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Background Acute necrotizing encephalopathy (ANE) is a severe encephalopathy associated with acute viral infection. While most ANE cases are sporadic, pathogenic variants in the gene RAN binding protein 2 (RANBP2) have been identified as a major cause of familial or recurrent ANE (ANE1). Although sporadic ANE predominantly affects Asian children, ANE1 is very rare in east Asia. Case report A 1-year-7-month-old boy, born to unrelated Japanese parents, presented with a seizure and impaired consciousness after 3 days of fever. Brain magnetic resonance imaging (MRI) showed a characteristic involvement of the bilateral thalami, external capsules, insular cortices, and brainstem, suggesting ANE. He received intravenous steroids. Two months later, he had another episode of acute encephalopathy during respiratory syncytial virus infection, from which he recovered relatively well. The recurrent encephalopathic episodes and the characteristic MRI suggested ANE1. Genetic analyses revealed two variants: a rare heterozygous missense variant of RANBP2 [c.1754C>T; p.Thr585Met], and a thermolabile polymorphism in carnitine palmitoyltransferase 2 (CPT2) [c. 1055T>G; p.Phe352Cys]. Conclusion This is the first case of recurrent ANE with an RANBP2 mutation in Japan. The patient also harbored a CPT2 polymorphism that is linked to acute encephalopathy in Japanese patients. Thus, he had a genetic background with two susceptibility variants for acute encephalopathy, RANBP2 (frequent in the Caucasians), and CPT2 (frequent in the Japanese). Further studies are needed to fully discover the genetic predisposition to familial or recurrent ANE in the Asian population.

While a number of approaches toward multicomponent metal-organic frameworks have been reported, new strategies affording greater structural versatility and molecular precision are needed to replicate the sophisticated active sites found in enzymes. Here, we outline a general method for templating functional groups within framework pores using thermolabile ligand cross-linkers. We show that tertiary ester-based cross-linkers can be used to install well-defined carboxylic acid pairs at precise relative distances and orientations. The tertiary ester linkages remain intact during framework formation but are readily cleaved to reveal free carboxylic acids upon microwave heating. Successful cross-linker synthesis, framework incorporation, and thermolysis is demonstrated using the mesoporous, terphenyl expanded analogues of MOF-74. When short cross-linkers are used, modeling studies show that the carboxylic acids are installed in a single configuration down the pore channels, spaced ∼7 Å apart. These precisely positioned acid pairs can be used as synthetic handles to build up more complex cooperative active sites.

Colibacteriosis is an acute zoonotic disease manifested by septicaemia, toxemia, enteritis, body dehydration, and central nervous system damage. Depending on the presence of virulence factors and the nature of interaction with the intestinal mucosa, enterotoxigenic, enteroinvasive, enteropathogenic, and enterohemorrhagic E. coli are isolated. Enterotoxigenic strains of E. coli occupy one of the leading places in the etiological structure of calf colibacteriosis in many livestock farms of the Republic of Belarus. The main reason why this disease develops is the presence of thermolabile and thermostable toxins in the causative strain. The thermolabile toxin subunit B is a potent antigen that allows pet immunity to be acquired against E. coli-induced cattle diarrhea. Many foreign vaccines used against intestinal infections of cattle contain either a native or recombinant variant of the subunit B. As a result of the work, we have created a new strain of E. coli 42eLTB – the producer of the recombinant subunit B of the thermolabile toxin E. coli. The producing capacity of the obtained strain is 480 mg with 1 culture liquid liter, which exceeds the already known strains 1.37 times.

Thermolabile nature of commercially available vaccines necessitates their storage, transportation, and dissemination under refrigerated condition. Maintenance of continuous cold chain at every step increases the final cost of vaccines. Any breach in the cold chain even for a short duration results in the need to discard the vaccines. As a result, there is a pressing need for the development of thermostable vaccines. In this proof-of-concept study, we showed that E. coli curli-green fluorescent fusion protein remains stable in freeze-dried yeast powder for more than 18 and 12 months when stored at 30 °C and 37 °C respectively. Stability of the heterologous protein remains unaffected during the process of heat-inactivation and lyophilization. The mass of lyophilized yeast powder remains almost unchanged during the entire period of storage and expressed protein remains intact even after two cycles of freeze and thaws. The protease-deficient strain appears ideal for the development of whole recombinant yeast-based vaccines. The cellular abundance of expressed antigen in dry powder after a year was comparable to freshly lyophilized cells. Scanning electron microscopy showed the intact nature of cells in powdered form even after a year of storage at 30 °C. Observation made in this study showed that freeze-dry yeast powder can play a vital role in the development of thermostable vaccines. Key Points • Yeast-based vaccines can overcome problem of cold chain associated with conventional vaccines • Lyophilized yeast powder can be a simple way for long-term storage of immunogen(s) • Protease deficient strain is important for whole recombinant yeast-based vaccines

The purpose of this research was to assess native Lactic Acid Bacteria (LAB) for antimicrobial activities in relation to fish preservation. LAB were isolated from intestine of Oreochromis sp. Listeria monocytogenes, being a big microbial hazard of fresh as well as preserved fish food, was chosen against LAB antimicrobials. In the present study sixteen LAB like isolates from fish intestine were judged for their anti-listeria activities by agar well diffusion assay. Thirteen of them were antagonized Listeria monocytogenes (ATCC 657) by creating inhibition zone in nutrient agar plate. Antagonistic activities with inhibitory zones but not with growth circles clearly marked that inhibition was due to antimicrobials but not for competitive exclusion. LAB like bacteria in different forms like live cells, cell-free supernatant, alkaline cell- free supernatant and heated cell-free supernatant conferred antibacterial activities. No acid and hydrogen peroxide inhibition was reported. Except few, most of the alkaline and catalase treated culture supernatant when being heated to 90oC exhibited strong antagonism against the pathogenic strain. Susceptibility of Listeria monocytogenes to LAB antimicrobials seemed to be caused by both thermostable and thermolabile compounds and could be practiced in fisheries industry for food hygiene and also as biopreservatives.

The role of the DnaKJE and Trigger Factor (TF) chaperones in folding and refolding of proteins in Bacillus subtilis is studied. Bacterial luciferases of Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that DnaKJE takes part in folding but not in refolding of the thermal inactivated proteins. It is shown that TF takes part in the synthesis of thermolabile P. leiognathi luciferase, but significantly decreases the level of synthesis of thermostable P. luminescens luciferase.

The purpose of the work is a comparative study of the molecular composition of thermolabile (volatilizes at 300°C) and thermostable (volatilizes at 500°C) fractions of organic matter from humus horizons of typical chernozems by double-shot pyrolysis followed by gas chromatographic separation and mass detection of the products. The objects of the study are typical chernozems under different land use types differing in the prevalent trends of organic matter transformation. As is shown, polysaccharides, furfural, and methylfurfural are the major contributors to the pyrolysis products of the chernozem thermolabile matter. The largest share of the pyrolysis products of thermostable matter belongs to nitrogen-containing compounds. It is demonstrated for the first time that the principal component analysis of the data on molecular composition of thermolabile organic matter makes it possible to divide the studied chernozems under different land uses into the groups reflecting the quasi-equilibrium state of soil organic matter, its degradation, or accumulation.

Objective: The inkjet printing (IP) and fused deposition modeling (FDM) technologies have emerged in the pharmaceutical field as novel and personalized formulation approaches. Specific manufacturing factors must be considered in each adopted methodology, i.e. the development of suitable substrates for IP and the incorporation of highly thermostable active pharmaceutical compounds (APIs) for FDM. In this study, IP and FDM printing technologies were investigated for the fabrication of hydroxypropyl methylcellulose-based mucoadhesive films for the buccal delivery of a thermolabile model drug.Significance: This proof-of-concept approach was expected to provide an alternative formulation methodology for personalized mucoadhesive buccal films.Methods: Mucoadhesive substrates were prepared by FDM and were subjected to sequential IP of an ibuprofen-loaded liquid ink. The interactions between these processes and the performance of the films were evaluated by various analytical and spectroscopic techniques, as well as by in vitro and ex vivo studies.Results: The model drug was efficiently deposited by sequential IP passes onto the FDM-printed substrates. Significant variations were revealed on the morphological, physicochemical and mechanical properties of the prepared films, and linked to the number of IP passes. The mechanism of drug release, the mucoadhesion and the permeation of the drug through the buccal epithelium were evaluated, in view of the extent of ink deposition onto the buccal films, as well as the distribution of the API.Conclusions: The presented methodology provided a proof-of-concept formulation approach for the development of personalized mucoadhesive films.

For the drying process of a liquid dispersed product layer on a horizontal substrate, a model for the formation of a solid phase particle residue is proposed. The model is based on the construction of a fractal cluster on a bounded spatial lattice according to given rules. The model is constructed on the basis of ideas about the mechanism of interaction of particles during the existence of a layer of free liquid in the drying product. The fractal model, which has the property of self-similarity of the forming structure, allows modeling spatial structures on bounded lattices and spreading the data obtained on bounded lattices to the entire volume of the simulated residue. The article provides a brief overview of the main works related to the application of the fractal concept in the modeling of micro and macroscopic structures arising during the drying of liquid products. It is shown that in order to calculate the kinetics of drying liquid dispersed products on substrates, in the case of the formation of a certain layer of surface film and the formation of a certain spatial structure of solid particles, an important task is to determine the critical moisture content. The proposed model allows modeling layers with fractal dimension lying in the range from 1.64 to 2. This range corresponds to layers in the porosity range from layers with dense monodisperse particle stacking with a coordination number of 12, to structures similar to dendrite fractals, with porosity much higher than the ordered simple cubic particle stacking. The results of fractal cluster modeling and comparison of calculated and experimental values of porosity and critical moisture content obtained in the process of drying a suspension of optical bleach CD-2 and polymethylene-B-naphthalene sulfonate under different modes of convective drying of the product layer on a fluoroplastic horizontal substrate as products characterized by a close value of the initial concentration of the solid phase (≈12%), but different values of particle sizes and the obtained values of the fractal dimension of the surface film and the dry residue are presented.

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket's accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutants as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.

The thermolabile acetylene dicarboxylic acid has been introduced as linker in UiO-66 topology, synthetizing the compound with formula [Ce6O4(OH)4(ADC)6] and denoted as Ce-UiO-66-ADC MOF. The characterization by multi-technique approach coupled with computational modelling revealed a peculiar intrinsic defective nature related to the nature of the linker.

Enhancing proteins' thermostability is an important aspect of enzyme engineering. Many studies have investigated the properties that determine the proteins' thermostability. However, no consensus has emerged. To understand the mechanisms underlying the high thermostability of thermophilic proteins, we evaluated the relative importance of the amino acid frequencies in protein sequences for discriminating thermophilic and non-thermophilic proteins based on machine learning algorithms together with a three-step feature selection procedure and a principal component (PC) analysis to remove noisy and redundant information. Our results showed that the frequencies of oppositely charged amino acids, i.e., Lys and Glu, were higher in thermophilic proteins, suggesting that electrostatic interactions are fundamentally important for protein stabilization at high temperatures. Further, we found that the frequencies of uncharged polar amino acids, which are thermolabile or actively interact with water molecules, were lower in thermophilic proteins. Moreover, the frequencies of β-branched aliphatic amino acids tended to increase with increasing thermostability. Overall, these results suggest that proteins' thermostability is determined by a few protein features, which were well captured by the first two PCs. A classifier based on only the first two PCs achieved a high accuracy of 90%, suggesting that our classifier could be an effective and efficient tool for engineering stable proteins.

High temperature during reproductive stages of cotton is a major constraint to achieve potential yield of cotton. The aim of the present study was to determine comparative thermo-sensitivity of squaring and flowering, to optimize exogenous selenium for heat-stressed cotton, and to explore correlation among physiochemical and morphological attributes. The experiment was conducted during 2013 and repeated during 2014. The experiment was laid out in randomized complete block design under split plot arrangement and replicated thrice. Treatments were comprised of heat stress (H0 = no heat imposition; H1 = heat imposition at squaring; and H2 = heat imposition at flowering) in main plot and varying foliar selenium concentrations (Se0 = water spray/control; Se50 = 50; Se100 = 100; and Se150 = 150 mg L-1 selenium) in split plot. Significantly, more antioxidants, chlorophyll contents, water relation attributes, seed cotton yield, and lesser hydrogen peroxide were recorded with 150 mg L-1 foliar selenium compared with other concentrations under H0. While under H1 and H2, statistically similar and significantly more antioxidants, chlorophyll contents, water relation attributes, seed cotton yield, and lesser hydrogen peroxide were observed for 100 and 150 mg L-1 selenium compared with other selenium concentrations. Conclusively, H2 proved more thermolabile compared with H1, and application of 150 mg L-1 foliar selenium effectively alleviated adverse effects of heat. Moreover, we observed strong and significant associations of all physiochemical attributes with seed cotton yield.

Ukraine has a high level of industry, agriculture and consumption. As a result of underutilization of recovered materials and their products into the environment returns significant amount of waste that contaminate soil, water, air. Dumps occupy large areas, long ground beneath them put out of production, they pollute the environment. The main causes of waste, lack of effective processes and capacities of processing residues, spatial and chronological dissociation processes of waste and their use, lack of sufficient market. Of all the organic waste in Ukraine is one of the main places belong chicken droppings. Fresh droppings quickly oxidizes the soil, humus and inhibits microorganisms, disrupting its natural ecosystem biological community. With some preparation and processing of chicken manure is a high quality fertilizer that contains chemical elements (N, P, K), macro- and micronutrients, humic acid and other components that are essential for growth and fertility of different plants. For decontamination litter widely used thermal drying at t = 600 – 800 °C. This product is well kept dry, easily transported, but using traditional drying facilities for its handling used large amount of heat. Today continually created and offered new modern technologies for processing chicken manure. At the Institute of Engineering Thermophysics NAS of Ukraine many years conducted investigations on the creation of modern technologies and equipment for processing organic waste. Analysis of the traditional intensification methods and processes of heat and mass transfer during drying of materials was presented in the paper. The author has proved the promising nature of the method of the combined processes of drying and dispersion using a mechanical rotor to intensify the process of drying of high–moisture thermolabile materials.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals that causes a significant economic burden globally. Vaccination is the most effective FMD control strategy. However, FMD virus (FMDV) particles are prone to dissociate when appropriate physical or chemical conditions are unavailable, such as an incomplete cold chain. Such degraded vaccines result in compromised herd vaccination. Therefore, thermostable FMD particles are needed for use in vaccines. This study generated thermostable FMDV mutants (M3 and M10) by serial passages at high temperature, subsequent amplification, and purification. Both mutants contained an alanine-to-threonine mutation at position 13 in VP1 (A1013T), although M3 contained 3 additional mutations. The selected mutants showed improved stability and immunogenicity in neutralizing antibody titers, compared with the wild-type (wt) virus. The sequencing analysis and cryo-electron microscopy showed that the mutation of alanine to threonine at the 13th amino acid in the VP1 protein (A1013T) is critical for the capsid stability of FMDV. Virus-like particles containing A1013T (VLPA1013T) also showed significantly improved stability to heat treatment. This study demonstrated that Thr at the 13th amino acid of VP1 could stabilize the capsid of FMDV. Our findings will facilitate the development of a stable vaccine against FMDV serotype O. IMPORTANCE Foot-and-mouth disease (FMD) serotype O is one of the global epidemic serotypes and causes significant economic loss. Vaccination plays a key role in the prevention and control of FMD. However, the success of vaccination mainly depends on the quality of the vaccine. Here, the thermostable FMD virus (FMDV) mutants (M3 and M10) were selected through thermal screening at high temperatures with improved stability and immunogenicity compared with the wild-type virus. The results of multisequence alignment and cryo-electron microscopy (cryo-EM) analysis showed that the Thr substitution at the 13th amino acid in the VP1 protein is critical for the capsid stability of FMDV. For thermolabile type O FMDV, this major discovery will aid the development of its thermostable vaccine.

Background Climate change and the associated risk for the occurrence of extreme temperature events or permanent changes in ambient temperature are important in the husbandry of farm animals. The aim of our study was to investigate the effects of permanent cultivation temperatures below (35 °C) and above (39 °C, 41 °C) the standard cultivation temperature (37 °C) on porcine muscle development. Therefore, we used our porcine primary muscle cell culture derived from satellite cells as an in vitro model. Neonatal piglets have limited thermoregulatory stability, and several days after birth are required to maintain their body temperature. To consider this developmental step, we used myoblasts originating from thermolabile (five days of age) and thermostable piglets (twenty days of age). Results The efficiency of myoblast proliferation using real-time monitoring via electrical impedance was comparable at all temperatures with no difference in the cell index, slope or doubling time. Both temperatures of 37 °C and 39 °C led to similar biochemical growth properties and cell viability. Only differences in the mRNA expression of myogenesis-associated genes were found at 39 °C compared to 37 °C with less MYF5, MYOD and MSTN and more MYH3 mRNA. Myoblasts grown at 35 °C are smaller, exhibit higher DNA synthesis and express higher amounts of the satellite cell marker PAX7, muscle growth inhibitor MSTN and metabolic coactivator PPARGC1A. Only permanent cultivation at 41 °C resulted in higher HSP expression at the mRNA and protein levels. Interactions between the temperature and donor age showed that MYOD, MYOG, MYH3 and SMPX mRNAs were temperature-dependently expressed in myoblasts of thermolabile but not thermostable piglets. Conclusions We conclude that 37 °C to 39 °C is the best physiological temperature range for adequate porcine myoblast development. Corresponding to the body temperatures of piglets, it is therefore possible to culture primary muscle cells at 39 °C. Only the highest temperature of 41 °C acts as a thermal stressor for myoblasts with increased HSP expression, but it also accelerates myogenic development. Cultivation at 35 °C, however, leads to less differentiated myoblasts with distinct thermogenetic activity. The adaptive behavior of derived primary muscle cells to different cultivation temperatures seems to be determined by the thermoregulatory stability of the donor piglets.

Objectives Acute encephalopathy is an acute brain dysfunction after preceding infection, consisting of multiple syndromes. Some syndromes, such as acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), are severe with poor outcome, whereas others, such as clinically mild encephalitis/encephalopathy with reversible splenial lesion (MERS), are mild with favorable outcome. Previous study reported the association of the thermolabile polymorphism in Carnitine Palmitoyltransferase 2 (CPT2) gene and severe syndromes of acute encephalopathy. To further explore the pathogenetic role of CPT2 in acute encephalopathy, we conducted a case-control association study of a typical thermolabile CPT2 polymorphism, rs2229291, in 416 patients of acute encephalopathy, including both severe and mild syndromes. Methods The case cohort consisted of 416 patients, including AESD, MERS, and other syndromes. The control subjects were 100 healthy Japanese. rs2229291 was genotyped by Sanger sequencing. Genetic distribution was compared between the patients and controls using Cochran-Armitage trend test. Results Minor allele frequency of rs2229291 was significantly higher in AESD (p = 0.044), MERS (p = 0.015) and entire acute encephalopathy (p = 0.044) compared to the controls. The polymorphism showed no significant association with influenza virus, or with outcome. Conclusions This study provided evidence that CPT2 is a susceptibility gene for overall acute encephalopathy, including both severe and mild syndromes, and suggested that impairment of mitochondrial metabolism is common to various syndromes of acute encephalopathy.

Cytotoxic necrotic factor (CNF) of Yersinia pseudotuberculosis (CNFY) is a 114-kDa toxin protein that is similar to CNF1, which is prevalent among pathogenic and saprophytic Escherichia coli isolates. In 2002, the CNF of Y. pseudotuberculosis YPIII strain (United States) was first described. The Rho family small GTP-binding proteins (GTPases) are inactivated by CNFY acting on eukaryotic cells of humans and mammals, which causes the formation of large multinucleated cells. An analysis of Y. pseudotuberculosis toxins isolated in Russia showed that the thermolabile toxin TLT described earlier was identified as identical to CNFY except for three amino acid substitutions. The TLT’s cytotoxic activity is similar to that of CNFY. Screening of Y. pseudotuberculosis strains collection isolated during the outbreaks of Far East scarletlike fever (FESLF) and in sporadic cases from patients with pseudotuberculosis/FESLF, rodents, and the environment in the Far East, Siberia, and the northwest of the Russian Federation showed that most isolates carry the specific cnfY gene allele encoding the inactive CNFY isoform. This allele contains a stop codon and two large deletions that block the production of the full-sized toxin. Interestingly, all isolates obtained from patients with FESLF, which is a severe form of pseudotuberculosis and endemic to the Far East region, contain inactivated CNFY. Possible causes of CNFY inactivation in strains associated with Far East scarletlike fever are discussed.

A mathematical model of the heat and mass transfer and the phase transformations in colloidal capillary-porous materials and a numerical method for calculating the dynamics of these processes in the indicated materials and the shrinkage of them in their drying in a continuous-belt drier have been developed for the purpose of optimization of the drying process. An energy-efficient, resource-conserving convective-condensation method of drying thermolabile materials, which makes it possible to decrease the drying time, is presented.

The heat and drought resistance of plants depend on their anatomical and biochemical features. In the present study, the adaptive features of three species of wild rose (Rosaceae, Rosales) under the short-term impact of high-temperature stress have been characterized. Plants of the species Rosa donetzica Dubovik, R. reversa Waldst. et Kit. and R. spinosissima L. were exposed to a temperature of 40 degrees C for 3 hours, following which peroxidase and superoxide dismutase activity, photosynthetic pigments and flavonoids’ content, and lipid peroxide oxidation level in the leaf were determined. In our investigation, the anatomical structure of leaves and drought resistance of three species of Rosa were studied. Xeromorphic features are the most expressed for R. reversa and R. spinosissima and almost absent for R. donetzica. It has been established that R. spinosissima is photophilous whereas R. donetzica is shade-tolerant. The relatively lower development of epidermic tissue in R. donetzica could probably contribute to more active destruction of the pigment complex under high temperature stress. The obtained data about changes in activity of peroxidase, superoxide dismutase, content of photosynthetic pigments and flavonoids, and level of lipid peroxidation indicate the low heat resistance of R. donetzica in comparison with the other two species. R. reversa, R. spinosissima were more tolerant to short-term hyperthermia. They showed faster antioxidant response, mainly due to the induction of peroxidase activity under stress. The species with the most expressed xerophytic features of anatomical structures have rapid antioxidant response and are more resistant to short-term hyperthermia. The induction of some activity of antioxidant enzymes "in reserve" is a less effective form of adaptation in wild roses. Such activation of enzymes is observed in plants with a more mesophytic structure. Flavonoids and superoxide dismutase were thermolabile to short-term influence of high temperature; therefore they play an insignificant role as antioxidants in the protecton against oxidative stress caused by high temperature stress in wild roses.

Because of their nontoxicity, economic applicability, and excellent performance on adsorptive desulfurization, the fabrication of Cu(I) sites onto porous supports has drawn much attention. However, high temperatures (usually ≥700 °C) are required for the formation of Cu(I) sites from Cu(II) species through the traditional autoreduction method, which is unworkable for thermolabile metal-organic frameworks (MOFs). Here, we report a strategy named vapor-induced reduction (VIR) to convert Cu(II) species to Cu(I) in MIL-101(Cr), in which ethanol is used as an environmentally benign reductant. The entire formation of Cu(I) from Cu(II) with more than 96% selectivity is allowed, at a relatively low temperature of 200 °C, and well-maintains the structure of the MOF. Moreover, the generated Cu(I) sites exhibit good performances in adsorption desulfurization with regard to both activity and reusability.

Prion diseases are caused by the conversion of physiological PrPC into the pathogenic misfolded protein PrPSc, conferring new properties to PrPSc that vary upon prion strains. In this work, we analyze the thermostability of three prion strains (BSE, RML and 22L) that were heated at 98 °C for 2 hours. PrPSc resistance to proteinase K (PrPres), residual infectivity by mouse bioassay and in vitro templating activity by protein misfolding cyclic amplification (PMCA) were studied. Heated strains showed a huge loss of PrPres and a radically different infectivity loss: RML was the most thermolabile strain (6 to 7 log10 infectivity loss), followed by 22L (5 log10) while BSE was the most thermostable strain with low or null infectivity reduction showing a clear dissociation between PrPres and infectivity. These results indicate that thermostability is a strain-specific feature, measurable by PMCA and mouse bioassay, and a great tool to distinguish prion strains.

The extraction of diverse thermolabile but highly value-added chemicals entrapped in the robust cell wall of algal biomass has remained a remarkable challenge. This study investigated the one-pot, ...

Background: Intravascular thrombosis is responsible for an increasing number of deaths every year. Lung blood clots in USA alone affect 1,000,000 patients annually. Clots formed from insoluble fibrin restrict the smooth flow of blood in blood vessels, leading to thrombosis and heart attacks. World-wide, over 29% of the total mortalities are due to thrombosis, thus antithrombotic therapy is of great interest. The three fibrinolytic (thrombolytic) agents that are currently being used for this purpose are urokinase, streptokinase and genet-ically engineered tissue-type Plasminogen Activator (t-PA). However, these enzymes are expensive, thermolabile, with low specificity and stability, large therapeutic doses and can produce undesirable side effects such as gastrointestinal bleeding, allergic reactions, and resistance to repercussion. This warrants the search for novel fibrinolytic enzymes from various sources with higher efficacy, safety, specificity and stability and preferably those direct acting activities. Though new fibrinolytic enzymes are being explored from microbes, microorganisms remain the preferred source due to their biochemical versatility, feasibility of mass culture and ease of genetic manipulation. Hence, many fibrinolytic enzymes have been isolated from a variety of microorganisms, including actinomycetes, bacteria, fungi, and algae.Aim of Study: To detect the characterization of fibrinolytic enzymes produced by the halophilic Streptomyces flaveolus and Streptomyces galtieri that were isolated from soil of Wady El-Natron region, in North Egypt, as a potential thrombolytic agent.Material and Methods: Fibrinolytic enzymes, extracted from Streptomyces flaveolus and Streptomyces galtieri, were purified by ammonium sulfate precipitation and gel filtration. By using SDS-Page electrophoresis, determining their molec-ular masses, classified by inhibitory acting materials, charac-terized by determining metal ions influences, anticoagulation clotting time delay with CaCl2, their proteolytic activity in units/mg, the active conc. in μg/ml and the least conc. in μl/ml of thrombolytic activity.Results: The fibrinolytic enzymes extracted from both Streptomyces flaveolus and Streptomyces galtieri are classified as metallo-protease enzymes, their molecular masses were 1 6kDa and 4 1kDa, their proteolytic activity are 1.4 and 2.3 units/mg of proteins, the anticoagulation clotting time assay showed 20 and 15min delay in clotting time with CaCl2, the active conc. of each enzyme was determined from the standard curve using streptokinase as standard fibrinolytic enzyme as 200 and 300μg/ml, and the least conc. of enzyme thrombolytic activity was measured by performing the modified Holmstrom method, as the crude thrombolytic activity were at concentra-tions of 80-100μl, whereas, precipitated enzymes, showed thrombolytic activity at concentrations 20μl and 30μl/ml respectively. Metal ions exhibited different influences on the fibrinolytic enzyme activity produced by Streptomyces flave-olus, such as Ca2+, Cu2+ and Fe2+ promoted the activity of the enzyme, whereas Mg2+ is strongly promoted it. But on the fibrinolytic enzyme activity produced by Streptomyces galtieri, Cu2+ promoted the activity of the enzyme, whereas Ca2+, partially inhibit the enzyme, but the Fe2+ and Mg2+ were strongly and completely inhibit it.Conclusion: These results suggest that the extracted enzymes from both isolated Streptomyces flaveolus and Streptomyces galtieri are with potent and potential applications as thrombolytic agent.

Enzyme is important as biocatalyst for industrial and biotechnological applications. Cold active enzymes have showed many advantages compare to mesophilic enzymes. Their cold active and thermolabile characteristics have shown potential benefits in many industries. In silico characterization of hydrolytic enzymes originated from polar and tropical regions was conducted. Amino acid sequences and molecular structures of mesophilic and psychrophilic bacterial enzyme homologues were compared thoroughly. Amino acid sequences from these two homologs do not showed any extraordinary differences. Overall, protein folds were highly similar when psychrophilic homologies were matched with mesophilic homologies. Active site residues located in catalytic domain of both psychrophilic and mesophilic enzymes were highly conserved. Thus, the hydrolytic mechanisms of these cold active enzymes still remain similar to mesophilic enzymes. However, the differences were indicated between these two enzymes at the substrate-binding sites. Cold active enzymes showed unique loop conformations and smaller side chains at the entrances. These characteristics provided larger active site of the enzymes. Larger entrance of active site was expected to stipulate substrates binding and products exiting with lower energy consumption. This part of the enzymes indicated one of the important features of cold active enzyme to work efficiently at lower temperature. Based on this in silico study, cold active enzymes have much more advantages compared to the mesophilic enzymes that made them valuable to be further researched and applied at industrial level.

The effects of convective and microwave heating at constant temperature (62.5, 66 and 70 °C) on the activity of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx) in pooled mature human milk were compared. Activities of the enzymes were determined using spectrophotometric kits. Activity of GPx decreased significantly in the first stage of heating when milk samples were warmed to pasteurisation temperature. CAT was the most thermolabile enzyme but microwave heating induced a smaller decrease in CAT activity than convective heating. SOD was most resistant to thermal pasteurisation, regardless of the heating method. SOD and GPx activity temporary increased during microwaves heating. Considering shorter pasteurisation period and lower demand for energy, it can be concluded that microwaves pasteurisation enjoys special merits. However, still there is no clear answer as to whether the microwave field itself can affect the antioxidant enzymes of human milk.

The development of plasma technology is associated with the unique features of non-equilibrium low-temperature plasma: high electron energy and high concentration of chemically active excited and charged particles at low gas temperature, which allows to process thermolabile materials and biological objects in gentle conditions when high temperatures are not required. The biological effects of low-temperature plasma exposure are considered. It was established that during plasma treatment, a combined effect on cells and tissues of living systems from UV radiation, ions and chemically active particles occurs. Depending on the plasma type, the significance of each of the listed mechanisms for increasing the effectiveness of plasma treatment may vary. However, all these mechanisms interact with each other and have a synergistic effect. It was shown that the conducted studies confirm the ability of low-temperature plasma to inactivate pathogenic microorganisms upon contact with biological objects and foods. The results of the studies are presented, the purpose of which was to study the effect of plasma treatment on nitrite concentration in the water treated by this method and to assess the possibility of its use as a source of nitrite when curing meat products.

Publisher's version (útgefin grein), The influence of lipid content and pretreatment methods (blanching and brining) on proteins conformation in fish (Capelin, Mallotus villosus) during drying and smoking were assessed. Soluble protein fractions were examined through changes in salt-soluble proteins, sulfhydryl groups, and disulfide bonds contents. The salt level and moisture content were also evaluated. Conformational changes in muscle protein were significant (p < 0.05) after blanching and during initial drying when moisture content and dryness rates were high. Salt-soluble proteins and total sulfhydryl groups contents reduced, whereas available sulfhydryl group and disulfide bonds contents intensified. The conformational changes were explained by muscle protein denaturation and aggregation ascribed to high temperature and dehydration during processing. The study reports fish protein denaturation to have reduced protein solubility as well as the nutritional value (loss of thermolabile compounds) and yield after smoking. More so, protein aggregation that was significantly (p < 0.05) higher in dried blanched fish could have reduced the dried fish sensory quality. Blanching pretreatment is thus not suitable for commercial capelin drying. Lipids were found to have protective effect against fish protein conformation., The authors acknowledge the United Nations University‐Fisheries Training Programme (UNU‐FTP, Reykjavik, Iceland) and Kenya coastal development project (KCDP, Kenya) for the financial support. The authors are also thankful to HB Grandi fishing company (Reykjavik, Iceland) for fish contribution. The Managers of Vestfirska Hardfisksalan fish drying company (Iceland) and Vigfus Asbjornsson (Matis Hornarfordur, Iceland) acknowledged for the drying and smoking facilities, respectively.

Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of l-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of l-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium responsible for gastrointestinal infection in human and vibriosis in aquatic animals. The thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and thermolabile hemolysin (tlh) positive strains of V. parahaemolyticus were identified from brackishwater aquaculture farms of West Bengal and Andhra Pradesh, India. Moreover, the presence of other virulent genes like vcrD1, vopD, vp1680 under type three secretion system 1 (T3SS1) and vcrD2 vopD2, vopB2, vopC2 under type three secretion system 2 (T3SS2) were detected in tdh positive strain of V. parahaemolyticus. Furthermore, the study revealed that the tdh and trh positive isolates were resistant to β-lactam antibiotics and were able to lyse more than 95% of human Red Blood Cells (RBCs). In addition, both the isolates showed high cytotoxicity in Human Embryonic Kidney (HEK) cell line compared to tlh positive strain. Additionally, intraperitoneal and oral administration of tdh and trh positive strain of V. parahaemolyticus in Indian Major Carp, Labeo rohita caused 100% mortality at the level of 2.0 × 108 CFU ml−1 and 1.6 × 108 CFU ml−1, respectively. In contrast, only 10% mortality was observed in the case of tlh positive strain at the level of 2.5× 108 CFU ml−1. The histopathological changes like infiltration of blood cells and degenerated hepatic tissue in the liver of L. rohita were observed after the experimental challenge. The changes like degeneration of glomeruli, necrosis of renal tubules and Bowman's capsule were observed in the kidney section. Ragged, irregular shaped villi and necrosis of the villus were observed in the intestinal lumen. Overall, the study demonstrates that isolated V. parahaemolyticus is a potent aquatic microbial pathogen. Additionally, as V. parahaemolyticus is also a human pathogen and might pose a threat to the human population, proper management strategies are required to prevent the possible occurrence of disease.

Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.

Recombinant protein (RP) production from Escherichia coli has been extensively studied to find strategies for increasing product yields. The thermoinducible expression system is commonly employed at the industrial level to produce various RPs, which avoids the addition of chemical inducers, thus minimizing contamination risks. Multiple aspects of the molecular origin and biotechnological uses of its regulatory elements (pL/pR promoters and cI857 thermolabile repressor) derived from bacteriophage λ provide knowledge to improve the bioprocesses using this system. Here, we discuss the main aspects of the potential use of the λpL/pR-cI857 thermoinducible system for RP production in E. coli, focusing on the approaches of investigations that have contributed to the advancement of this expression system. Metabolic and physiological changes that occur in the host cells caused by heat stress and RP overproduction are also described. Therefore, the current scenario and the future applications of systems that use heat to induce RP production are discussed to understand the relationship between the activation of the bacterial heat shock response, RP accumulation and its possible aggregation to form inclusion bodies.

Cold-adapted esterases are attracting increasing attention owing to their prospective use in biotechnology. In this study, a novel cold-adapted family Ⅳ esterase EstDR4 was identified and obtained from extremophile Deinococcus radiodurans (D. radiodurans). EstDR4 displayed significant substrate preference towards short and medium chain monoesters (C2–C12). It also showed regioselectivity, enantioselectivity and degradation effects on four insecticides. The optimum temperature and pH for EstDR4 activity were 30 °C and pH 8, respectively. Additionally, EstDR4 exhibited relatively high catalytic activity at 0 °C and high stability from 10–40 °C, with over 80% of its initial activity retained after 1 h of incubation. Moreover, EstDR4 activity was stimulated by Tween 80 and Triton X-100, and inhibited by metal ions such as Co2+, Cu2+ and Zn2+ and several organic solvents. Thus, this enzyme shows development potential for many industrial biotechnological applications, including the manufacture of thermolabile pharmaceutical products, cold-wash detergents and insecticide biodegradation.

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability‐landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4‐oxalocrotonate tautomerase (4‐OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature T m (up to 20 °C) and in half‐life at 80 °C (up to 111‐fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4‐OT variant (M45Y/F50A) afforded improved triple‐mutant enzyme variants showing an up to 39 °C increase in T m value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability‐landscape‐guided protein engineering for thermostabilizing enzymes., Hotspot for enzymes! The enzyme 4‐oxalocrotonate tautomerase (4‐OT) promiscuously catalyzes synthetically useful Michael‐type addition reactions. A mutability‐landscape approach was applied to identify “hotspot” positions within 4‐OT at which single mutations resulted in enzyme variants with strongly improved thermostability. This information was used to engineer a 4‐OT variant with greatly increased apparent T m but no reduction in activity or enantioselectivity.

To survive in cold environments, psychrophilic organisms produce enzymes endowed with high specific activity at low temperature. The structure of these enzymes is usually flexible and mostly thermolabile. In this work, we investigate the structural basis of cold adaptation of a GH42 β-galactosidase from the psychrophilic Marinomonas ef1. This enzyme couples cold activity with astonishing robustness for a psychrophilic protein, for it retains 23% of its highest activity at 5°C and it is stable for several days at 37°C and even 50°C. Phylogenetic analyses indicate a close relationship with thermophilic β-galactosidases, suggesting that the present-day enzyme evolved from a thermostable scaffold modeled by environmental selective pressure. The crystallographic structure reveals the overall similarity with GH42 enzymes, along with a hexameric arrangement (dimer of trimers) not found in psychrophilic, mesophilic, and thermophilic homologues. In the quaternary structure, protomers form a large central cavity, whose accessibility to the substrate is promoted by the dynamic behavior of surface loops, even at low temperature. A peculiar cooperative behavior of the enzyme is likely related to the increase of the internal cavity permeability triggered by heating. Overall, our results highlight a novel strategy of enzyme cold adaptation, based on the oligomerization state of the enzyme, which effectively challenges the paradigm of cold activity coupled with intrinsic thermolability. DATABASE: Structural data are available in the Protein Data Bank database under the accession number 6Y2K.

Prevention of haze formation in wines is challenging for winemakers. Thermolabile proteins in wines, notably thaumatin-like proteins (TLPs) and chitinases (CHIs), undergo structural changes under varying physicochemical conditions, resulting in protein aggregation and visible haze in bottled products. Peptidases are an alternative fining method, although an effective proteolysis under typical winemaking conditions (acidic pH and low temperature) is difficult to achieve. In this study, tryptic peptides from TLPs and CHIs were identified by MS-based peptidomics (top-down proteomics) after exposure of scissile bonds on the protein surface. As proposed by the theory of limited proteolysis, protein conformational changes following temperature and pH variations allowed the detection of enzyme-accessible regions. Protein structure visualization and molecular dynamics simulations were used to highlight cleavage spots and provide the scientific basis for haze formation mechanisms. The described method offers a tool to the search for ideal enzymes to prevent wine haze.

Thermolabile nature of commercially available vaccines necessitates their storage, transportation and dissemination under refrigerated condition. Maintenance of continuous cold chain at every step increases the final cost of vaccines. Any breach in the cold chain, even for a short duration results in the need to discard the vaccine. As a result, there is a pressing need for the development of thermostable vaccines. In this proof of concept study, we showed that E. coli curli-GFP fusion protein remains stable in freeze-dried yeast powder for more than a 13 and 6 months when stored at 30 °C and 37 °C respectively. Stability of the heterologous protein remains unaffected during the process of heat-inactivation and lyophilization. The mass of lyophilized yeast powder remains almost unchanged during the entire period of storage. Expressed protein remains intact even after two cycles of freeze and thaws. The protease deficient strain appears ideal for the development of whole recombinant yeast-based vaccines. The cellular abundance of expressed antigen in dry powder after a year was comparable to freshly lyophilized cells. SEM microscopy showed the intact nature of cells in powdered form even after a year of storage at 30 °C. Observation made in this study showed that freeze-dry yeast powder can play a vital role in the development of thermostable vaccines.

This study identifiedVibrio parahaemolyticusin oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyllawere measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. PresumptiveV.parahaemolyticuscolonies were counted and at least 20% of these colonies were selected for molecular chracterization.V.parahaemolyticusisolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), andV.parahaemolyticusmetalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptiveV.parahaemolyticuswere 9.63×103CFU/g and 1.85×103CFU/mL in the oyster and seawater samples, respectively, during the month of July.V.parahaemolyticuslevels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) weretlh+,vpm+, andtoxR+, respectively. Among theV.parahaemolyticus(tlh+) isolates, 7 (5.1%) and 15 (10.9%) weretdh+andtrh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessedtdhand/ortrhwere notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of totalV.parahaemolyticus(tlh+) was not necessarily proportional to the potential pathogenicV.parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential oftdhoccurrence withvpm. We believe exploring the role ofV.parahaemolyticusmetalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenicVibriodynamics in relation to environmental quality.