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Handwashing with soap at critical moments is one of the most important factors in controlling the spread of germs and preventing the spread of infection. The objective of this study was to determine the effectiveness of a low-cost, school-based intervention that simulated germs and their spread on hand hygiene knowledge, attitude and practice of primary school children. Five hundred and sixty-two students from 28 rural schools were enrolled in this pre-posttest study. Endline data was collected 4 weeks after conducting the intervention at baseline. The mean scores for knowledge, attitude and practice improved significantly after the intervention (p <.05). Significant positive gains were also observed in children's understanding of germs, the associated illness threat and washing hands with soap as a prevention mechanism. The present study suggests that entertainment-education-based interventions have the potential to improve hygiene behavior among children while being low-cost. [ABSTRACT FROM AUTHOR]

In this paper, loss due to staggered-via in SIW (Substrate Integrated Waveguide) is analyzed with the help of S-parameter responses and field diagrams. It is observed that staggered-via improves the response of an SIW structure, with higher via pitch (w) to via diameter (d) ratio (w/d), but the loss increases drastically at certain discrete frequencies for which λ / 2 n equals to w, where λ is the guided wavelength corresponding to the frequency and n is any integer value excluding zero. This study will be helpful in choosing between the use of single row of vias or staggered-vias as a sidewall, while designing SIWs at higher frequencies, where it is difficult to use conventional w/d ratio values. [ABSTRACT FROM AUTHOR]

World Health Organization (WHO) reports that one-third of the world's population is infected with a persistent form of Mycobacterium tuberculosis (M.tb), the causative bacterium responsible for causing the dreaded tuberculosis disease. Targeting mycobacterial persisters is important for achieving WHO's End TB target. The de-novo cysteine biosynthetic pathway is a novel target for addressing M.tb persistence. The two-step pathway comprises of serine acetyltransferase/CysE and O-acetyl-serinesulfhydrylase/OASS/CysK. The present study is an attempt to understand the structural features of mycobacterial CysE by investigating the divergence amongst orthologous through phylogenetic analysis. Mapping of mycobacterial CysE sequences on the whole orthologous (COG1045) tree segregated the species into four clusters and several isoforms leading to their descendants identification. Interestingly the analysis revealed that the extended C-terminal a-helix believed unique to M.tb is also present in other organisms such as: Campylobacter ureolyticus, Bacillus cereus, Geminocystis herdmanii and Paenibacillus borealis. Further, the Hidden Markov model search against the whole Uniprot database suggests a plausible role of C-terminal a-helix of CysE in strengthening the substrate and/or co-factor binding. In addition, phylogenetic analysis of CysE sequences from the Mycobacteriaceae family facilitates grouping them under ten well-formed and six monophyletic clades, each based on characteristic features with respect to domain architecture, oligomeric assembly, C-terminal tetra-peptide tail, regulatory and feedback mechanism etc. Employing molecular phylogeny in conjunction with structural analysis has provided detailed insights for mycobacterial CysEs as drug target. [ABSTRACT FROM AUTHOR]

The antimicrobial resistant strains of several pathogens are major culprits of hospital-acquired nosocomial infections. An active and urgent action is necessary against these pathogens for the development of unique therapeutics. The cysteine biosynthetic pathway or genes (that are absent in humans) involved in the production of L-cysteine appear to be an attractive target for developing novel antibiotics. CysE, a Serine Acetyltransferase (SAT), catalyzes the first step of cysteine synthesis and is reported to be essential for the survival of persistence in several microbes including Mycobacterium tuberculosis. Structure determination provides fundamental insight into structure and function of protein and aid in drug design/discovery efforts. This review focuses on the overview of current knowledge of structure function, regulatory mechanism, and potential inhibitors (active site as well as allosteric site) of CysE. Despite having conserved structure, slight modification in CysE structure lead to altered the regulatory mechanism and hence affects the cysteine production. Due to its possible role in virulence and vital metabolism of pathogens makes it a potential target in the quest to develop novel therapeutics to treat multi-drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

The antimicrobial resistant strains of several pathogens are major culprits of hospital-acquired nosocomial infections. An active and urgent action is necessary against these pathogens for the development of unique therapeutics. The cysteine biosynthetic pathway or genes (that are absent in humans) involved in the production of L-cysteine appear to be an attractive target for developing novel antibiotics. CysE, a Serine Acetyltransferase (SAT), catalyzes the first step of cysteine synthesis and is reported to be essential for the survival of persistence in several microbes including Mycobacterium tuberculosis. Structure determination provides fundamental insight into structure and function of protein and aid in drug design/discovery efforts. This review focuses on the overview of current knowledge of structure function, regulatory mechanism, and potential inhibitors (active site as well as allosteric site) of CysE. Despite having conserved structure, slight modification in CysE structure lead to altered the regulatory mechanism and hence affects the cysteine production. Due to its possible role in virulence and vital metabolism of pathogens makes it a potential target in the quest to develop novel therapeutics to treat multi-drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

In this work, we propose a heterogeneous committee (ensemble) of diverse members (classification approaches) to solve the problem of human epithelial (HEp-2) cell image classification using indirect Immunofluorescence (IIF) imaging. We hypothesize that an ensemble involving different feature representations can enable higher performance if individual members in the ensemble are sufficiently varied. These members are of two types: (1) CNN-based members, (2) traditional members. For the CNN members, we have employed the well-established ResNet, DenseNet, and Inception models, which have distinctive salient aspects. For the traditional members, we incorporate class-specific features which are characterized depending on visual morphological attributes, and some standard texture features. To select the members which are discriminating and not redundant, we use an information theoretic measure which considers the trade-off between individual accuracies and diversity among the members. For all selected members, a compelling fusion required to combine their outputs to reach a final decision. Thus, we also investigate various fusion methods that combine the opinion of the committee at different levels: maximum voting, product, decision template, Bayes, Dempster-Shafer, etc. The proposed method is evaluated using ICPR-2014 data which consists of more images than some previous datasets ICPR-2012 and demonstrate state-of-the-art performance. To check the effectiveness of the proposed methodology for other related datasets, we test our methodology with newly compiled large-scale HEp-2 dataset with 63K cell images and demonstrate comparable performance even with less number of training samples. The proposed method produces 99.80% and 86.03% accuracy respectively when tested on ICPR-2014 and a new large-scale data containing 63K samples. Graphical Abstract Overview of the proposed methodology. [ABSTRACT FROM AUTHOR]

In this article, a compact quad notched CPW fed ultrawideband (UWB) antenna of the size 35.4 × 26.6 × 1.6 mm3 with improved gain characteristic is proposed. The designed antenna has bandwidth ranging from 2.85 GHz to 11.45 GHz with the notching capability at WiMAX, C‐band, WLAN and downlink of X‐band for satellite communication. The gain of the antenna is improved by using two antennas in the stacked configuration without raising the overall size of the antenna in horizontal plane and maintaining the impedance matching over the ultrawideband range. The prototype of the proposed antenna has been fabricated on FR4 substrate and its performance parameters are measured and compared with the simulated results, which are in good agreement. The simulated and measured result shows the improvement of antenna peak gain by 4.2 dBi and thus improving the peak gain by 162%. The proposed antenna is an excellent candidate for the high gain UWB portable communication devices in the environment having different narrow interfering bands of WiMAX, C‐band, WLAN, and downlink X‐band of satellite communication. [ABSTRACT FROM AUTHOR]

This paper presents a two-element MIMO antenna configuration designed using magneto-dielectric (MD) material. The MD material has been designed such that its temperature coefficient value approaches zero. The microwave dielectric constant and permeability of the material is calculated by Nicholson-Ross-Weir conversion technique and found to be 8.02 and 1.64, respectively. Each element is designed so that it can operate at 5.71 GHz. The two elements of the MIMO configuration are separated by quarter wavelength and half wavelength and are compared. The designed antenna can give a quad-directional radiation property, which can be utilized for the diversity scheme. [ABSTRACT FROM AUTHOR]

ABSTRACT In this paper, a slot loaded ultra wideband monopole patch antenna with triple band-notched characteristic has been presented. The antenna consists of rectangular patch with three modified inverted U-shaped slots to create three notches at Wi-MAX (3.3-3.6 GHz), C-band (3.7-4.2 GHz), and WLAN (5.15-5.85 GHz) frequencies. Lumped-element equivalent circuit model and current distribution curves have been used to explain the triple band-rejection characteristics of the designed antenna. The proposed triple notch structure has been fabricated and tested against simulated data. © 2016 Wiley Periodicals, Inc. Microwave Opt Technol Lett 59:138-142, 2017 [ABSTRACT FROM AUTHOR]

ABSTRACT This article presents a metamaterial-based tri-band antenna for WiMAX (2.5/3.5/5.5 GHz) and WLAN (5.2/5.8 GHz) application. The antenna comprises of a metallic patch of area 22 mm × 14 mm on the top of a substrate. The patch is etched with a T-shaped slot and meandered lines. At the bottom of substrate we have a partial, rectangular shaped ground plane along with an electric-inductive-capacitive resonator. The proposed antenna gives a desired impedance bandwidth along with an omnidirectional radiation pattern. © 2016 Wiley Periodicals, Inc. Microwave Opt Technol Lett 58:558-561, 2016 [ABSTRACT FROM AUTHOR]

ABSTRACT This article presents quad-directional magneto-dielectric (MD) resonator antenna configuration, designed using two different MD materials with stack configuration. These materials have been designed such that their temperature coefficient is approximately zero. The microwave dielectric constant (εr) and permeability (μr) of both materials are calculated by Nicholson-Ross-Weir conversion technique and found to be 8.02, 1.64 and 10.47, 1.52, respectively. The designed antenna can radiates in four directions simultaneously at the resonant frequency of 5.84 GHz. The beam shaping has been achieved using single feed and this property can be utilized for the angle diversity. © 2016 Wiley Periodicals, Inc. Microwave Opt Technol Lett 58:62-64, 2016 [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex. Pull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation. Rv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation. This study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex. Partitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation. • Rv1915 and Rv1916 of Mtb H37Rv associate together to form in vitro protein-protein complex. • Deletion of disordered C-terminal of Rv1915 does not abrogate complex formation with Rv1916. • Site-directed mutagenesis studies show Y64 and Y65 residues of Rv1916 to be crucial for complex formation. • The presence of acetyl-CoA increases the ICL and MICL activities of Rv1916 and the complex. [ABSTRACT FROM AUTHOR]

Background Brassica juncea (AABB) is an allotetraploid species containing genomes of B. rapa (AA) and B. nigra (BB). It is a major oilseed crop in South Asia, and grown on approximately 6-7 million hectares of land in India during the winter season under dryland conditions. B. juncea has two well defined gene pools - Indian and east European. Hybrids between the two gene pools are heterotic for yield. A large number of qualitative and quantitative traits need to be introgressed from one gene pool into the other. This study explores the availability of SNPs in RNA-seq generated contigs, and their use for general mapping, fine mapping of selected regions, and comparative arrangement of gene blocks on B. juncea A and B genomes. Results RNA isolated from two lines of B. juncea - Varuna (Indian type) and Heera (east European type) - was sequenced using Illumina paired end sequencing technology, and assembled using the Velvet de novo programme. A and B genome specific contigs were identified in two steps. First, by aligning contigs against the B. rapa protein database (available at BRAD), and second by comparing percentage identity at the nucleotide level with B. rapa CDS and B. nigra transcriptome. 135,693 SNPs were recorded in the assembled partial gene models of Varuna and Heera, 85,473 in the A genome and 50,236 in the B. Using KASpar technology, 999 markers were added to an earlier intron polymorphism marker based map of a B. juncea Varuna x Heera DH population. Many new gene blocks were identified in the B genome. A number of SNP markers covered single copy homoeologues of the A and B genomes, and these were used to identify homoeologous blocks between the two genomes. Comparison of the block architecture of A and B genomes revealed extensive differences in gene block associations and block fragmentation patterns. Conclusions Sufficient SNP markers are available for general and specific -region fine mapping of crosses between lines of two diverse B. juncea gene pools. Comparative gene block arrangement and block fragmentation patterns between A and B genomes support the hypothesis that the two genomes evolved from independent hexaploidy events. [ABSTRACT FROM AUTHOR]

Nickel–cobalt–zinc ferrites, Ni0.2Co x Zn0.8−x Fe2O4 (with x=0, 0.2, 0.4, 0.6, 0.8,) have been prepared using the auto-combustion method. X-ray diffraction, Fourier transform infrared (FT-IR) and Raman spectroscopy confirmed the cubic spinel structure of the ferrites. Microwave dielectric constant (ε r ) and quality factor (Q×f) were measured by using the Hakki–Coleman technique. Dielectric constant calculated using the Clausius–Mossoitti relation showed good agreement with the experimental value. Relative permeability of the ferrites was calculated by using the Nicholson–Ross–Weir conversion technique. Dielectric constant (ε r ) decreased from 7.474 to 5.548 and temperature coefficient of resonant frequency (τ f ) increased from −75.85ppm/°C to −19.32ppm/°C, respectively, and relative permeability increased from 2.1085 to 3.2179 with increase in Cobalt content. The composition Ni0.2Co0.2Zn0.6Fe2O4 was used to develop ferrite resonator antennas (FRA) and was studied in cylindrical, triangular and cuboid shapes with co-axial probe feeding mechanisms. The antenna characteristics were simulated using high frequency structure simulation (HFSS) software and a good agreement has been achieved between the measured and simulated results. The cuboid shaped FRA show maximum bandwidth of 990MHz, while the cylindrical shaped FRA show a maximum gain of 3.48dB, respectively. [ABSTRACT FROM AUTHOR]

Esomeprazole is chemically bis(5methoxy-2-[(S)-[(4-methoxy-3,5-dimethyl-2-pyridinyl) methyl]sulfinyl]-1Hbenzimidazole- 1-yl). It is a gastric proton-pump inhibitor (PPI) used in treatment of gastric-acid related diseases. Simple, selective and accurate high performance liquid chromatographic (HPLC) methods were developed and validated for the analysis of esomeprazole magnesium. The objective of this review is to describe various analytical methods for estimation of Esomeprazole. All methods are validated as per ICH guidelines. [ABSTRACT FROM AUTHOR]

Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world's deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action. [ABSTRACT FROM AUTHOR]

Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs. [ABSTRACT FROM AUTHOR]

In this paper a novel feeding technique is presented using transition between microstrip-line segment and folded substrate integrated waveguide (FSIW), which provides very low insertion loss over wide frequency band and can be used for signal feeding in FSIW based waveguides and components. Concept of simultaneous transformation in mode and impedance by tapering central metal septum and removing upper ground plane precisely above the tapering over the transition section is used to design the novel transitions. The presented design procedures are applied on transitions for FSIW transmission lines operating in different frequency bands from C to K band. The concept is explained in detail with an example for transitions designed for FSIW operating in Ku band. All the designed structures are simulated using HFSS software. The designed FSIW with the transitions operating in Ku band is fabricated and the measured results are compared with simulated results and are found in good agreement. Dimensions of transitions designed for FSIWs operating in other frequency bands are tabulated. [ABSTRACT FROM AUTHOR]

A new compact square microstrip patch antenna is proposed using the optimization approach, namely, the genetic algorithm (GA). The antenna is designed at three different frequencies, 785 MHz., 1.57 GHz, and 2.46 GHz, for mobile communication, GPS, and Bluetooth applications, respectively. The simulations are carried out using IE3D from Zeland Software, which is based on the method of moments (MoM). The antenna consists of a probe-fed truncated-corner square patch with four inserted slits along the four diagonals and four angular grooves along the four edges of the patch. The proposed design has a reduced antenna size, as compared to the conventional microstrip antenna at a given operating frequency. The return loss, impedance, and axial-ratio bandwidth are determined and compared with the conventional square-patch antenna. © 2006 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2006. [ABSTRACT FROM AUTHOR]

Sml 1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnrlp) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml 1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys 14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml 1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml 1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C 14S Sml 1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml 1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnrlp activity. [ABSTRACT FROM AUTHOR]

Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (VL) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the VL domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable Vλ6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic Vλ6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

SUMMARY Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the northeastern regions of Africa. Although it is under limited cultivation, B. carinata is valuable as it is resistant/highly tolerant to most of the pathogens affecting widely cultivated Brassica species of the U's triangle. We report a chromosome‐scale genome assembly of B. carinata accession HC20 using long‐read Oxford Nanopore sequencing and Bionano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the long‐read genome assemblies of the U's triangle species and found extensive gene collinearity between the diploids and allopolyploids with no evidence of major gene losses. Therefore, B. juncea (AABB), B. napus (AACC), and B. carinata can be regarded as strict allopolyploids. We cataloged the nucleotide‐binding and leucine‐rich repeat immune receptor (NLR) repertoire of B. carinata and, identified 465 NLRs, and compared these with the NLRs in the other Brassica species. We investigated the extent and nature of early‐generation genomic interactions between the constituent genomes of B. carinata and B. juncea in interspecific crosses between the two species. Besides the expected recombination between the constituent B genomes, extensive homoeologous exchanges were observed between the A and C genomes. Interspecific crosses, therefore, can be used for transferring disease resistance from B. carinata to B. juncea and broadening the genetic base of the two allotetraploid species. [ABSTRACT FROM AUTHOR]

Allied species of crop Brassicas have potential value as donors of useful nuclear/organelle genomes. Eruca sativa, a member of subtribe Brassicineae, is resistant to white rust and well adapted to drought. Attempts to hybridize it with Brassica campestris by conventional methods were unsuccessful. However, hybrids were obtained by embryo rescue and the hybrid embryos were found to produce numerous secondary embryos in upto 7 to 8 subcultures. Plantlets developed from them exhibited morphological characteristics of both parents. The chromosome number of 2n = 42 showed that they were amphidiploids. The plants were allotetraploids (2n = 42) and showed 21 bivalents at M1 of meiosis. Hybridization of total DNA of the hybrids with two probes, a B. campestris tandem repeat DNA and 18s ribosomal DNA of wheat showed that it was derived from the genome of both parents. The hybrids are self-fertile and show the same high fertility even in A3 generation. They were selfed or crossed with B. juncea, B. campestris and B. nigra to screen for useful agronomical traits. Six more embryos of E. sativa x B. campestris have been obtained and their growth is being studied. Preliminary small scale field trials indicate that the selfed hybrid is comparable in yield with a high yielding commercial variety of B. juncea. [ABSTRACT FROM AUTHOR]

The characteristics of a bow-shaped microstrip patch antenna are analyzed and compared with a conventional rectangular microstrip antenna. The various parameters such as return loss, gain, and VSWR are determined and compared. It is found that the center frequency of the bow-shaped antenna is lower by about 12.82% than that of the conventional rectangular microstrip antenna. This implies that the size of the patch needs to be reduced in order to maintain the same resonant frequency. © 2004 Wiley Periodicals, Inc. Microwave Opt Technol Lett 40: 158–160, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.11314 [ABSTRACT FROM AUTHOR]

The emergence of drug resistance in Streptococcus pneumoniae (Spn) is a global health threat and necessitates discovery of novel therapeutics. The serine acetyltransferase (also known as CysE) is an enzyme of cysteine biosynthesis pathway and is reported to be essential for the survival of several pathogenic bacteria. Therefore, it appears to be a very attractive target for structure–function understanding and inhibitor design. This study describes the molecular cloning of cysE from Spn in the pET21c vector and efforts carried out for expression and purification of active recombinant CysE. Significant expression of recombinant Spn cysE could be achieved in codon optimized BL21(DE3)-RIL strain as opposed to conventional BL21(DE3) strain. Analysis of codon adaptation index (CAI) with levels of eukaryotic genes and prokaryotic cysEs expressed in heterologous E. coli host suggests that codon optimized E. coli BL21(DE3)-RIL may be a better host for expressing genes with low CAI. Here, an efficient protocol has been developed for recovery of recombinant Spn CysE in soluble and biologically active form by the usage of nonionic detergent Triton X-100 at a concentration as low as 1%. Altogether, this study reports a simple strategy for producing functionally active Spn CysE in E. coli. [ABSTRACT FROM AUTHOR]

Key message: Genetic mapping of some key plant architectural traits in a vegetable type and an oleiferous B. juncea cross revealed QTL and candidate genes for breeding more productive ideotypes. Brassica juncea (AABB, 2n = 36), commonly called mustard, is an allopolyploid crop of recent origin but contains considerable morphological and genetic variation. An F1-derived doubled haploid population developed from a cross between an Indian oleiferous line, Varuna, and a Chinese stem type vegetable mustard, Tumida showed significant variability for some key plant architectural traits—four stem strength-related traits, stem diameter (Dia), plant height (Plht), branch initiation height (Bih), number of primary branches (Pbr), and days to flowering (Df). Multi-environment QTL analysis identified twenty Stable QTL for the above-mentioned nine plant architectural traits. Though Tumida is ill-adapted to the Indian growing conditions, it was found to contribute favorable alleles in Stable QTL for five architectural traits—press force, Dia, Plht, Bih, and Pbr; these QTL could be used to breed superior ideotypes in the oleiferous mustard lines. A QTL cluster on LG A10 contained Stable QTL for seven architectural traits that included major QTL (phenotypic variance ≥ 10%) for Df and Pbr, with Tumida contributing the trait-enhancing alleles for both. Since early flowering is critical for the cultivation of mustard in the Indian subcontinent, this QTL cannot be used for the improvement of Pbr in the Indian gene pool lines. Conditional QTL analysis for Pbr, however, identified other QTL which could be used for the improvement of Pbr without affecting Df. The Stable QTL intervals were mapped on the genome assemblies of Tumida and Varuna for the identification of candidate genes. [ABSTRACT FROM AUTHOR]

Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea. [ABSTRACT FROM AUTHOR]

The present study is based on the citation pattern of the articles published for the period 2005-2014 of the Library Philosophy and Practice, E-Journal. 1180 articles are published in the journal during 2005-2014. Highest numbers (198) of articles were published in 2011. The EJournal contained 20978 references for the study period of which 15518 are documentary sources citations and 5460 are non-documentary source citations. Every year almost 118 articles are published and each article has an average of 13.15 documentary sources and 4.618 non-documentary citations. [ABSTRACT FROM AUTHOR]

The present study reveals the bibliometric study of the E-Journal, Library Philosophy and Practice from 2005 to 2014. The study covers the growth of literature and authorship patterns of the journal. Further, it analyzes various other bibliometrics aspects such as authors' degree of collaboration, geographical productivity in scholarly publications, subject wise distribution of articles and ranking pattern etc. For this study 1177 no. of articles was taken up for the evaluation. Necessary bibliometric measures are applied to analyze different publication parameters. In all with an average 117 articles were published each year. Single authorship is leading authorship trend but also two authored articles have shown good number of contribution with the 0.51 rate of degree of collaboration. [ABSTRACT FROM AUTHOR]

Objectives: Remote patient monitoring became critical for patients receiving cancer treatment during the COVID-19 pandemic. We sought to test feasibility of an electronic patient symptom management program implemented during a pandemic. We collected and analyzed the real-world data to inform practice quality improvement and understand the patient experience.Methods: Eligible patients had breast, lung, or ovarian cancers, multiple myeloma, or acute myeloid leukemia and 12 weeks of planned chemotherapy. Patients were notified that a symptom survey with common symptoms derived from the National Cancer Institute's Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events was available to complete using a smart phone, tablet, or computer. Patients recorded their symptoms and results were sent to the provider. Patients received care guidelines for mild/moderate severity symptoms and a phone call from the provider for severe reports.Results: A total of 282 patients generated > 119 088 data points. Patients completed 2860 of 3248 assigned surveys (88%), and 152 of 282 patients (54%) had symptom reports that generated an immediate notification to the provider. Longitudinal data were analyzed to determine whether previous reports predicted a notification alert and whether symptoms resolved after the alert was addressed.Conclusions: An electronic patient symptom management program was implemented in the midst of the COVID-19 pandemic. Enrollment of 282 patients and a high survey completion (88%) demonstrated feasibility/acceptance. Patients reported symptoms at severe levels of 54% of the time and received self-management instructions and provider phone calls that resolved or decreased the severity of the symptom. A standard approach and validated instrument provide opportunities for improving and benchmarking outcomes. [ABSTRACT FROM AUTHOR]

Key message: QTL mapping by two DH mapping populations deciphered allelic variations for five different seed glucosinolate traits in B. juncea. Abstract: Allelic variations for five different seed glucosinolate (GS) traits, namely % propyl, % butyl, % pentyl, aliphatics and total GS content were studied through QTL analysis using two doubled haploid (DH) mapping populations. While the high GS parents in two populations differed in their profiles of seed aliphatic GS, the low GS parents were similar. Phenotypic data of seed GS traits from three environments of the two populations were subjected to QTL analysis. The first population (referred to as DE population) detected a total of 60 QTL from three environments which upon intra-population meta-QTL analysis were merged to 17 S- QTL (Stable QTL) and 15 E- QTL (Environment QTL). The second population (referred to as VH population) detected 58 QTL from the three environments that were merged to 15 S- QTL and 16 E- QTL. In both the populations, majority of S- QTL were detected as major QTL. Inter-population meta-analysis identified three C- QTL (consensus QTL) formed by merging major QTL from the two populations. Candidate genes of GS pathway were co-localized to the QTL regions either through genetic mapping or through in silico comparative analysis. Parental allelic variants of QTL or of the co-mapped candidate gene(s) were determined on the basis of the significantly different R values of the component QTL from the two populations which were merged to form C- QTL. The results of the study are significant for marker-assisted transfer of the low GS trait and also for developing lines with lower GS than are present in Brassica juncea. [ABSTRACT FROM AUTHOR]

Brassica species (tribe Brassiceae) belonging to U's triangle—B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC)—originated via two polyploidization rounds: a U event producing the three allopolyploids, and a more ancient b genome-triplication event giving rise to the A-, B-, and C-genome diploid species. Molecular mapping studies, in situ hybridization, and genome sequencing of B. rapa support the genome triplication origin of tribe Brassiceae, and suggest that these three diploid species diversified from a common hexaploid ancestor. Analysis of plastid DNA has revealed two distinct lineages—Rapa/Oleracea and Nigra—that conflict with hexaploidization as a single event defining the tribe Brassiceae. We analysed an R-block region of A. thaliana present in six copies in B. juncea (AABB), three copies each on A- and B-genomes to study gene fractionation pattern and synonymous base substitution rates (Ks values). Divergence time of paralogues within the A and B genomes and homoeologues between the A and B genomes was estimated. Homoeologous R blocks of the A and B genomes exhibited high gene collinearity and a conserved gene fractionation pattern. The three progenitors of diploid Brassicas were estimated to have diverged approximately 12 mya. Divergence of B. rapa and B. nigra, calculated from plastid gene sequences, was estimated to have occurred approximately 12 mya, coinciding with the divergence of the three genomes participating in the b event. Divergence of B. juncea A and B genome homoeologues was estimated to have taken place around 7 mya. Based on divergence time estimates and the presence of distinct plastid lineages in tribe Brassiceae, it is concluded that at least two independent triplication events involving reciprocal crosses at the time of the b event have given rise to Rapa/Oleracea and Nigra lineages. [ABSTRACT FROM AUTHOR]

Identification of the candidate gene responsible for the seed coat colour variation in Brassica juncea was undertaken following an earlier study where two independent loci ( BjSc1 and BjSc2) were mapped to two linkage groups, LG A9 and B3 (Padmaja et al. in Theor Appl Genet 111:8-14, ). The genome search from BRAD data for the presence of flavonoid genes in B. rapa identified three candidate genes namely, DFR, TT1 and TT8 in the LG A9. Quantitative real-time PCR revealed absence of transcript for the late biosynthetic genes (LBGs) and showed significant reduction of transcript in the TT8 from the developing seeds of yellow-seeded line. While mapping of two DFR genes, the BjuA.DFR and BjuB.DFR did not show perfect co-segregation with the seed coat colour loci, that of the two TT8 genes, BjuA.TT8 and BjuB.TT8 showed perfect co-segregation with the seed coat colour phenotype. The BjuA.TT8 allele from the yellow-seeded line revealed the presence of an insertion of 1,279 bp in the exon 7 and did not produce any transcript as revealed by reverse transcriptase PCR. The BjuB.TT8 allele from the yellow-seeded line revealed the presence of an SNP (C→T) in the exon 7 resulting in a stop codon predicting a truncated protein lacking the C-terminal 8 amino acid residues and produced significantly low level of transcript than its wild-type counterpart. Hence, it is hypothesized that the mutations in both the TT8 genes are required for inhibiting the transcription of LBGs in the yellow-seeded mutant of B. juncea. [ABSTRACT FROM AUTHOR]

Background: Brassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra. Results: RNA-seq was carried out on the RNA isolated from young inflorescences containing unopened floral buds, floral axis and small leaves, using Illumina paired-end sequencing technology. Sequence assembly was carried out using the Velvet de-novo programme and the assembled contigs were organised against Chiifu gene models, available in the BRAD-CDS database. RNA-seq confirmed the presence of more than 17,000 single-copy gene models described in the BRAD database. The assembled contigs and the BRAD gene models were analyzed for the presence of SSRs and SNPs. While the number of SSRs was limited, more than 0.2 million SNPs were observed between Chiifu and the three oleiferous lines. Assays for SNPs were designed using KASPar technology and tested on a F7-RIL population derived from a Chiifu x Tetra cross. The design of the SNP assays were based on three considerations - the 50 bp flanking region of the SNPs should be strictly similar, the SNP should have a read-depth of ≥7 and no exon/intron junction should be present within the 101 bp target region. Using these criteria, a total of 640 markers (580 for genome-wide mapping and 60 for specific-region mapping) marking as many genes were tested for mapping. Out of 640 markers that were tested, 594 markers could be mapped unambiguously which included 542 markers for genome-wide mapping and 42 markers for fine mapping of the tet-o locus that is involved with the trait tetralocular ovary in the line Tetra. Conclusion: A large number of SNPs and PSVs are present in the transcriptome of B. rapa lines for genome-wide linkage mapping and specific-region fine mapping. Criteria used for SNP identification delivered markers, more than 93% of which could be successfully mapped to the F7-RIL population of Chiifu x Tetra cross. [ABSTRACT FROM AUTHOR]

INTRODUCTION: We sought to investigate the association between receipt of an opioid pain reliever (OPR) in the emergency department (ED) and downstream acute health care utilization. METHODS: Within Kaiser Permanente Northern California, we identified opioid-naïve patients, ages 18-64, who were treated and discharged from the ED for a painful, low-severity condition between January 1, 2017, and December 31, 2017. We also identified patients who received an OPR, either administered in the ED or obtained at a Kaiser Permanente Northern California pharmacy within 7 days of ED arrival, and investigated subsequent acute care utilization in cases with at least 1 ED, urgent care, or inpatient visit within 1 month or 3 months of the index encounter or 2 visits within 12 months. RESULTS: Of the 39,468 adults included in our study, 50.7% were female, 55.0% were non-White, and 25.2% received an OPR in association with their index ED encounter. After adjustment, we found that patients who received an OPR had greater odds of downstream acute care utilization than those who did not, with odds ratios of 1.68, 1.53, and 1.50 at 1, 3, and 12 months, respectively (all p < 0.05). CONCLUSION: Patients who received an OPR at their index encounter had substantially increased odds of a subsequent ED, urgent care, or inpatient visit. This effect was most pronounced early in followup and persisted for the duration of the study period. Receipt of an OPR among opioid-naïve adults for a painful, low-severity condition is associated with increased downstream acute care utilization. [ABSTRACT FROM AUTHOR]

Abstract: In this paper the structural and microwave dielectric properties of the (1− x)(Mg0.95Zn0.05)TiO3–(x)(Ca0.6La0.8/3)TiO3 ceramic composites have been investigated with the variation of x as well as sintering temperature. The grown samples have been characterized by means of X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectroscopy analysis. The Rietveld analysis of the XRD data has been carried out for structure refinement of the phases. The relative permittivity , quality factor (Q) and temperature coefficient of resonant frequency τf of the grown samples have been thoroughly studied. Out of all samples of this series, the sample with x =0.21 shows excellent dielectric properties with , Q × f ∼60,738GHz (at 6.44GHz) and a nearly zero τf ∼−0.8ppm/°C. Two types of dielectric resonator antennas with different feeding mechanisms have been fabricated using this sample to study their performance. The experimental results have been compared with the simulated results obtained using Ansoft High Frequency Structure Simulator software. [Copyright &y& Elsevier]

Abstract: This paper presents the design of a cylindrical dielectric resonator antenna (DRA) using a nearly temperature stable dielectric composite material. By the combination of compounds with positive and negative temperature coefficient, a dielectric composite series (1− x)(Mg0.95Zn0.05)TiO3 − x(Ca0.8Sr0.2)TiO3 has been developed. The structural and morphological properties of the grown samples have been characterized by means of X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive X-ray (EDX) spectroscopy analysis. Microwave dielectric properties of the composite samples have been investigated using the TE01δ dielectric resonator method. Relative permittivity (ɛ r ) of 21.9, dielectric loss tan δ of 0.0002 and temperature coefficient of resonant frequency (τ f ) of −0.15ppm/°C have been obtained for the x =0.08 sample, which was used for designing the DRA. The DRA resonates at 4.6GHz frequency and offers the bandwidth of 315MHz. The characteristics of proposed DRA have been simulated using the Ansoft high frequency structure simulator (HFSS). Comparison between the simulated and measured results shows a reasonably good agreement. [Copyright &y& Elsevier]

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard ( Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 ( FAD2), fatty acid desaturase 3 ( FAD3) or fatty acid elongase ( FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as 'weak' contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was 'masked' due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions. [ABSTRACT FROM AUTHOR]

We report in this study, strategies for enhancing and extending tissue-specific expression of a restorer gene (barstar) and their use in the development of improved fertility restorer lines for transgenic male sterile (barnase) lines for hybrid seed production. In the first strategy, a chimeric promoter was developed by combining regulatory elements of two overlapping tapetum-specific promoters (A9 and TA29) by placing a 275bp fragment of the A9 promoter (minus the TATA box) upstream to a functional 330bp fragment of the TA29 promoter. Analysis of chimeric promoter activity using the gusA gene showed that it enhanced transgene expression levels in anthers of transgenic Brassica juncea (Indian oilseed mustard) plants by two- to three-fold over those obtained with either promoter alone but retained the temporal expression profile of the TA29 promoter. In the second strategy, the A9 and TA29 promoters were used to independently express two copies of the wild type barstar gene sequence within the same T-DNA borders. Alternatively, one of the promoters was used to express the wild type sequence and the other was used to drive expression of a codon-modified version of the barstar gene designed for enhanced and stable expression in dicot plants. The efficiencies of these constructs for fertility restoration was determined by retransforming a male sterile TA29-barnase line of B. juncea and analyzing the frequency and pollen viability of male fertile (restored) plants thus obtained. A significantly higher frequency of restored plants was obtained with constructs containing two transcription units of the barstar gene (92.9% for constructs with two copies of the wild type gene and 89.8% for constructs using the wild type and codon-modified sequences) as compared to that obtained with the chimeric promoter-barstar construct (77.8%) or the TA29-barstar construct (65.6%). Pollen viability assays on restored plants showed that the desirable extent of restoration is obtained in a higher proportion of male fertile plants generated using the construct containing a combination of the wild type and modified barstar genes. These observations were corroborated by genetic analysis of F1 progeny of crosses between various barstar lines and four different male sterile barnase lines, three of which could not be restored in earlier studies using TA29-barstar constructs. The simultaneous use of the wild type and modified barstar genes with the TA29 and A9 promoters was found to be the most effective method for fertility restoration with efficient restorers being identified for all the male sterile barnase lines tested, including those which could not be restored earlier. The two gene-two promoter strategy could be successfully deployed for enhancing and extending the tissue-specific as well as constitutive expression of other agronomically important transgenes. [ABSTRACT FROM AUTHOR]

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)33Abbreviations used: RNR, ribonucleotide reductase; ORF, open reading frame; ES-FTICR-MS, electrospray Fourier transform ion cyclotron resonance mass spectrometry; NTA, nitrilotriacetic acid; IOD, image optical density; SORI-CAD, sustained off-resonance irradiation collision-activated dissociation; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; DTT, dithiothreitol., a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: () confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS–PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal–ion binding to the protein species, which may alter the function and binding of the protein partners. [Copyright &y& Elsevier]

Summary: Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species—B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single‐molecule real‐time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short‐read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping‐by‐sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome‐scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere. [ABSTRACT FROM AUTHOR]

The development of more productive crops will be key to addressing the challenges that climate change, population growth and diminishing resources pose to global food security. Advanced 'omics techniques can help to accelerate breeding by facilitating the identification of genetic markers for use in marker‐assisted selection. Here, we present the validation of a new Associative Transcriptomics platform in the important oilseed crop Brassica juncea. To develop this platform, we established a pan‐transcriptome reference for B. juncea, to which we mapped transcriptome data from a diverse panel of B. juncea accessions. From this panel, we identified 355 050 single nucleotide polymorphism variants and quantified the abundance of 93 963 transcripts. Subsequent association analysis of functional genotypes against a number of important agronomic and quality traits revealed a promising candidate gene for seed weight, BjA.TTL, as well as additional markers linked to seed colour and vitamin E content. The establishment of the first full‐scale Associative Transcriptomics platform for B. juncea enables rapid progress to be made towards an understanding of the genetic architecture of trait variation in this important species, and provides an exemplar for other crops. Significance Statement: We present the development of a new pan‐transcriptome reference for Brassica juncea, and the establishment of an Associative Transcriptomics platform for dissecting the genetic architecture of complex traits in this crop, which provides a valuable resource for molecular breeding in B. juncea. We validate this platform by identifying and testing predictive marker associations for important quality traits; seed size, seed colour and γ‐tocopherol content. [ABSTRACT FROM AUTHOR]

• Cloning, purification and biochemical characterization of CysE from Klebsiella pneumoniae • 3-D crystal structure of Kpn CysE complexed with L-cysteine at 2.8 Å • In silico identification and in vitro validation of inhibitory potential of quercetin • Identification of an allosteric site at the trimer-trimer interface in the hexameric CysE The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is one of the leading causes of death worldwide. Long duration of TB therapy, results in the persistence and development of drug resistant strains of causative organism Mycobacterium tuberculosis (Mtb). Novel drug targets against persistent Mtb is an immediate need for overcoming this global menace. Isocitrate lyase (ICL), the first enzyme of glyoxylate pathway, is essential for persistent Mtb and absent in humans, hence a propitious target for drug development. Pathogenic Mtb H37Rv, have two types of ICLs - ICL1 encoded by icl (Rv0467) is well characterized and homologous to eubacterial enzyme whereas ICL2 encoded by aceA is more related to eukaryotic isocitrate lyase. To compound it, the aceA gene is split into two ORFs namely rv1915 / aceAa and rv1916 / aceAb. No translational product has been reported for the later and therefore, in vivo existence of Rv1916/ICL2 b is debatable. This study reports recombinant production of Rv1916 in heterologous host E. coli BL21 (DE3) for structure function studies. The studies categorically demonstrate that akin to Mtb ICL1, recombinant Rv1916 also possess dual ICL and methylisocitrate lyase (MICL) activities in vitro. Based on in silico analysis, a putative function linked to secondary metabolite synthesis is assigned to unique mycobacterial domain IV. • This study is the first report of Rv1916 possessing in vitro ICL and MICL activities. • 3-Nitropropionate is an uncompetitive inhibitor of both the activities. • Phylogenetic analysis suggests, evolutionary relationship between Rv1916 and ICLs of some soil bacterial species. • Domain IV of Rv1916, is hypothesized to be involved in synthesis of secondary metabolites. [ABSTRACT FROM AUTHOR]

Key message: BjuWRR1, a CNL-type R gene, was identified from an east European gene pool line of Brassica juncea and validated for conferring resistance to white rust by genetic transformation. White rust caused by the oomycete pathogen Albugo candida is a significant disease of crucifer crops including Brassica juncea (mustard), a major oilseed crop of the Indian subcontinent. Earlier, a resistance-conferring locus named AcB1-A5.1 was mapped in an east European gene pool line of B. juncea—Donskaja-IV. This line was tested along with some other lines of B. juncea (AABB), B. rapa (AA) and B. nigra (BB) for resistance to six isolates of A. candida collected from different mustard growing regions of India. Donskaja-IV was found to be completely resistant to all the tested isolates. Sequencing of a BAC spanning the locus AcB1-A5.1 showed the presence of a single CC-NB-LRR protein encoding R gene. The genomic sequence of the putative R gene with its native promoter and terminator was used for the genetic transformation of a susceptible Indian gene pool line Varuna and was found to confer complete resistance to all the isolates. This is the first white rust resistance-conferring gene described from Brassica species and has been named BjuWRR1. Allelic variants of the gene in B. juncea germplasm and orthologues in the Brassicaceae genomes were studied to understand the evolutionary dynamics of the BjuWRR1 gene. [ABSTRACT FROM AUTHOR]

CysK (O-acetylserine sulfhydrylase) is a pyridoxal-5′ phosphate-dependent enzyme which catalyzes the second step of the de novo cysteine biosynthesis pathway by converting O-acetyl serine (OAS) into L-cysteine in the presence of sulfide. The first step of the cysteine biosynthesis involves formation of OAS from serine and acetyl CoA by CysE (serine acetyltransferase). Apart from role of CysK in cysteine biosynthesis, recent studies have revealed various additional roles of this enzyme in bacterial physiology. Other than the suggested regulatory role in cysteine production, other activities of CysK include involvement of CysK-in contact-dependent toxin activation in Gram-negative pathogens, as a transcriptional regulator of CymR by stabilizing the CymR-DNA interactions, in biofilm formation by providing cysteine and via another mechanism not yet understood, in ofloxacin and tellurite resistance as well as in cysteine desulfurization. Some of these activities involve binding of CysK to another cellular partner, where the complex is regulated by the availability of OAS and/or sulfide (H2S). The aim of this study is to present an overview of current knowledge of multiple functions performed by CysK and identifying structural features involved in alternate functions. Due to possible role in disease, promoting or inhibiting a "moonlighting" function of CysK could be a target for developing novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Structure-based drug discovery has emerged as a powerful tool in computational drug discovery and has gained rapid acceleration due to the development of better algorithms for high-end computation in an affordable time. Molecular docking and virtual screening methods are routinely used for the purpose but computing the ligand binding energies with inbuilt scoring functions accurately is still a limitation. Although, MMPBSA and MMGBSA are routinely employed tools for achieving accurate binding free energies, they are applied on well-equilibrated explicitly solvated systems and are computationally expensive and time-consuming. This study compares different post-processing protocols performed on an in-silico screened benchmarked P. falciparum Dihydrofolate reductase (PfDHFR) dataset with AutoDockVina. The docked and implicitly solvated complexes were subjected to (1) rescoring, (2) energy minimization and (3) Binding Estimation After Refinement (BEAR) algorithm. Subsequently, binding free energies were computed using three different tools—MMPBSA, MMGBSA and "g_mmpbsa". Surprisingly, rescoring alone displays lower accuracy than the inherent scoring function of the AutoDockVina. However, encouraging results were seen after post-processing with the other two protocols. The results suggest that MMPBSA applied on energy minimized conformations is able to achieve 42-fold reductions in computational time as opposed to the BEAR algorithm with comparable accuracy. [ABSTRACT FROM AUTHOR]

Drug resistance to almost all antibiotics of Shigella flexneri , a major cause of shigellosis in developing countries, necessitates continuous discovery of novel therapeutics. This study reports a structure-function analysis of a potential drug target serine acetyltransferase (CysE), an enzyme of de novo cysteine biosynthesis pathway that is absent in humans. Analysis of CysE sequences of S. flexneri species and serotypes displayed only two variants that differed by a single amino acid substitution at position 241. Structural inspection of the available crystal structure disclosed this site to be distinct from the substrate/cofactor binding pockets or dimer/trimer interfaces. This study discovers that V241 variant of S. flexneri CysE has nearly null enzymatic activity. The observation is explained by molecular dynamic studies which reveal that the disorder generated by A241V substitution is the basis of dissociation of the quaternary assembly of S. flexneri CysE leading to loss of enzymatic activity and stability. The study provides the first evidence that position 241 of CysE, affects the catalytic efficiency of enzyme and suggests this locus as a ‘hot spot’ for the propagation of conformational changes. It may be postulated that transient quaternary structure of CysE maybe another mechanism for regulating the intracellular level of cysteine. [ABSTRACT FROM AUTHOR]