Optimizing the Agrobacterium tumefaciens-mediated transformation conditions in Colletotrichum lindemuthianum: a step forward to unravel the functions of pathogenicity arsenals.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.

AKT is indispensable for coordinating Par-4/JNK cross talk in p21 downmodulation during ER stress.

The double-edged role of p21 to command survival and apoptosis is emerging. The current investigation highlights ER stress-mediated JNK activation that plausibly triggers cell death by attenuating endogenous p21 level. Here, we demonstrated that ER stress activator 3-AWA diminishes the p21 levels in cancer cells by averting the senescent phenotype to commence G2/M arrest. In essence, the deceleration in p21 level occurs through ER stress/JNK/Caspase-3 axis via activation/induction of proapoptotic Par-4 and inhibition of AKT. The molecular dynamics studies identified important interactions, which may be responsible for the AKT inhibition and efficacy of 3-AWA towards AKT binding pocket. Interestingly, the p21 deceleration was rescued by incubating the cells with 3-AWA in the presence of an ER stress inhibitor, Salubrinal. Furthermore, we demonstrated that p21 expression decreases solitarily in Par-4+/+ MEFs; albeit, ER stress-induced JNK activation was observed in both Par-4+/+ and Par-4-/- MEFs. Par-4 knockdown or overexpression studies established that ectopic Par-4 along with ER stress are not sufficient to downregulate p21 in PC-3 cells but are adequate for DU-145 cells and that the ER stress inflicted activation of JNK, inhibition of AKT and Par-4 induction are all crucial to p21 downmodulation by 3-AWA. By using isogenic cell lines, such as HCT-116 p53+/+ and HCT-116 p53-/-, we found that deceleration in p21 expression due to ER stress is p53 independent. Moreover, in orthotopic carcinogen-induced rat colorectal carcinoma model, we found that 3-AWA inhibits colorectal tumor growth and formation of colorectal polyps at a tolerable dose, similar to the first-line drug for colorectal cancer-5-fluorouracil.

Reprogramming of Molecular Switching Events in UPR Driven ER Stress: Scope for Development of Anticancer Therapeutics.

The incitement of unfolded protein response (UPR) during endoplasmic reticulum (ER) stress by diverse intracellular (hypoxia, nutrient deprivation, etc.) or extracellular (environmental or drug induced) stimuli is considered a major threat for perturbing cellular homeostasis leading to the aggregation of unfolded proteins inside the cell. The catastrophic UPR events emerge as a prime cellular adaptation by remodeling cancer cell signaling and restoring ER homeostasis in favor of tumor growth. The transient ER stress protects cancer cells from undergoing apoptosis, whereas the prolonged stress response further activates many cell death pathways. The present review summarizes the UPR mediated triggering of transcriptional and translational reprogramming, which will provide novel therapeutic strategies towards pro-death mechanisms rather than a cellular adaptation in tumorigenesis. Nonetheless, the current topic also points out the reprogramming of emerging molecular switching events by complex UPR-mediated signaling to trigger apoptosis. The novel agents from various natural, semi-synthetic and synthetic sources that target ER stress signaling pathway to modulate selectively the UPR phenomena with preclinical efficacy are outlined. Since major emphasis on ER stress-induced transcriptional and translational reprogramming remains to be explored, we believe that the current subject will instigate more attention from the biomedical researchers in this certain research direction.

Evaluation of deletion polymorphisms of glutathione S-transferase genes and colorectal cancer risk in ethnic Kashmiri population: A case-control study.

AIM: Glutathione S.transferases. (GSTs) are known to play a pivotal role in the detoxification of potential carcinogens, and their gene variation may alter susceptibility to colorectal cancer. (CRC). The aim of the study was to evaluate the genetic association of GSTM1 and GSTT1 gene deletion/null polymorphism with disease susceptibility and risk development in CRC patients of ethnic Kashmiri population. MATERIALS AND METHODS: Genotype frequencies of GSTM1 and GSTT1 gene deletion/null polymorphism were compared between 160 CRC patients and 200 healthy controls using polymerase chain reaction multiplex. RESULTS: The frequency of GSTM1-null was found to be 76.2% in cases and 81.5% in controls and odds ratio. (OR) = 1.37 (95% confidence interval. [CI]: 0.82-2.28). Likewise, the GSTT1-null genotype was found in 75.5% of cases and 77.5% of controls and the OR = 1.14 (95% CI: 0.76-1.8). The overall association between the GSTM1-null and GSTT1-null polymorphism and the CRC cases was found to be insignificant (P < 0.05). However, individuals with double-null genotype (GSTM1-/GSTT1-) were found to have 3.5-fold increased risk for the development of CRC. Further, the risk genotype (null) of GSTT1 was found to be associated with tumor grade (P = 0.001) and GSTM1 (null) genotype was significantly associated with smoking status (P = 0.004), when compared to the (present) genotype in CRC cases. CONCLUSION: Our results suggest that GSTM1 and GSTT1 gene deletion/null gene polymorphisms are not a key modulators of the risk of developing CRC in Kashmiri population.

IgE FcεR1β polymorphism and risk of developing chronic spontaneous urticaria: A study in an ethnic Kashmiri population

BACKGROUND: The pathogenesis of chronic spontaneous urticaria involves interplay between the genetic and environmental factors, most of which is still poorly understood. It is well-recognized that 30-40% of chronic spontaneous urticaria is autoimmune in nature. Chronic autoimmune urticaria is caused by anti-Fc¿R1β and less frequently, by anti-IgE auto antibodies that lead to mast cell and basophil activation, thereby giving rise to the release of histamine and other proinflammatory mediators. We investigated the association between SNP loci in Fc¿R1β and chronic spontaneous urticaria and to see its relation with serum IgE levels in Kashmiri population. METHODS: The autologous serum skin test was used as a screening test for chronic autoimmune urticaria. PCR-RFLP was used to detect the genotype of the SNP loci. Serum IgE levels were assessed by ELISA kit. RESULTS: No significant difference was found between the study population and control group in genotype distribution (wild and variant) among Fc¿R1β loci (P value = 0.06, odds ratio = 0.29). The frequency of c¿R1β (C109T) in autologous serum skin test positive chronic autoimmune urticaria patients with the CT genotype was found to be statistically non-significant when compared with the wild genotype (P = 0.35). Carriers of Fc¿R1β (T allele) had a more significant risk of developing CAU than those with C allele (P = 0.01). In our population serum total IgE levels did not find any statistical significance with regard to ASST positive & ASST negative patients (P = 0.26). CONCLUSIONS: There is statistically no significant association between Fc¿R1β gene polymorphism and CSU in Kashmiri population; however, there is a probability of developing CSU in patients carrying Fc¿R1β T allele. Furthermore, serum total IgE levels had no significant association with the development of CAU

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Relationship between Serum Soluble Interleukin-2 Receptor and Renal Allograft Rejection: A Hospital-Based Study in KashmirValley.

BACKGROUND: Even after adequate immunosuppression therapy, acute rejection continues to be the single most important cause of graft dysfunction after renal transplantation. Renal allograft biopsy continues to be the reference standard, though certain clinical and biochemical parameters are helpful in assessment of these patients. Renal allograft rejection is mediated by T lymphocytes, expressing cell surface interleukin-2 receptors (IL-2R) which has been suggested as a marker of acute rejection episodes after organ transplantation. OBJECTIVE: To determine the pre- and post-transplantation serum soluble IL-2R levels in live related kidney transplant patients to predict acute rejection episodes. METHODS: Serial serum samples from 75 recipients and 41 healthy controls were assessed for soluble IL-2R levels by ELISA. The outcome of the graft was also determined for each recipient. RESULTS: The mean±SD serum soluble IL-2R levels in renal allograft recipients with rejection were significantly (p<0.001) higher than those without rejection (329.85±59.22 vs 18.12±11.22 pg/mL). The elevation of serum soluble IL-2R was evident in acute rejection episodes and found before elevation of serum creatinine. The higher values of serum soluble IL-2R in the rejection group were significantly reduced after recovery of allograft function by adequate anti-rejection therapy. 36.4% of patients in the rejection group had proven positive biopsies for the rejection and higher creatinine values, which was found to be statistically significant (p<0.001). A cohort of 41 healthy controls showed significantly (p<0.05) lower serum soluble IL-2R concentrations (15.27±7.79 pg/mL) when compared with the rejection group. CONCLUSION: Serum soluble IL-2R concentrations showed significant correlation with the acute rejection episodes in the renal allograft recipients. Prediction of soluble IL-2R levels might help the early detection of rejection episodes, which may pave way for the management of immunosuppression regimes and better graft functioning.

IgE FcɛR1ß polymorphism and risk of developing chronic spontaneous urticaria: A study in an ethnic Kashmiri population.

BACKGROUND: The pathogenesis of chronic spontaneous urticaria involves interplay between the genetic and environmental factors, most of which is still poorly understood. It is well-recognized that 30-40% of chronic spontaneous urticaria is autoimmune in nature. Chronic autoimmune urticaria is caused by anti-FcɛR1ß and less frequently, by anti-IgE auto antibodies that lead to mast cell and basophil activation, thereby giving rise to the release of histamine and other proinflammatory mediators. We investigated the association between SNP loci in FcɛR1ß and chronic spontaneous urticaria and to see its relation with serum IgE levels in Kashmiri population. METHODS: The autologous serum skin test was used as a screening test for chronic autoimmune urticaria. PCR-RFLP was used to detect the genotype of the SNP loci. Serum IgE levels were assessed by ELISA kit. RESULTS: No significant difference was found between the study population and control group in genotype distribution (wild and variant) among FcɛR1ß loci (P value=0.06, odds ratio=0.29). The frequency of FcɛR1ß (C109T) in autologous serum skin test positive chronic autoimmune urticaria patients with the CT genotype was found to be statistically non-significant when compared with the wild genotype (P=0.35). Carriers of FcɛR1ß (T allele) had a more significant risk of developing CAU than those with C allele (P=0.01). In our population serum total IgE levels did not find any statistical significance with regard to ASST positive & ASST negative patients (P=0.26). CONCLUSIONS: There is statistically no significant association between FcɛR1ß gene polymorphism and CSU in Kashmiri population; however, there is a probability of developing CSU in patients carrying FcɛR1ß T allele. Furthermore, serum total IgE levels had no significant association with the development of CAU.

Free radical scavenging potential of in vitro raised and greenhouse acclimatized plants of Artemisia amygdalina.

AIM: Artemisia amygdalina Decne. (Asteraceae) is a critically endangered and endemic herb of Kashmir Himalayan sub-alpine region and Pakistan. Scientific research throughout the world has evidence to support the tremendous medicinal utility of the genus Artemisia. The natural resources of medicinal plants are being reduced day by day. This study provides the alternative way for medicinal resource utilization and conservation of A. amygdalina. METHODS: In vitro-raised plants and greenhouse acclimatized plants were obtained by culturing wild explants on Murashige and Skoog's medium. Plant extracts were obtained and subjected to different antioxidant assays: DPPH assay, riboflavin photo-oxidation assay, deoxy ribose assay, ferric thiocyanate assay, thiobarbituric acid assay, post mitochondrial supernatant assay and DNA damage on agarose gel. RESULTS: In vitro grown plants, as well as those acclimatized in the greenhouse reveals antioxidant activity against hydroxyl, superoxide, and lipid peroxyl radicals. CONCLUSION: This preliminary study revealed the free radical scavenging potential of tissue culture-raised plant extracts of A. amydalina.

Clinical and etiological profile of hyperprolactinemia--data from a tertiary care centre.

OBJECTIVE: To study the clinical presentation and etiology of hyperprolactinemia, a common disorder encountered in endocrine practice. METHODS: We analyzed the clinical data, hormone profile and imaging reports of 187 females with documented hyperprolactinemia, over a period of 6 years (5 years retrospective analysis and one year prospective study). RESULTS: Majority of the 187 subjects studied presented in 3rd or 4th decade. Galactorrhoea was the commonest presenting symptom occurring in 159 subjects (85%), followed by amenorrhea in 68.9%; both amenorrhea and galactorrhea were seen in 45.4%. A microprolactinoma was demonstrated in 67 patients (35.8%), a nonfunctioning pituitary macroadenoma with stalk hyperprolactinemia occurred in 30 patients (16%) and polycystic ovarian disease was documented in 24 (12.8%). In 52 patients (27.8%) no apparent cause could be ascertained. CONCLUSIONS: Syndrome of amenorrhea and/or galactorrhea is the commonest presentation in hyperprolactinemia. Microprolactinoma was the most frequent identifiable etiology followed by idiopathic and stalk hyperprolactinemia in our series.

Spatial sensitivity measurements of a prompt gamma IVNAA facility.

A technique has been developed for measuring the spatial sensitivity of a prompt gamma in vivo neutron activation analysis facility as used for determining total body nitrogen (i.e. protein). A water filled perspex phantom was used to simulate a patient. Relative spatial sensitivity of the system was measured at various positions in the tank by observing the prompt gamma rays from thermal neutron capture in the 35Cl of a carbon tetrachloride sample contained in a small glass phial.