Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

The common severe Z mutation (E342K) of α₁-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α₁-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P₁₄P₁₂ and P₄, but not P₁₀P₈ or P₂P_1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P₁₄P₁₂ region self-inserts early during activation, while the interaction between P₆P₄ and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P₆P₄, but not P₁₄P₁₂, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol⁻¹.     

Simulating Microdosimetry in a Virtual Hepatic Lobule

The liver plays a key role in removing harmful chemicals from the body and is therefore often the first tissue to suffer potentially adverse consequences. To protect public health it is necessary to quantitatively estimate the risk of long-term low dose exposure to environmental pollutants. Animal testing is the primary tool for extrapolating human risk but it is fraught with uncertainty, necessitating novel alternative approaches. Our goal is to integrate in vitro liver experiments with agent-based cellular models to simulate a spatially extended hepatic lobule. Here we describe a graphical model of the sinusoidal network that efficiently simulates portal to centrilobular mass transfer in the hepatic lobule. We analyzed the effects of vascular topology and metabolism on the cell-level distribution following oral exposure to chemicals. The spatial distribution of metabolically inactive chemicals was similar across different vascular networks and a baseline well-mixed compartment. When chemicals were rapidly metabolized, concentration heterogeneity of the parent compound increased across the vascular network. As a result, our spatially extended lobule generated greater variability in dose-dependent cellular responses, in this case apoptosis, than were observed in the classical well-mixed liver or in a parallel tubes model. The mass-balanced graphical approach to modeling the hepatic lobule is computationally efficient for simulating long-term exposure, modular for incorporating complex cellular interactions, and flexible for dealing with evolving tissues.     

Aspergillus Ficuum Phytase: Partial Primary Structure,Substrate Selectivity,and Kinetic Characterization

Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the K_cmt was affected. The enzyme was able to release more than 512 of the total available P₁ from phytate in a 3.0 hr assay at 58°C, but the K_cmt dropped to 15Z of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca^±± and Fe^±±produced no effect on the catalytic rate of the enzyme, Cu^±, Cu^±±, Zn^±±, and Fe^±±± were found to be inhibitory. Mn^±± was observed to enhance enzyme activity by 33Z at 50 μM. Known inhibitors of acid phosphatases e. g. L (±)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Profiling Deleterious Non-synonymous SNPs of Smoker's Gene CYP1A1

CYP1A1 gene belongs to the cytochrome P450 family and is known better as smokers’ gene due to its hyperactivation as a consequence of long term smoking. The expression of CYP1A1 induces polycyclic aromatic hydrocarbon production in the lungs, which when over expressed, is known to cause smoking related diseases, such as cardiovascular pathologies, cancer, and diabetes. Single nucleotide polymorphisms (SNPs) are the simplest form of genetic variations that occur at a higher frequency, and are denoted as synonymous and non-synonymous SNPs on the basis of their effects on the amino acids. This study adopts a systematic in silico approach to predict the deleterious SNPs that are associated with disease conditions. It is inferred that four SNPs are highly deleterious, among which the SNP with rs17861094 is commonly predicted to be harmful by all tools. Hydrophobic (isoleucine) to hydrophilic (serine) amino acid variation was observed in the candidate gene. Hence, this investigation aims to characterize a candidate gene from 159 SNPs of CYP1A1.     

Effect of organic carbon sources and environmental factors on cell growth and lipid content of Pavlova lutheri

The present study aimed to investigate the effects of organic carbon sources, cultivation methods, and environmental factors on growth and lipid content of Pavlova lutheri for biodiesel production. In the 250-mL flask bioreactors, P. lutheri was cultivated in the modified artificial seawater (ASW) medium containing glucose, glycerol, sodium acetate, or sucrose as an organic carbon substrate. The effects of different growth conditions (phototrophic, mixotrophic, and heterotrophic) and environmental factors such as photoperiod, light intensity, and salinity were evaluated. Growth of P. lutheri was inhibited under heterotrophy but was enhanced in mixotrophy as compared to phototrophy. Biomass and lipid content of P. lutheri were significantly (p < 0.05) affected by changing photoperiod, light intensity, and salinity. Higher biomass concentration and lipid content were observed at a light intensity of 100 ± 2 μmol photons m−2 s−1, 18 h photoperiod, and 30% salinity, in a modified ASW medium supplemented with 10 mmol sucrose. An increase in biomass concentration from 320 ± 25.53 to 1106 ± 18.52 mg L−1 and high lipid content of 31.11 ± 1.65% (w/w) were observed with the optimized culture conditions, demonstrating a significant (p < 0.05) enhancement in biomass and lipid content due to the improved culture conditions. The present study emphasizes the possible use of sucrose for biomass and lipid production with P. lutheri under the optimized culture conditions. Using low-cost and relatively easy accessible feedstock such as sucrose would be a valuable alternative for growing microalgae with enhanced lipid content.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Biosorption of lead by cotton shells powder: Characterization and equilibrium modeling study

Lead (Pb) is a toxic heavy metal causing serious health risks to humans and animals. In the present study, cotton (Gossypium hirsutum L.) shells powder was used as adsorbent for the treatment of synthetic Pb-contaminated water. The batch scale biosorption capacity of cotton shells powder was evaluated to study the effects of Pb concentrations, adsorbent doses and contact time at constant pH (6) and temperature (25?°C). Results revealed that sorption of Pb increased (q?=?0.09–9.60?mg/g) with increasing Pb concentration (1–15?mg/L) and contact time (15–90?min) while decreasing adsorbent dose (1–0.1?g/100?mL). The maximum Pb removal (90%) was achieved at Pb concentration (1?mg/L), contact time (90?min) and adsorbent dose (1?g/100?mL). Freundlich isotherm model proved best fit for Pb sorption (R²?=?0.99). The cotton shells powder has microporous structure confirmed by SEM, and has BET surface area (45 m²/g) and pore size (2.3 µm). These surface moieties along with various functional groups (C-H, C-O, C=O, O-H, S=O) confirmed by FTIR analysis might involve in Pb removal by complexation and ion exchange mechanisms. The cotton shells powder biomass could be considered as promising adsorbent for the removal of Pb from contaminated water.     

Synthesis of quinoline derivatives as diabetic II inhibitors and molecular docking studies

In searchof the potenttherapeutic agent as an α-glucosidase inhibitor, we have synthesized twenty-five analogs (1–25) of quinoline-based Schiff bases as an inhibitoragainst α-glucosidase enzyme under positive control acarbose (IC₅₀ = 38.45 ± 0.80 µM). From the activity profile it was foundthat analogs 1, 2, 3, 4, 11, 12 and 20with IC₅₀values 12.40 ± 0.40, 9.40 ± 0.30, 14.10 ± 0.40, 6.20 ± 0.30, 14.40 ± 0.40, 7.40 ± 0.20 and 13.20 ± 0.40 µMrespectively showed most potent inhibition among the series even than standard drug acarbose (IC₅₀ = 38.45 ± 0.80 µM). Here in the present study analog 4 (IC₅₀ = 6.20 ± 0.30 µM) was found with many folds better α-glucosidase inhibitory activity than the reference drug. Eight analogs like 5, 7, 8, 16, 17, 22, 24 and 25 among the whole series displayed less than 50% inhibition. The substituents effects on phenyl ring thereby superficially established through SAR study. Binding interactions of analogs and the active site of ligands proteins were confirmed through molecular docking study. Spectroscopic techniques like ¹H NMR, ¹³C NMR and ESIMS were used for characterization.