Ultrastructural Changes of Mitochondria in the Cultivated Skin Fibroblasts of Patients with Point Mutations in Mitochondrial DNA

Mitochondrial disorders represent a heterogeneous group of multisystem diseases with extreme variability in clinical phenotype. The diagnosis of mitochondrial disorders relies heavily on extensive biochemical and molecular analyses combined with morphological studies including electron microscopy. Although muscle is the tissue of choice for electron microscopic studies, the authors investigated cultivated human skin fibroblasts (HSF) harboring 3 different pathologic mtDNA mutations: 3243A > G, 8344A > G, 8993T > G. They addressed to the possibility of whether mtDNA mutations influence mitochondrial morphology in HSF and if ultrastructural changes of mitochondria may be used for differential diagnostics of mitochondrial disorders caused by mtDNA mutations. Ultrastructural analysis of patients' HSF revealed a heterogeneous mixture of mainly abnormal, partially swelling mitochondria with unusual and sparse cristae. The most characteristic cristal abnormalities were heterogeneity in size and shapes or their absence. Typical filamentous and branched mitochondria with numerous cristae as appeared in control HSF were almost not observed. In all lines of cultured HSF with various mtDNA mutations, similar ultrastructural abnormalities and severely changed mitochondrial interior were found, although no alterations in function and amount of OXPHOS were detected by routinely used biochemical methods in two lines of cultured HSF. This highlights the importance of morphological analysis, even in cultured fibroblasts, in diagnostics of mitochondrial disorders.     

Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR

Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100–1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.     

Genetic diversity of porcine sapoviruses, kobuviruses, and astroviruses in asymptomatic pigs: an emerging new sapovirus GIII genotype

Small, non-enveloped RNA viruses belonging to the genera Sapovirus, Kobuvirus, and Mamastrovirus are usually associated with gastroenteritis in humans and animals. These enteric pathogens are considered potential zoonotic agents. In this study, the prevalence and genetic diversity of sapoviruses (SaVs), kobuviruses (KoVs), and astroviruses (AstVs) in asymptomatic pigs were investigated using a PCR approach. KoV was found to be the most prevalent virus (87.3 %), followed by AstV (34.2 %) and SaV (10.2 %). Interestingly, the intra- and inter-cluster distances between porcine SaV capsid sequences revealed one strain (P38/11/CZ) that formed a new genotype within genogroup III of porcine SaVs, and it is tentatively called “P38/11-like” genotype. Moreover, this is the first report of porcine kobuvirus detection on Czech pig farms. The high prevalence rate of gastroenteritis-producing viruses in clinically healthy pigs represents a continuous source of infection of pigs, and possibly to humans.     

A case of homozygous familial hypercholesterolemia with an atypical phenotype and delayed clinical symptoms

We describe the casuistry of a homozygous familial hypercholesterolemia female patient with a biallelic missense variant (NM_000527.4:c.1775G>A, p.Gly592Glu) in the LDLR gene, severe hypertriglyceridemia and late manifestation of coronary heart disease not earlier than at the age of 45 years. An atypical phenotype led to a delayed diagnosis.     

TNF-α signalling and inflammation: interactions between old acquaintances

Introduction

Inflammation is a very important part of innate immunity and is regulated in many steps. One such regulating step is the cytokine network, where tumor necrosis factor α (TNF-α) plays one of the most important roles.

Methods

A PubMed and Web of Science databases search was performed for studies providing evidences on the role of TNF-α in inflammation, apoptosis, and cancer.

Results and Conclusion

This review concisely summarizes the role of this pro-inflammatory cytokine during inflammation. It is focused mainly on TNF-α intracellular signaling and its influence on the typical inflammatory features in the organism. Being one of the most important pro-inflammatory cytokines, TNF-α participates in vasodilatation and edema formation, and leukocyte adhesion to epithelium through expression of adhesion molecules; it regulates blood coagulation, contributes to oxidative stress in sites of inflammation, and indirectly induces fever. The connection between TNF-α and cancer is mentioned as well.     

Effect of clopidogrel pretreatment on angiographic and clinical outcomes in patients undergoing primary percutaneous coronary intervention for ST-elevation acute myocardial infarction

Pretreatment with clopidogrel before elective primary percutaneous coronary intervention (PCI) has been shown to reduce ischemic complications. There are limited data about the value of clopidogrel pretreatment in the setting of PCI for ST-elevation myocardial infarction (STEMI). We aimed to examine the effect of clopidogrel preloading on angiographic and clinical outcomes in patients with STEMI who were treated with PCI. We conducted a prospective registry of all patients treated with primary PCI for STEMI from March 2003 to June 2006. Excluded were patients with cardiogenic shock. For the current analysis, patients (n = 292) were allocated into 2 groups. One group received clopidogrel loading dose before PCI (in the emergency department or coronary care unit, n = 165); the other,immediately after PCI (n = 127). TIMI myocardial perfusion (TMP) grade at the end of PCI and 30-day and 6-month clinical outcomes were assessed. Clinical characteristics were similar among the groups. However, patients pretreated with clopidogrel were more likely to receive aspirin and beta blockers before the current admission. TMP grade 3 occurred in a higher proportion of patients in the clopidogrel pretreatment group than in the no-pretreatment group (85% vs 71%, p = 0.01). Multivariate logistic regression analysis showed that clopidogrel pretreatment was associated with an odds ratio of 2.2 for TMP grade 3 (1.2 to 3.9, p = 0.01). Furthermore, the incidence of reinfarction at 30 days was lower in the pretreatment group (0% vs 3.2%, respectively, p = 0.04). In conclusion, these findings support the early use of clopidogrel in patients with STEMI who are treated with primary PCI.