Contribution of proteomics to the study of the role of cytokeratins in disease and physiopathology

Cytokeratins (CKs), the most abundant group of cytoskeletal intermediate filaments, and proteomics are strongly connected. On the one hand, proteomics has been extremely useful to uncover new features and functions of CKs, on the other, the highly abundant CKs serve as an exceptional tool to test new technological developments in proteomics. As a result, proteomics has contributed to finding valuable associations of CKs with diseases as diverse as cancer, cystic fibrosis, steatohepatitis, viral and bacterial infection, keratoconus, vitreoretinopathy, preeclampsia or the chronic fatigue syndrome, as well as to characterizing their participation in a number of physiopathological processes, including drug resistance, response to toxicants, inflammation, stem cell differentiation, embryo development, and tissue repair. In some cases, like in cystic fibrosis, CKs have been described as potential therapeutic targets. The development of a specific field of proteomics where CKs become the main subject of research aims and hypotheses is suggested.     

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A

Non-canonical splice site variants are increasingly recognized as a relevant cause of the USH2A-associated diseases, non-syndromic autosomal recessive retinitis pigmentosa and Usher syndrome type 2. Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH2A non-canonical splice site variants and to study their potential pathogenicity. Eleven non-canonical splice site variants were selected based on four splice prediction tools. Ten different USH2A constructs were generated and minigene splice assays were performed in HEK293T cells. An effect on pre-mRNA splicing was observed for all 11 variants. Various events, such as exon skipping, dual exon skipping and partial exon skipping were observed and eight of the tested variants had a full effect on splicing as no conventionally spliced mRNA was detected. We demonstrated that non-canonical splice site variants in USH2A are an important contributor to the genetic etiology of the associated disorders. This type of variant generally should not be neglected in genetic screening, both in USH2A-associated disease as well as other hereditary disorders. In addition, cases with these specific variants may now receive a conclusive genetic diagnosis.     

Cellular Stress Assay in Peripheral Blood Mononuclear Cells: Factors Influencing Its Results

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep^® and Robosep^TM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.     

Synthesis,in vitro Antileishmanial Efficacy and Hit/Lead Identification of Nitrofurantoin-Triazole Hybrids

Leishmaniasis is a vector-borne neglected parasitic infection affecting thousands of individuals, mostly among populations in low- to moderate-income developing countries. In the absence of protective vaccines, the management of the disease banks solely on chemotherapy. However, the clinical usefulness of current antileishmanial drugs is threatened by their toxicity and the emergence of multidrug-resistant strains of the causative pathogens. This emphasizes the imperative for the development of new and effective antileishmanial agents. In this regard, we synthesized and evaluated in vitro the antileishmanial activity and cytotoxicity profile of a series of nitrofurantoin-triazole hybrids. The nitrofurantoin derivative 1 featuring propargyl moiety was distinctively the most active of all, was nontoxic to human cells and possessed submicromolar cellular activity selectively directed towards the pathogens of the life threatening visceral leishmaniasis. Hence it was identified as potential antileishmanial lead for further investigation into its prospective to act as alternative to therapies.     

Fatigue behavior of thermosetting-polyester-matrix sheet-molding compounds

Two sheet molding compounds, one containing 65 weight percent E-glass fiber and no filler (SMC-65) and the other containing 50 weight percent glass and 15 weight percent CaCO₃ filler were subjected to sinusoidal, load-control, tension-tension fatigue. The macroscopic behavior showed some notch sensitivity for both materials, but particularly for SMC-65. The extent of failure damage over the specimen length decreased with decreasing load amplitude. Different failure mechanisms appear to operate under high- and low-cycle fatigue conditions. Microscopic surface damage studies at stresses near the endurance limit showed several stages in the fatigue failure. For SMC-65, these stages are, sequentially, matrix crack initiation and growth, matrix pulverization, fiber fracture, and fiber pullout. A similar, but less distinct pattern was observed for SMC-50. Acoustic emission was also followed during fatigue cycling near the endurance limit. Distinct amplitude distribution peaks for each failure process were seen. It is suggested that matrix fracture, permanent deformation and Euler buckling are all important in high cycle fatigue failure.