Artificial intelligence and real-world data for drug and food safety - A regulatory science perspective.

In 2013, the Global Coalition for Regulatory Science Research (GCRSR) was established with members from over ten countries (www.gcrsr.net). One of the main objectives of GCRSR is to facilitate communication among global regulators on the rise of new technologies with regulatory applications through the annual conference Global Summit on Regulatory Science (GSRS). The 11th annual GSRS conference (GSRS21) focused on "Regulatory Sciences for Food/Drug Safety with Real-World Data (RWD) and Artificial Intelligence (AI)." The conference discussed current advancements in both AI and RWD approaches with a specific emphasis on how they impact regulatory sciences and how regulatory agencies across the globe are pursuing the adaptation and oversight of these technologies. There were presentations from Brazil, Canada, India, Italy, Japan, Germany, Switzerland, Singapore, the United Kingdom, and the United States. These presentations highlighted how various agencies are moving forward with these technologies by either improving the agencies' operation and/or preparing regulatory mechanisms to approve the products containing these innovations. To increase the content and discussion, the GSRS21 hosted two debate sessions on the question of "Is Regulatory Science Ready for AI?" and a workshop to showcase the analytical data tools that global regulatory agencies have been using and/or plan to apply to regulatory science. Several key topics were highlighted and discussed during the conference, such as the capabilities of AI and RWD to assist regulatory science policies for drug and food safety, the readiness of AI and data science to provide solutions for regulatory science. Discussions highlighted the need for a constant effort to evaluate emerging technologies for fit-for-purpose regulatory applications. The annual GSRS conferences offer a unique platform to facilitate discussion and collaboration across regulatory agencies, modernizing regulatory approaches, and harmonizing efforts.

Role of proteins in the biosynthesis and functioning of metallic nanoparticles.

Proteins are known to play important roles in the biosynthesis of metallic nanoparticles (NPs), which are biological substitutes for conventionally used chemical capping and stabilizing agents. When a pristine nanoparticle comes in contact with a biological media or system, a bimolecular layer is formed on the surface of the nanoparticle and is primarily composed of proteins. The role of proteins in the biosynthesis and further uptake, translocation, and bio-recognition of nanoparticles is documented in the literature. But, a complete understanding has not been achieved concerning the mechanism for protein-mediated nanoparticle biosynthesis and the role proteins play in the interaction and recognition of nanoparticles, aiding its uptake and assimilation into the biological system. This review critically evaluates the knowledge and gaps in the protein-mediated biosynthesis of nanoparticles. In particular, we review the role of proteins in multiple facets of metallic nanoparticle biosynthesis, the interaction of proteins with metallic nanoparticles for recognition and interaction with cells, and the toxic potential of protein-nanoparticle complexes when presented to the cell.

Recreating in vitro tripartite mycorrhizal associations through functional bacterial biofilms.

Arbuscular mycorrhizal fungi (AMF) and beneficial bacteria are found naturally associated with most terrestrial plant roots. While it is now well known that bacteria colonize AMF and can form aggregates and biofilms, little is known about how interactions between bacterial communities and AMF take place under both in situ and in vitro conditions. We investigated the impact of inoculation with AMF-associated bacteria (AABs) of AMF by in vitro recreation of the interaction on synthetic growth media in a two-compartment Petri plate system. The inoculated AABs were found to be associated with the mycorrhizal co-culture and were found to migrate along growing AMF hyphae and to be associated with the spore surface. AABs differentially influenced the growth of the AMF and their functional capability demonstrated by analysis of phosphate solubilization, nitrogen fixation, and biofilm formation. We have thus characterized these important interactions adding to a further understanding of the synergistic relationship between the two cross-kingdom microbial partners. KEY POINTS: • An in vitro assay was utilized to recreate functional biofilms with AMF-associated bacteria. • AMF-associated bacteria formed a biofilm and enhanced sporulation of Rhizophagus irregularis. • AMF-bacterial interactions through biofilm formation influence the functional capability of both partners.

Unveiling the dermatological potential of marine fungal species components: Antioxidant and inhibitory capacities over tyrosinase.

Marine fungi are a rich source of biologically active molecules, but a poorly explored bioresource for cosmeceutical products. This study evaluates the phytochemistry, antioxidant, and antityrosinase effects of the organic extracts of marine fungi isolated from various marine environments in India. Out of 35 screened fungal strains, methanol extracts of strains P2, Talaromyces stipitatus, and D4, Aspergillus terreus exhibited antityrosinase activity of 45% and 43%, respectively, at the lowest concentration of 0.5 mg/mL. The highest free radicals scavenging activity of 94% and 97% was observed at 500 mg/mL, respectively, of the same fungal extracts. The total phenolic content ranged from 8.20 to 20.30 mg/g of the dry weight of extract, expressed as gallic acid equivalent. GC-MS analysis of T. stipitatus and A. terreus extract identified seven and 10 major compounds, respectively. Some of the major compounds included azetidine, (3E)-3-[(3,5-dimethoxybenzoyl)hydrazono]-N-isobutyl butanamide, aziridine, and 3-methylcyclopentanone, 1,1-dimethylcyclohexane, cyclopentane carboxylic acid, N-allyl-4,5,6,7-tetrahydro-2-benzothiophene-1-carboxamide, cyclo(l-Pro-l-Val), and 3-phenylpropionitrile. In conclusion, this study showed abundant fungal resources in Indian marine environments. A correlation between total phenolic contents of the extracts confirmed that phenolic compounds play an important role in antioxidant as well as antityrosinase activity of the marine fungal extracts and can be viewed as new potential antityrosinase and antioxidant resources.

Review of patents for agricultural use of arbuscular mycorrhizal fungi.

Mycorrhizal biotechnology has emerged as a major component of sustainable agriculture and allied activities. Innovations related to its role in agriculture, land reclamation, forestry, and landscaping are well recognized. This review presents the evolution of innovations worldwide related to arbuscular mycorrhizal fungi (AMF) in the past two decades, from 2000 to April 2020, and maintains that such innovations must continue in the future. An analysis of 696 patents showed that AMF have been used consistently as a biofertilizer and bioremediator over that period, although an upsurge was noted in propagation technologies, next-generation production methods, and formulation technologies. This review will familiarize mycorrhizologists with novel and evolving trends and will convince them of the importance of applying for patents to safeguard their innovations and the use of those innovations by industry.

Harnessing the Untapped Catalytic Potential of a CoFe2O4/Mn-BDC Hybrid MOF Composite for Obtaining a Multitude of 1,4-Disubstituted 1,2,3-Triazole Scaffolds.

Metal-organic frameworks derived nanostructures with extraordinary variability, and many unprecedented properties have recently emerged as promising catalytic materials to address the challenges in the field of modern organic synthesis. In this contribution, the present work reports the fabrication of an intricately designed magnetic MOF composite based on Mn-BDC (manganese benzene-1,4-dicarboxylate/manganese terephthalate) microflakes via a facile and benign in situ solvothermal approach. Structural information about the as-synthesized hybrid composite has been obtained with characterization techniques such as TEM, SEM, XRD, FT-IR, AAS, EDX, ED-XRF, and VSM analysis. Upon investigation of catalytic performance, the resulting material unveils remarkable efficacy toward facile access of a diverse array of pharmaceutically active 1,2,3-triazoles from a multicomponent coupling reaction of terminal alkynes, sodium azide, and alkyl or aryl halides as coupling partners. In addition to a wide substrate scope, the catalyst with highly accessible active sites also possesses a stable catalytic metal center along with superb magnetic properties that facilitate rapid and efficient separation. The prominent feature that makes this protocol highly desirable is the ambient and greener reaction conditions in comparison to literature precedents reported to date. Further, a plausible mechanistic pathway is also proposed to rationalize the impressive potential of the developed catalytic system in the concerned reaction. We envision that findings from our study would not only provide new insights into the judicious design of advanced MOF based architectures but also pave the way toward greening of industrial manufacturing processes to tackle critical environmental and economic issues.

Chromium tolerance and accumulation in Aspergillus flavus isolated from tannery effluent.

Cr(VI) tolerance in Aspergillus flavus, strain SFL, isolated from tannery effluent was measured and compared with a reference strain of A. flavus, A1120. On solid medium, SFL had a high level of Cr(VI) tolerance (1,600 mg/L), which was 16 times that of A1120 and greater than most previously analyzed fungal strains. When in 100 mg/L of Cr(VI), SFL completely depleted Cr(VI) within 72 h while A1120 depleted 85% of Cr(VI). SFL was more effective in reducing extracellular Cr(VI) than A1120. While A1120 showed greater biosorption of Cr(VI) than SFL, intracellular accumulation was approximately 50% greater in SFL and was more energy-dependent than A1120. Cr(VI) modified the external surface of the hyphae. Cr speciation detected the presence of only Cr(III), corresponding to Cr(OH)3 , which precipitated on the hyphal surface. Cr(VI) bound to the functional groups carboxyl, amine, and hydroxyl in both SFL and A1120. Transmission electron microscopy energy-dispersive X-ray detected Cr on the fungal wall and within membrane-bound organelles of the cytoplasm. In conclusion, the greater tolerance of SFL to Cr(VI) relative to A1120 is due to more effective energy-dependant uptake of Cr(VI) into the cell and increased capacity of SFL to store Cr in intracellular vacuoles compared with A1120.

Understanding dynamics of Rhizophagus irregularis ontogenesis in axenically developed coculture through basic and advanced microscopic techniques.

Detailed information on structural changes that occur during ontogenesis of Rhizophagus irregularis in axenically developed coculture is limited. Our study aims to investigate the series of events that occur during mycorrhizal ontogenesis under axenic condition through basic and advanced microscopic techniques followed by comparison among these to identify the suitable technique for rapid and detailed analysis of mycorrhizal structures. Three stages were identified in mycorrhizal ontogenesis from initiation (preinfection stage of hyphae; its branching, infection and appressoria formation; epidermal opening; and hyphal entry), progression (arbuscular development; hyphal coils and vesicles) to maturity (extraradical spores). Scanning electron microscopy was found to be an efficient tool for studying spatial three-dimensional progression. Adding to the advantages of advanced microscopy, potential of autofluorescence to explore the stages of symbiosis nondestructively was also established. We also report imaging of ultrathin sections by bright field microscopy to provide finer details at subcellular interface. Owing to the merits of nondestructive sampling, ease of sample preparation, autofluorescence (no dye required), no use of toxic chemicals, rapid analysis and in depth characterization confocal laser scanning microscopy was identified as the most preferred technique. The method thus developed can be used for detailed structural inquisition of mycorrhizal symbiosis both in in planta and in an in vitro system.

Do environmentally induced DNA variations mediate adaptation in Aspergillus flavus exposed to chromium stress in tannery sludge?

BACKGROUND: Environmental stress induced genetic polymorphisms have been suggested to arbitrate functional modifications influencing adaptations in microbes. The relationship between the genetic processes and concomitant functional adaptation can now be investigated at a genomic scale with the help of next generation sequencing (NGS) technologies. Using a NGS approach we identified genetic variations putatively underlying chromium tolerance in a strain of Aspergillus flavus isolated from a tannery sludge. Correlation of nsSNPs in the candidate genes (n = 493) were investigated for their influence on protein structure and possible function. Whole genome sequencing of chromium tolerant A. flavus strain (TERIBR1) was done (Illumina HiSeq2000). The alignment of quality trimmed data of TERIBR1 with reference NRRL3357 (accession number EQ963472) strain was performed using Bowtie2 version 2.2.8. SNP with a minimum read depth of 5 and not in vicinity (10 bp) of INDEL were filtered. Candidate genes conferring chromium resistance were selected and SNPs were identified. Protein structure modeling and interpretation for protein-ligand (CrO4- 2) docking for selected proteins harbouring non-synonymous substitutions were done using Phyre2 and PatchDock programs. RESULTS: High rate of nsSNPs (approximately 11/kb) occurred in selected candidate genes for chromium tolerance. Of the 16 candidate genes selected for studying effect of nsSNPs on protein structure and protein-ligand interaction, four proteins belonging to the Major Facilitator Superfamily (MFS) and recG protein families showed significant interaction with chromium ion only in the chromium tolerant A. flavus strain TERIBR1. CONCLUSIONS: Presence of nsSNPs and subsequent amino-acid alterations evidently influenced the 3D structures of the candidate proteins, which could have led to improved interaction with (CrO4- 2) ion. Such structural modifications might have enhanced chromium efflux efficiency of A. flavus (TERIBR1) and thereby offered the adaptation benefits in counteracting chromate stress. Our findings are of fundamental importance to the field of heavy-metal bio-remediation.

In-vitro evaluation of marine derived fungi against Cutibacterium acnes.

Cutibacterium acnes (or Propionibacterium acnes) is the main target for the prevention and medical treatment of acne vulgaris. The aim of this study was to evaluate the in vitro anti-C. acnes and anti-S. epidermidis properties of some marine fungi isolated from different Indian marine environments. Seventy fungal isolates were obtained from samples collected from the west coasts and Andaman Island, India. Methanol extracts of 35 isolates were screened for their antibacterial properties and 5 out of the 35 isolates displayed significant inhibition as compared with tetracycline. DNA was successfully extracted from these five fungal isolates and phylogenetic analysis was performed. The methanol extracts possessed antibacterial activity against C. acnes and S. epidermidis with MIC values ranged from 0.8 mg/mL to 1 mg/mL. SEM analysis revealed that the extract induces deleterious morphological changes in the bacterial cell membrane. This study has identified some fungi extracts with significant antibacterial activity. The extracts may have potential for development as an antibacterial agent in the treatment of acne vulgaris.

An Aspergillus aculateus strain was capable of producing agriculturally useful nanoparticles via bioremediation of iron ore tailings.

Mining waste such as iron ore tailing is environmentally hazardous, encouraging researchers to develop effective bioremediation technologies. Among the microbial isolates collected from iron ore tailings, Aspergillus aculeatus (strain T6) showed good leaching efficiency and produced iron-containing nanoparticles under ambient conditions. This strain can convert iron ore tailing waste into agriculturally useful nanoparticles. Fourier-transform Infrared Spectroscopy (FT-IR analysis) established the at the particles are protein coated, with energy dispersive X-ray Spectroscopy (EDX analysis) showing strong signals for iron. Transmission Electron Microscopy (TEM analysis) showed semi-quasi spherical particles having average size of 15 ±â€¯5 nm. These biosynthesized nanoparticles when tested for their efficacy on seed emergence activity of mungbean (Vigna radiata) seeds, and enhanced plant growth at 10 and 20 ppm.

Development of a novel myconanomining approach for the recovery of agriculturally important elements from jarosite waste.

In this study, an ecofriendly and economically viable waste management approach have been attempted towards the biosynthesis of agriculturally important nanoparticles from jarosite waste. Aspergillus terreus strain J4 isolated from jarosite (waste from Debari Zinc Smelter, Udaipur, India), showed good leaching efficiency along with nanoparticles (NPs) formation under ambient conditions. Fourier-transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM) confirmed the formation of NPs. Energy dispersive X-ray spectroscopy (EDX analysis) showed strong signals for zinc, iron, calcium and magnesium, with these materials being leached out. TEM analysis and high resolution transmission electron microscopy (HRTEM) showed semi-quasi spherical particles having average size of 10-50nm. Thus, a novel biomethodology was developed using fungal cell-free extract for bioleaching and subsequently nanoconversion of the waste materials into nanostructured form. These biosynthesized nanoparticles were tested for their efficacy on seed emergence activity of wheat (Triticum aestivum) seeds and showed enhanced growth at concentration of 20ppm. These nanomaterials are expected to enhance plant growth properties and being targeted as additives in soil fertility and crop productivity enhancement.

Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

Intracellular Synthesis of Gold Nanoparticles Using an Ectomycorrhizal Strain EM-1083 of Laccaria fraterna and Its Nanoanti-quorum Sensing Potential Against Pseudomonas aeruginosa.

In this research work different shapes and sizes of gold nanoparticles (AuNPs) were synthesized through an intracellular biogenic approach, exploiting the chloroauric acid reducing and Au0 stabilizing potential of Laccaria fraterna EM-1083 mycelia. The intracellularly synthesized AuNPs exhibits anti-quorum sensing inhibitory potential against Pseudomonas aeruginosa. The synthesized AuNPs were characterized using UV-visible spectroscopy; transmission electron microscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. The characterization proved that the successful synthesis of highly stable crystalline AuNPs with various shapes. Here we tested inhibitory activity of AuNPs on QS-regulated biofilm development and pyocyanin production traits of P. aeruginosa. The qualitative and quantitative data demonstrated that AuNPs significantly inhibited the biofilm formation and pyocyanin production. In summary, our results signify the future use of intracellularly synthesized AuNPs in P. aeruginosa mediated diseases.

Phylogenetic analyses reveal molecular signatures associated with functional divergence among Subtilisin like Serine Proteases are linked to lifestyle transitions in Hypocreales.

BACKGROUND: Subtilisin-like serine proteases or Subtilases in fungi are important for penetration and colonization of host. In Hypocreales, these proteins share several properties with other fungal, bacterial, plant and mammalian homologs. However, adoption of specific roles in entomopathogenesis may be governed by attainment of unique biochemical and structural features during the evolutionary course. Due to such functional shifts Subtilases coded by different family members of Hypocreales acquire distinct features according to respective hosts and lifestyle. We conducted phylogenetic and DIVERGE analyses and identified important protein residues that putatively assign functional specificity to Subtilases in fungal families/species under the order Hypocreales. RESULTS: A total of 161 Subtilases coded by 10 species from five different families under the fungal order Hypocreales was included in the analysis. Based on the presence of conserved domains, the Subtilase genes were divided into three subfamilies, Subtilisin (S08.005), Proteinase K (S08.054) and Serine-carboxyl peptidases (S53.001). These subfamilies were investigated for phylogenetic associations, protein residues under positive selection and functional divergence among paralogous clades. The observations were co-related with the life-styles of the fungal families/species. Phylogenetic and Divergence analyses of Subtilisin (S08.005) and Proteinase K (S08.054) families of proteins revealed that the paralogous clades were clear-cut representation of familial origin of the protein sequences. We observed divergence between the paralogous clades of plant-pathogenic fungi (Nectriaceae), insect-pathogenic fungi (Cordycipitaceae/Clavicipitaceae) and nematophagous fungi (Ophiocordycipitaceae). In addition, Subtilase genes from the nematode-parasitic fungus Purpureocillium lilacinum made a unique cluster which putatively indicated that the fungus might have developed distinctive mechanisms for nematode-pathogenesis. Our evolutionary genetics analysis revealed evidence of positive selection on the Subtilisin (S08.005) and Proteinase K (S08.054) protein sequences of the entomopathogenic and nematophagous species belonging to Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae families of Hypocreales. CONCLUSIONS: Our study provided new insights into the evolution of Subtilisin like serine proteases in Hypocreales, a fungal order largely consisting of biological control species. Subtilisin (S08.005) and Proteinase K (S08.054) proteins seemed to play important roles during life style modifications among different families and species of Hypocreales. Protein residues found significant in functional divergence analysis in the present study may provide support for protein engineering in future.

Microbial nanowires: an electrifying tale.

Electromicrobiology has gained momentum in the last 10 years with advances in microbial fuel cells and the discovery of microbial nanowires (MNWs). The list of MNW-producing micro-organisms is growing and providing intriguing insights into the presence of such micro-organisms in diverse environments and the potential roles MNWs can perform. This review discusses the MNWs produced by different micro-organisms, including their structure, composition and mechanism of electron transfer through MNWs. Two hypotheses, metallic-like conductivity and an electron hopping model, have been proposed for electron transfer and we present a current understanding of both these hypotheses. MNWs not only are poised to change the way we see micro-organisms but also may impact the fields of bioenergy, biogeochemistry and bioremediation; hence, their potential applications in these fields are highlighted here.

Identification and topographical characterisation of microbial nanowires in Nostoc punctiforme.

Extracellular pili-like structures (PLS) produced by cyanobacteria have been poorly explored. We have done detailed topographical and electrical characterisation of PLS in Nostoc punctiforme PCC 73120 using transmission electron microscopy (TEM) and conductive atomic force microscopy (CAFM). TEM analysis showed that N. punctiforme produces two separate types of PLS differing in their length and diameter. The first type of PLS are 6-7.5 nm in diameter and 0.5-2 µm in length (short/thin PLS) while the second type of PLS are ~20-40 nm in diameter and more than 10 µm long (long/thick PLS). This is the first study to report long/thick PLS in N. punctiforme. Electrical characterisation of these two different PLS by CAFM showed that both are electrically conductive and can act as microbial nanowires. This is the first report to show two distinct PLS and also identifies microbial nanowires in N. punctiforme. This study paves the way for more detailed investigation of N. punctiforme nanowires and their potential role in cell physiology and symbiosis with plants.

Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system.

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.

Acidic Potassium Permanganate Chemiluminescence for the Determination of Antioxidant Potential in Three Cultivars of Ocimum basilicum.

Ocimum basilicum, a member of the family Lamiaceae, is a rich source of polyphenolics that have antioxidant properties. The present study describes the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative assessment of antioxidants in three cultivars of O. basilicum grown under greenhouse conditions. The chemiluminescence based assay was found to be a sensitive and efficient method for assessment of total and individual compound antioxidant potential. Leaves, flowers and roots were found to be rich reserves of the antioxidant compounds which showed intense chemiluminescence signals. The polyphenolics such as rosmarinic, chicoric, caffeic, p-coumaric, m-coumaric and ferulic acids showed antioxidant activity. Further, rosmarinic acid was found to be the major antioxidant component in water-ethanol extracts. The highest levels of rosmarinic acid was found in the leaves and roots of cultivars "holy green" (14.37; 11.52 mM/100 g DW respectively) followed by "red rubin" (10.02; 10.75 mM/100 g DW respectively) and "subja" (6.59; 4.97 mM/100 g DW respectively). The sensitivity, efficiency and ease of use of the chemiluminescence based assay should now be considered for its use as a primary method for the identification and quantification of antioxidants in plant extracts.

Whole genome annotation and comparative genomic analyses of bio-control fungus Purpureocillium lilacinum.

BACKGROUND: The fungus Purpureocillium lilacinum is widely known as a biological control agent against plant parasitic nematodes. This research article consists of genomic annotation of the first draft of whole genome sequence of P. lilacinum. The study aims to decipher the putative genetic components of the fungus involved in nematode pathogenesis by performing comparative genomic analysis with nine closely related fungal species in Hypocreales. RESULTS: de novo genomic assembly was done and a total of 301 scaffolds were constructed for P. lilacinum genomic DNA. By employing structural genome prediction models, 13, 266 genes coding for proteins were predicted in the genome. Approximately 73% of the predicted genes were functionally annotated using Blastp, InterProScan and Gene Ontology. A 14.7% fraction of the predicted genes shared significant homology with genes in the Pathogen Host Interactions (PHI) database. The phylogenomic analysis carried out using maximum likelihood RAxML algorithm provided insight into the evolutionary relationship of P. lilacinum. In congruence with other closely related species in the Hypocreales namely, Metarhizium spp., Pochonia chlamydosporia, Cordyceps militaris, Trichoderma reesei and Fusarium spp., P. lilacinum has large gene sets coding for G-protein coupled receptors (GPCRs), proteases, glycoside hydrolases and carbohydrate esterases that are required for degradation of nematode-egg shell components. Screening of the genome by Antibiotics & Secondary Metabolite Analysis Shell (AntiSMASH) pipeline indicated that the genome potentially codes for a variety of secondary metabolites, possibly required for adaptation to heterogeneous lifestyles reported for P. lilacinum. Significant up-regulation of subtilisin-like serine protease genes in presence of nematode eggs in quantitative real-time analyses suggested potential role of serine proteases in nematode pathogenesis. CONCLUSIONS: The data offer a better understanding of Purpureocillium lilacinum genome and will enhance our understanding on the molecular mechanism involved in nematophagy.