Isolation and Characteristics of Extracellular Vesicles Produced by Probiotics: Yeast Saccharomyces boulardii CNCM I-745 and Bacterium Streptococcus salivarius K12.

Numerous probiotic microorganisms have repeatedly been shown to produce nanometer-sized structures named extracellular vesicles (EVs). Recently, it has been suggested that similarly to whole microbial cells, EVs produced by probiotics may also demonstrate health benefits to the host, while their application does not involve the risk of infection caused by live microorganisms. In this work, we isolated EVs from two probiotic species originating from different taxonomic domains - yeast Saccharomyces boulardii CNCM I-745 and bacterium Streptococcus salivarius K12. The diameters of S. boulardii EVs were about 142 nm and for S. salivarius EVs about 123 nm. For S. boulardii EVs, 1641 proteins and for S. salivarius EVs, 466 proteins were identified with a liquid chromatography-coupled tandem mass spectrometry and then functionally classified. In both microbial species, metabolic proteins significantly contributed to the cargo of EVs comprising 25% and 26% of all identified vesicular proteins for fungi and bacteria, respectively. Moreover, enzymes associated with cell wall rearrangement, including enzymatically active glucanases, were also identified in EVs. Furthermore, probiotic EVs were shown to influence host cells and stimulate the production of IL-1ß and IL-8 by the human monocytic cell line THP-1, and, at the same time, did not cause any remarkable reduction in the survival rate of Galleria mellonella larvae in this invertebrate model commonly used to evaluate microbial EV toxicity. These observations suggest that the EVs produced by the investigated probiotic microorganisms may be promising structures for future use in pro-health applications.

Monitoring of age- and gender-related alterations of endocannabinoid levels in selected brain regions with the use of SPME probes.

INTRODUCTION: The endocannabinoid system consists of different types of receptors, enzymes and endocannabinoids (ECs), which are involved in several physiological processes, but also play important role in the development and progression of central nervous system disorders. OBJECTIVES: The purpose of this study was to apply precise and sensitive methodology for monitoring of four ECs, namely anandamide (AEA), 2-arachidonoyl glycerol (2-AG), N-arachidonoyl dopamine (NADA), 2-arachidonyl glyceryl ether (2-AGe) in selected brain regions of female and male rats at different stages of development (young, adult and old). METHODS: Biocompatible solid-phase microextraction (SPME) probes were introduced into the intact (non-homogenized) brain structures for isolation of four ECs, and the extracts were subjected to LC-MS/MS analysis. Two chemometric approaches, namely hierarchical cluster analysis (HCA) and Principal Component Analysis (PCA) were applied to provide more information about the levels of 2-AG and AEA in different brain structures. RESULTS: 2-AG and AEA were extracted and could be quantified in each brain region; the level of 2-AG was significantly higher in comparison to the level of AEA. Two highly unstable ECs, NADA and 2-AGe, were captured by SPME probes from intact brain samples for the first time. CONCLUSION: SPME probes were able to isolate highly unstable endogenous compounds from intact tissue, and provided new tools for precise analysis of the level and distribution of ECs in different brain regions. Monitoring of ECs in brain samples is important not only in physiological conditions, but also may contribute to better understanding of the functioning of the endocannabinoid system in various disorders.

Small Molecules Originated from Tryptophan and their Clinical Significance as Potential Biomarkers.

BACKGROUND: L-tryptophan is an essential amino acid necessary for the human body to function. Its degradation occurs through two metabolic pathways. Approximately 95 % of the Ltryptophan available in the body is converted via the kynurenine pathway, while the remainder is degraded via the serotonin pathway. Properly maintained balance between the concentrations of individual small molecular metabolites is extremely important to maintain homeostasis in the human body, and its disruption could lead to the development of numerous neurological, neurodegenerative, neoplastic, as well as cardiovascular diseases. Recent reports have suggested that by controlling the levels of selected L-tryptophan metabolites (potential biomarkers), it is possible to diagnose numerous diseases, monitor their course, and assess patient prognosis. OBJECTIVE: The aim of this paper is to review the currently important clinical applications of selected biomarkers from the L-tryptophan metabolism pathways that would be helpful in early diagnosis, monitoring the course and treatment of serious diseases of affluence, which ultimately could improve the patients' quality of life, as well as support targeted therapy of the aforementioned diseases. CONCLUSION: Since the biochemical biomarkers determination in body fluids presents the ideal minimally invasive tool in the patents' diagnosis and prognostication, this study emphasizes the current trends and perspectives of application of analysis of selected L-tryptophan metabolites named kynurenine and serotonin-derived small compounds in the routine medical procedures.

Digital Diagnostics and Mobile Health in Laboratory Medicine: An International Federation of Clinical Chemistry and Laboratory Medicine Survey on Current Practice and Future Perspectives.

BACKGROUND: A survey of IFCC members was conducted to determine current and future perspectives on digital innovations within laboratory medicine and healthcare sectors. METHODS: Questions focused on the relevance of digital diagnostic solutions, implementation and barriers to adopting digital technologies, and supplier roles in supporting innovation. Digital diagnostic market segments were defined by solution recipient (laboratory, clinician, patient/consumer, payor) and proximity to core laboratory operations. RESULTS: Digital solutions were of active interest for >90% of respondents. Although solutions to improve core operations were ranked as the most relevant currently, a future shift to technologies beyond core laboratory expertise is expected. A key area of potential differentiation for laboratory customers was clinical decision support. Currently, laboratories collaborate strongly with suppliers of laboratory integration software and information systems, with high expectations for future collaboration in clinical decision support, disease self-management, and population health management. Asia Pacific countries attributed greater importance to adopting digital solutions than those in other regions. Financial burden was the most commonly cited challenge in implementing digital solutions. CONCLUSIONS: Specialists in laboratory medicine are proactively approaching digital innovations and transformation, and there is high enthusiasm and expectation for further collaboration with suppliers and healthcare professionals beyond current core laboratory expertise.

The SWI/SNF complex in eosinophilic and non eosinophilic chronic rhinosinusitis.

INTRODUCTION: Chronic rhinosinusitis (CRS) can be classified as eosinophilic (eCRS) or non-eosinophilic (neCRS) based on infiltration type. The SWI/SNF complex may be involved in the pathophysiology of CRS. AIM: To assess the expression of the SWI/SNF complex in both CRS groups; to correlate blood eosinophil count (BEC), and histopathology eosinophil count (HPEC) with the SWI/SNF expression level in eCRS and neCRS. MATERIALS AND METHODS: The study population consisted of 96 patients (68 eCRS, 28 neCRS). Immunohistochemical staining was performed on sinonasal mucosa for assessment of SWI/SNF protein expression. Type of tissue infiltration was assessed in samples obtained from examined groups (HPEC). The diagnostic value of eCRS was 10 cells/HPF (high power field). Complete blood count was analysed in order to calculate BEC. RESULTS: BEC and HPEC correlated negatively with all the SWI/SNF subunits. HPEC and BEC correlated positively with clinical findings (L-M and SNOT-22), while SWI/SNF correlated negatively with clinical findings (L-M and SNOT-22). CONCLUSIONS: The SWI/SNF was observed in both eCRS and neCRS, with lower expression in former. The meaning of its negative correlation with BEC, HPEC and clinical findings in eCRS group remains to be understood.

Comparison of Intranasal Steroid Application Using Nasal Saline Irrigation and a Mucosal Atomization Device to Treat Chronic Rhinosinusitis.

Introduction: Chronic rhinosinusitis (CRS) is a disease that can significantly reduce patients' quality of life (QoL). Intranasal steroid therapy is the most commonly used treatment for CRS. There are many evaluation tools dedicated to assessing CRS patients' QoL, but none of them evaluates QoL during local steroid therapy. Mucosal atomization devices (MADs) and nasal saline irrigation (NSI) are effective and safe methods of applying intranasal steroids for CRS patients. Materials and Methods: The sample population for this prospective study comprised 43 CRS patients. Following endoscopic sinus surgery, all participants received intranasal steroids administered via an MAD, followed by NSI for 1.5 months. Each participant completed the SNOT-22 (22-item Sino-Nasal Outcomes Test) score and a new questionnaire, the Complementary Topical Nasal Drug Delivery Questionnaire (the Complementary Questionnaire), at the end of 3 months of intranasal steroid therapy. Results: The patients' responses in both the SNOT-22 score and the Complementary Questionnaire revealed significant differences in their adverse experiences. The patients who received intranasal steroid treatment using NSI experienced more frequently delayed nasal drainage, higher frequency of ear symptoms, and facial pain/pressure, while those whose therapy was administered using an MAD reported complaints such as nasal irritation, nasal dryness, and postnasal drip with unpleasant taste/smell. Conclusion: We used the Complementary Questionnaire as an effective tool for assessment of the QoL of CRS patients. The SNOT-22 score and the Complementary Questionnaire make it possible to select an intranasal applicator tailored to a CRS patient's specific complaints.

Role of chromatin remodeling complex SWI/SNF and VDR in chronic rhinosinusitis.

BACKGROUND: The SWI/SNF (SWItch/sucrose non-fermentable) chromatin remodeling complex enables glucocorticoid receptor (GR) and vitamin D receptor (VDR) to function correctly and is engaged in inflammation response. The SWI/SNF may play an important role in chronic rhinosinusitis (CRS). OBJECTIVES: The aim of this study was to assess the following: 1) the gene and protein expression of the SWI/SNF complex subunits in sinonasal mucosa; 2) relation of SWI/SNF complex and VDR expression; and 3) correlation with clinical data. MATERIAL AND METHODS: The study population consisted of 52 subjects with CRS without nasal polyps, 55 with CRS with nasal polyps and 59 controls. The SWI/SNF protein expression level was analyzed in immunohistochemical (IHC) staining. Human nasal epithelial cells (HNECs) was stimulated using lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and vitamin D3 (vitD3) in vitro. The transcript level of the SWI/SNF subunits was measured with polymerase chain reaction (PCR). RESULTS: In the control group, the intensity of the IHC staining for SWI/SNF subunits was significantly higher than in both groups of patients with CRS (p < 0.05). A positive correlation of the SWI/SNF protein expression was noticed with VDR expression level (p < 0.043). Association between SWI/SNF protein expression level and allergy, neutrophils and body mass index (BMI) has been observed (p < 0.05). The decreased transcript level of the SWI/SNF subunits genes in HNECs was observed after LPS stimulation and increased after vitD3 stimulation. CONCLUSIONS: The SWI/SNF complex may influence CRS through steroid hormone signaling and VDR. Thus, modification in therapy may be mandatory in patients with CRS and altered SWI/SNF signaling, reflecting resistance to steroids treatment.

Role of Vitamin D and Its Receptors in the Pathophysiology of Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) is a disease that represents a challenging therapeutic problem. Vitamin D and its receptors (VDR) are involved in the regulation of the immune system and may play role in CRS. Objectives of this study were to assess the relationships between the total concentration of vitamin D (25VD3) in sera, vitamin D receptor (VDR) expression, 1α-hydroxylase expression, and clinical data, including age, gender, Sino-Nasal Outcome Test (SNOT-22), computerized tomography (CT) scan, allergy status, and vitamin D supplementation in CRS patients with (CRSwNP) and without nasal polyps (CRSsNP), and in a control group. METHODS: The studied group comprised 52 patients with CRS without nasal polyps (sNP), 55 with CRS with nasal polyps (wNP), and 59 in the control group. The endpoints were determined by appropriate methods. We conducted immunohistochemical staining of gathered tissue from the ostiomeatal complex for determination of VDR and 1α-hydroxylase. Analytical results were compared with clinical data as already noted. RESULTS: A decrease in VDR nuclear staining occurred in CRS patients as compared to controls. Insignificant differences were observed in 1α-hydroxylase, expression in all studied groups, while VDR and cytochrome CYP27B1 protein expression (1α-hydroxylase) correlated with clinical data. CONCLUSIONS: The data provide evidence that indicates that vitamin D and its receptor and enzymes may play a role in CRS.

The role of pepsin in the laryngopharyngeal reflux.

Laryngopharyngeal reflux (LPR) is a common defect among laryngological and phoniatric patients. Although LPR is categorized as a superficial gastroesophageal reflux disease (GERD), differential diagnosis should treat these two diseases separately. LPR symptoms can be assessed in the interview using as a tool the reflux symptom index (RSI). In addition, changes in the larynx that occur during LPR might be seen during laryngoscopy and classified according to the reflux finding score (RFS). One of the main mucosal irritants in LPR is pepsin which digests proteins and impairs the functions of the upper respiratory tract cells by affecting carbonate anhydrase (CAIII) and the Sep 70 protein. Pepsin initiates inflammatory changes within the larynx, nasopharynx and nasal cavity. The use of pepsin detection in upper and lower throat secretions is a new direction in LPR diagnostics.

The Effect of Comprehensive Musculoskeletal Rehabilitation on Clinical Status of Ankylosing Spondylitis Patients.

BACKGROUND: This study aimed to compare the effectiveness of conventional and cryotherapy-based rehabilitation with respect to its impact on selected clinical parameters in AS patients. MATERIAL AND METHODS: Fifty working males aged 22-66 years were included in this study. Twenty-five of them underwent cryotherapy-based rehabilitation (cryogenic chamber, local cryotherapy; individual, instrumental, and nonweight-bearing exercises) for 3 weeks. The others received 3 weeks of conventional rehabilitation (magnetic field therapy; electrotherapy; individual and instrumental exercises). The patients were examined at three time points: before rehabilitation, immediately after its completion and at a three-month follow-up visit. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) was used to assess disease severity, the Bath Ankylosing Spondylitis Functional Index (BASFI) was used to assess musculoskeletal function and a visual analog scale (VAS) was used to assess pain. A global health index was also employed to assess patients' overall well-being. RESULTS: Cryotherapy-based rehabilitation improved the following parameters: BASDAI (P<0.001, P<0.001), BASFI (P<0.001, P=0.007), VAS (P<0.007, P=0.001) and global health index (P<0.001, P<0.001) at the second and third assessment, respectively. Conventional rehabilitation improved the BASDAI (P<0.001), VAS (P=0.029), and overall well-being (P<0.030) at the second assessment. Cryotherapy-based rehabilitation was more effective than conventional rehabilitation with respect to BASFI [F(2, 82)=6.571; P=0.004; eta2=0.120] and overall well-being [F(2, 96) =5.018; P=0.008; eta2=0.095)]. CONCLUSIONS: 1. Comprehensive rehabilitation in ankylosing spondylitis has a positive effect on patients' clinical status. 2. Rehabilitation involving cryotherapy is more effective in improving musculoskeletal function and overall well-being compared to conventional rehabilitation. 3. Cryotherapy-based rehabilitation significantly reduces the intensity of pain experienced by AS patients and decreases disease activity, with the positive effect maintained at 3 months post rehabilitation.

Complex rehabilitation and the clinical condition of working rheumatoid arthritis patients: does cryotherapy always overtop traditional rehabilitation?

AIM: Rehabilitation slows the progress of rheumatoid arthritis (RA) and prevents progression of disability. This study aimed to compare the impact of two rehabilitation programmes on pain, disease activity, locomotor function, global health and work ability forecast in RA patients. MATERIALS AND METHODS: Sixty-four employed women aged 24-65 years participated in the study. All patients underwent individual and instrumental kinesiotherapy. Thirty-two patients underwent cryogenic chamber therapy and local cryotherapy as well as non-weight-bearing, instrumental and individual kinesiotherapy. The remaining 32 patients received traditional rehabilitation in the form of electromagnetic and instrumental therapy, individual and pool-based non-weight-bearing kinesiotherapy. Rehabilitation lasted 3 weeks. Patients were examined three times: prior to rehabilitation, after 3 weeks of therapy and 3 months after completion of rehabilitation. The following study instruments were used: to assess disease activity: DAS-28; functional impairment: HAQ-DI; pain severity: VAS; patients' overall well-being: a scale from 0 to 100 (Global Health Index); and patients' own prognosis of fitness for work: the 6th question from Work Ability Index (WAI). Statistical analysis of data was performed using the STATISTICA 8.0 package. Mixed-design two-way analysis of variance was used for hypothesis testing. RESULTS: All patients improved after rehabilitation. The group of patients those who underwent cryotherapy had improved DAS-28, HAQ-DI, VAS and global health scores immediately following the 3-week rehabilitation programme (p < 0.001, p = 0.001, p = 0.007 and p < 0.001, respectively), as well as at the 3-month follow-up (p < 0.001, p < 0.001, p = 0.009 and p < 0.001, respectively). Rehabilitation using cryotherapy resulted in greater improvement in disease activity DAS-28 [F(2,105) = 5.700; p = 0.007; η(2) = 0.084] and HAQ-DI locomotor function scores [F(2,109) = 6.771; p = 0.003; η(2) = 0.098] compared to traditional rehabilitation. The impact of both forms of rehabilitation on patients' own prognosis of work ability in the next 2 years was not significant. Results of patients who underwent traditional approach showed decreased disease activity following the initial 3-week period; however, this improvement did not sustain to the end of follow-up, 3 months later. CONCLUSIONS: Complex rehabilitation in RA has a positive effect on patients' clinical condition. The rehabilitation programme that includes cryotherapy overtops traditional rehabilitation, particularly as regards improvement in locomotor function, disease activity and sustaining willingness to continue working and exerts long-lasting effect. IMPLICATIONS FOR REHABILITATION: Rehabilitation using cryotherapy is more effective in improving locomotor function, decreasing disease activity and sustaining willingness to continue working compared to traditional rehabilitation. Rehabilitation using cryotherapy significantly reduces the intensity of pain experienced by patients with RA, and this positive effect is maintained at 3 months post-rehabilitation. Complex rehabilitation, particularly treatment using cryotherapy, improves patients' subjective assessment of their overall well-being and perception of their disease. Complex rehabilitation in rheumatoid arthritis has a positive effect on patients' clinical condition.

The Paf1 complex represses small-RNA-mediated epigenetic gene silencing.

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the double-stranded RNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute-protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing and chromatin-dependent gene silencing. Although endogenous small RNAs have crucial roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, here we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by the continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model in which Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small-RNA-mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building epigenetic memory.

Accumulation of poly(ADP-ribose) polymerase inhibitors in children with chronic renal failure.

Nicotinamide, N-methyl-2-pyridone-5-carboxamide (Met2PY) and N-methyl-4-pyridone-3-carboxamide (Met4PY) are biological metabolites of the intracellular coenzyme nicotinamide adenine dinucleotide (NAD) that can potentially inhibit poly(ADP-ribose) polymerase 1 (PARP-1; DNA repair enzyme). Our research was aimed at establishing whether chronic renal failure (CRF) in children leads to the elevation of plasma NAD metabolites sufficient to inhibit PARP-1 activity. Nicotinamide, Met2PY and Met4PY plasma and erythrocyte concentrations were measured in 25 children with CRF and in 19 healthy children. The effect of these NAD metabolites on PARP-1 activity was studied in vitro. We found that plasma concentration of all NAD metabolites (nicotinamide, Met2PY, Met4PY) in children with CRF could reach the concentration of 2, 30 and 10 microM as compared to 0.2, 1 and 0.5 microM, respectively, in healthy children. The concentration of nicotinamide metabolites correlated positively with plasma creatinine concentration and negatively with creatinine clearance in children with CRF. We found that Met2PY, Met4PY and nicotinamide inhibited in vitro PARP-1 activity with IC50 values of 2.1, 0.18 and 0.12 mM, respectively. Our data indicate that NAD metabolites accumulate in plasma of children with CRF and their combined effect could lead to the inhibition of PARP-1 activity. NAD metabolites could be particularly harmful in children due to higher DNA turnover than in adults.