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Expression of transcription factor CITED4 is necessary for exercise-induced regional remodeling of the heart’s microstructural helicity, revealing how a key molecular regulator of cardiac remodeling mediates local tissue-level changes.

Clinical and basic research suggests that exercise is a safe behavioral intervention and is effective for improving cognitive function in cerebrovascular diseases, including subcortical ischemic vascular dementia (SIVD). However, most of the basic research uses young animals to assess the effects of exercise, although SIVD is an age-related disease. In this study, therefore, we used middle-aged mice to examine how treadmill exercise changes the cognitive function of SIVD mice. As a mouse model of SIVD, prolonged cerebral hypoperfusion was induced in 8-month-old male C57BL/6J mice by bilateral common carotid artery stenosis. A week later, the mice were randomly divided into two groups: a group that received 6-week treadmill exercise and a sedentary group for observation. After subjecting the mice to multiple behavioral tests (Y-maze, novel object recognition, and Morris water maze tests), the treadmill exercise training was shown to only be effective in ameliorating cognitive decline in the Y-maze test. We previously demonstrated that the same regimen of treadmill exercise was effective in young hypoperfused-SIVD mice for all three cognitive tests. Therefore, our study may indicate that treadmill exercise during cerebral hypoperfusion has only limited effects on cognitive function in aging populations.

Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1−/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1−/− BOECs which was observed in CCM1+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.

Autosomal dominant cerebral cavernous malformation (CCM) represents a genetic disorder with a high mutation detection rate given that stringent inclusion criteria are used and copy number variation analyses are part of the diagnostic workflow. Pathogenic variants in either CCM1 (KRIT1), CCM2 or CCM3 (PDCD10) can be identified in 87–98% of CCM families with at least two affected individuals. However, the interpretation of novel sequence variants in the 5′-region of CCM2 remains challenging as there are various alternatively spliced transcripts and different transcription start sites. Comprehensive genetic and clinical data of CCM2 patients with variants in cassette exons that are either skipped or included into alternative CCM2 transcripts in the splicing process can significantly facilitate clinical variant interpretation. We here report novel pathogenic CCM2 variants in exon 3 and the adjacent donor splice site, describe the natural history of CCM disease in mutation carriers and provide further evidence for the classification of the amino acids encoded by the nucleotides of this cassette exon as a critical region within CCM2. Finally, we illustrate the advantage of a combined single nucleotide and copy number variation detection approach in NGS-based CCM1/CCM2/CCM3 gene panel analyses which can significantly reduce diagnostic turnaround time.

Abstract Background The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard‐to‐transfect cells remains challenging. Methods In a proof‐of‐principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high‐fidelity Cas9 variant was then transfected into patient‐derived BOECs using a ribonucleoprotein complex and a single‐strand DNA oligonucleotide. In addition, patient‐specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation. Results Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9‐induced mutations in predicted off‐target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1‐deficient BOECs displayed increased resistance to stress‐induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1‐deficient BOECs largely replaced corrected as well as heterozygous BOECs. Conclusion We here demonstrate that a non‐viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard‐to‐transfect endothelial cells (ECs). Comparing patient‐derived isogenic CCM1+/+, CCM1+/−, and CCM1−/− ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.

Lentigo maligno é um melanoma in situ que mais comumente surge em áreas expostas à radiação ultravioleta, nos pacientes idosos. O tratamento é realizado, principalmente, para minimizar o risco de progressão para lentigo maligno melanoma. O presente relato se refere a uma paciente idosa com lesões recorrentes de lentigo maligno na face, tratada com sucesso com imiquimod tópico, mostrando que este pode ser um tratamento útil, em determinados casos da doença.Lentigno maligna is a melanoma in situ that most commonly appears on areas exposed to ultraviolet radiation, in elderly patients. Treatment is required mainly to minimize the risk of progression to lentigo maligna melanoma. The present report refers to an elderly patient with recurrent lesions of lentigo maligna in her face, who was successfully treated with topical imiquimod, which showed to be a useful therapy for some cases of the disease.

BACKGROUND: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host's primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. METHODS: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). RESULTS: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56(dim) NK cells. The production of GzmA in CD56(bright) NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. CONCLUSION: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation.

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p.

The double burden of under- and overnutrition profoundly affects human health globally. According to the World Health Organization, obesity and diabetes rates have almost doubled worldwide since 1980, and, in 2011, more than 40 million children under 5 years of age were overweight. Ecologic factors, parental genetics and fitness, and the intrauterine environment significantly influence the likelihood of offspring developing the dysmetabolic diathesis of obesity. This report examines the effects of these factors, including preconception, intrauterine and postnatal energy balance affecting programming of transgenerational transmission, and development of chronic diseases later in life-in particular, diabesity and its comorbidities.

Abstract Background Circulating tumour cells (CTCs) and tumour-derived extracellular vesicles (tdEVs) have great potential for monitoring therapy response and early detection of tumour relapse, facilitating personalized adjuvant therapeutic strategies. However, their low abundance in peripheral blood limits their informative value. In this study, we explored the presence of CTCs and tdEVs collected intraoperatively from a tumour-draining vein (DV) and via a central venous catheter (CVC) prior to tumour resection. Methods CellSearch analyses of 395 blood samples from 306 patients with gastrointestinal tumours and 93 blood samples from healthy donors were used to establish and validate gates for the automated detection of CTCs and tdEVs with ACCEPT software and R scripts. The selected gate settings were applied to 227 samples of 142 patients with colorectal cancer (CRC) from two independent collectives. Phenotypic features were obtained via numeric analysis of their fluorescence signals (e.g. size, shape, and intensity) and were used for calculating diversity using Shannon index (SI) of clusters generated via the k-means algorithm after Uniform Manifold Approximation and Projection (UMAP) pre-processing, and standard deviation (SD). Results CTCs and tdEVs were more abundant in the DV samples compared to CVC samples (p

Background/Introduction: Adipose tissue (AT) has been highlighted as a promising reservoir of infection for viruses, bacteria and parasites. Among them is Trypanosoma cruzi, which causes Chagas disease. The recommended treatment for the disease in Brazil is Benznidazole (BZ). However, its efficacy may vary according to the stage of the disease, geographical origin, age, immune background of the host and sensitivity of the strains to the drug. In this context, AT may act as an ally for the parasite survival and persistence in the host and a barrier for BZ action. Therefore, we investigated the immunomodulation of T. cruzi-infected human AT in the presence of peripheral blood mononuclear cells (PBMC) where BZ treatment was added. Methods: We performed indirect cultivation between T. cruzi-infected adipocytes, PBMC and the addition of BZ. After 72h of treatment, the supernatant was collected for cytokine, chemokine and adipokine assay. Infected adipocytes were removed to quantify T. cruzi DNA, and PBMC were removed for immunophenotyping. Results: Our findings showed elevated secretion of interleukin (IL)-6, IL-2 and monocyte chemoattractant protein-1 (MCP-1/CCL2) in the AT+PBMC condition compared to the other controls. In contrast, there was a decrease in tumor necrosis factor (TNF) and IL-8/CXCL-8 in the groups with AT. We also found high adipsin secretion in PBMC+AT+T compared to the treated condition (PBMC+AT +T+BZ). Likewise, the expression of CD80+ and HLA-DR+ in CD14+ cells decreased in the presence of T. cruzi. Discussion: Thus, our findings indicate that AT promotes up-regulation of inflammatory products such as IL-6, IL-2, and MCP-1/CCL2. However, adipogenic inducers may have triggered the downregulation of TNF and IL-8/ CXCL8 through the peroxisome proliferator agonist gamma (PPAR-g) or receptor expression. On the other hand, the administration of BZ only managed to reduce inflammation in the microenvironment by decreasing adipsin in the infected culture conditions. Therefore, given the findings, we can see that AT is an ally of the parasite in evading the host‘s immune response and the pharmacological action of BZ. [ABSTRACT FROM AUTHOR]