Intracellular Protective Functions and Therapeutical Potential of Trehalose.

Trehalose is a naturally occurring, non-reducing saccharide widely distributed in nature. Over the years, research on trehalose has revealed that this initially thought simple storage molecule is a multifunctional and multitasking compound protecting cells against various stress factors. This review presents data on the role of trehalose in maintaining cellular homeostasis under stress conditions and in the virulence of bacteria and fungi. Numerous studies have demonstrated that trehalose acts in the cell as an osmoprotectant, chemical chaperone, free radical scavenger, carbon source, virulence factor, and metabolic regulator. The increasingly researched medical and therapeutic applications of trehalose are also discussed.

Validation of Becton Dickinson Barricor™ tubes for use with Abbott Alinity™ and Siemens Atellica® highly sensitive cardiac troponin I measuring systems.

BACKGROUND: BD Barricor™ tubes have been proposed to decrease laboratory turnaround time (TAT). We analytically validated and then clinically verified these tubes for use with Abbott Alinity™ and Siemens Atellica® highly sensitive cardiac troponin I (hs-cTnI) assays. METHODS: hs-cTnI measurements were undertaken in paired Barricor™ and in-use PSTII™ tubes on both systems. 359 matched samples with hs-cTnI levels between 3 and 15,000 ng/L (Atellica® values) were used to assess the hemolysis rate and make method comparisons. 599 paired patient samples were collected on emergency department (ED) admission to compare the performance of the rapid acute myocardial infarction (AMI) rule-out strategy based on hs-cTnI concentrations lower than recommended thresholds (<4 ng/L Alinity™; <5 ng/L Atellica®) when different tubes and systems were employed. RESULTS: No between-tube differences in hemolysis rate were seen when free hemoglobin concentrations in plasma samples were ≥0.25 g/L, even if PSTII™ showed a significant increase of hemolysis rate vs. Barricor™ (31% vs. 22%, p=0.007) when a lower cut-off for hemolysis (≥0.11 g/L) was employed on the Atellica® detection system. The alternate use of these tubes did not influence the hs-cTnI results obtained from either of the two assays, which remained markedly biased (~40%) irrespective of the tube used. The expected optimal ability of very low hs-cTnI values on ED admission for ruling out AMI was confirmed by using both systems regardless of the tube type. CONCLUSIONS: Barricor™ and PSTII™ tubes can provide analytically equivalent hs-cTnI results when used on either Alinity™ or Atellica® hs-cTnI assays.

Influence of Nε-Lysine Acetylation on the Formation of Protein Aggregates and Antibiotic Persistence in E. coli.

Numerous studies indicate that reversible Nε-lysine acetylation in bacteria may play a key role in the regulation of metabolic processes, transcription and translation, biofilm formation, virulence, and drug resistance. Using appropriate mutant strains deficient in non-enzymatic acetylation and enzymatic acetylation or deacetylation pathways, we investigated the influence of protein acetylation on cell viability, protein aggregation, and persister formation in Escherichia coli. Lysine acetylation was found to increase protein aggregation and cell viability under the late stationary phase. Moreover, increased lysine acetylation stimulated the formation of persisters. These results suggest that acetylation-dependent aggregation may improve the survival of bacteria under adverse conditions (such as the late stationary phase) and during antibiotic treatment. Further experiments revealed that acetylation-favorable conditions may increase persister formation in Klebsiella pneumoniae clinical isolate. However, the exact mechanisms underlying the relationship between acetylation and persistence in this pathogen remain to be elucidated.

Applications of Silk Fibroin in Human and Veterinary Medicine.

The properties of silk make it a promising material for medical applications, both in human and veterinary medicine. Its predominant amino acids, glycine and alanine, exhibit low chemical reactivity, reducing the risk of graft rejection, a notable advantage over most synthetic polymers. Hence, silk is increasingly used as a material for 3D printing in biomedicine. It can be used to build cell scaffolding with the desired cytocompatibility and biodegradability. In combination with gelatine, silk can be used in the treatment of arthritis, and as a hydrogel, to regenerate chondrocytes and mesenchymal cells. When combined with gelatine and collagen, it can also make skin grafts and regenerate the integumentary system. In the treatment of bone tissue, it can be used in combination with polylactic acid and hydroxyapatite to produce bone clips having good mechanical properties and high immunological tolerance. Furthermore, silk can provide a good microenvironment for the proliferation of bone marrow stem cells. Moreover, research is underway to produce artificial blood vessels using silk in combination with glycidyl methacrylate. Silk vascular grafts have demonstrated a high degree of patency and a satisfactory degree of endothelial cells coverage.

Liquid-Liquid Phase Separation and Protective Protein Aggregates in Bacteria.

Liquid-liquid phase separation (LLPS) and the formation of membraneless organelles (MLOs) contribute to the spatiotemporal organization of various physiological processes in the cell. These phenomena have been studied and characterized mainly in eukaryotic cells. However, increasing evidence indicates that LLPS-driven protein condensation may also occur in prokaryotes. Recent studies indicate that aggregates formed during proteotoxic stresses may also play the role of MLOs and increase the fitness of bacteria under stress. The beneficial effect of aggregates may result from the sequestration and protection of proteins against irreversible inactivation or degradation, activation of the protein quality control system and induction of dormancy. The most common stress that bacteria encounter in the natural environment is water loss. Therefore, in this review, we focus on protein aggregates formed in E. coli upon desiccation-rehydration stress. In silico analyses suggest that various mechanisms and interactions are responsible for their formation, including LLPS, disordered sequences and aggregation-prone regions. These data support findings that intrinsically disordered proteins and LLPS may contribute to desiccation tolerance not only in eukaryotic cells but also in bacteria. LLPS-driven aggregation may be a strategy used by pathogens to survive antibiotic treatment and desiccation stress in the hospital environment.

New Strategies to Kill Metabolically-Dormant Cells Directly Bypassing the Need for Active Cellular Processes.

Antibiotic therapy failure is often caused by the presence of persister cells, which are metabolically-dormant bacteria capable of surviving exposure to antimicrobials. Under favorable conditions, persisters can resume growth leading to recurrent infections. Moreover, several studies have indicated that persisters may promote the evolution of antimicrobial resistance and facilitate the selection of specific resistant mutants; therefore, in light of the increasing numbers of multidrug-resistant infections worldwide, developing efficient strategies against dormant cells is of paramount importance. In this review, we present and discuss the efficacy of various agents whose antimicrobial activity is independent of the metabolic status of the bacteria as they target cell envelope structures. Since the biofilm-environment is favorable for the formation of dormant subpopulations, anti-persister strategies should also include agents that destroy the biofilm matrix or inhibit biofilm development. This article reviews examples of selected cell wall hydrolases, polysaccharide depolymerases and antimicrobial peptides. Their combination with standard antibiotics seems to be the most promising approach in combating persistent infections.

Protein aggregation and glycation in Escherichia coli exposed to desiccation-rehydration stress.

In natural environments, bacteria often enter a state of anhydrobiosis due to water loss. Multiple studies have demonstrated that desiccation may lead to protein aggregation and glycation both in vivo and in vitro. However, the exact effects of water-loss-induced proteotoxic stress and the interplay between protein glycation and aggregation in bacteria remain elusive. Our studies revealed that protein aggregates formation in Escherichia coli started during desiccation and continued during the rehydration stage. The aggregates were enriched in proteins prone to liquid-liquid phase separation. Although it is known that glycation may induce protein aggregation in vitro, the aggregates formed in E. coli contained low levels of glycation products compared to the soluble protein fraction. Carnosine, glycine betaine and trehalose diminished the formation of protein aggregates and glycation products, resulting in increased E. coli viability. Notably, although high concentrations of glycine-betaine and trehalose significantly enhanced protein aggregation, glycation was still inhibited and E. coli cells survived desiccation better than bacteria grown without osmolytes. Taken together, our results suggest that the aggregates might play protective functions during early desiccation-rehydration stress. Moreover, it seems glycation rather than protein aggregation is the main cause of E. coli death upon desiccation-rehydration stress.

Effectiveness and Safety of Transcatheter Atrial Septal Defect Closure in Adults with Systemic Essential Hypertension.

Concomitant systemic essential hypertension (HTN) in adults with a secundum atrial septal defect (ASD) can unfavorably affect the hemodynamics and transcatheter ASD closure (ASDC) effects. This study aims to assess the effectiveness and safety of ASDC in adults with HTN in real-world clinical practice. Right ventricular (RV) reverse remodeling (RVR) and the lack of a left-to-right interatrial residual shunt (NoRS) in echocardiography 24 h and 6 months (6 M) post-ASDC, and ASDC-related complications within 6 M were evaluated in 184 adults: 79 with HTN (HTN+) and 105 without HTN (HTN-). Compared to HTN-, HTN+ patients were older and had a greater RV size and the prevalence of atrial arrhythmias, chronic heart failure, nonobstructive coronary artery disease, diabetes, hyperlipidemia, and left ventricular diastolic dysfunction. ASDC was successful and resulted in RVR, NoRS, and a lack of ASDC-related complications in the majority of HTN+ patients both at 24 h and 6 M. HTN+ and HTN- did not differ in ASD size, a successful implantation rate (98.7% vs. 99%), RVR 24 h (46.8% vs. 46.7%) and 6 M (59.4% vs. 67.9%) post-ASDC, NoRS 24 h (79% vs. 81.5%) and 6 M (76.6% vs. 86.9%) post-ASDC, and the composite of RVR and NoRS at 6 M (43.8% vs. 57.1%). Most ASDC-related complications in HTN+ occurred within 24 h and were minor; however, major complications such as device embolization within 24 h and mitral regurgitation within 6 M were observed. No differences between HTN+ and HTN- were observed in the total (12.7% vs. 9.5%) and major (5.1% vs. 4.8%) complications. Transcatheter ASDC is effective and safe in adults with secundum ASD and concomitant HTN in real-world clinical practice; however, proper preprocedural management and regular long-term follow-up post-ASDC are required.

Initial Study on COMT and DRD2 Gene Polymorphisms as Well as the Influence of Temperament and Character Trait on the Severity of Alcohol Craving in Alcohol-Dependent Patients.

The main aim of this work was to determine the impact of COMT and DRD2 gene polymorphisms together with temperament and character traits on alcohol craving severity alcohol-dependent persons. The sample comprised of 89 men and 16 women (aged 38±7). For the sake of psychological assessment various analytic methods have been applied like the Short Alcohol Dependence Data Questionnaire (SADD), Penn Alcohol Craving Scale (PACS) or Temperament and Character Inventory (TCI) test. The SNP polymorphism of the analyzed genes was determined by Real Time PCR test. The results showed, that the COMT polymorphismmay have an indirected relationship with the intensity and changes in alcohol craving during abstinence. The DRD2 receptor gene polymorphisms are related with the intensity of alcohol craving. It seems that the character traits like "self-targeting", including "self-acceptance", are more closely related to the severity of alcohol craving and polymorphic changes in the DRD2 receptor than temperamental traits. Although this is a pilot study the obtained results appeared to be promising and clearly indicate the link betweengene polymorphisms alcohol craving and its severity.

Antibiotic Heteroresistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the most common pathogens responsible for infections, including pneumonia, urinary tract infections, and bacteremias. The increasing prevalence of multidrug-resistant K. pneumoniae was recognized in 2017 by the World Health Organization as a critical public health threat. Heteroresistance, defined as the presence of a subpopulation of cells with a higher MIC than the dominant population, is a frequent phenotype in many pathogens. Numerous reports on heteroresistant K. pneumoniae isolates have been published in the last few years. Heteroresistance is difficult to detect and study due to its phenotypic and genetic instability. Recent findings provide strong evidence that heteroresistance may be associated with an increased risk of recurrent infections and antibiotic treatment failure. This review focuses on antibiotic heteroresistance mechanisms in K. pneumoniae and potential therapeutic strategies against antibiotic heteroresistant isolates.

Scaling Risk Assessment in Nanofiltration of Mine Waters.

Nanofiltration can be applied for the treatment of mine waters. One of the main problems is the risk of crystallization of sparingly soluble salts on the membrane surface (scaling). In this work, a series of batch-mode nanofiltration experiments of the mine waters was performed in a dead-end Sterlitech® HP 4750X Stirred Cell. Based on the laboratory results, the concentration profiles of individual ions along the membrane length in a single-pass industrial-scale nanofiltration (NF) unit was calculated, assuming the tanks-in-series flow model inside the membrane module. These calculations also propose a method for estimating the maximum achievable recovery before the occurrence of the calcium sulfate dihydrate scaling in a single-pass NF 40″ length spiral wound module, simultaneously allowing metastable supersaturation of calcium sulfate dihydrate. The performance of three membrane types (NF270, NFX, NFDL) has been evaluated for the nanofiltration of mine water.

Mechanisms Protecting Acinetobacter baumannii against Multiple Stresses Triggered by the Host Immune Response, Antibiotics and Outside-Host Environment.

Acinetobacter baumannii is considered one of the most persistent pathogens responsible for nosocomial infections. Due to the emergence of multidrug resistant strains, as well as high morbidity and mortality caused by this pathogen, A. baumannii was placed on the World Health Organization (WHO) drug-resistant bacteria and antimicrobial resistance research priority list. This review summarizes current studies on mechanisms that protect A. baumannii against multiple stresses caused by the host immune response, outside host environment, and antibiotic treatment. We particularly focus on the ability of A. baumannii to survive long-term desiccation on abiotic surfaces and the population heterogeneity in A. baumannii biofilms. Insight into these protective mechanisms may provide clues for the development of new strategies to fight multidrug resistant strains of A. baumannii.

Evaluation of Risk Behavior in Gambling Addicted and Opioid Addicted Individuals.

Evidence suggests that both opioid addicted and gambling addicted individuals are characterized by higher levels of risky behavior in comparison to healthy people. It has been shown that the administration of substitution drugs can reduce cravings for opioids and the risky decisions made by individuals addicted to opioids. Although it is suggested that the neurobiological foundations of addiction are similar, it is possible that risk behaviors in opioid addicts may differ in detail from those addicted to gambling. The aim of this work was to compare the level of risk behavior in individuals addicted to opioid, with that of individuals addicted to gambling, using the Iowa Gambling Task (IGT). The score and response time during the task were measured. It was also observed, in the basis of the whole IGT test, that individuals addicted to gambling make riskier decisions in comparison to healthy individuals from the control group but less riskier decisions in comparison to individuals addicted to opioids, before administration of methadone and without any statistically significant difference after administration of methadone-as there has been growing evidence that methadone administration is strongly associated with a significant decrease in risky behavior.

New insight into the mechanisms protecting bacteria during desiccation.

Desiccation is a common stress that bacteria face in the natural environment, and thus, they have developed a variety of protective mechanisms to mitigate the damage caused by water loss. The formation of biofilms and the accumulation of trehalose and sporulation are well-known strategies used by bacteria to survive desiccation. Other mechanisms, including intrinsically disordered proteins and the anti-glycation defence, have been mainly studied in eukaryotic cells, and their role in bacteria remains unclear. We have recently shown that the impairment of trehalose synthesis results in higher glucose availability, leading to the accumulation of acetyl phosphate and enhanced protein acetylation, which in turn stimulates protein aggregation. In the absence of trehalose synthesis, excess glucose may stimulate non-enzymatic glycosylation and the formation of advanced glycation end products (AGEs) bound to proteins. Therefore, we propose that trehalose may prevent protein damage, not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation and glycation.

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation.

The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli ΔotsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild-type cells. The lack of trehalose synthesis boosted Nε-lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε-lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the ΔotsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress - the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.

Effect of Multi-Microbial Probiotic Formulation Bokashi on Pro- and Anti-Inflammatory Cytokines Profile in the Serum, Colostrum and Milk of Sows, and in a Culture of Polymorphonuclear Cells Isolated from Colostrum.

The use of probiotics in sows during pregnancy and lactation and their impact on the quality of colostrum and milk, as well as the health conditions of their offspring during the rearing period, are currently gaining the attention of researchers. The aim of the study was to determine the effect of Bokashi formulation on the concentrations of pro- and anti-inflammatory cytokines in the serum of sows during pregnancy, in their colostrum and milk, and in a culture of Con-A-stimulated polymorphonuclear cells (PMNs) isolated from the colostrum. The study was conducted on 60 sows aged 2-4 years. EM Bokashi were added to the sows' feed. The material for the study consisted of peripheral blood, colostrum, and milk. Blood samples were collected from the sows on days 60 and 114 of gestation. Colostrum and milk samples were collected from all sows at 0, 24, 48, 72, 96, 120, 144, and 168 h after parturition. The results indicate that the use of Bokashi as feed additives resulted in increased concentrations of pro-inflammatory cytokines TNF-α and IL-6, which increase the protective capacity of the colostrum by stimulating cellular immune mechanisms protecting the sow and neonates against infection. At the same time, the increased concentrations of cytokines IL-4, IL-10, TGF-ß, and of immunoglobulins in the colostrum and milk from sows in the experimental group demonstrate the immunoregulatory effect of Bokashi on Th2 cells and may lead to increased expression of regulatory T cells and polarization of the immune response from Th1 to Th2.

Effect of Zinc Sulfate and Zinc Glycine Chelate on Concentrations of Acute Phase Proteins in Chicken Serum and Liver Tissue.

The aim of the study was to determine how inorganic and organic forms of zinc affect the concentrations of C-reactive protein (CRP), serum amyloid A (SAA), alpha-1-acid glycoprotein (α-1-AGP), haptoglobin (Hp), and transferrin (TRF) in the blood and liver tissue of 450 1-day-old Ross 308 chicken. Four experimental groups received one the following: inorganic zinc (ZnSO4), a zinc phytase enzyme supplement (ZnSO4-F), organic zinc in combination with glycine (Zn-Gly), or organic zinc supplemented with phytase (Zn-Gly-F). The chicken serum and liver homogenates were assayed using an ELISA kit. The results of the study showed statistically significantly higher serum and liver concentration of SAA in the group of birds that received zinc sulfate in comparison to the group of birds receiving zinc in organic form. A statistically significantly higher serum concentration of CRP and α-1-AGP was also noted in the group receiving zinc sulfate as compared to the Zn-Gly group. Comparison of the serum concentration of TRF between the supplemented groups showed a statistically significant increase in this parameter in the Zn-Gly-F group as compared to the ZSO4-F group. The increase in the serum concentration of Hp in all groups in comparison to the control may indicate stimulation of local immune mechanisms. The results of this study showed an increase in the concentrations of APPs such as AGP and TRF following the administration of zinc glycine chelates, which may demonstrate their effect on metabolic processes in the liver and on immunocompetent cells that regulate the intensity of the immune response.

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs.

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I - control and II - experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

Proteomic analysis of protein homeostasis and aggregation.

Protein homeostasis (proteostasis) refers to the ability of cells to preserve the correct balance between protein synthesis, folding and degradation. Proteostasis is essential for optimal cell growth and survival under stressful conditions. Various extracellular and intracellular stresses including heat shock, oxidative stress, proteasome malfunction, mutations and aging-related modifications can result in disturbed proteostasis manifested by enhanced misfolding and aggregation of proteins. To limit protein misfolding and aggregation cells have evolved various strategies including molecular chaperones, proteasome system and autophagy. Molecular chaperones assist folding of proteins, protect them from denaturation and facilitate renaturation of the misfolded polypeptides, whereas proteasomes and autophagosomes remove the irreversibly damaged proteins. The impairment of proteostasis results in protein aggregation that is a major pathological hallmark of numerous age-related disorders, such as cataract, Alzheimer's, Parkinson's, Huntington's, and prion diseases. To discover protein markers and speed up diagnosis of neurodegenerative diseases accompanied by protein aggregation, proteomic tools have increasingly been used in recent years. Systematic and exhaustive analysis of the changes that occur in the proteomes of affected tissues and biofluids in humans or in model organisms is one of the most promising approaches to reveal mechanisms underlying protein aggregation diseases, improve their diagnosis and develop therapeutic strategies. Significance: In this review we outline the elements responsible for maintaining cellular proteostasis and present the overview of proteomic studies focused on protein-aggregation diseases. These studies provide insights into the mechanisms responsible for age-related disorders and reveal new potential biomarkers for Alzheimer's, Parkinson's, Huntigton's and prion diseases.

Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock.

Bacteria can form heterogeneous populations containing phenotypic variants of genetically identical cells. The heterogeneity of populations can be considered a bet-hedging strategy allowing adaptation to unknown environmental changes - at least some individual subpopulations or cells might be able to withstand future adverse conditions. Using Percoll gradient centrifugation, we demonstrated that in an Escherichia coli culture exposed to heat shock at 50 °C, two physiologically distinct subpopulations were formed. A high-density subpopulation (HD50) demonstrated continued growth immediately after its transfer to LB medium, whereas the growth of a low-density subpopulation (LD50) was considerably postponed. The LD50 subpopulation contained mainly viable but non-culturable bacteria and exhibited higher tolerance to sublethal concentrations of antibiotics or H2O2 than HD50 cells. The levels of aggregated proteins and main molecular chaperones were comparable in both subpopulations; however, a decreased number of ribosomes and a significant increase in protein oxidation were observed in the LD50 subpopulation as compared with the HD50 subpopulation. Interestingly, under anaerobic heat stress, the formation of the HD50 subpopulation was decreased and culturability of the LD50 subpopulation was significantly increased. In both subpopulations the level of protein aggregates formed under anaerobic and aerobic heat stress was comparable. We concluded that the formation of protein aggregates was independent of oxidative damage induced by heat stress, and that oxidative stress and not protein aggregation limited growth and caused loss of LD50 culturability. Our results indicate that heat stress induces the formation of distinct subpopulations differing in their ability to grow under standard and stress conditions.