Glucose-supplemented Medium Research Articles

CRP improves the survival and competitive fitness of Salmonella Typhimurium under starvation by controlling the cellular maintenance rate.

Catabolite repression is a mechanism of selectively utilizing preferred nutrient sources by redirecting the metabolic pathways. Therefore, it prevents non-essential energy expenditure by repressing the genes and proteins involved in the metabolism of other less favored nutrient sources. Catabolite repressor protein (CRP) is a chief mediator of catabolite repression in microorganisms. In this context, we investigated the role of CRP in starvation tolerance, at both cell physiology and molecular level, by comparing the growth, survival, competitive fitness, maintenance rate, and gene and protein expression of wild type (WT) and ∆crp of Salmonella Typhimurium, under nutrient-rich and minimal medium condition. The ∆crp shows slow growth upon the arrival of nutrient-limiting conditions, poor survival under prolong-starvation, and inability to compete with its counterpart WT strain in nutrient-rich [Luria broth (LB)] and glucose-supplemented M9 minimal medium. Surprisingly, we observed that the survival and competitive fitness of ∆crp are influenced by the composition of the growth medium. Consequently, compared to the glucose-supplemented M9 medium, ∆crp shows faster death and a higher maintenance rate in the LB medium. The comparative gene and protein expression studies of WT and ∆crp in LB medium show that ∆crp has partial or complete loss of repression from CRP-controlled genes, resulting in a high abundance of hundreds of proteins in ∆crp compared to WT. Subsequently, the addition of metabolizable sugar or fresh nutrients to the competing culture showed extended survival of ∆crp. Therefore, our results suggest that CRP-mediated gene repression improves starvation tolerance and competitive fitness of Salmonella Typhimurium by adapting its cellular maintenance rate to environmental conditions.IMPORTANCESalmonella Typhimurium is a master at adapting to chronic starvation conditions. However, the molecular mechanisms to adapt to such conditions are still unknown. In this context, we have evaluated the role of catabolite repressor protein (CRP), a dual transcriptional regulator, in providing survival and competitive fitness under starvation conditions. Also, it showed an association between CRP and nutrient composition. We observed that Δcrp growing on alternate carbon sources has lower survival and competitive fitness than Δcrp growing on glucose as a carbon source. We observed that this is due to the loss of repression from the glucose and CRP-controlled genes, resulting in elevated cellular metabolism (a high maintenance rate) of the Δcrp during growth in a medium having a carbon source other than glucose (e.g., Luria broth medium).

Influence of different light wavelengths and carbon sources on energy utilization efficiency by Chromochloris zofingiensis for cell growth and carotenoid synthesis

Chromochloris zofingiensis was cultured with different carbon sources (carbonate/glucose/acetate/pyruvate) and light conditions (dark/blue/red) under heterotrophic, autotrophic, and mixotrophic conditions to evaluate its energy utilization efficiency in terms of biomass and carotenoid accumulation. Enhanced specific growth rates of 0.58 d−1 and 0.51 d−1 were observed under blue- and red-light mixotrophic conditions, respectively, which both exceeded the rate of 0.44 d−1 obtained under heterotrophic conditions using a glucose-supplemented medium. The glucose medium showed superior cell synthesis compared to other carbon sources owing to its high utilization. Astaxanthin and neoxanthin accumulation were enhanced under mixotrophic conditions with blue-light. The energy efficiency for the synthesis of new cells was evaluated from a thermodynamic perspective. The efficiency was 1.1 to 2.1 % under autotrophic conditions, while 5.2 to 100 % was observed under heterotrophic conditions. Efficiencies in between were observed under mixotrophic conditions.

Effect of type of anticoagulant, transportation time, and glucose in the culture media on neutrophil viability and function test results in dairy cattle.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5’ untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Betulinic acid counteracts the lipid accumulation in Caenorhabditis elegans by modulation of nhr-49 expression

Obesity is an ingrained health problem with а multifactorial origin and а long history, thereby innovations in the treatment strategies are of great importance. In the search of a remedy for excessive weight gain, we have directed our investigations to phytochemicals as valuable bioactive compounds. Betulinic acid (BA), among the other triterpenoids, is known for its anti-inflammatory and anti-neoplastic properties. In addition, a previous study of ours has demonstrated а potent anti-adipogenic effect of BA in human adipocytes. Therefore, we aimed here to further verify the anti-obesogenic effect of BA in vivo in Caenorhabditis elegans. Induction of lipid accumulation in the nematodes was modelled with glucose-supplemented media, followed by treatment with BA (10–50 μM) or orlistat (12 μM) as a control anti-obesity medication. Oil red O and Nile red staining were applied to provide quantification of accumulated lipids. Analysis of the relative expression of genes, related to lipid metabolism suggested molecular mechanism of lipid-reducing action of BA in C. elegans. Treatment of nematodes with BA significantly decreased the lipid accumulation, downregulated desaturases involved in lipogenesis (fat-5, fat-6 and fat-7), modulated key transcription factors (nhr-49 and hlh-11) and microRNAs (miR-60, lin-4, let-7 and miR-786) associated with the lipid metabolism. Collectively, the current research provides additional insight on the molecular mechanism of the BA’s anti-obesogenic effect in vivo. Furthermore, it validates the potential of BA as a candidate compound in obesity management by reducing lipid accumulation.

Production of Sustainable Proteins Through the Conversion of Insects to Proteins Using Beauveria bassiana Cultures

Various strategies are being suggested to solve the challenges in the food system, such as changing the source of nutrients, including the use of non-traditional food sources such as insects. Although insects are promoted as a cheap and sustainable source of protein, consumers are reluctant to eat them. The mycoproteins produced by fungi, on the other hand, are very well received and appreciated by consumers. Thus, in this work we have studied the use of the entomopathogenic fungi Beauveria bassiana (Ascomycota: Hypocreales) for the production of protein using insects as feed. B. bassiana was cultivated in culture medium containing entire insects from the species Eurysacca and Hypothenemus or single carbon sources such as glucose or laminarin from Laminaria digitata. The results showed that B. bassiana can produce up to 16-fold more biomass and 8-fold more protein when grown in insect-based medium than when grown in glucose. The results also indicated that the production of proteins continuously increased when B. bassiana was grown in medium containing insects, reaching its maximum at 9 days (up to 3 mg/mL). On the other hand, when cultivated in glucose-supplemented medium, the production of proteins was constantly low (~0.5 mg/mL). In conclusion, B. bassiana was a large biomass producer and exuded a large amount of protein when grown in medium containing insect powder, making it an ideal intermediate link between insects and protein. Furthermore, the proteins produced by fungi such as B. bassiana can be used in the food, health, and cosmetic industries.

Isolation, screening, and molecular identification of pectinase producers from fruits, vegetables, and soil samples

Background and objective In this work, isolation, identification (morphological and chemical), and molecular characterization were done of local isolates of some pectinase-producing microorganisms such as bacteria, actinomycetes, fungi, and yeast.Materials and methods A total of 22 local bacterial isolates were obtained from various sources and were assayed for pectinolytic activity after optimization of conditions for pectinase production. Isolate no. 19 showed the highest pectinase-specific activity (6.73 U/ml) on glucose-supplemented medium, whereas isolate no. 5 gave the lowest pectinase productivity (3.21 U/ml). The identification of isolate no. 19 revealed that it belonged to the genus Bacillus based on morphological and biochemical characteristics. Based on molecular identification (16 S rRNA technique), isolate no. 19 was named Bacillus sp. strain NRBANKI-4 (with 99% similarity), with Gene Bank accession number OM540351.Results and conclusion A total of 14 local actinomycete isolates were obtained from soil samples. Isolate no. 13 showed the highest pectinase-specific activity (6.48 U/ml), whereas sample no. 10 gave the lowest pectinase-specific activity (3.07 U/ml). Based on molecular identification (16 S rRNA technique), isolate no. 13 was named Streptomyces sp. KP 12 (90.63% similarity), with Gene Bank accession number OM403596. A total of 10 fungal isolates were obtained from crop waste soil. Isolate no. 2 gave the highest pectinase productivity (21.20 U/ml). Based on molecular identification (internal transcribed spacer-PCR technique), isolate no. 2 was named Aspergillus niger F8121 (99.47% similarity), with Gene Bank accession number OM392061. Following the same trend, 10 yeast isolates were isolated from crop waste soil. The isolate that gave the highest pectinase productivity was no. 7, which gave 22.03 U/ml. The isolate that gave the lowest was no. 9 (20.74 U/ml). Isolate no. 7 was named Pichia barkeri Y1 (90.91% similarity), with Gene Bank accession number OM392066.

Phytochemical screening and antioxidant and antimicrobial activities of crude extracts of different filamentous fungi.

Five filamentous fungal strains that grew in different whey-based media under submerged fermentation were investigated for antioxidant properties and phytochemicals. Phytochemical analysis revealed the presence of alkaloids, tannin, flavonoids, glycosides, phenols, saponins, and terpenes in the crude intra- and extracellular ethyl acetate extracts of different strains. All fungal extracts exhibited effective antioxidant activities in terms of TPC, TFC, DPPH, FRAP, ABTS, reducing power, and metal chelating capacity. The activities of intracellular extracts were higher than the extracellular metabolites. Fermentation media with sugar and salt supplementation significantly influenced antioxidant production. Aspergillus niger in glucose-supplemented whey medium was found to exhibit the highest antioxidant properties. The antimicrobial activity of A. niger and Penicillium expansum extracts by microtiter plate assay showed a promising result against some pathogenic bacterial strains. Chromatographic analysis of the fungal extracts revealed the presence of chlorogenic acid, trans-cinnamic acid, ferulic acid quercetin, myricetin, kaempferol, and catechin which are known for their antioxidant properties. Accumulation of nutrients in fungal biomass under constraint environment produces secondary metabolites which has demonstrated efficacy towards alleviation of several degenerative diseases. The antioxidative enriched phytochemicals present in these five different fungal strains will provide a breakthrough in the utilisation of whey as inexpensive source of substrate for the growth of these fungi. Moreover, phytochemicals could be utilized as therapeutic agents in a cost-effective and environmentally friendly manner.

Annotation of chemotaxis gene clusters and proteins involved in chemotaxis of Bacillus subtilis strain MB378 capable of biodecolorizing different dyes.

This study explores the chemotactic potential of Bacillus subtilis MB378 against industrial dyes. Initial screening with swim plate assay showed significant movement of Bacillus subtilis MB378 towards test compounds. According to quantitative capillary assay, B. subtilis MB378 exhibited high chemotaxis potential towards Acid Orange 52 (CI: 9.52), followed by Direct Red 28 (CI: 8.39) and Basic Green 4 (CI: 5.21) in glucose-supplemented medium. Sequencing and gene annotation results evidently showed presence of chemotaxis genes and flagellar motor proteins in Bacillus subtilis draft genome. Methyl-accepting proteins (involved in chemotaxis regulation) belonged to pfam00672, pfam00072, and pfam00015 protein families. Annotated chemotaxis machinery of MB378 comprised 8 Che genes, 5 chemoreceptor genes, associated flagellar proteins, and rotary motors. Chemotaxis genes of B. subtilis MB378 were compared with genes of closely related Bacillus strains (168, WK1, and HTA426), depicting highly conserved regions showing evolutionary relation between them. Considering results of present study, it can be speculated that test compounds triggered chemotactic genes, which made these compounds bioavailable to the bacterium. Hence, the bacterium recognized and approached these compounds and facilitated biodegradation and detoxification of these compounds.

Comparative genomics of wild-type and laboratory-evolved biofilm-overproducing Deinococcus metallilatus strains.

Deinococcus metallilatus MA1002 was exposed to ultraviolet radiation to generate mutants with enhanced biofilm production. Two strains (nos 5 and 6) were then selected based on their high biofilm formation, as well as their possession of higher concentrations of extracellular matrix components (eDNA, protein and saccharides) than the wild-type (WT). Genomic sequencing revealed the presence of large genome deletions in a secondary chromosome in the mutants. Expression analyses of the WT and mutant strains indicated the upregulation of genes associated with exopolysaccharide synthesis and stress response. The mutant strains showed high mortality in glucose-supplemented (TYG) medium; however, cell death and biofilm formation were not increased in mutant cells grown under acetate- or glyoxylate-added media, suggesting that metabolic toxicity during glucose metabolism induced a high rate of cell death but improved biofilm formation in mutant strains. In damaged cells, eDNAs contributed to the enhanced biofilm formation of D. metallilatus .

Revealing the proteomic profiles of antianthracnose-related proteins in Serratia plymuthica UBCR_12

Capacity of Serratia plymuthica UBCR_12 in suppressing Colletotrichum gloeosporioides is regulated molecularly through protein expression. Efficacy of this bacteria is greatly affected by various environmental factors. This study was aimed to investigate the expression profile of antianthracnose-related proteins in S. plymuthica UBCR_12 under different modified medium. Antagonistic activity against C. gloeosporioides was assessed using agar spot method in PDA medium supplemented with several concentrations of peptone or glucose. Co-culture was performed under the same condition as an antagonistic assay to induce the extracellular proteomes expression related to this antianthracnose trait. Proteome profiles were visualized using SDS-PAGE and 2-DE, then a highly differential protein spot was further analyzed using MALDI-TOF-MS. Prediction of protein classification and possible hypothetical pathway were performed in-silico. The highest suppression (42.3% at 9th-day post application) resulted in 2% glucose-supplemented medium, however 2% peptone-supplemented medium conferred a quicker suppression (at 5th-day post application) with quite lower inhibition efficacy (40%). Differential expression of 42 kDa protein band recorded during peptone addition was predicted to be flagellin protein, which might correlate with a rapid stimulus of suppression activity. This protein involved in the quorum-sensing mechanism by triggering the greater rate of cell division resulting in bacterial colonization and motility approaching the fungal pathogen.

Biofilm production and distribution of pilus variants among Streptococcus agalactiae isolated from human and animal sources

Streptococcus agalactiae (group B Streptococcus, GBS) is a major pathogen in humans and animals. Pili and biofilm may be important virulence factors in this bacterial species. Here, biofilm production and the distribution of pilus variants among 134 GBS isolates from human and animal sources were evaluated. Biofilm production was significantly enhanced in 1% glucose-supplemented medium (p < 0.05). Using this medium, most GBS strains were strong biofilm producers. Biomass was mainly composed of proteins, followed by extracellular DNA, while polysaccharides represented a minor portion. All GBS strains presented at least one pilus variant. PI-2a was the most common among human GBS while PI-2b was the most common among animal isolates. Human GBS harboring PI-2b and animal GBS harboring PI-2a presented significantly reduced biofilm production (p = 0.0033). In conclusion, strong biofilm production seems to be a common characteristic in GBS, and association of the clinical source with the pilus variant may be crucial for this.

The role of GAD pathway for regulation of GABA accumulation and C/N balance in Synechocystis sp. PCC6803

Synechocystis sp. PCC6803 was engineered by interrupting the α-ketoglutarate decarboxylase (KGD) and overexpressing the glutamate decarboxylase (GAD) pathways to create ∆Kgd, GADox, and GADox/∆Kgd mutants with the aim to enhance GABA accumulation. Supplementation of 20 mM glutamate or 0.5 mM spermidine increased the GABA contents by several folds in engineered strains. Supplementation of glutamate not only increased the GABA accumulation of cells but also improved the growth rates of engineered strains, signifying that the GAD pathway plays a crucial role in maintaining the intracellular glutamate homeostasis in Synechocystis. The high GABA level was detected in GADox/∆Kgd grown in 0.1% glucose-supplemented medium, ~ 240-folds higher than that in WT grown without glucose. Overall results suggested that the optimization of carbon and nitrogen sources was effective in enhancing the GABA accumulation in Synechocystis overexpressing the GAD and lacking the functional KGD pathway. This work provides an insight into the strategy to improve GABA production in cyanobacteria and possibly in plants as well.

Phagocytosis and Epithelial Cell Invasion by Crohn's Disease-Associated Adherent-Invasive Escherichia coli Are Inhibited by the Anti-inflammatory Drug 6-Mercaptopurine.

Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn’s disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli, might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.

In Saccharomyces cerevisiae grown in synthetic minimal medium supplemented with non-fermentable carbon sources glutamate is synthesized within mitochondria

In Saccharomyces cerevisiae the export of 2-oxoglutarate from the mitochondria, catalyzed by Yhm2p, Odc1p and Odc2p or by at least one of these transporters, has recently been shown to be essential for glutamate biosynthesis in glucose-supplemented minimal synthetic (SM) medium without glutamate, because the triple mutant yhm2∆odc1∆odc2∆ displays a growth defect under these conditions. Surprisingly, in this study it was found that yhm2∆odc1∆odc2∆ cells grow like wild-type (WT) cells in the same medium supplemented with non-fermentable carbon sources. Direct transport assays of 2-oxoglutarate/2-oxoglutarate homoexchange activity in mitochondria from WT and yhm2∆odc1∆odc2∆ cells (solubilized and reconstituted into liposomes) showed that the mitochondrial extract from yhm2∆odc1∆odc2∆ was completely inactive at variance with that from WT cells, showing that S. cerevisiae mitochondria do not contain additional proteins capable of catalyzing 2-oxoglutarate transport efficiently besides Yhm2p, Odc1p and Odc2p. Furthermore, quantitative real-time PCR experiments showed that in both WT and yhm2∆odc1∆odc2∆ cells the expression of GDH1 is low on lactate and high on glucose and, vice versa, the expression of GDH3 is high on lactate and low on glucose. These results may be interpreted to indicate that in S. cerevisiae, grown in glucose-supplemented SM medium, glutamate is synthesized by Gdh1p in the cytosol, whereas in lactate-supplemented SM medium glutamate is synthesized by Gdh3p in the mitochondria; therefore, the pathway of ammonia assimilation under fermentative conditions requires export of 2-oxoglutarate from the mitochondria, whereas the alternative pathway under respiratory conditions does not.

The effects of model aromatic lignin compounds on growth and lipid accumulation of Rhodococcus rhodochrous

Lignocellulosic biomass is one of the most abundant and renewable organic materials in the world. The lignocellulosic complex is composed of cellulose, hemicellulose, and lignin, which can be pretreated to release sugars that can be utilized for microbial production of valued metabolites. Oleaginous microbes can accumulate over 20% of their cell dry weight as lipids, which are stored as intracellular energy reserves. The characterization of oleaginous bacteria creates opportunities for the development of alternative feedstocks and technologies. Rhodococcus rhodochrous is a bacterium recently determined to be oleaginous when grown in glucose-supplemented media. The purpose of this study was to evaluate model lignin phenolic compounds as substrates for lipid accumulation. Lipid accumulation in R. rhodochrous was evaluated using phenol, 4-hydroxybenzoic acid (HBA) and vanillic acid (VA) as model lignin compounds with and without glucose as a co-substrate. Cell dry weight increased in all treatments, indicating that growth was not impaired in these conditions. However, alterations were observed in the amount of lipids produced. Dry cell weight and lipids were analyzed daily. R. rhodochrous accumulated over 40% of its cell dry weight as lipids when grown in glucose with HBA and VA, but less than 20% when grown in HBA and VA alone. When grown in phenol and glucose, R. rhodochrous accumulated 35% of its dry weight at lipids, but did not accumulate lipids when grown in phenol alone. These data indicate that R. rhodochrous may have the capability to tolerate and utilize lignin-like aromatic compounds for lipid accumulation.

Autophagy protects human podocytes from high glucose-induced injury by preventing insulin resistance

ObjectiveInsulin resistance is correlated with the progress of albuminuria in diabetic patients, and podocytes are crucial for maintaining the normal function of the glomerular filtration barrier. In the present study, we aimed to investigate the high glucose-induced insulin resistance and cell injury in human podocytes and the putative role of autophagy in this process. MethodsHuman podocytes were cultured in high glucose-supplemented medium and low glucose and high osmotic conditions were used for the controls. Autophagy in the podocytes was regulated using rapamycin or 3-methyladenine stimulation. Next, autophagy markers including LC3B, Beclin-1, and p62 were investigated using western blot and qPCR, and the insulin responsiveness was analyzed based on glucose uptake and by using the phosphorylation of the insulin receptor with Nephrin as a podocyte injury marker. ResultsThe basal autophagy level decreased under the high glucose conditions, which was accompanied by a decrease in the glucose uptake and phosphorylation of the insulin receptor in the human podocytes. More interestingly, the glucose uptake and the phosphorylation of the insulin receptor were decreased by 3-MA stimulation and increased by rapamycin, illustrating that the responsiveness of insulin was regulated by autophagy. The activation of autophagy by rapamycin also ameliorated cell injury in the human podocytes. ConclusionsThe presence or activation of autophagy was found to play a protective role in human podocytes against high glucose-induced insulin resistance and cell injury, which indicates a novel cellular mechanism and provides a potential therapeutic target for diabetic nephropathy (DN).

Protective effect of hypoxic preconditioning on SH-SY5Y cells injured by oxygen-glucose deprivation

To investigate the protection effects of hypoxic preconditioning(HPC) on the SH-SY5Y cell injured by oxygen-glu-cose deprivation(OGD),and to discuss the possible mechanism. SH-SY5Y cells were randomly divided into 4 groups. In normal group,the cells were cultured without OGD treatment. In HPC group,the cultured SH-SY5Y cells were treated for 5 days by intermittently ex-posing to hypoxic gas mixture (2% O2,5% CO2) for 30 min in every day. In OGD group,the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O2 and 5% CO2 for 10 h. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In HPC+OGD group,the cultured SH-SY5Y cells were treated for 5 days by intermittently exposing to hypoxic gas mixture for 30 min in each day, then the cells were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by deter-mining lactate dehydrogenase (LDH) leakage. TUNEL staining were used to detect the variation of cell apoptosis. The expression of Caspase 3 and hypoxia inducible factor-1α(HIF-1α) at protein levels was examined by Western blot. Hypoxic preconditioning relieved the cells apoptosis,decreased the amount of LDH leakage and improved the viability of SH-SY5Y cells injured by OGD (P<0.05). Western blot showed that the expression of Caspase 3 protein in HPC+OGD group was significantly lower than that in OGD group (P<0.05); HIF-1α protein expression was significantly higher than that of OGD group (P<0.05). Hypoxic preconditioning has protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the increase of HIF-1a protein.

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii.

Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

BackgroundNext-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.ResultsTRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique.ConclusionsAmresco’s Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1726-3) contains supplementary material, which is available to authorized users.