Effect of post-harvest drying period on the chemical composition of Zingiber zerumbet Sm. Rhizomes essential oil and its biological activities.

Zingiber zerumbet Sm. (Family: Zingiberaceae) is an important perennial medicinal oil-bearing herb that is native to the Southeast Asia. This study examines the impact of different durations of post-harvest shade drying (ranging from 1 to 12 months) on essential oil yield and chemical composition of Z. zerumbet, in comparison to the freshly collected oil sample. This study explores how post-harvest shade drying impact the composition and longevity of Z. zerumbet rhizomes as well as its antimicrobial, antibiofilm activity. The oils were analyzed for their chemical composition analysis using a gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The post-harvest periods of drying (1-12 months) were discovered to enhance the concentration of marker constituents in the oil. The primary constituent, Zerumbone, was detected in concentrations ranging from 69.38 ± 5.63% to a maximum of 80.19 ± 1.53% as the drying duration of the rhizome was extended. The output of the essential oil was not significantly affected by drying times; however, it did have a noticeable impact on the proportions of monoterpenes. Both disc diffusion and broth microdilution assay were used in freshly collected Z. zerumbet oil for its antimicrobial potential against S. aureus, L. monocytogens, S. hominis, Salmonella enterica serovar Typhimurium, P. aeruginosa, S. intermedius, E. coli, and C. albicans. For the first time, the oil reported to exhibit antibiofilm activity against S. aureus which was validated using fluorescence microscopy, and effectively disrupts the biofilm by 47.38% revealing that essential oil was able to disintegrate the clusters of the pathogen. Z. zerumbet rhizome oil is effective to reduce food-borne microorganisms. Therefore, its essential oil, a natural source of bioactive zerumbone, may improve flavor, aroma, and preservation.

SARS-CoV, MERS-CoV, and 2019-nCoV viruses: an overview of origin, evolution, and genetic variations.

Coronaviruses are single stranded RNA viruses usually present in bats (reservoir hosts), and are generally lethal, highly transmissible, and pathogenic viruses causing sever morbidity and mortality rates in human. Several animals including civets, camels, etc. have been identified as intermediate hosts enabling effective recombination of these viruses to emerge as new virulent and pathogenic strains. Among the seven known human coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV) have evolved as severe pathogenic forms infecting the human respiratory tract. About 8096 cases and 774 deaths were reported worldwide with the SARS-CoV infection during year 2002; 2229 cases and 791 deaths were reported for the MERS-CoV that emerged during 2012. Recently ~ 33,849,737 cases and 1,012,742 deaths (data as on 30 Sep 2020) were reported from the recent evolver SARS-CoV-2 infection. Studies on epidemiology and pathogenicity have shown that the viral spread was potentially caused by the contact route especially through the droplets, aerosols, and contaminated fomites. Genomic studies have confirmed the role of the viral spike protein in virulence and pathogenicity. They target the respiratory tract of the human causing severe progressive pneumonia affecting other organs like central nervous system in case of SARS-CoV, severe renal failure in MERS-CoV, and multi-organ failure in SARS-CoV-2. Herein, with respect to current awareness and role of coronaviruses in global public health, we review the various factors involving the origin, evolution, and transmission including the genetic variations observed, epidemiology, and pathogenicity of the three potential coronaviruses variants SARS-CoV, MERS-CoV, and 2019-nCoV.

Nanosecond laser ablation enhances cellular infiltration in a hybrid tissue scaffold.

Hybrid tissue engineered (HTE) scaffolds constituting polymeric nanofibers and biological tissues have attractive bio-mechanical properties. However, they suffer from small pore size due to dense overlapping nanofibers resulting in poor cellular infiltration. In this study, using nanosecond (ns) laser, we fabricated micro-scale features on Polycaprolactone (PCL)-Chitosan (CH) nanofiber layered bovine pericardium based Bio-Hybrid scaffold to achieve enhanced cellular adhesion and infiltration. The laser energy parameters such as fluence of 25J/cm2, 0.1mm instep and 15 mark time were optimized to get structured microchannels on the Bio-Hybrid scaffolds. Laser irradiation time of 40µs along with these parameters resulted in microchannel width of ~50µm and spacing of ~35µm between adjacent lines. The biochemical, thermal, hydrophilic and uniaxial mechanical properties of the Bio-Hybrid scaffolds remained comparable after laser ablation reflecting extracellular matrix (ECM) stability. Human umbilical cord mesenchymal stem cells and mouse cardiac fibroblasts seeded on these laser-ablated Bio-Hybrid scaffolds exhibited biocompatibility and increased cellular adhesion in microchannels when compared to non-ablated Bio-Hybrid scaffolds. These findings suggest the feasibility to selectively ablate polymer layer in the HTE scaffolds without affecting their bio-mechanical properties and also describe a new approach to enhance cellular infiltration in the HTE scaffolds.

Biological and mechanical evaluation of a Bio-Hybrid scaffold for autologous valve tissue engineering.

Major challenge in heart valve tissue engineering for paediatric patients is the development of an autologous valve with regenerative capacity. Hybrid tissue engineering approach is recently gaining popularity to design scaffolds with desired biological and mechanical properties that can remodel post implantation. In this study, we fabricated aligned nanofibrous Bio-Hybrid scaffold made of decellularized bovine pericardium: polycaprolactone-chitosan with optimized polymer thickness to yield the desired biological and mechanical properties. CD44+, αSMA+, Vimentin+ and CD105- human valve interstitial cells were isolated and seeded on these Bio-Hybrid scaffolds. Subsequent biological evaluation revealed interstitial cell proliferation with dense extra cellular matrix deposition that indicated the viability for growth and proliferation of seeded cells on the scaffolds. Uniaxial mechanical tests along axial direction showed that the Bio-Hybrid scaffolds has at least 20 times the strength of the native valves and its stiffness is nearly 3 times more than that of native valves. Biaxial and uniaxial mechanical studies on valve interstitial cells cultured Bio-Hybrid scaffolds revealed that the response along the axial and circumferential direction was different, similar to native valves. Overall, our findings suggest that Bio-Hybrid scaffold is a promising material for future development of regenerative heart valve constructs in children.

Terrestrial Macrofungal Diversity from the Tropical Dry Evergreen Biome of Southern India and Its Potential Role in Aerobiology.

Macrofungi have long been investigated for various scientific purposes including their food and medicinal characteristics. Their role in aerobiology as a fraction of the primary biological aerosol particles (PBAPs), however, has been poorly studied. In this study, we present a source of macrofungi with two different but interdependent objectives: (i) to characterize the macrofungi from a tropical dry evergreen biome in southern India using advanced molecular techniques to enrich the database from this region, and (ii) to assess whether identified species of macrofungi are a potential source of atmospheric PBAPs. From the DNA analysis, we report the diversity of the terrestrial macrofungi from a tropical dry evergreen biome robustly supported by the statistical analyses for diversity conclusions. A total of 113 macrofungal species belonging to 54 genera and 23 families were recorded, with Basidiomycota and Ascomycota constituting 96% and 4% of the species, respectively. The highest species richness was found in the family Agaricaceae (25.3%) followed by Polyporaceae (15.3%) and Marasmiaceae (10.8%). The difference in the distribution of commonly observed macrofungal families over this location was compared with other locations in India (Karnataka, Kerala, Maharashtra, and West Bengal) using two statistical tests. The distributions of the terrestrial macrofungi were distinctly different in each ecosystem. We further attempted to demonstrate the potential role of terrestrial macrofungi as a source of PBAPs in ambient air. In our opinion, the findings from this ecosystem of India will enhance our understanding of the distribution, diversity, ecology, and biological prospects of terrestrial macrofungi as well as their potential to contribute to airborne fungal aerosols.

Engineering of a polymer layered bio-hybrid heart valve scaffold.

Current treatment strategy for end stage valve disease involves either valvular repair or replacement with homograft/mechanical/bioprosthetic valves. In cases of recurrent stenosis/ regurgitation, valve replacement is preferred choice of treatment over valvular repair. Currently available mechanical valves primarily provide durability whereas bioprosthetic valves have superior tissue compatibility but both lack remodelling and regenerative properties making their utility limited in paediatric patients. With advances in tissue engineering, attempts have been made to fabricate valves with regenerative potential using various polymers, decellularized tissues and hybrid scaffolds. To engineer an ideal heart valve, decellularized bovine pericardium extracellular matrix (DBPECM) is an attractive biocompatible scaffold but has weak mechanical properties and rapid degradation. However, DBPECM can be modified with synthetic polymers to enhance its mechanical properties. In this study, we developed a Bio-Hybrid scaffold with non-cross linked DBPECM in its native structure coated with a layer of Polycaprolactone-Chitosan (PCL-CH) nanofibers that displayed superior mechanical properties. Surface and functional studies demonstrated integration of PCL-CH to the DBPECM with enhanced bio and hemocompatibility. This engineered Bio-Hybrid scaffold exhibited most of the physical, biochemical and functional properties of the native valve that makes it an ideal scaffold for fabrication of cardiac valve with regenerative potential.

Enhanced cardiomyogenic lineage differentiation of adult bone-marrow-derived stem cells grown on cardiogel.

The extracellular matrix (ECM) and its components are known to promote growth and cellular differentiation in vitro. Cardiogel, a three-dimensional extracellular matrix derived from cardiac fibroblasts, is evaluated for its cardiomyogenic-differentiation-inducing potential on bone-marrow-derived stem cells (BMSC). BMSC from adult mice were grown on cardiogel and induced to differentiate into specific lineages that were validated by morphological, phenotypic and molecular assays. The data revealed that the cardiogel enhanced cardiomyogenic and adipogenic differentiation and relegated osteogenic differentiation following specific induction. More importantly, increased cardiomyogenic differentiation was also observed following BMSC growth on cardiogel without specific chemical (5-azacytidine) induction. This is the first report of an attempt to use cardiogel as a biomaterial on which to achieve cardiomyogenic differentiation of BMSC without chemical induction. Our study suggests that cardiogel is an efficient extracellular matrix that enhances the cardiomyogenic differentiation of BMSC and that it can therefore be used as a scaffold for cardiac tissue regeneration.

Natural ECM as biomaterial for scaffold based cardiac regeneration using adult bone marrow derived stem cells.

Cellular therapy using stem cells for cardiac diseases has recently gained much interest in the scientific community due to its potential in regenerating damaged and even dead tissue and thereby restoring the organ function. Stem cells from various sources and origin are being currently used for regeneration studies directly or along with differentiation inducing agents. Long term survival and minimal side effects can be attained by using autologous cells and reduced use of inducing agents. Cardiomyogenic differentiation of adult derived stem cells has been previously reported using various inducing agents but the use of a potentially harmful DNA demethylating agent 5-azacytidine (5-azaC) has been found to be critical in almost all studies. Alternate inducing factors and conditions/stimulant like physical condition including electrical stimulation, chemical inducers and biological agents have been attempted by numerous groups to induce cardiac differentiation. Biomaterials were initially used as artificial scaffold in in vitro studies and later as a delivery vehicle. Natural ECM is the ideal biological scaffold since it contains all the components of the tissue from which it was derived except for the living cells. Constructive remodeling can be performed using such natural ECM scaffolds and stem cells since, the cells can be delivered to the site of infraction and once delivered the cells adhere and are not "lost". Due to the niche like conditions of ECM, stem cells tend to differentiate into tissue specific cells and attain several characteristics similar to that of functional cells even in absence of any directed differentiation using external inducers. The development of niche mimicking biomaterials and hybrid biomaterial can further advance directed differentiation without specific induction. The mechanical and electrical integration of these materials to the functional tissue is a problem to be addressed. The search for the perfect extracellular matrix for therapeutic applications including engineering cardiac tissue structures for post ischemic cardiac tissue regeneration continues.

Generation of mesenchymal stem cell lines from murine bone marrow.

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

Impact of geographic range on genetic and chemical diversity of Indian valerian (Valeriana jatamansi) from northwestern Himalaya.

An effort was made to determine the impact of geographic range on genetic richness and chemical constituents of Valeriana jatamansi Jones, an herb indigenous to the northwestern Himalaya. The genetic structure of 16 accessions from two major divisions of Uttarakhand state (Kumaon and Garhwal) was analyzed by ISSR markers. Overall genetic diversity among the populations was 45 %, with a cumulative range of 35-92 % similarity for most of the high-altitude plants and a comparatively narrow range, 50-88 %, for the population below the altitude of 1,800 m. Likewise, a remarkable predictability was evident from the chemical constituents on an individual basis. In principal component analysis, most of the accessions fall into two major groups and are classified as chemotypes based on the percentage of similar chemical constituents; these are mostly correlated to altitude. Geographic distance seems to influence the genetic and chemical variability, indicating the genetic inbreeding within the population.

Microfluidic sorting with blinking optical traps.

It is shown that by appropriately choosing the periodicity of a blinking optical trap only larger sized colloidal spheres can be selectively trapped out of a mixed population. This happens because smaller sized, more agile, spheres escape out of the trap volume during the off period of the trap beam. Therefore, by scanning an array of blinking traps over a mixed sample, bigger spheres can be forced to move with the traps and eventually could be taken to the output side. Experimental demonstration of sorting between 1 µm and 2 µm diameter silica spheres is presented.

Synthesis and antibacterial activity of various substituted oxadiazole derivatives.

Some new 2-(2-(4(4-substitutedbenzoyl-2-methylphenoxy)acetyl)-N-(2-substitutedphenyl) hydrazinecarbothioamides (4a-4j) and (4-((5-(2-substitutedphenylamino)-1,3,4-oxadiazol-2-yl)methoxy)-3-substitutedphenyl)(phenyl)methanones (5a-5j) have been synthesized from 2-(4-(3-substitutedbenzoyl)-2-methylphenoxy)acetohydrazides (3a, 3b). These newly synthesized compounds (4a-4j and 5a-5j) were characterized by elemental and spectral (IR, (1)H-NMR and MS) analysis. All the synthesized compounds have been screened for their antibacterial activity against both types of Gram negative and Gram positive bacteria. The most potent antibacterial compound of this series was compound 5i which has the low MIC 3.75-0.9375 µg/mL value. Both minimal inhibitory concentration (MIC) and inhibition zones were determined in order to monitor the efficacy of the synthesized compounds. Certain compounds inhibit bacterial growth with low MIC (µg/mL) value.

Cardiogel supports adhesion, proliferation and differentiation of stem cells with increased oxidative stress protection.

Cultured murine bone marrow derived mesenchymal stem cells (BMSC) when grown along with cardiogel derived from mouse cardiac fibroblast, exhibited increased cell proliferation and differentiation and enhanced survival under oxidative stress induced by the exposure of H2O2 in vitro (similar to in vivo ischemia like condition). Adhesion of BMSC to the cardiogel occurred at a faster rate when compared to the cells grown on normal surface. BMSC attached to cardiogel showed an increased resistance to proteolytic (enzymatic) disassociation. This is the first report on an attempt to use an in house biomaterial for the growth of BMSC that led to their heightened resistance towards oxidative stress. These studies support that cardiogel is an efficient biodegradable three-dimensional extracellular matrix which supports better growth of BMSC and can be used as a scaffold for stem cell delivery, with potential therapeutic applications in cardiac tissue regeneration.

Seasonal Variation in Essential Oil Content and Composition of Thyme, Thymus serpyllum L. Cultivated in Uttarakhand Hills.

Thymus serpyllum L. grown in Kumaon region of Western Himalaya was investigated for essential oil content and composition in different seasons. The oils of fresh samples were obtained by hydrodistillation. The yield of essential oil (% v/w) during different seasons varied from 0.07 to 0.28% with the highest in summer season, at vegetative stage. The oils were analyzed by GC and GC-MS. Major components of all the samples were thymol (19.4-60.1%), γ-terpinene (0.3-13.8%) and p-cymene (3.5-10.4%). The results clearly indicated that season has significant effect on quality and quantity of thyme oil.

Chemical investigation of the essential oil of Thymus linearis (Benth. ex Benth) from western Himalaya, India.

Thymus linearis (Benth. ex Benth) was collected from five distinct locations of western Himalaya (India) during the summer season. The hydro-distilled essential oil (yield 0.84-0.95%) was analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). A total of 56 constituents, representing 81.55% to 98.11% of the total oil composition, were identified. Thymol (52.28-66.65%), p-cymene (1.81-21.60%) and γ-terpinene (1.94-12.48%) were the major constituents in all populations. Other constituents identified in significant amounts were carvacrol, p-cymen-8-ol, borneol, terpinen-4-ol and thymol methyl ether. The presence of high phenol and essential oil contents in this species make it a suitable substitute for common thyme oil.

Tumor cell imaging using the intrinsic emission from PAMAM dendrimer: a case study with HeLa cells.

HeLa 229 cells were treated with methotrexate (MTX) and doxorubicin (DOX), utilizing fourth generation (G4), amine terminated poly(amidoamine) {PAMAM} dendrimer as the drug carrier. In vitro kinetic studies of the release of both MTX and DOX in presence and absence of G4, amine terminated PAMAM dendrimers suggest that controlled drug release can be achieved in presence of the dendrimers. The cytotoxicity studies indicated improved cell death by dendrimer-drug combination, compared to the control experiments with dendrimer or drug alone at identical experimental conditions. Furthermore, HeLa 229 cells were imaged for the first time utilizing the intrinsic emission from the PAMAM dendrimers and drugs, without incorporating any conventional fluorophores. Experimental results collectively suggest that the decreased rate of drug efflux in presence of relatively large sized PAMAM dendrimers generates high local concentration of the dendrimer-drug combination inside the cell, which renders an easy way to image cell lines utilizing the intrinsic emission properties of PAMAM dendrimer and encapsulated drug molecule.

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus.

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.

Group-selective antibodies based fluorescence immunoassay for monitoring opiate drugs.

A novel carboxylic acid derivative of monoacetylmorphine (MAM-COOH) was synthesized and conjugated with bovine serum albumin (BSA) for generating polyclonal antibodies against the target molecule heroin and its major metabolites. The conjugate was characterized by fluorescence spectroscopy, polyacrylamide gel electrophoresis, and mass spectrometry to confirm the extent of haptenization of the carrier protein. A high titer (1:64,0000) of antibody was obtained by using the conjugate with an optimum protein/hapten molar ratio of 1:100. The generated antibody showed good binding affinity with heroin and its metabolites monoacetylmorphine (MAM) and morphine. The relative affinity constant (K (aff)) of the antibody was 3.1 x 10(7) l mol(-1), and the IC(50) values obtained for heroin, MAM, morphine, and codeine were 0.01, 0.013, 0.012, and 0.014 ng ml(-1), respectively. A fluorescence-based competitive inhibition immunoassay procedure was developed for the estimation of heroin and its major metabolites in standard and biofludic samples over a concentration range up to 0.01 ng ml(-1) with good signal reproducibility (p < 0.05). The method can be used as a convenient quantitative tool for the sensitive screening of major metabolites of heroin in biological samples.

Molecular typing of colonizing Streptococcus agalactiae strains by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) in a Chennai based hospital.

Streptococcus agalactiae is reported to be an asymptomatic vaginal colonizer in Indian women, although it is considered one of the major causes of neonatal infections in many European countries. DNA based molecular typing methods are more reliable than the conventional serotyping method for identification and typing of this pathogen. In the present study, we have evaluated genetic diversity among colonizing S. agalactiae strains (n=86) by using a PCR-based genotyping method i.e. Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). With ERIC-PCR fingerprinting at 60% similarity level in a dendrogram generated by UPGMA cluster analysis, 10 different ERIC groups were identified, which were subdivided into 62 distinct genotypes at ≥ 95% similarity level. Based on these findings, we demonstrate that ERIC-PCR is a simple, rapid, and inexpensive tool with sufficient discriminatory power and is applicable for characterization and genotyping of a large number of clinical isolates of S. agalactiae at molecular level.

Acute toxicity of copper sulphate to fresh water prawns.

Fresh water prawns, Macrobrachium lamarrei and Macrobrachium dayanum (Crustacea-Decapoda) were subjected to static bioassay tests to ascertain the LC50 values of copper sulphate. The 24, 48, 72 and 96 hr LC50 values of copper sulphate for M. lamarrei were 0.38, 0.361, 0.343 and 0.300 mg/l and for M. dayanum were 1.634, 0.988, 0.532 and 0.418 mg/l respectively. Behavioral responses and LC50 values indicate that M. lamarrei were more sensitive to copper sulphate than M. dayanum.