GL261 glioma tumor cells respond to ATP with an intracellular calcium rise and glutamate release

Necrotizing enterocolitis (NEC) is one of the most severe and unpredictable complications of prematurity. There are two possible mechanisms involved in the pathogenesis of NEC: individual inflammatory response and impaired blood flow in mesenteric vessels with secondary ischemia of the intestine. The aim of this study was to evaluate the possible relationship between polymorphisms: Il-1β 3953C>T, Il-6 ?174G>C and ?596G>A, TNFα ?308G>A, and 86 bp variable number tandem repeat polymorphism of interleukin-1 receptor antagonist (Il-1RN VNTR 86 bp) and three polymorphisms that may participate in arteries tension regulation and in consequence in intestine blood flow impairment: eNOS (894G>T and ?786T>C) and END-1 (5665G>T) and NEC in 100 infants born from singleton pregnancy, before 32 + 0 weeks of gestation, exposed to antenatal steroids therapy, and without congenital abnormalities. In study population, 22 (22%) newborns developed NEC. Surgery-requiring NEC was present in 7 children. Statistical analysis showed 20-fold higher prevalence of NEC in infants with the genotype TT [OR 20 (3.71–208.7); p = 0.0004] of eNOS 894G>T gene polymorphism. There was a higher prevalence of allele C carriers of eNOS 786T>C in patients with surgery-requiring NEC [OR 4.881 (1.33–21.99); p = 0.013]. Our investigation did not confirm any significant prevalence for NEC development in another studied genotypes/alleles. This study confirms the significant role of polymorphisms that play role in intestine blood flow. Identifying gene variants that increase the risk for NEC development may be useful in screening infants with inherent vulnerability and creating strategies for individualized care.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

White Lupin (Lupinus albus) response to phosphorus stress: evidence for complex regulation of LaSAP1

Proteoid roots are a unique adaptation that allow white lupin (Lupinus albus L. var Ultra) to survive under extreme phosphorus (P) deficient conditions. The cascade of events that signals P-deficiency induced gene expression in proteoid roots remains unknown. Through promoter::GUS analysis we showed that expression of acid phosphatase (LaSAP1) in P-deficient proteoid roots depends on DNA located from ?465 bp to ?345 bp 5′ of the ATG start codon and that the P1BS (PHR1 Binding Site) element, located at ?160 bp, also contributes regulatory control. DNA located within the ?414 bp to ?250 bp region of the LaSAP1 promoter was bound by nuclear proteins isolated from P-sufficient normal roots in electrophoretic mobility shift assays (EMSA), suggesting negative regulation. Competition experiments were performed with unlabeled oligonucleotides to further delineate the region of the LaSAP1 promoter bound by P-sufficient normal root nuclear proteins to a motif spanning ?361 bp to ?346 bp. The promoter motif characterized through EMSA spanning ?361 bp to ?345 bp was used as “bait” in a yeast one-hybrid (Y1H) experiment and 31 putative DNA binding proteins were isolated. Taken together, our results increase understanding of P-deficiency signaling by identifying regulatory regions and putative regulatory proteins for LaSAP1 expression.     

Endothelial nitric oxide synthase gene polymorphisms and the risk of acute myocardial infarction in a South Indian population

Myocardial infarction (MI) is a complex multi-factorial, polygenic disorder which results from an interaction between a person’s genetic makeup and various environmental factors. Nitric oxide (NO), a potent vasodilator produced by endothelial cells, plays an important role in the regulation of blood pressure, regional blood flow and also inhibits platelet aggregation, vascular smooth muscle cell proliferation and leukocyte adhesion to vascular endothelium. Our aim was to analyze the association of NOS3 (endothelial nitric oxide synthase 3) 894G>T and ?786T>C gene polymorphisms and MI risk in the South Indian population. A total of 287 MI patients, 279 risk control patients and 321 healthy controls were recruited for the retrospective study. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was no significant association observed between NOS3 894G>T, ?786T>C polymorphisms and MI. A significant difference was observed in the distribution of GT genotype of the NOS3 894G>T polymorphism between the cases and the risk controls (p = 0.05) but the odds ratio (0.6) did not show risk for MI. The present study showed lack of association between NOS3 gene polymorphisms and MI in South Indian population.     

A Point Mutation in <Emphasis Type="Italic">SCN1A</Emphasis> 5′ Genomic Region Decreases the Promoter Activity and Is Associated with Mild Epilepsy and Seizure Aggravation Induced by Antiepileptic Drug

The SCN1A gene with 1274 point mutations in the coding regions or genomic rearrangements is the most clinically relevant epilepsy gene. Recent studies have demonstrated that variations in the noncoding regions are potentially associated with epilepsies, but no distinct mutation has been reported. We sequenced the 5′ upstream region of SCN1A in 166 patients with epilepsy and febrile seizures who were negative for point mutations in the coding regions or genomic rearrangements. A heterozygous mutation h1u-1962 T?>?G was identified in a patient with partial epilepsy and febrile seizures, which was aggravated by oxcarbazepine. This mutation was transmitted from the patient’s asymptomatic mother and not found in the 110 normal controls. h1u-1962 T?>?G was located upstream the most frequently used noncoding exon and within the promoter sequences. Further experiments showed that this mutation decreased the promoter activity by 42.1 % compared with that of the paired haplotype (P?<?0.001). In contrast to the null expression that results in haploinsufficiency and severe phenotype, this mutation caused relatively less impairment, explaining the mild epilepsy with incomplete penetrance. The antiepileptic drug-induced seizure aggravation in this patient suggests clinical attention for mutations or variations in noncoding regions that may affect SCN1A expression.     

Next Generation Semiconductor Based-Sequencing of a Nutrigenetics Target Gene (GPR120) and Association with Growth Rate in Italian Large White Pigs

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3′-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.     

Molecular characterization of differentially expressed TXNIP gene and its association with porcine carcass traits

Thioredoxin interacting protein (TXNIP), which plays a regulatory role in lipid metabolism and immune regulation, is down-regulated expressed in F₁ hybrids Landrace?×?Yorkshire skeletal muscle. Here we described the molecular characterization of porcine TXNIP gene. The full-length cDNA contains a coding sequence of 1,176?bp nucleotides with untranslated regions of 263?bp at 5′-end and 441?bp at 3′-end, respectively. The predicted molecular mass and isoelectric point of porcine TXNIP is 43.81?kDa and 7.385, respectively. The deduced 391 amino acids exhibit high identity with other mammalian TXNIP. The TXNIP gene contains eight coding exons and seven non coding introns, spans approximately 3,348?bp. The expression of porcine TXNIP mRNA is almost absent in Landrace?×?Yorkshire and lower level in 6-month-old pigs during skeletal muscle development. Other stages and breeds were high level expressed. Statistical analysis showed the TXNIP gene polymorphism (c.575-4T>C) was different between F₁ hybrids and their parents, was highly associated with dressing percentage (DP) and thorax–waist fat thickness (TFT) in the Yorkshire?×?Meishan F₂ population. The possible role of TXNIP was discussed.     

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.     

Analysis of the associations between polymorphisms in GNAS complex locus and growth, carcass and meat quality traits in pigs

Imprinted genes are interesting candidates for marker assisted selection in farm animals. One of them—GNAS complex locus is engaged in obesity pathogenesis in humans and mice. In our study, we identified new polymorphism in porcine GNAS gene (variable number of CT repeats, accession number: rs196952953) and found that this polymorphism is in linkage disequilibrum with GNAS AM490165:g.324C>T. Statistical analysis (GLM procedure), performed on 552 animals (Large White n = 258 and Landrace n = 269), revealed that deduced haplotypes and GNAS AM490165:g.324C>T are associated with growth performance and a few carcass traits, but not with feed intake. We observed significant additive effects of GNAS AM490165:g.324C>T genotype and haplotype 2 (C/278 bp) on test daily gain (TDG), average daily gain (ADG), number of days on test, age of the slaughter (P < 0.01) and FCR ratio (P < 0.05). Animals with two copies of C/278 haplotype had significantly higher: TDG, ADG, lower feed:gain ratio and faster reached the weight of 100 kg. When carcass traits were considered, significant associations between GNAS AM490165:g.324C>T polymorphism, haplotype 2 (C/278) and weight of ham with and without backfat and skin (WH) (WH2), length of the carcass, height and the width of the loin, meat percentage, weight of the main cuts were identified. The significant dominance effects of GNAS AM490165:g.324C>T polymorphism and haplotype 2 on WH and WH2 were observed (P < 0.05). When the two breeds were analyzed separately significant associations were observed for most of the traits in Landrace while in Large White the same trends were present but the differences were mostly not significant. Among meat quality traits we found significant association between haplotype and IMF content in Landrace (P < 0.03). Our results show for the first time that GNAS complex locus may modulate economically important traits in pigs.     

Comparative Assessment of 5′ A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity

Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5′ A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5′ flaps. Results indicate that even if the performance of some primers can be improved substantially by 5′ flap addition, not all primers will be similarly improved. Optimal 5′ flap sequences are dependent on the primer sequences, and alter the primer’s T _m value. The manipulation of this feature may enhance primer’s efficiency to increase the PCR sensitivity and DNA yield.     

Investigation of four candidate genes (IGF2, JHDM1A, COPB1 and TEF1) for growth rate and backfat thickness traits on SSC2q in Large White pigs

As important quantitative traits, the growth rate and backfat thickness are controlled by multiple genes. The aim of this investigation was to evaluate the effect of the single and multiple SNPs of four candidate genes (IGF2, JHDM1A, COPB1 and TEF-1) on growth rate and backfat thickness. The four candidate genes were mapped on the p arm of SSC 2, and there are several QTLs, such as average daily gain, backfat thickness, an imprinted QTLs affecting muscle mass and fat deposition have been reported in this region. The polymorphisms of these genes were detected using PCR–RFLP methods, mixed procedure was used to analyze the single marker association with the growth and backfat thickness traits, and the gene–gene combination was investigated using multiple-markers analysis. The single marker association analysis indicated that the IGF2 intron-3 g.3072G > A and the substitution g.93G > A of TEF-1 gene were significantly associated with the age at 100 kg (P < 0.05). The JHDM1A 3′UTR g.224C > G, the c.3096C > T polymorphism of COPB1 gene and the substitution g.93G > A of TEF-1 gene were all significantly associated with the backfat at the shoulder (P < 0.05), backfat at the last rib, backfat at the lumbar, and the average backfat thickness, respectively. The multiple-markers analysis indicated that IGF2 and TEF-1 integrated gene networks for the age at 100 kg. Therefore, we can suggest that the polymorphism of IGF2 and TEF-1 gene could be used in marker-assisted selection for the age at 100 kg in Large White pigs.     

A family 5 β-mannanase from the thermophilic fungus Thielavia arenaria XZ7 with typical thermophilic enzyme features

A novel β-mannanase gene, man5XZ7, was cloned from thermophilic fungus Thielavia arenaria XZ7, and successfully expressed in Pichia pastoris. The gene (1,110 bp) encodes a 369-amino acid polypeptide with a molecular mass of approximately 40.8 kDa. The deduced sequence of Man5XZ7 consists of a putative 17-residue signal peptide and a catalytic module belonging to glycoside hydrolase (GH) family 5, and displays 76 % identity with the experimentally verified GH 5 endo-β-1,4-mannanase from Podospora anserina. Recombinant Man5XZ7 was optimally active at 75 °C and pH?5.0 and exhibited high activity at a wide temperature range (>50.0 % activity at 50–85 °C). Moreover, it had good adaptability to acidic to basic pH (>74.1 % activity at pH?4.0–7.0 and 25.6 % even at pH?9.0) and good stability from pH?3.0 to 10.0. These enzymatic properties showed that Man5XZ7 was a new thermophilic and alkali-tolerant β-mannanase. Further amino acid composition analysis indicated that Man5XZ7 has several characteristic features of thermophilic enzymes.     

Standardization of seed storage conditions in chilgoza pine (Pinus gerardiana Wall.): an endangered pine of Hind Kush Himalaya

The seeds of chilgoza pine (Pinus gerardiana) show moderate germination and not retain better germinability under normal ambient storage. In the present study, five storage containers [polythene bags (C₁), plastic jars (C₂), canvas bags (C₃), earthen pots (C₄) and tin boxes (C₅)] and four temperature regimes (19–22 °C) (room temperature, T₁), 0 ± 1 °C (T₂), ?4 ± 1 °C (T₃) and 4 ± 1 °C (T₄) were tested and the suitable seed storage conditions of chilgoza pine to retain viability were standardized. The storage devices preserve and retain viability trend of C₄ > C₃ > C₁ > C₂ > C₅ and T₂ > T₃ > T₄ > T₁ in the species throughout the storage period. However, the interaction treatment (C₄T₂) of earthen pots under 0 ± 1 °C temperature regime maintained significantly (P < 0.05) maximum germinability after 9 months (58.3 %) followed by C₃T₂ and C₄T₃ (47.5 %) as compared to other storage conditions. Notably, a sharp decline in germinability was recorded in seeds stored in tin boxes placed at room temperature. The implementation of these results for conservation management, especially nursery development and sustainable utilization of P. gerardiana in Himalayan region, has been suggested.