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.     

Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc⁺ (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.     

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.     

Co-administration of APD668, a G protein-coupled receptor 119 agonist and linagliptin,a DPPIV inhibitor,prevents progression of steatohepatitis in mice fed on a high trans-fat diet

Non-Alcoholic SteatoHepatitis (NASH) is the more severe form of Non-Alcoholic Fatty Liver Disease (NAFLD) and is characterized by the presence of hepatic steatosis, oxidative stress, inflammation, hepatocyte injury with or without fibrosis. Recently, GPR119 receptor has emerged as a novel therapeutic target for the treatment of dyslipidemia and non-alcoholic steatohepatitis. In the present study, we investigated the effect of APD668, a GPR119 agonist alone or in combination with linagliptin, a DPPIV inhibitor on the progression of steatohepatitis in mice fed on a high trans-fat diet. In this study, monotherapy with either APD668 or linagliptin caused a reduction in the levels of ALT, AST, glucose, cholesterol and epididymal fat mass but the effect was more pronounced upon treatment with combination of both drugs.On the other hand, combined treatment of APD668 with linagliptin demonstrated a non-significant additive effect in reduction of hepatic triglyceride (?78%) and cholesterol (?56%) compared to monotherapy groups. Moreover, co-administration of APD668 and linagliptin resulted in enhanced levels of active GLP-1 with additional benefit of significant synergistic decrease in body weight gain (?19%) in mice. We speculated that the enhanced effect observed with the combination treatment could be due to either 1) direct activation of GPR119 receptors present in liver and intestine or 2) enhanced active GLP-1 levels or 3) decreased degradation of GLP-1 in-vivo through DPPIV inhibition. Therefore, these findings clearly suggest that GPR119 receptor agonists in combination with DPPIV inhibitors may represent a promising therapeutic strategy for the treatment of non-alcoholic steatohepatitis.     

Lafora disease: from genotype to phenotype

The progressive myoclonic epilepsy of Lafora or Lafora disease (LD) is a neurodegenerative disorder characterized by recurrent seizures and cognitive deficits. With typical onset in the late childhood or early adolescence, the patients show progressive worsening of the disease symptoms, leading to death in about 10 years. It is an autosomal recessive disorder caused by the loss-of-function mutations in the EPM2A gene, coding for a protein phosphatase (laforin) or the NHLRC1 gene coding for an E3 ubiquitin ligase (malin). LD is characterized by the presence of abnormally branched water insoluble glycogen inclusions known as Lafora bodies in the neurons and other tissues, suggesting a role for laforin and malin in glycogen metabolic pathways. Mouse models of LD, developed by targeted disruption of the Epm2a or Nhlrc1 gene, recapitulated most of the symptoms and pathological features as seen in humans, and have offered insight into the pathomechanisms. Besides the formation of Lafora bodies in the neurons in the presymptomatic stage, the animal models have also demonstrated perturbations in the proteolytic pathways, such as ubiquitin-proteasome system and autophagy, and inflammatory response. This review attempts to provide a comprehensive coverage on the genetic defects leading to the LD in humans, on the functional properties of the laforin and malin proteins, and on how defects in any one of these two proteins result in a clinically similar phenotype. We also discuss the disease pathologies as revealed by the studies on the animal models and, finally, on the progress with therapeutic attempts albeit in the animal models.     

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora,Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al‐Adaid Lagoon in Qatar

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al‐Adaid Lagoon in Qatar were investigated based on live observation as well as protargol‐ and silver nitrate‐impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50–65 × 33–40 μm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral‐transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans–Euplotes nobilii–Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.     

Cerebrotendinous xanthomatosis: a case report

BACKGROUND: Cerebrotendinous xanthomatosis is a rare, autosomal recessive, inherited lipid storage disease characterized by accumulation of cholestanol and cholesterol in most tissues. The disease is caused by mutations in the sterol 27-hydroxylase gene, leading to a block in bile synthesis, with accumulation of substrates for this enzyme, including cholesterol, resulting in an increase in the conversion of cholesterol to cholestanol. CASE: A 26-year-old woman presented with gradually increasing bilateral ankle swelling. She had a history of bilateral cataracts and left-sided hemiparesis. She had mental retardation, with a history of delayed milestone development. Her serum cholesterol levels were elevated. Aspiration of both ankle swellings revealed histiocytes and many foreign body giant cells. There were numerous rectangular to rhomboid crystals in the background. CONCLUSION: Very few articles are available on the cytologic features of tendinous xanthomas; hence we tried to highlight these features.     

Prenatal onset of Axonopathy in Dystonia musculorum mice

Dystonia musculorum (dt) is a recessive hereditary neuropathy of the mouse. Affected animals display loss of limb coordination and twisting of the trunk. Sensory nerve fibers of these mice are severely reduced in number, and the remaining fibers present numerous axonal swellings. The gene defective in dt, dystonin (Dst), encodes a cytoskeletal linker protein that forms the bridge between F-actin and intermediate filaments. Dst is expressed during embryogenesis, whereas overt phenotype in dt mice only appears during the second week after birth. Here we show that axonal swellings are present in sensory nerve fibers of dt embryos as early as E15.5, before myelination and radial axonal growth have begun. Thus disease progression is gradual in dt mice, having begun during embryogenesis. In dt embryos, microtubule network disorganization and cytoplasmic organelle accumulation within axonal swellings were consistently observed. In addition, a few of the axonal swellings presented intermediate filament accumulation. These results demonstrate that dystonin is required for cytoskeleton organization during axonogenesis. They also suggest that axonal transport defects, through microtubule network perturbation, may be the primary mechanism of neurodegeneration in dt mice. Dev. Genet. 22:160–168, 1998. © 1998 Wiley-Liss, Inc.     

Efficient and Stable Tin–Lead Perovskite Photoconversion Devices Using Dual-Functional Cathode Interlayer

Tin–lead halide perovskites (TLHPs) are promising photoactive materials for photovoltaics (PVs) due to reduced toxicity and broad light absorption. However, their inherent ionic vacancies facilitate inward metal diffusion, accelerating device degradation. Here, efficient, stable TLHP-based PV and photoelectrochemical (PEC) devices are reported containing a chemically protective cathode interlayer—amine-functionalized perylene diimide (PDINN). Solution-processed PDINN effectively extract electrons and suppress inward-metal diffusion by forming tridentate metal complexes with its nucleophilic sites. The PV device achieved an efficiency of 23.21% (>81% retention after 750 h at 60 °C and >90% retention after 3100 h at 23 ± 4 °C), and the first demonstration of TLHP-based PEC devices exhibit a record-high bias-free solar hydrogen production rate (33.0 mA cm⁻²; ≈3.42 × 10⁻⁶ kg s⁻¹ m⁻²) when coupled with biomass oxidation, which is ≈1.7-fold higher than the ultimate target set by the U.S. Department of Energy for one-sun hydrogen production. These findings demonstrate the potential of TLHPs for efficient, stable photoconversion by the molecular design of the cathode interlayer.