Contribution of Artificial Intelligence to the Identification of Protein-Protein Interactions: A Case Study on PAR-3 and Its Partner Adapter Molecule Crk.

Protein-protein interactions (PPIs) are known to be involved in most cellular functions, and a detailed knowledge of such interactions is essential for studying their role in normal and pathological conditions. Significant progress is being made in the identification of PPIs through advances in computational methods. In particular, the AlphaFold2 machine learning-based model has been shown to accelerate drug discovery process by predicting the 3D structure of protein complexes. In this chapter, a straightforward protocol for predicting interprotein interactions between PAR-3 and its protein partner adapter molecule crk is provided. Such artificial intelligence-based and publicly available approaches can provide a resource for further investigation of therapeutic drug targets.

The role of body composition on cardio-respiratory fitness in futsal competitive athletes.

Futsal is an intermittent high intensity sport which has become popular worldwide. Body composition and physical fitness have been studied in different sports disciplines. However, little is known regarding body composition and cardio-respiratory performance in competitive futsal players. Body composition parameters were analyzed by electrical impedance body composition analyzer in 31 competitive male futsal players. All participants performed spirometry, handgrip strength and cardiopulmonary exercise testing. Significant correlations were observed between muscle mass and spirometry parameters and peak VO2 (p≤0.05). Fat mass resulted inversely correlated with peak VO2% predicted and hand grip strength (p≤0.05). Regression analysis showed that muscle mass significantly predicts respiratory parameters (p<0.01) and reduced fat mass is associated with increased peak VO2 % predicted and handgrip strength (p<0.01). In futsal competitive athletes increased muscle mass is associated with higher spirometry parameters and fat mass is inversely associated with lower cardiorespiratory fitness.

Self-reported side effects following COVID-19 vaccination in athletes: A retrospective study.

Billions of coronavirus disease 19 (COVID-19) vaccines have been administered worldwide. However, limited data on side effects have been reported in athletes. This study aimed to describe the incidence of side effects following COVID-19 vaccination in athletes and to identify the factors associated with the main side effects in this population. Information on COVID-19 vaccination, side effects, and overall symptom duration was retrospectively collected from recreational and competitive athletes. A total of 460 participants were included in this study. Fever and arm pain were more frequently reported after the first-dose vaccination, 9.6% vs 4.6%, p = .007 and 81.3% vs 24.9%, p ≤ .001. Myalgia was more common after the second-dose vaccination, 0.65% vs. 7.1% p ≤ .001. Males were more likely to present with arm pain after the first and second vaccinations. Those with SARS-CoV-2 infection before vaccination were less likely to present with arm pain after the first dose of vaccination (OR: 0.162, p ≤ .001) and more likely to present with fever after the second dose of vaccination (OR: 3.442, p = .046). First-dose vaccination with the BNT162b2 vaccine compared to other brands was characterized by lower odds of fever (OR: 0.394, p = .017). Our results indicated mild adverse effects and a short duration of symptoms in athletes following COVID-19 vaccination.

Coevolutionary analysis of Forkhead box protein P3 and its physical binary interactors E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α.

Forkhead box protein P3 (FOXP3) is known to orchestrate the development and maintenance of T regulatory cells, a cell population specialized in immune suppression and peripheral immune tolerance. FOXP3 activity is fine-tuned through its interaction with several protein-binding partners. By using IntAct database, we retrieved three physical binary interactors: E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α. Coevolution clusters between FOXP3 and its interactors were identified with the use of iBIS2 algorithm, the iterative version of BIS/BIS2. Most of the coevolving pairs came from some species of monotremes and marsupials, as well as from a group of bats, thus suggesting that protein interactions of FOXP3 with its partners may be changed and/or modulated during mammalian speciation. Furthermore, our analysis would suggest the occurrence of a determinant role of FOXP3 in suppressing pregnancy alloreactions in placental mammals. Similarly, FOXP3, through its interaction with different protein interaction mechanisms, would explain the unique control of inflammatory response to infections in bats. By identifying several inter-protein clusters between the different protein pairs, our findings may provide a guide for new therapeutic approaches to modulate T regulatory suppression and/or enhance immune tolerance.

Impact of Body Composition Parameters on Lung Function in Athletes.

Background: Given the potential risk of unhealthy weight management, the monitoring of body composition in athletes is advised. However, limited data reveal how body composition measurements can benefit athlete health and, in particular, respiratory function. The aim of this study is to evaluate the impact of body composition on pulmonary function in a population of adult athletes. Methods: Data from 435 competitive adult athletes regarding body compositions parameters and spirometry are retrospectively analyzed. Results: Our study population consists of 335 males and 100 female athletes. Muscle mass and fat-free mass are significantly and positively associated with forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) in the male and female population, while waist-to-height ratio is negatively associated with FEV1, FVC, and FEV1/FVC in the male population. In multivariable analysis, muscle mass and fat-free mass show significant association with FEV1 and FVC in both males and females (p < 0.05), and waist-to-height ratio is significantly and inversely associated with FEV1 and FVC in males (p < 0.05). Conclusions: Fat-free mass and muscle mass are positively and independently associated with FEV1 and FVC in athletes of both genders, and waist-to-height ratio is inversely associated with FEV1 and FVC only among male athletes. These findings suggest that body composition in athletes may be helpful in monitoring respiratory function.

Inverse correlation between the expression of AMPK/SIRT1 and NAMPT in psoriatic skin: A pilot study.

PURPOSE: Epidermal hyperplasia and the involvement of immune cells characterize the clinical picture of psoriasis. Among the several factors involved, attention has been focused on sirtuin 1 (SIRT1) - a deacetylase endowed with a variety of functions including the control of metabolic and inflammatory processes-, and on nicotinamide phosphoribosyltransferase (NAMPT), important for SIRT1 activation and involved in inflammatory events. The aim of the study was to analyze changes of SIRT1 and NAMPT expression in psoriatic skin. PATIENTS AND METHODS: Samples from healthy controls and psoriatic patients were subjected to immunohistochemical analysis. RESULTS: A strong downregulation of SIRT1 expression was observed in skin samples from psoriatic patients compared to healthy controls. This was accompanied by a parallel reduction of adenosine monophosphate-activated kinase (AMPK) expression and, more strikingly, by the disappearance of cells immunolabeled for its active, phosphorylated form (pAMPK). In both cases, analysis of the distribution of immunopositive cells revealed a shift towards reduced intensity of staining. In contrast, NAMPT expression was upregulated in psoriatic samples in line with its pro-inflammatory role. This was again more visible with an intensity-based distribution analysis that evidenced a shift towards more intensely immunostained cell populations. CONCLUSIONS: The present data correlate in the same samples the expression of SIRT1, pAMPK/AMPK and NAMPT in psoriasis and open the way for novel pharmacological targets in the treatment of the disease.

European primary datasets of alien bacteria and viruses.

Bacteria and viruses are a natural component of Earth biodiversity and play an essential role in biochemical and geological cycles. They may also pose problems outside their native range, where they can negatively impact on natural resources, wildlife, and human health. To address these challenges and develop sustainable conservation strategies, a thorough understanding of their invasion related- factors is needed: origin, country and year of introduction, and pathways dynamics. Yet, alien bacteria and viruses are underrepresented in invasion ecology studies, which limits our ability to quantify their impacts and address future introductions. This study provides primary datasets of alien bacteria and viruses of plants and animals present in the European environment. The datasets contain expert-revised data on 446 taxa and their invasion related- factors across terrestrial and aquatic environments. Taxa information are complemented with spatial occurrences. The datasets provide a basis for collaborative initiatives to improve the collection of alien bacteria and viruses' data, and a starting point for data-driven conservation practices.

The S100A7 nuclear interactors in autoimmune diseases: a coevolutionary study in mammals.

S100A7, a member of the S100A family of Ca2+-binding proteins, is considered a key effector in immune response. In particular, S100A7 dysregulation has been associated with several diseases, including autoimmune disorders. At the nuclear level, S100A7 interacts with several protein-binding partners which are involved in transcriptional regulation and DNA repair. By using the BioGRID and GAAD databases, S100A7 nuclear interactors with a putative involvement in autoimmune diseases were retrieved. We selected fatty acid-binding protein 5 (FABP5), autoimmune regulator (AIRE), cystic fibrosis transmembrane conductance regulator (CFTR), chromodomain helicase DNA-binding protein 4 (CHD4), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESR1), histone deacetylase 2 (HDAC2), v-myc avian myelocytomatosis viral oncogene homolog (MYC), protection of telomeres protein 1 (POT1), telomeric repeat-binding factor (NIMA-interacting) 1 (TERF1), telomeric repeat-binding factor 2 (TERF2), and Zic family member 1 (ZIC1). Linear correlation coefficients between interprotein distances were calculated with MirrorTree. Coevolution clusters were also identified with the use of a recent version of the Blocks in Sequences (BIS2) algorithm implemented in the BIS2Analyzer web server. Analysis of pair positions identified interprotein coevolving clusters between S100A7 and the binding partners CFTR and TERF1. Such findings could guide further analysis to better elucidate the function of S100A7 and its binding partners and to design drugs targeting for these molecules in autoimmune diseases.

Interaction between matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL): A recent evolutionary event in primates.

Matrix metalloproteases are known to represent an early step in the evolution of the immune system. Similarly, neutrophil gelatinase-associated lipocalin is known to be a key effector in immune response. MMP-9 interacts with NGAL, but their interaction mechanisms remain unclear. Functional interaction between proteins is ensured by coevolution. Protein coevolution was inferred by calculating the linear correlation coefficients between inter-protein distance matrices using MirrorTree. Among examined mammal species, we found a robust signal of MMP-9/NGAL coevolution exclusively within Primates (R = 0.96, p < 1e-06). Owing to the high conservation of these proteins among Mammals, we chose to utilize a recent version of Blocks in Sequences (BIS2) algorithm implemented in BIS2Analyzer webserver. Coevolution clusters between the two proteins were identified in MMP-9 fibronectin and hemopexin domains. Our results suggest that MMP-9/NGAL interaction is a recent evolutionary acquisition in Primates. Furthermore, MMP-9 hemopexin domain would represent a promising target for drug design against these molecules.

SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin's effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl2 (250 µM for 24 h). Melatonin (1 µM) significantly attenuated CoCl2 toxicity on microglia, an effect prevented by selective SIRT1 inhibitor EX527 (5 µM) and AMP-activated protein kinase (AMPK) inhibitor BML-275 (2 µM). CoCl2 did not modify SIRT1 expression, but prevented nuclear localization, while melatonin appeared to restore it. CoCl2 induced nuclear localization of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-kB), an effect contrasted by melatonin in an EX527-dependent fashion. Treatment of microglia with melatonin attenuated potentiation of neurotoxicity. Common carotid occlusion was performed in p7 rats, followed by intraperitoneal injection of melatonin (10 mg/kg). After 24 h, the number of Iba1+ microglia in the hippocampus of hypoxic rats was significantly increased, an effect not prevented by melatonin. At this time, SIRT1 was only detectable in the amoeboid, Iba1+ microglial population selectively localized in the corpus callosum. In these cells, nuclear localization of SIRT1 was significantly lower in hypoxic animals, an effect prevented by melatonin. NF-kB showed an opposite expression pattern, where nuclear localization in Iba1+ cells was significantly higher in hypoxic, but not in melatonin-treated animals. Our findings provide new evidence for a direct effect of melatonin on hypoxic microglia through SIRT1, which appears as a potential pharmacological target against hypoxic-derived neuronal damage.

S100A7/Ran-binding protein 9 coevolution in mammals.

S100A7 has been suggested to interact with Ran-binding protein 9. Both proteins are nowadays considered key effectors in immune response. Functional interaction between proteins is ensured by coevolution. The mechanisms of vertebrate coevolution between S100A7 and RanBP9 remain unclear. Several approaches for studying coevolution have been developed. Protein coevolution was inferred by calculating the linear correlation coefficients between inter-protein distance matrices using Mirrortree. We found an overall moderate correlation value (R = 0.53, p < 1e-06). Moreover, owing to the high conservation of RanBP9 protein among vertebrates, we chose to utilize a recent version of Blocks in Sequences (BIS2) algorithm implemented in BIS2Analyzer webserver. A coevolution cluster was identified between the two proteins (p < 8.10e-05). In conclusion, our coevolutionary analysis suggests that amino acid variations may modulate S100A7/RanBP9 interaction with potential pathogenic effects. Such findings could guide further analysis to better elucidate the function of S100A7 and RanBP9 and to design drugs targeting for these molecules in diseases.

Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte.

The application of immunohistological methods provides an invaluable contribution in revealing the protein colocalization, which may reflect the occurrence of molecular interaction processes.This chapter describes comprehensive protocols for detection of S100A7/JAB1 colocalization by immunohistochemistry in archival formalin-fixed and paraffin-embedded skin biopsies from healthy and psoriatic subjects. In addition, we provide a protocol for immunocytochemical detection of S100A7/JAB1 colocalization in S100A7 CRISPR-activated human keratinocyte cell line.

S100A7 in Psoriasis: Immunodetection and Activation by CRISPR technology.

Psoriasis, an inflammatory autoimmune skin disease, is the result of a chronic interaction between hyperproliferative keratinocytes and infiltrating activated immune cells. The mechanisms underlying psoriasis pathogenesis remain largely unknown, although a combination of genetic and environmental factors plays an important role in its development. S100A7 is overexpressed in psoriasis, and there is growing evidence that S100A7 may be involved in the pathogenesis of psoriasis. Since the mechanisms underlying S100A7 regulation and function remain elusive, a better understanding of these mechanisms may be useful to uncover additional treatment approaches for psoriasis. Immunohistology provides invaluable tools for a better understanding of the pathogenetic mechanisms of psoriasis. Here, we describe basic methods for immunofluorescence and immunohistochemistry analysis of S100A7 expression in psoriatic patients as well as in S100A7 CRISPR-activated human keratinocyte cell line.

S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line.

Psoriasis, a chronic immune-mediated inflammatory skin disease, is characterized by dysregulated keratinocyte proliferation. The EF-hand calcium binding protein S100A7 has been found to be overexpressed in psoriatic keratinocytes. It is know that S100A7 may interact with Jab1, a cofactor that stabilizes c-Jun. Jab1 is known to downregulate the expression of the cell cycle inhibitor p27Kip1 in some cancer models. In this study, we aimed to investigate the possible interaction between S100A7 and Jab1 and the downstream effects on p27 Kip1 expression in normal human keratinocyte cells transfected with S100A7 CRISPR activation plasmid and in archival psoriatic skin samples. Our results showed that the upregulated S100A7 colocalizes with Jab1 at the nuclear level in transfected cells and psoriatic skin samples. We also showed a differential protein expression of Jab1 between cytoplasmic and nuclear compartments, thus suggesting Jab1 translocation from nucleus to cytoplasm. p27 Kip1 protein expression patterns would imply a translocation from nucleus and a subsequent degradation of this protein. The upregulation of S1007 and its interaction with Jab1 would contribute to the p27 Kip1 -dependent impaired proliferation that characterizes psoriatic skin.

FOXP3 expression is modulated by TGF­ß1/NOTCH1 pathway in human melanoma.

Forkhead box protein 3 (FOXP3) transcription factor is expressed by immune cells and several human cancers and is associated with tumor aggressiveness and unfavorable clinical outcomes. NOTCH and transforming growth factor­ß (TGF­ß) protumorigenic effects are mediated by FOXP3 expression in several cancer models; however, their interaction and role in melanoma is unknown. We investigated TGF­ß­induced FOXP3 gene expression during NOTCH1 signaling inactivation. Primary (WM35) and metastatic melanoma (A375 and A2058) cell lines and normal melanocytes (NHEM) were used. FOXP3 subcellular distribution was evaluated by immuno-cytochemical analysis. Gene expression levels were assessed by reverse transcription­quantitative polymerase chain reaction. Protein levels were assessed by western blot analysis. The γ­secretase inhibitor (GSI) was used for NOTCH1 inhibition and recombinant human (rh)TGF­ß was used for melanoma cell stimulation. Cell proliferation and viability were respectively assessed by MTT and Trypan blue dye assays. FOXP3 mRNA and protein levels were progressively higher in WM35, A375 and A2058 cell lines compared to NHEM and their levels were further increased after stimulation with rh­TGF­ß. TGF­ß­mediated FOXP3 expression was mediated by NOTCH1 signaling. Inhibition of NOTCH1 with concomitant rh­TGF­ß stimulation determined the reduction in gene expression and protein level of FOXP3. Finally, melanoma cell line proliferation and viability were reduced by NOTCH1 inhibition. The results show that nn increase in FOXP3 expression in metastatic melanoma cell lines is a potential marker of tumor aggressiveness and metastasis. NOTCH1 is a central mediator of TGF­ß­mediated FOXP3 expression and NOTCH1 inhibition produces a significant reduction of melanoma cell proliferation and viability.

FOXP3, ICOS and ICOSL gene polymorphisms in systemic sclerosis: FOXP3 rs2294020 is associated with disease progression in a female Italian population.

Systemic sclerosis (SSc), an autoimmune disorder, is characterized by vasculopathy, inflammation, progressive perivascular and interstitial fibrosis. Its pathogenesis is largely unknown, however strong evidences suggest that genetic predisposition may contribute to SSc development. Several gene polymorphisms involved in regulatory T cell function have been identified in many autoimmune diseases, including SSc. Moreover, dysregulation of co-stimulatory and/or co-inhibitory signals, including ICOS signalling, can lead to autoimmunity. The aim of the present study was to investigate the association of the FOXP3 rs2294020, ICOS rs6726035 and ICOSL rs378299 SNPs with both the susceptibility and the progression to SSc in an Italian case-series of patients. SNP genotyping results were successfully obtained from a total of 350 subjects including 166 individuals with SSc and 184 healthy controls. Although analysis tests did not show any significant associations between the SNPs under study and susceptibility to SSc, the occurrence of FOXP3 rs2294020 in female patients was associated with decreased time to progression from early to definite SSc (allelic model: HR=1.43; CI=1.03-1.99; p=0.03; dominant model: HR=1.54; CI=1.04-2.28; p=0.03). The inclusion of presence of ACA autoantibodies in the model did not significantly change the estimates. No conclusions can be drawn for the susceptibility to the disease or the time to progression in men due to the low statistical power. This study provides evidence of the association of rs2294020 with SSc evolution in female patients, modulating the time of progression from the diagnosis of early SSc to the diagnosis of definite SSc, while no effect on SSc susceptibility per se was found. rs2294020 may be considered a disease-modifying gene-variant rather than a disease-susceptibility SNP in SSc.

Effects of Alpha Lipoic Acid on Multiple Cytokines and Biomarkers and Recurrence of Atrial Fibrillation Within 1 Year of Catheter Ablation.

Catheter ablation (CA) is a procedure commonly used to restore sinus rhythm in patients with atrial fibrillation (AF). However, AF recurrence after CA remains a relevant clinical issue. We tested the effects of an oral antioxidant treatment (alpha lipoic acid [ALA]) on AF recurrence post-CA. Patients with paroxysmal AF have been enrolled in a randomized, prospective, double-blind, controlled placebo trial. After CA, patients have been randomly assigned to receive ALA oral supplementation (ALA group) or placebo (control group) and evaluated at baseline and after a 12-month follow-up: 73 patients completed the 12-month follow-up (ALA: 33 and control: 40). No significant difference has been detected between the 2 groups at baseline. Strikingly, 1 year after CA, ALA therapy significantly reduced serum markers of inflammation. However, there was no significant difference in AF recurrence events at follow-up comparing ALA with placebo group. Multivariate analysis revealed that the only independent prognostic risk factor for AF recurrence after CA is age. In conclusion, ALA therapy reduces serum levels of common markers of inflammation in ablated patients. Nevertheless, ALA does not prevent AF recurrence after an ablative treatment.

Obesity, Type 1 Diabetes, and Psoriasis: An Autoimmune Triple Flip.

Obesity, type 1 diabetes, and psoriasis are wide-ranging health problems. Genetics, epigenetics, and environmental factors together with immune disturbances are involved in these diseases. The white adipose tissue is an active endocrine organ, secreting a wide variety of soluble mediators called adipokines that have a central role in the relationship between adipose tissue and immune system. Inflammatory cytokines, including the IL-23/IL-17 and IL-18 axes, and microRNAs are involved in many processes, including immunity and inflammation, thus having a major role in the onset of these three diseases. In this review, we present an overview of the roles of adipokines, cytokines, and microRNAs in the pathogenesis and the progression of these three diseases.

Association between rs2294020 in X-linked CCDC22 and susceptibility to autoimmune diseases with focus on systemic lupus erythematosus.

Autoimmune diseases often share common susceptibility genes. Most genetic variants associated with susceptibility to systemic lupus erythematosus are also associated with other autoimmune diseases. The X-linked variant rs2294020 is positioned in exon 7 of the CCDC22 gene. The encoded protein functions in the regulation of NF-κB, a master regulator in immune response. The aim of this study is to investigate whether the rs2294020 polymorphism may be a general susceptibility factor for autoimmunity. We evaluated case-control association between the occurrence of rs2294020 and different autoimmune diseases, including new data for systemic lupus erythematosus and previous genome-wide association studies (GWAS) (though most did not analyse the X chromosome) of psoriasis, celiac disease, Crohn's disease, ulcerative colitis, multiple sclerosis, vitiligo, type-1 diabetes, rheumatoid arthritis, and ankylosing spondylitis. Cases from patients affected by amyotrophic lateral sclerosis and type-2 diabetes were also included in the study. We detected nominal significant associations of rs2294020 with systemic lupus erythematosus (additive model test: p=0.01), vitiligo (p=0.016), psoriasis (p=0.038), and in only one of two studies of multiple sclerosis (p=0.03). Our results suggest that rs2294020 is associated with the risk of several autoimmune diseases in European populations, specifically with diseases that present themselves, among else, in the skin.