Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Morphometric and ultrastructural analysis of different pituitary cell populations in undernourished monkeys

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20% (control group) and a 10% (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings.

Performance of a Brazilian population sample in the Boston Diagnostic Aphasia Examination: A pilot study

Brazilian researchers and health professionals often face the challenge of having to use tests developed in foreign languages and standardized for populations of other countries, especially in the fields of Neuropsychology and Neurolinguistics. This fact promotes a feeling that some scoring systems may be inadequate for our sociocultural reality. In the present study, we describe the performance of a Brazilian population sample submitted to a translated and adapted version of the Boston Diagnostic Aphasia Examination (BDAE). Sixty normal volunteers (21 men and 39 women), all Portuguese native speakers, ranging in age from 15 to 78 years (average 43.7) and with an educational level of 2 to 16 years (average 9.9), were tested using a translated and adapted Portuguese version of the BDAE. Cut-off scores are suggested for our population and the performance of the Brazilian sample is compared to that of American and Colombian samples, with the results being closely similar in all tasks. We also performed a correlation analysis between age, gender and educational level and the influence of these variables on the performance of the subjects. We found no statistically significant differences between genders. Educational level correlated positively with performance, especially in the subtests involving reading and writing. There was a negative correlation between age and performance in two subtests (Visual Confrontation Naming and Sentences to Dictation), but a coexisting effect of educational level could not be ruled out.

Prevalence of antibodies to human herpesvirus-8 in populations with and without risk for infection in São Paulo State

Human herpesvirus 8 (HHV-8) is a newly described herpesvirus that is etiologically associated with all forms of Kaposi's sarcoma (KS). Seroepidemiological studies have shown high prevalence rates of HHV-8 antibodies among men who have sex with men (MSM) and AIDS patients, African children, Brazilian Amerindians, and elderly individuals in certain regions of Europe. The aim of the present study was to determine the prevalence of HHV-8 antibodies in healthy children and young adults from different cities in São Paulo State, and in a population at high risk for HHV-8 infection: HIV-negative MSM, and AIDS patients with and without KS. Antibodies to HHV-8 latency-associated nuclear antigen and lytic-phase antigens were detected by immunofluorescence assays. In 643 healthy children and young adults from the general population attending a vaccination program for yellow fever in ten different cities in São Paulo State, the prevalence of HHV-8 antibodies detected by the presence of latent or lytic antigens ranged from 1.0 to 4.1% in the different age groups (mean = 2.5%). In the MSM group, the prevalence was 31/95 (32.6%). In the group of patients with AIDS, the prevalence was 39.2% (51/130) for non-KS patients and 98.7% (77/78) for AIDS patients with the diagnosis of KS confirmed by histopathological examination. We conclude that HHV-8 has a restricted circulation among healthy children and young adults in the general population of São Paulo State and a high prevalence among MSM and AIDS patients.

Screening of antibacterial extracts from plants native to the Brazilian Amazon Rain Forest and Atlantic Forest

More than 20% of the world's biodiversity is located in Brazilian forests and only a few plant extracts have been evaluated for potential antibacterial activity. In the present study, 705 organic and aqueous extracts of plants obtained from different Amazon Rain Forest and Atlantic Forest plants were screened for antibacterial activity at 100 µg/ml, using a microdilution broth assay against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli. One extract, VO581, was active against S. aureus (minimum inhibitory concentration (MIC) = 140 µg/ml and minimal bactericidal concentration (MBC) = 160 µg/ml, organic extract obtained from stems) and two extracts were active against E. faecalis, SM053 (MIC = 80 µg/ml and MBC = 90 µg/ml, organic extract obtained from aerial parts), and MY841 (MIC = 30 µg/ml and MBC = 50 µg/ml, organic extract obtained from stems). The most active fractions are being fractionated to identify their active substances. Higher concentrations of other extracts are currently being evaluated against the same microorganisms.

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

Prevalence of the A1555G (12S rRNA) and tRNA Ser(UCN) mitochondrial mutations in hearing-impaired Brazilian patients

Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNA Ser(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNA Ser(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNA Ser(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

Frequency of CCR5delta32 in Brazilian populations

A sample of 103 randomly chosen healthy individuals from Alegrete, RS, Brazil, was tested for the CCR5delta32 allele, which is known to influence susceptibility to HIV-1 infection. The CCR5delta32 allele was identified by PCR amplification using specific primers flanking the region of deletion, followed by electrophoresis on a 3% agarose gel. The data obtained were compared to those reported for other populations and interpreted in terms of Brazilian history. The individuals studied came from a highly admixed population. Most of them were identified as white (N = 59), while blacks and browns (mulattoes) were N = 13 and N = 31, respectively. The observed frequencies, considering the white, black and brown samples (6.8, 3.8, and 6.4%, respectively), suggest an important European parental contribution, even in populations identified as black and brown. However, in Brazil as a whole, this allele shows gradients indicating a relatively good correlation with the classification based on skin color and other physical traits, used here to define major Brazilian population groups.

Spatial and temporal variation in fish populations and assemblages in coastal waters of the northern Baltic Proper

The distribution and traits of fish are of interest both ecologically and socio-economically. In this thesis, phenotypic and structural variation in fish populations and assemblages was studied on multiple spatial and temporal scales in shallow coastal areas in the archipelago of the northern Baltic Proper. In Lumparn basin in Åland Islands, the fish assemblage displayed significant seasonal variation in depth zone distribution. The results indicate that investigating both spatial and temporal variation in small scale is crucial for understanding patterns in fish distribution and community structure in large scale. The local population of Eurasian perch Perca fluviatilis L displayed habitat-specific morphological and dietary variation. Perch in the pelagic zone were on average deeper in their body shape than the littoral ones and fed on fish and benthic invertebrates. The results differ from previous studies conducted in freshwater habitats, where the pelagic perch typically are streamlined in body shape and zooplanktivorous. Stable isotopes of carbon and nitrogen differed between perch with different stomach contents, suggesting differentiation of individual diet preferences. In the study areas Lumparn and Ivarskärsfjärden in Åland Islands and Galtfjärden in Swedish east coast, the development in fish assemblages during the 2000’s indicated a general shift towards higher abundances of small-bodied lower-order consumers, especially cyprinids. For European pikeperch Sander lucioperca L., recent declines in adult fish abundances and high mortalities (Z = 1.06–1.16) were observed, which suggests unsustainably high fishing pressure on pikeperch. Based on the results it can be hypothesized that fishing has reduced the abundances of large predatory fish, which together with bottom-up forcing by eutrophication has allowed the lower-order consumer species to increase in abundances. This thesis contributes to the scientific understanding of aquatic ecosystems with new descriptions on morphological and dietary adaptations in perch in brackish water, and on the seasonal variation in small-scale spatial fish distribution. The results also demonstrate anthropogenic effects on coastal fish communities and underline the urgency of further reducing nutrient inputs and regulating fisheries in the Baltic Sea region.

Scientist-friendly policies for non-native English-speaking authors: timely and welcome

That English is the lingua franca of today's science is an indisputable fact. Publication in English in international journals is a pre-requisite for a research paper to gain visibility in academia. However, English proficiency appears to be taken for granted in the scientific community, though this language can be a hurdle for a number of authors, particularly from non-native English-speaking countries. The influence of English proficiency on the publication output of Brazilian authors has never been assessed. We report our preliminary data on the relationship between the English proficiency of 51,223 researchers registered in the CNPq database and their publication output in international journals. We have found that publication rates are higher for those authors with good command of English, particularly written English. Although our research is still underway and our results are preliminary, they suggest that the correlation between written English proficiency and research productivity should not be underestimated. We also present the comments of some Brazilian scientists with high publication records on the relevance of communication skills to the scientific enterprise.

High prevalence of the GSTM3*A/B polymorphism in sub-Sarahan African populations

A 3-bp insertion/deletion polymorphism in intron 6 of GSTM3 (rs1799735, GSTM3*A/*B) affects the activity of the phase 2 xenobiotic metabolizing enzyme GSTM3 and has been associated with increased cancer risk. The GSTM3*B allele is rare or absent in Southeast Asians, occurs in 5-20% of Europeans but was detected in 80% of Bantu from South Africa. The wide genetic diversity among Africans led us to investigate whether the high frequency of GSTM3*B prevailed in other sub-Saharan African populations. In 168 healthy individuals from Angola, Mozambique and the São Tomé e Príncipe islands, the GSTM3*B allele was three times more frequent (0.74-0.78) than the GSTM3*A allele (0.22-0.26), with no significant differences in allele frequency across the three groups. We combined these data with previously published results to carry out a multidimensional scaling analysis, which provided a visualization of the worldwide population affinities based on the GSTM3 *A/*B polymorphism.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Differences in prostate cancer detection between Canadian and Saudi populations

Few studies have addressed racial differences in prostate cancer (PCa) detection between Western and Arabian countries, although PCa has a significantly lower prevalence in Arabic populations compared to Western populations. Therefore, an explanation of this difference is lacking. Serum prostate-specific antigen (PSA) is a valuable marker used to select patients who should undergo prostate biopsies, although the manner in which it is used may require adjustments based on the ethnic population in question. We investigated racial differences in the PCa detection rate between Canadian and Saudi populations. A retrospective analysis was performed of data collected prospectively over 5 consecutive years in urology clinics at the McGill University Health Center (MUHC) and King Saud University Hospital (KSUH). Men who had high (>4'ng/mL) or rising PSA levels and a negative digital rectal examination were eligible. A total of 1403 Canadian and 414 Saudi patients were evaluated for the study; 717 and 158 men, median age 64 and 68 years, were included in the MUHC and KSUH cohorts, respectively, P<0.0001). Median serum PSA, prostate volume, and PSA density values were 6.1'ng/mL, 47.3 g, and 0.12'ng·mL−1·g−1, respectively, for MUHC patients and 5.2'ng/mL, 64.5'g, and 0.08'ng·mL−1·g−1, respectively, for KSUH patients (P<0.0001, t-test followed by one-way ANOVA). In addition, the KSUH group had a significantly lower PCa detection rate among patients younger than 60 years of age and with PSA values <10'ng/mL.

Chronic kidney disease in disadvantaged populations

The increased burden of chronic kidney disease (CKD) in disadvantaged populations is due to both global factors and population-specific issues. Low socioeconomic status and poor access to care contribute to health care disparities and exacerbate the negative effects of genetic or biological predisposition. Provision of appropriate renal care to these populations requires a two-pronged approach: expanding the reach of dialysis through development of low-cost alternatives that can be practiced in remote locations, and implementation and evaluation of cost-effective prevention strategies. Kidney transplantation should be promoted by expansion of deceased donor transplant programs and use of inexpensive, generic immunosuppressive drugs. The message of World Kidney Day 2015 is that a concerted attack against the diseases that lead to end-stage renal disease, by increasing community outreach, better education, improved economic opportunity, and access to preventive medicine for those at highest risk, could end the unacceptable relationship between CKD and disadvantage in these communities.

Genetic diversity and phenotypic variability of phytoplankton populations in the Baltic Sea

En av naturens mest grundläggande aspekter är den enorma mängd av variation som existerar mellan arter. Denna variation har lett oss till att klassificera olika organismer på basis av morfologiska skillnader och på senare tid till att jämföra genetiska skillnader på individens nivå. Den marina kiselalgen Skeletonema marinoi är en av de vanligaste växtplanktonarter i Östersjön under vårblomningen och anses viktig för den årliga produktionen. En av mina främsta målsättningar var att beskriva den intra-specifika diversiteten hos denna art längs med miljögradienter i Östersjön. Ett annat mål var att klargöra de faktorer som eventuellt är involverade i konfigurationen av genetisk diversitet och differentiering. Med hjälp av genetiska markörer visade jag att den genetiska diversiteten hos S. marinoi populationer i Östersjön är lägre jämfört med populationer i östra delen av Nordsjön. Arten är genetiskt uppdelad så att en utpräglad population förekommer i Östersjön och en annan, genetiskt åtskild population förekommer norr om de Danska sunden. Resultaten visar att de genetiskt åtskilda populationerna är anpassade till lokala salinitetsförhållanden. Genflödet mellan populationerna korrelerade kraftigt med havströmmar i området. Mina studier avslöjade även omfattande variation av fenotypiska, ekologiskt vikitga särdrag hos olika kloner. Djurplankton som äter kiselalger kunde modifiera den klonala mångfalden av fenotypiskt variabla S. marinoi populationer. En ökad klonal mångfald ledde till högre prestationsförmåga i fråga om primär produktion och stabiliserade ekofysiologiska funktioner. Som visas i denna avhandling består en art allt som oftast av åtskilliga genetiska varianter med fenotypiska skillnader. Kunskap om sådana intra-specifika skillnader är en förutsättning för att vi skall kunna förstå var och varför arter förekommer. Denna kunskap utgör även en grund för prognoser som siktar på att förutspå huruvida arter kan anpassa sig till framtida miljöförhållanden. ------------------------------------------------------ Suunnaton määrä variaatioita eliölajien välillä on perustavanlaatuinen ominaisuus luonnossa. Perinteisesti tätä monimuotoisuutta on käytetty organismien luokittelemiseen eri lajeihin niiden morfologisten eroavaisuuksien perusteella. Hiljattain myös geneettisten erojen huomioimista yksilötasolla on hyödynnetty lajien luokittelemisessa. Merialueilla esiintyvä piilevä, Skeletonema marinoi on yksi Itämeren tavallisimmista kasviplanktonlajeista kevätkukinnan aikana. Tavoitteenani oli selventää geneettistä ja fenotyyppistä monimuotoisuutta pitkin Itämeren ympäristögradienttejä. Geneettisen monimuotoisuuteen ja erkaantumiseen vaikuttavien tekijöiden selvittäminen oli tärkeä aspekti väitöstutkimuksessani. Geneettisiä markkereita käyttämällä pystyin toteamaan, että S. marinoi levän geneettinen monimuotoisuus on Itämeressä merkittävästi alhaisempi kuin läheisessä Pohjanmeren itäosassa. Tutkittu laji jakautuu geneettisesti yhteen erilliseen populaatioon Itämeressä ja toiseen selvästi erottuvaan populaatioon Tanskan salmien pohjoispuolella. Kokeellisten tulosten perusteella nämä geneettisesti erilaistuneet populaatiot ovat kumpikin sopeutuneet paikalliseen veden suolapitoisuuteen. Populaatioiden välisen geenivirran ja merivirtojen luoman yhteyden välillä havaittiin vahva korrelaatio. Tutkimukseni paljastivat myös laajaa vaihtelua Skeletonema-kloonien ekologisesti tärkeissä ominaisuuksissa. Kokeellisten tutkimusteni perusteella laiduntajat pystyivät muuttamaan geneettisten kloonien lukumäärää monimuotoisissa S. marinoi populaatioissa. Lisääntynyt kloonien lukumäärä paransi perustuotantokykyä ja vakautti ekofysiologisia toimintoja. Kuten tässä väitöstutkimuksessa osoitetaan, lajit koostuvat useimmiten lukuisista geneettisistä muunnelmista, jotka eroavat usein fenotyypeiltään. Ymmärtääksemme missä tietyt lajit esiintyvät ja miksi, tarvitsemme tietoa lajien sisäisistä vaihteluista. Tämä tieto on tarpeellista, jotta voimme ennustaa lajien sopeutumista tuleviin ympäristönmuutoksiin.