Mechanisms of Antifungal Drug Resistance.

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug- resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

Mechanisms of OGT-mediated HCF-1 protein maturation

Post-translational protein modifications are crucial for many fundamental cellular and extracellular processes and greatly contribute to the complexity of organisms. Human HCF-1 is a transcriptional co-regulator that undergoes complex protein maturation involving reversible and irreversible post-translational modifications. Upon synthesis as a large precursor protein, HCF-1 undergoes extensive reversible glycosylation with β-N-acetylglucosamine giving rise to O-linked-β-N-acetylglucosamine (O-GlcNAc) modified serines and threonines. HCF-1 also undergoes irreversible site-specific proteolysis, which is important for one of HCF-1's major functions - the regulation of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by a single enzyme with an unusual dual enzymatic activity, the O-GlcNAc transferase (OGT). HCF-1 is cleaved by OGT at any of six highly conserved 26 amino acid repeated sequences (HCF-1PRO repeats), but the mechanisms and the substrate requirements for OGT-mediated cleavage are not understood. In the present work, I characterized substrate requirements for OGT-mediated cleavage and O-GlcNAcylation of HCF-1. I identified key elements within the HCF-1PRO-repeat sequence that are important for proteolysis. Remarkably, an invariant single amino acid side-chain within the HCF-1PRO-repeat sequence displays particular OGT-binding properties and is essential for proteolysis. Additionally, I characterized substrate requirements for proteolysis outside of the HCF-1PRO repeat and identified a novel, highly O-GlcNAcylated OGT-binding sequence that enhances cleavage of the first HCF-1PRO repeat. These results link OGT association and its O-GlcNAcylation activities to HCF-1PRO-repeat proteolysis.

Fetal programming and epigenetic mechanisms in arterial hypertension.

PURPOSE OF REVIEW: To provide an overview of available evidence of the potential role of epigenetics in the pathogenesis of hypertension and vascular dysfunction. RECENT FINDINGS: Arterial hypertension is a highly heritable condition. Surprisingly, however, genetic variants only explain a tiny fraction of the phenotypic variation and the term 'missing heritability' has been coined to describe this phenomenon. Recent evidence suggests that phenotypic alteration that is unrelated to changes in DNA sequence (thereby escaping detection by classic genetic methodology) offers a potential explanation. Here, we present some basic information on epigenetics and review recent work consistent with the hypothesis of epigenetically induced arterial hypertension. SUMMARY: New technologies that enable the rigorous assessment of epigenetic changes and their phenotypic consequences may provide the basis for explaining the missing heritability of arterial hypertension and offer new possibilities for treatment and/or prevention.

Cenozoic evolution of eastern Iberia: structural data and dynamic model.

Iberia underwent intraplate deformation during the Mesozoic and Cenozoic. In eastem Ibena, compression took place during the Palaeogene and early Miocene, giving rise to the Iberian Chain, and extension started during the early Miocene in the coastal areas and the Valencia trough; during early Miocene compression continued in the western Iberian Chain whereas extension had started in the eastern Iberian Chain. From the kinematic data obtained from the major compressional and extensional structures formed dunng the Cenozoic, a simple dynamic model using Bott's (1959) formula is presented. The results show that both extension and compression may have been produced assuming a main horizontal stress-axis approximately N-S, in a similar direction that the convergence between Europe, Ibena and Afnca dunng the Cenozoic.

Hiperplasia nodular focal do fígado: apresentação de um caso e revisão da literatura

Neste trabalho apresentamos um caso de hiperplasia nodular focal que foi diagnosticado aos seis anos de idade e que está sendo acompanhado até o momento presente. Para o diagnóstico foram imprescindíveis as técnicas de imagem, tendo importância de realce a cintilografia hepatoesplênica e a tomografia computadorizada. Apresentamos, também, revisão da literatura sobre o assunto.

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a.

Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Focal pleural thickening mimicking pleural plaques on chest computed tomography: tips and tricks.

Diagnosis of pleural plaques (PPs) is commonly straightforward, especially when a typical appearance is observed in a context of previous asbestos exposure. Nevertheless, numerous causes of focal pleural thickening may be seen in routine practice. They may be related to normal structures, functional pleural thickening, previous tuberculosis, pleural metastasis, silicosis or other rarer conditions. An application of a rigorous technical approach as well as a familiarity with loco-regional anatomy and the knowledge of typical aspects of PP are required. Indeed, false-positive or false-negative results may engender psychological and medico-legal consequences or can delay diagnosis of malignant pleural involvement. Correct recognition of PPs is crucial, as they may also be an independent risk factor for mortality from lung cancer in asbestos-exposed workers particularly in either smokers or former/ex-smokers. Finally, the presence of PP(s) may help in considering asbestosis as a cause of interstitial lung disease predominating in the subpleural area of the lower lobes. The aim of this pictorial essay is to provide a brief reminder of the normal anatomy of the pleura and its surroundings as well as the various aspects of PPs. Afterwards, the common pitfalls encountered in PP diagnosis will be emphasized and practical clues to differentiate actual plaque and pseudoplaque will be concisely described.

Dosimetria de pacientes e médicos em intervenções coronárias percutâneas em Recife, Pernambuco, Brasil

OBJETIVO: Este trabalho teve como objetivo estimar os valores de doses de radiação recebidas por médicos e pacientes em procedimentos intervencionistas cardíacos realizados em um hospital público na cidade de Recife, Pernambuco. MATERIAIS E MÉTODOS: As medidas foram determinadas em 31 pacientes adultos, dos quais 22 tiveram acompanhamento clínico após o procedimento, e em dois cardiologistas com mais de dez anos de experiência. Parâmetros de irradiação para cada procedimento foram registrados. RESULTADOS: Os valores obtidos para a dose absorvida máxima na pele do paciente variaram entre 612 e 8.642 mGy, sendo que 53% foram maiores que 2.000 mGy, valores estes que podem causar efeitos determinísticos. Com relação aos médicos, a dose efetiva média por procedimento foi de 11 µSv e os valores médios do equivalente de dose nas extremidades, mais altos, foram: 923 µSv no pé esquerdo, 514 µSv no pé direito, 382 µSv na mão esquerda e 150 µSv no olho esquerdo. Dependendo do número de procedimentos, as doses recebidas pelos médicos podem exceder os valores limites de doses estabelecidos pelas normas nacionais e internacionais. CONCLUSÃO: Os resultados obtidos sinalizam a necessidade de adoção de estratégias para otimização da proteção radiológica tanto de pacientes quanto de médicos.

Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Cell cycle control in Alphaproteobacteria.

NlmCategory="UNASSIGNED">Alphaproteobacteria include many medically and environmentally important organisms. Despite the diversity of their niches and lifestyles, from free-living to host-associated, they usually rely on very similar mechanisms to control their cell cycles. Studies on Caulobacter crescentus still lay the foundation for understanding the molecular details of pathways regulating DNA replication and cell division and coordinating these two processes with other events of the cell cycle. This review highlights recent discoveries on the regulation and the mode of action of conserved global regulators and small molecules like c-di-GMP and (p)ppGpp, which play key roles in cell cycle control. It also describes several newly identified mechanisms that modulate cell cycle progression in response to stresses or environmental conditions.