Acidic NAADP-releasable Ca compartments in the megakaryoblastic cell line MEG01

Background

A novel family of intracellular Ca²⁺-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca²⁺ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca²⁺ release from the acidic compartments.

Objectives

The present study is aimed to investigate NAADP-mediated Ca²⁺ release from intracellular stores in the megakaryoblastic cell line MEG01.

Methods

Changes in cytosolic and intraluminal free Ca²⁺ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR.

Results

Treatment of MEG01 cells with the H⁺/K⁺ ionophore nigericin or the V-type H⁺-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca²⁺ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca²⁺ from acidic lysosomal-like Ca²⁺ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca²⁺ efflux from the acidic lysosomal-like Ca²⁺ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca²⁺-induced Ca²⁺ release (CICR) from the complementary Ca²⁺ compartment.

Conclusion

Our results show for the first time NAADP-evoked Ca²⁺ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.     

Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background

Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available.

Aim

This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates.

Methods

A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test.

Results

Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days.

Conclusion

Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.     

Evaluación breve del estado cognitivo de la demencia en estadios avanzados: resultados preliminares de la validación española del Severe Mini-Mental State Examination

Introduction

The cognitive assessment of patients with advanced dementia needs proper screening instruments that allow obtain information about the cognitive state and resources that these individuals still have. The present work conducts a Spanish validation study of the Severe Mini Mental State Examination (SMMSE).

Material and methods

Forty-seven patients with advanced dementia (Mini-Cognitive Examination [MEC]<11) were evaluated with the Reisberg's Global Deterioration Scale, MEC, SMMSE and Severe Cognitive Impairment Profile scales.

Results

All test items were discriminative. The test showed high internal ( α = 0.88), test-retest (0.64 to 1.00, P < .01) and between observers reliabilities (0.69-1.00, p < 0.01), both for scores total and for each item separately. Construct validity was tested through correlations between the instrument and MEC scores (r = 0.59, P < 0.01). Further information on the construct validity was obtained by dividing the sample into groups that scored above or below 5 points in the MEC and recalculating their correlations with SMMSE. The correlation between the scores in the SMMSE and MEC was significant in the MEC 0-5 group (r = 0.55, P < .05), but not in the MEC>5 group. Additionally, differences in scores were found in the SMMSE, but not in the MEC, between the three GDS groups (5, 6 and 7) (H = 11.1, P < .05).

Conclusions

The SMMSE is an instrument for the assessment of advanced cognitive impairment which prevents the floor effect through an extension of lower measurement range relative to that of the MEC.From our results, this rapid screening tool and easy to administer, can be considered valid and reliable.     

La fuerza de prensión manual: ¿puede ser un factor pronóstico de mortalidad en cuidados paliativos?

Objective

To determine whether hand grip strength (HGS) is a prognostic factor for mortality in a palliative care unit (PCU), using two variables: A1: The HP on admission; A2: The progression of the HGS in the first 12 days of admission.

Material and methods

A prospective, observational and comparative study of patients with advanced cancer admitted consecutively over a 4 month period into a PCU. A series of 4 determinations of HGS were made using a JAMAR^® 5030J1 dynamometer. A total of 78 patients fulfilled the inclusion criteria, of which 61 (78.2%) agreed to take part.

Results

Objective A1: Of the 61 enrolled patients, the survivors (n = 25) differed by -1.8 (Standard Deviation (SD) 0.8) from the reference values for age and gender, and for those that died (n = 36) it was -1.9 (1.1) (P = .6). A survival analysis was performed with this sample. The sample was subdivided into those who were > -2 SD (n = 34) and those < -2 SD (n = 27) (P = .3). Those patients who managed 4 determinations (n = 49) were included in objective A2. At discharge there were 26 deaths and 23 alive. There were no statistically significant differences between the determinations. Only the comparison between the difference between the 4th and 1st determination in the two groups showed a significant result (P = .01).

Conclusions

The HGS measured at admission, as well as in the first 12 days, was not a prognostic factor for mortality in the sample studied.     

Cryotherapy of the liver: A histological review

Introduction

Cryotherapy has been largely used in the past for palliation of unresectable liver tumors, but high local recurrence rates and peculiar systemic complications have determined its progressive abandonment. This review analyzes the histological changes produced to provide the basis for the local recurrences.

Materials and methods

A detailed literature search was performed on studies focusing on liver cryotherapy. Included were only those that described the histological characteristics in detail.

Results

A total of 22 studies were found, one clinical and the others in-vivo animal studies. Similar to other ablative techniques, cryotherapy produces a lesion which is composed by a central zone of coagulative necrosis surrounded with a transitional inflammatory zone. The lesion’s dimensions and morphology are influenced by numerous factors including the probe temperature, diameter, the duration of freezing time, fast cooling rate, slow thawing rate, the number of freezing cycles and the inflow occlusion (Pringle maneuver). The temporal evolution is consistent across studies and leads to a progressive inflammatory invasion of the necrosis with definitive fibrotic substitution.

Conclusions

Lesions obtained after cryotherapy seem similar and behave as those obtained after other techniques of liver ablation. However, controversial areas still exist and include the optimum number of freeze thaw cycles, the place of inflow occlusion, the potential corrupting effects of intra-lesional or proximal blood vessels on ablation morphology. The influence of these factors on the local recurrences are still not fully understood.     

Serum miR-146a and miR-223 as potential new biomarkers for sepsis

Objective

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

Methods

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay.

Results

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

Conclusion

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.)     

Disnea y psicopatología en el paciente anciano con enfermedad pulmonar obstructiva crónica

Chronic Obstructive Pulmonary Disease (COPD) is a progressive disease with dyspnoea perception as a main symptom. In severe stages, dyspnoea can constitute a risk factor for depression, anxiety and somatization disorders.

Objective

The objective was to evaluate the presence of these psychopathologies based on dyspnoea and severity stages in patients with COPD.

Materials and methods

Patients (n=51) were evaluated by means of the Hospital Anxiety and Depression Scale, the dyspnoea scale (MRC), the General Health Questionnaire (GHQ-28) and spirometric criteria.

Results

The increase in dyspnoea level and disease severity lead to a progressive worsening of anxiety, depressive and somatic symptoms with clinical relevance (P<0.05). There was a significant correlation between those parameters (P<0.05).

Conclusions

The early detection and treatment of these psychopathologies associated with dyspnoea and progression of the disease must be taken into account in this complex pathology.     

A computational tool for the simulation and optimization of microbial strains accounting integrated metabolic/regulatory information

Background and scope

Recently, a number of methods and tools have been proposed to allow the use of genome-scale metabolic models for the phenotype simulation and optimization of microbial strains, within the field of Metabolic Engineering (ME). One of the limitations of most of these algorithms and tools is the fact that only metabolic information is taken into account, disregarding knowledge on regulatory events.

Implementation and performances

This work proposes a novel software tool that implements methods for the phenotype simulation and optimization of microbial strains using integrated models, encompassing both metabolic and regulatory information. This tool is developed as a plug-in that runs over OptFlux, a computational platform that aims to be a reference tool for the ME community.

Availability

The plug-in is made available in the OptFlux web site (www.optflux.org) together with examples and documentation.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.     

Calidad de vida y sobrecarga de los cuidadores de pacientes con daño cerebral adquirido

Introduction

The health impact of acquired brain injury (ABI) is not only apparent in the patient, but also in the loss of health related quality of life (HRQol) of their carers. The objectives of this study were to measure the loss of HRQol as well as the burden of the carers of patients with ABI.

Material and methods

A retrospective study was conducted with 76 carers of patients with ABI. A questionnaire was used to collect information on the sociodemographic aspects, carer burden (Zarit Scale) and the HRQol (EuroQol Questionnaire) of the carers. A multiple linear regression model was constructed to analyse the effect of the different variables.

Results

The carers were predominantly women over 50 years, retired or dedicated to domestic tasks and who cared for their husband or one of their parents. One third showed a high risk of claudication. The mean HRQol obtained with the EuroQol went from a similar score to that of the general population (0.9) in the group without burden, to 0.67 in the group with risk of claudication. The regression models explained the burden better than the loss in quality of life.

Conclusions

Carers of patients with brain injury suffer a significant loss in HRQol compared to the general population. The deterioration arises from the mental dimensions and depends on the level of burden.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

High glucose stimulates macrophage SR-BI expression and induces a switch in its activity from cholesterol efflux to cholesterol influx

Aims

Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions.

Methods and results

ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5 mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI.

Conclusions

Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.     

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role

Background

The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors.

Scope of review

In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used.

Major conclusions

Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca²⁺.

General significance

Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.     

Síntomas depresivos en adultos mayores participantes de una universidad abierta a la tercera edad

Introduction

Although depressive disorders prevalence among older adults usually is less than in the younger population, the presence of significant symptoms of depression is common in elderly people. Studies show that taking part in social, educational and pleasure activities is associated with a reduction in depressive symptoms in this population. The purpose of this study was to examine the prevalence of depressive symptoms above the elderly participants of a Third Age Open University, taking the time taking part as a reference.

Material and methods

A cross-sectional design was implemented, with a participation of 95.2% (n = 140) of the total enrolled in the first trimester of 2009 in the activities of the Third Age Open University of the Schools of Arts, Sciences and Humanities of the University of São Paulo. They all answered a socio-demographic questionnaire and the Geriatric Depression Scale (GDS-15).

Results

The prevalence of depressive symptoms found was 3.57%. The statistical analysis showed a relationship between participation formore than one semester in the Third Age Open University and a lower index of depressive symptoms (p < .05).

Conclusions

Participating for longer than one academic semester is associated with less depressive symptoms, possibly being a protector factor against depression.     

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine

Background

There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients.

Methods

Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry.

Results

We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported.

Conclusions

Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.     

Interlaboratory comparison study of serum total testoserone measurements performed by mass spectrometry methods

Background

Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays.

Methods

Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis.

Results

The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range.

Conclusions

The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.     

Cancer thyroïdien de souche folliculaire avec persistance d’anticorps antithyroglobuline : signification pronostique et apport de la TEP/TDM au F-FDG

Aim

To investigate the rate of persistence or/and recurrence of follicular cell-derived thyroid cancer with elevated serum antithyroglobulin antibody (TgAb) one year after ablative therapy. We also evaluated the predictive factors of persistence or/and recurrence and the clinical impact of PET/CT with FDG.

Methods

This retrospective study involved 154 TgAb+ patients. PET/CT was performed in 22 patients with a pejorative anatomo-clinical type. The results of the PET/CT were compared with histology and clinical follow-up.

Results

In 23 cases, thyroglobulin (Tg) was positive (14% of TgAb+ Tg+) while in other 131 cases, stimulated Tg was negative (86% of TgAb+ Tg−). Twenty-nine patients (18%) were finally diagnosed with persistent or recurrent disease during follow-up. Independent predictive factors were: age higher than 60 years, Tg+, increase of TgAb concentration in the first year following surgery, and T3/T4/N1 status. PET/CT showed sensitivity of 66% and specificity of 90%. Positivity of PET/CT was significantly associated with a poor prognosis.

Discussion and conclusion

There is no significant association between persistence of TgAb and persistent/recurrent disease when stimulated Tg is negative. An increase of TgAb concentration in the first year of follow-up is an important prognostic indicator. PET/CT with FDG seems to be very useful in the therapeutic management of differentiated thyroid cancer, especially for the patients with pejorative anatomo-clinical types.