Measuring Retrograde Actin Flow in Neuronal Growth Cones.

Actin flow refers to the motion of the F-actin cytoskeleton and has been observed in many different cell types, especially in motile cells including neuronal growth cones. The direction of the actin flow is generally retrograde from the periphery toward the center of the cell. Actin flow can be harnessed for forward movement of the cell through substrate-cytoskeletal coupling; thus, a key function of actin flow is in cell locomotion. In this chapter, we illustrate three different methods of quantifying retrograde F-actin flow in growth cones derived from cultured Aplysia bag cell neurons. These methods include tracking the movement of surface marker beads as well as kymograph analysis of time-lapse sequences acquired by differential interference contrast (DIC) imaging or fluorescent speckle microscopy (FSM). Due to their large size, Aplysia neuronal growth cones are uniquely suited for these methods; however, they can also be applied to any other growth cones with clear F-actin-rich peripheral domains.

A modified motor-clutch model reveals that neuronal growth cones respond faster to soft substrates.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells.

Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.

The Prebiotic Effects of an Extract with Antioxidant Properties from Morus alba L. Contribute to Ameliorate High-Fat Diet-Induced Obesity in Mice.

Obesity is a global health issue, in which modifications in gut microbiota composition have a key role. Different therapeutic strategies are being developed in combination with diet and exercise, including the use of plant extracts, such as those obtained from Morus alba L. leaves. Recent studies have revealed their anti-inflammatory and antioxidant properties. The aim of the present work was to evaluate whether the beneficial effects of M. alba L. leaf extract in high-fat diet-induced obesity in mice is correlated with its impact on gut microbiota. The extract reduced body weight gain and attenuated lipid accumulation, as well as increased glucose sensitivity. These effects were associated with an amelioration of the obesity-associated inflammatory status, most probably due to the described antioxidant properties of the extract. Moreover, M. alba L. leaf extract mitigated gut dysbiosis, which was evidenced by the restoration of the Firmicutes/Bacteroidota ratio and the decrease in plasma lipopolysaccharide (LPS) levels. Specifically, the extract administration reduced Alistipes and increased Faecalibaculum abundance, these effects being correlated with the beneficial effects exerted by the extract on the obesity-associated inflammation. In conclusion, anti-obesogenic effects of M. alba L. leaf extract may be mediated through the amelioration of gut dysbiosis.

Study of the mechanism of ε-poly-l-lysine as an antifungal on Candida albicans and Saccharomyces cerevisiae.

The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 µg·mL-1; EPL produced a K+ and Ca2+ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.

Silk fibroin nanoparticles enhance quercetin immunomodulatory properties in DSS-induced mouse colitis.

Inflammatory bowel disease (IBD) is a chronic and idiopathic inflammatory disorder affecting the gastrointestinal tract. The pharmacological treatments used currently for its treatment lack efficacy, so new therapeutic strategies should be developed. In this context, flavonoids loaded in biopolymeric nanoparticles can be considered as novel promising candidates. The aim of the present study was to evaluate the intestinal anti-inflammatory effects of quercetin when is administered loaded in silk fibroin nanoparticles (QSFN) in the dextran sulphate sodium experimental model of mouse colitis, which displays some similarities to human IBD. Previously characterized quercetin-loaded silk fibroin nanoparticles (QSFN). QSFN showed a reversible aggregation profile induced by the acidification of the solution but did not affect the loaded quercetin. Daily administration of QSFN significantly reduced disease activity index values compared to the control colitic group. This beneficial effect was not only corroborated by the histological examination of the colonic specimens but also the improvement of the colonic expression of the different proinflammatory cytokines (Tnf-α, Il-1ß, Il-6, Mcp-1, Icam-1, Nlrp3 and iNOS). Therefore, these data suggest that QSFN could be a promising alternative to current treatments as a drug delivery system for IBD treatment.

Sonotherapy in the reduction of anxiety and postoperative pain in patients with regional anesthesia as a sole technique: randomized, controlled clinical trial.

OBJECTIVE: To determine if sonotherapy reduces the anxiety level and postoperative pain in adults undergoing outpatient orthopedic surgery under regional anesthesia. METHOD: A randomized, double-blind, placebo-controlled study in 80 adult patients undergoing orthopedic surgery, 40 received intervention with sonotherapy and 40 did not receive it. Pain, anxiety in the preoperative period, immediate postoperative, high, 24 and 48 hours later were measured. Hemodynamic parameters were measured in four moments. RESULTS: No significant differences were found between the two groups in the pain scales neither anxiety. A significant reduction of the systolic blood pressure was observed after the application of sonotherapy and in recovery. There was no difference in other hemodynamic variables such as heart rate, respiratory rate, arterial oxygen saturation or diastolic blood pressure between groups, however, the observed changes in some of them occurred faster in the intervention group. CONCLUSIONS: In adult patients undergoing outpatient orthopaedic surgery under regional anaesthesia, sound therapy is a novel strategy that significantly reduces systolic blood pressure, considered as an indicator of decreased anxiety. However, in our study, no difference could be demonstrated in terms of pain control or the need for additional sedation or analgesia.

OBJETIVO: Evaluar la eficacia de la sonoterapia en la diminución de la ansiedad y el dolor posoperatorio en adultos llevados a cirugía ambulatoria de ortopedia bajo anestesia regional. MÉTODO: estudio aleatorizado, doble ciego, controlado con placebo, en 80 pacientes adultos sometidos a cirugía ortopédica; 40 recibieron intervención con sonoterapia y 40 no la recibieron. Se midieron el dolor y la ansiedad en el preoperatorio, en el posoperatorio inmediato, al alta, y 24 y 48 horas después. Se midieron parámetros hemodinámicos en cuatro momentos. RESULTADOS: No se encontraron diferencias significativas entre ambos grupos en las escalas de dolor ni ansiedad. Se observó una reducción significativa de la presión arterial sistólica luego de la aplicación de la intervención y en recuperación. No se observaron diferencias en otras variables hemodinámicas entre los grupos, pero la reducción de algunas ocurre de forma más rápida en el grupo de intervención. CONCLUSIONES: En pacientes adultos llevados a cirugía ambulatoria de ortopedia bajo anestesia regional, la sonoterapia es una estrategia novedosa que reduce la presión arterial sistólica, y esto puede considerarse como un indicador de disminución de la ansiedad. En nuestro estudio no se pudo demostrar diferencia en términos de control del dolor ni de requerimiento de sedación o analgesia adicional.

Mutational analysis of BRCA1 and BRCA2 genes in women with familial breast cancer from different regions of Colombia.

PURPOSE: The main risk factor for familial breast cancer is the presence of mutations in BRCA1 and BRCA2 genes. The prevalence of mutations in these genes is heterogeneous and varies according to geographical origin of studied families. In Colombia mutations in these genes have been mainly studied on patients from Andean region. Bogotá and Medellin presented its own battery of mutations. This study aims to identify mutations in BRCA1-2 genes in women with familial breast cancer from different regions of Colombia. METHODS: One hundred four families with a history of breast cancer were sampled in different regions of Colombia, and the BRCA1 gene and exon 11 of the BRCA2 gene were sequenced. To predict the possible effects of sequence alterations found in protein function, different bioinformatics tools were used. RESULTS: A total of 33 variants were found; 18 in BRCA1 and 15 in BRCA2, of which 15 are unique variants of Colombia. In silico analysis established that alterations p.Thr790Ala, p.Arg959Lys and p.Glu1345Lys in the BRCA1 gene and variants p.Leu771Phe, p.Asn818Lys, p.Val859Ser*22 and p.Lys1032Ile in the BRCA2 gene are considered likely pathogenic. Both the mutations as the variants of unknown clinical significance, in their great majority, presented a specific region distribution and they were different from those reported in previous studies. CONCLUSIONS: In this study we report the BRCA1 and BRCA2 spectrum of mutations and their distribution by regions in Colombia. Our results may help to design a diagnostic test including recurrent mutations for screening high risk to breast cancer families in Colombia.

Septic Pulmonary Embolism Secondary to Klebsiella pneumoniae Epididymitis: Case Report and Literature Review.

BACKGROUND: Septic pulmonary embolism (SPE) is defined as the occurrence of septic thrombi in the pulmonary circulation. We report a case of SPE secondary to K. pneumoniae epididymitis. CASE PRESENTATION: A 74-year-old male with a history of diabetes mellitus experienced SPE secondary to epididymitis, with isolation of K. pneumoniae in blood and presence of lung nodules, with a chest computed tomography showing the halo and reversed halo signs. DISCUSSION: SPE is characterized by the presence of septic thrombi in the pulmonary circulation coming from an extrapulmonary infective focus. SPE secondary to K. pneumoniae epididymitis is an uncommon condition that is characterized by the presence of multiple bilateral nodules of peripheral distribution. CONCLUSION: SPE is an unusual complication of acute epididymitis. Suspicion of SPE should be considered in patients with a diagnosis of epididymitis, respiratory symptoms, and multiple nodules in chest imaging assessments.

Miocardiopatía de takotsubo desencadenada por la utilización o exposición a drogas de abuso, sustancias químicas o venenos de origen animal / Takotsubo cardiomyopathy following use or exposure to drugs of abuse, chemical substances or animal poison

Resumen Objetivo: Identificar drogas de abuso, sustancias químicas o venenos de origen animal asociados a cardiopatía de takotsubo. Método: Revisión estructurada en Pubmed/Medline, para la que se utilizaron los términos: Takotsubo cardiomyopathy, Tako-tsubo cardiomyopathy, stress cardiomyopathy, transient-leftventricular ballooning syndrome, ampulla cardiomyopathy, apical ballooning syndrome, OR broken heart syndrome; together with ''drugs abuse'', ''chemistry substance.'' ''iatrogenic'', ''drug-induced'' OR ''induced by''. Se seleccionaron artículos publicados entre el 01/01/1990 y 31/10/2015, en inglés, francés o español, con acceso a texto completo. Se incluyeron losartículos con reportes de drogas de abuso, sustancias químicas o venenos de origen animal, como posibles desencadenantes de la miocardiopatía. Adicionalmente, se incluyeron las referencias de los artículos seleccionados, valoradas como relevantes. Resultado: Se recuperaron 852 artículos diferentes y se seleccionaron 23, más 4 referencias relevantes, en las cuales se identificaron 38 casos de miocardiopatía de takotsubo, posiblemente asociados al uso o exposición a drogas de abuso, sustancias químicas o venenos de animales, especialmente, monóxido de carbono (n = 13; 34,2%), cocaína o crack (n = 5, 13,2%), abstinencia al alcohol (n = 5; 13,2%), venenos de origen animal (n = 5; 13,2%) y anfetaminas (n = 4; 10,5%). En total se identificaron 14 sustancias diferentes como posibles desencadenantes de la miocardiopatía en mención. Conclusión: La miocardiopatía de takotsubo podría ser desencadenada por el uso o la exposicióna 14 drogas de abuso, sustancias químicas o venenos de origen animal, las cuales, en la mayoría de los casos, generan sobreestimulación simpática. Por ello se recomienda considerar este tipo de situación en pacientes que presenten este tipo de miocardiopatía, especialmente en quienes no se identifique un desencadenante físico o emocional.

Abstract Motivation: To identify drugs of abuse, chemical substances or animal poisons associated to takotsubo cardiomyopathy. Methods: Structured review of Pubmed/Medline, where the following search terms were used: Takotsubo cardiomyopathy, Tako-tsubo cardiomyopathy, stress cardiomyopathy, transient-leftventricular ballooning syndrome, ampulla cardiomyopathy, apical ballooning syndrome, OR broken heart syndrome; together with ''drugs abuse'', ''chemistry substance.'' ''iatrogenic'', ''drug-induced'' OR ''induced by''. Articles in English, French or Spanish published between January 1st 1990 and October 31st 2015 and with access to the full text were selected. Articles reporting drugs of abuse, chemical substances or animal poisons as possible triggers of this cardiomyopathy were included. Additionally, references of those selected articles were included, valued as relevant. Results: 852 different articles were found, 23 were selected as well as 4 relevant references, where 38 takotsubo cardiomyopathy were identified, possibly associated to the use or exposure to drugs of abuse, chemical substances or animal poisons, especially carbon monoxide (n = 13; 34.2%), cocaine or crack (n = 5, 13.2%), alcohol withdrawal (n = 5; 13.2%), animal poison (n = 5; 13.2%) and amphetamines (n = 4; 10.5%). Overall 14 different substances were identified as possible triggers of this cardiomyopathy. Conclusion: Takotsubo cardiomyopathy could be triggered by the use or exposure to 14 drugs of abuse, chemical substances or animal poisons, which in most cases generate a sympathetic overstimulation. Thus it is recommended to consider this type of situation in patients presenting this kind of cardiomyopathy, especially in those where a physical or emotional trigger cannot be identified.

Drugs as Possible Triggers of Takotsubo Cardiomyopathy: A Comprehensive Literature Search - Update 2015.

BACKGROUND: In 2011 a list of 20 drugs linked to drug-induced Tako-Tsubo cardiomyopathy (TCM) was published. Thus, the objectives were both to identify cases of drug-induced TCM, and update the 2011 list of drugs as possible triggers of this cardiomyopathy. METHOD: As the 2011 report of drug-induced TCM, case reports of drug-induced TCM were identified by a comprehensive search in Medline/PubMed database. From December 2010 to March 2015 search terms were Takotsubo cardiomyopathy, Takotsubo cardiomyopathy, stress cardiomyopathy, transient-left-ventricular ballooning syndrome, ampulla cardiomyopathy, apical ballooning syndrome, OR broken heart syndrome; together with "iatrogenic", "drug-induced", OR "induced by". Publications limited to English, Spanish, and French, in humans, and with full texts were retrieved. Articles that recognized any drug as a possible trigger of TCM were selected. RESULTS: Overall, 405 different references were retrieved and 67 were selected (62 case reports and 5 case series) involving 78 patient cases with TCM possibly associated to drugs were reviewed. At total of 44 drugs were recognized as possible drug-induced TCM, mainly with sympathetic effect, and 37 (84.1%) were different to those 20 identified in the 2011 review; therefore, a list of 57 drugs associated to TCM was obtained. CONCLUSION: There are new case reports that linked drug-use with the development of TCM. The list of 57 drugs obtained is principally integrated by drugs that generate sympathetic overstimulation. Consequently, the recommendation "drug-induced TCM would be considered in patients with TCM, particularly those in which no clear emotional or stress trigger could be identified" is endorsed.