Novel extended-release transdermal formulations of the psychedelic N,N-dimethyltryptamine (DMT).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

Effects of Soy Protein Isolate on Fragile X Phenotypes in Mice.

Obesity is a pediatric epidemic that is more prevalent in children with developmental disabilities. We hypothesize that soy protein-based diets increase weight gain and alter neurobehavioral outcomes. Our objective herein was to test matched casein- and soy protein-based purified ingredient diets in a mouse model of fragile X syndrome, Fmr1KO mice. The experimental methods included assessment of growth; 24-7 activity levels; motor coordination; learning and memory; blood-based amino acid, phytoestrogen and glucose levels; and organ weights. The primary outcome measure was body weight. We find increased body weight in male Fmr1KO from postnatal day 6 (P6) to P224, male wild type (WT) from P32-P39, female Fmr1KO from P6-P18 and P168-P224, and female Fmr1HET from P9-P18 as a function of soy. Activity at the beginning of the light and dark cycles increased in female Fmr1HET and Fmr1KO mice fed soy. We did not find significant differences in rotarod or passive avoidance behavior as a function of genotype or diet. Several blood-based amino acids and phytoestrogens were significantly altered in response to soy. Liver weight was increased in WT and adipose tissue in Fmr1KO mice fed soy. Activity levels at the beginning of the light cycle and testes weight were greater in Fmr1KO versus WT males irrespective of diet. DEXA analysis at 8-months-old indicated increased fat mass and total body area in Fmr1KO females and lean mass and bone mineral density in Fmr1KO males fed soy. Overall, dietary consumption of soy protein isolate by C57BL/6J mice caused increased growth, which could be attributed to increased lean mass in males and fat mass in females. There were sex-specific differences with more pronounced effects in Fmr1KO versus WT and in males versus females.

Pharmacokinetic analysis of acute and dietary exposure to piperonyl butoxide in the mouse.

Piperonyl butoxide (PBO) is a popular insecticide synergist present in thousands of commercial, agricultural, and household products. PBO inhibits cytochrome P450 activity, impairing the ability of insects to detoxify insecticides. PBO was recently discovered to also inhibit Sonic hedgehog signaling, a pathway required for embryonic development, and rodent studies have demonstrated the potential for in utero PBO exposure to cause structural malformations of the brain, face, and limbs, or more subtle neurodevelopmental abnormalities. The current understanding of the pharmacokinetics of PBO in mice is limited, particularly with respect to dosing paradigms associated with developmental toxicity. To establish a pharmacokinetic (PK) model for oral exposure, PBO was administered to female C57BL/6J mice acutely by oral gavage (22-1800 mg/kg) or via diet (0.09 % PBO in chow). Serum and adipose samples were collected, and PBO concentrations were determined by HPLC-MS/MS. The serum concentrations of PBO were best fit by a linear one-compartment model. PBO concentrations in visceral adipose tissue greatly exceeded those in serum. PBO concentrations in both serum and adipose tissue decreased quickly after cessation of dietary exposure. The elimination half-life of PBO in the mouse after gavage dosing was 6.5 h (90 % CI 4.7-9.5 h), and systemic oral clearance was 83.3 ± 20.5 mL/h. The bioavailability of PBO in chow was 41 % that of PBO delivered in olive oil by gavage. Establishment of this PK model provides a foundation for relating PBO concentrations that cause developmental toxicity in the rodent models to Sonic hedgehog signaling pathway inhibition.

In vivo validation of psilacetin as a prodrug yielding modestly lower peripheral psilocin exposure than psilocybin.

Introduction: The use of the psychedelic compound psilocybin in conjunction with psychotherapy has shown promising results in the treatment of psychiatric disorders, though the underlying mechanisms supporting these effects remain unclear. Psilocybin is a Schedule I substance that is dephosphorylated in vivo to form an active metabolite, psilocin. Psilacetin, also known as O-acetylpsilocin or 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), is an unscheduled compound that has long been suggested as an alternative psilocin prodrug, though direct in vivo support for this hypothesis has thus far been lacking. Methods: This study employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess the time-course and plasma concentrations of psilocin following the intraperitoneal (IP) administration of psilacetin fumarate or psilocybin to male and female C57Bl6/J mice. Results: Direct comparisons of the time courses for psilocin exposure arising from psilocybin and psilacetin found that psilocybin led to 10-25% higher psilocin concentrations than psilacetin at 15-min post-injection. The half-life of psilocin remained approximately 30 min, irrespective of whether it came from psilocybin or psilacetin. Overall, the relative amount of psilocin exposure from psilacetin fumarate was found to be approximately 70% of that from psilocybin. Discussion: These findings provide the first direct support for the long-standing assumption in the field that psilacetin functions as a prodrug for psilocin in vivo. In addition, these results indicate that psilacetin fumarate results in lower peripheral psilocin exposure than psilocybin when dosed on an equimolar basis. Thoughtful substitution of psilocybin with psilacetin fumarate appears to be a viable approach for conducting mechanistic psychedelic research in C57Bl6/J mice.

Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach for Microscale Cell Culture.

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

Proteomic Correlates of Enhanced Daptomycin Activity following ß-Lactam Preconditioning in Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus.

Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus ß-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro ß-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected ß-lactam pretreatments on DAP plus ß-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring ß-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each ß-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without ß-lactam preconditioning. Relative proteomic quantitation comparing ß-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with ß-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to ß-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

A randomized, double-blind, dose-ranging, pilot trial of piperine with resveratrol on the effects on serum levels of resveratrol.

Resveratrol (3,4,5-trihydroxystilbene) is a naturally occurring phytoalexin with purported health-promoting effects, but with limited oral bioavailability. Our prior murine modeling research observed enhanced resveratrol bioavailability with piperine co-administration. In this study, single-dose pharmacokinetics of resveratrol with or without piperine and the associated toxicities were studied on a cohort of healthy volunteers. We performed a double-blind, randomized, three-arm pilot study. Participants were randomized to receive a single dose of resveratrol 2.5 g, with piperine in 0 mg, 5 mg, or 25 mg dose. An improved liquid chromatography/mass spectrometry assay was used to determine serum levels of resveratrol and resveratrol-glucuronide. Baseline through 24 h post-study drug serum analyses were performed and adverse events were followed for 30 days. Twenty-four participants were enroled. No significant relationship between dose and pharmacokinetic values were found. In the sex stratified analysis, Cmax for resveratrol in women showed a trend (P = 0.057) toward an increase with piperine. Pharmacokinetic values for resveratrol were: Cmax - 18.5 ± 16 ng/mL resveratrol alone, 29 ± 29 resveratrol + 5 mg piperine, 16 ± 13 resveratrol + 25 mg piperine; area under the concentration × time curve - 1270 ± 852 ng/h/mL resveratrol alone, 2083 ± 2284 resveratrol + 5 mg piperine, 1132 ± 222 resveratrol + 25 mg piperine. All subjects tolerated their protocol therapy with minimal to no toxicity and no evidence of differences between the three groups. The co-administration of resveratrol with piperine at 5 and 25 mg doses did not sufficiently alter the pharmacokinetics of resveratrol or resveratrol-glucuronide to demonstrate the significant enhancement observed in murine modeling.

Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum.

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum-infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR).

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

Low-fat yogurt consumption reduces biomarkers of chronic inflammation and inhibits markers of endotoxin exposure in healthy premenopausal women: a randomised controlled trial.

The anti-inflammatory mechanisms of low-fat dairy product consumption are largely unknown. The objective of this study was to determine whether low-fat yogurt reduces biomarkers of chronic inflammation and endotoxin exposure in women. Premenopausal women (BMI 18·5-27 and 30-40 kg/m2) were randomised to consume 339 g of low-fat yogurt (yogurt non-obese (YN); yogurt obese (YO)) or 324 g of soya pudding (control non-obese; control obese (CO)) daily for 9 weeks (n 30/group). Fasting blood samples were analysed for IL-6, TNF-α/soluble TNF II (sTNF-RII), high-sensitivity C-reactive protein, 2-arachidonoyl glycerol, anandamide, monocyte gene expression, soluble CD14 (sCD14), lipopolysaccharide (LPS), LPS binding protein (LBP), IgM endotoxin-core antibody (IgM EndoCAb), and zonulin. BMI, waist circumference and blood pressure were also determined. After 9-week yogurt consumption, YO and YN had decreased TNF-α/sTNFR-RII. Yogurt consumption increased plasma IgM EndoCAb regardless of obesity status. sCD14 was not affected by diet, but LBP/sCD14 was lowered by yogurt consumption in both YN and YO. Yogurt intervention increased plasma 2-arachidonoylglycerol in YO but not YN. YO peripheral blood mononuclear cells expression of NF-κB inhibitor α and transforming growth factor ß1 increased relative to CO at 9 weeks. Other biomarkers were unchanged by diet. CO and YO gained approximately 0·9 kg in body weight. YO had 3·6 % lower diastolic blood pressure at week 3. Low-fat yogurt for 9 weeks reduced biomarkers of chronic inflammation and endotoxin exposure in premenopausal women compared with a non-dairy control food. This trial was registered as NCT01686204.

Molecular signatures of sanguinarine in human pancreatic cancer cells: A large scale label-free comparative proteomics approach.

Pancreatic cancer remains one of the most lethal of all human malignancies with its incidence nearly equaling its mortality rate. Therefore, it's crucial to identify newer mechanism-based agents and targets to effectively manage pancreatic cancer. Plant-derived agents/drugs have historically been useful in cancer therapeutics. Sanguinarine is a plant alkaloid with anti-proliferative effects against cancers, including pancreatic cancer. This study was designed to determine the mechanism of sanguinarine's effects in pancreatic cancer with a hope to obtain useful information to improve the therapeutic options for the management of this neoplasm. We employed a quantitative proteomics approach to define the mechanism of sanguinarine's effects in human pancreatic cancer cells. Proteins from control and sanguinarine-treated pancreatic cancer cells were digested with trypsin, run by nano-LC/MS/MS, and identified with the help of Swiss-Prot database. Results from replicate injections were processed with the SIEVE software to identify proteins with differential expression. We identified 37 differentially expressed proteins (from a total of 3107), which are known to be involved in variety of cellular processes. Four of these proteins (IL33, CUL5, GPS1 and DUSP4) appear to occupy regulatory nodes in key pathways. Further validation by qRT-PCR and immunoblot analyses demonstrated that the dual specificity phosphatase-4 (DUSP4) was significantly upregulated by sanguinarine in BxPC-3 and MIA PaCa-2 cells. Sanguinarine treatment also caused down-regulation of HIF1α and PCNA, and increased cleavage of PARP and Caspase-7. Taken together, sanguinarine appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of sanguinarine against pancreatic cancer.

Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAF(V600E) mutant melanoma cells.

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAF(V600E) mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC-MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1's role in cancer cell survival.

Measurement of NMDA Receptor Antagonist, CPP, in Mouse Plasma and Brain Tissue Following Systematic Administration Using Ion-Pair LCMS/MS.

(RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation for CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 18:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.

Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis.

Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway. BIOLOGICAL SIGNIFICANCE: The development and functional activity of natural killer (NK) cells is regulated by interleukin (IL)-2 which stimulates the proliferation of NK cells and increases NK cell activity. With the development of IL-2-based immunotherapeutic strategies that rely on the IL-2-mediated activation of NK cells to target human cancers, it is important to understand the global molecular events triggered by IL-2 in human NK cells. The differentially expressed proteins in human primary NK cells following IL-2 activation identified in this study confirmed the activation of JAK-STAT signaling pathway and cell proliferation by IL-2 as expected, but also led to the discovery and identification of other factors that are potentially important in IL-2 signaling. These new factors warrant further investigation on their potential roles in modulating NK cell biology. The results from this study suggest that the activation of NK cells by IL-2 is a dynamic process through which proteins with various functions are regulated. Such findings will be important for the elucidation of molecular pathways involved in IL-2 signaling in NK cells and provide new targets for future studies in NK cell biology.

Stability of carboplatin, paclitaxel, and docetaxel with acetyl-l-carnitine during simulated Y-site administration.

The compatibility of acetyl-l-carnitine with three chemotherapy agents was studied during simulated Y-site administration. Acetyl-l-carnitine 30 mg/mL in 5% dextrose for injection (D5W) was combined with carboplatin 4 mg/mL, paclitaxel 2 mg/mL, and docetaxel 0.74 mg/mL in glass vials. Physical compatibility over the 4-hour storage at room temperature was assessed by visual examinations with unaided eye under fluorescent light and by measuring the percent transmittance at 600 nm. Chemical compatibility was measured by the percent of initial concentration remaining using stability-indicating high-performance liquid chromatographic-ultraviolet and high-performance liquid chromatographic-mass spectrometry methods. No visible particulate matter, haze, or color change was observed, and the percent transmittance was >95% for all admixtures. After a 4-hour incubation, 93.2% of the paclitaxel and 96.5% of docetaxel remained in separate mixtures with acetyl-l-carnitine. Carboplatin 4 mg/mL, paclitaxel 1.2 mg/mL, and docetaxel 0.74 mg/mL are physically and chemically compatible with acetyl-l-carnitine 30 mg/mLin D5W during a simulated 4-hour Y-site administration.

Enhancing the bioavailability of resveratrol by combining it with piperine.

SCOPE: Resveratrol (3,5,4'-trihydroxystilbene) is a phytoalexin shown to possess a multitude of health-promoting properties in pre-clinical studies. However, the poor in vivo bioavailability of resveratrol due to its rapid metabolism is being considered as a major obstacle in translating its effects in humans. In this study, we examined the hypothesis that piperine will enhance the pharmacokinetic parameters of resveratrol via inhibiting its glucuronidation, thereby slowing its elimination. METHODS AND RESULTS: Employing a standardized LC/MS assay, we determined the effect of piperine co-administration with resveratrol on serum levels resveratrol and resveratrol-3-O-ß-D-glucuronide in C57BL mice. Mice were administered resveratrol (100 mg/kg; oral gavage) or resveratrol (100 mg/kg; oral gavage)+piperine (10 mg/kg; oral gavage), and the serum levels of resveratrol and resveratrol-3-O-ß-D-glucuronide were analyzed at different times. We found that the degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serum concentration (C(max)) was increased to 1544% with the addition of piperine. CONCLUSION: Our study demonstrated that piperine significantly improves the in vivo bioavailability of resveratrol. However, further detailed research is needed to study the mechanism of improved bioavailability of resveratrol via its combination with piperine as well as its effect on resveratrol metabolism.

Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry.

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis.

Data Self-Recalibration and Mixture Mass Fingerprint Searching (DASER-MMF) to enhance protein identification within complex mixtures.

A novel algorithm based on Data Self-Recalibration and a subsequent Mixture Mass Fingerprint search (DASER-MMF) has been developed to improve the performance of protein identification from online 1D and 2D-LC-MS/MS experiments conducted on high-resolution mass spectrometers. Recalibration of 40% to 75% of the MS spectra in a human serum dataset is demonstrated with average errors of 0.3 +/- 0.3 ppm, regardless of the original calibration quality. With simple protein mixtures, the MMF search identifies new proteins not found in the MS/MS based search and increases the sequence coverage for identified proteins by six times. The high mass accuracy allows proteins to be identified with as little as three peptide mass hits. When applied to very complex samples, the MMF search shows less dramatic performance improvements. However, refinements such as additional discriminating factors utilized within the search space provide significant gains in protein identification ability and indicate that further enhancements are possible in this realm.

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta.

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.

Three male germline-specific aldolase A isozymes are generated by alternative splicing and retrotransposition.

Enzymes in the glycolytic pathway of mammalian sperm are modified extensively and are localized in the flagellum, where several are tightly bound to the fibrous sheath. This study provides the first evidence for three novel aldolase isozymes in mouse sperm, two encoded by Aldoart1 and Aldoart2 retrogenes on different chromosomes and another by Aldoa_v2, a splice variant of Aldoa. Phylogenetic analyses and comparative genomics indicate that the retrogenes and splice variant have remained functional and have been under positive selection for millions of years. Their expression is restricted to the male germline and is tightly regulated at both transcriptional and translational levels. All three isozymes are present only in spermatids and sperm and have distinctive features that may be important for localization in the flagellum and/or altered metabolic regulation. Both ALDOART1 and ALDOA_V2 have unusual N-terminal extensions not found in other aldolases. The N-terminal extension of ALDOA_V2 is highly conserved in mammals, suggesting a conserved function in sperm. We hypothesize that the N-terminal extensions are responsible for localizing components of the glycolytic pathway to the fibrous sheath and that this localization is required to provide sufficient ATP along the length of the flagellum to support sperm motility.