Preparation and application of hydroxypropyl methylcellulose blended with beeswax and essential oil edible coating to enhance the shelf life of sweet cherries.

The study involved preparing and applying edible nano-emulsion coatings containing hydroxypropyl methylcellulose (HPMC), beeswax (BW), and essential oils (thyme, cinnamon, clove, and peppermint) onto sweet cherries. The application was conducted at 4 °C, and the coated cherries were stored for 36 days. This research examines synthesized nano-emulsions physicochemical properties and antibacterial and antifungal activities (C1, C2, and C3). Additionally, it evaluates the quality parameters of control and coated sweet cherry samples. The features of the three edible coatings were assessed, and the findings from the zeta sizer, zeta potential, FTIR, and SEM analyses were deemed satisfactory. It was observed that the application of nano-emulsion coating C1 yielded positive results in maintaining quality attributes such as total suspended solids (TSS), total solids (TS), color, weight loss, respiration rate, firmness, total phenolic contents, and sensory evaluations. Nano-emulsion coating C1 demonstrated efficacy as an antibacterial and antifungal agent against foodborne pathogens E. coli and A. niger, respectively. The current research results are promising and applicable in food industries. The implications suggest that composite nano-emulsion, specifically nano-emulsion edible coatings, can be extensively and effectively used to preserve the quality and shelf life of fruits and vegetables. Furthermore, the environmental waste from conventional food packaging will be minimized using edible packaging applications.

Association of Trace Metals in Patients with Schizophrenia.

OBJECTIVE: To compare the serum concentrations of copper, iron and zinc in schizophrenic patients with healthy individuals. STUDY DESIGN: Cross-sectional comparison study. PLACE AND DURATION OF STUDY: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan. The study was conducted from July to December 2020. METHODOLOGY: Among 115 participants in the study, schizophrenic patients and healthy subjects were 35 and 80, respectively. Copper and zinc were measured by using atomic absorption spectrophotometry. Serum Iron measurement was done on ADVIA 1800 chemistry auto analyser. Comparison of these trace metals among patients with schizophrenia and healthy subjects, by using Mann-Whitney U-test and correlation with the duration of disease, was analysed by the application of Spearman's correlation. RESULTS: Males accounted for 25 (71.4%) and females were 10 (28.6%) in diseased group; while in healthy subjects, males accounted for 54 (67.5%) and females 26 (32.5%). Copper, iron and zinc levels were noted to be significantly reduced in schizophrenic patients, when compared with healthy subjects (p <0.001). CONCLUSION: This study could offer an additional clue in the diagnosis and possible role of trace metals in pathophysiology and progression of many psychiatric illnesses, particularly schizophrenia. Key Words: Psychiatric illness, Schizophrenia, Trace metals.

Novel Lysine-Based Thioureas as Mechanism-Based Inhibitors of Sirtuin 2 (SIRT2) with Anticancer Activity in a Colorectal Cancer Murine Model.

Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile synthesis of novel lysine-derived thioureas as mechanism-based SIRT2 inhibitors with anticancer activity. Compounds AF8, AF10, and AF12 selectively inhibited SIRT2 with IC50 values of 0.06, 0.15, and 0.08 µM, respectively. Compounds AF8 and AF10 demonstrated broad cytotoxicity amongst cancer cell lines, but minimal toxicity in noncancerous cells. AF8 and AF10 inhibited the anchorage-independent growth of human colorectal cancer cell line HCT116 with GI50 values of ∼7 µM. Furthermore, AF8 potently inhibited tumor growth in a HCT116 xenograft murine model, supporting that SIRT2 is a viable therapeutic target for colorectal cancer.

Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor.

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with subcellular and temporal resolution. Ratiometric sensors hold the additional promise of meaningful quantification of intracellular hydrogen peroxide levels as a function of time, a longstanding goal in the field of redox signaling. To date, studies that have connected the magnitudes of observed ratios with peroxide concentrations have either examined suspensions of cells or small numbers of adherent cells (∼10). In this work, we examined the response of all cells in several microscopic fields of view to an identical perturbation and observed a striking degree of heterogeneity of fluorescence ratios from individual cells. The expression level of the probe and phase within the cell cycle were each examined as potential contributors to the observed heterogeneity. Higher ratiometric responses correlated with greater expression levels of the probe and phase in the cell cycle were also shown to influence the magnitude of response. To aid in the interpretation of experimental observations, we incorporated the reaction of the reduced probe with peroxide and the reactions of the oxidized probe with glutathione and glutaredoxin into a larger kinetic model of peroxide metabolism. The predictions of the kinetic model suggest possible explanations for the experimental observations. This work highlights the importance of a systems-level approach to understanding the output of genetically encoded sensors that function via redox reactions involving thiol and disulfide groups.

Exploring nicotinamide cofactor promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) using natural variation within the phosphate binding loop. Structure and activity of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13.

Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg-Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln-His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872-878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr-Ser and Gln-Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix-loop-helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.

Mutations of an NAD(P)H-dependent flavoprotein monooxygenase that influence cofactor promiscuity and enantioselectivity.

The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2'-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(k cat/K m NADH)/k cat/K m NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced K m for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e., compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.

Genome sequence of Stenotrophomonas maltophilia PML168, which displays Baeyer-Villiger monooxygenase activity.

Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase.

A flavoprotein monooxygenase that catalyses a Baeyer-Villiger reaction and thioether oxidation using NADH as the nicotinamide cofactor.

A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD-containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin-containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer-Villiger oxidation of the model substrate bicyclo[3.2.0]hept-2-en-6-one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The K(M) and k(cat) values for NADH were 23.7±9.1 µM and 0.029 s(-1) and 27.3±5.3 µM and 0.022 s(-1) for NADPH. However, k(cat) /K(M) value for the ketone substrate in the presence of 100 µM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2-oxa and 3-oxa lactone products of Baeyer-Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15 %. SMFMO also catalysed the NADH-dependent transformation of prochiral aromatic thioethers, giving in the best case, 80 % ee for the transformation of p-chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2'-phosphate on the NADPH ribose in related NADPH-dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single-component, flavoprotein monooxygenases that catalyse NADH-dependent oxidations from which possible sequences and strategies for developing NADH-dependent biocatalysts for asymmetric oxygenation reactions might be identified.

Coccolithophores: functional biodiversity, enzymes and bioprospecting.

Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

The psychological and psychosocial impact of the Pakistan Kashmir earthquake after 8 months: a preliminary evaluation by PACTT.

Natural disasters are complex events that challenge the coping abilities of individuals and communities (Alexander, 2005). They are characterised by substantial loss, physical injury and economic hardship, as well as by extensive internal displacement and damage to the infrastructure, as exemplified by the Pakistan Kashmir earthquake of 8 October 2005. Measuring 7.6 on the Richter scale, it affected an area of 30 000 square miles and a population of 3.6 million. Approximately 90 000 were killed, 200 000 were injured and 3.5 million were left homeless (Khan, 2006). Based on a literature review and estimates from the World Health Organization (WHO), the National Plan of Action for Mental Health and Psychosocial Relief of Earthquake Survivors anticipated high levels of trauma-related psychopathology (Rana et al, 2006).

Psychological trauma in a relief worker--a case report from earthquake-struck areas of north Pakistan.

Vicarious traumatization is now a well-known entity and may have negative influences on those that are involved in rescue efforts in any disaster or traumatic events. Healthcare workers work with trauma survivors and witness an immense array of gruesome and ghastly images. This work has the potential to cause those engaged in rescue efforts to become affected subconsciously. Job-related stress may cause psychological symptoms in care providers who provide support and listen to the survivors' account of trauma. A therapist working in disaster situations may become a victim of psychological anguish--undermining their physical and mental well-being as well as their profession, adversely affecting their traumatized patients, and leading to a counter-productive therapist-survivor relationship. This significant theme of secondary trauma must be recognized in relief workers at early stages and must be addressed at an individual as well as organizational level. The key may lie in turning to social supports, adapting positive coping mechanisms, and subsequently seeking mental health consultation. Further research is required in this area to determine the best resolution.

The structure of chorismate synthase reveals a novel flavin binding site fundamental to a unique chemical reaction.

The crystal structure of chorismate synthase (CS) from Streptococcus pneumoniae has been solved to 2.0 A resolution in the presence of flavin mononucleotide (FMN) and the substrate 5-enolpyruvyl-3-shikimate phosphate (EPSP). CS catalyses the final step of the shikimate pathway and is a potential therapeutic target for the rational design of novel antibacterials, antifungals, antiprotozoals, and herbicides. CS is a tetramer with the monomer possessing a novel beta-alpha-beta fold. The interactions between the enzyme, cofactor, and substrate reveal the structural reasons underlying the unique catalytic mechanism and identify the amino acids involved. This structure provides the essential initial information necessary for the generation of novel anti-infective compounds by a structure-guided medicinal chemistry approach.

Novel targets for antibiotic drug design.

The urgent need for new antibiotics has led to an explosion in the number and diversity of antibiotic drug targets under investigation. The majority of such targets are enzymes required for essential cellular functions. Often, such novel targets are completely unexploited for antibiotic therapy and therefore have the advantage of avoiding current resistance mechanisms. In general, the most advanced novel targets are drawn from processes where an existing antibacterial compound has validated that process for antibiotic therapy. This review describes a number of the more promising targets under development.