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Slavic populations, such as those in Poland, are considered to have a low prevalence of giant cell arteritis (GCA), although epidemiological data are sparse. The study aimed to compare the reported frequency of GCA in various regions of Poland and analyze the differences between them. We conducted a multicenter, retrospective study of all GCA patients included in the POLVAS registry—the first large multicenter database of patients with vasculitis in Poland. The data from the POLVAS registry were compared with the reported prevalence provided by national insurers from the corresponding regions. A 10-fold increase in the diagnostic rates of GCA was observed in Poland between 2008 and 2019, reaching 8.38 per 100,000 population > 50 years old. It may be attributed to increased interest accompanied by improved diagnostic modalities with the introduction of ultrasound-based, fast-track diagnostic pathways in some centers. However, regional inequities are present, resulting in 10-fold differences (from 2.57 to 24.92) in reported prevalence between different regions. Corticosteroid (CS) monotherapy was the main stem of treatment. Further cooperation and education are needed to minimize regional inequities. This observational study suggests some potential for further increase of the recognizability of GCA and wider use of other than CS monotherapy treatment regimens. We hope that the Polish experience might be interesting and serve as some guidance for the populations where GCA is underdiagnosed.

Objective. To characterize the eosinophilic granulomatosis with polyangiitis (EGPA) population from the POLVAS registry depending on ANCA status and diagnosis onset, including their comparison with the granulomatosis with polyangiitis (GPA) subset with elevated blood eosinophilia (min. 400/μl) (GPA HE) to develop a differentiating strategy. Methods. A retrospective analysis of the POLVAS registry. Results. The EGPA group comprised 111 patients. The ANCA-positive subset (n = 45 [40.54%]) did not differ from the ANCA-negative one in clinics. Nevertheless, cardiovascular manifestations were more common in ANCA-negative patients than in those with anti-myeloperoxidase (MPO) antibodies (46.97% vs. 26.92%, p = 0.045). Patients diagnosed before 2012 (n = 70 [63.06%]) were younger (median 41 vs. 49 years, p

Machine learning (ML) algorithms can handle complex genomic data and identify predictive patterns that may not be apparent through traditional statistical methods. They become popular tools for medical applications including prediction, diagnosis or treatment of complex diseases like rheumatoid arthritis (RA). RA is an autoimmune disease in which genetic factors play a major role. Among the most important genetic factors predisposing to the development of this disease and serving as genetic markers are HLA-DRB and non-HLA genes single nucleotide polymorphisms (SNPs). Another marker of RA is the presence of anticitrullinated peptide antibodies (ACPA) which is correlated with severity of RA. We use genetic data of SNPs in four non-HLA genes (PTPN22, STAT4, TRAF1, CD40 and PADI4) to predict the occurrence of ACPA positive RA in the Polish population. This work is a comprehensive comparative analysis, wherein we assess and juxtapose various ML classifiers. Our evaluation encompasses a range of models, including logistic regression, k-nearest neighbors, naïve Bayes, decision tree, boosted trees, multilayer perceptron, and support vector machines. The top-performing models demonstrated closely matched levels of accuracy, each distinguished by its particular strengths. Among these, we highly recommend the use of a decision tree as the foremost choice, given its exceptional performance and interpretability. The sensitivity and specificity of the ML models is about 70% that are satisfying. In addition, we introduce a novel feature importance estimation method characterized by its transparent interpretability and global optimality. This method allows us to thoroughly explore all conceivable combinations of polymorphisms, enabling us to pinpoint those possessing the highest predictive power. Taken together, these findings suggest that non-HLA SNPs allow to determine the group of individuals more prone to develop RA rheumatoid arthritis and further implement more precise preventive approach.

IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Systemic sclerosis (SSc) represents a rare and intricate autoimmune connective tissue disease, the pathophysiology of which has not been fully understood. Its key features include progressive fibrosis of the skin and internal organs, vasculopathy and aberrant immune activation. While various anti-nuclear antibodies can serve as biomarkers for the classification and prognosis of SSc, their direct role in organ dysfunction remains unclear. Anti-Th/To antibodies are present in approximately 5% of SSc patients, and are particularly prevalent among those with the limited subtype of the disease. Although the presence of these autoantibodies is associated with a mild course of the disease, there is a strong connection between them and severe clinical manifestations of SSc, including interstitial lung disease, pulmonary arterial hypertension and gastrointestinal involvement. Also, the additional clinical correlations, particularly with malignancies, need further research. Moreover, the disease’s course seems to be influenced by antibodies, specific serum cytokines and TLR signaling pathways. Understanding the relationships between presence of anti-Th/To, its molecular aspects and response to treatment options is crucial for the development of novel, personalized therapeutic techniques and should undergo profound analysis in future studies.

Objective Still’s disease is more frequently observed in the paediatric context, but a delayed onset is not exceptional both in the adulthood and in the elderly. However, whether paediatric-onset, adult-onset and elderly-onset Still’s disease represent expressions of the same disease continuum or different clinical entities is still a matter of controversy. The aim of this study is to search for any differences in demographic, clinical features and response to treatment between pediatric-onset, adult-onset and elderly-onset Still’s disease.Methods Subjects included in this study were drawn from the International AutoInflammatory Disease Alliance Network registry for patients with Still’s disease.Results A total of 411 patients suffering from Still’s disease were enrolled; the disease occurred in the childhood in 65 (15.8%) patients, in the adult 314 (76.4%) patients and in the elderly in 32 (7.8%) patients. No statistically significant differences at post-hoc analysis were observed in demographic features of the disease between pediatric-onset, adult-onset and elderly-onset Still’s disease. The salmon-coloured skin rash (p=0.004), arthritis (p=0.009) and abdominal pain (p=0.007) resulted significantly more frequent among paediatric patients than in adult cases, while pleuritis (p=0.015) and arthralgia (p

Background Different patient clusters were preliminarily suggested to dissect the clinical heterogeneity in Still’s disease. Thus, we aimed at deriving and validating disease clusters in a multicentre, observational, prospective study to stratify these patients.Methods Patients included in GIRRCS AOSD-study group and AIDA Network Still Disease Registry were assessed if variables for cluster analysis were available (age, systemic score, erythrocyte sedimentation rate (ESR), C reactive protein (CRP) and ferritin). K-means algorithm with Euclidean metric and Elbow plot were used to derive an adequate number of clusters.Results K-means clustering assessment provided four clusters based on means standardised according to z-scores on 349 patients. All clusters mainly presented fever, skin rash and joint involvement. Cluster 1 was composed by 115 patients distinguished by lower values of age and characterised by skin rash myalgia, sore throat and splenomegaly. Cluster 2 included 128 patients identified by lower levels of ESR, ferritin and systemic score; multiorgan manifestations were less frequently observed. Cluster 3 comprised 31 patients categorised by higher levels of CRP and ferritin, they were characterised by fever and joint involvement. Cluster 4 contained 75 patients derived by higher values of age and systemic score. Myalgia, sore throat, liver involvement and life-threatening complications, leading to a high mortality rate, were observed in these patients.Conclusions Four patient clusters in Still’s disease may be recognised by a multidimensional characterisation (‘Juvenile/Transitional’, ‘Uncomplicated’, ‘Hyperferritinemic’ and ‘Catastrophic’). Of interest, cluster 4 was burdened by an increased rate of life-threatening complications and mortality, suggesting a more severe patient group.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8–355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7–613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3–1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

ABSTRACT This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 μM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

ObjectiveThe aim of this paper is to present the AutoInflammatory Disease Alliance (AIDA) international Registry dedicated to Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, describing its design, construction, and modalities of dissemination.MethodsThis Registry is a clinical, physician-driven, population- and electronic-based instrument designed for the retrospective and prospective collection of real-life data. Data gathering is based on the Research Electronic Data Capture (REDCap) tool and is intended to obtain real-world evidence for daily patients' management. The Registry may potentially communicate with other on-line tools dedicated to VEXAS syndrome, thus enhancing international collaboration and data sharing for research purposes. The Registry is practical enough to be easily modified to meet future needs regarding VEXAS syndrome.ResultsTo date (April 22nd, 2022), 113 Centers from 23 Countries in 4 continents have been involved; 324 users (114 Principal Investigators, 205 Site Investigators, 2 Lead Investigators, and 3 data managers) are currently able to access the registry for data entry (or data sharing) and collection. The Registry includes 4,952 fields organized into 18 instruments designed to fully describe patient's details about demographics, clinical manifestations, symptoms, histologic details about skin and bone marrow biopsies and aspirate, laboratory features, complications, comorbidities, therapies, and healthcare access.ConclusionThis international Registry for patients with VEXAS syndrome will allow the achievement of a comprehensive knowledge about this new disease, with the final goal to obtain real-world evidence for daily clinical practice, especially in relation to the comprehension of this disease about the natural history and the possible therapeutic approaches. This Project can be found on https://clinicaltrials.gov NCT05200715.

Psoriatic arthritis is a heterogenous chronic inflammatory disease that develops over time in some patients with psoriasis. The course of the disease is variable, with a broad clinical spectrum. The management of PsA has changed tremendously over the last decade, thanks to earlier diagnosis, a multidisciplinary approach and progress in pharmacological therapies. Therefore, screening for risk factors and the early signs of arthritis is highly important and recommended. Currently, research is focused on finding soluble biomarkers and developing imaging techniques that can improve the prediction of psoriatic arthritis. Among imaging modalities, ultrasonography seems to be the most accurate in detecting subclinical inflammation. Early intervention is based on the assumption that it is possible to prevent or delay psoriatic arthritis if systemic treatment for psoriasis can be administered early enough. This review article provides an overview of the current perspectives and evidence regarding the diagnosis, management and prevention of psoriatic arthritis.

Single nucleotide polymorphisms in non-HLA genes are involved in the development of rheumatoid arthritis (RA). SNPS in genes: PADI4 (rs2240340), STAT4 (rs7574865), CD40 (rs4810485), PTPN22 (rs2476601), and TRAF1 (rs3761847) have been described as risk factors for the development of autoimmune diseases, including RA. This study aimed to assess the prevalence of polymorphisms of these genes in the Polish population of patients with rheumatoid arthritis as compared to healthy controls. 324 subjects were included in the study: 153 healthy subjects and 181 patients from the Department of Rheumatology, Medical University of Lodz who fulfilled the criteria of rheumatoid arthritis diagnosis. Genotypes were determined by Taqman SNP Genotyping Assay. rs2476601 (G/A, OR = 2.16, CI = 1.27–3.66; A/A, OR = 10.35, CI = 1.27–84.21), rs2240340 (C/T, OR = 4.35, CI = 2.55–7.42; T/T, OR = 2.80, CI = 1.43–4.10) and rs7574865 (G/T, OR = 1.97, CI = 1.21–3.21; T/T, OR = 3.33, CI = 1.01–11.02) were associated with RA in the Polish population. Rs4810485 was also associated with RA, however after Bonferroni’s correction was statistically insignificant. We also found an association between minor alleles of rs2476601, rs2240340, and rs7574865 and RA (OR = 2.32, CI = 1.47–3.66; OR = 2.335, CI = 1.64–3.31; OR = 1.88, CI = 1.27–2.79, respectively). Multilocus analysis revealed an association between CGGGT and rare (below 0.02 frequency) haplotypes (OR = 12.28, CI = 2.65–56.91; OR = 3.23, CI = 1.63–6.39). In the Polish population, polymorphisms of the PADI4, PTPN22, and STAT4 genes have been detected, which are also known risk factors for RA in various other populations.

Abstract Background The course of asthma may differ between elderly asthmatics (EA) and non-elderly asthmatics (nEA), which may be partially associated with an age-dependent aberrant immune response. The aim of the study was to determine the influence of serum miRNA expression on asthma characteristics and systemic inflammation markers in EA and nEA. Methods Control and severity of asthma, pulmonary function and FeNO were assessed in 28 EA and 31 nEA patients. The control group included 59 elderly and non-elderly healthy individuals. The expression of selected miRNAs in serum was measured with rt-PCR, and proinflammatory cytokine activity was assayed by ELISA or flow cytometry. Results No difference in serum miRNA expression was observed between the asthmatics and healthy controls. EA demonstrated lower expression of miRNA-106a and miRNA-126a than nEA (p = 0.003 and p = 0.02) and EC had lower expression of miRNA-146a, -126a, -106a and 19b than nEC (p = 0.001, p = 0.003, p = 0.005 and p

ObjectiveAim of this paper is to present the design, construction, and modalities of dissemination of the AutoInflammatory Disease Alliance (AIDA) International Registry for patients with systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD), which are the pediatric and adult forms of the same autoinflammatory disorder.MethodsThis Registry is a clinical, physician-driven, population- and electronic-based instrument implemented for the retrospective and prospective collection of real-world data. The collection of data is based on the Research Electronic Data Capture (REDCap) tool and is intended to obtain evidence drawn from routine patients' management. The collection of standardized data is thought to bring knowledge about real-life clinical research and potentially communicate with other existing and future Registries dedicated to Still's disease. Moreover, it has been conceived to be flexible enough to easily change according to future scientific acquisitions.ResultsStarting from June 30th to February 7th, 2022, 110 Centers from 23 Countries in 4 continents have been involved. Fifty-four of these have already obtained the approval from their local Ethics Committees. Currently, the platform counts 290 users (111 Principal Investigators, 175 Site Investigators, 2 Lead Investigators, and 2 data managers). The Registry collects baseline and follow-up data using 4449 fields organized into 14 instruments, including patient's demographics, history, clinical manifestations and symptoms, trigger/risk factors, therapies and healthcare access.ConclusionsThis international Registry for patients with Still's disease will allow a robust clinical research through collection of standardized data, international consultation, dissemination of knowledge, and implementation of observational studies based on wide cohorts of patients followed-up for very long periods. Solid evidence drawn from “real-life” data represents the ultimate goal of this Registry, which has been implemented to significantly improve the overall management of patients with Still's disease. NCT 05200715 available at https://clinicaltrials.gov/.

Biological agents such as monoclonal antibodies and fusion proteins are widely used for the treatment of patients with various rheumatic disorders, influencing the quality of life, disability and even mortality in patients. However, biological agents can evoke adverse reactions of different grades of severity. Although drug avoidance remains a gold standard in the care of patients hypersensitive to medication, in certain clinical situations the culprit drug is the drug of choice and cannot be replaced by another equally effective compound. In such cases, desensitization can allow the patient to be treated within current guidelines and with the most effective treatment. The authors searched Medline and Scopus databases for English-language sources using the following key words: hypersensitivity, desensitization, biologicals, adalimumab, etanercept, adalimumab, certolizumab, golimumab, rituximab, infliximab, ixekizumab, tocilizumab, anakinra and canakinumab. The aim of our review is to present the current knowledge about desensitization to biological agents and some guidelines according to patient inclusion, contraindications, procedures, and safety requirements. Drug desensitization is a new issue in rheumatology, and the solution to the problem of allergic reactions to biological drugs, which gives patients with rheumatic diseases the opportunity to extend and prolong their therapy. The present article is one of the first widely discussing this topic in the biological treatment of rheumatic diseases.

Monogenic autoinflammatory diseases (AIDs, formerly known as hereditary periodic fever syndromes) cover a spectrum of diseases which lead to chronic or recurrent inflammation caused by activation of the innate immune system. The most common monogenic AID is familial Mediterranean fever. Monogenic autoinflammatory diseases are generally considered intracellular signalling defects. Some stereotypical knowledge may be misleading; e.g. monogenic AIDs are not exclusively found in children, family history is often negative, fever frequently is not a leading manifestation and frequency of attacks in adults is usually variable. Lack of genetic confirmation should not stop anti-inflammatory ex juvantibus therapy. The pattern of tissue injury in AIDs is basically different from that observed in autoimmunity. There is no autoaggression against organ-specific antigens, but substantial damage (amyloidosis, cachexia, premature cardiovascular disease) is secondary to long-lasting inflammation. The Polish national programme of anti-interleukin 1 treatment opens new possibilities for the treatment. However, monogenic AIDs are frequently misdiagnosed and more awareness is needed.

The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.

Supraglottic stenosis is a rare symptom, particularly in fibroinflammatory multifocal diseases, such as IgG4-related disease (IgG4-RD). There is still an inconsistency in the diagnosis of less-common locations of IgG4-RD, which causes a delay in the diagnosis and treatment. Our paper aims to analyze different aspects of IgG4-RD presenting as supraglottic stenosis, including the possible overlap with ANCA-associated vasculitis. We compare the usefulness of the recently revised ACR/EULAR and Comprehensive criteria and discuss treatment options. The review was performed according to PRISMA guidelines using the MEDLINE Pubmed and Scopus databases. The analysis includes nine papers describing supraglottic laryngeal stenosis in 13 patients. Furthermore, we present a case of a woman with ongoing supraglottic stenosis presenting with cough, temporary dyspnea and stridor as the symptoms of localized IgG4-RD. At the time of writing, the patient remains in remission while receiving treatment with cyclophosphamide and methylprednisolone. The symptoms of supraglottic localization of IgG4-RD may be severe; however, at that point, clinicians should suspect autoimmune etiology and attempt to modulate the autoimmune response instead of performing dilatation surgery—the effects of which may not result in extended intervals between interventions. The ACR/EULAR criteria show great specificity; however, when IgG4-RD is presumed, the specific treatment should be implemented.

The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 = −7.35 ± 0.09 kcal mol−1; ΔH(ITC)2 = 4.00 ± 0.14 kcal mol−1; TΔS(ITC)2 = 11.3 kcal mol−1). The formation of the {[B(Ph)4]−}–BSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Abstract One of the major concerns in the practice of allergy is related to the safety of procedures for the diagnosis and treatment of allergic disease. Management (diagnosis and treatment) of hypersensitivity disorders involves often intentional exposure to potentially allergenic substances (during skin testing), deliberate induction in the office of allergic symptoms to offending compounds (provocation tests) or intentional application of potentially dangerous substances (allergy vaccine) to sensitized patients. These situations may be associated with a significant risk of unwanted, excessive or even dangerous reactions, which in many instances cannot be completely avoided. However, adverse reactions can be minimized or even avoided if a physician is fully aware of potential risk and is prepared to appropriately handle the situation. Information on the risk of diagnostic and therapeutic procedures in allergic diseases has been accumulated in the medical literature for decades; however, except for allergen specific immunotherapy, it has never been presented in a systematic fashion. Up to now no single document addressed the risk of the most commonly used medical procedures in the allergy office nor attempted to present general requirements necessary to assure the safety of these procedures. Following review of available literature a group of allergy experts within the World Allergy Organization (WAO), representing various continents and areas of allergy expertise, presents this report on risk associated with diagnostic and therapeutic procedures in allergology and proposes a consensus on safety requirements for performing procedures in allergy offices. Optimal safety measures including appropriate location, type and required time of supervision, availability of safety equipment, access to specialized emergency services, etc. for various procedures have been recommended. This document should be useful for allergists with already established practices and experience as well as to other specialists taking care of patients with allergies.

An amendment to this paper has been published and can be accessed via the original article.

Gout, known as “the disease of the kings”, is the most frequent type of arthritis. It results from sustained hyperuricemia that leads to monosodium urate crystal deposition in joint structures and soft tissue. Environmental factors such as diet affect the incidence of gout; there is a known relationship between the occurrence of an acute attack of gout and the consumption of alcohol and meat; and a low purine diet is a widely recognized nonpharmacological method of supplementing the treatment and preventing recurrence of arthritis. This review aims to summarize the current knowledge about the role of vitamin C in prevention and treatment of gout. A PubMed/Medline database search on the role of vitamin C in purine metabolism was done. Reports from in vitro and animal studies seem to be promising and to allow explanation of the physiological relationship between vitamin C and uric acid. Most epidemiological studies indicate a significant correlation between high vitamin C intake and lower serum uric acid levels. Despite promising observations, there are few observational and interventional studies, and their results do not clearly define the benefits of a high daily intake of vitamin C in preventing the development and recurrence of gout.

In the following paper, we present the results of our studies on the interactions of the Aβ1-42 peptide and its three short fragments, namely Aβ5-16 (RHDSGYEVHHQK; HZ1), Aβ8-13 (SGYEVH; HZ2), and Aβ8-12 (SGYEV; HZ3) with selected painkillers (ibuprofen and aspirin) and compounds of natural origin (anabasine and epinephrine). Steady-state fluorescence spectroscopy was used to study the binding properties of the selected systems. Additionally, based on molecular dynamics (MD) calculations supported by NMR-derived restrains, we have proposed the most likely area of the interactions of Aβ1-42 and Aβ5-16 peptides with the investigated compounds. The influence of symmetrically oriented side chains of amino acid residues present in the first part of the Aβ1-42 sequence on the stability of the resulting complexes has been discussed. Finally, the changes in the peptide structures on account of complex formation were analyzed.

Osteoporosis and associated low energy fractures are a significant clinical problem, especially in the elderly population. The occurrence of a hip fracture is associated with significant mortality and a high risk of disability. For this, apart from the treatment of osteoporosis, effective prevention of both the development of the disease and related fractures is extremely important. One aspect of osteoporosis prevention is proper dietary calcium intake and normal vitamin D3 levels. However, there is some evidence for a potential role of vitamin C in osteoporosis and fracture prevention, too. This review aims to summarize the current knowledge about the role of vitamin C in osteoporosis development, prevention and treatment. The PubMed/Medline search on the role of vitamin C in bone metabolism database was performed for articles between 2000 and May 2020. Reports from in vitro and animal studies seem promising. Epidemiological studies also indicate the positive effect of high vitamin C content in the daily diet on bone mineral density. Despite promising observations, there are still few observational and intervention studies and their results do not allow for unequivocal determination of the benefits of high daily intake of vitamin C or its long-term supplementation.

Rheumatoid arthritis (RA) is a systemic, inflammatory disease of the joints and surrounding tissues. RA manifests itself with severe joint pain, articular inflammation, and oxidative stress. RA is associated with certain types of cancer. We have assumed that RA patients’ increased susceptibility to cancer may be linked with genomic instability induced by impaired DNA repair and sensitivity to DNA damaging agents. The aim of this work was to analyze the sensitivity of peripheral blood mononuclear cells (PBMCs) isolated from RA patients to DNA damaging agents: tert-butyl hydroperoxide (TBH), bleomycin, ultraviolet (UV) radiation, and methyl methanesulfonate (MMS) and calculate the repair efficiency. TBH induce oxidative DNA lesions repaired mainly by base excision repair (BER). Bleomycin induced mainly DNA double-strand breaks repaired by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). We included 20 rheumatoid arthritis patients and 20 healthy controls and used an alkaline version of the comet assay with modification to measure sensitivity to DNA damaging agents and DNA repair efficiency. We found an increased number of DNA breaks and alkali-labile sites in the RA patients compared to those in the controls. Exposure to DNA damaging agents evoked the same increased damage in both groups, but we observed statistically higher PMBC sensitivity to TBH, MMS, bleomycin as well as UV. Examination of the repair kinetics of both groups revealed that the DNA lesions induced by TBH and bleomycin were more efficiently repaired in the controls than in the patients. These data suggest impaired DNA repair in RA patients, which may accelerate PBMC aging and/or lead to higher cancer incidence among RA patients.

Herein, we have investigated principally with the use of UV and fluorescence (steady-state and time-resolved) spectroscopy the interactions between selected pentapeptides with tyrosine residue (EYHHQ, EHYHQ, EHHQY, and KYHHE) and various metal ions (Cu2+, Mn2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+, Ag+, Pb2+, Sr2+, Ba2+, Ca2+, Mg2+, Al3+, Fe2+, and Ga3+) in order to establish the relationship between the position of a tyrosine residue in the peptide sequence and the metal ion-binding properties. Among the peptides studied, EHYHQ was evaluated as an efficient and selective ligand for developing a chemosensor for the detection of copper(II) ions. While significant fluorescence emission quenching was observed for that peptide in the presence of Cu2+ cations, other metal cations used at the same and at considerably higher concentrations caused a negligible change of the fluorescence emission spectrum, indicating a high selectivity of EHYHQ for Cu2+ ions. Under optimum conditions, fluorescence intensity was inversely proportional to the concentration of Cu2+ ions. The limit of detection of Cu2+ ions with the use of EHYHQ was determined at the level of 26.6 nM. The binding stoichiometry of the complexes of the studied peptides with Cu2+ ions was evaluated spectrophotometrically and fluorimetrically (as in the case of EHYHQ confirmed by mass spectrometry) and found to be 1:2 (Cu2+-peptide) for all the investigated systems. Furthermore, the stability constant (K) values of these complexes were determined. The reversibility of the proposed Cu2+ ions sensor was confirmed, the pH range where the sensor acts was determined, while its analytical performance was compared with some other reported recently fluorescent sensors. The mechanism of the interactions between EHYHQ and Cu2+ was proposed on the basis of NMR spectroscopy investigations.

It is shown that probably three residues: His6, His14 and His16 in the original sequence Aβ(1−42) serve as metal-binding sites for Cu2+ ions. On the other hand, there is a possibility that only one of them plays a crucial role in the formation of the {Aβ(1−42) --Cu2+} complex. The isothermal titration calorimetry (ITC) measurements supported by molecular dynamic simulation (MD) with the NMR derived restrains were used to investigate the interactions of Cu2+ with Aβ(5−16), a fragment of the Aβ(1−42) protein, with the following sequence: Ac--Arg--His--Asp--Ser--Gly--Tyr--Glu--Val--His--His--Gln--Lys--NH2, termed HZ1. The conditional thermodynamic parameters suggest that the formation of the Cu2+--HZ1 complex is both an enthalpy and entropy driven process under the experimental conditions. The studies presented here (after comparison with our previous results) show that the affinity of peptides to copper metal ions depends on two factors: the primary structure (amino acid composition) and the shape of the peptide conformation adopted.

In forensic laboratories, the most commonly analyzed microtraces are microscopic fragments of single fibers. One of the main goals of the examination of fragments of fibers a few millimeters long is to determine their characteristic physicochemical properties and compare them with fibers originating from a known source (e.g., a suspect’s clothes). The color and dyes of fiber microtraces play an important role in their research and evaluation, being analyzed by means of microscopic, spectroscopic, and chromatographic methods. The results of examinations conducted with the use of spectroscopic techniques might be ambiguous due to overlapping bands of absorption and the transmission and dispersion of electromagnetic radiation corresponding to the specific chemical structure of the fibers and their dyes. For this reason, it is very important to improve currently available spectroscopic methods and/or to propose new ones that allow evidential materials to be analyzed in a much more reliable way. In this review, the possibility of the use of chromatographic techniques with different detection systems for such analyses is underlined. This review covers the different analytical methods used in the forensic analysis of polyester fibers dyed with disperse dyes. Polyester fibers occupy the first position among synthetic fibers in their use for a variety of purposes, and disperse dyes are commonly applied for dyeing them.

Background: Impaired regeneration of airway epithelium may lead to persistence of inflammation and remodelling. Regeneration of injured epithelium is a complex phenomenon and the role of toll-like receptors (TLRs) in the stimulation of respiratory virus products in this process has not been established. Objective: This study was undertaken to test the hypothesis that the wound repair process in airway epithelium is modulated by microbial products via toll-like receptors. Methods: Injured and not-injured bronchial epithelial cells (ECs) (BEAS-2B line) were incubated with the TLR agonists poly(I:C), lipopolisacharide (LPS), allergen Der p1, and supernatants from virus-infected epithelial cells, either alone or in combination with TLR inhibitors. Regeneration and immune response in injured and not-injured cells were studied. Results: Addition of either poly(I:C) or LPS to ECs induced a marked inhibition of wound repair. Supernatants from RV1b-infected cells also decreased regeneration. Preincubation of injured and not-injured ECs with TLR inhibitors decreased LPS and poly(I:C)-induced repair inhibition. TGF-β and RANTES mRNA expression was higher in injured ECs and IFN-α, IFN-β, IL-8, and VEGF mRNA expression was lower in damaged epithelium as compared to not-injured. Stimulation with poly(I:C) increased IFN-α and IFN-β mRNA expression in injured cells, and LPS stimulation decreased interferons mRNA expression both in not-injured and injured ECs. Conclusion: Regeneration of the airway epithelium is modulated by microbial products via toll-like receptors.

Background: Flavonoids exert anti-inflammatory properties and modulate oxidative stress in vitro, suggesting a protective effect on lung function, but epidemiological studies examining this association are scarce. Methods: A stratified random sample was drawn from the GA2LEN screening survey, in which 55,000 adults aged 15 to 75 answered a questionnaire on respiratory symptoms. Post-bronchodilator spirometry was obtained from 2850 subjects. Forced vital capacity (FVC), the ratio between the forced exhaled volume in 1 second (FEV1) and FVC (FEV1/FVC), FVC below lower limit of normal (FVC < LLN), and FEV1/FVC < LLN were calculated. Intake of the six main subclasses of flavonoids was estimated using the GA2LEN Food Frequency Questionnaire. Adjusted associations between outcomes and each subclass of flavonoids were examined with multivariate regressions. Simes’ procedure was used to test for multiple comparisons. Results: A total of 2599 subjects had valid lung function and dietary data. A lower prevalence of FVC < LLN (airway restriction) was observed in those with higher total flavonoid (adjusted odds ratio (aOR), higher vs. lowest quintile intake 0.58; 95% Confidence Interval (CI) 0.36, 0.94), and pro-anthocyanidin intakes (aOR 0.47; 95% CI 0.27, 0.81). A higher FEV1/FVC was associated with higher intakes of total flavonoids and pro-anthocyanidins (adjusted correlation coefficient (a β-coeff 0.33; 0.10, 0.57 and a β-coeff 0.44; 95% CI 0.19, 0.69, respectively). After Simes’ procedure, the statistical significance of each of these associations was attenuated but remained below 0.05, with the exception of total flavonoids and airway restriction. Conclusions: This population-based study in European adults provides cross-sectional evidence of a positive association of total flavonoid intake and pro-anthocyanidins and ventilatory function, and a negative association with spirometric restriction in European adults.

Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (Tm = 99.82°C, ΔHcal = 4.58 × 10(4) cal mol(-1)). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.

We examined the effect of like-charged residues on the conformation of an original nine amino-acid-residue fragment of the human Pin1 WW domain (hPin1) with the following sequence: Ac-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-NH2 (U9). This was facilitated by CD and NMR spectroscopic measurements, and molecular dynamics calculations. Our earlier studies suggested that the presence of like charged residues at the end of a short polypeptide chain composed of nonpolar residues could induce a chain reversal. For the U9 peptide, canonical MD simulations with NMR-derived restraints demonstrated the presence of ensembles of structures with a tendency to form a β-chain reversal. Additionally, thermal stabilities of the peptide under study were measured using differential scanning calorimetry (DSC). The estimated well defined phase transition point showed that conformational equilibria in the U9 peptide were strongly dependent on temperature.