An efficient and universal Agrobacterium-mediated transformation protocol in rice

For development of transgenic varieties of interest in rice, we have developed a simple, efficient and universal Agrobacterium mediated transformation protocol. Mature seeds of two indica (IR64 and Jaya), one each from japonica (AC41039) and aromatic (Basmati370) varieties were used as explants in the present study. Agrobacterium tumefaciens strain EHA 105 carrying Ti plasmid (pBI121) with the selectable marker npt ^II along with the reporter gene uidA encoding ??-glucuronidase (GUS) was successfully integrated with rice genome without use of acetosyringone. Sterile distilled water washing in place of cefotaxime in the elimination process has been used to control excess growth of Agrobacterium. All the material after transformation germinated in 2 to 3?days of co-cultivation on MS basal medium. Germinated seeds transferred to the selection medium i.e. plain MS medium with 50?mg/l of kanamycin, produced two to three primary tillers within 2?weeks. Mesocotyls from 2?week old in vitro grown plants were taken and cultured in the multiplication medium (MS supplemented with 0.5?mg/l BA and 50?mg/l Kanamycin) where within 6?C8?days they produced 3?C4 secondary tillers. All the five to six tiller shoots so produced in the process developed roots on plain MS and grew well when transferred to pots containing autoclaved soil and vermicompost in the proportion of 4:1. Transient expression of GUS was observed in all the tissues of recipient plants. Integration of the transgene was confirmed by employing southern blotting and real-time PCR technique. The transformation protocol developed can be efficiently used across the two major subspecies/ecotypes of the Asian rice cultivar Oryza sativa.     

On the relevance of using laboratory selection to study the adaptive value of circadian clocks

The phrase ‘as sure as night comes after day’ highlights the most conspicuous phenomenon on earth (i.e. the daily cycling of geophysical factors). It is hypothesized that life‐forms on earth have evolved timekeeping mechanisms (circadian clocks) as adaptations to cope with such cyclic variations in their environment and, to test this hypothesis, diverse strategies have been employed. In this review, these different approaches, including comparative, clinal, ecology and/or trait manipulation and laboratory selection strategies are discussed, aiming to evaluate the adaptive value of circadian clocks. The limitations of each of these methods are assessed, and it is suggested that laboratory selection is an ideal, potent and suitable strategy for examining whether circadian clocks are indeed adaptations. In support of this, laboratory selection strategies are highlighted and critically reviewed in a discussion of studies that demonstrate the evolution of circadian clocks and life‐history traits in response to selection for the timing of rhythmic behaviours, as well as those studies that demonstrate the evolution of circadian clocks in response to selection for life‐history traits. Finally, newer approaches are proposed that involve the use of mutants, simultaneous manipulation of multiple environmental factors and genomic technologies in conjunction with laboratory selection to further explore the adaptive significance of circadian clocks.     

Change in dysphagia and laryngeal function after radical radiotherapy in laryngo pharyngeal malignancies — a prospective observational study

BackgroundIntensity modulated radiotherapy (IMRT) has the perceived advantage of function preservation by reduction of toxicities in the treatment of laryngo-pharyngeal malignancies. The aim of the study was to assess changes in dysphagia from baseline (i.e. prior to start of treatment) at three and six months post treatment in patients with laryngo-pharyngeal malignancies treated with radical radiotherapy ± chemotherapy. Functional assessment of other structures involved in swallowing was also studied.Materials and methods40 patients were sampled consecutively. 33 were available for final analysis. Dysphagia, laryngeal edema, xerostomia and voice of patients were assessed at baseline and at three and six months after treatment. Radiation was delivered with simultaneous integrated boost (SIB) using volumetric modulated radiation therapy (VMAT). Concurrent chemotherapy was three weekly cisplatin 100 mg/m².ResultsProportion of patients with dysphagia rose significantly from 45.5% before the start of treatment to 57.6% at three months and 60.6% at six months post treatment (p = 0.019). 67% patients received chemotherapy and addition of chemotherapy had a significant correlation with dysphagia (p = 0.05, r = −0.336). Severity of dysphagia at three and six months correlated significantly with the mean dose received by the superior constrictors (p = 0.003, r = 0.508 and p = 0.024, r = 0.391) and oral cavity (p = 0.001, r = 0.558 and p = 0.003, r = 0.501). There was a significant worsening in laryngeal edema at three and six months post treatment (p < 0.01) when compared to the pre-treatment examination findings with 60.6% of patients having grade two edema at six months. Significant fall in the mean spoken fundamental frequency from baseline was seen at 6 months (p = 0.04), mean fall was 21.3 Hz (95% CI: 1.5–41 Hz) with significant increase in roughness of voice post treatment (p = 0.01).ConclusionThere was progressive worsening in dysphagia, laryngeal edema and voice in laryngo-pharyngeal malignancies post radical radiotherapy ± chemotherapy.     

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.     

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.     

Non-Invasive Clinical Parameters for the Prediction of Urodynamic Bladder Outlet Obstruction: Analysis Using Causal Bayesian Networks

Purpose

To identify non-invasive clinical parameters to predict urodynamic bladder outlet obstruction (BOO) in patients with benign prostatic hyperplasia (BPH) using causal Bayesian networks (CBN).

Subjects and Methods

From October 2004 to August 2013, 1,381 eligible BPH patients with complete data were selected for analysis. The following clinical variables were considered: age, total prostate volume (TPV), transition zone volume (TZV), prostate specific antigen (PSA), maximum flow rate (Qmax), and post-void residual volume (PVR) on uroflowmetry, and International Prostate Symptom Score (IPSS). Among these variables, the independent predictors of BOO were selected using the CBN model. The predictive performance of the CBN model using the selected variables was verified through a logistic regression (LR) model with the same dataset.

Results

Mean age, TPV, and IPSS were 6.2 (±7.3, SD) years, 48.5 (±25.9) ml, and 17.9 (±7.9), respectively. The mean BOO index was 35.1 (±25.2) and 477 patients (34.5%) had urodynamic BOO (BOO index ≥40). By using the CBN model, we identified TPV, Qmax, and PVR as independent predictors of BOO. With these three variables, the BOO prediction accuracy was 73.5%. The LR model showed a similar accuracy (77.0%). However, the area under the receiver operating characteristic curve of the CBN model was statistically smaller than that of the LR model (0.772 vs. 0.798, p = 0.020).

Conclusions

Our study demonstrated that TPV, Qmax, and PVR are independent predictors of urodynamic BOO.     

Breeding lines of the Indian mega-rice variety,MTU 1010, possessing protein kinase <Emphasis Type="Italic">OsPSTOL</Emphasis> (<Emphasis Type="Italic">Pup1</Emphasis>), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F₁ and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC₂F₁, the best backcross plant was selfed to generate BC₂F₂s. Among them, the plants homozygous for Pup1 (n?=?22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC₂F₆ generation. A total of five elite BC₂F₆ lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (>?390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.     

Improvement of blast resistance of the popular high-yielding,medium slender-grain type,bacterial blight resistant rice variety,Improved Samba Mahsuri by marker-assisted breeding

Improved Samba Mahsuri (ISM) is a popular, high-yielding, bacterial blight resistant rice variety possessing medium-slender grain type. As ISM is highly susceptible to blast disease of rice, through the present study we have transferred two major blast resistance genes, Pi2 and Pi54 into the elite variety by marker-assisted backcross breeding. The two blast resistance genes were transferred to ISM through sets of backcrosses. In every backcross generation, PCR-based markers, specific for the blast resistance genes (Pi2 and Pi54) and bacterial blight resistance genes (Xa21, xa13 and xa5) were utilized for foreground selection, while a set of 144 parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC₂ generation. A solitary BC₂F₁ plant possessing Pi2 or Pi54 along with Xa21, xa13 and xa5 and >?90% recovery of ISM genome was selected from the two sets of backcrosses were crossed and the intercross F₁s (ICF₁s) thus obtained were selfed to generate ICF₂s. Homozygous ICF₂ plants carrying all the five resistance genes were identified through markers and advanced through selfing till ICF₅ generation by adopting pedigree method of selection. Three best lines at ICF₅, possessing excellent resistance against bacterial blight and blast and closely resembling or superior to ISM in terms of grain quality: yield and agro-morphological traits have been identified and advanced for multi-location trials.     

Identification of function for CD44 intracytoplasmic domain (CD44-ICD): modulation of matrix metalloproteinase 9 (MMP-9) transcription via novel promoter response element

CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.