Update on Myocarditis: From Etiology and Clinical Picture to Modern Diagnostics and Methods of Treatment.

Although the frequency of myocarditis in the general population is very difficult to accurately determine due to the large number of asymptomatic cases, the incidence of this disease is increasing significantly due to better defined criteria for diagnosis and the development of modern diagnostic methods. The multitude of different etiological factors, the diversity of the clinical picture, and the variability of the diagnostic findings make this disease often demanding both for the selection of the diagnostic modality and for the proper therapeutic approach. The previously known most common viral etiology of this disease is today overshadowed by new findings based on immune-mediated processes, associated with diseases that in their natural course can lead to myocardial involvement, as well as the iatrogenic cause of myocarditis, which is due to use of immune checkpoint inhibitors in the treatment of cancer patients. Suspecting that a patient with polymorphic and non-specific clinical signs and symptoms, such as changes in ECG and echocardiography readings, has myocarditis is the starting point in the diagnostic algorithm. Cardio magnetic resonance imaging is non-invasive and is the gold standard for diagnosis and clinical follow-up of these patients. Endomyocardial biopsy as an invasive method is the diagnostic choice in life-threatening cases with suspicion of fulminant myocarditis where the diagnosis has not yet established or there is no adequate response to the applied therapeutic regimen. The treatment of myocarditis is increasingly demanding and includes conservative methods of treating heart failure, immunomodulatory and immunospressive therapy, methods of mechanical circulatory support, and heart transplantation. The goal of developing new diagnostic and therapeutic methods is to reduce mortality from this complex disease, which is still high.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Novel chemical-physical autopsy investigation in sudden infant death and sudden intrauterine unexplained death syndromes.

Aim: Verify the presence of inorganic nanoparticle entities in brain tissue samples from sudden infant death syndrome (SIDS)/sudden intrauterine unexplained death syndrome (SIUDS) cases. The presence of inorganic debris could be a cofactor that compromises proper brain tissue functionality. Materials & methods: A novel autopsy approach that consists of neuropathological analysis procedures combined with energy dispersive spectroscopy/field emission gun environmental scanning electron microscopy investigations was implemented on 10 SIDS/SIUDS cases, whereas control samples were obtained from 10 cases of fetal/infant death from known cause. Results: Developmental abnormalities of the brain were associated with the presence of foreign bodies. Although nanoparticles were present as well in control samples, they were not associated with histological brain anomalies, as was the case in SIDS/SIUDS. Conclusion: Inorganic particles present in brain tissues demonstrate their ability to cross the hemato-encephalic barrier and to interact with tissues and cells in an unknown yet pathological fashion. This gives a rationale to consider them as cofactors of lethality.

Prevalence of intestinal parasites in dogs from public shelters in Serbia.

Data on endoparasitic infections in dogs from dog shelters in Southeastern Europe are limited; thus, this study aimed to add to the existing knowledge on this topic by reporting on the prevalence of intestinal parasites in dogs from public dog shelters in the Republic of Serbia. In 2017 and 2018, individual and pooled fecal samples, were collected from 1267 dogs from six shelters. All samples were qualitatively examined for parasites using flotation tests. Seven taxa of intestinal parasites were identified: Cystoisospora spp., ascarids: Toxocara canis and Toxascaris leonina, hookworms, Trichuris vulpis, taeniids and Dipylidium caninum. The overall prevalence of intestinal parasites was 58. 3 % (78. 1 % in young dogs and 53.1 % in adult dogs). The parasites detected in both young (<1 year old) and adult dogs (>1 year old) were Cystoisospora spp. (20 % and 4.9 %), T. canis (33.5 % and 14.7 %), T. leonina (7.7 % and 2.3 %), and hookworms (16.9 % and 15 %), respectively. However, T. vulpis (9.6 %), taeniids (1.3 %), and D. caninum (5.4 %) were detected only in adult dogs. In the Belgrade shelter, young dogs had a higher prevalence of endoparasitic infections (18.9 %, 49/260) than adult dogs (14.8 %, 149/1007). In the Subotica, Jagodina and Nis shelters, young dogs had significantly higher (p < 0.001 and p < 0.05, respectively) prevalence of endoparasitic infections (10 %, 12.3 % and 14.6 %) than adult dogs (5.3 %, 8 % and 7.2 %). These results will be useful for establishing health care programs in dog shelters and implementing effective strategies for the control of intestinal parasites.

Predicting the Effect of Chemical Factors on the pH of Crystallization Trials.

In macromolecular crystallization, success is often dependent on the pH of the experiment. However, little is known about the pH of reagents used, and it is generally assumed that the pH of the experiment will closely match that of any buffering chemical in the solution. We use a large dataset of experimentally measured solution pH values to show that this assumption can be very wrong and generate a model that can be used to successfully predict the overall solution pH of a crystallization experiment. Furthermore, we investigate the time dependence of the pH of some polyethylene glycol polymers widely used in protein crystallization under different storage conditions.

Cinder: keeping crystallographers app-y.

The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds (or thousands) of individual crystallization trials, each of which must be repeatedly examined for crystals or hints of crystallinity. Currently, the only real way to address this bottleneck is to use an automated imager to capture images of the trials. However, the images still need to be assessed for crystals or other outcomes. Ideally, there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result. However, as yet no such tool exists in wide usage, despite this being a well recognized problem. One of the issues in creating robust automatic image-analysis software is the lack of reliable data for training machine-learning algorithms. Here, a mobile application, Cinder, has been developed which allows crystallization images to be scored quickly on a smartphone or tablet. The Cinder scores are inserted into the appropriate table in a crystallization database and are immediately available to the user through a more sophisticated web interface, allowing more detailed analyses. A sharp increase in the number of scored images was observed after Cinder was released, which in turn provides more data for training machine-learning tools.

Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway.

The detection of protein-protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein-protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein-protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.

Molecular Dissection of Renal Ischemia-Reperfusion: Oxidative Stress and Cellular Events.

Ischemic reperfusion kidney injury (IRKI) is a complex pathophysiological event, which is the most common cause of the acute kidney injury. The key characteristic of IRKI is a reduction in glomerular filtration rate, which implies an underlying impairment in hemodynamic regulation. In recent decades, convincing evidence illuminated the molecular and pathological events in the acute kidney injury, revealing the role of ischemia/reperfusion, oxidative stress, apoptosis, inflammation, fibrosis and changes in gene expression which activate different signaling pathways. The cascade of inflammation events is a key mediator of IRKI, which includes the inflammation process, complement activation and mobilization of innate immunity. Oxidative stress represents the increased presence of various free radicals that cannot be buffered by the antioxidant capacity which comprises of enzymatic and non-enzymatic components. Renal tissue injury during ischemia/reperfusion comes as a result of membrane lipids peroxidation, oxidative damage of proteins and DNA and results in apoptosis and necrosis. It is evident from many studies that augmentation of the antioxidant defense mechanisms has a protective role on kidney tissue. In recent years, the importance of heat-shock proteins and MicroRNAs in the pathogenesis of IRKI has been revealed and there are promising indications that in future they could serve as diagnostic biomarkers or therapeutic targets. Striking changes in global gene expression were shown, providing a great potential for fundamental understanding and clinical management of IRKI. The clinical outcome among patients with kidney transplantation will have the furthermost advance from the better understanding of the underlying molecular pathology of IRKI.

Antibacterial effect of Herniaria hirsuta, Prunus avium, Rubia tinctorum and Sempervivum tectorum plant extracts on multiple antibiotic resistant Escherichia coli / Efeito antibacteriano de Herniaria hirsuta, Prunus avium, Rubia tinctorum e Sempervivum tectorum extratos vegetais em vários resistentes a antibióticos Escherichia coli

The emergence of Escherichia coli isolates with multiple antibiotic resistant phenotypes is considered as a severe health concern. In the present work the antibacterial effect of following plants (Herniaria hirsuta, Prunus avium, Rubia tinctorum and Sempervivum tectorum) was examined. The bacterial model used for estimation of bacterial susceptibility is hospital multiple antibiotic resistant E. coli strain. E. coli ATCC 25922 was used for standard comparison of bacterial susceptibility. Leaves of H. hirsuta, R. tinctorum and S. tectorum as well as petioles of P. avium were collected. Ethanol and aqueous extract of each plant was prepared. Antibacterial activity was examined using the agar well diffusion method. Concentration of total phenols, flavonoids, tannins, antocyanins and saponins was determined in plant extracts. E. coli strain is resistant to four unrelated families of antibiotics. Antibacterial effect is proven for all examined plants. Ethanol extracts of H. hirsuta and P. avium have a more potent antibacterial effect than their aqueous extracts. Aqueous extracts of R. tinctorum and S. tectorum have higher antibacterial potential than theirs ethanol extracts. Examined plant extracts represent good candidates for more extensive research in view of their application in the treatment of multiple antibiotic resistant E.coli strains.

O surgimento de Escherichia coli isoladas com vários fenótipos resistentes aos antibióticos é considerado como um grave problema de saúde. No presente trabalho o efeito antibacteriano das seguintes plantas (Herniaria hirsuta, Prunus avium, Rubia tinctorum e Sempervivum tectorum) foi analisado. O agente bacteriano modelo utilizado para estimativa de susceptibilidade bacteriana é o hospital vários resistentes a antibióticos E. coli. E. coli ATCC 25922 padrão foi utilizado para comparação de antibiogramas. Folhas de H. hirsuta, R. tinctorum e S. tectorum bem como pecíolos de P. avium foram coletados. Etanol e extrato aquoso de cada planta foi preparado. Atividade antibacteriana foi analisada através do método de difusão em ágar-bem. Total Concentração de fenóis, flavonóides, taninos e saponinas antocyanins determinou-se em extratos de plantas. E. coli estirpe é resistente às quatro famílias de antibióticos independentes. Efeito antibacteriano é comprovado para todas as plantas examinadas. Os extratos etanólicos de H. hirsuta e P. avium têm um efeito mais potente antibacteriano de seus extratos aquosos. Extratos aquosos de R. tinctorum e S. tectorum têm maior potencial antibacteriano que os extratos etanólicos. Extratos vegetais examinados representam bons candidatos para pesquisa mais ampla em vista de sua aplicação no tratamento de vários antibióticos resistentes a cepas de E. coli.

Formulation screening by differential scanning fluorimetry: how often does it work?

There is strong evidence to suggest that a protein sample needs to be well folded and uniform in order to form protein crystals, and it is accepted knowledge that the formulation can have profound effects on the behaviour of the protein sample. The technique of differential scanning fluorimetry (DSF) is a very accessible method to determine protein stability as a function of the formulation chemistry and the temperature. A diverse set of 252 soluble protein samples was subjected to a standard formulation-screening protocol using DSF. Automated analysis of the DSF results suggest that in over 35% of cases buffer screening significantly increases the stability of the protein sample. Of the 28 standard formulations tested, three stood out as being statistically better than the others: these included a formulation containing the buffer citrate, long known to be `protein friendly'; bis-tris and ADA were also identified as being very useful buffers in protein formulations.

Meltdown: A Tool to Help in the Interpretation of Thermal Melt Curves Acquired by Differential Scanning Fluorimetry.

The output of a differential scanning fluorimetry (DSF) assay is a series of melt curves, which need to be interpreted to get value from the assay. An application that translates raw thermal melt curve data into more easily assimilated knowledge is described. This program, called "Meltdown," conducts four main activities--control checks, curve normalization, outlier rejection, and melt temperature (T(m)) estimation--and performs optimally in the presence of triplicate (or higher) sample data. The final output is a report that summarizes the results of a DSF experiment. The goal of Meltdown is not to replace human analysis of the raw fluorescence data but to provide a meaningful and comprehensive interpretation of the data to make this useful experimental technique accessible to inexperienced users, as well as providing a starting point for detailed analyses by more experienced users.

The ROS/SUMO axis contributes to the response of acute myeloid leukemia cells to chemotherapeutic drugs.

Chemotherapeutic drugs used in the treatment of acute myeloid leukemias (AMLs) are thought to induce cancer cell death through the generation of DNA double-strand breaks. Here, we report that one of their early effects is the loss of conjugation of the ubiquitin-like protein SUMO from its targets via reactive oxygen species (ROS)-dependent inhibition of the SUMO-conjugating enzymes. Desumoylation regulates the expression of specific genes, such as the proapoptotic gene DDIT3, and helps induce apoptosis in chemosensitive AMLs. In contrast, chemotherapeutics do not activate the ROS/SUMO axis in chemoresistant cells. However, pro-oxidants or inhibition of the SUMO pathway by anacardic acid restores DDIT3 expression and apoptosis in chemoresistant cell lines and patient samples, including leukemic stem cells. Finally, inhibition of the SUMO pathway decreases tumor growth in mice xenografted with AML cells. Thus, targeting the ROS/SUMO axis might constitute a therapeutic strategy for AML patients resistant to conventional chemotherapies.