Assessment of goat activin receptor type IIB knockdown by short hairpin RNAs in vitro.

BACKGROUND: Targeted knockdown of ACVR2B, a receptor for TGF beta superfamily, has been seen as a potential candidate to enhance the muscle mass through RNAi approach. METHODS: We have evaluated the potential short hairpin RNAs targeting goat ACVR2B in human HEK293T cells and goat myoblasts cells by transient transfection and measured their knockdown efficiency and possible undesired interferon response by quantitative real-time PCR. RESULTS: We observed a significant silencing (64-81%) of ACVR2B in 293T cells with all seven shRNAs (sh1 to sh7) constructs and 16-46% silencing with maximum of 46% by sh6 (p = 0.0318) against endogenous ACVR2B whereas up to 66% (p = 0.0002) silencing by sh6 against exogenously expressed ACVR2B in goat myoblasts cells. Transient knockdown of ACVR2B in goat myoblasts cells by shRNAs did not show significant correlation with the expression of MyoD (r = 0.547; p = 0.102), myogenin (r = 0.517; p = 0.126) and Myf5 (r = 0.262; p = 0.465). As reported earlier, transfection of plasmid DNA induced potent interferon response in 293T and goat myoblasts cells. CONCLUSIONS: The present study demonstrates the targeted knockdown of ACVR2B by shRNAs in HEK293T and goat myoblasts cells in vitro. The transient knockdown of ACVR2B by shRNAs in goat myoblasts did not alter the myogenic gene expression program. However, shRNAs showing significant knockdown efficiency in our study may further be tested for long term and stable knockdown to assess their potential to use for enhancing muscle mass in vivo. As reported earlier, expression of shRNAs through plasmid expression vectors induces potent interferon response raising the concern of safety of its application in vivo.

Identification of novel splice variants in horn cancer by RNA-Seq analysis in Zebu cattle.

Horn cancer accounts for nearly 83% of total tumors found in Indian Zebu cattle, which results in chronic suffering and causes heavy economic losses. Alternative splicing has been frequently implicated in the various types of cancer progression. Utilizing the transcriptome sequence generated by next generation sequencing, we analyzed the transcript data for the presence of alternative splicing using BLAT program and identified 27 alternatively spliced genes, of which 12 spliced variants appeared to be the novel spliced candidates. Protein prediction of these novel spliced variants revealed that splice variation has caused either truncation of protein, insertion/deletion of stretch of amino acids or formation of unique carboxy terminus. The RT-PCR analysis confirmed the expression of 8 of the 12 novel spliced variants observed by transcriptome sequencing. Additionally, altered splicing/expression of these novel candidates between cancer and normal tissues revealed by qPCR suggests their potential involvement in the development of horn cancer.

Use of real-time PCR technique in determination of major fibrolytic and non fibrolytic bacteria present in Indian Surti buffaloes (Bubalus bubalis).

In the milk industry in India, buffalo breeds are most commonly used for milk production. Efficiency of fiber digestion in ruminants is critical for animal productivity. Bacteria play an important role in fiber digestion and utilization. Absolute quantification real-time PCR was used to quantify ten bacterial species in rumen fluid of Surti buffalo fed green fodder, dry roughage and compound concentrate mixture. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific primers. Bacterial populations showed a clear predominance of Ruminococcus albus, which comprised 5.66% of the bacterial rRNA gene copies in the samples. However, only 0.9% to 4.24% of the bacterial rRNA gene copies were represented by the ruminal Fibrobacter succinogenes, Ruminococcus flavefaciens and Prevotella species. The proportion of rRNA gene copies attributable to Selenomonas ruminantium, Streptococcus bovis, Ruminobacter amylophilus, Treponema bryantii and Anaerovibrio lipolytica was even less abundant, each comprising < 0.11% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.

Human leukocyte antigen alleles, genotypes and haplotypes frequencies in renal transplant donors and recipients from West Central India.

BACKGROUND: Human leukocyte antigen (HLA) is comprised of a highly polymorphic set of genes which determines the histocompatibility of organ transplantation. The present study was undertaken to identify HLA class I and class II allele, genotype and haplotype frequencies in renal transplant recipients and donors from West Central India. MATERIALS AND METHODS: HLA typing was carried out using Polymerase Chain Reaction-Sequence Specific Primer in 552 live related and unrelated renal transplant recipients and donors. RESULTS: The most frequent HLA class I and class II alleles and their frequencies in recipients were HLA-AFNx0101 (0.1685) and AFNx0102 (0.1649), HLA-BFNx0135 (0.1322), and HLA-DR beta 1 (DRB 1)FNx0115 (0.2192), whereas in donors, these were HLA-AFNx0102 (0.1848) and AFNx0101 (0.1667), HLA-BFNx0135 (0.1359), and HLA-DRB1FNx0115 (0.2409). The two-locus haplotype statistical analysis revealed HLA-AFNx0102-B61 as the most common haplotype with the frequency of 0.0487 and 0.0510 in recipients and donors, respectively. Further, among the three locus haplotypes HLA-AFNx0133-BFNx0144-DRB1FNx0107 and HLA-AFNx0102-BFNx0161-DRB1FNx0115 were the most common haplotypes with frequencies 0.0362 and 0.0326, respectively in recipients and 0.0236 and 0.0323, respectively in donors. Genotype frequency revealed a high prevalence of genotype HLA-AFNx0102/AFNx0124 in recipients (0.058) compared to donors (0.0109) whereas low prevalence of HLA-AFNx0101/AFNx0102 in recipients (0.0435) than in donors (0.0797). The phylogenetic and principal component analysis of HLA allele and haplotype frequency distribution revealed genetic similarities of various ethnic groups. Further, case control analysis provides preliminary evidence of association of HLA-A genotype (P < 0.05) with renal failure. CONCLUSION: This study will be helpful in suitable donor search besides providing valuable information for population genetics and HLA disease association analysis.

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study.

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.

Somatotropin-mediated gene expression profiling of differentially displayed ESTs during lactation in Indian buffalo (Bubalus bubalis).

The study of bovine mammary gland functional genomics requires appropriate cDNA library collections to access gene expression patterns from different developmental and physiological stages. The present study was undertaken with the objective to identify candidate genes involved in the process of increased milk synthesis following 0, 48 and 96 h of recombinant bovine somatotropin (rbST) treatment to Surti buffalo (Bubalus bubalis) through differential display reverse transcriptase PCR (DDRT-PCR). Of a total 50 sequenced DD bands, 64% of ESTs were differentially expressed (appeared only in post-treatment samples, i.e. 48 h and 96 h) and 36% were up-regulated after rbST treatment. Of the ESTs 32%were found to be located on Bos taurus chromosome 24 (equivalent to buffalo chromosome 22), whereas 16% of ESTs could not be mapped, indicating that they are specific to buffalo. Quantitative real time PCR assay of 15 ESTs revealed transcript level surge in 13 ESTs, and decline in one EST, while one showed up-regulation in expression level at 48 h while down-regulation at 96 h. This study indicates more than 30 novel transcripts, with unknown function, involved in increased milk synthesis and also the involvement of many more genes in the physiology of milk production than once thought.

Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.

Transport of Non-Transferrin Bound Iron to the Brain: Implications for Alzheimer's Disease.

A direct correlation between brain iron and Alzheimer's disease (AD) raises questions regarding the transport of non-transferrin-bound iron (NTBI), a toxic but less researched pool of circulating iron that is likely to increase due to pathological and/or iatrogenic systemic iron overload. Here, we compared the distribution of radiolabeled-NTBI (59Fe-NTBI) and transferrin-bound iron (59Fe-Tf) in mouse models of iron overload in the absence or presence of inflammation. Following a short pulse, most of the 59Fe-NTBI was taken up by the liver, followed by the kidney, pancreas, and heart. Notably, a strong signal of 59Fe-NTBI was detected in the brain ventricular system after 2 h, and the brain parenchyma after 24 h. 59Fe-Tf accumulated mainly in the femur and spleen, and was transported to the brain at a much slower rate than 59Fe-NTBI. In the kidney, 59Fe-NTBI was detected in the cortex after 2 h, and outer medulla after 24 hours. Most of the 59Fe-NTBI and 59Fe-Tf from the kidney was reabsorbed; negligible amount was excreted in the urine. Acute inflammation increased the uptake of 59Fe-NTBI by the kidney and brain from 2-24 hours. Chronic inflammation, on the other hand, resulted in sequestration of iron in the liver and kidney, reducing its transport to the brain. These observations provide direct evidence for the transport of NTBI to the brain, and reveal a complex interplay between inflammation and brain iron homeostasis. Further studies are necessary to determine whether transient increase in NTBI due to systemic iron overload is a risk factor for AD.

Prion Protein-Hemin Interaction Upregulates Hemoglobin Synthesis: Implications for Cerebral Hemorrhage and Sporadic Creutzfeldt-Jakob Disease.

Hemin is known to induce endocytosis of prion-protein (PrP(C)) from the neuronal plasma membrane, potentially limiting propagation of the disease causing PrP-scrapie (PrP(Sc)) isoform. Hemin is therefore an attractive disease-modifying option for sporadic Creutzfeldt-Jakob disease (sCJD), a human prion disorder with no effective treatment. The hemin-PrP(C) interaction is also of interest in cerebral-hemorrhage (CH), a condition where potentially toxic hemin molecules come in contact with neuronal PrP(C). Interestingly, PrP(C) is upregulated in penumbric neurons surrounding CH and is known to confer neuroprotection in a dose-dependent manner. The underlying mechanism, however, is not clear. Here, we report that hemin binds PrP(C) on diverse cell lines, resulting in its aggregation or degradation in a cell-type specific manner. Surprisingly, the hemin-PrP(C) interaction upregulates Hb synthesis in hematopoietic cells, a response reversed by deleting the hemin-binding octa-peptide repeat region of PrP(C). A similar response is noted in brain organotypic cultures where exposure to hemin induces significantly more α-globin in wild-type (PrP(+/+)) relative to PrP-knock-out (PrP(-/-)) samples. Furthermore, red blood cells and brain tissue from PrP(-/-) mice show significantly less α-globin relative to PrP(+/+) controls, indicating a positive effect of PrP(C) on Hb synthesis under physiological conditions as well. Surprisingly, levels of α-globin are significantly higher in sCJD brain tissue relative to controls, suggesting compensatory upregulation of Hb synthesis by surviving neurons or misregulation in diseased brains. These observations reveal a unique function of PrP(C) that is likely to impact the therapeutic management of CH and sCJD.

Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: Implications for visual manifestations of Parkinson's disease.

Aggregation of α-synuclein (α-syn) in neurons of the substantia nigra is diagnostic of Parkinson's disease (PD), a neuro-motor disorder with prominent visual symptoms. Here, we demonstrate that α-syn, the principal protein involved in the pathogenesis of PD, is expressed widely in the neuroretina, and facilitates the uptake of transferrin-bound iron (Tf-Fe) by retinal pigment epithelial (RPE) cells that form the outer blood-retinal barrier. Absence of α-syn in knock-out mice (α-syn(-/-)) resulted in down-regulation of ferritin in the neuroretina, indicating depletion of cellular iron stores. A similar phenotype of iron deficiency was observed in the spleen, femur, and brain tissue of α-syn(-)(/-) mice, organs that utilize mainly Tf-Fe for their metabolic needs. The liver and kidney, organs that take up significant amounts of non-Tf-bound iron (NTBI), showed minimal change. Evaluation of the underlying mechanism in the human RPE47 cell line suggested a prominent role of α-syn in the uptake of Tf-Fe by modulating the endocytosis and recycling of transferrin (Tf)/transferrin-receptor (TfR) complex. Down-regulation of α-syn in RPE cells by RNAi resulted in the accumulation of Tf/TfR complex in common recycling endosomes (CREs), indicating disruption of recycling to the plasma membrane. Over-expression of exogenous α-syn in RPE cells, on the other hand, up-regulated ferritin and TfR expression. Interestingly, exposure to exogenous iron increased membrane association and co-localization of α-syn with TfR, supporting its role in iron uptake by the Tf/TfR complex. Together with our observations indicating basolateral expression of α-syn and TfR on RPE cells in vivo, this study reveals a novel function of α-syn in the uptake of Tf-Fe by the neuroretina. It is likely that retinal iron dyshomeostasis due to impaired or altered function of α-syn contributes to the visual symptoms associated with PD.

The prion-ZIP connection: From cousins to partners in iron uptake.

Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrP(C)), a plasma membrane glycoprotein, from α-helical to a ß-sheet rich PrP-scrapie (PrP(Sc)) isoform. Biochemical evidence indicates that PrP(C) facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrP(C) and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrP(C) with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrP(C) and ZIP14 suggest that PrP(C) functions as a FR partner for certain members of this family. The connection between PrP(C) and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrP(C) in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1.

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts.

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p = 0.003) and 54% (p = 0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p = 0.001), MYOG (94%, p = 0.000), and MYF5 (36%, p = 0.000), cell cycle regulator p21 (62%, p = 0.000), MSTN receptor ACVR2B (23%, p = 0.061), MSTN antagonist follistatin (81%, p = 0.000), and downstream signaling mediators SMAD2 (20%, p = 0.060) and SMAD3 (49%, p = 0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p = 0.354) and 79% compared to untransduced myoblasts (p = 0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p = 0.000)- and sh2 (1.71-fold; p = 0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.

Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson's disease.

SIGNIFICANCE: Intracellular and extracellular aggregation of a specific protein or protein fragments is the principal pathological event in several neurodegenerative conditions. We describe two such conditions: sporadic Creutzfeldt-Jakob disease (sCJD), a rare but potentially infectious and invariably fatal human prion disorder, and Parkinson's disease (PD), a common neurodegenerative condition second only to Alzheimer's disease in prevalence. In sCJD, a cell surface glycoprotein known as the prion protein (PrP(C)) undergoes a conformational change to PrP-scrapie, a pathogenic and infectious isoform that accumulates in the brain parenchyma as insoluble aggregates. In PD, α-synuclein, a cytosolic protein, forms insoluble aggregates that accumulate in neurons of the substantia nigra and cause neurotoxicity. RECENT ADVANCES: Although distinct processes are involved in the pathogenesis of sCJD and PD, both share brain iron dyshomeostasis as a common associated feature that is reflected in the cerebrospinal fluid in a disease-specific manner. CRITICAL ISSUES: Since PrP(C) and α-synuclein play a significant role in maintaining cellular iron homeostasis, it is important to understand whether the aggregation of these proteins and iron dyshomeostasis are causally related. Here, we discuss recent information on the normal function of PrP(C) and α-synuclein in cellular iron metabolism and the cellular and biochemical processes that contribute to iron imbalance in sCJD and PD. FUTURE DIRECTIONS: Improved understanding of the relationship between brain iron imbalance and protein aggregation is likely to help in the development of therapeutic strategies that can restore brain iron homeostasis and mitigate neurotoxicity.

Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq.

Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. In this study, we performed whole transcriptome profiling of adult caprine skeletal muscle derived myoblast and fused myotubes. Using Ion Torrent PGM sequencing platform, a total of 948,776 and 799,976 reads were generated in myoblasts and fused myotubes, respectively. The sequence reads were analyzed on CLC Genomics Workbench using Bos taurus RNA database to study the gene expression in both stages to study different genes responsible for muscle development and regeneration. The up and down-regulated genes were analyzed for gene ontology (GO) and KEGG pathways by Database for Annotation, Visualization and Integrated Discovery (DAVID) database. We found many genes exclusive to multinuclear fused myotubes and contractile nature of skeletal muscle, whereas up-regulated genes in myoblast stage were related to cell division and transcriptional regulation. Out of 27 genes selected for expression validation by RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), 19 genes showed the expression pattern comparable with CLC Genomics Workbench findings. Further, mRNA originated muscle specific microRNAs (miRNA-1 and miRNA-133b) were also observed in the fused myotubes along with other miRNAs with possible importance in muscle development. This study highlights important genes responsible for muscle development and differentiation in adult skeletal muscle system.

Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

H19 gene methylation study in Indian buffalo (Bubalus bubalis).

The methylation of DNA at cytosine residues within CpG dinucleotides is associated with transcriptional repression and is implicated in maintaining genomic stability and also the silencing of repetitive elements. These imprinted genes are unique as they are expressed exclusively from one parental allele. The present study was carried out to detect methylation status in H19 gene promoter CTCF III region in three Indian buffalo breeds (Jaffarabadi, Surti and Mehsani) by bisulfite sequencing. Methylation percent in Jaffarabadi, Surti and Mehsani buffaloes were found to be 50.19, 70.85 and 52.24, respectively, with mean incidence of methylation percent in H19 in all three breeds as 57.36. Apart from CpG methylation, unexpected nucleotide conversion (T>C, A>G, G>A) and deletion (A and G) after bisulfite sequencing were also observed. We observed no significant relationships in milk yield and milk fat per cent with methylation pattern in H19 gene in any of the three breeds.

Short hairpin RNA-induced myostatin gene silencing in caprine myoblast cells in vitro.

Myostatin (MSTN) belongs to the transforming growth factor (TGF)-ß superfamily and is a potent negative regulator of skeletal muscle development and growth. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the remarkable muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. We utilized three antisense RNA expressing vectors with six constructs to knockdown MSTN gene in in vitro caprine myoblast cell culture system. We observed that all six shRNA constructs were successful in MSTN silencing with efficiency ranging from 7 to 46 % by quantitative real-time PCR and up to 19 % by western blotting. The significant upregulation of interferon response gene OAS1 (5- to 11-fold) in cells transfected with shRNA constructs were indicative of induction of interferon response. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for improving the production traits and economic properties of livestock.

In vitro silencing of myostatin gene by shRNAs in chicken embryonic myoblast cells.

RNA interference represents one of the potential mechanisms of regulation of gene expression. Selective downregulation of myostatin (MSTN), a member of transforming growth factor-ß (TGF-ß) superfamily and a negative regulator of myogenesis, has been demonstrated to enhance skeletal muscle growth. In this study, we studied short hairpin RNA (shRNA)-induced myostatin gene silencing in chicken embryonic myoblast cells using seven different shRNA-expressing constructs by reverse transcription-quantitative real time PCR (RT-qPCR). Myostatin-silencing efficiency of all shRNA constructs were first evaluated in human embryonic kidney cell line 293T (HEK293T) cells, where we observed 30-75.6% reduction in myostatin expression, followed by chicken embryo myoblast cells that revealed up to 55% reduction in myostatin expression along with upregulation of MyoD by 4.65-folds. Consistent with the earlier observations, the transfection of cells with plasmids led to significant increase in interferon responsive genes OAS1 and IFN ß (2-112-folds), independent of myostatin silencing in both HEK293T and chicken embryonic myoblast cells. Our study suggests that apart from shRNA sequences, cell type-specific factors may play a significant role in determining the knockdown efficiency of shRNAs.