Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

Veni, Vidi, Vici: Immobilized Peptide-Based Conjugates as Tools for Capture, Analysis, and Transformation

Analysis of peptide biomarkers of pathological states of the organism is often a serious challenge, due to a very complex composition of the cell and insufficient sensitivity of the current analytical methods (including mass spectrometry). One of the possible ways to overcome this problem is sample enrichment by capturing the selected components using a specific solid support. Another option is increasing the detectability of the desired compound by its selective tagging. Appropriately modified and immobilized peptides can be used for these purposes. In addition, they find application in studying the specificity and activity of proteolytic enzymes. Immobilized heterocyclic peptide conjugates may serve as metal ligands, to form complexes used as catalysts or analytical markers. In this review, we describe various applications of immobilized peptides, including selective capturing of cysteine-containing peptides, tagging of the carbonyl compounds to increase the sensitivity of their detection, enrichment of biological samples in deoxyfructosylated peptides, and fishing out of tyrosine–containing peptides by the formation of azo bond. Moreover, the use of the one-bead-one-compound peptide library for the analysis of substrate specificity and activity of caspases is described. Furthermore, the evolution of immobilization from the solid support used in peptide synthesis to nanocarriers is presented. Taken together, the examples presented here demonstrate immobilized peptides as a multifunctional tool, which can be successfully used to solve multiple analytical problems.

Hyaluronidase treatment of synovial fluid is required for accurate detection of inflammatory cells and soluble mediators

Background Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis. Methods SF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry. Results In all patients, there was less variation in IgG, 17-HDHA, leukotriene B4 (LTB4), and prostaglandin E2 (PGE2) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7–85% (median 22%) and 7–88% (median 23 %), respectively. Conclusions Homogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.

Natural Bioactive Compounds of Sechium spp. for Therapeutic and Nutraceutical Supplements

Natural products are in great demand because certain secondary metabolites (SMs) are sources of antioxidants, flavorings, active substances, or anticancer agents with less aggressiveness and selectivity, among which triterpenes and flavonoids are of importance because they inhibit carcinogenesis. For Sechium spp. P. Br. (chayotes), there is scientific evidence of antiproliferative activity that has occurred when cancer cell lines have been treated with this fruit. In order to compare future therapeutic designs and identify new and ancestral characteristics, triterpenes and flavonoids were determined in contrasting Sechium genotypes. The obtained data were analyzed via a cladistics approach, with the aim of identifying the characteristics and state of phytochemicals and genetic variables. The concentrations of flavonoids and triterpenes were determined, and a more complex composition of secondary metabolites was found in the wild types as compared to their domesticated genotypes. Bitter fruits contained a higher number of SMs, followed by those with a neutral and sweet flavor. A cladogram showed the differentiation of the three groups based on the flavor of the fruits. The diversity of SMs decreases in evolutionary terms, in response to domestication and environmental adaptation. Therefore, genotypes can be feasibly selected based on fruit flavor for gross-breeding, and cytotoxicity can be reduced without losing possible therapeutic effects.

Verification of Possibility of Molten Steels Decopperization with ZnAl2O4

In the previous research works, ZnAl2O4 material was considered as one of the solutions for the decopperization process of molten steels; up to 33% of decopperization efficiency was reported by utilising the ZnAl2O4 filter. In order to verify the decopperization possibility of ZnAl2O4 materials, iron-based alloys with various copper and carbon contents were interacted with ZnAl2O4 substrates in a heating microscope under an argon gas atmosphere at 1600 °C. Fe-Cu alloys were found to react with the ZnAl2O4 substrate during the interaction process, and a reaction layer with a complex composition around the alloy droplet was formed; however, Cu was not detected in the reaction layer. Cu was later found diffused inside of the ZnAl2O4 substrates. Furthermore, the Cu-Zn compounds were detected when the copper content in Fe-Cu alloys was 10 wt% Cu. After interaction experiments, copper was decreased in all cases. Thereby, the copper evaporation and infiltration into the ZnAl2O4 substrate were considered as the reasons for copper loss. Moreover, oxygen dissolved in melt was found to have a great effect on the copper evaporation process.

Biopolymers in Aerobic Granular Sludge—Their Role in Wastewater Treatment and Possibilities of Re-Use in Line with Circular Economy

Aerobic granular sludge (AGS) technology for wastewater treatment ensures better quality effluent and higher process sustainability than wastewater treatment systems based on activated sludge. One of the reasons for the better operational results of AGS systems is the high content of extracellular polymers (EPS) in the granule structures. EPS produced during granulation have a very complex composition with a predo minance of polysaccharides and proteins, and one of the main components, alginate, may have a wide variety of practical applications. This review summarizes up-to-date information on the composition of EPS in AGS, the manner in which their production and composition are affected by the operational parameters of wastewater treatment, and the effects of EPS in biomass on wastewater treatment and sludge management. Additionally, the possibility of polymer recovery from AGS is presented together with information regarding potential applications based on the newest findings. Re-use of AGS-derived polymers will increase the sustainability of wastewater treatment processes by making them more economical and reducing the amount of sludge that requires management.

Methodological Bases of Optimal Load Distribution at a Thermal Power Plant with a Complex Composition of Equipment, Taking into Account Market Requirements

The features of a mathematical model for optimizing the distribution of heat and electricity at a large thermal power plant with a complex composition of equipment as part of traditional heating units and a heating CCGT are considered. The selection and justification of optimization criteria at different stages of preparation and entry of the station to the electricity and capacity market is given. The disadvantages of the previously proposed optimal distribution algorithms are analyzed in relation to thermal power plants with a complex composition of equipment and with a complex scheme for the supply of electricity and heat. A method and algorithm for solving the problem are proposed based on the equivalence of the CHP equipment and the decomposition of the problem taking into account the schemes of electricity and heat output. The description of mathematical optimization methods is given, taking into account the peculiarities of the CCGT operating modes at reduced loads. The requirements for information support when integrating the developed algorithm into the application software of the automated process control system based on the PTC are given.

PGU-450 network heaters modes modelling with CCGT participation in regulating the electrical load in the heating mode

The features of a mathematical model for optimizing the distribution of heat and electricity at a large thermal power plant with a complex composition of equipment as part of traditional heating units and a heating CCGT are considered. The selection and justification of optimization criteria at different stages of preparation and entry of the station to the electricity and capacity market is given. The disadvantages of the previously proposed optimal distribution algorithms are analyzed in relation to thermal power plants with a complex composition of equipment and with a complex scheme for the supply of electricity and heat. A method and algorithm for solving the problem are proposed based on the equivalence of the CHP equipment and the decomposition of the problem taking into account the schemes of electricity and heat output. The description of mathematical optimization methods is given, taking into account the peculiarities of the CCGT operating modes at reduced loads. The requirements for information support when integrating the developed algorithm into the application software of the automated process control system based on the PTC are given.

Transition of Serotype 35B Pneumococci From Commensal to Prevalent Virulent Strain in Children

In our community-based prospective cohort study in young children, we observed a significant increase in pneumococcal serotype 35B nasopharyngeal (NP) commensal colonization during the 2011–2014 timeframe, but these strains were not associated with disease. Beginning in 2015 and continuing through to the present, the serotype 35B virulence changed, and it became the dominant bacteria isolated and associated with pneumococcal acute otitis-media (AOM) in our cohort. We performed comparative analyses of 250 35B isolates obtained from 140 children collected between 2006 and 2019. Changes in prevalence, clonal-complex composition, and antibiotic resistance were analyzed. Seventy-two (29%) of 35B isolates underwent whole-genome sequencing to investigate genomic changes associated with the shift in virulence that resulted in increased rates of 35B-associated AOM disease. 35B strains that were commensals and AOM disease-causing were mainly associated with sequence type (ST) 558. Antibiotic concentrations of β-lactams and ofloxacin necessary to inhibit growth of 35B strains rose significantly (2006–2019) (p<0.005). However, only isolates from the 35B/ST558 showed significant increases in MIC50 of penicillin and ofloxacin between the years 2006–2014 and 2015–2019 (p=0.007 and p<0.0001). One hundred thirty-eight SNPs located in 34 different genes were significantly associated with post-2015 strains. SNPs were found in nrdG (metal binding, 10%); metP and metN (ABC transporter, 9%); corA (Mg2+ transporter, 6%); priA (DNA replication, 5%); and on the enzymic gene ldcB (LD-carboxypeptidase, 3%). Pneumococcal serotype 35B strains was a common NP commensal during 2010–2014. In 2015, a shift in increasing number of AOM cases occurred in young children caused by 35B, that was associated with changes in genetic composition and antibiotic susceptibility.