Evaluation of cardiovascular events and progression to end-stage renal disease in patients with dyslipidemia and chronic kidney disease from the North-Eastern area of Romania

International Urology and Nephrology - The aim of this prospective cohort study was: to identify the association between different biomarkers [proprotein convertase subtilisin/kexin 9-PCSK9,...     

Use of MLPA test in the detection of subtelomeric rearrangements--case report

Genetic causes of mental retardation (MR) are heterogeneous, but subtelomeric rearrangements are an important one. MLPA technique provides the best results in case detection. We have used MLPA to identify subtelomeric rearrangements in children with idiopatic MR. The protocol included: clinical selection; karyotype; antiFMRP test; MLPA. We have selected cases using de Vries diagnostic score. Patient data were recorded in a database. The group was formed of 142 MR children. In 24 (16.9%) the karyotype was abnormal. 16 cases (11.3%) presented speech delay/autism, but antiFMRP test was normal. 60 MLPA tests were done: 46 cases (76.7% were normal, 6 (10%) abnormal, 4 (6.7%) had polymorphism and 4 (6.7%) could not be interpreted. Clinical features of the cases identified are illustrated. In conclusion, the diagnostic score is useful in case selection for further testing and MLPA proves to be efficient in diagnosing subtelomeric rearrangements as a possible cause of idiopatic MR.     

Large‐conductance calcium‐activated potassium channels modulate vascular tone in experimental cirrhosis

Background: Large‐conductance calcium‐activated potassium (BK_Ca) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BK_Ca channels. The aim of this study was to evaluate the role of BK_Ca channels in the regulation of vascular tone in control (CT) and carbon tetrachloride‐cirrhotic (CH) rat livers. Methods: Changes in intrahepatic vascular resistance were assessed by evaluating the portal perfusion pressure (PP) response to methoxamine (Mtx) in the presence of Iberiotoxin (Ibtx; a BK_Ca channel blocker), NS1619 (a BK_Ca channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, N^G‐nitro‐l ‐arginine (l ‐NNA) or l ‐NNA alone. In addition, in CH livers, PP dose–response curves to the NO donor, S‐nitroso‐N‐acetyl‐d,l ‐penicillamine (SNAP), were performed after pre‐incubation with Ibtx or its vehicle. BK_Ca mRNA expression was assessed in liver homogenates, and BK_Ca protein expression in HSC isolated from CT and CH livers. Results: In CH livers, Ibtx significantly increased baseline PP and exacerbated the PP response to Mtx. Conversely, NS1619 induced a mild nonsignificant decrease of baseline PP and attenuated the hyperresponse to Mtx. CH livers exhibited an upregulation of both mRNA and protein of the α‐subunit of BK_Ca. Conclusion: Large‐conductance calcium‐activated potassium channels are overexpressed in CH livers and might represent a compensatory mechanism modulating the increased hepatic vascular tone of cirrhosis.     

Epidemiology of severe Streptococcus pyogenes disease in Europe

The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.     

Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative

Cyclic nucleotide-gated (CNG) ion channels play a key role in the sensory transduction of vision and olfaction. The channels are opened by the binding of cyclic nucleotides. Native olfactory CNG channels are heterotetramers of CNGA2, CNGA4, and CNGB1b subunits. Upon heterologous expression, only CNGA2 subunits can form functional homotetrameric channels. It is presently not known how the binding of the ligands to the four subunits is translated to channel opening. We studied activation of olfactory CNG channels by photolysis-induced jumps of cGMP or cAMP, two cyclic nucleotides with markedly different apparent affinity. It is shown that at equal degree of activation, the activation time course of homotetrameric channels is similar with cGMP and cAMP and it is also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Kinetic models were globally fitted to activation time courses of homotetrameric channels. While all models containing equivalent binding sites failed, a model containing three binding sites with a ligand affinity high–low–high described the data adequately. Only the second binding step switches from a very low to a very high open probability. We propose a unique gating mechanism for homotetrameric and heterotetrameric channels that involves only three highly cooperative binding steps.     

Effects of permeating ions and cGMP on gating and conductance of rod-type cyclic nucleotide-gated (CNGA1) channels

Cyclic nucleotide-gated (CNG) channels are tetrameric non-specific cation channels. They mediate the receptor potentials in photoreceptors and cells of the olfactory epithelium and they are activated by the binding of cyclic nucleotides such as cGMP and cAMP. Previous studies in homotetrameric CNGA1 channels, activated with covalently bound cGMP, presented evidence that partially liganded channels cause partial channel opening ( Ruiz & Karpen, 1997, 1999 ). Here, homotetrameric CNGA1 channels were expressed in Xenopus oocytes. Conductance and gating of these channels were studied as a function of the concentration of freely diffusible cGMP and with different permeating ions. At saturating cGMP the current levels distributed around a single mean in a Gaussian fashion and the open times were long. At low cGMP, however, the current levels were heterogeneous: they were smaller than those at saturating cGMP, equal, or larger. The open times were short. Ions generating the larger single-channel currents (Na⁺ > K⁺ > Rb⁺) concomitantly increased the heterogeneity of current levels and decreased the open probability and open times. The results suggest that the activation of CNGA1 channels by cGMP and ions staying longer in the pore is associated with less extensive and less frequent conformational fluctuations of the channel pore.     

Preprocessing of collagenous sources with the aim of extracting minimally denatured collagen

Biomedical applications of collagen are conditioned by its use in a state very close to the native state, as it concerns conformation and reactivity. Minimal denaturation implies that both the collagen structure and the side amino acid groups are kept intact. The bulk of alterations take place in phases preceding the extraction itself. MATERIAL AND METHOD: The authors have established an optimal protocol for preprocessing of young bovine hides, i.e. to obtain a maximum solubilisation yield when a standard proteases-based solubilisation technique was applied. RESULTS: The sequence: unhairing--loosing--cleaning--labilisation of corium tissue was taken in account. The effect of the treating composition upon the deamination degree of the extracted collagen was determined through a binary gradient HPLC technique, on a ProPac WCX-10 analytical column. The optimal range of loosing--cleaning recipe components was found to be between 18 ... 27% dithiotreitol (DDT), 12 ... 20% N-ethyl-acetamide (NEM) and 30 ... 53% iodide-acetamide (IDAM).     

Relationship diabetes mellitus-periodontal disease: etiology and risk factors

The interrelation between diabetes mellitus and inflammatory periodontal disease has been intensively studied for more than 50 years, a real bidirectional influence existing between patient's glycemic level disorder and periodontal territories alteration. Several studies developed in this direction emerged to the evidences that reveal a general increase of prevalence, extent and severity of gingivitis and periodontitis. Inflammation plays an important role in this interrelation, orchestrating both the periodontal disease and diabetes mellitus pathogeny and complications. Conversely, periodontal disease--infectious disease characterized by a significant inflammatory component--can seriously impair metabolic control of some diabetic patient. Moreover, treatment of periodontal disease and reduction of oral signs of inflammation may have a beneficial result on the diabetic condition (1). Less clear are the mechanisms governing this interrelation (even the literature is abundant in this direction), and, very probably, periodontal diseases serve as initiators of insulin resistance (in a way similar to obesity), thereby aggravating glycemic control. Further research is so imposed in order to clarify this aspect of the relationship between diabetes and periodontal disease.     

How cells flow in the spreading of cellular aggregates

Like liquid droplets, cellular aggregates, also called “living droplets,” spread onto adhesive surfaces. When deposited onto fibronectin-coated glass or polyacrylamide gels, they adhere and spread by protruding a cellular monolayer (precursor film) that expands around the droplet. The dynamics of spreading results from a balance between the pulling forces exerted by the highly motile cells at the periphery of the film, and friction forces associated with two types of cellular flows: (i) permeation, corresponding to the entry of the cells from the aggregates into the film; and (ii) slippage as the film expands. We characterize these flow fields within a spreading aggregate by using fluorescent tracking of individual cells and particle imaging velocimetry of cell populations. We find that permeation is limited to a narrow ring of width ξ (approximately a few cells) at the edge of the aggregate and regulates the dynamics of spreading. Furthermore, we find that the subsequent spreading of the monolayer depends heavily on the substrate rigidity. On rigid substrates, the migration of the cells in the monolayer is similar to the flow of a viscous liquid. By contrast, as the substrate gets softer, the film under tension becomes unstable with nucleation and growth of holes, flows are irregular, and cohesion decreases. Our results demonstrate that the mechanical properties of the environment influence the balance of forces that modulate collective cell migration, and therefore have important implications for the spreading behavior of tissues in both early development and cancer.Tissue spreading is a fundamental phenomenon in many biological processes. Examples include wound healing (1–4) where the surrounding tissue spreads to close the injury, or the development of the embryo (5–7), which requires the orchestrated movement of cells to specific locations. It is also present in the progression of cancer (8–10). For example, glioblastomas grow and spread aggressively to invade surrounding regions and may lead to dramatic damages (11). The first step of cancer propagation (invasion) is characterized by a loss of cell–cell adhesion associated with an increase in cell motility. Increased cell motility is followed by entry into the blood circulation (intravasation), and subsequent escape from the circulation into distal tissue (extravasation). From this distal site, cell proliferation leads to a secondary tumor (11). Thus, it is crucial to understand how noninvasive tumor cells become metastatic by the loss of cell–cell adhesion and increased migration, which leads to malignancy. Further investigation into this topic requires the design and analysis of model in vitro experimental systems suitable for recapitulating these early stage events.Model in vitro systems in 3D are essential to recapitulating the early stages of cancer progression. For example, it has been reported that the efficiency of medical drugs tested on 2D cell culture systems is not transposable to 3D in more than 50% cases (12). Recently, 3D cellular aggregates have been identified as a model system that approximates living tissues and tumors (13–15). Cell aggregates in culture consist of hundreds to thousands of cells. Structurally, cell aggregates resemble concentrated emulsions, such as the honeycomb organization of foams (16). However, contrary to foam, which is solid, cell aggregates flow like a liquid when placed onto an adhesive surface or in contact with each other. For example, cell aggregates in solution form spheroids to minimize their surface energy, and fuse when in contact with each other like liquid droplets (15). Since the pioneering work by Steinberg in the 1960s (17), several studies have shown that the mechanical behavior of tissues and cellular aggregates can be characterized through their liquid-like behavior (18, 19). The analogy between tissues and liquids has been very fruitful in describing the spreading of tissues, using the physics of capillarity and wetting (13, 20). The statics of wetting has been previously described in ref. 13, where interfacial energies involved in the wetting of droplets are replaced by intercellular and cell–substrate adhesion strengths. By varying both the intercellular adhesion (W_cc) and the adhesion with the substrate (W_cs), two aggregate spreading regimes have been observed as characterized by the sign of the effective spreading parameter, S = W_cs – W_cc. If S < 0, partial wetting occurs and the aggregate does not spread. If S > 0, complete wetting occurs and a precursor film made of a cellular monolayer flows outwards from the edge of the aggregate. The transition between partial and complete wetting is induced experimentally by varying W_cc, by tuning the level of expression of the cadherins, or W_cs, chemically using PEG–fibronectin substrate coating (21) or soft gels (22–24). A number of observations suggest that the strength of adhesion depends on both the substrate’s chemical receptors and its rigidity (25, 26). On a single-cell level, matrix rigidity has been shown to strongly affect myriad cellular physiological functions, including differentiation (27), spreading (28), and migration (29). On a multicellular level, the transition from complete (S > 0) to partial wetting (S < 0) of cell aggregates can be induced by decreasing the substrate elastic modulus below a critical value E_c ≈ 5–8 kPa (24). Thus, as the rigidity of the substrate is a key parameter that controls the statics of cell aggregate wetting, we focus here on the dynamics of wetting in substrates of varying rigidity (10³ to 10⁹ Pa).A cellular aggregate deposited on a wettable substrate spreads as a stratified droplet with a single monolayer (30). The dynamics of spreading results from a balance between friction forces and driving forces S (per unit length). In simple liquids, the driving forces are capillary forces. In the spreading aggregate, the driving forces are due to motile cells pulling at the periphery of the film (31). Previously, De Gennes and Cazabat described the dynamics of growth of a stratified precursor film (32). The precursor film has a 2D horizontal velocity field. The aggregate acts as a reservoir to feed the precursor film in a process named permeation, introduced by Helfrich to describe the flows of smectic liquid crystals normal to the layers (33). In our model system, permeation is the process by which the cells from the aggregates enter into the cell monolayer. During spreading, we expect two types of viscous dissipation, due to (i) slippage of the expanding monolayer with the substrate and (ii) permeation normal to the layer. De Gennes and Cazabat (32) predicted that the permeation flow is limited to a narrow ribbon near the contact line, characterized by the permeation width ξ. For simple liquids, they conclude that the friction with the substrate is dominant to permeation. By contrast, with the spreading of cellular aggregates, we will show here that the permeation of cells from the aggregates into the film is the factor limiting the dynamics of spreading (34). For example, aggregates deposited on adhesive stripes spread at constant velocity (21), which can be explained only if permeation is dominant. Thus, to date, the permeation of cells within aggregates has only been introduced as a theoretical hypothesis. Permeation has never been directly visualized for cell aggregates and we propose to directly observe this phenomenon using E-cadherin (Ecad)–expressing cells with a fluorescent nucleus.Studies on the collective motion of multicellular systems have been mainly restricted to 2D cell culture, confluent monolayers (4, 35), and wound healing assays (1). Our aim in this paper is to assess the collective motion of cells in 3D aggregates as they spread onto a 2D adhesive substrate. Using fluorescence ubiquitination cell cycle indicator (FUCCI) to identify the position of each cell within the aggregate, the migration trajectories of the cells will be monitored over time. Dynamical mapping of the velocity field is performed to fully characterize the collective mode of spreading. To this end, we use particle imaging velocimetry (PIV), an efficient technique already used for cell migration (36–38). PIV is a cross-correlation technique initially developed for characterizing fluid flow (39), by calculating the local displacements of embedded particles imaged in real time. Using PIV for cell migration, we have monitored for the first time, to our knowledge, the evolution of the velocity field within the migrating monolayer that emerges from the aggregate as an analogy to the early stages of tumor invasion. With this analysis, we have observed two regimes: a viscous liquid-like regime with a quasiradial flow of the cells at short times, where the aggregate acts as a reservoir, followed by a chaotic state at long times, once the aggregate has been depleted into the monolayer film. The latter is characterized by the formation of long range rotating velocity fields (“swirls”). In the case of deformable substrates, the cell monolayer is unstable during spreading, with growth of holes in the monolayer leading to rupture.The aim of this paper is to relate the macroscopic spreading behavior of cellular populations to the migration of individual cells using simple physical laws, such as the conservation of matter, permeation, and balance between viscous and driving forces. From these laws, we intend to shed light on the dynamics of tissue spreading, as it occurs during the early stages of cancer progression and during tissue development.     

Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration,invasion, and metastasis in early‐stage endometrioid endometrial cancer

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell–cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early‐stage patients (N = 134), recurrence‐free survival was poorer in patients with ALCAM‐positive compared to ALCAM‐negative tumours (HR 4.237; 95% CI 1.01–17.76). This difference was more significant in patients with early‐stage moderately–poorly differentiated tumours (HR 9.259; 95% CI 2.12–53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early‐stage disease (HR 6.027; 95% CI 1.41–25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss‐of‐function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM‐depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM‐mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early‐stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.