Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Magnesium Supplementation Is Associated with a Lower Cardio-Metabolic Risk in Patients Submitted to Bariatric Surgery.

PURPOSE: Literature has demonstrated an inverse relation between magnesium (Mg) consumption and development of type 2 diabetes mellitus (T2DM), hypertension (HT), and dyslipidemia. After bariatric surgery (BS), micronutrients deficiencies are common, it being important to ensure appropriate supplementation. There is no recommendation about Mg supplementation and to our knowledge, its effect has not been studied to date. Our aim was to evaluate the effect of Mg supplementation in cardio-metabolic risk factors on post-bariatric patients. MATERIALS AND METHODS: A retrospective observational study of patients with obesity who underwent BS was performed. Data was assessed preoperatively and yearly (4-year follow-up). RESULTS: A total of 3363 patients were included. In the first year of follow-up, 79.8% (n = 2123) of the patients were supplemented with Mg, with evidence of slightly decreased percentages in the following years. Mg deficiency (serum Mg < 1.52 mEq/L) was more common among patients who were not supplemented during each year of follow-up (p < 0.05). Among those who underwent Mg supplementation, the percentage of T2DM, HT, or low-density lipoprotein cholesterol (LDL-C) > 130 mg/dL was significantly lower. In the first year post-surgery, the supplementation group had a lower risk of T2DM (OR = 0.545, p < 0.0001), LDL-C > 130 mg/dL (OR = 0.612, p < 0.0001), and HT (OR = 0.584, p < 0.0001). The OR for having these metabolic comorbidities persisted lower during the 4 years' follow-up. Patients who had Mg deficiency had higher prevalence of T2DM and HT. CONCLUSION: Mg supplementation seems to have a protective effect on the development of T2DM, HT, and LDL-C > 130 mg/dL in post-bariatric patients.

Environment driven oscillation in an off-lattice May-Leonard model.

Cyclic dominance of competing species is an intensively used working hypothesis to explain biodiversity in certain living systems, where the evolutionary selection principle would dictate a single victor otherwise. Technically the May-Leonard models offer a mathematical framework to describe the mentioned non-transitive interaction of competing species when individual movement is also considered in a spatial system. Emerging rotating spirals composed by the competing species are frequently observed character of the resulting patterns. But how do these spiraling patterns change when we vary the external environment which affects the general vitality of individuals? Motivated by this question we suggest an off-lattice version of the tradition May-Leonard model which allows us to change the actual state of the environment gradually. This can be done by introducing a local carrying capacity parameter which value can be varied gently in an off-lattice environment. Our results support a previous analysis obtained in a more intricate metapopulation model and we show that the well-known rotating spirals become evident in a benign environment when the general density of the population is high. The accompanying time-dependent oscillation of competing species can also be detected where the amplitude and the frequency show a scaling law of the parameter that characterizes the state of the environment. These observations highlight that the assumed non-transitive interaction alone is insufficient condition to maintain biodiversity safely, but the actual state of the environment, which characterizes the general living conditions, also plays a decisive role on the evolution of related systems.

Herbivory and leaf traits of Amazonian tree species as affected by irradiance.

Herbivory is one of the major biotic stress factors that affect the establishment of plants. However, the main factors that drive herbivory in seedlings of Amazonian tree species are still not well understood. Here we investigated whether contrasting levels of irradiance influence herbivory according to different herbivory indicators and which leaf traits are most related to interspecific variation in herbivory under contrasting irradiance conditions. We measured the leaf area lost as a result of insect herbivory in five tree species planted in a silvicultural system of secondary forest enrichment according to two indicators, herbivore damage (accumulated since plant germination) and herbivory rate (measured over time), and two irradiance conditions, understorey PPFD 2.6 mol·m-2 ·day-1 ) and gap PPFD 33.1 mol·m-2 ·day-1 . Furthermore, we related the interspecific variation in herbivory to a set of leaf traits: SLA, RWC, sclerophylly, phenolic compound content, tannins, condensed tannins and non-structural carbohydrates. Herbivore damage was significantly affected by light availability and species, with the highest percentage variation observed in the Meliaceae (Carapa guianensis and Swietenia macrophylla). For the herbivory rate, only the interspecific variation was significant, with Bertholletia excelsa having the lowest rates. Chemical characteristics (phenolic compounds and tannins) were most related to herbivory rates, as well as highly influenced by light conditions. Non-structural carbohydrates (starch and sucrose) were also related to the interspecific variation in herbivory. The phenolic compounds and starch, as affected by light quantity, are species dependent. Thus, the selective pressure on herbivores may be driven by species-dependent responses to light conditions.

ARMC5 Primary Bilateral Macronodular Adrenal Hyperplasia Associated with a Meningioma: A Family Report.

Primary bilateral adrenal macronodular hyperplasia is characterized by functioning adrenal macronodules and variable cortisol secretion. Familial clustering suggests a genetic cause that has been confirmed with the identification of some genetic mutations, including inactivating germline mutations, in armadillo repeat containing 5 (ARMC5) gene. The identification of the pathogenic variant enables the physician to identify and treat these patients earlier and more effectively. It has also been noticed that patients with germline causative variants show a different clinical spectrum, presenting specific clinical characteristics, as the association with the presence of meningiomas.

Succinyl-ß-cyclodextrin: Influence of the substitution degree on albendazole inclusion complexes probed by NMR.

Succinyl-ß-CD derivatives were obtained by green synthesis with degrees of substitution (DS) 1.3 and 2.9. The spray-drying technique was used to obtain albendazole (ABZ):succinyl-ß-CD inclusion complexes. Phase solubility diagrams indicated that both succinyl-ß-CD derivatives formed 1:1 molar ratio ABZ complexes, but the complex with DS 2.9 has a lower formation constant. The presence of stable inclusion complexes in aqueous solution was confirmed by NMR. For both complexes the aromatic moiety is encapsulated into the host cavity. In the solid-state, 13C and 15N NMR spectral differences between ABZ and ABZ included in spray-dried systems showed that strong structural changes occurred in the systems. At least two different ABZ amorphous species were identified based on DS. ABZ species were stable over more than six months based on spectral data. Finally, the influence of DS in the number and type of the inclusion complexes was elucidated.

Interaction of Cytotoxic and Cytoprotective Bile Acids with Model Membranes: Influence of the Membrane Composition.

To understand the role of bile acids (BAs) in cell function, many authors have investigated their effect on biomembrane models which are less complex systems, but there are still many open questions. The present study aims to contribute for the deepening of the knowledge of the interaction between BAs and model membranes, in particular, focusing on the effect of BA mixtures. The cytotoxic deoxycholic acid (DCA), the cytoprotective ursodeoxycholic acid (UDCA), and the equimolar mixture (DCA + UDCA) were investigated. Monolayers and liposomes were taken as model membranes with two lipid compositions: an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), and cholesterol (Chol)) traditionally associated with the formation of lipid rafts and an equimolar POPC/SM binary mixture. The obtained results showed that DCA causes the fluidization of monolayers and bilayers, leading to the eventual rupture of POPC/SM liposomes at high concentration. UDCA may provide a stabilization of POPC/SM membranes but has a negligible effect on the Chol-containing liposomes. In the case of equimolar mixture DCA/UDCA, the interactions depend not only on the lipid composition but also on the design of the experiment. The BA mixture has a greater impact on the monolayers than do pure BAs, suggesting a cooperative DCA-UDCA interaction that enhances the penetration of UDCA in both POPC/SM and POPC/SM/Chol monolayers. For the bilayers, the presence of UDCA in the mixture decreases the disturbing effect of DCA.

The importance of the Abn2 calcium cluster in the endo-1,5-arabinanase activity from Bacillus subtilis.

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic ß-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.

Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells.

Nineteen terpenoids, including macrocyclic diterpenes, diterpenic lactones and other polycyclic diterpenes, steroids and a triterpene isolated from the methanolic extracts of Euphorbia species, were evaluated for their potential antineoplastic activity in various human cancer cell lines that were derived from three tumor entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) carcinomas. Furthermore, different multidrug-resistant variants of these cancer cell lines with over-expression of MDR1/P-gp or no MDR1/P-gp expression were also investigated. In parental drug-sensitive cell lines, the tested compounds showed a moderate/weak antiproliferative effect or were inactive. Most of them were found more effective in drug-resistant cells than in the parental, drug-sensitive ones, and some of them showed high antineoplastic efficacy in classical or atypical drug-resistant cells. The most active compounds were the lathyrane diterpenes latilagascenes C and D, and the diterpenic lactones 3beta-acetoxy-helioscopinolide B and helioscopinolide E which exhibited high antineoplastic activities against the drug-resistant subline EPG85-257RDB derived from gastric carcinoma. In addition, the macrocyclic lathyrane diterpene jolkinol B was found to be highly effective in the multidrug-resistant variant HT-29RNOV.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

Influence of the multidrug transporter inhibitors on the activity of Kv1.3 voltage-gated potassium channels.

Using the whole-cell patch-clamp technique, the influence of selected multidrug resistance modulators, both plant-derived compounds and derivatives on the activity of voltage-gated potassium channels Kv1.3 was investigated. Twelve compounds with phenolic and terpenic structures were tested: the stilbenes piceatannol (1) and its tetramethoxy (2) and tetracetoxy (3) derivatives, the flavonoids naringenin (4) and its methylated derivatives: naringenin-4',7-dimethylether (5) and naringenin-7-methylether (6), and aromadendrin (7), the coumarins esculetin (8) and scopoletin (9) and ent-abietane diterpenes, helioscopinolide B (10) and its 3beta-acetoxy derivative (11) and helioscopinolide E (12). The studies were performed on a model system with Kv1.3 channels endogenously expressed in human T lymphocytes. Obtained data provide evidence that compounds 2, 5 and 6 applied at 30 microM inhibited the amplitude of recorded currents to 31%, 4% and 29% of its control value, respectively. On the other hand, compounds 3, 4, 7-12 (at 30 microM) and compound 1 (at 40 microM) did not affect significantly the channel activity. These results indicate that some methoxy-derivatives of the tested compounds are effective inhibitors of Kv1.3 channels. Since the inhibition of Kv1.3 channels may inhibit the proliferation of prostate, breast and colon cancer cells expressing these channels, the channel inhibitors may exert an antiproliferative action. This action combined with a simultaneous modulation of the multidrug resistance may be significant for a potential application of these compounds in cancer chemotherapy.

Prenatal sonographic diagnosis of a rare Cantrell's pentalogy variant with associated open neural tube defect - a case report.

Cantrell's pentalogy is a very rare congenital anomaly. The hallmark of this condition is the presence of ectopia cordis and an abdominal wall defect. Its association with craniorachischisis is even more infrequent with just a few cases reported in the literature. The authors describe a case of a prenatal ultrasound diagnosis of Cantrell's pentalogy with posterior encephalocele and spinal dysraphism associated.

Evaluation of the antiviral and antimicrobial activities of triterpenes isolated from Euphorbia segetalis.

A phytochemical reinvestigation of the whole plant of Euphorbia segetalis yielded five tetracyclic triterpenes: 3beta-hydroxy-cycloart-25-en-24-one (1), cycloart-25-ene-3beta,24-diol (2), cycloart-23-ene-3beta,25-diol (3), lanosta-7,9(11),24-trien-3beta-ol (4) and lanosta-7,9(11),24(31)-trien-3beta-ol (5). beta-acetoxy-cycloart-25-en-24-one (1a) and glutinol (6), lupenone (7), dammaranodienol (9), cycloartenol acetate (10), 24-methylenecycloartanol acetate (11) and beta-sitosterol (12), isolated previously, were evaluated for their antiviral activities against Herpes simplex virus (HSV) and African swine fever virus (ASFV). Lupenone exhibited strong viral plaque inhibitory effect against HSV-1 and HSV-2. The in vitro antifungal and antibacterial activities of la, cycloart-23-ene-3beta,25-diol, 3-acetate (3a) and 6-12 were also investigated.

Essential oils from Piper cernuum and Piper regnellii: antimicrobial activities and analysis by GC/MS and 13C-NMR.

The essential oils of Piper cernuum and Piper regnellii leaves were analyzed by gas chromatography-mass spectrometry (GC-MS) and the results were compared to that obtained by means of a program designed to analyse (13)C-NMR data of complex mixtures. Bicyclogermacrene (21.88 %)/beta-caryophyllene (20.69 %) and myrcene (52.60 %)/linalool (15.89 %) were the major constituents in essential oil from leaves of P. cernuum and P. regnellii, respectively. Both essential oils presented growth inhibitory activities against Staphylococcus aureus and Candida albicans.

Cycloartane triterpenes from Euphorbia tuckeyana.

Investigation of the acetone extract of the whole plant of Euphorbia tuckeyana afforded a new cycloartane-type triterpene named as cyclotucanol. Its structure was established as cycloartane-24-methylene-3beta,25-diol (1). The known cycloartane triterpenes cycloeucalenol (2), 3beta-hydroxycycloart-25-en-24-one (3), cycloart-25-ene-3beta,24-diol (4), 25,26,27-trisnor-3beta-hydroxycycloartan-24-al (5) and cycloart-23-ene-3beta,25-diol (6) were also isolated and identified.

The role of nuclear cardiology for preoperative risk assessment prior to noncardiac surgery.

Preoperative risk assessment, before noncardiac surgery, aims to reduce mortality and morbidity, during the perioperative period. Cardiac risk could be minimised through treatment and stabilisation of the underlying disease, careful monitoring and prophylactic medications. However, cardiac complications, especially, ischemic events are still frequent causes of death during surgery and hospital recovery. A teamwork approach that involves cardiologist, anaesthesiologist and surgeon is required for optimal risk assessment and monitoring in the perioperative period. The incidence of coronary artery disease increases with age and with the presence of risk factors such as diabetes or hypertension. There is also a strong relation between coronary and vascular disease as they are part of the same pathophysiology and have common risk factors. Careful cardiac evaluation is essential when vascular surgery is planned. Clinical evaluation is the first step of preoperative risk assessment and allows the identification of low and high-risk patients. If there is absent or low cardiac risk, surgery may be carried out. In the presence of high-risk markers and if the surgery is elective, coronary arteriography should be considered before non-cardiac surgery. The information provided by non-invasive diagnostic testing is essential in those patients with an intermediate clinical risk for cardiac events. Concerning coronary artery disease, myocardial function and ischemic burden are strong prognostic markers and in this particular setting, nuclear cardiology can play an important role. The extent and localisation of ischemia is well defined by myocardial perfusion scintigraphy and ventricular function can be evaluated by radionuclide ventriculography or by gated perfusion studies.

The additive values of left ventricular function and extent of myocardium at risk to dipyridamole perfusion imaging for optimal risk stratification prior to vascular surgery.

Although the increased risk of cardiac complications in surgical patients with diminished left ventricular ejection fraction (LVEF) is well-established, this method has been supplanted in recent years by assessment of ischaemic burden using myocardial perfusion imaging (MPI). This study was conducted to determine if MPI and LVEF determination provide complementary or redundant information in preoperative evaluation of vascular surgery patients. A total of 101 patients were studied with dipyridamole MPI and radionuclide ventriculography before surgery. Single photon emission tomographic MPI images were scored for defect severity and categorized as either fixed or reflecting ischaemia. Resting left ventricular cavity was also categorized as normal or dilated. LVEF was subdivided into normal (> or = 50%) and abnormal (< 50%). Seventeen patients had cardiac events. Events were more frequent in patients with ischaemia, in patients with a LVEF < 50% and in those with dilated left ventricular chambers. The mean number of ischaemic segments was also higher in the cardiac event group. Higher event rates were seen when a combination of these factors was present. A history of myocardial infarct, congestive heart failure or coronary artery disease was also a significant predictor of subsequent events. Thus, both abnormal left ventricular function and extent of ischaemic myocardium have independent and complementary predictive power for cardiac events in vascular surgery patients.