Mitochondria Metabolomics Reveals a Role of β-Nicotinamide Mononucleotide Metabolism in Mitochondrial DNA Replication

Summary Mitochondrial DNA (mtDNA) replication is tightly regulated and necessary for cellular homeostasis; however, its relationship with mitochondrial metabolism remains unclear. Advances in metabolomics integrated with the rapid isolation of mitochondria will allow for remarkable progress in analyzing mitochondrial metabolism. Here, we propose a novel methodology for mitochondria-targeted metabolomics, which employs a quick isolation procedure using a hemolytic toxin from Streptococcus pyogenes streptolysin O (SLO). SLO-isolation of mitochondria from cultured HEK293 cells is time- and labor-saving for simultaneous multi-sample processing and has been applied to various other cell lines in this study. Furthermore, our method can detect the time-dependent reduction in mitochondrial ATP in response to a glycolytic inhibitor 2-deoxyglucose, indicating the suitability to prepare metabolite analysis-competent mitochondria. Using this methodology, we searched for specific mitochondrial metabolites associated with mtDNA replication activation, and nucleotides and NAD+ were identified to be prominently altered. Most notably, treatment of β-Nicotinamide Mononucleotide (β-NMN), a precursor of NAD+, to HEK293 cells activated and improved the rate of mtDNA replication by increasing nucleotides in mitochondria and decreasing their degradation products: nucleosides. Our results suggest that β-NMN metabolism play a role in supporting mtDNA replication by maintaining the nucleotide pool balance in the mitochondria.

Determination of anti-streptolysin – O titer in suspected cases of streptococcal infection

The Group A beta hemolytic streptococcus has remained a major human infective agent for hundreds of years. Group 'A' beta hemolytic streptococcus related ailment and sequelae keep on affecting general public and national economy as they mostly influence kids and youthful grown-ups. Current research aimed to determination of anti-streptolysin –o titer in suspected cases of streptococcal infection. : Determination of anti-Streptolysin-O (ASO) titer in suspected cases of streptococcal infection. The measurement of ASO levels was done by semi-quantitative analyzer analyzer on photometric and colorimetric systems. 107 blood samples were taken in our study. This study was conduct in serological section of microbiology department of Teerthanker Mahaveer Hospital & research centre Moradabad. A total 107 samples were tested in this study. Of these, 23(21.5%) were found to be positive for the presence of ASO having titre of >200IU/mL. This study will be useful to evaluate utility of sero-diagnosis in our catering population and found useful in early diagnosis and treatment of these pathogens. Treatment can be initiated at an early stage leading to reduction in complications and associated mortality.

Signaling Peptide SpoV Is Essential for Streptococcus pyogenes Virulence, and Prophylaxis with Anti-SpoV Decreases Disease Severity

Streptococcal peptide of virulence (SpoV) is a Streptococcus pyogenes (group A streptococcus (GAS))-specific peptide that is important for GAS survival in murine blood, and the expression of the virulence factors streptolysin O (slo) and streptolysin S (sagA). We used a spoV mutant in isolate MGAS315 to assess the contribution of the SpoV peptide to virulence by using a murine model of invasive disease and an ex vivo human model (Lancefield assay). We then used antibodies to SpoV in both models to evaluate their ability to decrease morbidity and mortality. Results showed that SpoV is essential for GAS virulence, and targeting the peptide has therapeutic potential.

Clinical Features in Patients With PANDAS/PANS and Therapeutic Approaches: A Retrospective Study

Objective: The clinical characteristics of patients with PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection) and PANS (pediatric acute-onset neuropsychiatric syndrome) and the efficacy of antibiotic therapy with psychotherapy and antipsychotics were investigated to improve neurological symptoms as well as obsessive compulsive disorder (OCD).Methods: We retrospectively analyzed 62 patients with a clinical diagnosis of PANDAS/PANS enrolled from May 14, 2013 to September 15, 2020 in the Neurology Childhood Division, Department of Pediatrics at Sapienza, Rome. Clinical manifestations, neurological and psychiatric, laboratory investigations, and familiar history were collected to evaluate the differences between the two groups. The effects of various therapeutic approaches were examined. Descriptive and comparative statistical analyses were performed.Results: The mean age at onset of PANDAS/PANS symptoms was 6.2 ± 1.2 years. The most common diagnosis was PANDAS, followed by PANS. Neurological and psychiatric symptoms were mostly evident in both groups (>70% of the population), with no significant difference between them (P = 0.52 and P = 0.15, respectively). Irritability, aggressivity, and food restriction were more prevalent in children with PANS than in those with PANDAS (P = 0.024 and P = 0.0023, respectively). The levels of anti-streptolysin O and anti-DNAse B 10-fold higher in PANDAS than those in PANS (P < 0.0001). Antibiotics or psychotherapy were administered in most cases (90.3 and 53.2%, respectively), followed by antipsychotic treatments (24.2%). In the multivariate analysis, among the therapies used, psychotherapy significantly resulted in the most efficacious relief of OCD, reducing stress in patients and their parents (P = 0.042).Conclusion: Our findings confirm a clear clinical difference between the two groups, PANDAS and PANS, using different approaches. In fact, irritability, aggressivity, and food restriction were significantly more frequent in children with PANS and the levels of anti-streptolysin O and anti-DNAse B were higher in PANDAS. Another relevant finding is the efficacy of psychotherapy, especially for obsessive-compulsive disorder, and of antibiotic prophylaxis in managing acute neurological symptoms.

Streptolysin O concentration and activity is central to in vivo phenotype and disease outcome in Group A Streptococcus infection

Group A Streptoccocus (GAS) is among the most diverse of all human pathogens, responsible for a range of clinical manifestations, from mild superficial infections such as pharyngitis to serious invasive infections such as necrotising fasciitis and sepsis. The drivers of these different disease phenotypes are not known. The GAS cholesterol-dependent cytolysin, Streptolysin O (SLO), has well established cell and tissue destructive activity. We investigated the role of SLO in determining disease outcome in vivo, by using two different clinical lineages; the recently emerged hypervirulent outbreak emm type 32.2 strains, which result in sepsis, and the emm type 1.0 strains which cause septic arthritis. Using clinically relevant in vivo mouse models of sepsis and a novel septic arthritis model, we found that the amount and activity of SLO was vital in determining the course of infection. The emm type 32.2 strain produced large quantities of highly haemolytic SLO that resulted in rapid development of sepsis. By contrast, the reduced concentration and lower haemolytic activity of emm type 1.0 SLO led to translocation of bacteria from blood to joints. Importantly, sepsis associated strains that were attenuated by deletion or inhibition of SLO, then also translocated to the joint, confirming the key role of SLO in determining infection niche. Our findings demonstrate that SLO is key to in vivo phenotype and disease outcome. Careful consideration should be given to novel therapy or vaccination strategies that target SLO. Whilst neutralising SLO activity may reduce severe invasive disease, it has the potential to promote chronic inflammatory conditions such as septic arthritis.

Analysis of ASO Titres among Paediatric Age Group Patients Attending a Tertiary Care Hospital, Secunderabad, Telangana

BACKGROUND Streptococcus pyogenes can affect most often in children with peak age related incidence between 11 to 15 years of age and all races of the world. ASO estimation as an early diagnosis can help to start early & accurate treatment. The objectives of this study were to determine the ASO levels among paediatric age group patients and to estimate the age and sex relation to ASO levels. METHODS A total of 200 patient samples were considered under study population for investigating Anti streptolysin O titres. All the serum samples were processed using Tulip Diagnostics ASO Kits. All blood samples of study population were collected and processed immediately by centrifugation at 3000 rpm for 15 minutes. Serum sample is extracted and processed by semi quantitative method for detection of Anti Streptolysin – O. RESULTS Majority of the patients were observed in the age group of 11 - 15 years i.e., 104 out of 200 (52 %) followed by 5-10 years makes up to 41 %. Male preponderance was noted which accounts for 56.5 % of total cases. Out of 68 male positive patients, 21 (30.8 %) had ASO titres of 200-400 IU / mL, 27 (39.7 %) had ASO titres of 400-800 IU / mL and 20 (29.4 %) had ASO titres of > 800 IU / mL. Out of 31 female ASO positive patients, 5 had (19.2 %) of ASO 200 - 400 IU / mL, 17 had (38.6 %) of ASO 400-800 IU/mL and 9 had (31.03 %) ASO titres of > 8 00 IU / mL. CONCLUSIONS Increase in ASO titres indicate recent infection, which can be tested easily and timely report. Many hospitals are choosing ASO test as a priority investigation to diagnose GAS infections. KEYWORDS Anti-Streptolysin O, Children

Utility of Human Immune Responses to GAS Antigens as a Diagnostic Indicator for ARF: A Systematic Review

Background: Previous studies have established that streptococcal antibody titer is correlated with a diagnosis of acute rheumatic fever (ARF). However, results vary in the usefulness of GAS antibodies, particularly anti-streptolysin-O (ASO) and anti-DNase B, in confirming a recent GAS infection. Therefore, we sought to provide, from published studies, an evidence-based synthesis of the correlation of streptococcal serology to establish the usefulness of immunological data in aiding the diagnosis of ARF. These findings are anticipated to have implications where echocardiography is not freely available, especially where ARF is rampant.Methods: We conducted a comprehensive search across a number of databases. Applying a priori criteria, we selected articles reporting on studies, regardless of study design, that evaluate the levels of antibodies against GAS-specific antigens in ARF subjects against control values or a published standard. Data were extracted onto data extraction forms, captured electronically, and analyzed using Stata software. Risk of bias was assessed in included studies using the Newcastle-Ottawa Scale (NOS).Results and Conclusion: The search strategy yielded 534 studies, from which 24 met the inclusion criteria, reporting on evaluation of titers for SLO (n = 10), DNase B (n = 9), anti-streptokinase (ASK) (n = 3) amongst others. Elevation in titers was determined by comparison with controls and upper limit of normal (ULN) antibody values as determined in healthy individuals. Meta-analysis of case-controlled studies revealed moderate odds ratio (OR) correlations between ARF diagnosis and elevated titers for SLO (OR = 10.57; 95% CI, 3.36–33.29; 10 studies) and DNAse B (OR = 6.97; 95% CI, 2.99–16.27; 7 studies). While providing support for incorporating SLO and DNase B in the diagnosis of ARF, we present the following reflections: an elevation in SLO and DNase B levels are not consistently associated with an ARF diagnosis; increasing the number of GAS proteins in the test is warranted to improve sensitivity; paired (acute and convalescent) samples could provide a more accurate indication of a rising titer. Use of community-based controls as a standard is not a reliable marker by which to gauge recent GAS infection.