Development of principle culture medium formulations for Saccharomyces cerevisiae

Saccharomyces cerevisiae as well as other microorganisms are frequently used in industry with the purpose of obtain different kind of products that can be applied in several areas (research investigation, pharmaceutical compounds, etc.). In order to obtain high yields for the desired product, it is necessary to make an adequate medium supplementation during the growth of the microorganisms. The higher yields are typically reached by using complex media, however the exact formulation of these media is not known. Moreover, it is difficult to control the exact composition of complex media, leading to batch-to-batch variations. So, to overcome this problem, some industries choose to use defined media, with a defined and known chemical composition. However these kind of media, many times, do not reach the same high yields that are obtained by using complex media. In order to obtain similar yield with defined media the addition of many different compounds has to be tested experimentally. Therefore, the industries use a set of empirical methods with which it is tried to formulate defined media that can reach the same high yields as complex media. In this thesis, a defined medium for Saccharomyces cerevisiae was developed using a rational design approach. In this approach a given metabolic network of Saccharomyces cerevisiae is divided into a several unique and not further decomposable sub networks of metabolic reactions that work coherently in steady state, so called elementary flux modes. The EFMtool algorithm was used in order to calculate the EFM’s for two Saccharomyces cerevisiae metabolic networks (amino acids supplemented metabolic network; amino acids non-supplemented metabolic network). For the supplemented metabolic network 1352172 EFM’s were calculated and then divided into: 1306854 EFM’s producing biomass, and 18582 EFM’s exclusively producing CO2 (cellular respiration). For the non-supplemented network 635 EFM’s were calculated and then divided into: 215 EFM’s producing biomass; 420 EFM’s producing exclusively CO2. The EFM’s of each group were normalized by the respective glucose consumption value. After that, the EFMs’ of the supplemented network were grouped again into: 30 clusters for the 1306854 EFMs producing biomass and, 20 clusters for the 18582 EFM’s producing CO2. For the non-supplemented metabolic network the respective EFM’s of each metabolic function were grouped into 10 clusters. After the clustering step, the concentrations of the other medium compounds were calculated by considering a reasonable glucose amount and by accounting for the proportionality between the compounds concentrations and the glucose ratios. The approach adopted/developed in this thesis may allow a faster and more economical way for media development.

Nutritional assessment and lipid profile in HIV-infected children and adolescents treated with highly active antiretroviral therapy

INTRODUCTION: HIV-infected children and adolescents treated with highly active antiretroviral therapy (HAART) regimens that include a protease inhibitor (PI) can show significant improvements in clinical outcomes, nutritional status and quality of life. The study aimed to report nutritional and metabolic alterations for pediatric patients continuously exposed to HAART and for healthy controls for up to 1 year. METHODS: Clinical, anthropometric, lipid profile and food intake data were collected prospectively over approximately 12-months for each patient. RESULTS: Fifty-one individuals were studied, of these, 16 were healthy. After 12 months follow-up, HIV-positive individuals remained below the healthy control group parameters. No change was observed concerning food intake. Triglyceride serum levels were higher in patients using protease inhibitor at the onset of the study [PI groups: 114 (43 - 336), and 136 (63 - 271) versus control group: 54.5 (20 - 162); p = 0.003], but after twelve months follow-up, only the group using protease inhibitor for up to two months presented higher values [140 (73 - 273) versus 67.5 (33 - 117); p = 0.004]. HDL-cholesterol was lower in HIV-positive individuals [HIV-positive groups: 36 (27 - 58) and 36 (23 - 43); control 49.5 (34 - 69); p = 0.004]. CONCLUSIONS: HIV-infected children and adolescents treated with highly active antiretroviral therapy showed compromised nutritional parameters compared to a paired healthy control group. Individuals using protease inhibitor presented worse triglyceride serum levels compared to their healthy counterparts.

Anti-HBs levels among children and adolescents with complete immunization schedule against hepatitis B virus. A cross-sectional study in Blumenau, State of Santa Catarina, Brazil, 2007-2008

INTRODUCTION: Vaccination is the main tool for preventing hepatitis B virus (HBV) infection; however, following the completion of the vaccination series, the concentrations of anti-HBs can decline over the years and reach levels less than 10mIU/mL. The persistence of protection in these individuals is still unknown. The present study aimed to determine the anti-HBs antibody levels among children and adolescents who had received a complete vaccination course for hepatitis B. METHODS: Antibodies against HBV surface antigen (anti-HBs) were tested in 371 individuals aged 10 to 15 years-old. RESULTS: Volunteers who showed undetectable quantities of anti-HBs accounted for 10.2% of the population studied and 39.9% presented antibody titers of less than 10mIU/mL. Anti-HBs > 10mIU/mL were verified in 49.9%. CONCLUSIONS: These results corroborate other studies indicating levels of anti-HBs below 10mIU/mL in vaccinated individuals. Additional studies are required to assess whether this indicates susceptibility to HBV infection and the need and age for booster doses.

Hepatitis B marker seroprevalence and vaccination coverage in adolescents in the City of Itajaí, State of Santa Catarina, Southern Brazil, in 2008

INTRODUCTION: Hepatitis B infection constitutes an important cause of morbidity and mortality worldwide. In Brazil, however, the current epidemiological situation is not clear. Considering the importance of establishing this prevalence, the aim of this study was to determine the prevalence of HBV markers in voluntary adolescents, junior high (secondary school) students, in the City of Itajaí, State of Santa Catarina, Brazil. METHODS: A seroepidemiological, transverse study was conducted with 353 randomly chosen adolescents from elementary school in 2008. Blood samples were analyzed for HBsAg, anti-HBc and anti-HBs. All analyses were conducted by automated microparticle enzyme immunosorbent assay (Abbott®, AxSYM system, Deerfield, IL, USA), according to the manufacturer's instructions. RESULTS: The prevalence of HBsAg was 0.6% (CI 95% 0.1 - 2.0), that of anti-HBc was 1.1% (CI 95% 0.3 - 2.9) and that of detectable anti-HBs was 83.6% (CI 95% 79.3 - 87.3). Hepatitis B vaccination coverage was 97.5% (CI 95% 95.2 - 98.8). CONCLUSIONS: These results demonstrate the success of the vaccination program against hepatitis B in the region studied and indicate that prevention strategies must be maintained and, if possible, expanded to contribute to the establishment of positive prevalence rates in all age groups.

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.

Relationship between national culture, democracy and environmental performance: A panel data analysis

This paper demonstrates the significance of culture in examining the relationshipbetween democratic capital and environmental performance.The aim is to examine the relationship among scores on the Environmental Performance Index and the two dimensions of cross cultural variation suggested by Ronald Inglehart and Christian Welzel. Significantional interrelationships among democracy, cultural and environmental sustaintability measures could be found, following the regression results. Firstly, higher levels of democratic capital stock are associated with better environmental performance. Secondly importance to distinguish between cultural groups could be confirmed.

Hepatitis B virus infection in children, adolescents, and their relatives: genotype distribution and precore and core gene mutations

INTRODUCTION:The objectives of this study were evaluate hepatitis B virus (HBV) serological markers in children and adolescents followed up at the Child Institute of the Hospital das Clínicas, Faculdade de Medicina de São Paulo, Universidade de São Paulo; identify chronic HBV carriers and susceptible individuals in the intrafamilial environment; characterize HBV genotypes; and identify mutations in the patients and household contacts. METHODS: Ninety-five hepatitis B surface antigen-positive children aged <19 years and 118 household contacts were enrolled in this study. Commercial kits were used for the detection of serological markers, and PCR was used for genotyping. RESULTS: Hepatitis B e antigen (HBeAg) was detected in 66.3% (63/95) of cases. Three of the 30 HBeAg-negative and anti-HBeAg-positive patients presented with precore mutations and 11 presented with mutations in the basal core promoter (BCP). Genotype A was identified in 39 (43.8%) patients, genotype D in 45 (50.6%), and genotype C in 5 (5.6%). Of the 118 relatives, 40 were chronic HBV carriers, 52 presented with the anti-HBc marker, 19 were vaccinated, and 7 were susceptible. Among the relatives, genotypes A, D, and C were the most frequent. One parent presented with a precore mutation and 4 presented with BCP mutations. CONCLUSIONS: Genotypes A and D were the most frequent among children, adolescents, and their relatives. The high prevalence of HBV in the families showed the possibility of its intrafamilial transmission.

Contributions of culture and antimicrobial susceptibility tests to the retreatment of patients with pulmonary tuberculosis

Introduction This study evaluated the efficacy of retreatment of pulmonary tuberculosis (TB) with regard to treatment outcomes and antimicrobial susceptibility testing (ST) profiles. Methods This retrospective cohort study analyzed 144 patients treated at a referral hospital in Brazil. All of them had undergone prior treatment, were smear-positive for TB and received a standardized retreatment regimen. Fisher's 2-tailed exact test and the χ2 test were used; RRs and 95% CIs were calculated using univariate and multivariate binary logistic regression. Results The patients were cured in 84 (58.3%) cases. Failure was associated with relapsed treatment and abandonment (n=34). Culture tests were obtained for 103 (71.5%) cases; 70 (48.6%) had positive results. ST results were available for 67 (46.5%) cases; the prevalence of acquired resistance was 53.7%. There were no significant differences between those who achieved or not therapeutic success (p=0.988), despite being sensitive or resistant to 1 or more drugs. Rifampicin resistance was independently associated with therapeutic failure (OR: 4.4, 95% CI:1.12-17.37, p=0.034). For those cases in which cultures were unavailable, a 2nd model without this information was built. In this, return after abandonment was significantly associated with retreatment failure (OR: 3.59, 95% CI:1.17-11.06, p=0.026). Conclusions In this cohort, the general resistance profile appeared to have no influence on treatment outcome, except in cases of rifampicin resistance. The form of reentry was another independent predictor of failure. The use of bacterial culture identification and ST in TB management must be re-evaluated. The recommendations for different susceptibility profiles must also be improved.

Dengue infection in children and adolescents: Clinical profile in a reference hospital in northeast Brazil

Introduction This study aimed to describe the clinical spectrum of dengue in children and adolescents from a hyperendemic region who were admitted for hospitalization. Methods A retrospective study was conducted on patients diagnosed with dengue infection upon admission to a reference center in Fortaleza, Brazil. Results Of the 84 patients included, 42 underwent confirmatory testing. The main symptoms were fever, abdominal pain and vomiting. The median level of serum aspartate aminotransferase was 143.5±128mg/dL. Conclusions A peculiar clinical profile was evident among children and adolescents with dengue infection in a reference center in northeast Brazil, including gastrointestinal symptoms and liver involvement.

In vitro antimalarial activity of tigecycline against Plasmodium falciparum culture-adapted reference strains and clinical isolates from the Brazilian Amazon

Introduction: We evaluated the in vitro antimalarial activity of tigecycline as an alternative drug for the treatment of severe malaria. Methods: A chloroquine-sensitive Plasmodium falciparum reference strain, a chloroquine-resistant reference strain, and three clinical isolates were tested for in vitro susceptibility to tigecycline. A histidine-rich protein in vitro assay was used to evaluate antimalarial activity. Results: The geometric-mean 50% effective concentration (EC50%) of tigecycline was 535.5 nM (confidence interval (CI): 344.3-726.8). No significant correlation was found between the EC50% of tigecycline and that of any other tested antimalarial drug. Conclusions: Tigecycline may represent an alternative drug for the treatment of patients with severe malaria.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Urinary tract infection in full-term newborn infants: value of urine culture by bag specimen collection

OBJETIVE: to evaluate the efficacy of urine culture by bag specimen for the detection of neonatal urinary tract infection in full-term newborn infants. Retrospective study (1997) including full-term newborn infants having a positive urine culture (>100,000 CFU/ml) by bag specimen collection. The urinary tract infection diagnosis was confirmed by positive urine culture (suprapubic bladder aspiration method). The select cases were divided into three groups, according to newborn infant age at the bag specimen collection: GI (< 48 h, n = 17), GII (48 h to 7 d, n = 35) and GIII (> 7 d, n = 9). Sixty one full-term newborn infants were studied (5.1 % of total infants). The diagnosis was confirmed on 19/61 (31.1 %) of full-term infants born alive. Distribution among the groups was: GI = 2/17 (11.8 %), GII = 10//35 (28.6 %), and GIII = 7/9 (77.7 %). The most relevant clinical symptoms were: fever (GI - 100 %, GII - 91.4 %) and weight loss (GI - 35.3 %, GII - 45.7 %). Urine culture results for specimens collected by suprapubic aspiration were: E. coli GI (100 %), GII (40 %) and GIII (28.6 %), E. faecalis GI (30%), Staphylococcus coagulase-negative GII (20 %) and GIII (42.8 %), and Staphylococcus aureus GII (10 %). Correlation between positive urine culture collection (bag specimen method) and urinary tract infection diagnosis, using relative risk analysis, produced the following results: GI=0.30 (CI95% 0.08-1.15), GII=0.51 (CI 95% 0.25-1.06) and GIII=3.31 (CI95% 1.8-6.06) The most frequent urinary tract infection clinical signs in the first week were fever and weight loss, while non-specific symptomatology occurred later. E. coli was most frequent infectious agent, although from the 7th day of life, staphylococcus was noted. The urine culture (bag specimen method) was effective in detecting urinary tract infection only after the 7th day of life.

Analysis of human leukocyte antigens class II-DR in Brazilian children and adolescents with systemic lupus erythematosus

OBJECTIVE: To analyze the frequency of human leukocyte antigens class II-DR in children and adolescents with systemic lupus erythematosus. PATIENTS AND METHODS: Fifty-fiveBrazilian systemic lupus erythematosus children and adolescents and 308 healthy individuals were studied. Gender, race, and age of onset of systemic lupus erythematosus were recorded. The human leukocyte antigens typing of class II-DR was carried out by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP). Data were analyzed statistically using the chi square test with Yates' correction, Fisher's exact test, and Bonferroni's correction. RESULTS: Human leukocyte antigen-DR 15 was the most frequently detected antigen in this group of children and adolescents, and it also occurred more frequently in the female group, in children with onset of systemic lupus erythematosus between 0 and 9 years and between 10 to 14 years, and in the Black race group, but these associations were not statistically significants. CONCLUSION: In this group of children and adolescents with a high degree of racial admixture, we could not verify a significant association between human leukocyte antigens class II-DR and systemic lupus erythematosus.

Antiphospholipid antibodies in 57 children and adolescents with systemic lupus erythematosus

OBJECTIVE: To investigate the frequencies and behavior of antiphospholipid antibodies in 57 children and adolescents with systemic lupus erythematosus. METHODS: Anticardiolipin antibodies were investigated by ELISA and lupus anticoagulant antibodies by the international tests recommended. The antiphospholipid antibodies analyses were performed in frozen samples (mean of 5.3 samples per patient obtained during a mean follow-up period of 3 years and 7 months) and on blood samples collected between January 1997 and November 1998 (mean of 2.5 samples per patient during a 2-year follow-up period). RESULTS: The frequencies of antiphospholipid antibodies (anticardiolipin and lupus anticoagulant) were similar in the samples collected prospectively and in the frozen samples (retrospective study): 63.2% and 75.4% respectively. Positivity for these antibodies fluctuated during the follow-up period and was not associated with any clinical or laboratory parameters of lupus erythematosus, including autoantibodies and also including disease activity and/or severity scores. CONCLUSIONS: The frequencies of antiphospholipid antibodies in children and adolescents with lupus erythematosus were similar to those observed in adults. The positivity fluctuated during the follow-up and was not correlated with clinical and/or laboratory disease parameters.