Understanding the nature of blast resistance in combined bacterial leaf blight and blast gene pyramided lines of rice variety tellahamsa.

BACKGROUND: Rice blast and bacterial leaf blight (BLB) are the most limiting factors for rice production in the world which cause yield losses typically ranging from 20 to 30% and can be as high as 50% in some areas of Asia especially India under severe infection conditions. METHODS AND RESULTS: An improved line of Tellahamsa, TH-625-491 having two BLB resistance genes (xa13 and Xa21) and two blast resistance genes (Pi54 and Pi1) with 95% Tellahamsa genome was used in the present study. TH-625-491 was validated for all four target genes and was used for backcrossing with Tellahamsa. Seventeen IBC1F1 plants heterozygous for all four target genes, 19 IBC1F2 plants homozygous for four, three and two gene combinations and 19 IBC1F2:3 plants also homozygous for four, three and two gene combinations were observed. Among seventeen IBC1F1 plants, IBC1F1-62 plant recorded highest recurrent parent genome (97.5%) covering 75 polymorphic markers. Out of the total of 920 IBC1F2 plants screened, 19 homozygous plants were homozygous for four, three and two target genes along with bacterial blight resistance. Background analysis was done in all 19 homozygous IBC1F2 plants possessing BLB resistance (possessing xa13, Xa21, Pi54 and Pi1 in different combinations) with five parental polymorphic SSR markers. IBC1F2-62-515 recovered 98.5% recurrent parent genome. The four, three and two gene pyramided lines of Tellahamsa exhibited varying resistance to blast. CONCLUSIONS: Results show that there might be presence of antagonistic effect between bacterial blight and blast resistance genes since the lines with Pi54 and Pi1 combination are showing better resistance than the combinations with both bacterial blight and blast resistance genes.

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.

BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of ß-thalassemia and sickle cell disease using a next-generation sequencing platform.

ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.

Accelerated charge transfer in well-designed S-scheme Fe@TiO2/Boron carbon nitride heterostructures for high performance tetracycline removal and selective photo-reduction of CO2 greenhouse gas into CH4 fuel.

Designing and fabrication of smart hybrid multifunctional materials for energy/fuel production and environmental detoxification is indeed of great significance for sustainable development. Herein, we synthesized a new well-structured S-scheme heterostructure Fe@TiO2/Boron Carbon nitride (FT/BCN) with high performance tetracycline degradation and selective CO2 photo-reduction to CH4. Under visible light irradiation, 96.3% tetracycline was degraded in 60 min using best performing FT30/BCN sample with a high 83.2% total organic carbon removal in 2 h. The tetracycline degradation rate for FT30/BCN composite catalyst was ∼7 times than bare boron carbon nitride (BCN). The impact of reaction parameters as pH, presence of interfering electrolytes, light source and water matrix was also investigated. The FT30/BCN photocatalyst shows dramatic improvement in CO2 photoreduction as exhibited in 24.7 µmol g-1 h-1 CH4 and 2.4 µmol g-1 h-1 CO evolutions with optimal 91.1% CH4 selectivity. Pure BCN shows a poor 39.1% selectivity. Further, effect of alkali activation, CO2/H2O feed ratio, reducing agent and light source onto CH4 production and selectivity was also investigated. The CH4 evolution and selectivity was improved because of enhanced visible light absorption, high adsorption potential, charge carrier separation and high reducing power of photogenerated electrons induced by an effective S-scheme heterojunction between Fe@TiO2 and boron carbon nitride. An S-scheme (step-scheme) charge transfer mechanism is here operative both during tetracycline removal and CO2 reduction. The drug degradation route and photocatalytic mechanism for antibiotic removal and CO2 reduction was also predicted.

A Simple, Cost-Effective, and Extraction-Free Molecular Diagnostic Test for Sickle Cell Disease Using a Noninvasive Buccal Swab Specimen for a Limited-Resource Setting.

Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting.

Establishment and characterization of induced pluripotent stem cell line (IGIBi002-A) from a ß-thalassemia patient with IVS1-5 mutation by non-integrating reprogramming approach.

ß-thalassemia (BT) is a hereditary blood disorder caused by mutations in the ß-globin (HBB) gene leading to severely reduced or no synthesis of the ß-chain of adult hemoglobin. IVS1-5 (G > C) is the most common BT mutation in Indian population and yet no patient-specific cellular models have been generated. Here, we have established an induced pluripotent stem cell (iPSC) line, IGIBi002-A from a thalassemia patient with a homozygous IVS1-5(G > C) mutation. Characterization of IGIBi002-A demonstrated that these iPSCs are free of exogenous reprogramming genes and expressed pluripotent stem cell markers, exhibited a normal karyotype and were potential of three germ layer differentiation.

Fluorescence behavior of cysteine-mediated Ag@CdS nanocolloids.

Ag@CdS quantum dots were synthesized by a colloidal method. CdS nanoparticles stabilized by L-cysteine were doped with varying silver concentrations. Ag@CdS nanoparticles were characterized by UV-visible spectroscopy, steady-state and time-resolved fluorescence, X-ray diffraction, Fourier transform infrared resonance (FTIR), and transmission electron microscopy. The effect of dopant concentration and effect of aging of Ag@CdS colloids were studied. At low silver content, emission was found to enhance, while at high silver content, emission quenching was observed after aging. Ag-CdS nanocolloids exhibited multiexponential decay with tau2 being a long-lived component. X-ray diffraction spectra of as prepared samples indicated that silver occupies an interstitial position, while for annealed samples, Ag+ ions get oxidized to Ag2+ and substituted for Cd2+ in the CdS lattice. FTIR studies have shown that cysteine acts as a monodentate ligand for cadmium and silver for as-prepared sample, whereas it acts as a tridentate ligand for annealed samples toward silver. Transmission electron microscopy of as-prepared samples showed spherical-shaped composites with a uniform layer of capping molecules.

Structural phase behavior and vibrational spectroscopic studies of biofunctionalized CdS nanoparticles.

Biomodified CdS nanoparticles were synthesized using l-cysteine as a capping agent in the colloidal state as a function of pH. The role of pH on the size and structure of CdS nanoparticles was investigated in detail. At pH 7.4 and 9.1, X-ray diffraction spectra of as prepared samples showed the presence of a mixture of cubic and hexagonal phases while cubic phase was formed at pH 11.2. A gradual transition to the hexagonal phase was observed for refluxed samples at pH 9.1 and 11.2. Whereas, at pH 7.4, the sample remains in a mixture of cubic and hexagonal phase even after refluxing. The particle size of as prepared samples was about 2 nm, and for refluxed samples the size increased up to 10 nm. The binding of cadmium through thiol group is evidenced by infrared spectra. An intense band due to C-C-N vibration was observed after 24 h of reflux. The formation of a specific molecular cluster determines the growth of a particular phase. Transmission electron microscopy (TEM) studies support the X-ray diffraction (XRD) studies and exhibit well separated spherical particles while refluxed samples show clustering.

Antenatal 3-dimensional sonographic diagnosis of fetal oropharyngeal teratoma.

We report a case of a large fetal oropharyngeal teratoma in a 19-week fetus evaluated with 3-dimensional (3D) sonography. The 3D sonographic surface and maximum mode rendering of the tumor allowed detailed visualization of the mass lesion and thus enabled active patient participation in the management of the pregnancy.