Dietary intake from complementary feeding is associated with intestinal barrier function and environmental enteropathy in Brazilian children from the MAL-ED cohort study.

A child's diet contains nutrients and other substances that influence intestinal health. The present study aimed to evaluate the relations between complementary feeding, intestinal barrier function and environmental enteropathy (EE) in infants. Data from 233 children were obtained from the Brazilian site of the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project cohort study. Habitual dietary intake from complementary feeding was estimated using seven 24-h dietary recalls, from 9 to 15 months of age. Intestinal barrier function was assessed using the lactulose-mannitol test (L-M), and EE was determined as a composite measure using faecal biomarkers concentrations - α-1-antitrypsin, myeloperoxidase (MPO) and neopterin (NEO) at 15 months of age. The nutrient adequacies explored the associations between dietary intake and the intestinal biomarkers. Children showed adequate nutrient intakes (with the exception of fibre), impaired intestinal barrier function and intestinal inflammation. There was a negative correlation between energy adequacy and L-M (ρ = -0·19, P < 0·05) and between folate adequacy and NEO concentrations (ρ = -0·21, P < 0·01). In addition, there was a positive correlation between thiamine adequacy and MPO concentration (ρ = 0·22, P < 0·01) and between Ca adequacy and NEO concentration (ρ = 0·23; P < 0·01). Multiple linear regression models showed that energy intakes were inversely associated with intestinal barrier function (ß = -0·19, P = 0·02), and fibre intake was inversely associated with the EE scores (ß = -0·20, P = 0·04). Findings suggest that dietary intake from complementary feeding is associated with decreased intestinal barrier function and EE in children.

Infant feeding practices and determinant variables for early complementary feeding in the first 8 months of life: results from the Brazilian MAL-ED cohort site.

OBJECTIVE: The present study aimed to describe breast-feeding, complementary feeding and determining factors for early complementary feeding from birth to 8 months of age in a typical Brazilian low-income urban community. DESIGN: A birth cohort was conducted (n 233), with data collection twice weekly, allowing close observation of breast-feeding, complementary feeding introduction and description of the WHO core indicators on infant and young child feeding. Infant feeding practices were related to socio-economic status (SES), assessed by Water/sanitation, wealth measured by a set of eight Assets, Maternal education and monthly household Income (WAMI index). Two logistic regression models were constructed to evaluate risk factors associated with early complementary feeding. RESULTS: Based on twice weekly follow-up, 65 % of the children received exclusive breast-feeding in the first month of life and 5 % in the sixth month. Complementary feeding was offered in the first month: 29 % of the children received water, 15 % infant formulas, 13 % other milks and 9·4 % grain-derived foods. At 6 months, dietary diversity and minimum acceptable diet were both 47 % and these increased to 69 % at 8 months. No breast-feeding within the first hour of birth was a risk factor for the early introduction of water (adjusted OR=4·68; 95 % CI 1·33, 16·47) and low WAMI index a risk factor for the early introduction of other milks (adjusted OR=0·00; 95 % CI 0·00, 0·02). CONCLUSIONS: Data suggest local policies should promote: (i) early breast-feeding initiation; (ii) SES, considering maternal education, income and household conditions; (iii) timely introduction of complementary feeding; and (iv) dietary diversity.

Frequency of SNP -336A/G in the promoter region of CD209 in a population from northeastern Brazil.

Dendritic cells (DCs) mediate the initiation of the immune response against a variety of pathogens. The DC-SIGN receptor is encoded by the gene CD209 and is expressed on the surface of DCs. It binds to mannose-rich carbohydrates and enables the recognition of bacteria, fungi, parasites, and viruses. SNP -336A/G in the promoter region of CD209 influences the expression of the DC-SIGN receptor. Several studies have associated this SNP with an increased susceptibility to infectious diseases and the development of more severe forms of disease. Therefore, the aim of this study was to determine the prevalence of SNP -336A/G in a population from northeastern Brazil. We analyzed 181 individuals from the general population of Parnaíba, Piauí, Brazil, of which 37% were men and 63% were women. SNP -336A/G was detected by polymerase chain reaction and treatment with the restriction enzyme MscI and visualized by electrophoresis on an 8% polyacrylamide gel stained with silver nitrate. Of the individuals analyzed, 116 (64.1%) were homozygous AA, 57 (31.5%) were heterozygous (AG), and 8 (4.4%) were homozygous GG. The allele frequency of -336G was 20.2%. Genotype frequencies were in Hardy-Weinberg equilibrium. To the best of our knowledge, this is the first report to describe the frequency of the CD209 SNP -336A/G in a population in the State of Piauí. Further studies are needed to determine the relationship between this SNP and the vulnerability of this population to major infectious diseases.

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Prevalence of variants that confer risk for venous thromboembolism in an elderly population of northeastern Brazil.

Venous thromboembolism (VTE) is an important cause of morbidity and mortality stemming from cardiovascular disease. It is a multifactorial disease caused by a combination of acquired risk factors, of which advanced age is the most significant, and genetic factors, including the variants FV G1691A, FII G20210A, and MTHFR C677T. We estimated the prevalence of these genomic variants in an elderly population of northeastern Brazil. The study included 188 elderly persons (65-93 years), of which 68 (36.2%) were men and 120 (63.8%) were women. Variants were detected by polymerase chain reaction-restriction fragment length polymorphism analysis, and subsequent electrophoresis on an 8% polyacrylamide gel stained with silver nitrate. The study population was in Hardy-Weinberg equilibrium for the 3 loci. Of the individuals analyzed, none carried variants of FV or FII (0%), and 24.7% had the MTHFR C677T polymorphism: 59 subjects (31.4%) were heterozygous (CT) and 17 subjects (9%) were homozygous (TT). Based on the analysis of these particular genes, we conclude that the study population does not present an increased risk for the development of VTE. Faced with a growing aging population worldwide, similar studies in other countries will help in the prevention of VTE in older individuals.

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism.

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.

In the facultative sulphate/nitrate reducer Desulfovibrio desulfuricans ATCC 27774, the nine-haem cytochrome c is part of a membrane-bound redox complex mainly expressed in sulphate-grown cells.

The bacterium Desulfovibrio desulfuricans ATCC 27774 belongs to the group of sulphate reducers also capable of utilising nitrate as its terminal electron acceptor for anaerobic growth. One of the complex multihaem proteins found in nitrate- or sulphate-grown cells of Desulfovibrio desulfuricans ATCC 27774 is the nine-haem cytochrome c. The present work shows that the gene encoding for Desulfovibrio desulfuricans ATCC 27774 nine-haem cytochrome c is part of an operon formed by the gene cluster 9hcA-D. Besides 9hcA, the gene encoding for the nine-haem cytochrome c, genes 9hcB to D encode for a protein containing four [4Fe-4S](2+/1+) centres, for a dihaem transmembrane cytochrome b and for an unknown hydrophobic protein, respectively. The four proteins have a predicted topology that is in accordance with the formation of a membrane-bound redox complex. Furthermore, the transcriptional studies show that not only the expression of the 9HcA-D complex is dependent on the growth phase, but also is markedly increased in sulphate-grown cells.

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas.

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.

Expression of a Desulfovibrio tetraheme cytochrome c in Escherichia coli.

A tetraheme cytochrome c was successfully overexpressed for the first time in Escherichia coli. Desulfovibrio desulfuricans ATCC 27774 tetraheme cytochrome c(3) was expressed in aerobically grown Escherichia coli cotransformed with Escherichia coli ccm gene cluster (Arslan et al. (1998) Bioch. Biophys. Res. Commun. 251, 744-747). The analysis of the produced cytochrome showed that the signal peptide was correctly cleaved, the four heme groups were inserted and the electronic structure around the heme irons was conserved, i.e., the recombinant tetraheme cytochrome was identical to that isolated from the native source. Contradicting previous results which indicated that Escherichia coli was only capable of producing apocytochrome c(3) (Pollock et al. (1989) J. Gen. Microbiol. 135, 2319-2328), the present work proves unequivocally that the holoform can also be obtained.

Sequencing the gene encoding desulfovibrio desulfuricans ATCC 27774 nine-heme cytochrome c.

Contradicting early suggestions, the sequencing of the gene encoding the Desulfovibrio desulfuricans (ATCC 27774) nine-heme cytochrome c proves that this cytochrome is not the product of the degradation of the 16-heme containing cytochrome c [Coelho et al. (1996) Acta Cryst. D52, 1202-1208]. However, preliminary data indicate that the cytochrome gene is part of an operon similar to the DvH hmc operon, which contains the gene coding for the 16-heme cytochrome c [Rossi et al. (1993) J. Bacteriol. 175, 4699-4711]. Also, the amino acid sequence deduced from the DNA sequence shows four residues in the C-terminal not predicted in the amino acid sequence obtained by X-ray methods [Matias et al. (1999) Structure 7, 119-130].

Replacement of lysine 45 by uncharged residues modulates the redox-Bohr effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.