To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.

Patterns of Mycobacterium tuberculosis-complex excretion and characterization of super-shedders in naturally-infected wild boar and red deer

Wild boar (Sus scrofa) and red deer (Cervus elaphus) are the main maintenance hosts for bovine tuberculosis (bTB) in continental Europe. Understanding Mycobacterium tuberculosis complex (MTC) excretion routes is crucial to define strategies to control bTB in free-ranging populations, nevertheless available information is scarce. Aiming at filling this gap, four different MTC excretion routes (oronasal, bronchial-alveolar, fecal and urinary) were investigated by molecular methods in naturally infected hunter-harvested wild boar and red deer. In addition MTC concentrations were estimated by the Most Probable Number method. MTC DNA was amplified in all types of excretion routes. MTC DNA was amplified in at least one excretion route from 83.0% (CI95 70.8-90.8) of wild ungulates with bTB-like lesions. Oronasal or bronchial-alveolar shedding were detected with higher frequency than fecal shedding (p < 0.001). The majority of shedders yielded MTC concentrations <10(3) CFU/g or mL. However, from those ungulates from which oronasal, bronchial-alveolar and fecal samples were available, 28.2% of wild boar (CI95 16.6-43.8) and 35.7% of red deer (CI95 16.3-61.2) yielded MTC concentrations >10(3) CFU/g or mL (referred here as super-shedders). Red deer have a significantly higher risk of being super-shedders compared to wild boar (OR = 11.8, CI95 2.3-60.2). The existence of super-shedders among the naturally infected population of wild boar and red deer is thus reported here for the first time and MTC DNA concentrations greater than the minimum infective doses were estimated in excretion samples from both species.

Widespread environmental contamination with Mycobacterium tuberculosis complex revealed by a molecular detection protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique

BACKGROUND: The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. RESULTS: Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98 %) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit - Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7 %) cases, was the main cause of TBLN and 66.7 % of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n?=?19; 43.2 %). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. CONCLUSIONS: M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.

Transmission and progression to disease of Mycobacterium tuberculosis phylogenetic lineages in The Netherlands

The aim of this study was to determine if mycobacterial lineages affect infection risk, clustering, and disease progression among Mycobacterium tuberculosis cases in The Netherlands. Multivariate negative binomial regression models adjusted for patient-related factors and stratified by patient ethnicity were used to determine the association between phylogenetic lineages and infectivity (mean number of positive contacts around each patient) and clustering (as defined by number of secondary cases within 2 years after diagnosis of an index case sharing the same fingerprint) indices. An estimate of progression to disease by each risk factor was calculated as a bootstrapped risk ratio of the clustering index by the infectivity index. Compared to the Euro-American reference, Mycobacterium africanum showed significantly lower infectivity and clustering indices in the foreign-born population, while Mycobacterium bovis showed significantly lower infectivity and clustering indices in the native population. Significantly lower infectivity was also observed for the East African Indian lineage in the foreign-born population. Smear positivity was a significant risk factor for increased infectivity and increased clustering. Estimates of progression to disease were significantly associated with age, sputum-smear status, and behavioral risk factors, such as alcohol and intravenous drug abuse, but not with phylogenetic lineages. In conclusion, we found evidence of a bacteriological factor influencing indicators of a strain's transmissibility, namely, a decreased ability to infect and a lower clustering index in ancient phylogenetic lineages compared to their modern counterparts. Confirmation of these findings via follow-up studies using tuberculin skin test conversion data should have important implications on M. tuberculosis control efforts.

Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.

Designation of natural monuments by the local administration: the example of Viana do Castelo municipality and its engagement with geoconservation (NW Portugal)

"First online: 11 April 2016"

Procesos de plasticidad sin��ptica en gyrus dentado hipocampal durante la administraci��n cr��nica y el s��ndrome de abstinencia a coca��na. Synaptic plasticity in hippocampal dentate gyrus during chronic administration and withdrawal from cocaine.

Los procesos neuronales adaptativos que se observan como consecuencia de la administraci��n cr��nica de drogas de abuso, son similares a los procesos pl��sticos que subyacen al aprendizaje y la memoria. Por otra parte, el hipocampo forma parte del circuito neuronal responsable de los cambios conductuales observados como consecuencia de la administraci��n cr��nica de diferentes drogas de abuso. De acuerdo con esto, resultados previos de nuestro laboratorio demostraron que la plasticidad sin��ptica en el hipocampo y las claves contextuales relacionadas con la administraci��n de la droga, son relevantes para el incremento de la plasticidad hipocampal por la administraci��n cr��nica de diazepam. Espec��ficamente en el gyrus dentado hipocampal se han descripto fen��menos pl��sticos relacionados con la exposici��n cr��nica a psicof��rmacos, tales como facilitaci��n en la transmisi��n sin��ptica, disminuci��n de la proliferaci��n celular y el aumento del factor de transcripci��n ?Fos B. Debido a la correlaci��n existente entre los mecanismos de plasticidad neuronal, los aprendizaje asociativos y formaci��n de memorias y aquellos responsables de la adicci��n, el objetivo general de este trabajo es caracterizar los cambios inducidos por la exposici��n repetida de coca��na y durante el periodo de abstinencia, en la excitabilidad neuronal de las c��lulas del gyrus dentado hipocampal, los canales i��nicos afectados y los posibles mecanismos bioqu��micos involucrados en dichos cambios, que podr��an explicar las alteraciones conductuales observadas despu��s de dicho tratamiento. Con este prop��sito, se estudiar��: 1) la plasticidad sin��ptica (potenciaci��n a largo plazo, LTP y depotenciaci��n a largo plazo, LTD) en el gyrus dentado, mediante registros electrofisiol��gios multiunitarios; 2)la excitabilidad de las c��lulas granulares del gyrus dentado y la actividad de los canales i��nicos, utilizando la t��cnica de patch clamp; 3) las alteraciones en la neurotransmisi��n glutamatergica, midiendo los niveles del neurotransmisor <i>in vivo</i>, utilizando la t��cnica de microdi��lisis; el tr��fico de receptores glutamat��rgicos, utilizando la t��cnica de western-blott, 4) la participaci��n del ��xido n��trico en los cambios adaptativos observados como consecuencia de la sensibilizaci��n a coca��na. Adem��s, mediante la utilizaci��n de t��cnicas comportamentales (avoidance inhibitorio), se estudiar��n las posibles alteraciones de conductas que se sabe dependen de la integridad funcional del hipocampo.En relaci��n a los resultados del presente proyecto se espera obtener un incremento en la plasticidad sin��ptica, en la excitabilidad neuronal de las c��lulas granulares del gyrus dentado de la formaci��n hipoc��mpica, en la liberaci��n extracelular de glutamato <i>in vivo</i>, como as�� tambi��n en el tr��fico de receptores glutamat��rgicos. Adem��s se espera obtener un aumento de las v��as de se��alizaci��n activadas por la acci��n de glutamato, como la de ��xido n��trico/GMPc, como consecuencia de la administraci��n cr��nica de coca��na. Con este aumento global de la plasticidad sin��ptica hipocampal, las conductas dependientes de esta estructura debieran estar facilitadas, demostrando as�� una participaci��n activa del hipocampo en los procesos de sensibilizaci��n y posiblemente en la adicci��n a psicoestimulantes. La caracterizaci��n del impacto del desarrollo de sensibilizaci��n a coca��na en la excitabilidad neuronal en el hipocampo, sobre los sistemas de neurotransmisi��n y las v��as de se��alizaci��n involucradas contribuir��an a dilucidar los mecanismos que contribuyen al desarrollo de sensibilizaci��n a coca��na, los cuales podr��an representar potenciales blancos terap��uticos para el tratamiento de la adicci��n, considerando principalmente aspectos espec��ficos de la actividad el��ctrica neuronal y la plasticidad sin��ptica asociada con las diferentes fases del ciclo de la adicci��n.

Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

AbstractBackground:One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes).Objective:This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats.Method:In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 ��g.kg-1.day-1, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage.Results:Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 �� 4 beat/min, 0.71 �� 0.02 mv vs. hypothyroid 145 �� 9 beat/min, 0.563 �� 0.015 mv; respectively).Conclusion:The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU&#8209;induced hypothyroid rats.

Fishery bulletin / U.S. Dept. of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service.

v.81 (1983)

Fishery bulletin / U.S. Dept. of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service.

v.91 (1993)

Fishery bulletin / U.S. Dept. of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service.

v.74 (1976)

Fishery bulletin / U.S. Dept. of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service.

v.71 (1973)

Fishery bulletin / U.S. Dept. of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service.

v.69 (1971)