Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression.

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

Complex Analysis of Vanillin and Syringic Acid as Natural Antimicrobial Agents against Staphylococcus epidermidis Biofilms.

The presence of Staphylococcus epidermidis biofilms on medical devices is a major cause of nosocomial diseases and infections. Extensive research is directed at inhibiting the formation and maturation of such biofilms. Natural plant-derived phenolic compounds have promising antimicrobial effects against drug-resistant bacteria. The anti-biofilm activity of two selected phenolic compounds (vanillin and syringic acid) was tested against three biofilm-forming methicillin-resistant S. epidermidis strains with different genotypes. Resazurin assay combining crystal violet staining and confocal microscopy was used for biofilm and extracellular polymer substance (EPS) inhibition tests. Effects on EPS compounds such as proteins, extracellular DNA, and polysaccharides were also examined. Combined with quantitative real-time PCR of selected agr quorum-sensing systems and biofilm genetic determinants, our complex analysis of vanillin and syringic acid showed similar biofilm and EPS inhibition effects on S. epidermidis strains, reducing biofilm formation up to 80% and EPS up to 55%, depending on the genotype of the tested strain. Natural antimicrobial agents are thus potentially useful inhibitors of biofilms.

Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms.

Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms in biofilm formation can help find new ways to treat resistant strains and lower the prevalence of nosocomial infections. In order to examine the role of RNAIII regulated by the agr quorum sensing system and to what extent it influences biofilm resistance to antimicrobial agents, deletion mutant S. epidermidis RP62a-ΔRNAIII deficient in repressor domains with a re-maining functional hld gene was created. A deletion strain was used to examine the influence of oxacillin in combination with vanillin on biofilm resistance and cell survival was determined. Utilizing real-time qPCR, confocal laser scanning microscopy (CLSM), and crystal violet staining analyses, we found that the RNAIII-independent controlled phenol soluble modulins (PSMs) and RNAIII effector molecule have a significant role in biofilm resistance to antibiotics and phenolic compounds, and it protects the integrity of biofilms. Moreover, a combination of antibiotic and antimicrobial agents can induce methicillin-resistant S. epidermidis biofilm formation and can lead to exceedingly difficult medical treatment.

Alzheimer's Disease-Associated SNP rs708727 in SLC41A1 May Increase Risk for Parkinson's Disease: Report from Enlarged Slovak Study.

SLC41A1 (A1) SNPs rs11240569 and rs823156 are associated with altered risk for Parkinson's disease (PD), predominantly in Asian populations, and rs708727 has been linked to Alzheimer's disease (AD). In this study, we have examined a potential association of the three aforementioned SNPs and of rs9438393, rs56152218, and rs61822602 (all three lying in the A1 promoter region) with PD in the Slovak population. Out of the six tested SNPs, we have identified only rs708727 as being associated with an increased risk for PD onset in Slovaks. The minor allele (A) in rs708727 is associated with PD in dominant and completely over-dominant genetic models (ORD = 1.36 (1.05-1.77), p = 0.02, and ORCOD = 1.34 (1.04-1.72), p = 0.02). Furthermore, the genotypic triplet GG(rs708727) + AG(rs823156) + CC(rs61822602) might be clinically relevant despite showing a medium (h ≥ 0.5) size difference (h = 0.522) between the PD and the control populations. RandomForest modeling has identified the power of the tested SNPs for discriminating between PD-patients and the controls to be essentially zero. The identified association of rs708727 with PD in the Slovak population leads us to hypothesize that this A1 polymorphism, which is involved in the epigenetic regulation of the expression of the AD-linked gene PM20D1, is also involved in the pathoetiology of PD (or universally in neurodegeneration) through the same or similar mechanism as in AD.

Enhancement of solubility of recombinant alcohol dehydrogenase from Rhodococcus ruber using predictive tool.

Solubility is one of key factors influencing the heterologous production of recombinant proteins in biotechnology. Among many aggregation-prone proteins, alcohol dehydrogenase (ADH-A) from Rhodococcus ruber (in this work abbreviated RrADH) shows a great potential in processes involved in the biotransformation of natural compounds. As ADH-A is a potentially high value asset in industrial biotransformation processes, improvement of its solubility would be of major commercial benefit. Predictive tools and in silico analysis provide a fast means for improving protein properties, for selecting appropriate changes, and ultimately for saving costs. We have therefore focused on enhancement of the solubility of RrADH using an online accesible predictive tool Aggrescan 3D 2.0. Selected mutations were introduced into the protein amino acid sequence by using site-directed PCR. This led to a 17% increase in the protein solubility of RrADHmut1 and a 98% increase for RrADHmut2. Moreover, the basic kinetics of the enzyme reaction were positively affected, further optimizing the overall performance of the production process.

Tellurium: A Rare Element with Influence on Prokaryotic and Eukaryotic Biological Systems.

Metalloid tellurium is characterized as a chemical element belonging to the chalcogen group without known biological function. However, its compounds, especially the oxyanions, exert numerous negative effects on both prokaryotic and eukaryotic organisms. Recent evidence suggests that increasing environmental pollution with tellurium has a causal link to autoimmune, neurodegenerative and oncological diseases. In this review, we provide an overview about the current knowledge on the mechanisms of tellurium compounds' toxicity in bacteria and humans and we summarise the various ways organisms cope and detoxify these compounds. Over the last decades, several gene clusters conferring resistance to tellurium compounds have been identified in a variety of bacterial species and strains. These genetic determinants exhibit great genetic and functional diversity. Besides the existence of specific resistance mechanisms, tellurium and its toxic compounds interact with molecular systems, mediating general detoxification and mitigation of oxidative stress. We also discuss the similarity of tellurium and selenium biochemistry and the impact of their compounds on humans.

Current state and prospects of biotechnology in Central and Eastern European countries. Part I: Visegrad countries (CZ, H, PL, SK).

Innovation is a key determinant of sustainable growth. Biotechnology (BT) is one such industry that has witnessed a revolution in innovative ideas leading to the founding of many new companies based on providing products, solutions and services, stretching from the food industry to environmental remediation, and new medicines. BT holds much promise for the development of national and local economies, however, this requires a strategic approach involving actors within government, industry, and academia working in concert to maximize this potential. This first article reviews the current "state of play" in the field of BT within the Central Eastern European (CEE) countries. For the purposes of this article, CEE refers to the countries of Czech Republic, Hungary, Poland, and Slovakia (the so-called Visegrad - V4 countries). We examine the components that support the creation and development of a BT sector in CEE and also highlight the barriers to these objectives. Clearly setting priorities for the countries' policy agenda, as well as the alignment of Smart Specialization Strategy will help to focus efforts. Recent investments in R&D infrastructure within CEE have been substantial, but conditions will need to be optimized to harness these largely European investments for effective use towards SME high-tech development.

Evaluation of thermostable catalase-peroxidase AfKatG supplementation on toxicity of residual hydrogen peroxide in cultivation media of lactic acid bacteria from starter cultures.

Lactic acid bacteria (LAB) are exceptionally important strains in food industry. It is a heterogeneous group sharing same metabolic and physiological properties. They are usually catalase-negative strains, which represents a big disadvantage in food production in comparison with pathogenic bacteria as staphylococci and listeria existing in the same environment, because of the use of hydrogen peroxide as a disinfection agent which is utilized by catalases. We focused on increase in LAB surviving through the disinfection without any positive effect on growth of pathogenic bacteria. In our functional test hydrogen peroxide was used for disinfection. Ten mM thermostable catalase-peroxidase AfKatG was added to solid media to cultivate bacteria afterwards. As predicted there was no difference in the growth of pathogenic bacteria with or without catalase-peroxidase addition to media. However, we showed a huge positive effect on surviving LAB. With addition of AfKatG to solid media we gained 2-38 times higher CFU/ml than in control samples without it. We can assume AfKatG as an excellent supplement for growth media of food strains.

Simultaneous analysis of carbohydrates, polyols and amines in urine samples using chemical ionization gas chromatography with tandem mass spectrometry.

A simple method for the simultaneous derivatization of carbohydrates, polyols, amines and amino acids using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide was developed. This method allows the direct derivatization of urine samples without sample pretreatment before derivatization. The method was successfully used for analysis of the selected metabolites in urine samples of healthy individuals and neonates suffering from galactosemia. The limits of detection by positive chemical ionization gas chromatography with tandem mass spectrometry analysis were in the range of 1.0 mgL-1 for mannitol to 4.7 mg/L for glucose.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis.

By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.

Validation of dopamine receptor DRD1 and DRD2 antibodies using receptor deficient mice.

Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.

Apoptosis-induced ectodomain shedding of hypoxia-regulated carbonic anhydrase IX from tumor cells: a double-edged response to chemotherapy.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli.

The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain α-thrombin precursor prethrombin-2 and its shortened form prethrombin-2∆13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate.

Production of Reverse Transcriptase and DNA Polymerase in Bacterial Expression Systems.

DNA amplification and reverse transcription enzymes have proven to be invaluable in fast and reliable diagnostics and research applications because of their processivity, specificity, and robustness. Our study focused on the production of mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase in the expression hosts Vibrio natriegens and Escherichia coli under various expression conditions. We also examined nonspecific extracellular production in V. natriegens. Intracellularly, M-MLV was produced in V. natriegens at the level of 11% of the total cell proteins (TCPs) compared with 16% of TCPs in E. coli. We obtained a soluble protein that accounted for 11% of the enzyme produced in V. natriegens and 22% of the enzyme produced in E. coli. Taq pol was produced intracellularly in V. natriegens at the level of 30% of TCPs compared with 26% of TCPs in E. coli. However, Taq pol was almost non-soluble in E. coli, whereas in V. natriegens, we obtained a soluble protein that accounted for 23% of the produced enzyme. We detected substantial extracellular production of Taq pol. Thus, V. natriegens is a suitable alternative host with the potential for production of recombinant proteins.

Analysis of the DNA region mediating increased thermotolerance at 58°C in Cronobacter sp. and other enterobacterial strains.

Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. The increased stress tolerance, including thermoresistance, of some Cronobacter strains can promote their survival in production facilities and thus raise the possibility of contamination of dried infant milk formula, which has been identified as a potential source of infection. In this study, we characterized a DNA region which is present in some Cronobacter strains and which contributes to their prolonged survival at 58°C. The 18 kbp long region containing 22 open reading frames was sequenced in Cronobacter sakazakii ATCC 29544. The major feature of the region contained a cluster of conserved genes, most of them having significant homologies with bacterial proteins involved in some type of stress response, including heat, oxidation and acid stress. The same thermoresistance DNA region was detected in strains belonging to the genera Cronobacter, Enterobacter, Citrobacter and Escherichia and its presence positively correlated with increased thermotolerance.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation.

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

Comparison of simple expression procedures in novel expression host Vibrio natriegens and established Escherichia coli system.

Marine bacterium Vibrio natriegensis a novel host platform for different applications in molecular biology and biotechnology. It has one of the fastest growth rates of any known microorganisms and its extremely short doubling time indicates a high level of proteosynthetic activity. Regarding the necessity of developing new high-level protein expression systems it represents an extremely interesting subject. V. natriegens fulfills many important features for a suitable host including non- pathogenicity, easy scale-up process, potential for using alternative carbon sources (compared to E. coli), growth media and potential for further genetic and metabolic engineering with employment of a wide range of genetic tools. This work compares V. natriegens as an expression host for production of recombinant human growth hormone (hGH), yeast alcohol dehydrogenase (ADH) and archaeal catalase-peroxidase (AfKatG) to E. coliand establishes the basis for future development of this platform. The selected proteins are of different origins, sizes and intended applications. Our results have shown that cultures of V. natriegens using sucrose as a main carbon source can be used for the production of industrially applicable proteins, where it offers higher biomass productions compared to E. coli. In case of human growth hormone production, produced amounts were lower compared to those of E. coli (38 % of total cell protein (TCP) for V. natriegens vs. 58 % of TCP for E. coli, with similar solubility of around 40 % in both cases). In case of yeast alcohol dehydrogenase, V. natriegens produced 26 % of TCP vs. 42 % of TCP in E. coli, but with severely decreased solubility in case of V. natriegens cultures. Finally V. natriegens cultures were able to produce catalase-peroxidase AfKatG at the level of 33 % of TCP compared to 26 % of TCP in E. coli. Obtained results suggest that there are still significant differences in reliability and ease of use between E. coli and V. natriegens, with latter being more susceptible to condition changes and producing inconsistent results.

Seasonal changes of circulating 25-hydroxyvitamin D correlate with the lower gut microbiome composition in inflammatory bowel disease patients.

Higher probability of the development of Crohn's disease (CD) and ulcerative colitis (UC) as a possible consequence of the north-south gradient has been recently suggested. Living far north or south of the equator is manifested in fluctuation of vitamin D (vitD) levels depending on the season in both healthy and affected individuals. In the present study we investigate the possible link between the seasonal serum vitD level to the microbial composition of the lower gut of Inflammatory Bowel disease (IBD) patients using 16S rRNA sequencing. Decrease of serum vitD level in winter/spring season in a cohort of 35 UC patients and 39 CD patients was confirmed. Low gut microbiota composition of patients with IBD correlated with the serum level of 25(OH)D that directly coupled to seasonal variability of the sunshine in the central European countries. It is supposed to be related to increased abundance of Actinobacteria and Proteobacteria in UC and Actinobacteria, Fusobacteria, Firmicutes and Bacteroidetes in CD. In summer/autumn period, we observed a reduction in abundance of bacterial genera typical for inflammation like Eggerthella lenta, Fusobacterium spp., Bacteroides spp., Collinsella aerofaciens, Helicobacter spp., Rhodococcus spp., Faecalibacterium prausnitzii; and increased abundance of Pediococcus spp. and Clostridium spp. and of Escherichia/Shigella spp.

Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.