Andrographis Echioides Molecular Characterization and Phylogenetic Study Using DNA Barcoding Technique

With the advancement in various molecular diagnostic tools, DNA Barcoding has emerged as a gold standard molecular diagnostic tool across the globe. Since ancient times, medicinal plants have been widely used in Indian Ayurvedic medicine for treating a variety of ailments. Plants of the genus Andrographis have been extensively used for treating different types of ailments. In this study, rarely studied medicinal plant species were isolated, sequenced at the genetic level and studied for their evolutionary characteristics using phylogenetic analysis. In the present study, the identity of A. echioides was confirmed by targeting different barcoding genes such as ribulose-bisphosphate carboxylase, internal transcribed spacer, RNA polymerase-beta subunit, maturase K, and photosystem II protein D1 genes using a phylogenetic approach. After successful isolation and amplification of genomic DNA, specific primers were utilised for sequencing of each barcoding gene, followed by nucleotide BLAST analysis to determine the sequence percent identity of each gene with that from other plant species. The best homologs were then utilised for conducting phylogenetic analysis which confirmed the identity of the plant as Andrographis echioides.

Anti-oxidant and Anti-microbial Activities of Silver Nanoparticles of Essential Oil Extracts from Leaves of Zanthoxylum ovalifolium

Aim: The present study was aimed to synthesis and characterization of silver nanoparticles (AgNPs) using Zanthoxylum ovalifolium leaf essential oil extracts and to screen the anti-oxidant and antimicrobial potential of the same. Place and Duration of Study: The studies were carried out at Department of Botany, AVK College for Women, Hassan and Department of Biochemistry, Aurora’s Degree & PG College, Hyderabad from July 2017 to June 2018 Methodology: The essential oil from leaves of Zanthoxylum ovalifolium was obtained by hydrodistillation and analyzed by GC and GC-MS. Synthesis of silver nanoparticles of essential oil extract was carried out and characterized by using UV-VIS spectroscopy and transmission electron microscopy (TEM). The total phenolic and flavonoid contents were estimated in all the extracts. Furthermore, all the extracts were evaluated for anti-microbial activity against two gram-positive and two gram-negative bacteria and four pathogenic fungi using agar disc diffusion technique. Subsequently the minimum inhibitory concentration (MIC) was also determined. Results: The major compounds identified were Limonene, isofenchol, Geijerene, isothujanol, Borneol, dihydrocarveol, isobornyl acetate, pregeijerene, b-elemene, trans-caryophyllene and Germacrene D. The TEM analysis of nanoparticles synthesized showed a size of 8 to 14 nm with a lmax of 450nm. All the extracts were evaluated for in vitro anti-oxidant activity where the AgNP of essential oil extract showed maximum activity of 89.61% and 84.92% respectively for both DPPH free radical scavenging and Hydrogen peroxide free radical scavenging assays at a concentration of 100µg/ml. Among all the bacteria tested, B. subtilis was most susceptible at 100µg/ml with zone of inhibition of 22.5mm. While among all the fungi tested, A. niger inhibited more effectively by the AgNP of essential oil extract at 100µg/ml with a zone of inhibition of 16.2mm. Conclusion: The results obtained were remarkable suggesting that AgNP of essential oil extract possess excellent anti-oxidant and anti-microbial activity and can be an alternative bio-friendly source for various pharmaceutical industries.

Genomic profiling in a homogeneous molecular subtype of non‑small cell lung cancer: An effort to explore new drug targets.

BACKGROUND: Patients’ who are positive for kinase domain activating mutations in epidermal growth factor receptor (EGFR) gene, constitute 30–40% of non‑small cell lung cancer (NSCLC), and are suitable candidates for Tyrosine Kinase Inhibitor based targeted/personalized therapy. In EGFR non‑mutated subset, 8–10% that show molecular abnormalities such as EML4‑ALK, ROS1‑ALK, KIP4‑ALK, may also derive the benefit of targeted therapy. However, 40% of NSCLC belong to a grey zone of tumours that are negative for the clinically approved biomarkers for personalized therapy. This pilot study aims to identify and classify molecular subtypes of this group to address the un‑met need for new drug targets in this category. Here we screened for known/novel oncogenic driver mutations using a 46 gene Ampliseq Panel V1.0 that includes Ser/Thr/ Tyr kinases, transcription factors and tumor suppressors. METHODS: NSCLC with tumor burden of at least 40% on histopathology were screened for 29 somatic mutations in the EGFR kinase domain by real‑time polymerase chain reaction methods. 20 cases which were EGFR non‑mutated for TK domain mutations were included in this study. DNA Quality was verified from each of the 20 cases by fluorimeter, pooled and subjected to targeted re‑sequencing in the Ion Torrent platform. Torrent Suite software was used for next generation sequencing raw data processing and variant calling. RESULTS: The clinical relevance and pathological role of all the mutations/variants that include SNPs and Indels was assessed using polyphen‑2/SIFT/PROVEAN/mutation assessor structure function prediction programs. There were 10 pathogenic mutations in six different oncogenes for which annotation was available in the COSMIC database; C420R mutation in PIK3CA, Q472H mutation in vascular endothelial growth factor receptor 2 (VEGFR2) (KDR), C630W and C634R in RET, K367M mutation in fibroblast growth factor receptor 2 (FGFR2), G12C in KRAS and 4 pathogenic mutations in TP53 in the DNA binding domain (E285K, R213L, R175H, V173G). CONCLUSION: Results suggest, a potential role for PIK3CA, VEGFR2, RET and FGFR2 as therapeutic targets in EGFR non‑mutated NSCLC that requires further clinical validation.

Prevalence of KRAS mutations in metastatic colorectal cancer: A retrospective observational study from India.

BACKGROUND: One of the genetic alterations implicated in tumor progression in colorectal cancers (CRCs) are abnormalities in Kristen Rat Sarcoma (KRAS) gene. Evaluation of KRAS mutation status is an important prognostic factor and has predictive value in deciding first line therapy based on monoclonal antibodies such as Cetuximab and Panitumumab in metastatic CRCs. MATERIALS AND METHODS: In this retrospective study, we analyzed 7 different somatic mutations in Exon 2 of KRAS gene in 299 unselected incidental CRC patients who visited the hospital for clinical management during the period 2009–2013. Most of the tumors were primarily originating from colon and rectum; nevertheless, there were a few from rectosigmoid, sigmoid, ceacum and anal canal in the study group. Genomic DNA extracted from paraffin embedded tumor tissues was screened for 7 point mutations located in Codons 12 and 13 of KRAS gene, using Scorpions amplified refractory mutation system real time polymerase chain reaction technology. Statistical analysis was performed to assess bivariate relationship between different variables that includes: mutation status, mutation type, tumor location, tumor morphology, age and sex. RESULTS: Prevalence of mutation in Codons 12 and 13 was 42.8% in the study group. Well‑differentiated tumors had significantly more mutation positivity than moderately and poorly differentiated tumors (P = 0.001). 92% of the mutations were from Codon 12 and 8% in Codon 13. Glycine to Arginine was relatively more common in rectosigmoid followed by ceacum, while Glycine to Alanine mutation was relatively more prevalent in sigmoid, followed by rectum and rectosigmoid. CONCLUSION: The results suggest a prevalence of KRAS mutation at 42.8% in Indian population indicating that this testing is very crucial for targeted therapy management in metastatic CRC in India. Further analysis on mutation status of other homologues such as NRAS and downstream partner, v‑raf murine sarcoma viral oncogene homolog B1, would add value to understanding the role of anti‑epidermal growth factor receptor therapy in CRC management.

A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca2+, Mn2+ and Mg2+. Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca2+ than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Epidermal growth factor receptor mutation in non-small-cell lung carcinomas: A retrospective analysis of 1036 lung cancer specimens from a network of tertiary cancer care centers in India.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation plays a vital role in the prognosis of patients with lung cancer. However, there is a dearth of studies on EGFR mutation in Indian population. In this retrospective study conducted at a network of tertiary cancer care centers across India, we evaluated the proportion of EGFR mutation in patients with non‑small‑cell lung carcinomas (NSCLC). MATERIALS AND METHODS: A total of 1036 cases of non‑small lung cancer were assessed for EGFR mutation status using Scorpion amplified refractory mutation system real time polymerase chain reaction method from fine needle aspiration cytology core biopsy, pleural fluid and cell blocks. For a few cases, macro dissection of tumor from H and E slides was also performed for EGFR analysis. EGFR Status was assessed for the most commonly known driver mutations in Exons 18, 19, 20 and 21, which contributes to a total of 29 somatic mutations including the resistance mutation T790M. RESULTS: Around 39% of the cohort was female and 61% were male. Mutation was positive in 40.3% and negative (wild type) in 59.7%. There was 1.8% mutation in exon 18, 24.6% in exon 19, 1.6% in exon 20 and 12.8% in exon 21. 38.2% had a mutation in a single site and 1.1% had a mutation in two sites. Overall mutation was significant in females (50.5% vs. 33.9%) compared with males (c2 = 28.3, P < 0.001). Mutation was significant in exon 21 (16.8% vs. 10.3%, c2 = 9.44, P = 0.002) and exon 19 (30.7% vs. 20.7%, c2 = 13.2, P < 0.001) in females compared with males. CONCLUSION: EGFR is expressed differentially/ mutated in patients with NSCLC. Further studies to unravel the predictors for acquired genetic alterations of EGFR are needed.

Pediatricians' views about oral health care.

Pediatricians and family practitioners have the responsibility of providing the crucial primary care for the children. They are considered to be in a unique position to contribute to the dental health of their young patients. In view of this a study was conducted in Mangalore city (India) to assess pediatricians' views about oral health care and their advice regarding oral hygiene maintenance. Fifty pediatricians were personally interviewed with a questionnaire. All pediatricians acknowledged their role in oral hygiene maintenance of their patients. This study showed that there is a need for more communication between the two pediatric specialties of medicine and dentistry so as to deliver better child health care.

Rhizoctonia wilt suppression of brinjal (Solanum melongena L) and plant growth activity by Bacillus BS2.

An antibiotic-producing and hydrogen-cyanide-producing rhizobacteria strain Bacillus BS2 showed a wide range of antifungal activity against many Fusarium sp. and brinjal wilt disease pathogen Rhizoctonia solani. Seed bacterization with the strain BS2 promoted seed germination and plant growth in leguminous plants Phaseolus vulgaris and non-leguminous plants Solanum melongena L, Brassica oleracea var. capitata, B. oleraceae var. gongylodes and Lycopersicon esculentum Mill in terms of relative growth rate, shoot height, root length, total biomass production and total chlorophyll content of leaves. Yield of bacterized plants were increased by 10 to 49% compared to uninoculated control plants. Brinjal sapling raised through seed bacterization by the strain BS2 showed a significantly reduced wilt syndrome of brinjal caused by Rhizoctonia solani. Control of wilt disease by the bacterium was clue to the production of antibiotic-like substances, whereas plant growth-promotion was due to the activity of hydrogen cyanide. Root colonization study confirmed that the introduced bacteria colonized the roots and occupied 23-25% of total aerobic bacteria, which was confirmed using dual antibiotic (nalidixic acid and streptomycin sulphate) resistant mutant strain. The results obtained through this investigation suggested the potentiality of the strain BS2 to be used as a plant growth promoter and suppressor of wilt pathogen.

Influence of metham sodium on suppression of collar rot disease of peanut, in vitro antibiosis, siderophore production and root colonization by a fluorescent pseudomonad strain FPO4.

A hydroxamate type siderophore producing fluorescent Pseudomonas strain, isolated from the rhizoplane of paddy root showing plant growth promoting activity, exhibited a decreased in vitro antibiosis, production of siderophore and suppression of collar rot in presence of metham sodium. Use of herbicide had a detrimental effect on the plant growth promoting activity of this organism. The multiple drug resistant mutant strain derived from this rhizobacteria colonized the roots, but the herbicide application had a negative effect on their population. HPLC analysis of the siderophore showed five peaks of which the peak number three confirmed the antifungal activity.

Left ventricular mass index in patients of end stage renal disease (non-diabetic) on regular haemodialysis.

Echocardiography in patients of end stage renal failure (non diabetic) on regular haemodialysis revealed increase in left ventricular mass index in comparison to healthy controls. Anaemia, chronic uraemic state and hypertension were the probable contributory factors.

Hepatic cysts in adult polycystic kidney disease.

Of ninety two adult polycystic kidney patients 18.5% had liver cysts. Mostly they were multiple. Liver cysts were more common in men.

Crop improvement and disease suppression by a Bacillus spp. SR2 from pea nut rhizosphere.

An antibiotic producing Bacillus strain SR2, isolated from pea nut rhizosphere, showed in vitro antibiosis against many known plant pathogens. Seed bacterization with this strain showed an enhancement in seed germination, shoot height, root length, fresh and dry weights in four crop plants. A multiple drug resistant strain, SR2+, used to monitor root colonization confirmed the root colonization by the organism. Seed bacterization, reduced the number of chick pea wilted plants in wilt-sick soil, making the organism a potential bio-control agent against chick pea wilt.

Fungicidal and insect controlling properties of Proteus strain RRLJ 16, isolated from tea, Camellia sinensis (L) O Kuntze, plantations.

A strain of Proteus RRLJ 16, isolated from acid (pH 4.5-5.5) iron rich tea (Camellia sinensis) plantation soil, produced siderophores with single absorption peak at 370 nm when cultured in iron free medium. Extracted crude fraction of siderophores and live cultures showed in vitro antibiosis against five species of pathogenic fungi. When the strain was cultured in a normal bacteriological medium without iron stress, it produced an insect repellent compound. The culture filtrate sprayed (dilution of 1:100 in water) on tea plants infected with pink mites or green flies, the insect populations disappeared from the plants. Termite galleries sprayed similarly showed no termite activity after 24 hr of spraying. No reinfestation by insects was observed on treated plants for more than one year.

Termination of photorefractoriness in the brahminy myna, Sturnus pagodarum: role of photoperiods and gonadal hormones.

In brahminy myna a photosensitive species, long days caused full gonadal development followed by rapid regression, whereas short days inhibited these responses. Experiments were performed to investigate the effects of duration of photoperiod and gonadal hormones on the recovery of photosensitivity to long photoperiods in male birds. Groups of photorefractory birds were subjected to 8-, 9- or 11-hr daily photoperiods for 45 (6.5 weeks) or 63 (9 weeks) days and then transferred to 15 h daily photoperiods for 60 days to check for the regaining of photoresponsivity. A control group was held under 15L:9D throughout the period of study. Another experiment included three groups of photorefractory males, which were maintained on 9L:15D for 9 weeks and administered with, birth-1day-1 alternately for first 30 days olive oil or different doses (10 or 100 micrograms) of testosterone propionate (TP)/bird/day alternately for first 30 days, and then transferred to 15L:9D for another 30 days to test the recovery of photosensitivity. The results indicated that (i) a period of exposure to short daylengths is required to dissipate photorefractoriness, (ii) termination of photorefractoriness is dependent on the length and duration of photoperiods and (iii) TP inhibits the recovery of photosensitivity in a dose dependent manner.

Effect of testosterone on testes, body weight and plumage regeneration in photorefractory male redheaded bunting Emberiza bruniceps.

Various doses of testosterone propionate (TP) were administered for 10 days to refractory male redheaded buntings exposed to 15L:9D in last week of June, shortly before the onset of molt, to determine the effect of exogenous male hormone on the testes, body weight and plumage regeneration in photorefractory birds. While 5-, 10-, or 25 micrograms of TP bird-1 day-1 did not affect the testes, body weight or feather regeneration, testicular growth was stimulated but body weight declined and plumage regeneration prevented in birds that received 150- or 250 micrograms of TP bird-1 day-1. In contrast, a 50 micrograms treatment, although unable to induce the growth of the testes, caused loss in body weight and feather regeneration. Also, a 25 micrograms dose of TP administered to photostimulated birds induced complete testicular atrophy. These results suggests that testosterone exerts dose-dependent effects on the testes, body weight and plumage regeneration in buntings. A positive correlation is found between the amount of hormone and the testes growth, while the increasing amount of hormone has negative effect on the body weight and regeneration of feathers.