RSM and ANN-Based Optimized Ultrasound-Assisted Extraction of Functional Components from Olive Fruit (cv Arbequina): Assessment of Antioxidant Attributes and GC-MS Metabolites Profiling.

The current study devises an optimized ethanolic extraction for efficient recovery of high-value components from Pakistani olives (cv. Arbequina) using response surface methodology (RSM) and artificial neural networking (ANN). Four factors such as time, temperature, solvent concentration, and solute weight (g/100 mL) were evaluated as independent variables for determining the response (% yield). The results obtained under optimum extraction conditions such as duration (25 min), temperature (45 °C), solvent concentration (65 %; ethanol: water v/v), and solute (7.50 g/100 mL) offered bioactives extract yield of 40.96 % from Arbiquina olives. The analysis of variance (ANOVA) for the RSM model showed significant p-values and a correlation coefficient (R2) of 0.9960, confirming model's reliability. The results of ANN, which employed the multilayer perceptron design, were fairly in line with the findings of the experiments. The antioxidant characteristics and GC-MS metabolite profile of the obtained extracts were examined. Arbequina olive extract (AOE) demonstrated very good antioxidant ability in terms of total phenolic, total flavonoid contents, and DPPH radical scavenging. The GC-MS analysis of AOE confirmed the presence of several bioactives, including oleic acid (36.22 %), hydroxytyrosol (3.95 %), tyrosol (3.32 %), ß-sitosterol (2.10 %), squalene (1.10 %), sinapic acid (0.67 %), α-tocopherol (0.66 %), vanillic acid (0.56 %), 3,5-di-tert-butylcatechol (0.31 %), and quercetin (0.21 %). The suggested optimized extraction method can be employed to efficiently extract a wide variety of high-value components from olives with potential for nutraceutical applications.

Kinetic Barrier to Enzyme Inhibition Is Manipulated by Dynamical Local Interactions in E. coli DHFR.

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Genetic variation and population structure of Fasciola hepatica: an in silico analysis.

Fasciola hepatica is a trematode leading to heavy economic setbacks to the livestock sector globally. The population's genetic information and intimate kinship level are frequently assessed using analysis of mitochondrial DNA. In this analysis, we retrieved cox1 (n = 247) and nad1 (n = 357) sequences of F. hepatica from the NCBI GenBank database and aligned the sequences with the respective reference sequences using MEGA software. The median joining network was drawn using PopArt software while neutrality and diversity indices were estimated with the help of DnaSp software. Neighbor-joining phylogenetic tree was constructed using the MEGA software package. A total of 46 and 98 distinctive haplotypes were observed for cox1 and nad1 genes, respectively. Diversity indices indicated high haplotype and nucleotide diversities in both genes. Positive Tajima's D and Fu's Fs values were found for the entire population of both the genes under study. The cox1 and nad1 gene segments in this study showed high Tajima's D values, suggesting a low likelihood of future population growth. The Tajima's D value of the nad1 gene sequence is lower (2.14910) than that of the cox1 gene sequence (3.40314), which suggests that the former is growing at a slower rate. However, the region-wise analysis revealed that both the cox1 and nad1 genes showed deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. Furthermore, the overall host-wise analysis showed positive and significant Tajima's D values for the cox1 and nad1 gene sequences. To the best of our knowledge, this is the first attempt to provide insights into genetic variations and population structure of F. hepatica at a global scale using cox1 and nad1 genes. Our findings suggest the existence of specific variants of F. hepatica in different parts of the world and provide information on the molecular ecology of F. hepatica. The results of this study also mark a critical development in upcoming epidemiological investigations on F. hepatica and will also contribute to understanding the global molecular epidemiology and population structure of F. hepatica.

DHFR Mutants Modulate Their Synchronized Dynamics with the Substrate by Shifting Hydrogen Bond Occupancies.

Antibiotic resistance is a global health problem in which mutations occurring in functional proteins render drugs ineffective. The working mechanisms of the arising mutants are seldom apparent; a methodology to decipher these mechanisms systematically would render devising therapies to control the arising mutational pathways possible. Here we utilize Cα-Cß bond vector relaxations obtained from moderate length MD trajectories to determine conduits for functionality of the resistance conferring mutants of Escherichia coli dihydrofolate reductase. We find that the whole enzyme is synchronized to the motions of the substrate, irrespective of the mutation introducing gain-of-function or loss-of function. The total coordination of the motions suggests changes in the hydrogen bond dynamics with respect to the wild type as a possible route to determine and classify the mode-of-action of individual mutants. As a result, nine trimethoprim-resistant point mutations arising frequently in evolution experiments are categorized. One group of mutants that display the largest occurrence (L28R, W30G) work directly by modifying the dihydrofolate binding region. Conversely, W30R works indirectly by the formation of the E139-R30 salt bridge which releases energy resulting from tight binding by distorting the binding cavity. A third group (D27E, F153S, I94L) arising as single, resistance invoking mutants in evolution experiment trajectories allosterically and dynamically affects a hydrogen bonding motif formed at residues 59-69-71 which in turn modifies the binding site dynamics. The final group (I5F, A26T, R98P) consists of those mutants that have properties most similar to the wild type; these only appear after one of the other mutants is fixed on the protein structure and therefore display clear epistasis. Thus, we show that the binding event is governed by the entire enzyme dynamics while the binding site residues play gating roles. The adjustments made in the total enzyme in response to point mutations are what make quantifying and pinpointing their effect a hard problem. Here, we show that hydrogen bond dynamics recorded on sub-µs time scales provide the necessary fingerprints to decipher the various mechanisms at play.

LR-AKAP: A Lightweight and Robust Security Protocol for Smart Home Environments.

For the betterment of human life, smart Internet of Things (IoT)-based systems are needed for the new era. IoT is evolving swiftly for its applications in the smart environment, including smart airports, smart buildings, smart manufacturing, smart homes, etc. A smart home environment includes resource-constrained devices that are interlinked, monitored, controlled, and analyzed with the help of the Internet. In a distributed smart environment, devices with low and high computational power work together and require authenticity. Therefore, a computationally efficient and secure protocol is needed. The authentication protocol is employed to ensure that authorized smart devices communicate with the smart environment and are accessible by authorized personnel only. We have designed a novel, lightweight secure protocol for a smart home environment. The introduced novel protocol can withstand well-known attacks and is effective with respect to computation and communication complexities. Comparative, formal, and informal analyses were conducted to draw the comparison between the introduced protocol and previous state-of-the-art protocols.

Molecular markers for the detection of pathogenic and food poisoning potential of methicillin resistant Staphylococcus aureus isolated from wounds of hospitalized patients.

Pathogenic strains of Staphylococcus aureus are mostly resistant to methicillin and they can cause severe infections. The current study was planned to assess the food poisoning potential of pathogenic, methicillin resistant Staphylococcus aureus by molecular detection of enterotoxin A (Eta) gene. A total of 100 septic wound samples from patients admitted in surgical ward (n=50) and burn unit (n=50) of Mayo Hospital Lahore were collected aseptically. These samples were processed primarily for bacterial growth on nutrient agar and purified on mannitol salt agar where twenty (20) samples showed pin-point colonies with yellow discoloration of media. Moreover, isolates were further characterized on the basis of microscopic appearance and biochemical assays where fourteen (14) isolates were declared Staphylococcus. DNA of these isolates were subjected to 16S rRNA gene amplification and sequences of S. aureus were submitted to NCBI GenBank viz., MW344063.1, MW341438.1, MW344064.1, MW344065.1, MW341439.1, MW341440.1, MW345971.1, MW345972.1, MW345973.1, MW716458.1. All the isolates (n=10) demonstrated molecular confirmation of pathogenicity and methicillin resistance by amplification of Coa and mecA gene. Out of these ten isolates, three amplified enterotoxin A (Eta) gene were confirmed. It is concluded that enterotoxin A of S. aureus which causes food poisoning is present in pathogenic, methicillin resistant S. aureus isolated from various wounds infections.

Tuning the Surface Chemistry of Second-Harmonic-Active Lithium Niobate Nanoprobes Using a Silanol-Alcohol Condensation Reaction.

The surface functionalization of nanoparticles (NPs) is of great interest for improving the use of NPs in, for example, therapeutic and diagnostic applications. The conjugation of specific molecules with NPs through the formation of covalent linkages is often sought to provide a high degree of colloidal stability and biocompatibility, as well as to provide functional groups for further surface modification. NPs of lithium niobate (LiNbO3) have been explored for use in second-harmonic-generation (SHG)-based bioimaging, expanding the applications of SHG-based microscopy techniques. The efficient use of SHG-active LiNbO3 NPs as probes will, however, require the functionalization of their surfaces with molecular reagents such as polyethylene glycol and fluorescent molecules to enhance their colloidal and chemical stability and to enable a correlative imaging platform. Herein, we demonstrate the surface functionalization of LiNbO3 NPs through the covalent attachment of alcohol-based reagents through a silanol-alcohol condensation reaction. Alcohol-based reagents are widely available and can have a range of terminal functional groups such as carboxylic acids, amines, and aldehydes. Attaching these molecules to NPs through the silanol-alcohol condensation reaction could diversify the reagents available to modify NPs, but this reaction pathway must first be established as a viable route to modifying NPs. This study focuses on the attachment of a linear alcohol functionalized with carboxylic acid and its use as a reactive group to further tune the surface chemistry of LiNbO3 NPs. These carboxylic acid groups were reacted to covalently attach other molecules to the NPs using copper-free click chemistry. This derivatization of the NPs provided a means to covalently attach polyethylene glycols and fluorescent probes to the NPs, reducing NP aggregation and enabling multimodal tracking of SHG nanoprobes, respectively. This extension of the silanol-alcohol condensation reaction to functionalize the surfaces of LiNbO3 NPs can be extended to other types of nanoprobes for use in bioimaging, biosensing, and photodynamic therapies.

N-Terminus of the Third PDZ Domain of PSD-95 Orchestrates Allosteric Communication for Selective Ligand Binding.

PDZ domains constitute common models to study single-domain allostery without significant structural changes. The third PDZ domain of PSD-95 (PDZ3) is known to have selective structural features that confer unique modulatory roles to this unit. In this model system, two residues, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T-H372A mutations change ligand preferences from class I (T/S amino acid at position -2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for wild-type, H372A, and G330T single mutants and a double mutant of PDZ3 in the absence and presence of both types of ligands. With the combination of free-energy difference calculations and a detailed analysis of MD trajectories, "class switching" and "class bridging" behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. We show that the dynamics of the charged N-terminus plays a fundamental role in determining the binding preferences in PDZ3 by altering the electrostatic energy. These findings are corroborated by simulations on N-terminus-truncated versions of these systems. The dynamical allostery orchestrated by the N-terminus offers a fresh perspective to the study of communication pathways in proteins.

Distribution and antibiotic sensitivity pattern of Mycobacterium tuberculosis isolates from children, enrolled in a tertiary care hospital.

The present study was planned to assess the distribution of tuberculosis in children and evaluate the antimycobacterial sensitivity pattern of Mycobacterium tuberculosis (MTB) isolates from pediatric patients. A total number of 1718 pediatric patients suspected of Mycobacterium tuberculosis were enrolled in the Institute of Child Health and Children's Hospital, Lahore during 2016-17. Out of 1718, only 710 different types of samples were tested for MTB. The samples were processed using bacteriology and GeneXpert along with the chest X-ray and clinical picture of the patients. The sensitivity pattern of Streptomycin, Isoniazid, Rifampicin and Ethambutol (SIRE) was determined using BACTEC MGIT 960. Total patients were divided into four groups including group A (birth to 12 months), B (1 to 5 years), C (6 to 10 years), and D (11 to 15 years). Out of 710, 106 (55 females and 51 males) were declared positive and 604 negative for tuberculosis. Out of 106 positive cases, 89 (83.96%) were sensitive to Rifampicin and 17 (16.04%) were resistant. Only, 04 (3.77%) were resistant to both Rifampicin and Isoniazid and declared as multidrug-resistant (MDR). It was concluded that children of age 11 to 15 years were more prone to MTB and a minimum percentage of MDR isolates was recorded in age group A (birth to 12 months).

Appraisal of anti-mycobacterial potential against MDR-MTB in pediatric patients, cytotoxicity and mutagenicity of Aloe vera and Allium sativum.

The purpose of the studies was to evaluate an in-vitro anti-mycobacterial activity of Aloe vera and Allium sativum against MDR-MTB, their cytotoxicity and mutagenicity. Four extracts of Aloe vera and Allium sativum were prepared by Soxhlet apparatus and their minimum inhibitory concentrations (MIC's) were determined by BACTEC MGIT960 system against multi drug resistant Mycobacterium tuberculosis (MDR-MTB) isolates, collected from pediatric patients. Fractions of Aloe vera and Allium sativum extracts were separated using glass column chromatography, followed by evaluation of cytotoxicity and mutagenicity by tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Ames test, respectively. Out of four extracts, ethanol extracts of Aloe vera and Allium sativum exhibited activity at MIC 5mg/mL to 7mg/mL and 3mg/mL to 5mg/mL, respectively and IC50 by MTT assay for combination of all fractions were 278.3mcg/100µL and 270.8mcg/100µL and in Ames assay M.I of TA98 were 0.14 and 0.07 and M.I of TA100 were 1.14 and 0.44, respectively. Aloe vera and Allium sativum extracts showed anti-mycobacterial activity against MDR-MTB isolates so, MIC of ethanol extracts of each plant and fractions of column chromatography had been checked. The MTT and Ames tests depicted that ethanol extracts of Aloe vera and Allium sativum were non-cytotoxic and non-mutagenic, and can be used in treatment of patients suffering from MDR-MTB.

High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection.

Evolutionary fitness landscapes of several antibiotic target proteins have been comprehensively mapped showing strong high-order epistasis between mutations, but understanding these effects at the biochemical and structural levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim-induced selection. To facilitate this, we developed a new in vitro assay for rapidly characterizing DHFR steady-state kinetics. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct changes in the catalytic efficiencies of mutated DHFR enzymes. Next, we measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the high-order epistasis in catalytic power of DHFR (kcat and Km) creates a rugged fitness landscape under trimethoprim selection. Taken together, our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.

Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2.

The sphingosine-1-phosphate receptors (S1PRs) are a well-studied class of transmembrane G protein-coupled sphingolipid receptors that mediate multiple cellular processes. However, S1PRs have not been previously reported to be involved in the genetic etiology of human traits. S1PR2 lies within the autosomal-recessive nonsyndromic hearing impairment (ARNSHI) locus DFNB68 on 19p13.2. From exome sequence data we identified two pathogenic S1PR2 variants, c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys). Each of these variants co-segregates with congenital profound hearing impairment in consanguineous Pakistani families with maximum LOD scores of 6.4 for family DEM4154 and 3.3 for family PKDF1400. Neither S1PR2 missense variant was reported among ∼120,000 chromosomes in the Exome Aggregation Consortium database, in 76 unrelated Pakistani exomes, or in 720 Pakistani control chromosomes. Both DNA variants affect highly conserved residues of S1PR2 and are predicted to be damaging by multiple bioinformatics tools. Molecular modeling predicts that these variants affect binding of sphingosine-1-phosphate (p.Arg108Pro) and G protein docking (p.Tyr140Cys). In the previously reported S1pr2(-/-) mice, stria vascularis abnormalities, organ of Corti degeneration, and profound hearing loss were observed. Additionally, hair cell defects were seen in both knockout mice and morphant zebrafish. Family PKDF1400 presents with ARNSHI, which is consistent with the lack of gross malformations in S1pr2(-/-) mice, whereas family DEM4154 has lower limb malformations in addition to hearing loss. Our findings suggest the possibility of developing therapies against hair cell damage (e.g., from ototoxic drugs) through targeted stimulation of S1PR2.

MODE-TASK: large-scale protein motion tools.

Summary: MODE-TASK, a novel and versatile software suite, comprises Principal Component Analysis, Multidimensional Scaling, and t-Distributed Stochastic Neighbor Embedding techniques using Molecular Dynamics trajectories. MODE-TASK also includes a Normal Mode Analysis tool based on Anisotropic Network Model so as to provide a variety of ways to analyse and compare large-scale motions of protein complexes for which long MD simulations are prohibitive. Beside the command line function, a GUI has been developed as a PyMOL plugin. Availability and implementation: MODE-TASK is open source, and available for download from https://github.com/RUBi-ZA/MODE-TASK. It is implemented in Python and C++. It is compatible with Python 2.x and Python 3.x and can be installed by Conda. Supplementary information: Supplementary data are available at Bioinformatics online.

Unraveling the Motions behind Enterovirus 71 Uncoating.

Enterovirus 71 can be a severe pathogen in small children and immunocompromised adults. Virus uncoating is a critical step in the infection of the host cell; however, the mechanisms that control this process remain poorly understood. We applied normal mode analysis and perturbation response scanning to several complexes of the virus capsid and present a coarse-graining approach to analyze the full capsid. We show that our method offers an alternative to expressing the system as a set of rigid blocks and accounts for the interconnection between nodes within each subunit and protein interfaces across the capsid. In our coarse-grained approach, the modes associated with capsid expansion are captured in the first three nondegenerate modes and correspond to the changes observed in structural studies of the virus. We show that the resolution of the analysis may be modified without losing information on the global motions leading to uncoating. Perturbation response scanning revealed that a protomer cannot serve as a functional unit to explain deformations of the capsid. Instead, we define a pentamer as the minimum functional unit to investigate changes within the capsid. From the modal analysis and perturbation response scanning, we locate a hotspot region surrounding the fivefold axis. The range of the effect of these single, hotspot residues extend to 140 Å. The perturbation of internal capsid residues in this region displayed greatest propensity to capsid expansion, thus indicating the significant role that the RNA genome may play in triggering uncoating.

MD-TASK: a software suite for analyzing molecular dynamics trajectories.

SUMMARY: Molecular dynamics (MD) determines the physical motions of atoms of a biological macromolecule in a cell-like environment and is an important method in structural bioinformatics. Traditionally, measurements such as root mean square deviation, root mean square fluctuation, radius of gyration, and various energy measures have been used to analyze MD simulations. Here, we present MD-TASK, a novel software suite that employs graph theory techniques, perturbation response scanning, and dynamic cross-correlation to provide unique ways for analyzing MD trajectories. AVAILABILITY AND IMPLEMENTATION: MD-TASK has been open-sourced and is available for download from https://github.com/RUBi-ZA/MD-TASK , implemented in Python and supported on Linux/Unix. CONTACT: o.tastanbishop@ru.ac.za.

Mutations in TBC1D24, a gene associated with epilepsy, also cause nonsyndromic deafness DFNB86.

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.

FbpA iron storage and release are governed by periplasmic microenvironments.

Ferric binding protein (FbpA) is part of an elaborate iron piracy mechanism evolved in Gram-negative bacteria, shuttling iron in the periplasmic space, from the outer to the cytoplasmic membrane side. We address how the dissociation process of iron is facilitated, since the binding constant of iron is on the order of 1018 M-1 at 6.5 pH and 200 mM ionic strength (IS). We monitor the conformational preferences of FbpA by extensive molecular dynamics (MD) simulations under conditions where IS, charge states of iron coordinating tyrosines and pH are varied, as well as when a mutation is introduced at an allosteric site. Steered MD is utilized to predict the binding affinity of iron. After triggering lobe opening by changing the charge states of tyrosines, the conformations adopted and the iron binding affinity still depend on pH, IS and allosteric interactions. To relate the observed conformational changes to the environmental conditions that might be encountered in the periplasmic space, we offer a plausible model that couples electrostatic potential distribution to the mechanical motions invoked. Although low pH/IS and allosteric perturbations decrease the affinity of iron, it remains high for spontaneous dissociation. However, the conformational changes modulated by the environmental conditions expose iron for chelation. Our study provides a quantitative dimension and molecular details to interpret the contribution of possible environmental conditions present in the periplasmic space to iron dissociation from FbpA, opening up the opportunity of modulating function via allosteric mutations or altering environmental conditions, thus offering a new route to developing strategies towards antibiotic resistance by targeting nutritional requirements.

Increased substrate affinity in the Escherichia coli L28R dihydrofolate reductase mutant causes trimethoprim resistance.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme with an essential role in cell metabolism. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is a precursor for purine and thymidylate synthesis. Several DHFR targeting antifolate drugs including trimethoprim, a competitive antibacterial inhibitor, have therefore been developed and are clinically used. Evolution of resistance against antifolates is a common public health problem rendering these drugs ineffective. To combat the resistance problem, it is important to understand resistance-conferring changes in the DHFR structure and accordingly develop alternative strategies. Here, we structurally and dynamically characterize Escherichia coli DHFR in its wild type (WT) and trimethoprim resistant L28R mutant forms in the presence of the substrate and its inhibitor trimethoprim. We use molecular dynamics simulations to determine the conformational space, loop dynamics and hydrogen bond distributions at the active site of DHFR for the WT and the L28R mutant. We also report their experimental kcat, Km, and Ki values, accompanied by isothermal titration calorimetry measurements of DHFR that distinguish enthalpic and entropic contributions to trimethoprim binding. Although mutations that confer resistance to competitive inhibitors typically make enzymes more promiscuous and decrease affinity to both the substrate and the inhibitor, strikingly, we find that the L28R mutant has a unique resistance mechanism. While the binding affinity differences between the WT and the mutant for the inhibitor and the substrate are small, the newly formed extra hydrogen bonds with the aminobenzoyl glutamate tail of DHF in the L28R mutant leads to increased barriers for the dissociation of the substrate and the product. Therefore, the L28R mutant indirectly gains resistance by enjoying prolonged binding times in the enzyme-substrate complex. While this also leads to slower product release and decreases the catalytic rate of the L28R mutant, the overall effect is the maintenance of a sufficient product formation rate. Finally, the experimental and computational analyses together reveal the changes that occur in the energetic landscape of DHFR upon the resistance-conferring L28R mutation. We show that the negative entropy associated with the binding of trimethoprim in WT DHFR is due to water organization at the binding interface. Our study lays the framework to study structural changes in other trimethoprim resistant DHFR mutants.

Efficient Synthesis of Novel Pyridine-Based Derivatives via Suzuki Cross-Coupling Reaction of Commercially Available 5-Bromo-2-methylpyridin-3-amine: Quantum Mechanical Investigations and Biological Activities.

The present study describes palladium-catalyzed one pot Suzuki cross-coupling reaction to synthesize a series of novel pyridine derivatives 2a-2i, 4a-4i. In brief, Suzuki cross-coupling reaction of 5-bromo-2-methylpyridin-3-amine (1) directly or via N-[5-bromo-2-methylpyridine-3-yl]acetamide (3) with several arylboronic acids produced these novel pyridine derivatives in moderate to good yield. Density functional theory (DFT) studies were carried out for the pyridine derivatives 2a-2i and 4a-4i by using B3LYP/6-31G(d,p) basis with the help of GAUSSIAN 09 suite programme. The frontier molecular orbitals analysis, reactivity indices, molecular electrostatic potential and dipole measurements with the help of DFT methods, described the possible reaction pathways and potential candidates as chiral dopants for liquid crystals. The anti-thrombolytic, biofilm inhibition and haemolytic activities of pyridine derivatives were also investigated. In particular, the compound 4b exhibited the highest percentage lysis value (41.32%) against clot formation in human blood among all newly synthesized compounds. In addition, the compound 4f was found to be the most potent against Escherichia coli with an inhibition value of 91.95%. The rest of the pyridine derivatives displayed moderate biological activities.