices of accessory sex organ function in males, diminished. Microscopic examination of the sperm derived from the cauda epididymides of treated animals showed only a marginal change in vitality. However, sperm numbers dwindled and slackness in their motility was observed, factors that may impede fertility. Toxicity testing in blood did not point to distress in any of the vital organs. Taken together, it is inferred that the seed extracts of V. negundo interfere with male reproductive function without producing adverse toxicity in other vital organs.

A comprehensive evaluation of the reproductive toxicity of Quassia amara in male rats.

Chloroform extracts of the bark of Quassia amara in different dilutions was used to assess its impact on the male reproductive system of albino rats. Single daily intramuscular injections of the extract for 15 days resulted in a significant reduction in the weight of testis and epididymis but not that of the seminal vesicles and prostate (all lobes). A marked decrease in the sperm count, motility, viability was also observed in sperm collected from the cauda epididymis of treated animals. A number of abnormalities like double heads, double tails, detached heads and fragile tails were frequently seen. Epididymal alpha-glucosidase activity was drastically reduced. However, prostatic acid phosphatase activity and citric acid levels and seminal vesicle fructose concentrations remained unchanged following treatment. Thus, it appears that the prime site of action is at the level of both the testis and the epididymis. Examination of the blood showed that cell counts and hemoglobin levels were in the normal range. Bilirubin, SGPT, SGOT, protein and urea were also not altered by the herbal extract. From the selective action on the male reproductive tract we suggest that the chloroform extract of the bark of Quassia amara has potential for use as an antifertility agent.