Comprehensive Assessment of the Virulence Factors sub 3, sub 6 and mcpA in the Zoonotic Dermatophyte Trichophyton benhamiae Using FISH and qPCR

Skin infections by keratinophilic fungi are commonly referred to as dermatophytosis and represent a major health burden worldwide. Although patient numbers are on the rise, data on virulence factors, their function and kinetics are scarce. We employed an ex vivo infection model based on guinea pig skin explants (GPSE) for the zoonotic dermatophyte Trichophyton (T.) benhamiae to investigate kinetics of the virulence factors subtilisin (sub) 3, sub 6, metallocarboxypeptidase A (mcpA) and isocitrate lyase (isol) at gene level for ten days. Fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to detect and quantify the transcripts, respectively. Kingdom-spanning, species-specific and virulence factor-specific probes were successfully applied to isolated fungal elements showing inhomogeneous fluorescence signals along hyphae. Staining results for inoculated GPSE remained inconsistent despite thorough optimization. qPCR revealed a significant increase of sub 3- and mcpA-transcripts toward the end of culture, sub 6 and isol remained at a low level throughout the entire culture period. Sub 3 is tightly connected to the de novo formation of conidia during culture. Since sub 6 is considered an in vivo disease marker. However, the presented findings urgently call for further research on the role of certain virulence factors during infection and disease.

Heterogeneity in biomarkers, mitogenome and genetic disorders of Arab population with special emphasis on large-scale whole-exome sequencing

More than 25 million DNA variations were discovered as novel including major alleles from Arab population. Exome studies on Arabs discovered >3000 novel nucleotide variants associated with >1200 rare genetic disorders. Reclassification of many pathogenic variant into benign through the Arab database enhance building a detailed and comprehensive map of Arab morbid genome. Intellectual disability stands first with the combined and observed carrier frequency. Genome studies and advanced computational biology discovered interesting novel candidate disease marker variations in many genes from consanguineous families with intellectual disability, neurogenetic disorders, blood and bleeding disorder and rare genetic diseases. Pathogenic variants in C12orf57 gene are prominently associated with the etiology of developmental delay/intellectual impairment. Arab mitogenome exposed hundreds of variations in mtDNA genome and its association with obesity. Further study is needed in genomics to fully comprehend the molecular abnormalities and associated pathogenesis that cause inherited disorders in Arab ancestries.

Decrowding Expansion Pathology: Unmasking Previously Invisible Nanostructures and Cells in Intact Human Brain Pathology Specimens

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here we present an ExM variant, decrowding expansion pathology (dExPath), which can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and diagnosis.

DOSE-DEPENDENT PROTECTIVE FEATURES OF LOBOPHORA VARIEGATA METHANOLIC EXTRACT (LVME) IN N-NITROSODIETHYLAMINE INDUCED EXPERIMENTAL HEPATOCARCINOGENESIS IN RATS

Objective: This study explores the anti-cancer property of Lobophora variegata, also an effective dose to treat hepatocarcinoma in Male Albino Wistar rats in N-nitrosodiethylamine induced hepatocarcinoma paradigm and its possible mechanism of action. Methods: In this study, rats were segregated into five groups; group-1 (control), group-2 treated with 0.01% NDEA through drinking water for 15 w, group-3 NDEA+treated with Lobophora variegata methanolic extract (LVME) (100 mg/kg b.w.), group-4 NDEA+treated with (LVME) (200 mg/kg b.w.) and group-5 NDEA+treated with (LVME) (400 mg/kg b.w.). Results: After the experimental period, functional and morphological changes in the liver were observed both macro and microscopically, the activities of liver marker enzymes, alkaline phosphatase (ALP), aspartate and alanine transaminases (AST and ALT) were analyzed. Administration of LVME as 200 mg/kg b.w. (to NDEA treated rats) significantly (i) reduced the preneoplastic lesions alleviated lipid peroxidation through scavenging free radicals, (ii) enhanced antioxidant status and reverted liver/disease marker enzymes plausibly by modulating xenobiotics metabolizing enzymes (XMEs) and by exhibiting antiproliferative and cytoprotective effects. Conclusion: LVME doses higher than 200 mg/kg b.w. are not effective in quenching the free radicals and restoring the liver functions as saturation level could have been reached; also, doses lower than 200 mg/kg b.w. could not be effective as they are below the optimum dose required to exhibit the pharmacological effects.

Proteomic Analysis of Glomerular Proteins in Primary and Bucillamine-induced Membranous Nephropathy

The aim of this study was to characterize glomerular proteins in primary membranous nephropathy (pMN) and drug-induced secondary MN (sMN) by laser microdissection and comparative proteomic analysis. We used renal biopsy specimens from 6 patients with anti-phospholipase A2 receptor autoantibody (PLA2R Ab) (+) pMN, 6 patients with PLA2R Ab (‒) pMN, 6 patients with bucillamine (BCL)-induced sMN, and 5 control cases (time 0 transplant biopsies). Proteins were extracted from laser-microdissected glomeruli and analyzed using mass spectrometry. The quantification values of protein abundance in each MN group were compared with those in the control group. More than 800 proteins with high confidence were identified. Principal component analysis revealed a different distribution between the pMN and sMN groups. For further analysis, 441 proteins matched with ≥3 peptides were selected. Among the pMN and sMN groups, we compared the profiles of several protein groups based on the structural and functional characteristics, such as immunoglobulins, complements, complement-regulating proteins, podocyte-associated proteins, glomerular basement membrane proteins, and several proteins that are known to be associated with kidney diseases, including MN. Between the pMN and BCL-induced sMN groups, we observed common and different alterations in protein levels such as known disease-associated proteins and potential disease marker proteins.

PKM2 Is a Potential Diagnostic and Therapeutic Target for Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

Myocardial Work Does Not Have Additional Diagnostic Value in the Assessment of ATTR Cardiac Amyloidosis

Background: Reduced LV longitudinal strain (GLS) and increased relative apical sparing (RELAPS) and increased wall thickness have been proposed as features for transthyretin cardiac amyloidosis (ATTR-CA). Myocardial work (MW) has recently been shown as useful afterload independent disease marker, hence we aimed to investigate its use in differentiating ATTR-CA from heart failure with increased septal thickness but no cardiac amyloidosis (CA) (HFnCA). Methods: This study included patients with HF and increased septal thickness ≥ 14 mm. We included 59 patients with hereditary (ATTRv) and 27 wild type transthyretin amyloidosis (ATTRwt) described as ATTR-CA based on DPD scintigraphy. We also enrolled 30 non-amyloidosis heart failure patients with negative DPD scintigraphy, as a control group. Myocardial work (MW) was used to assess the index (GWI), constructive (GCW) and wasted (GWW) work. Relative wall thickness (RWT) and relative apical sparing (RELAPS) were tested as conventional measures. Results: The RWT and RELAPS were higher in ATTR-CA (p < 0.001) and predicted ATTR-CA (RWT; AUC = 0.84, p < 0.001) and RELAPS (AUC = 0.81, p < 0.001). MW; GWI (p = 0.04), GCW (p = 0.03), GWW (p = 0.001) were all lower in ATTR-CA compared with HFnCA but only GWW predicted ATTR-CA, (AUC = 0.75, p < 0.001). Binary logistic univariate regression analysis showed RWT (p = 0.003, β = 16.2) and RELAPS (p = 0.003, β = 2.3) to be associated with ATTR-CA but not MW. GWI and GCW correlated with NT-proBNP (p < 0.05) and Troponin (p < 0.01), but not RWT or RELAPS. Conclusion: Myocardial work had lower accuracy, compared to RWT or RELAPS, in identifying ATTR-CA but was better related to biomarkers. Thus, MW assessment is unlikely to have additional value in improving the diagnosis of heart failure due to ATTR-CA.

Welcoming teleretinography into diabetes integrated care

Integrated Care (IC) is a perfect fit for people with diabetes. Fundus examination (FE) is a disease marker for diabetologists and identifies potentially blinding complications (Diabetic Retinopathy, DR). In our Diabetes Clinic (DC) in Pescara, Italy, FE is possibly provided with telemedicine in same day as other exams, avoiding it to be a standalone clinical one; images taken with a retinal digital camera are graded by a remote ophthalmologist within a shared Electronic Health Record (EHR), immediately readable by other stakeholders; a dedicated care path to the Eye Clinic, University of Chieti-Pescara is provided for urgent cases. Personnel’s worktime shortening allows gaining time for ophthalmologists’ eye examinations in outpatient settings and other stakeholders’ work in the DC. The need for a DR digital screening system is growing worldwide: our experience confirms the ease of implementation, and the advantage of sharing clinical data with all stakeholders when working within an EHR, aiming to optimize an IC effective system.