Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart.

B-cell lymphoma 2-associated athanogene-3 (Bag3) is expressed in all animal species, with Bag3 levels being most prominent in the heart, the skeletal muscle, the central nervous system, and in many cancers. Preclinical studies of Bag3 biology have focused on animals that have developed compromised cardiac function; however, the present studies were performed to identify the pathways perturbed in the heart even before the occurrence of clinical signs of dilatation and failure of the heart. These studies show that hearts carrying variants that knockout one allele of BAG3 have significant alterations in multiple cellular pathways including apoptosis, autophagy, mitochondrial homeostasis, and the inflammasome.

Inhibition of RUNX1 blocks the differentiation of lung fibroblasts to myofibroblasts.

Pathological fibrosis contributes to progression of various diseases, for which the therapeutic options are limited. Idiopathic pulmonary fibrosis (IPF) is one such progressive and fatal interstitial fibrotic disease that is often characterized by excessive accumulation of extracellular matrix (ECM) proteins leading to stiff lung tissue and impaired gas exchange. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined the role of Runt-related transcription factor 1 (RUNX1), an evolutionarily conserved transcription factor, in the differentiation of human lung fibroblasts (HLFs) in vitro and in an animal model of bleomycin (BLM)-induced lung fibrosis. We observed that the expression of RUNX1 was significantly increased in the lungs of BLM-injected mice as compared to saline-treated mice. Furthermore, HLFs stimulated with transforming growth factor ß (TGF-ß) showed significantly higher RUNX1 expression at both mRNA and protein levels, and compartmentalization in the nucleus. Inhibition of RUNX1 in HLFs (using siRNA) showed a significant reduction in the differentiation of fibroblasts into myofibroblasts as evidenced by reduced expression of alpha-smooth muscle actin (α-SMA), TGF-ß and ECM proteins such as fibronectin 1 (FN1), and collagen 1A1 (COL1A1). Mechanistic studies revealed that the increased expression of RUNX1 in TGF-ß-stimulated lung fibroblasts is due to enhanced mRNA stability of RUNX1 through selective interaction with the RNA-binding profibrotic protein, human antigen R (HuR). Collectively, our data demonstrate that increased expression of RUNX1 augments processes involved in lung fibrosis including the differentiation of fibroblasts into collagen-synthesizing myofibroblasts. Our study suggests that targeting RUNX1 could limit the progression of organ fibrosis in diseases characterized by abnormal collagen deposition.

MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages.

OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

Increased m6A-RNA methylation and FTO suppression is associated with myocardial inflammation and dysfunction during endotoxemia in mice.

Endotoxemia triggers life-threatening immune and cardiovascular response that leads to tissue damage, multi-organ failure, and death. The understanding of underlying molecular mechanisms is still evolving. N6-methyladenosine (m6A)-RNA modification plays key regulatory role in numerous biological processes. However, it remains unclear whether endotoxemia alters RNA methylation in the myocardium. In the current study, we investigated the effect of lipopolysaccharide (LPS)-induced endotoxemia on m6A-RNA methylation and its implications on myocardial inflammation and left ventricular (LV) function. Following LPS administration, mice showed increases in m6A-RNA methylation in the myocardium with a corresponding decrease in the expression of fat mass and obesity-associated protein (FTO, an m6A eraser/demethylase). The changes were associated with a significant increase in expression of myocardial inflammatory cytokine genes, such as IL-6, TNF-α, IL-1ß, and reduced LV function. Moreover, rat cardiomyoblasts (H9c2) exposed to LPS showed similar changes (with increase in m6A-RNA methylation and inflammatory cytokine genes, whereas downregulation of FTO). Furthermore, methylated RNA immunoprecipitation assay showed hypermethylation and increase in the expression of IL-6 and TNF-α genes in LPS-treated H9c2 cells as compared to untreated cells. Interestingly, FTO knockdown in cardiomyocytes mimicked the above effects. Taken together, these data suggest that endotoxemia-induced m6A methylation might play a critical role in expression of cardiac proinflammatory cytokines, and modulation of m6A methylation might limit myocardial inflammation and dysfunction during endotoxemia.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

[Figure: see text].

microRNA-377 Signaling Modulates Anticancer Drug-Induced Cardiotoxicity in Mice.

Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.

Evaluation of therapeutic potential of recombinant buffalo lactoferrin N-lobe expressed in E. coli.

Lactoferrin (Lf) is a multifunctional bi-lobate iron-binding glycoprotein belonging to transferrin family with a mass of approximately 80 kD. Being ubiquitously present in almost all biological secretions, it performs important biological functions. One of the earliest and very well-documented functions of Lf is the antibacterial effect against broad spectrum Gram-negative and Gram-positive bacteria. In this study, buffalo Lf N-lobe cDNA was amplified, cloned and expressed as a fusion protein in Escherichia coli cells using pQE30 expression vector. After post-induction confirmation of expressed protein by SDS-PAGE, purification of recombinant protein using Ni-NTA was attempted and the yield of recombinant buffalo N-lobe Lf was estimated to be 1 mg/ml. Antibacterial activity of recombinant buffalo Lf N-lobe was assessed on pathogenic E. coli and Staphylococcus aureus strains. Peptic digest of recombinant N-lobe buffalo Lf showed antibacterial activity comparable to commercially available bovine Lf. The successful expression and characterization of functional recombinant N-lobe of buffalo Lf expressed in E. coli opens new vistas for developing alternate therapeutics, particularly against the diseases caused by Gram-negative microbes such as septicemia and diarrhea in newborn calves and mastitis in dairy animals.

TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a.

The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.

The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis.

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.

A conformational analysis of mouse Nalp3 domain structures by molecular dynamics simulations, and binding site analysis.

Scrutinizing various nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) genes in higher eukaryotes is very important for understanding the intriguing mechanism of the host defense against pathogens. The nucleotide-binding domain (NACHT), leucine-rich repeat (LRR), and pyrin domains (PYD)-containing protein 3 (Nalp3), is an intracellular innate immune receptor and is associated with several immune system related disorders. Despite Nalp3's protective role during a pathogenic invasion, the molecular features and structural organization of this crucial protein is poorly understood. Using comparative modeling and molecular dynamics simulations, we have studied the structural architecture of Nalp3 domains, and characterized the dynamic and energetic parameters of adenosine triphosphate (ATP) binding in NACHT, and pathogen-derived ligands muramyl dipeptide (MDP) and imidazoquinoline with LRR domains. The results suggested that walker A, B and extended walker B motifs were the key ATP binding regions in NACHT that mediate self-oligomerization. The analysis of the binding sites of MDP and imidazoquinoline revealed LRR 7-9 to be the most energetically favored site for imidazoquinoline interaction. However, the binding free energy calculations using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method indicated that MDP is incompatible for activating the Nalp3 molecule in its monomeric form, and suggest its complex interaction with NOD2 or other NLRs accounts for MDP recognition. The high binding affinity of ATP with NACHT was correlated to the experimental data for human NLRs. Our binding site prediction for imidazoquinoline in LRR warrants further investigation via in vivo models. This is the first study that provides ligand recognition in mouse Nalp3 and its spatial structural arrangements.

Detection of polymorphism and sequence characterization of Toll-like receptor 7 gene of Indian goat revealing close relationship between ruminant species.

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.

RGD modified albumin nanospheres for tumour vasculature targeting.

OBJECTIVES: Cyclic arginine-glycine-aspartic acid (RGD) peptide-anchored sterically stabilized albumin nanospheres (RGD-SN) have been investigated for the selective and preferential presentation of carrier contents at angiogenic endothelial cells overexpressing a(v) b(3) integrins on and around tumour tissue. Their targetability was assessed. METHODS: Albumin nanospheres were formulated, conjugated with RGD/RAD peptide and characterized on the basis of size and size distribution. The control Arginine-Alanine-Aspartic acid (RAD) peptide-anchored sterically stabilized nanospheres (RAD-SN) and nanosphere with 5 mol% PEG (SN) without peptide conjugate were used for comparison with RGD-SN for in vitro cell binding, in vivo organ distribution and tumor angiogenesis studies. KEY FINDINGS: The average size of all nanospheres prepared was approximately 100 nm and maximum drug entrapment was 67.2 ± 5.2%. In-vitro endothelial cell binding of nanospheres exhibited 8-fold higher binding of RGD-SN to human umbilical vein endothelial cells in comparison with the SN and RAD-SN. RGD peptide-anchored nanospheres were significantly (P ≤ 0.01) effective in the prevention of lung metastasis, angiogenesis and in effective regression of tumours compared with free fluorouracil, SN and RAD-SN. Results indicated that cyclic RGD peptide-anchored sterically stabilized nanospheres bearing fluorouracil were significantly (P ≤ 0.01) active against primary tumour and metastasis than the nontargeted sterically stabilized nanospheres and free drug. CONCLUSIONS: Cyclic RGD peptide-anchored sterically stabilized nanospheres appears promising for targeted cancer chemotherapeutics.

Polymeric nanospheres modified with YIGSR peptide for tumor targeting.

YIGSR peptide anchored pegylated nanospheres (YIGSR-SN) loaded with 5-fluorouracil (5-FU) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells over-expressing laminin receptors on and around tumor tissue and thus for assessing their targetability. Pegylated nanosphere (SN) without peptide conjugate were used for comparison. The average size of all nanosphere preparations prepared was approximately 108 nm and maximum drug entrapment was 68.5 +/- 5.2%. In vitro endothelial cell binding of nanospheres exhibited 8-fold higher binding of YIGSR-SN to HUVEC in comparison to the SN. Spontaneous lung metastasis and angiogenesis assays show that YIGSR peptide anchored nanospheres are significantly (p

Seven novel single nucleotide polymorphisms identified within river buffalo (Bubalus bubalis) lactoferrin gene.

The present study aimed at identifying single-nucleotide polymorphic (SNP) sites in different coding and non-coding regions of lactoferrin gene in Indian riverine buffaloes. A total of 102 animals from six different river buffalo breeds were screened at six bubaline lactoferrin gene loci. Single-strand conformation polymorphism (SSCP) analysis revealed monomorphic patterns at three loci LtfE2, LtfE11, and LtfE14 while a total of eight distinct patterns were observed in the other three loci viz. LtfE5, LtfE10, and LtfE16 which correspond to respective exons and their flanking regions. Sequence analysis of different SSCP variants revealed the presence of two SNP sites within the coding (exon 16) region and five SNP sites in flanking non-coding regions (intron 4 and intron 9). Both SNPs within exon 16 were found to be synonymous. The SNPs and haplotypes identified in the present study could serve as potential markers for association with susceptibility/resistance to mastitis in buffaloes.

Liposomes modified with YIGSR peptide for tumor targeting.

YIGSR peptide anchored sterically stabilized liposomes (YIGSR-SL) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells overexpressing laminin receptors on and around tumor tissue and thus for assessing their targetabilty. In vitro endothelial cell binding of liposomes exhibited 7-fold higher binding of YIGSR-SL to HUVEC in comparison to the nontargeted sterically stabilized liposomes (SL). Spontaneous lung metastasis and angiogenesis assays show that YIGSR peptide anchored liposomes are significantly (P

Cationic transfersomes based topical genetic vaccine against hepatitis B.

DNA vaccines have been shown to elicit both cellular and humoral immune responses and to be effective in a variety of preclinical bacterial, viral, and parasitic animal models. We have recently described a needle-free method of vaccination, transcutaneous immunization, based on topical application of vaccine antigens on intact skin using a novel carrier system, namely transfersomes. In the present study, a novel modified version of transfersomes, i.e., cationic transfersomes for topical DNA vaccine delivery has been developed. Cationic transfersomes composed of cationic lipid DOTMA and sodium deoxycholate as constitutive lipids were prepared and optimized for their size, shape, zeta potentials, deformability and loading efficiency. Plasmid DNA encoding hepatitis B surface antigen (HBsAg) was loaded in the cationic transfersomes using charge neutralization method. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cytokines level (IL-2 and IFN-gamma) following topical applications of plasmid DNA loaded cationic transfersomes in Balb/c mice and results were compared with naked DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. Results revealed that DNA loaded cationic transfersomes elicited significantly (*P<0.05) higher anti-HBsAg antibody titer and cytokines level as compared to naked DNA. It was also observed that topical application of DNA loaded cationic transfersomes elicited a comparable serum antibody titer and endogenous cytokines levels as produced after intramuscular recombinant HBsAg administration. The study signifies the potential of cationic transfersomes as DNA vaccine carriers for effective topical immunization.

Liposomes modified with cyclic RGD peptide for tumor targeting.

Cyclic RGD peptide anchored sterically stabilized liposomes (RGD-SL) were investigated for selective and preferential presentation of carrier contents at angiogenic endothelial cells overexpressing alphavbeta3 integrins on and around tumor tissue and thus for assessing their targetabilty. Liposomes were prepared using distearoylphosphatidylcholine (DSPC), cholesterol and distearoylphosphatidylethanolamine-polyethyleneglycol-RGD peptide conjugate (DSPE-PEG-RGD) in a molar ratio 56:39:5. The control RAD peptide anchored sterically stabilized liposomes (RAD-SL) and liposome with 5 mol% PEG (SL) without peptide conjugate which had similar lipid composition were used for comparison. The average size of all liposome preparations prepared was approximately 105 nm and maximum drug entrapment was 10.5+/- 1.1%. In vitro endothelial cell binding of liposomes exhibited 7-fold higher binding of RGD-SL to HUVEC in comparison to the SL and RAD-SL. Spontaneous lung metastasis and angiogenesis assays show that RGD peptide anchored liposomes are significantly (p<0.01) effective in the prevention of lung metastasis and angiogenesis compared to free 5-FU, SL and RAD-SL. In therapeutic experiments, 5-FU, SL, RGD-SL and RAD-SL were administered intravenously on day 4 at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing BALB/c mice resulting in effective regression of tumors compared with free 5-FU, SL and RAD-SL. Results indicate that cyclic RGD peptide anchored sterically stabilized liposomes bearing 5-FU are significantly (p<0.01) active against primary tumor and metastasis than the non-targeted sterically stabilized liposomes and free drug. Thus cyclic RGD peptide anchored sterically stabilized liposomes hold potential of targeted cancer chemotherapeutics.