The combined effects of binder addition and different sintering methods on the mechanical properties of bovine hydroxyapatite.

Hydroxyapatite (HA) from bovine bones has been used as a biomaterial in dentistry due to its biocompatibility and bioactivity. However, dense HA bioceramics still present inadequate properties for applications that require high mechanical performance, such as infrastructure. Microstructural reinforcements and control of ceramic processing steps are methods to improve these shortcomings. The present study assessed the effects of polyvinyl butyral (PVB) addition in combination with two sintering methodologies (2-step and conventional), on the mechanical properties of polycrystalline bovine HA bioceramics. The samples were divided into four groups (with 15 samples per group): conventional sintering with binder (HBC) and without binder (HWC) and 2-step sintering with (HB2) and without binder (HW2). HA was extracted from bovine bones, turned into nanoparticles in a ball mill, and subjected to uniaxial and isostatic pressing into discs, according to ISO 6872 standards. All groups were characterized by x-ray diffractometry (XRD), differential thermal analysis (DTA) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and relative density. Besides, mechanical analyses (biaxial flexural strength (BFS) and modulus of elasticity) were also performed. The characterization results demonstrated that adding agglutinants or the sintering method did not affect HA's chemical and structural characteristics. Even so, the HWC group showed the highest mechanical values for BFS and modulus of elasticity being 109.0 (98.0; 117.0) MPa and 105.17 ± 14.65 GPa, respectively. The HA ceramics submitted to conventional sintering and without the addition of binders achieved better mechanical properties than the other groups. The impacts of each variable were discussed and correlated to the final microstructures and mechanical properties.

Mild-to-moderate COVID-19 impact on the cardiorespiratory fitness in young and middle-aged populations.

The goal of the present study was to compare pulmonary function test (PFT) and cardiopulmonary exercise test (CPET) performance in COVID-19 survivors with a control group (CG). This was a cross-sectional study. Patients diagnosed with COVID-19, without severe signs and symptoms, were evaluated one month after the infection. Healthy volunteers matched for sex and age constituted the control group. All volunteers underwent the following assessments: i) clinical evaluation, ii) PTF; and iii) CPET on a cycle ergometer. Metabolic variables were measured by the CareFusion Oxycon Mobile device. In addition, heart rate responses, peak systolic and diastolic blood pressure, and perceived exertion were recorded. Twenty-nine patients with COVID-19 and 18 healthy control subjects were evaluated. Surviving patients of COVID-19 had a mean age of 40 years and had higher body mass index and persistent symptoms compared to the CG (P<0.05), but patients with COVID-19 had more comorbidities, number of medications, and greater impairment of lung function (P<0.05). Regarding CPET, patients surviving COVID-19 had reduced peak workload, oxygen uptake (V̇O2), carbon dioxide output (V̇CO2), circulatory power (CP), and end-tidal pressure for carbon dioxide (PETCO2) (P<0.05). Additionally, survivors had depressed chronotropic and ventilatory responses, low peak oxygen saturation, and greater muscle fatigue (P<0.05) compared to CG. Despite not showing signs and symptoms of severe disease during infection, adult survivors had losses of lung function and cardiorespiratory capacity one month after recovery from COVID-19. In addition, cardiovascular, ventilatory, and lower limb fatigue responses were the main exercise limitations.

Reference equations of oxygen uptake for the step test in the obese population.

The aim of this study was to establish reference equations for the six-minute step test (6MST) based on demographic, anthropometric, body composition, and performance variables able to predict oxygen uptake (V̇O2) in obese individuals. Seventy-three obese adults (42±14 years old, body mass index >30 kg/m2) from both sexes were included. They underwent anamnesis, body composition evaluation, and the 6MST with expired gases registered simultaneously. Three equations were developed for the obese population (n=73; 59% female). The first equation was composed of the up-and-down step cycles (UDS), sex, and age as predictors; the second equation was composed of the UDS, age, and lean mass (LM). Both equations collectively explained 68.1% of the V̇O2 variance in the 6MST, while the third equation, composed of the UDS, age, and body mass, accounted for 67.7% of the V̇O2 variance. UDS, sex, age, LM, and body mass were important V̇O2 predictors of 6MST in these obese individuals. This study contributes to the dissemination of a simple, inexpensive, and fast evaluation method that can provide important indicators of cardiorespiratory fitness and guide strategies for rehabilitation.

Mild-to-moderate COVID-19 impact on the cardiorespiratory fitness in young and middle-aged populations

The goal of the present study was to compare pulmonary function test (PFT) and cardiopulmonary exercise test (CPET) performance in COVID-19 survivors with a control group (CG). This was a cross-sectional study. Patients diagnosed with COVID-19, without severe signs and symptoms, were evaluated one month after the infection. Healthy volunteers matched for sex and age constituted the control group. All volunteers underwent the following assessments: i) clinical evaluation, ii) PTF; and iii) CPET on a cycle ergometer. Metabolic variables were measured by the CareFusion Oxycon Mobile device. In addition, heart rate responses, peak systolic and diastolic blood pressure, and perceived exertion were recorded. Twenty-nine patients with COVID-19 and 18 healthy control subjects were evaluated. Surviving patients of COVID-19 had a mean age of 40 years and had higher body mass index and persistent symptoms compared to the CG (P<0.05), but patients with COVID-19 had more comorbidities, number of medications, and greater impairment of lung function (P<0.05). Regarding CPET, patients surviving COVID-19 had reduced peak workload, oxygen uptake (V̇O2), carbon dioxide output (V̇CO2), circulatory power (CP), and end-tidal pressure for carbon dioxide (PETCO2) (P<0.05). Additionally, survivors had depressed chronotropic and ventilatory responses, low peak oxygen saturation, and greater muscle fatigue (P<0.05) compared to CG. Despite not showing signs and symptoms of severe disease during infection, adult survivors had losses of lung function and cardiorespiratory capacity one month after recovery from COVID-19. In addition, cardiovascular, ventilatory, and lower limb fatigue responses were the main exercise limitations.

Reference equations of oxygen uptake for the step test in the obese population

The aim of this study was to establish reference equations for the six-minute step test (6MST) based on demographic, anthropometric, body composition, and performance variables able to predict oxygen uptake (V̇O2) in obese individuals. Seventy-three obese adults (42±14 years old, body mass index >30 kg/m2) from both sexes were included. They underwent anamnesis, body composition evaluation, and the 6MST with expired gases registered simultaneously. Three equations were developed for the obese population (n=73; 59% female). The first equation was composed of the up-and-down step cycles (UDS), sex, and age as predictors; the second equation was composed of the UDS, age, and lean mass (LM). Both equations collectively explained 68.1% of the V̇O2 variance in the 6MST, while the third equation, composed of the UDS, age, and body mass, accounted for 67.7% of the V̇O2 variance. UDS, sex, age, LM, and body mass were important V̇O2 predictors of 6MST in these obese individuals. This study contributes to the dissemination of a simple, inexpensive, and fast evaluation method that can provide important indicators of cardiorespiratory fitness and guide strategies for rehabilitation.

Reliability and validity of six-minute step test in patients with heart failure.

Exercise intolerance is the hallmark consequence of advanced chronic heart failure (HF). The six-minute step test (6MST) has been considered an option for the six-minute walk test because it is safe, inexpensive, and can be applied in small places. However, its reliability and concurrent validity has still not been investigated in participants with HF with reduced ejection fraction (HFrEF). Clinically stable HFrEF participants were included. Reliability and error measurement were calculated by comparing the first with the second 6MST result. Forty-eight hours after participants underwent the 6MST, they were invited to perform a cardiopulmonary exercise test (CPET) on a cycle ergometer. Concurrent validity was assessed by correlation between number of steps and peak oxygen uptake (V̇O2 peak) at CPET. Twenty-seven participants with HFrEF (60±8 years old and left ventricle ejection fraction of 41±6%) undertook a mean of 94±30 steps in the 6MST. Intra-rater reliability was excellent for 6MST (ICC=0.9), with mean error of 4.85 steps and superior and inferior limits of agreement of 30.6 and -20.9 steps, respectively. In addition, strong correlations between number of steps and CPET workload (r=0.76, P<0.01) and peak V̇O2 (r=0.71, P<0.01) were observed. From simple linear regression the following predictive equations were obtained with 6MST results: V̇O2 peak (mL/min) = 350.22 + (7.333 × number of steps), with R2=0.51, and peak workload (W) = 4.044 + (0.772 × number of steps), with R2=0.58. The 6MST was a reliable and valid tool to assess functional capacity in HFrEF participants and may moderately predict peak workload and oxygen uptake of a CPET.

Impact of chronic obstructive pulmonary disease on linear and nonlinear dynamics of heart rate variability in patients with heart failure

The objective of this study was to investigate the impact of chronic obstructive pulmonary disease (COPD)-heart failure (HF) coexistence on linear and nonlinear dynamics of heart rate variability (HRV). Forty-one patients (14 with COPD-HF and 27 HF) were enrolled and underwent pulmonary function and echocardiography evaluation to confirm the clinical diagnosis. Heart rate (HR) and R-R intervals (iRR) were collected during active postural maneuver (APM) [supine (10 min) to orthostasis (10 min)], respiratory sinus arrhythmia maneuver (RSA-M) (4 min), and analysis of frequency domain, time domain, and nonlinear HRV. We found expected autonomic response during orthostatic changes with reduction of mean iRR, root mean square of successive differences between heart beats (RMSSD), RR tri index, and high-frequency [HF (nu)] and an increased mean HR, low-frequency [LF (nu)], and LF/HF (nu) compared with supine only in HF patients (P<0.05). Patients with COPD-HF coexistence did not respond to postural change. In addition, in the orthostatic position, higher HF nu and lower LF nu and LF/HF (nu) were observed in COPD-HF compared with HF patients. HF patients showed an opposite response during RSA-M, with increased sympathetic modulation (LF nu) and reduced parasympathetic modulation (HF nu) (P<0.05) compared with COPD-HF patients. COPD-HF directly influenced cardiac autonomic modulation during active postural change and controlled breathing, demonstrating an autonomic imbalance during sympathetic and parasympathetic maneuvers compared with isolated HF.

Reliability and validity of six-minute step test in patients with heart failure

Exercise intolerance is the hallmark consequence of advanced chronic heart failure (HF). The six-minute step test (6MST) has been considered an option for the six-minute walk test because it is safe, inexpensive, and can be applied in small places. However, its reliability and concurrent validity has still not been investigated in participants with HF with reduced ejection fraction (HFrEF). Clinically stable HFrEF participants were included. Reliability and error measurement were calculated by comparing the first with the second 6MST result. Forty-eight hours after participants underwent the 6MST, they were invited to perform a cardiopulmonary exercise test (CPET) on a cycle ergometer. Concurrent validity was assessed by correlation between number of steps and peak oxygen uptake (V̇O2 peak) at CPET. Twenty-seven participants with HFrEF (60±8 years old and left ventricle ejection fraction of 41±6%) undertook a mean of 94±30 steps in the 6MST. Intra-rater reliability was excellent for 6MST (ICC=0.9), with mean error of 4.85 steps and superior and inferior limits of agreement of 30.6 and -20.9 steps, respectively. In addition, strong correlations between number of steps and CPET workload (r=0.76, P<0.01) and peak V̇O2 (r=0.71, P<0.01) were observed. From simple linear regression the following predictive equations were obtained with 6MST results: V̇O2 peak (mL/min) = 350.22 + (7.333 × number of steps), with R2=0.51, and peak workload (W) = 4.044 + (0.772 × number of steps), with R2=0.58. The 6MST was a reliable and valid tool to assess functional capacity in HFrEF participants and may moderately predict peak workload and oxygen uptake of a CPET.

Impact of chronic obstructive pulmonary disease on linear and nonlinear dynamics of heart rate variability in patients with heart failure.

The objective of this study was to investigate the impact of chronic obstructive pulmonary disease (COPD)-heart failure (HF) coexistence on linear and nonlinear dynamics of heart rate variability (HRV). Forty-one patients (14 with COPD-HF and 27 HF) were enrolled and underwent pulmonary function and echocardiography evaluation to confirm the clinical diagnosis. Heart rate (HR) and R-R intervals (iRR) were collected during active postural maneuver (APM) [supine (10 min) to orthostasis (10 min)], respiratory sinus arrhythmia maneuver (RSA-M) (4 min), and analysis of frequency domain, time domain, and nonlinear HRV. We found expected autonomic response during orthostatic changes with reduction of mean iRR, root mean square of successive differences between heart beats (RMSSD), RR tri index, and high-frequency [HF (nu)] and an increased mean HR, low-frequency [LF (nu)], and LF/HF (nu) compared with supine only in HF patients (P<0.05). Patients with COPD-HF coexistence did not respond to postural change. In addition, in the orthostatic position, higher HF nu and lower LF nu and LF/HF (nu) were observed in COPD-HF compared with HF patients. HF patients showed an opposite response during RSA-M, with increased sympathetic modulation (LF nu) and reduced parasympathetic modulation (HF nu) (P<0.05) compared with COPD-HF patients. COPD-HF directly influenced cardiac autonomic modulation during active postural change and controlled breathing, demonstrating an autonomic imbalance during sympathetic and parasympathetic maneuvers compared with isolated HF.

Cardiorespiratory and metabolic determinants during moderate and high resistance exercise intensities until exhaustion using dynamic leg press: comparison with critical load.

The objective of this study was to assess cardiovascular, respiratory, and metabolic responses during a commonly used dynamic leg press resistance exercise until exhaustion (TEx) at different intensities and compare with critical load (CL). This was a prospective, cross-sectional, controlled, and crossover study. Twelve healthy young men (23±2.5 years old) participated. The subjects carried out three bouts of resistance exercise in different percentages of 1 repetition maximum (60, 75, and 90% 1RM) until TEx. CL was obtained by means of hyperbolic model and linearization of the load-duration function. During all bout intensities, oxygen uptake (VO2), carbon dioxide production (VCO2), ventilation (VE), and respiratory exchange ratio (RER) were obtained. Variations (peak-rest=Δ) were corrected by TEx. In addition, systolic and diastolic blood pressure (SBP and DBP), blood lactate concentration [La-] and Borg scores were obtained at the peak and corrected to TEx. CL induced greater TEx as well as number of repetitions when compared to all intensities (P<0.001). During CL, Borg/TEx, ΔSBP/TEx, ΔDBP/TEx, and [La-] were significantly lower compared with 90% load (P<0.0001). In addition, VO2, VCO2, VE, and RER were higher during CL when compared to 90 or 75%. TEx was significantly correlated with VO2 on CL (r=0.73, P<0.05). These findings support the theory that CL constitutes the intensity that can be maintained for a very long time, provoking greater metabolic and ventilatory demand and lower cardiovascular and fatigue symptoms during resistance exercise.

Influence of expiratory positive airway pressure on cardiac autonomic modulation at rest and in submaximal exercise in COPD patients.

The aim of this study was to evaluate the effect of expiratory positive airway pressure (EPAP) on heart rate variability (HRV) indices at rest and during 6-min walk test (6MWT) in chronic obstructive pulmonary disease (COPD) patients. Fifteen moderate to severe COPD patients were randomized and evaluated with and without (Non-EPAP) a 5 cmH2O EPAP device. Respiratory rate (RR) was collected at rest (5 min), during the 6MWT (5 min), and at recovery (5 min). Indices of HRV were computed in the time domain, in the frequency domain, and nonlinear analysis. For EPAP and Non-EPAP during the 6MWT, we found an increased mean heart rate (HR) (P=0.001; P=0.001) while mean RR (P=0.001; P=0.015) and RR tri index decreased (P=0.006; P=0.028). Peripheral oxygen saturation (P=0.019) increased at rest only in the EPAP group. In EPAP, correlations were found between forced expiratory volume in 1 s (FEV1) and low frequency (LF) sympathetic tonus (P=0.05; r=-0.49), FEV1 and high frequency (HF) parasympathetic tonus at rest (P=0.05; r=0.49), lactate at rest and LF during the 6MWT (P=0.02; r=-0.57), and lactate at rest and HF during 6MWT (P=0.02; r=0.56). Through a linear regression model, we found that lactate at rest explained 27% of the alterations of LF during 6MWT. The use of 5 cmH2O EPAP improved autonomic cardiac modulation and its complexity at rest in COPD patients. Although it did not influence the performance of the 6MWT, the EPAP device caused alterations in resting lactate concentration with an effect on sympatho-vagal control during the test.

Cardiorespiratory and metabolic determinants during moderate and high resistance exercise intensities until exhaustion using dynamic leg press: comparison with critical load

The objective of this study was to assess cardiovascular, respiratory, and metabolic responses during a commonly used dynamic leg press resistance exercise until exhaustion (TEx) at different intensities and compare with critical load (CL). This was a prospective, cross-sectional, controlled, and crossover study. Twelve healthy young men (23±2.5 years old) participated. The subjects carried out three bouts of resistance exercise in different percentages of 1 repetition maximum (60, 75, and 90% 1RM) until TEx. CL was obtained by means of hyperbolic model and linearization of the load-duration function. During all bout intensities, oxygen uptake (VO2), carbon dioxide production (VCO2), ventilation (VE), and respiratory exchange ratio (RER) were obtained. Variations (peak-rest=Δ) were corrected by TEx. In addition, systolic and diastolic blood pressure (SBP and DBP), blood lactate concentration [La-] and Borg scores were obtained at the peak and corrected to TEx. CL induced greater TEx as well as number of repetitions when compared to all intensities (P<0.001). During CL, Borg/TEx, ΔSBP/TEx, ΔDBP/TEx, and [La-] were significantly lower compared with 90% load (P<0.0001). In addition, VO2, VCO2, VE, and RER were higher during CL when compared to 90 or 75%. TEx was significantly correlated with VO2 on CL (r=0.73, P<0.05). These findings support the theory that CL constitutes the intensity that can be maintained for a very long time, provoking greater metabolic and ventilatory demand and lower cardiovascular and fatigue symptoms during resistance exercise.

Influence of expiratory positive airway pressure on cardiac autonomic modulation at rest and in submaximal exercise in COPD patients

The aim of this study was to evaluate the effect of expiratory positive airway pressure (EPAP) on heart rate variability (HRV) indices at rest and during 6-min walk test (6MWT) in chronic obstructive pulmonary disease (COPD) patients. Fifteen moderate to severe COPD patients were randomized and evaluated with and without (Non-EPAP) a 5 cmH2O EPAP device. Respiratory rate (RR) was collected at rest (5 min), during the 6MWT (5 min), and at recovery (5 min). Indices of HRV were computed in the time domain, in the frequency domain, and nonlinear analysis. For EPAP and Non-EPAP during the 6MWT, we found an increased mean heart rate (HR) (P=0.001; P=0.001) while mean RR (P=0.001; P=0.015) and RR tri index decreased (P=0.006; P=0.028). Peripheral oxygen saturation (P=0.019) increased at rest only in the EPAP group. In EPAP, correlations were found between forced expiratory volume in 1 s (FEV1) and low frequency (LF) sympathetic tonus (P=0.05; r=-0.49), FEV1 and high frequency (HF) parasympathetic tonus at rest (P=0.05; r=0.49), lactate at rest and LF during the 6MWT (P=0.02; r=-0.57), and lactate at rest and HF during 6MWT (P=0.02; r=0.56). Through a linear regression model, we found that lactate at rest explained 27% of the alterations of LF during 6MWT. The use of 5 cmH2O EPAP improved autonomic cardiac modulation and its complexity at rest in COPD patients. Although it did not influence the performance of the 6MWT, the EPAP device caused alterations in resting lactate concentration with an effect on sympatho-vagal control during the test.

A protein chimera including PspA in fusion with PotD is protective against invasive pneumococcal infection and reduces nasopharyngeal colonization in mice.

Despite the success of the available polysaccharide-based vaccines against Streptococcus pneumoniae in preventing invasive diseases, this bacterium remains a major cause of death in many parts of the world. New vaccine strategies are needed in order to increase protection. Thus, the utilization of fusion proteins is being investigated as an alternative to the current formulations. In the present work, we demonstrate that a chimeric protein, composed of PspA and PotD in fusion is able to maintain the protective characteristics of both parental proteins, providing protection against systemic infection while reducing nasal colonization. The hybrid was not able to improve the response against invasive disease elicited by PspA alone, but the inclusion of PotD was able to reduce colonization, an effect never observed using subcutaneous immunization with PspA. The mechanisms underlying the protective efficacy of the rPspA-PotD hybrid protein were investigated, revealing the production of antibodies with an increased binding capacity to pneumococcal strains of diverse serotypes and genetic backgrounds, enhanced opsonophagocytosis, and secretion of IL-17 by splenocytes. These findings reinforce the use of chimeric proteins based on surface antigens as an effective strategy against pneumococcal infections.

Rational selection of broadly cross-reactive family 2 PspA molecules for inclusion in chimeric pneumococcal vaccines.

Pneumococcal surface protein A (PspA) is a widely studied pneumococcal protein, exposed at the surface of all strains. It is an important virulence factor, preventing complement deposition as well as inhibiting the lytic effects of lactoferrin over pneumococci. Several studies have investigated the use of PspA as a candidate in alternative pneumococcal vaccines, with great success. However, PspA presents sequence variability - there are six clades, grouped in three families - and PspAs within the same clade exhibit different levels of cross-reactivity. Therefore, the aim of this work was to select, from a panel of eight pneumococcal isolates expressing family 2 PspAs, the molecule with the broadest reactivity within this family. Antisera to these PspA fragments were initially screened by immunoblot against thirteen pneumococcal extracts; the three most cross-reactive antisera were tested for their ability to enhance the deposition of complement factor C3b on the bacterial surface and to promote their phagocytosis in vitro. PspA from strain P490 was the most effective, increasing phagocytosis of all but one pneumococcal isolate. Thus, this molecule was selected for inclusion in chimeric protein-based pneumococcal vaccines. In conclusion, the rational selection of cross-reactive molecules is an important step in the development of vaccines with broad coverage.

Systemic immunization with rPotD reduces Streptococcus pneumoniae nasopharyngeal colonization in mice.

Streptococcus pneumoniae (pneumococcus) is a human pathogen that can cause otitis media, pneumonia and, in severe cases, meningitis and bacteremia. The pneumococcus expresses PotD, a protein belonging to the polyamines transporter complex called PotABCD. PotD is a membrane-associated protein that binds polyamines and has been shown to be important for virulence. In this work we demonstrate that subcutaneous immunization with rPotD reduces the bacterial load in the nasal tissue of mice, following intranasal challenge with a type 6B pneumococcus. The protective effect correlated with the induction of high levels of antibodies in the immunized group; the antibodies were able to increase bacterial phagocytosis by mouse peritoneal cells. The cellular immune response was characterized by the production of gamma-interferon, IL-2 and IL-17 by splenocytes and nitric oxide by peritoneal cells of immunized mice, upon stimulation with rPotD. Taken together our results suggest that PotD is a promising candidate to be included in a protein based pneumococcal vaccine, able to induce phagocytic antibodies, a Th1 cellular immune response and production of IL-17, reducing nasopharyngeal colonization, the main event responsible for transmission of pneumococci in humans.

Effect of l-glutamic acid supplementation on performance and nitrogen balance of broilers fed low protein diets.

The aim of this study was to evaluate the effect of protein reduction and supplementation of l-glutamic acid in male broiler diets. A total of 648 chicks of the Cobb 500 strain were distributed in a completely randomized design with six treatments and six replications with eighteen birds per experimental unit. The study comprised pre-starter (1-7 days), starter (8-21 days), growth (22-35 days) and final (36-45 days) phases. The first treatment consisted of a control diet formulated according to the requirements of essential amino acids for each rearing phase. The second and third treatments had crude protein (CP) reduced by 1.8 and 3.6 percentage points (pp) in relation to the control diet respectively. In the fourth treatment, l-glutamic acid was added to provide the same glutamate level as the control diet, and in the last two treatments, the broilers were supplemented with 1 and 2 pp of glutamate above that of the control diet respectively. The reduction in CP decreased the performance of broilers and the supplementation of l-glutamic acid did not influence performance when supplied in the diets with excess of glutamate. The lowest excreted nitrogen values were observed in the control diet, and treatments 2 and 3, respectively, in comparison with treatments with the use of l-glutamic acid (5 and 6). Retention efficiency of nitrogen was better in the control diet and in the treatment with a reduction of 1.8 pp of CP. It was verified that the serum uric acid level decreased with the CP reduction. A reduction in CP levels of up to 21.3%, 18.8%, 18.32% and 17.57% is recommended in phases from 1 to 7, 8 to 21, 22 to 35 and at 36 to 42 days, respectively, with a level of glutamate at 5.32%, 4.73%, 4.57%, 4.38%, also in these phases.

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong P-L5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovis BCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response

Exigência nutricional de proteína bruta para codornas japonesas (Coturnix coturnix japonica) na fase de postura / Nutritional requirement of crude protein for japanese quail (Coturnix coturnix japonica) in the production phase

O objetivo do trabalho foi determinar o melhor nível de proteína bruta na alimentação de codornas japonesas em fase de produção. Foram utilizadas 300 codornas com 16 semanas de idade, alojadas em gaiolas, em um delineamento inteiramente casualizado, com cinco tratamentos, seis repetições e dez aves por unidade experimental. Os níveis avaliados foram 14, 17, 20, 23 e 26 por cento de proteína bruta, e as dietas foram formuladas para serem isoenergéticas. Foi verificado efeito quadrático para consumo de ração, peso de ovo, massa de ovo, ingestão de energia, eficiência energética por dúzia de ovo, coeficiente de digestibilidade de nitrogênio, retenção de nitrogênio, peso de gema, casca e albúmen. Efeito linear foi verificado para produção de ovos, ingestão de proteína, conversão alimentar por massa de ovos, eficiência energética por massa de ovo em kg, peso final e gravidade específica. Não houve efeito significativo para conversão alimentar por dúzia e concentração sérica de ácido úrico. Recomenda-se nível de 20 por cento de proteína bruta para codornas japonesas em fase de postura...

The aim of this study was to determine the best level of crude protein in the diet of Japanese quails in the production phase. A total of 300 quails at 16 weeks of age in cages, in a completely randomized design with five treatments and six replicates of ten birds per experimental unit was used. The levels evaluated were 14, 17, 20, 23 and 26 percent crude protein and diets were formulated to be isoenergetic. A quadratic effect was observed for feed intake, egg weight, egg mass, energy intake, energy efficiency per dozen eggs, nitrogen digestibility, shell percentage, nitrogen retention, weight of yolk, albumen and shell. A linear effect was observed for egg production, protein intake, feed conversion by egg mass, energy efficiency per egg mass in kg, final weight and yolk percentage and specific gravity. There was no significant effect on feed conversion per dozen, percentage of albumen, shell thickness and serum uric acid. The recommendation is of a 20 percent crude protein level for Japanese quail during the laying phase...