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Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges to scientific research and monitoring of wastewaters to predict the spread of the virus in the community. Our study investigated the COVID-19 disease in Bratislava, based on wastewater monitoring from September 2020 until March 2021. Samples were analyzed from two wastewater treatment plants of the city with reaching 0.6 million monitored inhabitants. Obtained results from the wastewater analysis suggest significant statistical dependence. High correlations between the number of viral particles in wastewater and the number of reported positive nasopharyngeal RT-qPCR tests of infected individuals with a time lag of 2 weeks/12 days (R2 = 83.78%/R2 = 52.65%) as well as with a reported number of death cases with a time lag of 4 weeks/27 days (R2 = 83.21%/R2 = 61.89%) was observed. The obtained results and subsequent mathematical modeling will serve in the future as an early warning system for the occurrence of a local site of infection and, at the same time, predict the load on the health system up to two weeks in advance.

Background The aim of this study was to provide an information about the homogeneity on the level of enterokinase productivity in P. pastoris depending on different suppliers of the media components. Results In previous studies, we performed the optimisation process for the production of enterokinase by improving the fermentation process. Enterokinase is the ideal enzyme for removing fusion partners from target recombinant proteins. In this study, we focused our optimization efforts on the sources of cultivation media components. YPD media components were chosen as variables for these experiments. Several suppliers for particular components were combined and the optimisation procedure was performed in 24-well plates. Peptone had the highest impact on enterokinase production, where the difference between the best and worst results was threefold. The least effect on the production level was recorded for yeast extract with a 1.5 fold difference. The worst combination of media components had a activity of only 0.15 U/ml and the best combination had the activity of 0.88 U/ml, i.e., a 5.87 fold difference. A substantially higher impact on the production level of enterokinase was observed during fermentation in two selected media combinations, where the difference was almost 21-fold. Conclusions Results demonstrated in the present study show that the media components from different suppliers have high impact on enterokinase productivity and also provide the hypothesis that the optimization process should be multidimensional and for achieving best results it is important to perform massive process also in terms of the particular media component supplier .

COVID-19 pandemic caused by β-coronavirus SARS-CoV-2 emerges to intensive scientific research and monitoring of wastewaters because of their possible important role in identifying and early warning of a spread of the virus in the community. In our study, we investigated the prevalence of the COVID-19 disease in the population of the capital city of Slovakia, Bratislava, based on wastewater monitoring from September 2020 until March 2021. Samples were analyzed from two major wastewater treatment plants of the city with reaching nearly 0.6 million monitored inhabitants. Obtained results from the wastewater analysis suggest significant statistical dependence. High correlations between the number of viral particles in wastewater and the number of reported positive nasopharyngeal RT-qPCR tests of infected individuals with a time lag of 2 weeks / 12 days (R2 = 83.78% / R2 = 52.65%) as well as with a reported number of death cases with a time lag of 4 weeks / 27 days (R2 = 83.21% / R2 = 61.89%) was observed. The obtained results and subsequent mathematical modeling will serve in the future as an early warning system for the occurrence of a local site of infection and, at the same time, predict the load on the health system up to two weeks in advance.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE “SARS-CoV-2 in sewage” database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice. Validerad;2021;Nivå 2;2021-05-21 (alebob);Finansiär: För finansiärsinformation se Acknowledgements https://www.sciencedirect.com/science/article/pii/S0043135421003651?via%3Dihub#ack0001 DRIZZLE

The overuse of antibiotics is strongly associated with the development of the bacterial resistance which means that novel antimicrobial agents with different mode of action must be prepared. Antimicrobial peptides (AMP) can serve as almost an ideal candidate for the new generation of antimicrobial compounds. In this work the interactions of recombinantly prepared cathelicidin LL-37 towards various types and mixtures of supported bilayer lipid membranes (s-BLMs) are studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). All of these methods have approved excellent efficiency of LL-37 towards membranes that are typical for prokaryotic organisms composed mainly of phosphatidylglycerol and lipid A. On the contrary, almost zero activity towards phosphatidylcholine membranes that are more frequently found in the structure of eukaryotic organisms was observed. A ‘carpet model’ where the peptides interact primarily with the lipid head groups, have been proved as a mode of LL-37 action. This finding is based on the results obtained by CV and EIS techniques and was revealed by the visualization of the electrode surface by AFM technique.

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% &plusmn, 4.9%, 11.2% &plusmn, 4.2%, and 36.6% &plusmn, 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

Yeast Candida utilis is considered to be a potentially advantageous expression system for production of recombinant proteins utilizable for industrial and pharmaceutical purposes. As the scientific literature is not consistent in the ploidy of this yeast, in this work, we focused on resolving the problem via several methods such as the copy number determination of maltase gene by multiplex PCR, measuring α-glucosidase activity, the characterization of maltase gene copy number in deletion mutants using qPCR and flow cytometry. In context with the published data and results obtained in this study about the copy number of the maltase gene on C. utilis genome, we attempted to hypothesise and made conclusion about the ploidy of C. utilis. The results of this work, besides the biotechnological aspect, contribute to the elementary knowledge of C. utilis. The exact information about the ploidy or more specifically about the copy number of appropriate gene is essential for expression cassette dosage determination integrated into the chromosome of the host. In this study, we come to the conclusion that the maltase gene is present in C. utilis genome in four alleles, and in combination with flow cytometry, published information and the published genome sequences, the observations support the theory about tetraploidy of C. utilis.

Basic biochemical properties such as Michaelis constant (K M), turnover number (k cat), and the ratio between these two kinetic parameters of enzyme enteropeptidase were determined by electrochemical impedance spectroscopy. This method is developed for an easy, fast, and economic determination of mention enzyme characteristics and can be used for wide spectrum of biochemical systems. All values obtained by impedance measurements are compared and are in good agreement with those determined by traditional spectroscopic techniques and literature data. Therefore, one can suppose that electrochemical methods can be successful for such measurements also in the cases where usual ones (UV–Vis, fluorescence spectroscopy) are not able to determine these enzyme characteristics.

Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations.

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60–70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4 U/mL of wet matrix.

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.

Recently, a new gene encoding beta-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37 degrees C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52 degrees C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of beta-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of beta-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified beta-glucuronidase from SEZ glcHis was four times as low as beta-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal beta-glucuronidase from E. coli with E. coli beta-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equi subsp. zooepidemicus is used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are beta-glucuronidase deficient. Comparing the wild-type strain, which is positive in beta-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that beta-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free beta-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by beta-glucuronidase. The presence of free beta-glucuronic acid would likely induce the expression of the beta-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.

An enzyme KfoG with unknown function is coded by the gene kfoG. Gene kfoG belongs to genes from region 2, which are responsible for structure of capsular polysaccharide. Only two enzymes, KfoG and KfoC, coded by genes from region 2, have a glycosyltransferase motif. KfoC is the bifunctional enzyme, which is able to add both GalNAc and GlcUA on nascent polysaccharide, termed chondroitin polymerase. KfoG was predicted to be a fructosyltransferase. The gene that codes the KfoG enzyme was disrupted using homological recombination and absence of this gene was confirmed on both DNA and RNA levels. After disruption no structural changes have been observed, what indicates that fructose branching of the chondroitin backbone is not caused by enzymes, which are coded by genes from region 2 of the K4 capsular gene cluster.

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.

Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. The current study presents preliminary characterization of the upstream regulatory regions controlling the carbon source-dependent expression of the C. utilis maltase gene. Cellobiose and soluble starch were recognised as inducers of maltase promoter, besides maltose. We successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in polyploid yeast C. utilis. Furthermore, the strength and minimal functional region of the promoter was evaluated by measuring β-galactosidase activity. Our results suggest, the qPCR was shown itself a very smart method for relatively easy characterization of the transformants and correlation of the expression levels with gene dosage.

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.

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: −43, −44, −49, and −50 bp. To facilitate measuring of the recombinant promoter relative strength, s-glucuronidase gene was used as a reporter gene. Three mutations located in positions −49 and −50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.

Supporting material (scripts and tables) for pre-print of "COVID-19 epidemiology based on wastewater monitoringin Slovakia" &nbsp