Optimization of processing and storage of clinical samples to be used for the molecular diagnosis of pertussis

Coqueluche o pertussis es una infección respiratoria aguda, muy contagiosa y particularmente grave en menores de un año. En la enfermedad clásica, el diagnóstico clínico puede realizarse sin dificultad. En otros casos se requiere la confirmación por el laboratorio. Los métodos usados son el cultivo, la serología y la PCR. Para esta última, la muestra de elección es el aspirado nasofaríngeo y la metodología más sencilla de procesamiento utiliza proteinasa K. Aunque por lo general los resultados son satisfactorios, frecuentemente se presentan dificultades relacionadas con la naturaleza mucosa de los especímenes. Por otra parte, existen incertidumbres respecto de las condiciones óptimas de conservación de las muestras. En el presente estudio se evaluaron distintas técnicas de procesamiento y diversas condiciones de conservación de los materiales. Los resultados obtenidos permitieron seleccionar un método que optimiza el procesamiento de estas muestras, con un desempeño comparable al de los métodos comerciales y costos muy inferiores. Asimismo, las experiencias diseñadas para evaluar la conservación de las muestras nos permitieron elaborar una serie de recomendaciones, que pueden resultar muy valiosas cuando se debe guiar la derivación desde los lugares donde se atienden los pacientes hasta los centros donde se realizan las determinaciones basadas en metodologías moleculares.Pertussis or whooping cough is an acute, highly contagious respiratory infection, which is particularly severe in infants under one year old. In classic disease, clinical diagnosis may present no difficulties. In other cases, it requires laboratory confirmation. Generally used methods are: culture, serology and PCR. For the latter, the sample of choice is a nasopharyngeal aspirate, and the simplest method for processing these samples uses proteinase K. Although results are generally satisfactory, difficulties often arise regarding the mucosal nature of the specimens. Moreover, uncertainties exist regarding the optimal conditions for sample storage. This study evaluated various technologies for processing and storing samples. Results enabled us to select a method for optimizing sample processing, with performance comparable to commercial methods and far lower costs. The experiments designed to assess the conservation of samples enabled us to obtain valuable information to guide the referral of samples from patient care centres to laboratories where such samples are processed by molecular methods.Instituto de Biotecnologia y Biologia Molecula

Lactic acid fermentation of pomegranate juice as a tool to improve antioxidant activity

An increasing consumer demand for pomegranate has been globally observed, mainly thanks to the scientific evidence related to its functional and health-promoting features. Pomegranate fruits from twenty accessions identified in Southeastern Italy were characterized according to morphological and chemical features. Juices extracted from pomegranate fruits were fermented with selected Lactobacillus plantarum PU1 and the antioxidant activity investigated. Whey was added to juices to promote the microbial growth. Fermentation led to the increase of the radical scavenging activity (up to 40%) and significant inhibition of the linoleic acid peroxidation. The three fermented juices showing the highest antioxidant activity, and the corresponding unfermented controls, were further characterized. In detail, the cytotoxicity and the protective role toward artificially induced oxidative stress were determined on murine fibroblasts Balb 3T3 through the determination of the viability and the intracellular ROS (reactive oxygen species) scavenging activity (RSA). RSA reached values of ca. 70% in fermented juices, being ca. 40% higher than the unfermented and control samples. Phenols compounds of the pomegranate juices obtained from accessions “Bitonto Piscina,” “Sanrà nero,” and “Wonderful (reference cultivar) were analyzed through ultrahigh pressure liquid chromatography coupled with mass spectrometry, showing that a marked increase (up to 60%) of the ellagitannins derivatives occurred during fermentation. Sensory analysis showed suitability of the fermented juices to be used as beverage and food ingredient

Neurobiological Dysfunctional Substrates for the Self-Medication Hypothesis in Adult Individuals with Attention-Deficit Hyperactivity Disorder and Cocaine Use Disorder:A Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography Study

Objectives: Attention-deficit hyperactivity disorder (ADHD) in adulthood shows high co-occurrence rates with cocaine use disorder (CoUD). The self-medication hypothesis (SMH) provides a theoretical explanation for this comorbidity. This study investigates the neurobiological mechanisms that could support SMH in adult patients with attention-deficit hyperactivity disorder with cocaine use disorder (ADHD–CoUD).Materials and Methods: We included 19 ADHD–CoUD patients (84.2% male; age: 32.11 years [7.18]) and 16 CoUD patients (68.7% male; age: 36.63 years [8.12]). All subjects underwent a fluorine-18-fluorodeoxyglucose positron emission tomography (18F-FDG PET) brain scan. We tested brain metabolism differences between ADHD–CoUD and CoUD patients using voxel-based and regions of interest (ROIs)-based analyses. The correlation between dependence/abstinence duration and regional brain metabolism was also assessed in the two groups. Lastly, we investigated the integrity of brain metabolic connectivity of mesocorticolimbic and nigrostriatal dopaminergic systems, and large-scale brain networks involved in ADHD and addictions.Results: The voxel-wise and ROIs-based approaches showed that ADHD–CoUD patients had a lower metabolism in the thalamus and increased metabolism in the amygdala and parahippocampus, bilaterally, than CoUD subjects and healthy controls (HCs). Metabolism in the thalamus negatively correlated with years of dependence in ADHD–CoUD patients. Moreover, connectivity analyses revealed that ADHD–CoUD patients had a more preserved metabolic connectivity than CoUD patients in the dopaminergic networks and large-scale networks involved in self-regulation mechanisms of attention and behaviors (i.e., anterior default mode network [ADMN], executive network [ECN], and anterior salience network [aSAN]).Conclusions: We demonstrated distinct neuropathological substrates underlying substance-use behaviors in ADHD–CoUD and CoUD patients. Furthermore, we provided neurobiological evidence in support of SMH, demonstrating that ADHD–CoUD patients might experience short-term advantages of cocaine assumption (i.e., compensation of dopaminergic deficiency and related cognitive-behavioral deficits).</p

Neurobiological Dysfunctional Substrates for the Self-Medication Hypothesis in Adult Individuals with Attention-Deficit Hyperactivity Disorder and Cocaine Use Disorder:A Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography Study

Objectives: Attention-deficit hyperactivity disorder (ADHD) in adulthood shows high co-occurrence rates with cocaine use disorder (CoUD). The self-medication hypothesis (SMH) provides a theoretical explanation for this comorbidity. This study investigates the neurobiological mechanisms that could support SMH in adult patients with attention-deficit hyperactivity disorder with cocaine use disorder (ADHD–CoUD).Materials and Methods: We included 19 ADHD–CoUD patients (84.2% male; age: 32.11 years [7.18]) and 16 CoUD patients (68.7% male; age: 36.63 years [8.12]). All subjects underwent a fluorine-18-fluorodeoxyglucose positron emission tomography (18F-FDG PET) brain scan. We tested brain metabolism differences between ADHD–CoUD and CoUD patients using voxel-based and regions of interest (ROIs)-based analyses. The correlation between dependence/abstinence duration and regional brain metabolism was also assessed in the two groups. Lastly, we investigated the integrity of brain metabolic connectivity of mesocorticolimbic and nigrostriatal dopaminergic systems, and large-scale brain networks involved in ADHD and addictions.Results: The voxel-wise and ROIs-based approaches showed that ADHD–CoUD patients had a lower metabolism in the thalamus and increased metabolism in the amygdala and parahippocampus, bilaterally, than CoUD subjects and healthy controls (HCs). Metabolism in the thalamus negatively correlated with years of dependence in ADHD–CoUD patients. Moreover, connectivity analyses revealed that ADHD–CoUD patients had a more preserved metabolic connectivity than CoUD patients in the dopaminergic networks and large-scale networks involved in self-regulation mechanisms of attention and behaviors (i.e., anterior default mode network [ADMN], executive network [ECN], and anterior salience network [aSAN]).Conclusions: We demonstrated distinct neuropathological substrates underlying substance-use behaviors in ADHD–CoUD and CoUD patients. Furthermore, we provided neurobiological evidence in support of SMH, demonstrating that ADHD–CoUD patients might experience short-term advantages of cocaine assumption (i.e., compensation of dopaminergic deficiency and related cognitive-behavioral deficits).</p

Mice carrying a complete deletion of the talin2 coding sequence are viable and fertile

AbstractMice homozygous for several Tln2 gene targeted alleles are viable and fertile. Here we show that although the expression of talin2 protein is drastically reduced in muscle from these mice, other tissues continue to express talin2 albeit at reduced levels. We therefore generated a Tln2 allele lacking the entire coding sequence (Tln2cd). Tln2cd/cd mice were viable and fertile, and the genotypes of Tln2cd/+ intercrosses were at the expected Mendelian ratio. Tln2cd/cd mice showed no major difference in body mass or the weight of the major organs compared to wild-type, although they displayed a mildly dystrophic phenotype. Moreover, Tln2cd/cd mouse embryo fibroblasts showed no obvious defects in cell adhesion, migration or proliferation. However, the number of Tln2cd/cd pups surviving to adulthood was variable suggesting that such mice have an underlying defect

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML

Prevention of Asbestos Exposure in Latin America within a Global Public Health Perspective

Background: Asbestos consumption in Latin America (LA) amounts to 10% of yearly global production. Little is known about the impact of asbestos exposure in the region. Objective: To discuss scientific and socio-economic issues and conflicts of interest and to summarize epidemiological data of asbestos health effects in LA. Discussion: Recent data on chrysotile strengthened the evidence of its carcinogenicity and showed an excessive risk of lung cancer at cumulative exposure levels as low as 1.5 fibre-years/ml. Technology for substitution is available for all asbestos-containing products and ceasing asbestos production and manufacturing will not result in unemployment and loss of income, except for the mining industry. The flawed arguments used by the industry to maintain its market, both to the public and in courtrooms, strongly relies on the lack of local evidence of the ill effects and on the invisibility of asbestos-related diseases in LA, due to the limited number of studies and the exposed workers’ difficulty accessing health services. The few epidemiological studies available show clear evidence of clusters of mesothelioma in municipalities with a history of asbestos consumption and a forecasted rise in its incidence in Argentina and Brazil for the next decade. In Brazil, non-governmental organizations of asbestos workers were pivotal to counterbalance misinformation and inequities, ending recently in a Supreme Court decision backing an asbestos ban. In parallel, continuous efforts should be made to stimulate the growth of competent and ethical researchers to convey adequate information to the scientific community and to the general public

Melatonina revierte el daño oxidativo en glándula submandibular de ratas tratadas con Ciclofosfamida

OBJETIVO: Ciclofosfamida (Cf) produce daño oxidativo en glándula submandibular (GSM) de ratas. En el presente trabajo se evaluó el efecto protector antioxidante de melatonina (MLT) en GSM de ratas tratadas con Cf. METODO: Se utilizaron 40 ratas Wistar machos adultas divididas en 5 grupos (G): G1: control; G2: Control+Etanol: tratados con etanol al 1% durante 10 días consecutivos. Los días 11 y 12 recibieron una dosis de solución salina; G3: Cf: tratados con etanol al 1% durante 12 días, días 11 y 12 recibieron una dosis intraperitoneal (i.p.) de Cf de 50 mg/Kg de pc; G4: Cf + MLT: se administró diariamente MLT (5 mg/Kg pc, intraperitoneal, disuelta en etanol al 1%), días 11 y 12 recibieron Cf igual que G3; G5: MLT: tratamiento 12 días consecutivos con MLT (igual dosis de G4). Los animales fueron anestesiados, extirpándose ambas GSM y sacrificados, previo ayuno 24 hs. Se midió la concentración de ácido úrico (AU), peróxidos lipídicos (PL) y acuosos (PA) y actividad de superóxido dismutasa (SOD) en homogenato de GSM. Análisis estadístico: ANOVA y test de Bonferroni, considerando significativo p<0,05. RESULTADOS: El tratamiento con Cf disminuyó la concentración de AU y la actividad de SOD (AU, mg/mg prot., G1: 2,50±0,68; G2: 2,18±0,13; G3: 0,54±0,09* G4: 1,95±0,24#, G5: 2,64±0,47, *p< 0,01 G3 vs G1, G2, G4; #p< 0,01 G4 vs G3 y G5; SOD, U/mg prot., G1: 4,57±0.95, G2: 4,79±0,94, G3: 2,18±0,53*, G4: 5,13±1,10, G5: 5,09±0,39, *p< 0,01 G3 vs G1, G2, G4 y G5). El tratamiento con MLT previno esos efectos. Además, Cf aumentó la formación PL y PA. CONCLUSION: MLT mejoró el estado redox en GSM de ratas tratadas con Cf. MLT podría prevenir los procesos oxidativos en GSM producidos por Cf

Anxiety and depression in Charcot-Marie-Tooth disease: data from the Italian CMT national registry

Background There is little information about neuropsychiatric comorbidities in Charcot-Marie-Tooth disease (CMT). We assessed frequency of anxiety, depression, and general distress in CMT.Methods We administered online the Hospital Anxiety-Depression Scale (HADS) to CMT patients of the Italian registry and controls. HADS-A and HADS-D scores &gt;= 11 defined the presence of anxiety/depression and HADS total score (HADS-T) &gt;= 22 of general distress. We analysed correlation with disease severity and clinical characteristics, use of anxiolytics/antidepressants and analgesic/anti-inflammatory drugs.Results We collected data from 252 CMT patients (137 females) and 56 controls. CMT patient scores for anxiety (mean +/- standard deviation, 6.7 +/- 4.8), depression (4.5 +/- 4.0), and general distress (11.5 +/- 8.1) did not differ from controls and the Italian population. However, compared to controls, the percentages of subjects with depression (10% vs 2%) and general distress (14% vs 4%) were significantly higher in CMT patients. We found no association between HADS scores and disease duration or CMT type. Patients with general distress showed more severe disease and higher rate of positive sensory symptoms. Depressed patients also had more severe disease. Nineteen percent of CMT patients took antidepressants/anxiolytics (12% daily) and 70% analgesic/anti-inflammatory drugs. Patients with anxiety, depression, and distress reported higher consumption of anxiolytics/antidepressants. About 50% of patients with depression and/or general distress did not receive any specific pharmacological treatment.Conclusions An appreciable proportion of CMT patients shows general distress and depression. Both correlated with disease severity and consumption of antidepressants/anxiolytics, suggesting that the disease itself is contributing to general distress and depression

Frequency, entity and determinants of fatigue in Charcot-Marie-Tooth disease

Fatigue, a disabling symptom in many neuromuscular disorders, has been reported also in Charcot-Marie-Tooth disease (CMT). The presence of fatigue and its correlations in CMT was investigated