De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc²-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4⁺ and CD8⁺ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc²-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc²-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis.     

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

A molecular insight into papaya leaf curl—a severe viral disease

Papaya leaf curl disease (PaLCuD) caused by papaya leaf curl virus (PaLCuV) not only affects yield but also plant growth and fruit size and quality of papaya and is one of the most damaging and economically important disease. Management of PaLCuV is a challenging task due to diversity of viral strains, the alternate hosts, and the genomic complexities of the viruses. Several management strategies currently used by plant virologists to broadly control or eliminate the viruses have been discussed. In the absence of such strategies in the case of PaLCuV at present, the few available options to control the disease include methods like removal of affected plants from the field, insecticide treatments against the insect vector (Bemisia tabaci), and gene-specific control through transgenic constructs. This review presents the current understanding of papaya leaf curl disease, genomic components including satellite DNA associated with the virus, wide host and vector range, and management of the disease and suggests possible generic resistance strategies.     

Characterization of a metal resistant Pseudomonas sp. isolated from uranium mine for its potential in heavy metal (Ni2+, Co2+, Cu2+, and Cd2+) sequestration

Heavy metal sequestration by a multimetal resistant Pseudomonas strain isolated from a uranium mine was characterized for its potential application in metal bioremediation. 16S rRNA gene analysis revealed phylogenetic relatedness of this isolate to Pseudomonas fluorescens. Metal uptake by this bacterium was monophasic, fast saturating, concentration and pH dependent with maximum loading of 1048 nmol Ni²⁺ followed by 845 nmol Co²⁺, 828 nmol Cu²⁺ and 700 nmol Cd²⁺ mg^?1 dry wt. Preferential metal deposition in cell envelope was confirmed by TEM and cell fractionation. FTIR spectroscopy and EDX analysis revealed a major role of carboxyl and phosphoryl groups along with a possible ion exchange mechanism in cation binding. Binary system demonstrated selective metal binding affinity in the order of Cu²⁺ > Ni²⁺ > Co²⁺ > Cd²⁺. A comparison with similar metal uptake reports considering live bacteria strongly indicated the superiority of this strain in metal sequestration, which could be useful for developing efficient metal removal system.     

Temperature dependent 2nd derivative absorbance spectroscopy of aromatic amino acids as a probe of protein dynamics

Proteins display a broad peak in 250–300 nm region of their UV spectrum containing multiple overlapping bands arising from the aromatic rings of phenylalanine, tyrosine, and tryptophan residues. Employing high resolution 2^nd derivative absorbance spectroscopy, these overlapping absorption bands can be highly resolved and therefore provide a very sensitive measure of changes in the local microenvironment of the aromatic side chains. This has traditionally been used to detect both subtle and dramatic (i.e., unfolding) conformational alterations of proteins. Herein, we show that plots of the temperature dependent 2^nd derivative peak positions of aromatic residues have measurable slopes before protein unfolding and that these slopes are sensitive to the dielectric properties of the surrounding microenvironment. We further demonstrate that these slopes correlate with hydration of the buried aromatic residues in protein cores and can therefore be used as qualitative probes of protein dynamics.     

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.