Molecular Identification and Antioxidant Activity Determination among Coffee Varieties Cultivated in Nepal.

Coffee is the most popular beverage containing numerous phytochemical components that have antioxidant activity capable of scavenging free radicals. Antioxidant and phenolic contents have considerable benefits for human health. The aim of this study was the molecular identification of 9 coffee samples from the Nepal Agricultural Research Council, Lalitpur, Nepal, and the determination of the antioxidant activity and total phenolic content of green and roasted coffee beans. Molecular identification was performed using ITS-specific PCR followed by sequencing and phylogenetic tree construction using the maximum parsimony method. The DPPH assay was used to determine the antioxidant activity, and the Folin-Ciocalteu (F-C) assay was used to determine the total phenolic content. All the samples belonged to the taxa Coffea arabica. The antioxidant activity in roasted beans varied from 2.49 to 4.62 AAE mg/g and from 1.4 to 3.9 AAE mg/g in green beans. The total phenolic content varied from 2.58 to 3.38 GAE mg/g and from 4.16 to 5.36 GAE mg/g for the roasted beans and green beans, respectively. The data revealed that the highest antioxidant content (4.62 AAE mg/g) was found in roasted coffee and that the highest phenolic content (5.36 GAE mg/g) was found in green coffee. The study concludes that roasting increases the antioxidant activity but decreases the phenolic content of coffee.

Ticagrelor Versus Prasugrel in Patients With Acute Coronary Syndrome: A Systematic Review and Meta-Analysis.

Limited data comparing prasugrel and ticagrelor in acute coronary syndrome are available. Online databases, including MEDLINE and Cochrane Central, were queried to compare these drugs. The primary outcomes of this meta-analysis are myocardial infarction (MI), all-cause mortality, cardiovascular mortality, noncardiovascular mortality, stent thrombosis, and stroke. The secondary outcome is major bleeding. A total of 9 studies, including 94,590 patients (prasugrel group = 32,759; ticagrelor group = 61,831), were included in this meta-analysis. The overall mean age was 62.73 years, whereas the mean age for the ticagrelor and prasugrel groups was 63.80 and 61.65 years, respectively. Prasugrel is equally effective as compared with ticagrelor in preventing MI. There was no difference between the 2 groups regarding all-cause mortality, stent thrombosis, stroke, or major bleeding. In patients with acute coronary syndrome, prasugrel is equally effective when compared with ticagrelor in preventing MI.

Fast response and recovery polyaniline montmorillonite reduce graphene oxide polymer nanocomposite material for detection of hydrogen cyanide gas.

In the present work, we have developed a polymer based gas sensor. The polymer nanocomposites are synthesized by the chemical oxidative polymerization of aniline with ammonium persulfate and sulfuric acid. The fabricated sensor is able to achieve a sensing response of 4.56% for PANI/MMT-rGO at 2 ppm of hydrogen cyanide (HCN) gas. The sensitivity of the sensors PANI/MMT and PANI/MMT-rGO are 0.89 ppm-1 and 1.1174 ppm-1 respectively. The increase in the sensitivity of the sensor may be due to an increase in the surface area provided by MMT and rGO which provided more binding sites for the HCN gas. The sensing response of the sensor increases as the concentration of the gas exposed increases but saturates after 10 ppm. The sensor recovers automatically. The sensor is stable and can work for 8 months.

IL1 Pathway in HPV-Negative HNSCC Cells Is an Indicator of Radioresistance After Photon and Carbon Ion Irradiation Without Functional Involvement.

Background: Treatment of locally advanced HPV-negative head and neck squamous cell carcinoma (HNSCC) with photon radiation is the standard of care but shows only moderate success. Alterations in response toward DNA DSB repair, apoptosis, and senescence are underlying determinants of radioresistance in the tumor cells. Recently, senescence and the associated secretory phenotype (SASP) came into the focus of research and raised the need to identify the tumor-promoting molecular mechanisms of the SASP. The aim of this project was to unravel more of this process and to understand the impact of the IL1 pathway, which plays a major role in SASP. The studies were performed for photon and 12C-ion irradiation, which strongly vary in their effect on radioresistance. Materials and Methods: A panel of five HPV-negative HNSCC cell lines was treated with photon and 12C-ion irradiation and examined for clonogenic survival, DNA DSB repair, and senescence. SASP and IL1 gene expressions were determined by RNA sequencing and activation of the IL1 pathway by ELISA. A functional impact of IL1A and IL1B was examined by specific siRNA knockdown. Results: Cell killing and residual DSBs were higher after 12C-ion than after photon irradiation. 12C-ion induced more senescence with a significant correlation with cell survival. The impact on radioresistance appears to be less than after photon irradiation. The expression of SASP-related genes and the IL1 pathway are strongly induced by both types of irradiation and correlate with radioresistance and senescence, especially IL1A and IL1B which exhibit excellent associations. Surprisingly, knockdown of IL1A and IL1B revealed that the IL1 pathway is functionally not involved in radioresistance, DSB repair, or induction of senescence. Conclusions: IL1A and IL1B are excellent indicators of cellular radioresistance and senescence in HNSCC cells without functional involvement in these processes. Clearly more research is needed to understand the molecular mechanisms of senescence and SASP and its impact on radioresistance.

Anthocyanins: Multi-Target Agents for Prevention and Therapy of Chronic Diseases.

Anthocyanins, a flavonoid class of polyphenols, are water soluble dark colored natural pigments found in fruits and vegetables. Owing to their wide distribution in plant materials, dietary consumption of anthocyanins is high compared to other flavonoids. Anthocyanins, due to their multifaceted medicinal properties are the active components in many herbal folk medicines. As in vitro and in vivo results, animal models, and clinical trials in various cell lines suggest, anthocyanins possess antioxidant, antidiabetic, antihyperlipidemic, anti-inflammatory, anticarcinogenic, antiulcer, and preventive activities against cardiovascular diseases. Additionally, anthocyanins exhibit chemotherapeutic, cardioprotective, hepatoprotective, and neuroprotective activities. In the diet, anthocyanins are absorbed in the stomach and intestinal cells and rapidly detected in the plasma. These promising properties of anthocyanins may well provide health benefits against chronic diseases.

Bisphenol A Induces Oxidative Stress in Bone Marrow Cells, Lymphocytes, and Reproductive Organs of Holtzman Rats.

Bisphenol A (BPA) is an estrogenic chemical used in the production of polycarbonate plastics and epoxy resins. Our earlier studies have demonstrated that BPA is a potent reproductive and genotoxic agent and affects the normal physiological functions. The objective of this study was to evaluate whether exposure to BPA induces oxidative stress. The male Holtzman rats were orally gavaged with BPA (0.01 mg and 5.0 mg/kg/bw) over the period of 6 days. Animals were euthanized by cervical dislocation at the end of the treatments; bone marrow cells and blood lymphocytes were aspirated; testis and epididymis were collected, immediately frozen in liquid nitrogen, and stored at -80°C. These samples were utilized for the determination of lipid peroxidation and various antioxidant enzymes such as superoxide dismutase, catalase, and nonenzymatic reduced glutathione. The results demonstrated that BPA caused an increase in lipid peroxidation and a decrease in activity of various enzymatic and nonenzymatic antioxidants in bone marrow cells, blood lymphocytes, and testicular and epididymal tissues. The findings of the current study suggest that BPA exposure induced oxidative stress, which could be one of the possible mechanisms causing reproductive and genetic toxicity.

Cloning, localization and differential expression of Neuropeptide-Y during early brain development and gonadal recrudescence in the catfish, Clarias gariepinus.

Neuropeptide-Y (NPY) has diverse physiological functions which are extensively studied in vertebrates. However, regulatory role of NPY in relation to brain ontogeny and recrudescence with reference to reproduction is less understood in fish. Present report for the first time evaluated the significance of NPY by transient esiRNA silencing and also analyzed its expression during brain development and gonadal recrudescence in the catfish, Clarias gariepinus. As a first step, full-length cDNA of NPY was cloned from adult catfish brain, which shared high homology with its counterparts from other teleosts upon phylogenetic analysis. Tissue distribution revealed dominant expression of NPY in brain and testis. NPY expression increased during brain development wherein the levels were higher in 100 and 150days post hatch females than the respective age-matched males. Seasonal cycle analysis showed high expression of NPY in brain during pre-spawning phase in comparison with other reproductive phases. Localization studies exhibited the presence of NPY, abundantly, in the regions of preoptic area, hypothalamus and pituitary. Transient silencing of NPY-esiRNA directly into the brain significantly decreased NPY expression in both the male and female brain of catfish which further resulted in significant decrease of transcripts of tryptophan hydroxylase 2, catfish gonadotropin-releasing hormone (cfGnRH), tyrosine hydroxylase and 3ß-hydroxysteroid dehydrogenase in brain and luteinizing hormone-ß/gonadotropin-II (lh-ß/GTH-II) in pituitary exhibiting its influence on gonadal axis. In addition, significant decrease of several ovary-related transcripts was observed in NPY-esiRNA silenced female catfish, indicating the plausible role of NPY in ovary through cfGnRH-GTH axis.

A cost-effective anaerobic culture method & its comparison with a standard method.

Twenty six anaerobes were recovered from 150 deep-seated abscess samples cultured by the proposed two-step combustion-modified candle-jar system and Anoxomat. The degree of growth and colony size were similar in both systems, except for Clostridium difficile. The modified candle-jar system was found to be a sensitive and cost-effective alternative that might be used in resource-limited settings.

Microbiology and outcomes of peritonitis in northern India.

BACKGROUND: Peritoneal dialysis (PD) is an established treatment modality for end-stage renal disease (ESRD). Peritonitis remains a serious complication in PD patients and an important cause of drop-out from the program. Types of pathogens and their drug resistance patterns may determine the outcome of peritonitis. The present study was undertaken to determine the microbiology of peritonitis in PD patients, antibiotic resistance in commonly isolated bacterial pathogens and clinical outcomes. METHOD: We enrolled 211 patients with ESRD undergoing PD who developed peritonitis during 2002 to 2011. PD fluids were cultured and antibiotic susceptibility test of the bacterial isolates was performed. RESULT: A total of 303 peritonitis episodes with an overall incidence of 0.41 episodes per patient-year were recorded. Gram-positive, gram-negative, fungi, Mycobacterium tuberculosis and ≥ 2 organisms were isolated from 102 (33.7%), 89 (29.4%), 41 (13.5%), 11 (3.6%) and five (1.6%) episodes respectively; 55 (18.2%) episodes were culture negative. Coagulase-negative Staphylococcus spp. (CONS) was the most common isolate. Catheter loss and hospital admission in gram-negative peritonitis were significantly higher than in gram-positive peritonitis (36/89 (40.4%) vs 20/102 (19.6%), p < 0.001; and 56/89 (62.9%) vs 42/102 (41.2%), p = 0.004 respectively). Antibiotic susceptibility tests showed 54.3% of Enterobacteriaceae isolates were extended spectrum ß-lactamase (ESBL) producers, 23.5% of Acinetobacter species and 11.5% of Pseudomonas aeruginosa were metallo-ß-lactamase (MBL) producers; 15.4% of enterococci and 28.6% of staphylococci were resistant to vancomycin and methicillin respectively. Mortality was significantly higher in patients having peritonitis due to vancomycin-resistant enterococci, ESBL- and MBL-producing bacteria. CONCLUSION: Emerging antimicrobial resistance calls for prompt diagnosis and aggressive empiric therapy based on the local sensitivity data.

Mutagenic effect of Bisphenol A on adult rat male germ cells and their fertility.

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10 µg, and 5.0 mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

Genotoxicity evaluation of asymmetric lipid polymer hybrid nanoparticles of doxycycline hydrochloride following intravenous administration.

Nanoparticles, being small (<1,000 nm) in size, provide high surface area-to-volume ratio as compared with the bulk materials which increase the concern about their potential toxicities. The present investigation was undertaken to evaluate the genotoxic potential of asymmetric lipid polymer hybrid nanoparticles of doxycycline hydrochloride (DH lipomer) following intravenous route. DH lipomer was prepared by modified nano-precipitation method as reported earlier. Doxycyline loading was found to be 20 ± 2.5 %. Average particle size of DH lipomer and blank lipomer was 512 ± 8 and 520 ± 6 nm, respectively. Micronucleus (MN) assay was performed in adult healthy Swiss mice whereas chromosomal aberration (CA) test and comet assay were performed in healthy Holtzman rats following intravenous administration. Animals were divided into two sets, male and female, each set comprising of six groups (n = 5/group), viz., three test groups, blank lipomer (BL), vehicle control (VC), and positive control. Groups treated with 1.5 mg/kg BW DH lipomer did not show micronuclei formation in bone marrow cell, DNA damage, and CA, respectively, as compared with VC, suggesting no genotoxicity. On the other hand 3 and 6 mg/kg BW revealed significant (P > 0.001) increase in micronuclei formation, DNA damage, and chromosomal aberrations. Furthermore, BL (6 mg/kg BW) did not reveal genotoxic response in any of the tests, suggesting lipomer components as non-genotoxic. No sex-dependent variation in genotoxicity was observed. This study therefore suggests the potential safety of the proposed dose of DH lipomer at 1 mg/kg BW. An interesting highlight of the study is safety of lipomer matrix which could be exploited for other biomedical application.

Clastogenic and mutagenic effects of bisphenol A: an endocrine disruptor.

Bisphenol A (BPA) is a well-known endocrine disruptor (ED) which represents a major toxicological and public health concern due to its widespread exposure to humans. BPA has been reported to induce DNA adduct and aneuploidy in rodents. Recent studies in humans depicted its association with recurrent miscarriages and male infertility due to sperm DNA damage indicating that BPA might have genotoxic activity. Hence, the present study was designed to determine genotoxic and mutagenic effects of BPA using in-vivo and in-vitro assays. The adult male and female rats were orally administered with various doses of BPA (2.4 µg, 10 µg, 5mg and 50mg/kgbw) once a day for six consecutive days. Animals were sacrificed, bone marrow and blood samples were collected and subjected to series of genotoxicity assay such as micronucleus, chromosome aberration and single cell gel electrophoresis (SCGE) assay respectively. Mutagenicity was determined using tester strains of Salmonella typhimurium (TA 98, TA 100 and TA 102) in the presence and absence of metabolically active microsomal fractions (S9). Further, we estimated the levels of 8-hydroxydeoxyguanosine, lipid per-oxidation and glutathione activity to decipher the potential genotoxic mechanism of BPA. We observed that BPA exposure caused a significant increase in the frequency of micronucleus (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations in bone marrow cells and DNA damage in blood lymphocytes. These effects were observed at various doses tested except 2.4 µg compared to vehicle control. We did not observe the mutagenic response in any of the tester strains tested at different concentrations of BPA. We found an increase in the level of 8-hydroxydeoxyguanosine in the plasma and increase in lipid per-oxidation and decrease in glutathione activity in liver of rats respectively which were exposed to BPA. In conclusion, the data obtained clearly documents that BPA is not mutagenic but exhibit genotoxic activity and oxidative stress could be one of the mechanisms leading to genetic toxicity.

Curcumin-loaded hydrogel nanoparticles: application in anti-malarial therapy and toxicological evaluation.

The present investigation involved preparation of hydrogel nanoparticles using a combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone. The objective was to exploit the size and hydrophilic nature of the formulated nanocarriers to enhance absorption and prolong the rapid clearance of curcumin due to possible evasion of the reticulo-endothelial system. Reproducible nanoparticles of size around 100 nm, a fairly narrow distribution and encapsulation efficiency of 72%, were produced by the solvent emulsion-evaporation technique. This optimized system was further subjected to freeze-drying. The freeze-dried product was readily reconstituted with distilled water. The reconstituted product exhibited a size and distribution similar to that before freeze-drying, drug content of greater than 99% and presence of amorphous drug when analyzed by differential scanning calorimetry (DSC) which may result in possible improved absorption of curcumin. In vivo anti-malarial studies revealed significant superior action of nanoparticles over curcumin control suggesting the possibility of the formulation being employed as an adjunct anti-malarial therapy along with the standard therapy. Acute and subacute toxicity studies confirmed the oral safety of the formulation. A battery of genotoxicity studies was conducted to evaluate the nongenotoxic potential of the developed formulation thus indicating the possibility of the formulation being employed for prolonged duration.

Toxicological evaluation of pH-sensitive nanoparticles of curcumin: acute, sub-acute and genotoxicity studies.

Research in nanotoxicology is still in nascent stages. This hampers the design of appropriate regulatory policies for these beneficial nano-drug delivery systems thus affecting their routine employment as therapeutics. Establishing the entire toxicological profile is thus indispensable for proving the human safety of nanocarriers, which was the primary objective of the current investigation. The developed curcumin loaded polymeric nanoparticles of Eudragit S100 were subjected to various toxicological evaluations which included acute-toxicity study, sub-acute-toxicity study (28 days) and various genotoxicity studies like in vivo Micronucleus assay, in vivo Chromosomal Aberration assay and in vivo Comet assay. The formulation was found to be non-toxic at the dose equivalent to 2000 mg/kg of body weight of curcumin in the acute-toxicity study. Sub-acute-toxicity study proved the safety of the formulation for prolonged administration at the commonly used therapeutic dose of 100mg/kg of body weight of curcumin and at twice the therapeutic dose. Genotoxicity studies proved the cellular safety of the developed formulation at the therapeutic dose, and even at doses equivalent to thrice the therapeutic dose. Thus the developed curcumin loaded polymeric nanoparticles of Eudragit S100 were found to be safe for oral administration for a short as well as a prolonged duration.

Lipopolysaccharide alters the vaginal electrical resistance in cycling and pregnant mice.

PROBLEM: Lipopolysaccharide (LPS) has been postulated to exert harmful biologic effects during pregnancy. The objective of present investigation is to measure the vaginal electrical resistance (VER) in LPS-treated normal cycling and pregnant female mice. METHOD OF STUDY: Minimum dose (MD) of LPS (250 microg/kg body weight) was injected in pregnant female mice through i.p. route on day 0.5 of pregnancy. VER was measured during different phases of reproductive cycle in female mice, which were pre-exposed to LPS and in untreated cycling female mice. VER was also measured in control pregnant female mice (saline-treated mice) through whole pregnancy and LPS-treated female mice in early stages of pregnancy. RESULTS: Vaginal electrical resistance was significantly higher during proestrous or early estrous stage as compared with any other stages of reproductive cycle in mouse. One peak of VER was observed during peri-implantation period of pregnancy in control female mice. The significant differences in the pattern of VER were found between LPS-treated and control female mice during peri-implantation period of pregnancy, and between cycling female mice, which were pre-exposed to LPS and untreated cycling female mice during proestrus. CONCLUSION: The presented results demonstrate, for the first time, that LPS exposure during pregnancy may be determined by measuring VER in mothers without any adverse effect on ongoing pregnancy and may help in refining the assisted reproduction techniques.

Emerging vancomycin resistance in enterococci in India.

Infection caused by vancomycin resistant enterococci (VRE) leads to adverse outcome and is a real challenge. Despite increasing reports of VRE in different countries, there is scanty data on this issue from India. A total of 685 enterococci were isolated from various clinical samples from January to December 2004. Antimicrobial susceptibility was performed as prescribed by National Committee for Clinical Laboratory Standards (NCCLS). Vancomycin resistance was confirmed by minimum inhibitory concentration (MIC). Resistant phenotype was determined by Polymerase chain reaction (PCR). Of 685, 456 (67%) were E. faecalis and 229 (33%) were E. faecium. Resistance to various antibiotics in E. faecalis and E. faecium was as follows: ampicillin 33% and 54%, erythromycin 91% and 86%, ciprofloxacin 69% and 81%, tetracycline 50% and 54% and high level gentamicin resistance in 62% and 77% respectively. Vancomycin resistance was confirmed in 10 (1.4%) cases by MIC and all had Van A phenotype by PCR. Emergence of vancomycin resistant enterococci is of great concern because of its epidemic potential and scanty therapeutic options. Prompt diagnosis and efficient infection control measures can restrict its spread.

A comparative evaluation of phenotypic and molecular methods for the detection of oxacillin resistance in coagulase-negative staphylococci.

Detection of oxacillin resistance in coagulase-negative staphylococci (C-NS) by phenotypic methods is often difficult. The present study compared the National Committee for Clinical Laboratory Standards (NCCLS) revised guidelines of phenotypic methods with a mecA-based polymerase chain reaction (PCR) for C-NS. Ninety clinical C-NS isolates were tested for oxacillin resistance by disk diffusion (1-microg disk), minimum inhibitory concentration (MIC) breakpoint (0.5 microg/ml) after 24 h, and mecA-based PCR. The sensitivity and specificity of disk diffusion was 80% and 93%, and the sensitivity and specificity of the MIC breakpoint after 24 h was 84% and 91%, respectively, against PCR as gold standard. Eleven strains (7 mecA-positive and 4 mecA-negative) showed discordant results between MIC breakpoint after 24 h and PCR. Six of the 7 mecA-positive and all 4 mecA-negative discordant strains had inducible oxacillin resistance and beta-lactamase hyperproduction, respectively. The present study concludes that inducible oxacillin resistance and beta-lactamase hyperproduction are the major causes of discordant results between phenotypic methods and mecA-based PCR, and need special attention.