Rational design of a bacterial import system for new-to-nature molecules.

Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system in vivo and showcase its functionality by importing an impermeant non-canonical amino acid.

The use of Bayesian Belief Networks (BBNs) to probe deeper into railway safety management systems - Two studies from Great Britain and Italy.

The importance of Safety Management Systems (SMS) to the railway industry is underlined by the fact that all organisations operating on UK railways are required by law to have one. Analysing SMSs can provide a reliable systemic tool to identify hazards and weaknesses within complex systems like the railway, making it possible to significantly increase safety, reducing the odds of near misses and accidents. However, there is little empirical research evidence to determine the impact on safety of a structured SMS. The current paper describes two studies which use Bayesian Belief Networks (BBN) to conceptualise SMSs and their impact on front-line performance. The paper presents the usefulness of BBNs to compare complex systems and reconcile cultural differences within the railway industry, identifying factors that are deemed vital within Italy and Britain. The two studies allowed us to identify the most influential factors within a SMS and how they interact with each other, as well as the strength of the identified relationships. A BBN is particularly useful in estimating how changing some of the node states (e.g., by making safety leadership present) affected the other factors. The current study showed that safety leadership has an impact on the SMSs of the British and Italian railway industries.

Noncanonical DNA polymerization by aminoadenine-based siphoviruses.

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution.

Impact of Maternal Nutrition and Perinatal Factors on Breast Milk Composition after Premature Delivery.

(1) Background: Premature infants require mothers' milk fortification to meet nutrition needs, but breast milk composition may be variable, leading to the risk of inadequate nutrition. We aimed at determining the factors influencing mothers' milk macronutrients. (2) Methods: Milk samples were analyzed for the first five weeks after premature delivery by infrared spectroscopy. Mothers' nutritional intake data were obtained during standardized interviews with dieticians, and then analyzed with reference software. (3) Results: The composition of 367 milk samples from 81 mothers was (median (range) g/100 mL): carbohydrates 6.8 (4.4⁻7.3), lipids 3.4 (1.3⁻6.4), proteins 1.3 (0.1⁻3.1). There was a relationship between milk composition and mothers' carbohydrates intake only (r = 0.164; p < 0.01). Postnatal age was correlated with milk proteins (r = -0.505; p < 0.001) and carbohydrates (r = +0.202, p < 0.001). Multiple linear regression analyses showed (coefficient) a relationship between milk proteins r = 0.547 and postnatal age (-0.028), carbohydrate intake (+0.449), and the absence of maturation (-0.066); associations were also found among milk lipids r = 0.295, carbohydrate intake (+1.279), and smoking (-0.557). Finally, there was a relationship among the concentration of milk carbohydrates r = 0.266, postnatal age (+0.012), and smoking (-0.167). (4) Conclusions: The variability of mothers' milk composition is differentially associated for each macronutrient with maternal carbohydrate intake, antenatal steroids, smoking, and postnatal age. Improvement in milk composition could be achieved by the modification of these related factors.

Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

Low-penetrance alleles predisposing to sporadic colorectal cancers: a French case-controlled genetic association study.

BACKGROUND: Sporadic colorectal cancers (CRC) are multifactorial diseases resulting from the combined effects of numerous genetic, environmental and behavioral risk factors. Genetic association studies have suggested low-penetrance alleles of extremely varied genes to be involved in susceptibility to CRC in Caucasian populations. METHODS: Through a large genetic association study based on 1023 patients with sporadic CRC and 1121 controls, we tested a panel of these low-penetrance alleles to find out whether they could determine "genotypic profiles" at risk for CRC among individuals of the French population. We examined 52 polymorphisms of 35 genes - drawn from inflammation, xenobiotic detoxification, one-carbon, insulin signaling, and DNA repair pathways - for their possible contribution to colorectal carcinogenesis. The risk of cancer associated with these polymorphisms was assessed by calculation of odds ratios (OR) using multivariate analyses and logistic regression. RESULTS: Whereas all these polymorphisms had previously been found to be associated with CRC risk, especially in Caucasian populations, we were able to replicate the association for only five of them. Three SNPs were shown to increase CRC risk: PTGS1 c.639C>A (p.Gly213Gly), IL8 c.-352T>A, and MTHFR c.1286A>C (p.Ala429Glu). On the contrary, two other SNPs, PLA2G2A c.435+230C>T and PPARG c.1431C>T (p.His477His), were associated with a decrease in CRC risk. Further analyses highlighted genotypic combinations having a greater predisposing effect on CRC (OR 1.97, 95%CI 1.31-2.97, p = 0.0009) than the allelic variants that were examined separately. CONCLUSION: The identification of CRC-predisposing combinations, composed of alleles PTGS1 c.639A, PLA2G2A c.435+230C, PPARG c.1431C, IL8 c.-352A, and MTHFR c.1286C, highlights the importance of inflammatory processes in susceptibility to sporadic CRC, as well as a possible crosstalk between inflammation and one-carbon pathways.

Prognostic factors and long-term results of pulmonary metastasectomy for pediatric histologies.

OBJECTIVE: Pulmonary resection in metastatic pediatric solid tumors is an accepted method of treatment. The purpose of this study was to determine the clinical course, outcome and prognostic factors after surgery. METHODS: A retrospective analysis from 1985 to 2006 of 52 patients less than 17 years old at the time of tumor diagnosis and submitted to thoracotomy for pulmonary metastatic disease was performed. Three had nodules excised which proved to be benign at histology and were excluded from further analysis. There were 28 males and 21 females with median age of 13.2 years [2-36] at the time of metastasectomy. The primaries were osteosarcoma (25), Ewing's sarcoma (6), Wilms' tumors (4), hepatoblastoma (3) and miscellaneous (11). Pulmonary metastases were encountered either at the time of initial diagnosis (18%) or occurring within 1 month to 22 years. Disease free interval (DFI) was equal or less than 2 years in 31 (63%) patients and more than 2 years in 18 (37%). RESULTS: Patients had one (24), two (16), three (2), four or more (7) metastasectomies resulting in a total of 95 thoracotomies. Wedges (75%) were performed more frequently than anatomic resections (25%). The number of resected metastatic nodules ranged from 1 to 45, median 3. There were no perioperative deaths. There were six complications: pneumothoraces requiring chest tube drainage in two cases. Resection was incomplete in four cases. The mean drainage time and hospital length of stay were 2.7 and 5 days, respectively. Using the date of pulmonary resection as the starting point, 5-year survival rate was 25%. By univariate analysis, we found that a significantly longer survival was observed for patients with a complete resection (p=0.004), with two or less metastases (p=0.0004), with unilateral metastases (p=0.001) or when the DFI was more than 2 years (p=0.003). By multivariate analysis, we showed that the number of pulmonary metastases was an independent predictor of survival. CONCLUSION: We conclude that resection of pulmonary metastases of pediatric solid tumors is a safe and effective treatment that offers improved survival benefit in carefully selected patients within a multidisciplinary approach for pediatric cancer. Prognosis related criteria that support patient selection for surgery are identified.

Growth and bone mineralization in preterm infants fed preterm formula or standard term formula after discharge.

OBJECTIVE: To evaluate growth and bone mineralization in very low birth weight (VLBW) infants fed preterm formula (PF) or term formula (TF). STUDY DESIGN: In a double-blind prospective study, 49 preterm infants of gestational age 33 weeks or less were randomly fed PF or TF for 2 months after discharge, then all of the infants were fed TF for the next 2 months. Anthropometric and dual-energy x-ray absorptiometry data were collected at discharge and at 2 months and 4 months after discharge. Anthropometric data also were collected at 12 months postterm. RESULTS: Four months after discharge, both body weight (6139 +/- 1254 g vs 5540 +/- 863 g; P = .03) and bone mineral content (104.4 +/- 29.2 g vs 87.5 +/- 17.1 g; P = .01) were significantly higher in the PF group compared with the TF group. At 12 months postterm, mean body weight, length, and head circumference remained higher in the PF group than in the TF group, and body mass index was similar and within the normal range in the 2 groups. CONCLUSIONS: At 4 months after discharge, growth and mineralization were better in the VLBW infants who were fed PF during the first 2 months after discharge compared with those who were fed TF, suggesting that PF may be particularly valuable at this early stage of development.

Partial splenectomy for portal hypertension in cystic fibrosis related liver disease.

AIMS: To review the middle- and long-term effects of partial splenectomy (PS) on portal hypertension (PHT) and its complications in patients with cystic fibrosis (CF) related liver disease risky PHT. METHOD: Over a 20 years period, 19 patients aged 7-23 years underwent partial PS for massive splenomegaly, hypersplenism, and/ or severe PHT. RESULTS: In all but three cases, PHT and hypersplenism have improved for long periods. Noticeable improvement of hepatic tests occurred simultaneously. In all patients PS resolved abdominal discomfort. Fifteen patients are alive and a stabilization of the liver disease occurred with a follow-up of 1-20 years (mean 7.9). One patient died following respiratory insufficiency 10 years after PS although PHT was stable. Manifestations recurred in 2 patients 5 and 6 years after PS. In two patients, the course of the disease evolved to hepatic insufficiency without recurrence of PHT 3 and 8 years after PS. PS did not give the expected results in three cases only, in which PHT was not modified or reoccurred during the following year. No severe complication was observed. Early (three patients) or late (one patient) eventration required surgical procedure. CONCLUSIONS: Our results show that PS is a reliable and well-tolerated technique. Therefore, it is a therapeutic option for the management of PHT in CF patients with a preserved liver function. It can prevent and significantly delay a liver transplantation and its constraints.

The thorough screening of the MUTYH gene in a large French cohort of sporadic colorectal cancers.

The MUTYH gene encodes a key glycosylase of the base-excision repair system that is involved in maintaining genomic DNA stability against oxidative damage. Biallelic germline MUTYH mutations have been proved to greatly predispose to non-familial adenomatous polyposis (FAP) and non-hereditary non-polyposis colorectal cancer (HNPCC) familial recessive forms of colorectal cancer with multiple adenomas. To date, there is still much debate over the impact of monoallelic germline MUTYH mutations on colorectal carcinogenesis. To evaluate their role in the susceptibility to sporadic colon and rectum cancers, we screened 1024 French sporadic colorectal cancer cases and 1121 French healthy controls for Caucasian MUTYH-associated polyposis mutations, including already known mutations p.Gly382Asp and p.Tyr165Cys, and new mutation p.Val479Phe. We observed a nonstatistically significant association between these MUTYH mutations at a heterozygous state and an increase in colorectal cancer risk (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.70-2.27). As a result, we conclude that heterozygous MUTYH mutations do not play a major role in sporadic colorectal carcinogenesis although a modest effect on this process cannot be ruled out.

Combinations of cytochrome P450 gene polymorphisms enhancing the risk for sporadic colorectal cancer related to red meat consumption.

Susceptibility to sporadic colorectal cancers (CRC) is generally thought to be the sum of complex interactions between environmental and genetic factors, all of which contribute independently, producing only a modest effect on the whole phenomenon. However, to date, most research has concealed the notion of interaction and merely focused on dissociate analyses of risk factors to highlight associations with CRC. By contrast, we have chosen a combinative approach here to explore the joint effects of several factors at a time. Through an association study based on 1,023 cases and 1,121 controls, we examined the influence on CRC risk of environmental factors coanalyzed with combinations of six single nucleotide polymorphisms located in cytochrome P450 genes (c.-163A>C and c.1548T>C in CYP1A2, g.-1293G>C and g.-1053C>T in CYP2E1, c.1294C>G in CYP1B1, and c.430C>T in CYP2C9). Whereas separate analyses of the SNPs showed no effect on CRC risk, three allelic variant combinations were found to be associated with a significant increase in CRC risk in interaction with an excessive red meat consumption, thereby exacerbating the intrinsic procarcinogenic effect of this dietary factor. One of these three predisposing combinations was also shown to interact positively with obesity. Provided that they are validated, our results suggest the need to develop robust combinative methods to improve genetic investigations into the susceptibility to CRC.

Adequacy and safety of an infant formula with a protein/energy ratio of 1.8 g/100 kcal and enhanced protein efficiency for term infants during the first 4 months of life.

OBJECTIVE: Excess protein in infant formula may lead to renal overload and play a role in later obesity. The objective of this controlled, prospective, randomized, double-blind study was to assess the suitability and safety of a modified protein content infant formula and its noninferiority as compared to a conventional formula. PATIENTS AND METHODS: Healthy term infants age < 7 days were either breast-fed or randomized to be fed exclusively with a conventional casein-predominant formula (protein/energy ratio: 2.6 g/100 kcal) or the isocaloric whey-predominant study formula (protein/energy ratio: 1.8 g/100 kcal) for 120 days. Primary outcome was daily weight gain between D0 and D120 (noninferiority criterion: difference in daily weight gain < or = 4 g). Secondary outcomes were daily gain in weight, length, head circumference and body mass index at monthly intervals. Tolerance and safety were assessed at each visit. RESULTS: 162 infants were enrolled, 84% of the formula-fed infants and 36% of the breast-fed infants completing the study. Mean daily weight gain from D0 to D120 in the formula-fed groups differed by 0.38 g/day [95% CI: -2.59; 1.83] signifying the noninferiority of the study formula. Secondary outcomes did not differ between the 2 groups at any time and were comparable to outcomes in the breast-fed group. Tolerance was good and adverse events were not different between study groups. CONCLUSIONS: The whey-predominant study infant formula with a protein/energy ratio of 1.8 g/100 kcal and enhanced protein efficiency is safe and not inferior to a conventional formula in ensuring normal growth during the first four months of life.

Selection and evolution of NTP-specific aptamers.

ATP occupies a central position in biology, for it is both an elementary building block of RNA and the most widely used cofactor in all living organisms. For this reason, it has been a recurrent target for in vitro molecular evolution techniques. The exploration of ATP-binding motifs constitutes both an important step in investigating the plausibility of the 'RNA world' hypothesis and a central starting point for the development of new enzymes. To date, only two RNA motifs that bind ATP have been characterized. The first one is targeted to the adenosine moiety, while the second one recognizes the 'Hoogsteen' face of the base. To isolate aptamers that bind ATP in different orientations, we selected RNAs on an affinity resin that presents ATP in three different orientations. We obtained five new motifs that were characterized and subsequently submitted to a secondary selection protocol designed to isolate aptamers specific for cordycepin. Interestingly, all the ATP-binding motifs selected specifically recognize the sugar-phosphate backbone region of the nucleotides. Three of the aptamers show some selectivity for adenine derivatives, while the remainder recognize any of the four nucleotides with similar efficiency. The characteristics of these aptamers are discussed along with implications for in vitro molecular evolution.

Acidified milk formula supplemented with bifidobacterium lactis: impact on infant diarrhea in residential care settings.

OBJECTIVES: Probiotics may be useful in preventing acute infectious diarrhea. Bifidobacteria are particularly attractive as probiotics agent because they constitute the predominant colonic flora of breastfed infants and are thought to play a role in the decreased incidence of diarrhea in breastfed infants. METHODS: This was a multicenter, double-blind, controlled study to evaluate the efficacy of a milk formula supplemented with viable Bifidobacterium lactis strain Bb 12 (BbF) in the prevention of acute diarrhea in infants younger than 8 months living in residential nurseries or foster care centers. RESULTS: Ninety healthy children received either the BbF or a conventional formula (CF) daily. The mean duration of the stay in the residential center was similar (137 v 148 days). At enrollment, there were no differences between the two groups with respect to age (3.7 +/- 2.1 months), gender, anthropometric data, history of allergy or gastrointestinal disease, frequency of breast-feeding in the neonatal period or timing of introduction of solid food. Altogether, 28.3% of the BbF infants had diarrhea during the study compared with 38.7% of controls (NS). There was a statistically insignificant trend for shorter episodes of diarrhea in the BbF group (5.1 +/- 3.3 days v 7 +/- 5.5 days, NS). The number of days with diarrhea was 1.15 +/- 2.5 in the BbF group with a daily probability of diarrhea of 0.84 versus 2.3 +/- 4.5 days and 1.55, respectively, in the CF group (P = 0.0002 and 0.0014). Feeding infants with the BbF reduced their risk of getting diarrhea by a factor of 1.9 (range, 1.33-2.6). Analysis of the cumulative incidence of diarrheal episodes showed a trend that the first onset of diarrhea occurred later in the BbF group. CONCLUSION: These results provide some evidence that viable Bifidobacterium lactis strain Bb 12, added to an acidified infant formula, has some protective effect against acute diarrhea in healthy children.