Fraping: A computational tool for detecting slight differences in fluorescence recovery after photobleaching (FRAP) data for actin polymerization analysis.

Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.

ROS produced by NOX promote the neurite growth in a PI3K/Akt independent manner.

Reactive oxygen species (ROS) function as signaling molecules in several physiologic and pathologic processes. In central nervous system, ROS are critical for differentiation, migration, polarization, and neurite growth. These actions are mediated by reversible oxidation of target proteins. On the other hand, PI3K/Akt signaling pathway is susceptible to be modulated by ROS and it has been implicated in neurite growth. In this study, we evaluated the participation of ROS in the neurite growth of cultured rat cerebellar granule neurons (CGN), as well as the possible regulation of the PI3K/Akt pathway by ROS during neurite outgrowth. For this purpose, CGN were treated with cellular or mitochondrial antioxidants, or an NOX inhibitor and neurite growth was evaluated. Moreover, to assess the participation Akt in this process, the p-Akt levels were measured in CGN treated with antioxidants or a NOX inhibitor. The effect of antioxidants on the neurite growth in the presence of a PI3K inhibitor was also measured. We found that cellular antioxidants and the NOX inhibitor decreased the neurite growth, but not the mitochondrial antioxidant. Interestingly, the antioxidants increased the p-Akt levels; however, the effect of antioxidants on neurite growth was no dependent on the Akt activity since the inhibitor of PI3K did not modify the antioxidant action on neurite growth. Our results show that the PI3K/Akt pathway participates in neurite growth and that ROS produced by NOX could function as signals in this process; however, this action is not mediated by a redox regulation of Akt activity.

Tibolone Improves Locomotor Function in a Rat Model of Spinal Cord Injury by Modulating Apoptosis and Autophagy.

Spinal cord injury (SCI) harms patients' health and social and economic well-being. Unfortunately, fully effective therapeutic strategies have yet to be developed to treat this disease, affecting millions worldwide. Apoptosis and autophagy are critical cell death signaling pathways after SCI that should be targeted for early therapeutic interventions to mitigate their adverse effects and promote functional recovery. Tibolone (TIB) is a selective tissue estrogen activity regulator (STEAR) with neuroprotective properties demonstrated in some experimental models. This study aimed to investigate the effect of TIB on apoptotic cell death and autophagy after SCI and verify whether TIB promotes motor function recovery. A moderate contusion SCI was produced at thoracic level 9 (T9) in male Sprague Dawley rats. Subsequently, animals received a daily dose of TIB orally and were sacrificed at 1, 3, 14 or 30 days post-injury. Tissue samples were collected for morphometric and immunofluorescence analysis to identify tissue damage and the percentage of neurons at the injury site. Autophagic (Beclin-1, LC3-I/LC3-II, p62) and apoptotic (Caspase 3) markers were also analyzed via Western blot. Finally, motor function was assessed using the BBB scale. TIB administration significantly increased the amount of preserved tissue (p < 0.05), improved the recovery of motor function (p < 0.001) and modulated the expression of autophagy markers in a time-dependent manner while consistently inhibiting apoptosis (p < 0.05). Therefore, TIB could be a therapeutic alternative for the recovery of motor function after SCI.

Corrigendum: Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons.

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Differential ROS-Mediated Phosphorylation of Drp1 in Mitochondrial Fragmentation Induced by Distinct Cell Death Conditions in Cerebellar Granule Neurons.

Reactive oxygen species (ROS) production has been associated with neuronal death. ROS are also involved in mitochondrial fission, which is mediated by Dynamin-related protein 1 (Drp1). The regulation of mitochondrial fragmentation mediated by Drp1 and its relationship to mitochondrial ROS (mtROS) in neuronal death have not been completely clarified. The aim of this study is to evaluate the role of mtROS in cell death and their involvement in the activation of Drp1 and mitochondrial fission in a model of cell death of cultured cerebellar granule neurons (CGN). Neuronal death of CGN induced by potassium deprivation (K5) and staurosporine (ST) triggers mitochondrial ROS production and mitochondrial fragmentation. K5 condition evoked an increase of Drp1 phosphorylation at Ser616, but ST treatment led to a decrease of Drp1 phosphorylation. Moreover, the death of CGN induced by both K5 and ST was markedly reduced in the presence of MitoTEMPO; however, mitochondrial morphology was not recovered. Here, we show that the mitochondria are the initial source of ROS involved in the neuronal death of CGN and that mitochondrial fragmentation is a common event in cell death; however, this process is not mediated by Drp1 phosphorylation at Ser616.

A mini-incision carpal tunnel release technique to prevent pillar pain: A technical note.

Pillar pain represents one of the most common complications of classic open carpal tunnel release (CTR). This complication causes a sense of discomfort worse than the compression syndrome itself. We, herein, introduce a new treatment method for CTR through a mini-incision, which allows subcutaneously cutting the transverse carpal ligament (TCL) and releasing the median nerve without neurovascular complications. This mini-incision approach can allow the direct visualization and preservation of the thenar motor branch in those rare cases where it has an aberrant origin. For the past 10 years, we have consecutively performed this technique in the surgical treatment of 318 patients with the diagnosis of primary CTS, without developing any neurovascular and tendon injuries as well as pillar pain.

An accessory abductor digiti minimi as possible risk factor of the median and ulnar nerves compression / Músculo abductor digiti minimi accesorio como posible factor de riesgo de la compresión de los nervios mediano y ulnar

SUMMARY: During routine dissection of a left upper limb of a 68-year-old male human cadaver, an unusual muscle was observed originating from the radius and flexor retinaculum, and continued in the hypothenar region with the muscle belly of the abductor digiti minimi. We checked that it was an accessory abductor digiti minimi (ADM). Its muscular belly was in close relation to the median and ulnar nerves. We review the literature regarding such muscle variations and discuss the potential for compression of the median and ulnar nerves. Although the accessory ADM is usually asymptomatic and only rarely results in nerve compression, it should be taken into account by surgeons when establishing a differential diagnosis in the compression neuropathies of the median and ulnar nerves. An ultrasound scanning can help establish the differential diagnosis.

RESUMEN: Durante la disección de rutina de un miembro superior izquierdo de un cadáver humano masculino de 68 años, se observó un músculo inusual que se originaba en el radio y el retináculo flexor del carpo, y continuuaba en la región hipotenar con el vientre muscular del abductor digiti minimi manus. Verificamos que se trataba del músculo abductor digiti minimi accessorius (ADMA). Su vientre muscular se encontraba en estrecha relación con los nervios mediano y ulnar. Revisamos la literatura sobre variaciones musculares y discutimos la potencial compresión de los nervios mediano y ulnar. Aunque el ADMA suele ser asintomático y rara vez produce compresión nerviosa, los cirujanos deben tenerlo en cuenta al establecer un diagnóstico diferencial en las neuropatías de compresión de los nervios mediano y ulnar. Una ecografía puede ayudar a establecer el diagnóstico diferencial.

Protein-conformational diseases in childhood: Naturally-occurring hIAPP amyloid-oligomers and early ß-cell damage in obesity and diabetes.

BACKGROUND AND AIMS: This is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when ß-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early ß-cell damage. MATERIALS AND METHODS: We performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis. RESULTS: We demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early ß-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk. CONCLUSIONS: RIAO level increases as the number of complications rises; RIAOs > 3.35 µg/ml is a predictor of changes in the current indicators of ß-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Caspase-3 Activation Correlates With the Initial Mitochondrial Membrane Depolarization in Neonatal Cerebellar Granule Neurons.

In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca2+]ER), changes in the cytoplasmic calcium concentration ([Ca2+]i), alteration of the mitochondrial membrane potential, and the ER stress in the activation of caspase-3 in neonatal cerebellar granule cells (CGN). The cells were loaded with Fura-2 to detect changes in the [Ca2+]i and with Mag-fluo-4 to measure variations in the [Ca2+]ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca2+]i, but only K5 immediately reduced the [Ca2+]ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca2+]i and they decreased both the [Ca2+]ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca2+]i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca2+ and this Ca2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca2+]ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

Evolution of the innate and adaptive immune response in women with acute Zika virus infection.

Zika virus (ZIKV) is a flavivirus that is closely related to other human pathogens, such as dengue virus (DENV)1. Primary transmission usually involves Aedes aegypti, which has expanded its distribution range considerably2, although rarer infection routes, including mother-to-fetus transmission, sexual contact and blood transfusion, have also been observed3-7. Primary ZIKV infection is usually asymptomatic or mild in adults, with quickly resolved blood viraemia, but ZIKV might persist for months in saliva, urine, semen, breast milk and the central nervous system8-12. During a recent ZIKV outbreak in South America, substantial numbers of neurological complications, such as Guillain-Barré syndrome, were reported13,14 together with cases of microcephaly and associated developmental problems in infants born to women infected with ZIKV during pregnancy15-20, highlighting the clinical importance of this infection. Analyses of the human immune response to ZIKV are lacking21-28, but the recent outbreak has provided an opportunity to assess ZIKV immunity using current immunological methods. Here, we comprehensively assess the acute innate and adaptive immune response to ZIKV infection in ten women who were recruited during early infection and followed through reconvalescence. We define a cascade of events that lead to immunological control of ZIKV, with previous exposure to DENV impacting some, but not all, mediators of antiviral immunity.

Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

Thyroid Function in Health Care Workers Exposed to Ionizing Radiation.

OBJECTIVES: To analyze possible alterations of thyroid function related to dosimetric values in health care workers exposed to ionizing radiation. MATERIALS AND METHODS: Forty-six health care workers exposed to ionizing radiation at a tertiary hospital previously exposed to ionizing radiation were included in the study. Age, sex, history of thyroid diseases, thyroid hormones, work post, service, dosimetric values of previous year, and 5 y period were considered. Alterations of thyroid function and other variables were analyzed by exact logistic regression univariate model. RESULTS: 7.1% workers showed an increased serum thyroid-stimulating hormone without free T3 or free T4 alteration. A significant relationship between workers with increased thyroid-stimulating hormone and dosimetric values of previous year (odds ratio 6.35, 95% confidence interval 1.20-98.1, p = 0.021) and previous 5 y period of radiation exposure (odds ratio 1.72, 95% confidence interval 1.12-3.34, p = 0.007) was obtained. CONCLUSION: An increased risk of subclinical hypothyroidism related to radiation doses was observed in this pilot study on a group of health care workers exposed to ionizing radiation.

Effect of the comprehensive smoke-free law on time trends in smoking behaviour in primary healthcare patients in Spain: a longitudinal observational study.

OBJECTIVE: This study aimed to analyse the impact of comprehensive smoke-free legislation (SFL) on the prevalence and incidence of adult smoking in primary healthcare (PHC) patients from three Spanish regions, overall and stratified by sex. DESIGN: Longitudinal observational study conducted between 2008 and 2013. SETTING: 66 PHC teams in Catalonia, Navarre and the Balearic Islands (Spain). PARTICIPANTS: Population over 15 years of age assigned to PHC teams. PRIMARY AND SECONDARY OUTCOMES MEASURES: Quarterly age-standardised prevalence of non-smoker, smoker and ex-smoker and incidence of new smoker, new ex-smoker and ex-smoker relapse rates were estimated with data retrieved from PHC electronic health records. Joinpoint analysis was used to analyse the trends of age-standardised prevalence and incidence rates. Trends were expressed as annual percentage change and average annual percent change. RESULTS: The overall standardised smoker prevalence rate showed a significant downward trend (higher in men than women) and the overall standardised ex-smoker prevalence rate showed a significant increased trend (higher in women than men) in the three regions. Standardised smoker and ex-smoker prevalence rates were higher for men than women in all regions. With regard to overall trends of incidence rates, new smokers decreased significantly in Catalonia and Navarre and similarly in men and women, new ex-smokers decreased significantly and more in men in Catalonia and the Balearic Islands, and ex-smoker relapse increased in Catalonia (particularly in women) and decreased in Navarre. CONCLUSIONS: Trends in smoking behaviour in PHC patients remain unchanged after the implementation of comprehensive SFL. The impact of the comprehensive SFL might have been lessened by the effect of the preceding partial SFL.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages.

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host-pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.

ROS as Regulators of Mitochondrial Dynamics in Neurons.

Mitochondrial dynamics is a complex process, which involves the fission and fusion of mitochondrial outer and inner membranes. These processes organize the mitochondrial size and morphology, as well as their localization throughout the cells. In the last two decades, it has become a spotlight due to their importance in the pathophysiological processes, particularly in neurological diseases. It is known that Drp1, mitofusin 1 and 2, and Opa1 constitute the core of proteins that coordinate this intricate and dynamic process. Likewise, changes in the levels of reactive oxygen species (ROS) lead to modifications in the expression and/or activity of the proteins implicated in the mitochondrial dynamics, suggesting an involvement of these molecules in the process. In this review, we discuss the role of ROS in the regulation of fusion/fission in the nervous system, as well as the involvement of mitochondrial dynamics proteins in neurodegenerative diseases.

Effect of comprehensive smoke-free legislation on asthma and coronary disease trends in Spanish primary care patients.

Background: To examine the impact of comprehensive smoke-free legislation (SFL) (Law 42/2010) on the incidence and prevalence of adult asthma and coronary disease in primary health care (PHC) patients from three Spanish regions, overall and stratified by sex. Methods: Longitudinal observational study conducted between 2007 and 2013 in the population over 15 years of age assigned to 66 PHC teams in Catalonia, Navarre and the Balearic Islands. Crude rates and age-standardized (truncated: asthma ≥ 16 years and coronary disease ≥ 35 years) incidence and prevalence rates using the direct method based on the European Standard Population were estimated based on data from PHC electronic health records. Joinpoint analysis was used to analyse the trends of age-standardized incidence and prevalence rates. Trends were expressed as annual percentage change and average annual percent change (AAPC). Results: The standardized asthma incidence rate showed a non-significant downward trend and the standardized prevalence rates rose significantly in the three regions. Standardized coronary disease incidence and prevalence rates were considerably higher for men than for women in all regions. The standardized coronary disease incidence rates in Catalonia (AAPC: -8.00%, 95% CI: -10.46; -5.47) and Navarre (AAPC: -3.66%, 95% CI: -4.95;-2.35) showed a significant downward trend from 2007 to 2013, overall and by sex. The standardized coronary disease prevalence trend rate increased significantly in the whole period in Catalonia and the Balearic Islands, although a non-significant downward trend was observed from 2010 in Catalonia. Conclusion: No changes in the trends of adult asthma and coronary disease in PHC Spanish patients were detected after the introduction of comprehensive SFL.