Allicin extracted from Allium sativum shows potent anti-cancer and antioxidant properties in zebrafish.

Garlic (Allium sativum) is an important flavouring component in Indian cuisine. Allicin, a sulphur containing compound, is the most abundant component of garlic and has been widely studied for its antimicrobial and antioxidant properties. It is also known to play a role in the regulation of blood pressure and cholesterol levels. Despite the known health benefits associated with allicin, systematic studies on its anti-cancer properties using animal models are very limited. This study aimed to develop a simple method for the extraction of allicin from fresh garlic, study the stability of the extracted compound at various temperatures, and evaluate the antioxidant, anti-proliferative, pro-apoptotic and anti-angiogenic properties in zebrafish. A five-month stability study indicated that allicin remains significantly stable at temperatures 4 °C and below but shows extensive degradation if stored at room temperature. The in vivo studies in zebrafish using a combination of mutants and transgenic lines demonstrated the antioxidant, anti-proliferative, apoptotic and anti-angiogenic properties of allicin. The study highlights the importance of natural bioactive compounds as potential anti-cancer agents that can be studied further.

Investigation of anti-proliferative and anti-angiogenic properties of Parkia javanica bark and fruit extracts in zebrafish.

The use of herbal products as traditional medicines has been a practice in India for centuries. Due to high ethnic diversity, the pool of herbal medicines is enormous, and they are often preferred over modern medicines in certain parts of the country. Cancer is one of the major non-communicable diseases affecting people worldwide. Despite considerable research, cancer is a disease that is still not understood completely, and there have been constant efforts towards the identification of novel drugs or approaches in cancer management. Parkia javanica, an important medicinal plant and a rich source of flavonoids and terpenoids, is widely studied for its antioxidant and anti-inflammatory activities. Traditionally, the fruit and bark extracts of P. javanica find use as home remedy for dysentery and piles in NE India. Moreover, the fruits are consumed by the people of North-East (NE) India as vegetables, either in steamed or cooked form. In this study, crude extracts of P. javanica fruit and bark were obtained, the sub-lethal dose was determined and were then analyzed for anti-proliferative and anti-angiogenic properties using a battery of assays in zebrafish embryos. The sub-lethal concentration 50 (LC50) was found to be 28.66 mg/L and 346.66 mg/L for bark and fruit extract respectively, indicating a decreased toxicity of the fruit extract compared to that of the bark. The anti-proliferative and anti-angiogenic properties were more pronounced for the fruit extract compared to the bark extract. Although preliminary, the results of the study suggest that P. javanica fruits possess potent anti-angiogenic and anti-proliferative properties, which can be further studied for the isolation of active phytochemicals for use as therapeutic agents.

Endospores associated with deep seabed geofluid features in the eastern Gulf of Mexico.

Recent studies have reported up to 1.9 × 1029 bacterial endospores in the upper kilometre of deep subseafloor marine sediments, however, little is understood about their origin and dispersal. In cold ocean environments, the presence of thermospores (endospores produced by thermophilic bacteria) suggests that distribution is governed by passive migration from warm anoxic sources possibly facilitated by geofluid flow, such as advective hydrocarbon seepage sourced from petroleum deposits deeper in the subsurface. This study assesses this hypothesis by measuring endospore abundance and distribution across 60 sites in Eastern Gulf of Mexico (EGM) sediments using a combination of the endospore biomarker 2,6-pyridine dicarboxylic acid or 'dipicolinic acid' (DPA), sequencing 16S rRNA genes of thermospores germinated in 50°C sediment incubations, petroleum geochemistry in the sediments and acoustic seabed data from sub-bottom profiling. High endospore abundance is associated with geologically active conduit features (mud volcanoes, pockmarks, escarpments and fault systems), consistent with subsurface fluid flow dispersing endospores from deep warm sources up into the cold ocean. Thermospores identified at conduit sites were most closely related to bacteria associated with the deep biosphere habitats including hydrocarbon systems. The high endospore abundance at geological seep features demonstrated here suggests that recalcitrant endospores and their chemical components (such as DPA) can be used in concert with geochemical and geophysical analyses to locate discharging seafloor features. This multiproxy approach can be used to better understand patterns of advective fluid flow in regions with complex geology like the EGM basin.

Isolation and identification of quorum sensing antagonist from Cinnamomum verum leaves against Pseudomonas aeruginosa.

PURPOSE: The study aimed at isolating and identifying potential anti-quorum sensing (QS) compounds from Cinnamomum verum leaves against Pseudomonas aeruginosa. METHODOLOGY: Isolation of anti-QS compounds from C. verum leaf ethanol extract was carried out by column chromatography. The bioactive fraction was analysed by UV, IR, and GCMS spectroscopy. Various virulence assays were performed to assess the QS quenching ability of the purified compounds. In vivo toxicity of the purified compounds was examined in zebrafish model. The expression of the virulence genes was evaluated by qPCR analysis and in silico assessment was accomplished to check the binding ability of the compounds with the autoinducer molecule. KEY FINDINGS: The QS inhibitors isolated and identified showed a remarkable ability in reducing the production of elastase, pyocyanin, swarming motility and biofilm formation in P. aeruginosa. In the presence of the characterized compounds, the expression of virulence genes of P. aeruginosa was significantly reduced. Toxicity studies in zebrafish model indicated no effects on development and organogenesis at a concentration below 100 mg/l. Further, in silico analysis demonstrated the binding efficiency of the anti-QS compounds to AHL molecules, thus proving the QS quenching ability of the isolated compounds. SIGNIFICANCE: To the best of our knowledge this is the first report of isolation of anti-QS compounds from C. verum leaves against P. aeruginosa. The identified compounds qualify as potential QS antagonists. Further studies on these compounds can pave way for an effective and attractive anti-pathogenic therapy, to overcome the emergence of antibiotic resistance in bacteria.

Hydrocarbon seepage in the deep seabed links subsurface and seafloor biospheres.

Marine cold seeps transmit fluids between the subseafloor and seafloor biospheres through upward migration of hydrocarbons that originate in deep sediment layers. It remains unclear how geofluids influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. Here we analyzed 172 marine surficial sediments from the deep-water Eastern Gulf of Mexico to assess whether hydrocarbon fluid migration is a mechanism for upward microbial dispersal. While 132 of these sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well-known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the uncultivated bacterial phyla Atribacteria and Aminicenantes and the archaeal order Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum, in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of nonrespiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. These results point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.

Natural attenuation of spilled crude oil by cold-adapted soil bacterial communities at a decommissioned High Arctic oil well site.

Romulus C-42 is a decommissioned oil well on Ellesmere Island in the Canadian High Arctic, and is the northernmost well to have produced oil and gas anywhere in the world. The remote site has been untouched since a crude oil spill in 1972, offering a rare opportunity to examine natural attenuation in Arctic soils >40 years after a pollution event. Bacterial community composition in crude oil contaminated soils was significantly different from adjacent background soils. Two members of the genus Rhodanobacter (Alphaproteobacteria) were found consistently in contaminated soils, whereas two members of the genus Sphingomonas (Gammaproteobacteria) appeared opposite to each other, one consistently within the oil contaminated soil and the other consistently in non-oil contaminated soils. GC of soil hydrocarbon extracts revealed moderate levels of biodegradation relative to the original oil produced in 1972. Despite conditions permissive for bacterial activity (>0 °C) being limited to only a few months each year, natural attenuation by cold adapted soil microbial communities has taken place since the oil spill over 40 years ago. Rhodanobacter and Sphingomonas lineages are associated with contaminated and baseline conditions in this extreme environment, revealing the utility of bacterial diversity measurements for assessing long-term responses of Arctic soils to pollution. ORIGINALITY-SIGNIFICANCE STATEMENT: Romulus C-42, the northernmost onshore drilling well in the world, was decommissioned following a small crude oil spill in 1972. Soil bacterial diversity profiles obtained >40 years later revealed significant differences in oil contaminated soils relative to adjacent non-oil contaminated background soils, consistent with evidence for moderate biodegradation of spilled crude oil having taken place since 1972. The results indicate that microbial diversity profiling is an effective tool for assessing natural attenuation in remote High Arctic soils with a history of oil pollution.

Metabolic potential of uncultured bacteria and archaea associated with petroleum seepage in deep-sea sediments.

The lack of microbial genomes and isolates from the deep seabed means that very little is known about the ecology of this vast habitat. Here, we investigate energy and carbon acquisition strategies of microbial communities from three deep seabed petroleum seeps (3 km water depth) in the Eastern Gulf of Mexico. Shotgun metagenomic analysis reveals that each sediment harbors diverse communities of chemoheterotrophs and chemolithotrophs. We recovered 82 metagenome-assembled genomes affiliated with 21 different archaeal and bacterial phyla. Multiple genomes encode enzymes for anaerobic oxidation of aliphatic and aromatic compounds, including those of candidate phyla Aerophobetes, Aminicenantes, TA06 and Bathyarchaeota. Microbial interactions are predicted to be driven by acetate and molecular hydrogen. These findings are supported by sediment geochemistry, metabolomics, and thermodynamic modelling. Overall, we infer that deep-sea sediments experiencing thermogenic hydrocarbon inputs harbor phylogenetically and functionally diverse communities potentially sustained through anaerobic hydrocarbon, acetate and hydrogen metabolism.

Thermophilic endospores associated with migrated thermogenic hydrocarbons in deep Gulf of Mexico marine sediments.

Dormant endospores of thermophilic bacteria (thermospores) can be detected in cold marine sediments following high-temperature incubation. Thermospores in the cold seabed may be explained by a dispersal history originating in deep biosphere oil reservoir habitats where upward migration of petroleum fluids at hydrocarbon seeps transports viable cells into the overlying ocean. We assessed this deep-to-shallow dispersal hypothesis through geochemical and microbiological analyses of 111 marine sediments from the deep water Eastern Gulf of Mexico. GC-MS and fluorescence confirmed the unambiguous presence of thermogenic hydrocarbons in 71 of these locations, indicating seepage from deeply sourced petroleum in the subsurface. Heating each sediment to 50 °C followed by 16S rRNA gene sequencing revealed several thermospores with a cosmopolitan distribution throughout the study area, as well as thermospores that were more geographically restricted. Among the thermospores having a more limited distribution, 12 OTUs from eight different lineages were repeatedly detected in sediments containing thermogenic hydrocarbons. A subset of these were significantly correlated with hydrocarbons (p < 0.05) and most closely related to Clostridiales previously detected in oil reservoirs from around the world. This provides evidence of bacteria in the ocean being dispersed out of oil reservoirs, and suggests that specific thermospores may be used as model organisms for studying warm-to-cold transmigration in the deep sea.

Melamine-DNA encoded periodicity of quantum dot arrays.

Formation of QD-array in solution phase guided by the self-assembly with DNA-melamine hybrid molecules is reported here. Melamine was conjugated with ssDNA using phosphoramidate chemistry. Aqueous soluble ZnSe/ZnS QDs conjugated to complementary ssDNA was self-assembled with the DNA-melamine hybrid molecules by DNA-hybridization. The self-assembly leads to the precise positioning of the QDs in QDs array where the inter QD distance is being maintained by the DNA sequence length. The QD array was characterized by gel electrophoresis, UV-visible and fluorescence spectrophotometry and circular dichroism. Direct visualization of the DNA-melamine hybrid molecule mediated QD array was made possible by atomic force microscopy (AFM) and transmission electron microscopy (TEM) analysis. Substantial increase in the fluorescence intensity and lifetime of the QDs was observed on array formation by DNA self-assembly.

Simple Detection Methods for Antinutritive Factor ß-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography.

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease "neurolathyrism", present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for ß-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed ß-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100µg/ml and exhibited linear response with r2 > 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 µg/ml and 16.86 µg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of ß-ODAP is 0.6µg and for its substrate, L-1,2-diaminopropionic acid is 5µg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops.

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding ß-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.

In vitro and in vivo genotoxicity of silver nanoparticles.

The biocidal effect of silver nanoparticles (Ag-np) has resulted in their incorporation into consumer products. While the population exposed to Ag-np continues to increase with ever new applications, Ag-np remains a controversial research area with regard to their toxicity in biological systems. Here a genotoxic and cytotoxic approach was employed to elucidate the activity of Ag-np in vitro and in vivo. Characterization of Ag-np using scanning electron microscopy revealed a size range of 90-180nm. Cytotoxic potential of Ag-np was evaluated in human lymphocytes via cell viability assay (Trypan blue dye exclusion method, MTT and WST assay). The uptake and incorporation of Ag-np into the lymphocytes was confirmed by flow cytometry. Additionally apoptosis (AnnexinV-FITC-PI staining) and DNA strand breaks (comet assay) in human lymphocytes revealed that Ag-np at concentration 25µg/ml can cause genotoxicity. In vivo experiments on plants (Allium cepa and Nicotiana tabacum) and animal (Swiss albino male mice) showed impairment of nuclear DNA. Induction of oxidative stress was also studied. The DNA damage and chromosomal aberrations raise the concern about the safety associated with applications of the Ag-np. A single ip administration of Ag-np gave a significant (P≤0.05) increase in the frequency of aberrant cells and Tail DNA percent at concentrations 10mg/kg body weight and above. Results of comet assay in A. cepa and N. tabacum demonstrated that the genotoxic effect of Ag-np was more pronounced in root than shoot/leaf of the plants. The present study indicated a good correlation between the in vitro and in vivo experiments. Therefore the biological applications employing Ag-np should be given special attention besides adapting the antimicrobial potential.

Comparative study of hypocholesterolemic and hypolipidemic effects of conjugated linolenic acid isomers against induced biochemical perturbations and aberration in erythrocyte membrane fluidity.

AIM OF STUDY: The purpose of the study was to evaluate hypolipidemic and hypocholesterolemic activities of conjugated linolenic acid (CLnA) isomers, present in bitter gourd and snake gourd seed, in terms of amelioration of plasma lipid profile, lipoprotein oxidation and erythrocyte membrane fluidity after oral administration. METHODS: Male albino rats were divided into six groups. Group 1 was control, and others were induced with oxidative stress by oral gavage of sodium arsenite (Sa). Group 2 was kept as treated control, and groups 3-6 were further treated with different oral doses of seed oils to maintaining definite concentration of CLnA isomers (0.5 and 1.0% of total lipid for each CLnA isomer). RESULTS: CLnA isomers normalized cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride contents in plasma and body weight of experimental rats and decreased cholesterol synthesis by reducing hepatic HMG-CoA reductase activity. Administration of Sa caused alteration in erythrocyte membrane fluidity due to increase in cholesterol and decrease in phospholipid content. Tissue cholesterol and lipid contents were also increased by Sa administration. These altered parameters were reversed by experimental oil administration. Protective effect of CLnA isomers on erythrocyte morphology was observed by atomic force microscopy (AFM). Fatty acid composition of erythrocyte membrane showed decrease in polyunsaturated fatty acid (PUFA) and increase in arachidonic acid content after Sa administration, which was normalized with the treatment of these oils. Supplementation of CLnA isomers restored erythrocyte membrane (EM) lipid peroxidation and lipoprotein oxidation. CONCLUSION: CLnA isomers, present in vegetable oils, showed potent hypolipidemic and hypocholesterolemic activities against biochemical perturbations.