Structural insights of Labeo catla (catla) myxovirus resistance protein,GTP binding recognition and constitutive expression induced with Poly I:C.

The Myxovirus resistance (Mx) proteins are critical effectors belonging to the super-family of guanidine triphosphatase, often stimulated by type I interferon (IFN) and mediates antiviral responses to restrict the replication of numerous viral genes in fishes. In teleosts, Mx proteins display diverse and complicated antiviral activity in different species. The present investigation seeks to characterize the Mx gene from Labeo catla upon induction by double-stranded (ds) RNA, polyinosinic-polycytidylic acid, (poly I: C). Molecular modeling and all-atoms molecular dynamics (MD) simulations were employed to understand the architecture of the GTPase domain and its plausible mode of GTP recognition in Mx protein. The full-length L. catla Mx (LcMx) gene sequence (1821 bp nucleotides) encodes an open reading frame of 606 amino acids. Domain search indicated conserved tripartite domain architecture of LcMx and forms a major cluster with the Mx from other teleosts. The positively charged Arginine and polar Glutamine residues from helix 3 and 4 of stalk region LcMx aid in homo-oligomerization. MD simulation portrayed the role of conserved critical residues aid in GTP recognition by the GTPase domain which perfectly corroborates with experimental findings and prior MD studies. After injection of poly I:C, the temporal mRNA profile showed that LcMx expression was significantly elevated in the spleen, brain, kidney, liver, muscle, heart, intestine, and gill tissues. Collectively, these results suggest that the elevated expression of the major innate immune defense gene Mx was able to inhibit the poly I: C mediated virulence in fish.Communicated by Ramaswamy H. Sarma.

Breast cancer in young Indian women: factors, challenges in screening, and upcoming diagnostics.

Breast cancer management for young Indian women are full of challenges. The National Cancer Registry Programme (NCRP) has predicted that nearly 2,30,000 cases of breast cancer will be reported annually by 2025; with a steady increase in cases of young women (< 45 years of age) with breast cancer. In this review, the available literature is evaluated to understand the various risk factors contributing to the rise in cases of breast cancer in young women in India. Further, the challenges that are faced by the technicians in early diagnosis (e.g., physiology of young breasts, limited trained professionals, and awareness among patients, and cost of the treatment) of breast cancer. This review also focuses on the upcoming diagnostics like serum biomarkers and nanosensors for the early identification of the disease. For better prognosis and to reduce the chances of disease reoccurrence and metastasis, it is important that the disease has to be identified at an early stage.

Molecular characterization, constitutive expression and GTP binding mechanism of Cirrhinus mrigala (Hamilton, 1822) Myxovirus resistance (Mx) protein.

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.

Molecular cloning, GTP recognition mechanism and tissue-specific expression profiling of myxovirus resistance (Mx) protein in Labeo rohita (Hamilton) after Poly I:C induction.

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.

Expression of Mx Gene in Cirrhinus mrigala (Hamilton, 1822) to OmpC Protein of Aeromonas hydrophila and Bacterial Infection.

The aims of this study were to identify alternative myxovirus (Mx) stimulatory compounds in Cirrhinus mrigala and to characterize the kinetics and intensity of their stimulated responses by semi-quantitative RT-PCR. Mx transcripts were measured in C. mrigala injected with Aeromonas OmpC (outer membrane protein) at a dose 0.4 mg/fish. At day 1, day 2, day 3, day 5, day 10, day 20 and day 30, samples were collected from kidney, spleen, liver, heart brain, gill, intestine and muscle for the study of Mx transcript and housekeeping gene ß-actin. Similarly, Mx gene expression was also studied in Aeromonas hydrophila-infected fish for a period of 10 days. Mx/ß-actin ratio was constitutively expressed from all the organs of OmpC-vaccinated fish. The expression was significantly highest (P ≤ 0.05) in spleen, followed by liver, kidney, intestine, gill, heart, muscle and brain. The expression was highest in day 2 except spleen (on day 3) and subsequently reduced up to day 30. Control fish also showed Mx expression. Similarly, A. hydrophila-infected fish showed Mx/ß-actin ratio upregulated significantly in the spleen and kidney on day 5, liver on day 2 and intestine on day 3. This study revealed that OmpC of A. hydrophila and its infection could stimulate the antiviral Mx gene of C. mrigala.

Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein.

PROBLEM: It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility. METHOD OF STUDY: An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. RESULTS: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. CONCLUSION: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.