Preparation of surface-enhanced resonance Raman scattering-active substrates: applications to heme-containing proteins

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy combines the advantages unique to resonance Raman scattering (RRS) with those of surface-enhanced Raman scattering (SERS). Although much of the initial studies concentrated on elucidating the mechanism (s), considerable attention has recently been directed at exploiting the enormous potential of the technique for analytical and biological purposes. However, the practical applicability of SERRS to many investigations remains limited because experimental conditions corresponding to the effect have not been fully characterized;This problem is especially apparent when SERRS is used for the study of biological systems. In many cases, the biomolecule species is perturbed upon adsorption onto a SERRS-active metal substrate. Hence, SERRS is not a viable method if the resulting spectra correlate to perturbed biological structures. It is the aim of our research to develop procedures for preserving the native structure and activity of proteins at SERRS-active substrates including, silver island-films, electrochemically-roughened silver electrodes, and citrate-reduced sols. In particular, our work has focused on the heme-containing proteins, cytochrome c, cytochrome P-450[subscript] b, myoglobin, and cytochrome c[subscript]3. Depending upon the biomolecule, the native state of the protein can be retained by choosing the appropriate adsorption conditions;Another aspect of our research has been concerned with optimizing and characterizing the experimental parameters in the preparation of SERRS/SERS-active silver-island films. Our results show that for both SERS and SERRS, the intensity of the observed signals is dependent upon the evaporation rate used in the preparation of the silver-island film. The optical densities of the films as well as the resulting surface morphologies were also studied as a function of deposition rate. Specific applications of silver-island films as SERRS-active substrates included Raman microprobe SERRS investigations and the characterization of covalently modified electrodes by using the SERRS technique

Off target effects of sulforaphane include the de-repression of long-terminal repeats through histone acetylation events

Sulforaphane is a naturally occurring isothiocyanate in cruciferous vegetables. Sulforaphane inhibits histone deacetylases, leading to the transcriptional activation of genes including tumor suppressor genes. The compound has attracted considerable attention in the chemoprevention of prostate cancer. Here we tested the hypothesis that sulforaphane is not specific for tumor suppressor genes but also activates loci such as long terminal repeats (LTRs), which might impair genome stability. Studies were conducted using chemically pure sulforaphane in primary human IMR-90 fibroblasts and in broccoli sprout feeding studies in healthy adults. Sulforaphane (2.0 μM) caused an increase in LTR transcriptional activity in cultured cells. Consumption of broccoli sprouts (34, 68, or 102g) by human volunteers caused a dose dependent elevation in LTR mRNA in circulating leukocytes, peaking at more than a 10-fold increase. This increase in transcript levels was associated with an increase in histone H3 K9 acetylation marks in LTR 15 in peripheral blood mononuclear cells from subjects consuming sprouts. Collectively, this study suggests that sulforaphane has off-target effects that warrant further investigation when recommending high levels of sulforaphane intake, despite its promising activities in chemoprevention

Pinto Beans (\u3ci\u3ePhaseolus vulgaris\u3c/i\u3e L.) as a Functional Food: Implications on Human Health

Most foods are considered functional in terms of providing nutrients and energy to sustain daily life, but dietary systems that are capable of preventing or remediating a stressed or diseased state are classified as functional foods. Dry beans (Phaseolus vulgaris L.) contain high levels of chemically diverse components (phenols, resistance starch, vitamins, fructooligosaccharides) that have shown to protect against such conditions as oxidative stress, cardiovascular disease, diabetes, metabolic syndrome, and many types of cancer, thereby positioning this legume as an excellent functional food. Moreover, the United States has a rich dry bean history and is currently a top producer of dry beans in the world with pinto beans accounting for the vast majority. Despite these attributes, dry bean consumption in the US remains relatively low. Therefore, the objective of this manuscript is to review dry beans as an important US agricultural crop and as functional food for the present age with an emphasis on pinto beans

Development of an internet based system for modeling biotin metabolism using Bayesian networks

Biotin is an essential water-soluble vitamin crucial for maintaining normal body functions. The importance of biotin for human health has been under-appreciated but there is plenty of opportunity for future research with great importance for human health. Currently, carrying out predictions of biotin metabolism involves tedious manual manipulations. In this paper, we report the development of BiotinNet, an internet based program that uses Bayesian networks to integrate published data on various aspects of biotin metabolism. Users can provide a combination of values on the levels of biotin related metabolites to obtain the predictions on other metabolites that are not specified. As an inherent feature of Bayesian networks, the uncertainty of the prediction is also quantified and reported to the user. This program enables convenient in silico experiments regarding biotin metabolism, which can help researchers design future experiments while new data can be continuously incorporated

Evaluation of Metals in a Defined Medium for \u3ci\u3ePichia pastoris\u3c/i\u3e Expressing Recombinant β-Galactosidase

Culture growth and recombinant protein yield of the Pichia pastoris GS115 methanol utilization positive system were studied in response to the types and levels of metals present in the growth medium and the supplemental salts typically used for these fermentations. Magnesium and zinc were both required to support cell growth but at significantly reduced levels compared to the control. However, supplementation with calcium, cobalt, iron, manganese, iodine, boron, and molybdenum were not required to sustain cell mass. When the medium was reformulated with only zinc and magnesium, the cells grew to 12–15 generations, which are expected for high cell density fed-batch fermentations. Product yields of the recombinant protein β-galactosidase were significantly influenced by the trace metal concentrations. By using response surface and full factorial designs, maximum protein yield occurred when the concentration of zinc salt was limited to the level necessary only to support cell mass while protein yield positively correlated to increasing levels of the remaining trace metal salts. These studies are the first to show that excess trace metals must be optimized when developing P. pastoris based fed-batch fermentations

Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects

BACKGROUND: To systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals. METHODS AND FINDINGS: Ten human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18-30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS. CONCLUSION: Our results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches

Essential Oil Yield, Composition, and Bioactivity of Sagebrush Species in the Bighorn Mountains

Sagebrush (Artemisia spp.) are dominant wild plants in large areas of the U.S., Canada and Mexico, and they include several species and subspecies. The aim was to determine if there are significant differences in essential oil (EO) yield, composition, and biological activity of sagebrush within the Bighorn Mountains, U.S. The EO yield in fresh herbage varied from 0.15 to 1.69% for all species, including 0.25–1.69% in A. tridentata var. vaseyana, 0.64–1.44% in A. tridentata var. tridentata, 1% in A. tridentata var. wyomingensis, 0.8–1.2% in A. longifolia, 0.8–1% in A. cana, and 0.16% in A. ludoviciana. There was significant variability in the EO profile between species, and subspecies. Some EO constituents, such as α-pinene (0–35.5%), camphene (0–21.5%), eucalyptol (0–30.8%), and camphor (0–45.5%), were found in most species and varied with species and subspecies. The antioxidant capacity of the EOs varied between the species and subspecies. None of the sagebrush EOs had significant antimicrobial, antimalarial, antileishmanial activity, or contained podophyllotoxin. Some accessions yielded EO with significant concentrations of compounds including camphor, eucalyptol, cis-thujone, α-pinene, α-necrodol-acetate, fragranol, grandisol, para-cymene, and arthole. Therefore, chemotypes can be selected and possibly introduced into culture and be grown for commercial production of these compounds to meet specific industry needs

Histone acetylation increases in response to ferulic, gallic, and sinapic acids acting synergistically in vitro to inhibit \u3ci\u3eCandida albicans\u3c/i\u3e yeast‐to‐hyphae transition

Novel treatments are needed to prevent candidiasis/candidemia infection due to the emergence of Candida species resistant to current antifungals. Considering the yeast-to‐hyphae switch is a critical factor to Candida albicans virulence, phenols common in plant sources have been reported to demonstrating their ability to prevent dimorphism. Therefore, phenols present in many agricultural waste stress (ferulic (FA) and gallic (GA) acid) were initially screened in isolation for their yeast‐to‐hyphae inhibitory properties at times 3, 6, and 24 hr. Both FA and GA inhibited 50% of hyphae formation inhibitory concentration (IC50) but at a concentration of 8.0 ± 0.09 and 90.6 ± 1.05 mM, respectively, at 24 hr. However, the inhibitory effect of FA increased by 1.9–2.6 fold when combined with different GA concentrations. GA and FA values decreased even lower when sinapic acid (SA) was added as a third component. As evidenced by concave isobolograms and combination indexes less than 1, both GA:F A and GA:FA:SA combinations acted synergistically to inhibit 50% hyphae formation at 24 hr. Lastly, acetylation of histone H3 lysine 56 acetylation (H3K56) was higher in response to the triple phenolic cocktail (using the IC50 24 hr inhibitory concentration level) comparable with the nontreated samples, indicating that the phenols inhibited hyphal growth in part by targeting H3K56 acetylation

Identification and assessment of markers of biotin status in healthy adults

Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here, we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency and supplementation in sixteen healthy men and women. A total of twenty possible markers of biotin status were assessed, including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing biotin-deficient and biotin-sufficient individuals. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid detected some biotin-deficient subjects, but produced a meaningful number of false-negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase and biotin-dependent carboxylases in lymphocytes were not different among the treatment groups. Generally, datasets were characterised by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in the present study

Grain sorghum whole kernel oil lowers plasma and liver cholesterol in male hamsters with minimal wax involvement

The lipid fraction of the grain sorghum whole kernel (GS-WK) (i.e., phytosterol rich oil or policosanol rich wax) responsible for lowering cholesterol in hamsters fed the crude lipid (wax + oil) was determined. As expected, hamsters fed an atherogenic diet for a four week period presented with higher plasma non-HDL plasma and liver esterified cholesterol than those on the low fat diet. However, the atherogenic diet containing 5% (w/w) oil significantly lowered non-HDL plasma and liver cholesterol. Although the 5% wax supplement did not affect either plasma or liver cholesterol, excreted neutral sterol and bile acid were slightly higher than produced by the atherogenic diet. Still, cholesterol excretion negatively correlated with liver cholesterol concentration (r = –0.681, p \u3c 0.001) across diets with the oil fraction producing the greatest impact. These combined results indicate that oil plays the most significant role in modulating cholesterol, most likely by inhibiting absorption, but subtle interactions by the wax may be involved. However, the sorghum oil would be the most potent component to serve as a possible heart health ingredient in functional foods