Single-nucleotide polymorphisms in one-carbon metabolism genes,Mediterranean diet and breast cancer risk: a case–control study in the Greek-Cypriot female population

Single-nucleotide polymorphisms (SNPs) within genes of the one-carbon metabolism pathway have been shown to interact with dietary folate intake to modify breast cancer (BC) risk. Our group has previously demonstrated that the Mediterranean dietary pattern, rich in beneficial one-carbon metabolism micronutrients, protects against BC in Greek-Cypriot women. We aimed to investigate whether SNPs in the MTHFR (rs1801133 and rs1801131) and MTR (rs1805087) genes modify the effect of the Mediterranean dietary pattern on BC risk. Dietary intake data were obtained using a 32-item food-frequency questionnaire. A dietary pattern specific to the Greek-Cypriot population, which closely resembles the Mediterranean diet, was derived using principal component analysis (PCA) and used as our dietary variable. Genotyping was performed on subjects from the MASTOS study, a case–control study of BC in Cyprus, using TaqMan assays. Adjusted odds ratios (ORs) were estimated using logistic regression analyses. High adherence to the PCA-derived Mediterranean dietary pattern further reduced BC risk with increasing number of variant MTHFR 677T alleles (OR_Q4vs.Q1 for 677TT = 0.37, 95 % CI 0.20–0.69, for 677 CT = 0.60, 95 % CI 0.42–0.86). Additionally, high adherence to the Mediterranean dietary pattern decreased BC risk in subjects with at least one MTR 2756A allele (OR_Q4vs.Q1 for 2756AA = 0.59, 95 % CI 0.43–0.81, for 2756AG = 0.59, 95 % CI 0.39–0.91) and in subjects with the MTHFR 1298CC genotype (OR_Q4vs.Q1 0.44, 95 % CI 0.30–0.65). Overall P-interaction values, however, were not statistically significant. Our study suggests that these MTHFR and MTR SNPs may act as effect modifiers, highlighting their biological significance in the association between Mediterranean diet, the one-carbon metabolism pathway and BC.

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Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Analysis of arsenic induced physiological and biochemical responses in a medicinal plant,Withania somnifera

Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India. However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources. In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 μM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes. Plants showed more As accumulation upon exposure to AsIII (320 μg g⁻¹ DW in roots and 161 μg g⁻¹ DW in leaves) than to AsV (173 μg g⁻¹ DW in roots and 100 μg g⁻¹ DW in leaves) after 10 days of treatment. Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations. Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1–5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols. In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.     

Maternal dietary intake of folate,vitamin B12 and MTHFR 677C>T genotype: their impact on newborn’s anthropometric parameters

In this study, we evaluated the effects of dietary intake of vitamin B₁₂ and folate during pregnancy and their interactions with maternal polymorphism of MTHFR (677C>T; 1298A>C) on intrauterine development. Anthropometric parameters were obtained from 231 newborns that belong to a prospective birth cohort in Morelos, Mexico. Maternal dietary intake of vitamin B₁₂ and folate was assessed using a semi-quantitative questionnaire administered during the first and third trimesters of the pregnancy. Maternal MTHFR 677C>T and 1298 A>C genotypes were determined by PCR–RFLP. The associations between deficient dietary intake of vitamin B₁₂ (<2.0 μg/d) and folate (<400 μg/d) in the first and third trimesters and maternal polymorphisms of MTHFR on anthropometric parameters at birth were estimated using a multivariate linear regression model. During pregnancy, the deficient dietary intake was roughly 60 % for folate and 19 % for vitamin B₁₂. Allelic frequencies of 677T and 1298C were 59 and 10 %, respectively. After adjusting for confounders, deficiency in maternal dietary intake of vitamin B₁₂ (<2.0 μg/d) was associated with a significant reduction in length (β ~ −2.4; 95 % CI −4.3; −0.6) and length-for-age at birth (β ~ −1.2; 95 % CI −2.3; −0.1) among infants whose mothers were carriers of the 677TT genotype (p for interaction = 0.02). In contrast, no association was observed between deficiency in maternal dietary intake of folate (<400 μg/d) and any anthropometric parameter of newborns. These results suggest that supplementation with vitamin B₁₂ during pregnancy could have a favorable impact on intrauterine fetal development mainly in populations that are genetically susceptible.     

Preliminary Insights into the Impact of Dietary Starch on the Ciliate,Neobalantidium coli,in Captive Chimpanzees

Infections caused by the intestinal ciliate Neobalantidium coli are asymptomatic in most hosts. In humans and captive African great apes clinical infections occasionally occur, manifested mainly by dysentery; however, factors responsible for development of clinical balantidiasis have not been fully clarified. We studied the effect of dietary starch on the intensities of infection by N. coli in two groups of captive chimpanzees. Adult chimpanzees infected by N. coli from the Hodonín Zoo and from the Brno Zoo, Czech Republic, were fed with a high starch diet (HSD) (average 14.7% of starch) for 14 days, followed by a five-day transition period and subsequently with a period of low starch diet (LoSD) (average 0.1% of starch) for another 14 days. We collected fecal samples during the last seven days of HSD and LoSD and fixed them in 10% formalin. We quantified trophozoites of N. coli using the FLOTAC method. The numbers of N. coli trophozoites were higher during the HSD (mean ± SD: 49.0±134.7) than during the LoSD (3.5±6.8). A generalized linear mixed-effects model revealed significantly lower numbers of the N. coli trophozoites in the feces during the LoSD period in comparison to the HSD period (treatment contrast LoSD vs. HSD: 2.7±0.06 (SE), z = 47.7; p<<0.001). We conclude that our data provide a first indication that starch-rich diet might be responsible for high intensities of infection of N. coli in captive individuals and might predispose them for clinically manifested balantidiasis. We discuss the potential nutritional modifications to host diets that can be implemented in part to control N. coli infections.     

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.     

Diversity of the Intestinal Bacteria of Cattle Fed on Diets with Different Doses of Gelatinized Starch-Urea

Gelatinized starch-urea (Starea, SU) is an effective and economical source of urea for ruminants. Here we assessed the influence of dietary supplementation with gelatinized starch-urea on the diversity of intestinal bacteria in finishing cattle. Fifty steers were randomly allotted to five treatments with diets supplemented with different doses of Starea [0 % (SU0), 8 % (SU8), 16 % (SU16), 24 % (SU24), and 32 % (SU32) of urea-N in total nitrogen]. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes was used to examine the effect of dietary supplementation of Starea on intestinal bacterial flora. Shannon–Weaver and Simpson diversity indices consistently showed the lowest bacterial diversity in the SU0 treatment. Increasing doses of Starea increased the diversity up to SU24 after which, diversity decreased. Cluster analysis of 16S rRNA gene DGGE profiles indicates that the intestinal bacterial communities associated with cattle that were not supplemented with Starea in feed differed in composition and structure from those supplemented with Starea. The amount of Starea supplemented in cattle diets influenced the abundance of several key species affiliated with Lachnospiraceae, Ruminococcaceae, Peptostreptococcaceae, Comamonadaceae and Moraxellaceae. These results suggest that Starea influences the composition and structure of intestinal bacteria which may play a role in promoting ruminant health and production performance.

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The PNPLA3 Ile148Met interacts with overweight and dietary intakes on fasting triglyceride levels

The Ile148Met (rs738409, G-allele) in the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) associates with liver fat content and may lead to loss-of-function (hydrolysis) or gain-of-function (CoA-dependent lysophosphatidic acid acyltransferase) defects. PNPLA3 is up-regulated by dietary carbohydrates, and interactions between rs738409 and carbohydrates, and sugar and ω6:ω3-polyunsaturated fatty acid (PUFA) ratio on hepatic fat accumulation have been reported. We examined interaction between rs738409 and overweight, and between rs738409 and dietary intakes (carbohydrates, sucrose and ω6:ω3-PUFA ratio), on fasting triglyceride levels. From the Malmo Diet and Cancer Study-Cardiovascular Cohort, 4,827 individuals without diabetes aged 58 ± 6 years, 2,346 with BMI ≤ 25 kg/m² and 2,478 with BMI > 25 kg/m², were included in cross-sectional analyses. Dietary data were collected by a modified diet history method. Overweight modified the association between rs738409 and fasting triglyceride levels (P_interaction = 0.003). G-allele associated with lower triglycerides only among overweight individuals (P = 0.01). Nominally, significant interaction on triglyceride levels was observed between rs738409 and sucrose among normal-weight individuals (P_interaction = 0.03). G-allele associated with lower triglycerides among overweight individuals in the lowest tertiles of carbohydrate and ω6:ω3-PUFA ratio (P = 0.04 and P = 0.001) and with higher triglycerides among normal-weight individuals in the highest tertile of sucrose (P = 0.001). We conclude that overweight and dietary sucrose may modify the association between rs738409 and fasting triglyceride levels.

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A randomized,double-blind,placebo-controlled,clinical trial on probiotic soy milk and soy milk: effects on epigenetics and oxidative stress in patients with type II diabetes

This clinical trial aimed to discover the effects of probiotic soy milk and soy milk on MLH1 and MSH2 promoter methylation, and oxidative stress among type II diabetic patients. Forty patients with type II diabetes mellitus aged 35–68 years were assigned to two groups in this randomized, double-blind, controlled clinical trial. Patients in the intervention group consumed 200 ml/day of probiotic soy milk containing Lactobacillus plantarum A7, while those in the control group consumed 200 ml/d of conventional soy milk for 8 weeks. Fasting blood samples, anthropometric measurements, and 24-h dietary recalls were collected at the baseline and at the end of the study, respectively. Probiotic soy milk significantly decreased promoter methylation in proximal and distal MLH1 promoter region (P < 0.01 and P < 0.0001, respectively) compared with the baseline values, while plasma concentration of 8-hydroxy-2′-deoxyguanosine (8-OHdG) decreased significantly compared with soy milk (P < 0.05). In addition, a significant increase in superoxide dismutase (SOD) activity was observed in probiotic soy milk group compared with baseline value (P < 0.01). There were no significant changes from baseline in the promoter methylation of MSH2 within either group (P > 0.05). The consumption of probiotic soy milk improved antioxidant status in type II diabetic patients and may decrease promoter methylation among these patients, indicating that probiotic soy milk is a promising agent for diabetes management.

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抗口臭中药复方的筛选

目的筛选有效、安全的抗口臭中药处方,为抗口臭护理用品提供研发实验依据。方法选用9味可食性中药用口臭主要相关菌种的敏感试验及硫化物检测仪（Hallimeter）检测含硫代谢产物。按单味中药及复合配方两步筛选,确定有效处方。结果9味中药敏感试验证实乌梅、丁香抑菌作用强于其它中药,菊花降低硫化物较强,特别在无抑菌浓度作用下仍有减低硫化物作用。结论乌梅、丁香、菊花配方有较强抗口臭作用,待进一步临床证实。     

Complexities of complex II: Sulfide metabolism in vivo

High levels of H₂S produced by gut microbiota can block oxygen utilization by inhibiting mitochondrial complex IV. Kumar et al. have shown how cells respond to this inhibition by using the mitochondrial sulfide oxidation pathway and reverse electron transport. The reverse activity of mitochondrial complex II (succinate-quinone oxidoreductase, i.e., fumarate reduction) generates oxidized coenzyme Q, which is then reduced by the mitochondrial sulfide quinone oxidoreductase to oxidize H₂S. This newly identified redox circuitry points to the importance of complex II reversal in mitochondria during periods of hypoxia and cellular stress.     

Induction and Resuscitation of Viable but Nonculturable Salmonella enterica Serovar Typhimurium DT104†

Salmonella enterica serovar Typhimurium DT104 11601was tested for its ability to maintain viability in minimal, chemically defined solutions. Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms (~235 days) at 5°C. Organisms maintained at a higher temperature (21°C) showed continuous, equivalent CFU per milliliter (~10⁶) up to 400 days after inoculation. Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity. Temperature upshift (56°C ± 0.5, 15 s) of the nonculturable organisms (5°C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation. Interestingly, S. enterica serovar Typhimurium DT104 from the microcosms at either 5°C (1 to 200 days) or 21°C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA. Furthermore, cells from 21°C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells. It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans. The fact that S. enterica serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that S. enterica serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions.     

Dietary polyunsaturated fatty acids and the Pro12Ala polymorphisms of PPARG regulate serum lipids through divergent pathways: a randomized crossover clinical trial

Human and animal studies suggest an interaction between the Pro12Ala polymorphism of PPARG and dietary fat. In this randomized crossover clinical trial, we investigated whether subjects with the Pro12Pro and Ala12Ala genotypes of PPARG respond differently to a diet supplemented with high saturated (SAFA) or polyunsaturated fatty acid (PUFA).We recruited non-diabetic men from a population-based METSIM study (including 10,197 men) to obtain men with the Ala12Ala and the Pro12Pro genotypes matched for age and body mass index. Seventeen men with the Pro12Pro genotype and 14 with the Ala12Ala genotype were randomized to both a PUFA diet and a SAFA diet for 8 weeks in a crossover setting. Serum lipids and adipose tissue mRNA expression were measured during the diet intervention. At baseline, subjects with the Ala12Ala genotype had higher levels of HDL cholesterol and lower levels of LDL cholesterol, total triglycerides, and apolipoprotein B compared to those subjects with the Pro12Pro genotype (P < 0.05, FDR < 0.1). The Ala12Ala genotype also associated with higher mRNA expression of PPARG2, LPIN1, and SREBP-1c compared to participants with the Pro12Pro genotype (FDR < 0.001). On the other hand, PUFA diet resulted in lower levels of fasting glucose, total cholesterol, total triglycerides, and apolipoprotein B (P < 0.05, FDR < 0.1) but did not affect PPARG2 mRNA expression in adipose tissue. We conclude that individuals with the Pro12Pro genotype, with higher triglyceride levels at baseline, are more likely to benefit from the PUFA diet. However, the beneficial effects of dietary PUFA and the Ala12Ala genotype of PPARG on serum lipids are mediated through divergent mechanisms.

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High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

Survival of Clinical and Poultry-Derived Isolates of Campylobacter jejuni at a Low Temperature (4°C)

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31°C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4°C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at −20°C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4°C) tolerance.     

Effect of high versus low doses of fat and vitamin A dietary supplementation on fatty acid composition of phospholipids in mice

Dietary fat and vitamin A provide important precursors for potent bioactive ligands of nuclear hormone receptors, which regulate various enzymes involved in lipid homeostasis, metabolism and inflammation. We determined the effects of dietary fat and dietary vitamin A on hepatic expression of two fatty acid metabolizing enzymes, elongase 6 (ELOVL6) and stearoyl-coenzyme A desaturase 1 (SCD1) and the concentration of saturated fatty acids (SAFA) and monounsaturated fatty acid (MUFA) of phospholipids in serum and liver. Mice (n = 6) were fed 4 weeks with diets containing 2, 5 and 25 % of fat or vitamin A (0, 2,500 and 326,500 RE/kg as retinyl palmitate). MUFAs and SAFAs were measured using GC and ESI–MS/MS. Hepatic expression of metabolizing enzymes was determined using QRT-PCR. ELOVL6 was significantly down-regulated in response to a high-fat diet (p < 0.001) and significantly up-regulated in response to low-fat diet (p < 0.05). SCD1 expression was significantly lower in high- versus low-fat diet (p < 0.05). The vitamin A content in the diet did not influence the hepatic expression of both enzymes. In plasma, the amounts of MUFAs bound to phospholipids significantly decreased in response to a high-fat diet and increased after a low-fat diet. This tendency was also observed in the liver for various phospholipids sub-classes. In summary, this study shows that fat content in the diet has a stronger impact than the content of vitamin A on hepatic gene expression of SCD1 and ELOVL6 and thereby on MUFA and SAFA concentrations in liver and plasma.