Tested Stress Conditions Research Articles

Chromosomal Type II Toxin–Antitoxin Systems May Enhance Bacterial Fitness of a Hybrid Pathogenic Escherichia coli Strain Under Stress Conditions

The functions of bacterial plasmid-encoded toxin–antitoxin (TA) systems are unambiguous in the sense of controlling cells that fail to inherit a plasmid copy. However, its role in chromosomal copies is contradictory, including stress-response-promoting fitness and antibiotic treatment survival. A hybrid pathogenic Escherichia coli strain may have the ability to colonize distinct host niches, facing contrasting stress environments. Herein, we determined the influence of multiple environmental stress factors on the bacterial growth dynamic and expression profile of previously described TA systems present in the chromosome of a hybrid atypical enteropathogenic and extraintestinal E. coli strain. Genomic analysis revealed 26 TA loci and the presence of five type II TA systems in the chromosome. Among the tested stress conditions, osmotic and acid stress significantly altered the growth dynamics of the hybrid strain, enhancing the necessary time to reach the stationary phase. Using qPCR analyses, 80% of the studied TA systems were differentially expressed in at least one of the tested conditions, either in the log or in the stationary phase. These data indicate that type II TA systems may contribute to the physiology of pathogenic hybrid strains, enabling their adaptation to different milieus.

Stability Indicating Analytical Method, Characterization of Degradation Impurity by UPLC-Q-TOF-MS/MS, and Establishment of Degradation Mechanism of Evodiamine

Aim: The current research aims to establish a stability-indicating analytical method (SIAM) for the quantification of evodiamine (EVO), characterization of its degradation impurity, and establishment of possible degradation pathways. Background: None of the degradation impurities of EVO is known and the mechanism of their formation has not been reported in any literature to date. Moreover, a SIAM for EVO is not available in any public domain. Objective: The objective of this study is to characterize the degradation impurity of EVO by LC-MS/MS, proposing its molecular structure, identifying possible degradation pathways of generation of its impurity, and establishing a SIAM. Method: To assist future product development, a degradation study of EVO was performed and an RPHPLC- based SIAM was developed. The major degradation product was characterized by LC-Q-TOFMS/ MS. In addition, in silico toxicity prediction was performed using the ProTox-ІI toxicity predictor. Result: The method was found to be linear, accurate, precise, and robust over the range of 12.5 to 100 μg /mL of EVO. The method met all the acceptance criteria as specified in the ICH guideline. Only one degradation product (9% of the drug area) of EVO was generated in acidic hydrolytic conditions. The degradation product was found to be potentially inactive for hepatotoxicity and immunotoxicity, with a confidence score of more than 0.7 (70%). Moreover, the confidence score for carcinogenicity, mutagenicity, and cytotoxicity was less than 0.7, indicating it was moderately inactive for these toxicities. Conclusion: The molecule was found to be stable in the majority of the tested stress conditions. However, the degradation product generated in acidic hydrolytic stress was characterized using LC-Q-TOF-MS/MS, which was unknown to date. The novelty of this research can be justified by the unavailability of any SIAM of EVO and the absence of any report on its susceptibility to degradation in the presence of different potential stressors. Moreover, the potential toxicity of the molecule and its impurity was not known previously. The reported degradation impurity may be useful to set the quality control acceptance criteria for EVO. Additionally, pharmaceutical industries and research laboratories may use the developed method for the analysis of quality control and stability samples of EVO.

Molecular and Physiological Diversity of Indigenous Yeasts Isolated from Spontaneously Fermented Wine Wort from Ilfov County, Romania.

(1) Background: Wine yeast research offers the possibility of isolating new strains with distinct metabolic properties due to the geographical location of the vineyard and the processes used in winemaking. Our study deals with the isolation and identification of six yeasts from spontaneously fermented wine wort from Romania and their characterization as new potential starter culture for traditional beverages, for food industry or biomedicine. (2) Materials and methods: The isolates were identified using conventional taxonomy tests, phenotypic phylogeny analysis (Biolog YT), MALDI-TOF mass spectrometry, PCR-RFLP, and sequencing of the ITS1-5,8S-ITS2 rDNA region. The capacity of the yeasts to grow under thermal, ionic, and osmotic stress was determined. The safe status was confirmed by testing virulence and pathogenicity factors. Assays were performed in order to evaluate the growth inhibition of Candida strains and determine the antimicrobial mechanism of action. (3) Results and discussions: The yeast isolates were identified as belonging to the Metschinikowia, Hanseniaspora, Torulaspora, Pichia, and Saccharomyces genera. All the isolates were able to develop under the tested stress conditions and were confirmed as safe. With the exception of S. cerevisiae CMGB-MS1-1, all the isolates showed good antimicrobial activity based on competition for iron ions or production of killer toxins. (4) Conclusions: The results revealed the resistance of our yeasts to environmental conditions related to industrial and biomedical applications and their high potential as starter cultures and biocontrol agents, respectively.

The combination of hypoxia and high temperature affects heat shock, anaerobic metabolism, and pentose phosphate pathway key components responses in the white shrimp (Litopenaeus vannamei).

Due to global warming, world water bodies have higher temperatures and lower oxygen concentrations that affect aquatic species including the white shrimp Litopenaeus vannamei. This species withstands these conditions, but the information of the physiological responses that allow them to survive are scarce. We analyzed the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on the relative expression of selected genes: HSF1, Hsp70, p53, TIGAR, HIF-1α, and VEGF1-3 in gills of L. vannamei. Additionally, glucose, lactate, NADP, and NADPH were determined. HSF1 was up-regulated in the high temperature and oxygen stress conditions, but Hsp70 was up-regulated only in reoxygenation at both temperatures. HIF-1α was also up-regulated by reoxygenation in both temperatures. Meanwhile, the VEGF genes were not altered by the stress conditions, since none of them changed expression drastically. p53 relative expression remained stable at the tested stress conditions, which prompts to the maintenance of antioxidant defenses. TIGAR expression was induced in normoxia and hypoxia at high temperature, which induced NADPH content helping to scavenge reactive oxygen species (ROS). Additionally, high temperature caused higher glucose and lactate content in normoxia and hypoxia, indicating carbohydrate mobilization and a switch to anaerobic metabolism. The results showed that HSF1, the anaerobic metabolism and the pentose phosphate pathway (PPP) are crucial for the shrimp response to these abiotic stress conditions and contribute to their survival.

Accelerated Life Test Technology Based on Unilateral Confidence Estimation

The test principle, test type and test model of accelerated life test, as well as the data processing method of unilateral confidence estimation are introduced. Taking the enhanced field effect transistor as an example, a reasonable accelerated life test scheme is formulated by selecting the appropriate accelerated test model and stress conditions. After the test, the life characteristic value of the device under normal stress level is calculated according to Arrhenius equation and unilateral confidence estimation method. Through research, an accelerated life test method for processing non failure data is provided.

DNA methylation dynamics during stress response in woodland strawberry (Fragaria vesca).

SummaryEnvironmental stresses can result in a wide range of physiological and molecular responses in plants. These responses can also impact epigenetic information in genomes, especially at the level of DNA methylation (5-methylcytosine). DNA methylation is the hallmark heritable epigenetic modification and plays a key role in silencing transposable elements (TEs). Although DNA methylation is an essential epigenetic mechanism, fundamental aspects of its contribution to stress responses and adaptation remain obscure.We investigated epigenome dynamics of wild strawberry (Fragaria vesca) in response to variable ecologically relevant environmental conditions at the DNA methylation level. F. vesca methylome responded with great plasticity to ecologically relevant abiotic and hormonal stresses. Thermal stress resulted in substantial genome-wide loss of DNA methylation. Notably, all tested stress conditions resulted in marked hot spots of differential DNA methylation near centromeric or pericentromeric regions, particularly in the non-symmetrical DNA methylation context. Additionally, we identified differentially methylated regions (DMRs) within promoter regions of transcription factor (TF) superfamilies involved in plant stress-response and assessed the effects of these changes on gene expression.These findings improve our understanding on stress-response at the epigenome level by highlighting the correlation between DNA methylation, TEs and gene expression regulation in plants subjected to a broad range of environmental stresses.

Functional characterization of late embryogenesis abundant genes and promoters in pearl millet (Pennisetum glaucum L.) for abiotic stress tolerance.

Late embryogenesis abundant (LEA) genes display distinct functions in response to abiotic stresses in plants. In pearl millet (Pennisetum glaucum L.), a total of 21 PgLEA genes were identified and classified into six groups including LEA1, LEA2, LEA3, LEA5, LEA7, and dehydrins (DHN). Open reading frames (ORFs) of PgLEAs range from 291 bp (PgLEA1-1) to 945 bp (PgLEA2-11) and distributed randomly among the seven chromosomes. Phylogenetic analysis revealed that all PgLEA proteins are closely related to sorghum LEA proteins. The PgLEAs were found to be expressed differentially under high progressive vapor pressure deficit (VPD), PgLEA7 was significantly expressed under high VPD and was selected for functional validation. In silico analysis of the PgLEA promoter regions revealed abiotic stress-specific cis-acting elements such as ABRE, CCAAT, MYBS, and LTRE. Based on the type of motifs, PgLEAPC promoter (758 bp), its deletion 1 (PgLpd1, 349 bp) and deletion 2 (PgLpd2, 125 bp) were cloned into the plant expression vector pMDC164 having the promoter-less uidA gene. All the three plant expression vectors were introduced into tobacco through Agrobacterium tumefaciens-mediated transformation to obtain T1 and T2 generations of transgenic plants. Based on expression of the uidA gene, tissue-specific expression was observed in mature stems, roots and seedlings of PgLEAPC and PgLpd1 carrying transgenics only. While the transgenic PgLEAPC plants displayed significantly higher uidA expression in the stem and root tissues under salt, drought, heat, and cold stresses, very low or no expression was observed in PgLpd1 and PgLpd2 transgenics under the tested stress conditions. The results of this study indicate that the complete promoter of PgLEAPC plays a role in developing abiotic stress tolerance in plants.

Cultivation and biochemical characterization of isolated Sicilian microalgal species in salt and temperature stress conditions

In the last years, the possibility to exploit autochthone microalgae in regional applications has been explored. The regional-based microalgal industry may bring several benefits, as autochthone microalgae are already adapted to the biotic and abiotic stresses of their environment. In this work, this concept was applied to Sicily, in which three microalgal strains were collected from the coastline. Monoalgal strains were then isolated and molecular characterization was performed for the species determination. Three of them, two strains of Chlorella and one of Dunaliella, were cultivated in lab-scale in four different conditions: Low Temperature-Low Salt (LT-LS), High-Temperature-Low Salt (HT-LS), Low Temperature-High Salt (LT-HS) and High Temperature-High Salt (HT-HS) to investigate the role of each condition on the growth performance, the productivity and the biochemical composition of the microalgal biomass. In particular, lipid, fatty acid composition and antioxidant capacity were assessed. Results indicated that one of the Sicilian strains of Chlorella has a better growth performance at a higher temperature while the Dunaliella strain is tolerant to high-salt stress. Moreover, the biochemical composition appears to be strongly influenced by temperature and salt stresses: the lipid content decreased in all the strains and a significant shift in fatty acid composition was observed, with an increase in the content of n-3 PUFAs in some cases. Results indicated that also the carotenoids content decreased in some of the tested stress conditions. The results obtained in this research represent a first step for developing a regional-based microalgal industry in Sicily by exploiting the natural biodiversity of the Sicilian environment.

Brittle Crack Arrestability and Inverse Fracture in DWTT

Abstract The drop-weight tear test (DWTT) has been widely used to evaluate the resistance of linepipe steels against brittle crack propagation, and the shear area fraction SA% in the DWTT has been adopted in the requirement for the linepipe steels. However, recent studies have pointed out the issue of ambiguity in evaluation of the DWTT when a ductile crack initiates from the notch and then transits to a brittle crack during ductile crack propagation. This fracture behavior is termed “inverse fracture.” According to the API Recommended Practice 5L3 (API RP 5L3), a test is considered invalid when a DWTT specimen shows inverse fracture. In this case, it is difficult to examine the acceptance criterion (85% shear area transition temperature) for linepipe steels. Because the purpose of the DWTT is to evaluate the brittle crack arrestability of the steels in a pressurized linepipe, the DWTT results should be examined with a propagating brittle crack arrest test. A large-scale brittle crack arrest test called the West Jefferson test is generally conducted to reproduce the crack propagation and arrest behavior in actual linepipes. However, it is somewhat difficult to control the lower test temperature and to initiate brittle crack in recent high-toughness steels in this burst test. Although the test stress conditions of the uniaxial tension in the plate tension brittle crack arrest test and the biaxial tension in a pressurized pipe are different, the plate tension brittle crack arrest test has the advantages of accurate control of the test temperature, test stress, and brittle crack initiation in comparison with the actual pipe burst test. Therefore, in this study, the brittle crack arrestability of linepipe steel which showed inverse fracture in the DWTT was investigated by conducting plate tension brittle crack arrest tests under an isothermal condition (crack arrest temperature test (CAT test)), which simulates the condition of the actual pipelines in service. This study also investigated the local shear lip thickness fraction in the CAT tests together with the shear area fraction SA% measured in DWTTs. Based on the results, the effect of brittle crack arrestability on inverse fracture appearance in the DWTTs was discussed in comparison with the brittle crack arrest behavior in the CAT tests.

Ocean acidification, hypoxia and warming impair digestive parameters of marine mussels

Global change and anthropogenic activities have driven marine environment changes dramatically during the past century, and hypoxia, acidification and warming have received much attention recently. Yet, the interactive effects among these stressors on marine organisms are extremely complex and not accurately clarified. Here, we evaluated the combined effects of low dissolved oxygen (DO), low pH and warming on the digestive enzyme activities of the mussel Mytilus coruscus. In this experiment, mussels were exposed to eight treatments, including two degrees of pH (8.1, 7.7), DO (6, 2 mg/l) and temperature (30 °C and 20 °C) for 30 days. Amylase (AMS), lipase (LPS), trypsin (TRY), trehalase (TREH) and lysozyme (LZM) activities were measured in the digestive glands of mussels. All the tested stress conditions showed significant effects on the enzymatic activities. AMS, LPS, TRY, TREH showed throughout decreased trend in their activities due to low pH, low DO, increased temperature and different combinations of these three stressors with time but LZM showed increased and then decreased trend in their activities. Hypoxia and warming showed almost similar effects on the enzymatic activities. PCA showed a positive correlation among all measured biochemical parameters. Therefore, the fitness of mussel is likely impaired by such marine environmental changes and their population may be affected under the global change scenarios.

Interplay and Targetome of the Two Conserved Cyanobacterial sRNAs Yfr1 and Yfr2 in Prochlorococcus MED4

The sRNA Yfr1 and members of the Yfr2 sRNA family are almost universally present within cyanobacteria. The conserved motifs of these sRNAs are nearly complementary to each other, suggesting their ability to participate in crosstalk. The conserved motif of Yfr1 is shared by members of the Yfr10 sRNA family, members of which are otherwise less conserved in sequence, structure, and synteny compared to Yfr1. The different structural properties enable the discrimination of unique targets of Yfr1 and Yfr10. Unlike most studied regulatory sRNAs, Yfr1 gene expression only slightly changes under the tested stress conditions and is present at high levels at all times. In contrast, cellular levels of Yfr10 increase during the course of acclimation to darkness, and levels of Yfr2 increase when cells are shifted to high light or nitrogen limitation conditions. In this study, we investigated the targetomes of Yfr2, Yfr1, and Yfr10 in Prochlorococcus MED4, establishing CRAFD-Seq as a new method for identifying direct targets of these sRNAs that is applicable to all bacteria, including those that are not amenable to genetic modification. The results suggest that these sRNAs are integrated within a regulatory network of unprecedented complexity in the adjustment of carbon and nitrogen-related primary metabolism.

Separation and characterization of forced degradation products in homoharringtonine injection by UHPLC-Q-TOF-MS

A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed for separation and characterization of the degradation products in HHT injection. Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 HD column (2.1 × 100 mm, 1.8μm) using methanol- ammonium formate (pH 3.0; 30 mM) (30:70, v/v) as mobile phase in an isocratic mode of elution. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, photolytic and thermal stress conditions as described in ICH. A total of eleven forced degradation products were detected and the drug was found to be susceptible to all the tested stress conditions. The degradation products were characterized through Q-TOF fragmentation studies and their fragmentation pathways were proposed. Seven of them have not been reported or described as degradation product before, and one of them was further confirmed by reference substance. In addition, plausible mechanisms for the formation of the degradation products were proposed.

Differential expression of acetylcholinesterase 1 in response to various stress factors in honey bee workers

The honey bee acetylcholinesterase 1 (AmAChE1) has been suggested to be related to stress response as judged from its elevated expression level under brood rearing-suppressed conditions. To further investigate the involvement of AmAChE1 expression in the stress response and its physiological functions, we analyzed altered expression profiles of AmAChE1 induced by diverse stress factors. In addition, transcription profiles of several heat shock protein (Hsp) genes (hsps) and the vitellogenin (Vg) gene (vg) known as general stress markers were investigated as positive references. Among the tested stress conditions, AmAChE1 expression was induced under the brood rearing-suppressed, crowding and heat shock conditions. The hsps, particularly hsp70 and hsp90, responded to seven of nine stress conditions tested, confirming that hsp expression profiles can serve as a general stress marker. Taken together, AmAChE1 expression is not suitable for using as a stress marker due to its limited response. Nevertheless, AmAChE1 expression appears to be connected, at least in part, to heat shock response and other pathways. Considering that AmAChE1 likely regulates the ACh titer particularly in non-neuronal tissues, thereby modulating the signal cascades mediated by mAChR, the AmAChE1 expression profile under different conditions likely provides important information on its physiological roles in honey bees.

Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes—ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)—were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.

The Carotenogenic Dunaliella salina CCAP 19/20 Produces Enhanced Levels of Carotenoid under Specific Nutrients Limitation

Dunaliella salina is the popular microalga for β-carotene production. There is still a growing demand for the best strain identification and growth conditions optimization for maximum carotenoids production. Some strains are noncarotenogenic while other strains may respond differently to applied growth conditions and produce enhanced carotenoid levels. This study tested the carotenogenic ability of Dunaliella salina CCAP 19/20 under sixteen stress conditions and certain biochemical changes in response to specific stress were investigated. This study identified the above strain as carotenogenic, which produces maximum carotenoids under high light (240 μmol photons m−2 sec−1) when combined nitrogen and micronutrients (Cu or CuMn) were limited. Based on the intensity of extracted ions chromatograms, lutein (m/z 568.4357) appears as the major carotenoid followed by β-carotene (m/z 536.4446) and α-carotene (m/z 536.4435). A polypeptide of 28.3 kDa appeared while another polypeptide of 25.5 kDa disappeared in stress cells as compared to noncarotenogenic cells. Expression levels of antioxidative-enzyme superoxide dismutase-1 (SOD1, H2O2-resistant) remained identical, while the prominent H2O2-sensitive isoforms SOD2 and SOD3 were downregulated during carotenogenic conditions. Overall, increased carotenoids levels might be due to the response of differential expression of specific polypeptides and retention of H2O2-resistant SOD, which eventually might help the organism to thrive in the tested stress conditions.

Triaxial Wetting Test on Rockfill Materials under Stress Combination Conditions of Spherical Stress p and Deviatoric Stress q

A GCTS medium‐sized triaxial apparatus is used to conduct a single‐line method wetting test on three kinds of rockfill materials of different mother rocks such as mixture of sandstone and slate, and dolomite and granite, and the test stress conditions is the combination of spherical stress p and deviatoric stress q. The test results show that (1) for wetting shear strain, the effects of spherical stress p and deviatoric stress q are equivalent, and wetting shear strain and deviatoric stress q show the power function relationship preferably. (2) For wetting volumetric strain, the effect of deviatoric stress q can be neglected because it is extremely insignificant, and spherical stress p is the main influencing factor and shows the power function relationship preferably. (3) The wetting strains decrease significantly with the increase in initial water content and sample density generally, but the excessively high dry density will increase the wetting deformation. Also, the wetting strains will decrease with the increase in the saturated uniaxial compressive strength and average softening coefficient of the mother rock. Based on the test results, a wetting strain model is proposed for rockfill materials. The verification results indicate that the model satisfactorily reflects the development law of wetting deformation.

Evaluation and modelling of permanent deformation behaviour of asphalt mixtures using dynamic creep test in uniaxial mode

Permanent deformation behaviour of asphalt mixtures is likely to be examined in laboratory prior to laying those mixtures in field. This study aimed at relative evaluation and modelling of permanent deformation behaviour of various wearing course mixtures using advanced statistical techniques. Thirty wearing course mixtures with variable combinations of constituent materials and gradations were tested at three temperature conditions (i.e. 25, 40 and 50 °C) and two stress conditions (i.e. 300 and 500 kPa) using uniaxial dynamic creep test (DCT). Consistent ranking has been observed at various test temperatures and stress conditions. Variation of permanent strain slope with number of loading cycles was found to be a useful parameter for asphalt mixture laboratory performance. It has been observed that permanent strain slope followed nearly similar pattern when stress was increased from 300 to 500 kPa, at the temperature condition of 25 °C. The waviness among permanent strain slope values has been increased significantly with the increase in stress from 300 to 500 kPa for the results at temperature conditions of 40 °C and higher. Non-linear regression and artificial neural networking (ANN) have been found as effective techniques to model the permanent strain in uniaxial DCT. Temperature, number of cycles, bitumen penetration, aggregate flakiness index, and stress condition are considered as significant independent variables included in the model. Normalised importance plots indicated temperature as most significant variable for the developed models. It has been found that non-linear regression model most accurately predicts permanent strain data of NHA-A graded mixtures while, ANN model most precisely estimates permanent strain data for SP-B graded mixtures as translated by larger R2 values of 0.88 and 0.99, respectively. The statistical parameters of R2, RMSE, VAF and MAPE indicated high prediction performances of artificial neural network (ANN) modelling technique as compared to nonlinear regression modelling technique.

Elimination of the last reactions in ergosterol biosynthesis alters the resistance of Saccharomyces cerevisiae to multiple stresses.

The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion. Compared with the wild type, the mutants showed altered resistances to different exogenous stresses regarding the specific growth rate and duration of lag phase. The erg5 deletion mutant whose sterol has a saturated side chain exhibited overall robust growth under the tested stress conditions. The thermotolerant phenotype of erg5 deletion mutant was reproduced in filamentous fungus Penicillium oxalicum. These results highlight the important role of sterols in the response of yeast cells to environmental stresses, and suggest the possibility of improving the robustness of industrial yeast strains by engineering their sterol composition.

Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme.

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4. On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.