Possible Role of Metal(II) Octacyanomolybdate(IV) in Chemical Evolution: Interaction with Ribose Nucleotides

We have proposed that double metal cyanide compounds (DMCs) might have played vital roles as catalysts in chemical evolution and the origin of life. We have synthesized a series of metal octacyanomolybdates (MOCMos) and studied their interactions with ribose nucleotides. MOCMos have been shown to be effective adsorbents for 5′-ribonucleotides. The maximum adsorption level was found to be about 50 % at neutral pH under the conditions studied. The zinc(II) octacyanomolybdate(IV) showed larger adsorption compared to other MOCMos. The surface area seems to important parameter for the adsorption of nucleotides. The adsorption followed a Langmuir adsorption isotherms with an overall adsorption trends of the order of 5′-GMP > 5′-AMP > 5′-CMP > 5′-UMP. Purine nucleotides were adsorbed more strongly than pyrimidine nucleotides on all MOCMos possibly because of the additional binding afforded by the imidazole ring in purines. Infrared spectral studies of adsorption adducts indicate that adsorption takes place through interaction between adsorbate molecules and outer divalent ions of MOCMos.     

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K _m and k _cat values of the enzyme were obtained as 1.7 mg/ml and 66 s^?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.     

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin‐induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

The postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.     

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

An efficient Agrobacterium‐mediated site‐specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.