Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.     

Plant growth promotion and suppression of charcoal‐rot fungus (Macrophomina phaseolina) in velvet bean (Mucuna pruriens L.) by root nodule bacteria

Root‐nodulating bacteria are intimate associates of legumes. From a pool of rhizobia isolated from root nodules of Mucuna pruriens (Velvet bean/Kaunch), RMP66 and BMP17 were found to be capable of promoting siderophore and IAA production and phosphate solubilization (insoluble tri‐calcium). Both symbionts were studied further to determine their abilities to promote plant growth and to control root‐rot in Mucuna pruriens caused by the pathogenic plant fungus Macrophomina phaseolina. RMP66 and BMP17 were selected based on their excellent inhibitory activities against M. phaseolina (by 78% and 71%, respectively) in dual culture and in agar‐well assays using cell‐free culture filtrate (CFCF) (by 76% and 62%, respectively). Both strains inhibited fungal growth to a greater extent in iron‐deficient medium (51% and 69%) than in iron‐supplemented medium (37% and 0%), respectively. CFCFs of RMP66 and BMP17 obtained from Pikovskaya's broth and tryptophan‐amended YEM broth inhibited fungal growth by 80%‐55% and 70%‐43%, respectively, and were identified as Sinorhizobium meliloti RMP66 and Bradyrhizobium diazoefficiens BMP17 by 16S rDNA sequencing. Centrifuged and pelleted cells harvested from exponentially grown cultures of S. meliloti RMP66 and B. diazoefficiens BMP17 were used to bacterize seeds of M. pruriens, which then showed enhanced seed germination (by up to 17% and 12%, respectively), and subsequent increases in other plant growth parameters in field trials. Considerable increases in seedling vigour indices (62%: 53% and 110%: 130%) and biomass (8%: 13% and 25%: 28%) were also observed for bacterial treatments. Tn5‐mediated antibiotic‐resistant marker strains showed enhanced nodule occupancy by up to 72% and 68%, respectively. This study describes a multifunctional legume nodule rhizobia that could be utilized in multicropping systems under different agroclimatic conditions as a bioinoculant and alternative to fertilizers.     

Membrane skeletal association and post‐translational allosteric regulation of Toxoplasma gondii GAPDH1

When Toxoplasma gondii egresses from the host cell, glyceraldehyde‐3‐phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure–function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S‐loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S‐loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N‐terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post‐translational modifications.     

Bacterial Virulence Factors: Secreted for Survival

Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.     

Effect on lycopene, β-carotene,ascorbic acid and phenolic content in tomato fruits infected by Alternaria alternata and its toxins (TeA,AOH and AME)

Tomato is considered as one of the most important sources of nutrients such as lycopene, β-carotene, flavonoids, ascorbic acid (vitamin C) and hydroxyl-cinnamic acid derivatives. The quality and quantity of nutrients in tomato fruits were decreased during the severe infection of Alternaria alternata. The present study deals with the estimation of lycopene, β-carotene, phenolic and ascorbic acid content in tomato fruits which were infected with A. alternata and its toxins such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lycopene, β-carotene, ascorbic acid and phenolic content were found lowest in pathogen-infected fruits i.e. (0.66 ± 0.03 mg/g), (0.14 ± 0.01 mg/g), (1.89 ± 0.2 mg/g) and (0.58 ± 0.05 mg/g), respectively, followed by toxins-treated samples as compared to the control. The results concluded that A. alternata mostly affects the nutritional values of tomato fruits due to the combined effect of the toxins.     

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.     

Sex determination in 6 bovid species by duplex PCR

Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae,viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, andOvis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including theSRY gene and theGAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.     

Metal Cu(II) and Zn(II) bipyridyls as inhibitors of lactate dehydrogenase

Metal complex–protein interaction is an evolving concept for determining cellular targets of metallodrugs. Lacatate dehydrogenase (LDH) is critically implicated in tumor growth and therefore, considered to be an important target protein for anti-tumor metal complexes. Due to efficient biocompatibility of copper (Cu²⁺) and zinc (Zn²⁺), we synthesized CubpyAc₂ · H₂O (Cu-bpy) and ZnbpyAc₂ · H₂O (Zn-bpy; where bpy = 2,2′ bipyridine, Ac = CH₃COO⁻) complexes and evaluated their interaction with and modulation of LDH in mouse tissues. The increasing concentration of both the complexes showed a significant shift in UV–Vis spectra of LDH. The binding constant data (Kc = 1 × 10³ M⁻¹ for Cu-bpy and 7 × 10⁶ M⁻¹ for Zn-bpy) suggested that Zn-bpy-LDH interaction is stronger than that of Cu-bpy-LDH. LDH modulating potential of the complexes were monitored by perfusing the mice tissues with non-toxic doses of Cu-bpy and Zn-bpy followed by activity measurement and analysis of LDH isozymes on non-denaturing polyacrylamide gel electrophoresis (PAGE). As compared to the control sets, Cu-bpy caused a significant decline (P < 0.05–0.001) in the activity of LDH in all the tissues studied. However, Zn-bpy showed inhibition of LDH only in liver (P < 0.01), kidney (P < 0.001) and heart (P < 0.01), but with no effect in spleen, brain and skeletal muscle tissues. PAGE analysis suggested that all the five LDH isozymes are equally sensitive to both the complexes in the respective tissues. The results suggest that Cu- and Zn-bpy are able to interact with and inhibit LDH, a tumor growth supportive target protein at tissue level.