Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Design,synthesis, and structure-activity relationship of a new class of amidinophenylurea-based factor VIIa inhibitors

Selective inhibition of coagulation factor VIIa has recently gained attraction as interesting approach towards antithrombotic treatment. Using parallel synthesis supported by structure-based design and X-ray crystallography, we were able to identify a novel series of amidinophenylurea derivatives with remarkable affinity for factor VIIa. The most potent compound displays a K(i) value of 23 nM for factor VIIa.     

QSAR study on solubility of alkanes in water and their partition coefficients in different solvent systems using PI index

The aqueous solubility (S(w)) of liquids and solids, expressed as log S(w) as well as their partition coefficients in different solvent systems viz. P(oct) (partition coefficient in octanol-water), P(16) (partition coefficient in water-hexadecane), P(alk) (partition coefficient in water-alkane), and P(cyc) (partition coefficient in water-cyclohexane), and aqueous solubility (S(w)) have been estimated using the PI (Padmakar-Ivan) index and the results compared with those obtained using the widely used Wiener index (W). Regression analysis of the data using n-alkanes show that the PI index gives better results than the W index.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

I n Vivo Evaluation of 5-ASA Colon-Specific Tablets Using Experimental-Induced Colitis Rat Animal Model

Colonic drug delivery is intended not only for local treatment in inflammatory bowel disease (IBD) but also for systemic delivery of therapeutics. Intestinal myeloperoxidase (MPO) determination could be used to estimate the average level of inflammation in colon as well as to determine the efficacy of drugs to be used in the treatment of inflammatory bowel diseases or study the specificity of dosage forms to be used for colonic targeting of anti-inflammatory drugs. Colonic prodrug sulfasalazine (SASP) gets metabolized to give 5-aminosalicylic acid (5-ASA), which is the active portion of SASP. However, when given orally, 5-ASA is absorbed in upper part of gastrointestinal tract (GIT) and not made available in colon. In the present study, colon-targeted delivery of 5-ASA was achieved by formulating tablets with two natural polymers namely guar gum and pectin using compression coating method. Colonic specificity of 5-ASA tablets (prepared using guar gum and pectin as polymers) was evaluated in vitro using simulated fluids mimicking in vivo environment as well as in vivo method using chemically (2,4,6-trinitrobenzenesulfonic acid and acetic acid)-induced colitis rat model. Both colon-specific formulations of 5-ASA (guar gum and pectin) were observed to be more effective in reducing inflammation in chemically induced colitis rat models when compared to colon-specific prodrug sulfasalazine as well as conventional 5-ASA administered orally.KEY WORDS: colitis, colon-specific drug delivery, myeloperoxidase     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

The DNA Replication,Repair, and Recombination Pathway Genes Modulating Yield and Stress Tolerance Traits in Chickpea

DNA replication, repair, and recombination (DRRR) are the fundamental processes required for faithful transmission of genetic information within and between generations. The DRRR genes protect the cells from potential mutations and damage during the developmental phases and stress conditions. Thus, these genes indirectly regulate diverse important agronomic traits in a crop plant. A genome-wide survey of six DRRR pathway genes, namely, DNA replication, Base Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Mismatch Excision Repair, and Non-Homologous End-Joining, identified 157 DRRR genes in chickpea. Phylogenetic analysis of these genes within the legume clades and model plant Arabidopsis identified 42 conserved DRRR genes exhibiting clade-specific evolutionary patterns. Integrating the gene-based association mapping with differential expression profiling identified the natural alleles of the potential DRRR genes, primarily regulating flowering and maturation time and involved in drought tolerance of chickpea. Identifying and understanding DRRR genes’ roles in regulating yield and stress tolerance traits in a vital grain legume like chickpea is requisite for its future crop improvement endeavors. Manipulation of promising functionally relevant DRRR genes will pave the way for simultaneous improvement in multiple beneficial agronomic traits in chickpea.

     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.     

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R² at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.