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

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

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

Hydrodistillation Extraction Kinetics Regression Models for Essential Oil Yield and Composition in \u3ci\u3eJuniperus virginiana, J. excelsa\u3c/i\u3e, and \u3ci\u3eJ. sabina\u3c/i\u3e

The chemical profile and antioxidant capacity of Juniperus virginiana, J. excelsa, and J. sabina essential oil (EO) fractions as a function of time was the subject of this study. The hypothesis was that, capturing EO in sequential timeframes during hydrodistillation would generate fractions containing unique compositions and antioxidant capacity. In J. virginiana, the highest limonene (43%) was found in the 0–5 min oil fraction, with safrole (37%) being highest in the 10–20 and 20–40 min fractions, and elemol (34%) being highest in the 160–240 min fraction. In J. excelsa, α-pinene (34-36%) was the highest in the 0–5 min fraction and in the control (non-stop 0–240 min distillation) oil, limonene (39%) was the highest in the 0–10 min fractions and cedrol (50-53%) was the highest in the 40–240 min fractions. In J. sabina, sabinene (80%) was highest in the 0–3 min fraction. The highest antioxidant capacity of J. virginiana was demonstrated by the 5–10 min fraction; the one in J. sabina by the 3–10 min fraction; and, the one in J. excelsa, by the control. The kinetics regression models that were developed can predict EO composition of the three juniper species eluted at different timeframes. Various industries could benefit from the results from this study

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 intersubject variations in the response to RS indicates that such strategies might benefit from more personalized approaches

Distillation Time as Tool for Improved Antimalarial Activity and Differential Oil Composition of Cumin Seed Oil

A steam distillation extraction kinetics experiment was conducted to estimate essential oil yield, composition, antimalarial, and antioxidant capacity of cumin (Cuminum cyminum L.) seed (fruits). Furthermore, regression models were developed to predict essential oil yield and composition for a given duration of the steam distillation time (DT). Ten DT durations were tested in this study: 5, 7.5, 15, 30, 60, 120, 240, 360, 480, and 600 min. Oil yields increased with an increase in the DT. Maximum oil yield (content, 2.3 g/100 seed), was achieved at 480 min; longer DT did not increase oil yields. The concentrations of the major oil constituents α-pinene (0.14–0.5% concentration range), β-pinene (3.7–10.3% range), γ-cymene (5–7.3% range), γ-terpinene (1.8–7.2% range), cumin aldehyde (50–66% range), α-terpinen-7-al (3.8–16% range), and β-terpinen-7-al (12–20% range) varied as a function of the DT. The concentrations of α-pinene, β-pinene, γ-cymene, γ-terpinene in the oil increased with the increase of the duration of the DT; α-pinene was highest in the oil obtained at 600 min DT, β-pinene and γ-terpinene reached maximum concentrations in the oil at 360 min DT; γ-cymene reached a maximum in the oil at 60 min DT, cumin aldehyde was high in the oils obtained at 5–60 min DT, and low in the oils obtained at 240–600 min DT, α-terpinen-7-al reached maximum in the oils obtained at 480 or 600 min DT, whereas β-terpinen-7-al reached a maximum concentration in the oil at 60 min DT. The yield of individual oil constituents (calculated from the oil yields and the concentration of a given compound at a particular DT) increased and reached a maximum at 480 or 600 min DT. The antimalarial activity of the cumin seed oil obtained during the 0–5 and at 5–7.5 min DT timeframes was twice higher than the antimalarial activity of the oils obtained at the other DT. This study opens the possibility for distinct marketing and utilization for these improved oils. The antioxidant capacity of the oil was highest in the oil obtained at 30 min DT and lowest in the oil from 360 min DT. The Michaelis-Menton and the Power nonlinear regression models developed in this study can be utilized to predict essential oil yield and composition of cumin seed at any given duration of DT and may also be useful to compare previous reports on cumin oil yield and composition. DT can be utilized to obtain cumin seed oil with improved antimalarial activity, improved antioxidant capacity, and with various compositions