Development and validation of multiplex-PCR assay to simultaneously detect favourable alleles of shrunken2, opaque2, crtRB1 and lcyE genes in marker-assisted selection for maize biofortification

Journal of Plant Biochemistry and Biotechnology - Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A,...     

Hepatic neovascularization after partial portal vein ligation: novel mechanism of chronic regulation of blood flow

The present study was undertaken to investigate hepatic microcirculatory response following partial portal vein ligation (PPVL) in rats. Portal pressure was markedly increased 2-6 wk after PPVL, but no significant reduction in sinusoidal perfusion and hepatocellular injury were detected. However, marked neovascularization was observed in PPVL rats using intravital microscopy and scanning electron microscopy (SEM). Extremely high red blood cell velocity (2,000-4,900 microm/s) was seen in these vessels. Injection of fluorescein sodium via the carotid artery revealed that the neovessels originated from the hepatic arterial vasculature. This was further confirmed by clamping the common hepatic artery and phenylephrine injection from the carotid artery. These vessels maintained sufficient flow after massive sinusoidal shutdown elicited by the portal infusion of endothelin receptor B agonist IRL-1620. SEM also showed extensive neovascularization at the hilum. Additionally, clamping the portal vein decreased sinusoidal perfusion only by 9.5% in PPVL, whereas a 71.2% decrease was observed in sham. These results strongly suggest that the liver maintains its microcirculatory flow by vascular remodeling from the hepatic arterial vasculature following PPVL.     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.     

Microsatellite-based genetic diversity analyses of <Emphasis Type="Italic">sugary1</Emphasis>-, <Emphasis Type="Italic">shrunken2</Emphasis>- and double mutant- sweet corn inbreds for their utilization in breeding programme

Sweet corn has recently experienced sharp rise in demand worldwide. Recessive sugary1 (su1) and shrunken2 (sh2) that enhances kernel sweetness have been abundantly used in sweet corn breeding. Analyses of genetic diversity among sweet corn inbreds assume great significance for their effective utilization in hybrid breeding. A set of 48 diverse sweet corn genotypes encompassing su1su1, sh2sh2 and su1su1/sh2sh2 types were analyzed using 56 microsatellite markers. A total of 213 alleles with mean of 3.8 alleles per locus were generated. Two unique- and 12 rare- alleles were identified. The average PIC and genetic dissimilarity was 0.50 and 0.73, respectively. Cluster analysis grouped the inbreds into three major clusters, with each of the su1su1-, sh2sh2- and su1su1/sh2sh2-types were broadly clustered together. Principal coordinate analyses also depicted the diverse origin of the genotypes. The study identified inbreds for synthesis of pools and pedigree populations to develop novel inbreds. The study led to the identification of prospective heterotic combinations in various genetic backgrounds (sh2sh2 × sh2sh2, su1su1 × su1su1, su1su1/sh2sh2 × su1su1/sh2sh2, sh2sh2 × su1su1/sh2sh2 and su1su1 × su1su1/sh2sh2).     

Role of thromboxane A2 in early BDL-induced portal hypertension

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A(2) (TXA(2)) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA(2) in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B(2) (TXB(2)), the stable metabolite of TXA(2). Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB(2) in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 +/- 803 vs. 10,210 +/- 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB(2) release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA(2) release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA(2) in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA(2), which is associated with upregulation of the COX-2 enzyme.