Label-free multiple reaction monitoring, a promising method for quantification analyses of specific proteins in bacteria

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity-and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9-and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture

Diversity and adaptations of escherichia coli strains: Exploring the intestinal community in crohn’s disease patients and healthy individuals

Crohn’s disease (CD) is characterized by a chronic, progressive inflammation across the gastrointestinal tract with a series of exacerbations and remissions. A significant factor in the CD pathogenesis is an imbalance in gut microbiota composition, particularly the prevalence of Escherichia coli. In the present study, the genomes of sixty-three E. coli strains from the gut of patients with CD and healthy subjects were sequenced. In addition, eighteen E. coli-like metagenomeassembled genomes (MAGs) were reconstructed from the shotgun-metagenome sequencing data of fecal samples. The comparative analysis revealed the similarity of E. coli genomes regardless of the origin of the strain. The strains exhibited similar genetic patterns of virulence, antibiotic resistance, and bacteriocin-producing systems. The study showed antagonistic activity of E. coli strains and the metabolic features needed for their successful competition in the human gut environment. These observations suggest complex bacterial interactions within the gut which may affect the host and cause intestinal damage

Targeting pathogenic fungi, bacteria and fungal-bacterial biofilms by newly synthesized quaternary ammonium derivative of pyridoxine and terbinafine with dual action profile

© 2020 Elsevier Inc. Many pathogenic bacteria and microscopic fungi form rigid polymicrobial biofilms this way enhancing their resistant to treatment. A series of novel pyridoxine-based quaternary ammonium derivatives of terbinafine characterized by both antifungal and antibacterial activities was designed. The leading compound named KFU-127 exhibits promising antifungal and antibacterial activities against various bacteria and micromycetes in both planktonic and biofilm-embedded forms demonstrating MIC values comparable with those of conventional antifungals and antimicrobials. Similar to other antiseptics like benzalkonium chloride and miramistin, KFU-127 is considerably toxic for eukaryotic cells that limits is application to topical treatment options. On the other hand, KFU-127 reduces the number of viable biofilm-embedded bacteria and C. albicans by 3 orders of magnitude at concentrations 2–4 times lower than those of reference drugs and successfully eradicates S. aureus-C. albicans mixed biofilms. The mechanism of antimicrobial action of KFU-127 is bimodal including both membrane integrity damage and pyridoxal-dependent enzymes targeting. We expect that this bilateral mechanism would result in lower rates of resistance development in both fungal and bacterial pathogens. Taken together, our data suggest KFU-127 as a new promising broad spectrum topical antimicrobial capable of one-shot targeting of bacterial and fungal-bacterial biofilms

Serum cytokine profile, beta-hexosaminidase a enzymatic activity and gm<inf>2</inf> ganglioside levels in the plasma of a tay-sachs disease patient after cord blood cell transplantation and curcumin administration: A case report

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder that occurs due to a deficiency of a β hexosaminidase A (HexA) enzyme, resulting in the accumulation of GM2 gangliosides. In this work, we analyzed the effect of umbilical cord blood cell transplantation (UCBCT) and curcumin administration on the course of the disease in a patient with adult TSD. The patient’s serum cytokine profile was determined using multiplex analysis. The level of GM2 gangliosides in plasma was determined using mass spectrometry. The enzymatic activity of HexA in the plasma of the patient was assessed using a fluorescent substrate assay. The HexA α-subunit (HexA) concentration was determined using ELISA. It was shown that both UCBCT and curcumin administration led to a change in the patient’s cytokine profile. The UCBCT resulted in an increase in the concentration of HexA in the patient’s serum and in an improvement in the patient’s neurological status. However, neither UCBCT nor curcumin were able to alter HexA activity and the level of GM2 in patient’s plasma. The data obtained indicate that UCBCT and curcumin administration can alter the immunity of a patient with TSD, reduce the level of inflammatory cytokines and thereby improve the patient’s condition

Insights into the health effects of acrolein and crotonaldehyde in Russian smokers switching from regular cigarettes to heated tobacco products

The effects of acrolein and crotonaldehyde on the health of Russian smokers upon switching to heated tobacco product use were analyzed in a five-day randomized clinical trial. The findings suggest that heated tobacco products significantly reduce exposure to these toxicants, with the adverse effects becoming less pronounced in just one day and comparable to complete cessation of smoking. The dynamics of nicotine intake and metabolism in the smokers who switched to heated tobacco products remained stable throughout the study and was similar to that in the group of regular cigarette smokers. Therefore, our study, which has been performed for the first time among the Russian population, shows that smokers switching to heated tobacco products are less exposed to harmful chemicals like acrolein and crotonaldehyde and thus may be protected against some of the negative health effects often associated with regular cigarettes

Inflammatory Bowel Disease-Associated Changes in the Gut: Focus on Kazan Patients

Background: Several studies have highlighted the role of host-microbiome interactions in the pathogenesis of inflammatory bowel disease (IBD), resulting in an increasing amount of data mainly focusing on Western patients. Because of the increasing prevalence of IBD in newly industrialized countries such as those in Asia, the Middle East, and South America, there is mounting interest in elucidating the gut microbiota of these populations. We present a comprehensive analysis of several IBD-related biomarkers and gut microbiota profiles and functions of a unique population of patients with IBD and healthy patients from Kazan (Republic of Tatarstan, Russia). Methods: Blood and fecal IBD biomarkers, serum cytokines, and fecal short-chain fatty acid (SCFA) content were profiled. Finally, fecal microbiota composition was analyzed by 16S and whole-genome shotgun sequencing. Results: Fecal microbiota whole-genome sequencing confirmed the presence of classic IBD dysbiotic features at the phylum level, with increased abundance of Proteobacteria, Actinobacteria, and Fusobacteria and decreased abundance of Firmicutes, Bacteroidetes, and Verrucomicrobia. At the genus level, the abundance of both fermentative (SCFA-producing and hydrogen (H2)-releasing) and hydrogenotrophic (H2-consuming) microbes was affected in patients with IBD. This imbalance was confirmed by the decreased abundance of SCFA species in the feces of patients with IBD and the change in anaerobic index, which mirrors the redox status of the intestine. Conclusions: Our analyses highlighted how IBD-related dysbiotic microbiota - which are generally mainly linked to SCFA imbalance - may affect other important metabolic pathways, such as H2 metabolism, that are critical for host physiology and disease development