Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept.

The environmental impact of agricultural waste from the processing of food and feed crops is an increasing concern worldwide. Concerted efforts are underway to develop sustainable practices for the disposal of residues from the processing of such crops as coffee, sugarcane, or corn. Coffee is crucial to the economies of many countries because its cultivation, processing, trading, and marketing provide employment for millions of people. In coffee-producing countries, improved technology for treatment of the significant amounts of coffee waste is critical to prevent ecological damage. This mini-review discusses a multi-stage biorefinery concept with the potential to convert waste produced at crop processing operations, such as coffee pulping stations, to valuable biofuels and bioproducts using biochemical and thermochemical conversion technologies. The initial bioconversion stage uses a mutant Kluyveromyces marxianus yeast strain to produce bioethanol from sugars. The resulting sugar-depleted solids (mostly protein) can be used in a second stage by the oleaginous yeast Yarrowia lipolytica to produce bio-based ammonia for fertilizer and are further degraded by Y. lipolytica proteases to peptides and free amino acids for animal feed. The lignocellulosic fraction can be ground and treated to release sugars for fermentation in a third stage by a recombinant cellulosic Saccharomyces cerevisiae, which can also be engineered to express valuable peptide products. The residual protein and lignin solids can be jet cooked and passed to a fourth-stage fermenter where Rhodotorula glutinis converts methane into isoprenoid intermediates. The residues can be combined and transferred into pyrocracking and hydroformylation reactions to convert ammonia, protein, isoprenes, lignins, and oils into renewable gas. Any remaining waste can be thermoconverted to biochar as a humus soil enhancer. The integration of multiple technologies for treatment of coffee waste has the potential to contribute to economic and environmental sustainability.

Light to moderate drinking and therapeutic doses of acetaminophen: An assessment of risks for renal dysfunction.

This study investigated the potential effect of therapeutic doses of acetaminophen (APAP) in combination with light-moderate amounts of alcohol on kidney functions controlling for factors such as hypertension, diabetes and obesity that may predispose the kidney to APAP and/or alcohol toxicity. Secondary analysis of the 2003-2004 National Health and Nutrition Examination Survey data was performed using SAS 9.4. Odds ratios (OR) and 95% confidence intervals (CI) comparing the likelihood that individuals who ingested therapeutic doses of APAP and light-moderate amount of alcohol, compared to those who did not, would have kidney dysfunction were generated from multiple logistics regression models by further controlling for potential predisposing factors namely hypertension, diabetes and obesity. Kidney dysfunction was defined based on self-reports and laboratory examination of serum creatinine (SCr), blood urea nitrogen (BUN), glomerular filtration rate (GFR) and albumin creatinine ratio (ABCR). Statistically significant increased odds of renal dysfunction were noted among respondents who reported use of therapeutic doses of APAP and light-moderate amount of alcohol [OR(95% CI) = 1.64(1.28-2.10) self-report, 2.18(1.81-2.63) SCr, 4.60(3.03-7.00) BUN, 3.14(2.42-4.07) GFR, and 1.71(1.36-2.14) ALBCR)] even after adjusting for hypertension, diabetes and obesity [Adjusted OR (95% CI) = 1.78 (1.22-2.58) self-report, 2.05 (1.07-3.92) GFR]. The toxic effects of APAP and alcohol on the kidney were hypothesized. The threshold doses at which these effects begin to occur are unknown. The findings of this study suggest that even therapeutic doses of APAP and light-moderate amount of alcohol could be health problematic if consumed concomitantly.

Metabolic interaction between cyclosporine and sirolimus.

BACKGROUND: When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone. Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects. Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp). METHODS: To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats. Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion. RESULTS: CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels. SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA. With combined dosing, however, there was a much greater reduction. Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing. Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase. CONCLUSIONS: Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats. Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.

Cyclosporine and bromocriptine-induced suppressions of CYP3A1/2 and CYP2C11 are not mediated by prolactin.

The purpose of this study was to determine if the suppression of hepatic CYP3A1/2 (cytochrome P450 3A1/2) and CYP2C11 (cytochrome P450 2C11) by cyclosporine is mediated by prolactin. Male intact rats were given subcutaneous doses of either 15 mg/kg/day of cyclosporine or 1 ml/kg/day of cyclosporine vehicle concomitantly with one of the following: 500 mg/kg prolactin, 1 ml/kg prolactin vehicle, 4 mg/kg bromocriptine, or 1 ml/kg bromocriptine vehicle for 14 days. Protein expressions were measured using Western blot analysis and activities were measured using an in vitro testosterone hydroxylation assay. The administration of prolactin did not significantly alter CYP3A1/2 protein expression. Hypoprolactinemia, produced by bromocriptine, caused a significant suppression of CYP3A1/2 activity levels when bromocriptine was administered alone and in combination with cyclosporine (P<0.001, P<0.05; respectively). However, the cause of the suppression by bromocriptine was likely not the result of lowering prolactin levels. Bromocriptine administration also lowered CYP2C11 protein expression and activity, while prolactin administration had virtually no effect on CYP2C11. Chronic cyclosporine administration caused a 140% increase in prolactin area under the curve (AUC) (P<0.001). Bromocriptine caused a significant decline in endogenous prolactin secretion, however, concurrent cyclosporine administration did not recover these levels. Overall, while both cyclosporine and bromocriptine, separately, can significantly alter the fate of hepatic P450 enzymes, the suppression is likely not due to an alteration in prolactin levels.

Altered cytochrome P450 and P-glycoprotein levels in rats during simulated weightlessness.

BACKGROUND: In order to investigate the effects of simulated weightlessness on drug metabolism, liver, kidney, and small intestine, microsomal proteins from tail-suspended rats were analyzed to determine cytochrome P450 (CYP) and P-glycoprotein levels following varying durations of tail-suspension. HYPOTHESIS: P-glycoprotein and CYP levels would both decrease similar to previous findings from actual spaceflight data. METHODS: Six groups of four Sprague-Dawley rats each were tail-suspended for up to 21 d; CYP and P-glycoprotein levels in the liver, kidney and small intestine were then measured by Western blotting. The results were compared with a control group of unsuspended rats. RESULTS: Our data showed there were significant changes in the levels of hepatic CYP2C11, 2E1, 4A1, and P-glycoprotein and significant changes in the levels of P-glycoprotein and CYP4A1 in the kidney. However, there were no significant changes detected in the levels of CYP3A2 in the liver or small intestine. CONCLUSIONS: We conclude that simulated weightlessness, using the tail-suspended rat model, showed significant suppressive effects on levels of CYP2C11, 2E1, and P-glycoprotein in the liver and CYP4A1 in the kidney, while demonstrating no significant effect on the levels of CYP3A2 in the liver or small intestine. Thus, generalized predictions on the effect of simulated microgravity on drug metabolism cannot be made and the overall effect of spaceflight on individual enzymes should be investigated.

The effect of lipoprotein-associated cyclosporine on drug metabolism and toxicity in rats.

PURPOSE: The objective of the study was to examine the effect of lipoprotein-associated cyclosporine on hepatic metabolism, hepatic lipoprotein receptors, and renal toxicity in comparison to the current commercially available cyclosporine (CSA) product. METHODS: Rats within the same group were given one of the following treatments: 10 mg/kg of CSA, plasma-CSA, very low-density lipoprotein (VLDL)-CSA, low-density lipoprotein (LDL)-CSA, LDL, high-density lipoprotein (HDL)-CSA, 1 mL/kg of vehicle, or saline intravenously for 14 days. Urine and blood samples were evaluated for renal function. Hepatic microsomes were prepared for immunoblotting and in vitro catalytic assays of CYP activity. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine genetic regulation. RESULTS: (1) There were no statistical differences in cholesterol levels in lipoprotein-associated CSA groups as compared with vehicle controls. (2) A significant decrease in creatinine clearance was seen in the plasma-CSA treated group (56%; P < 0.05). (3) No suppressions of CYP3A protein, activity or mRNA were found in the VLDL-CSA treated group. (4) CYP3A mRNA was suppressed to a greater degree in the LDL- and HDL-CSA treated groups as compared with the suppression caused by CSA alone. (5) A significant suppression of hepatic low-density lipoprotein receptor (LDL-R) mRNA levels was found in the LDL-CSA (50%; P = 0.0333) and plasma-CSA (40%; P = 0.1138), which was not attributed to LDL alone. (6) Significant suppression of scavenger-receptors class B type I (SR-BI) mRNA levels was found in the plasma-CSA group, although no significant differences in SRBI protein levels were seen between groups. CONCLUSIONS: Specific lipoprotein-CSA complexes appear to alter metabolic responses differently in comparison to CSA alone, indicating that the metabolism of CSA is dependent on the in vivo disposition of lipoprotein-CSA. Furthermore, LDL-R is one regulatory factor responsible for altering CSA metabolism as a result of an increase in uptake of CSA into hepatocytes.

Effect of high-density lipoprotein associated cyclosporine on hepatic metabolism and renal function.

Cyclosporine (CSA), in both humans and animals, is associated with plasma lipoproteins. It has been demonstrated that CSA-lipoprotein association is partly responsible for the distribution and toxicity related to CSA use. Altered plasma lipoprotein profiles are often seen in transplantation recipients undergoing CSA treatment. In the present study, daily 0.1 mg/kg intravenous injections of either high-density lipoprotein-associated CSA (HDL-CSA), plasma-associated CSA (Plasma-CSA), or CSA in Cremophor (CSA) were administered to adult male rats for 14 days. Vehicle controls included daily administrations of 0.5 ml/kg of Cremophor or saline. Serum creatinine levels, a marker of renal function, increased in rats administered Plasma-CSA as compared with control rats treated with CSA. CYP3A and CYP2C11 protein expression was suppressed by 27% and 39%, respectively, in the HDL-CSA treatment group and by 38% and 40% in the Plasma-CSA treated group as compared with CSA controls. In addition, 6beta-hydroxytestosterone, a marker of CYP3A activity, was reduced by 33% and 34% in the HDL-CSA and the Plasma-CSA treatment groups, respectively, as compared with the CSA control group. CYP2C11 activity was measured by the in vitro formation of 2alpha-hydroxytestosterone. Activity levels in rats treated with HDL-CSA and Plasma-CSA were slightly induced as compared to CSA controls, however these differences were not found to be statistically significant. In summary, Plasma-CSA treatment resulted in renal dysfunction and suppressed CYP3A and CPY2C11 protein expression. These results demonstratethat intravenous lipoprotein-associated CSA alters the metabolism of CSA in the rat differently than CSA alone.

Team-based learning in pharmacy education.

Instructors wanting to engage students in the classroom seek methods to augment the delivery of factual information and help students move from being passive recipients to active participants in their own learning. One such method that has gained interest is team-based learning. This method encourages students to be prepared before class and has students work in teams while in the classroom. Key benefits to this pedagogy are student engagement, improved communication skills, and enhanced critical-thinking abilities. In most cases, student satisfaction and academic performance are also noted. This paper reviews the fundamentals of team-based learning in pharmacy education and its implementation in the classroom. Literature reports from medical, nursing, and pharmacy programs are also discussed.

Impact of transgene expression on drug metabolism following systemic adenoviral vector administration.

BACKGROUND: Systemic administration of a first-generation adenovirus expressing E. coli beta-galactosidase (AdlacZ) alters expression and function of two hepatic drug-metabolizing enzymes, cytochrome P450 (CYP) 3A2 and 2C11, for 14 days. The objective of these studies was to determine how the transgene cassette influences CYP expression and function. METHODS: Sprague-Dawley rats were given 5.7 x 10(12) viral particles (vp)/kg of either: AdlacZ, Ad expressing murine erythropoietin (Epo), Ad without a transgene (Null), or phosphate-buffered saline (Vehicle). Hepatic CYP protein expression, activity, mRNA and alanine aminotransferase (ALT) levels were analyzed 0.25, 1, 4, and 14 days following a single intravenous injection. RESULTS: Administration of Epo did not alter CYP3A2 activity, but induced RNA levels by a factor of 2 at 4 and 14 days (P< or =0.01). This vector suppressed CYP2C11 activity levels by 45% at 1 day (P< or =0.05) and RNA levels throughout the study period (P< or =0.05). The Null vector suppressed CYP3A2 activity by 36, 63, 34, and 45% at 0.25, 1, 4 and 14 days, respectively (P< or =0.05). CYP2C11 activity was suppressed 1 day after administration (41%) and RNA levels were suppressed at 6 h (53%) and 1 day (36%, P< or =0.05). In contrast, AdlacZ suppressed both CYP3A2 and 2C11 at all time points. CONCLUSIONS: The immunogenic and biological nature of the transgene cassette can influence changes in CYP3A2, but not the 2C11 isoform. The shift in transcription and translation of protein for maintenance of physiologic homeostasis to production of viral proteins and transgene product and their associated toxicity during viral infection may explain our observations.

Considerations for use of recombinant adenoviral vectors: dose effect on hepatic cytochromes P450.

Recombinant adenovirus (Ad) serotype 5 is a vector commonly used for gene delivery. Although this vector has a natural tropism for the liver, there is a limited understanding of how Ad administration affects one of the primary hepatic processes, drug metabolism. The effects of systemic administration of a model recombinant adenoviral vector on two hepatic cytochrome P450 (P450) enzymes, CYP3A2 and 2C11, were investigated. Sprague-Dawley rats were treated with one of six vector doses, ranging from 5.7 x 10(6) to 5.7 x 10(12) virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 x 10(10)-5.7 x 10(12) vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 x 10(12) vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P < or = 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P < or = 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 x 10(6), 5.7 x 10(8), and 5.7 x 10(9) vp/kg of Ad significantly increased both CYP2C11 protein expression by 86, 71, and 107% and activity 110, 118, and 53%, respectively, above those of animals treated with saline (P < or = 0.01). These results clearly indicate that a single dose of adenovirus significantly alters key drug metabolizing enzymes for an extended period of time and should be investigated further in the context of the design and implementation of clinical trial protocols.

Effect of gender and estrous cycle on the pharmacokinetics of ethanol in the rat brain.

BACKGROUND: Aspects of ethanol pharmacokinetics in the blood, such as elimination, are known to vary by gender in the rat. Ethanol concentrations reaching the brain, which are relevant in affecting behavior, have not been measured rigorously by gender. This study used quantitative microdialysis to measure ethanol pharmacokinetics in the nucleus accumbens after a moderate dose of ethanol, comparing males with females and females across the estrous cycle. METHODS: We administered 1 g/kg ethanol intravenously or intragastrically to male and female rats. We measured ethanol concentrations in the nucleus accumbens by use of microdialysis with in vivo probe calibration and compared them with concentrations in jugular venous blood. RESULTS: After intravenous delivery, apparent ethanol elimination was approximately 15% faster in females than males from both brain and blood. After intragastric delivery, blood ethanol concentrations peaked faster in females than males by approximately 20 min, suggesting faster absorption or diffusion from the stomach. Likewise, accumbal ethanol concentrations peaked faster in females than males by approximately 25 min, indicating faster distribution to the brain. These pharmacokinetic parameters did not vary across the estrous cycle, nor were they influenced significantly by body water. CONCLUSIONS: This study provides the first quantitative measurement of ethanol concentrations and, thus, pharmacokinetics in the brain of awake rats. If ethanol distribution to the brain varies between genders, as these results suggest, then differences in the pharmacological response to ethanol can be expected, providing a rationale for studying the pharmacodynamics of ethanol in the brain of the self-administering rat.

Suppression of hepatic CYP3A1/2 and CYP2C11 by cyclosporine is not mediated by altering growth hormone levels.

Cyclosporine (CsA) suppresses drug metabolism by decreasing cytochrome P450 (P450) enzyme levels in rat liver. Growth hormone (GH) is known to pretranslationally regulate P450 expression. Thus, the suppression of P450 by CsA may involve GH as an intermediate. To address this question, we examined the effects of administering exogenous GH via twice daily subcutaneous injections and in conjunction with chronic subcutaneous CsA administration for 14 days on hepatic P450 expression. CsA alone decreased CYP3A1/2 and CYP2C11 significantly, in a manner similar to that previously found. When administered in the absence of CsA, GH also suppressed CYP3A1/2 and CYP2C11 protein levels as compared with GH vehicle. In the presence of CsA, GH did not cause further suppression of either CYP3A1/2 or CYP2C11 expression when compared with CsA treatment with GH vehicle. Testosterone in vitro catalytic assays confirmed that CsA and GH separately cause significant decreases in activity levels. Also, the concomitant administration of GH and CsA caused lowered production of 16alpha-, 2alpha-, 6beta-, and 2beta-hydroxytestosterone as compared with the administration of GH with CsA vehicle and as compared with the administration of GH vehicle with CsA. This study shows that GH is a dominating factor over CsA in determining hepatic P450 expression and activity. In addition, CsA does not seem to alter GH levels as a mediating event in suppressing P450 expression and activity. Since CsA given in combination with GH further suppressed P450 activity as compared with CsA given in combination with vehicle, this suggests that changes in hormonal status are likely to be one of the many factors that is responsible for the lack of a clear association between cyclosporine dosing and markers of toxicity.

Effect of sodium alterations on hepatic cytochrome P450 3A2 and 2C11 and renal function in rats.

Numerous dietary supplements are known to modulate cytochrome P450 (CYP)-mediated metabolism and subsequently alter drug toxicity or efficacy in animals and humans. In the present study we investigated the effect of varying amounts of sodium intake on renal function and the metabolic activity of the hepatic CYP3A2 and CYP2C11 isoforms. Rats were maintained on standard rodent chow or a low-salt rice diet. Within each of these groups rats received either a single intraperitoneal injection of furosemide to initiate salt depletion, or saline. Additional groups included salt supplementation of 500 mg/300 g body weight/day and 1.25 g/300 g body weight/day of sodium chloride solution. Rats receiving the low-salt diet, both with and without a concomitant furosemide administration, had a significant reduction in creatinine clearance without changes in serum creatinine. In addition, urine flow rate was markedly reduced in rats maintained on the low-salt diet. Western blot analysis indicated that neither sodium supplementation nor deprivation altered hepatic microsomal CYP3A2 levels; however, hepatic CYP2C11 levels significantly increased in rats receiving the largest sodium supplement. In vitro metabolic activity of CYP3A2 was unchanged as compared with controls. Activity of CYP2C11 was significantly reduced in both rat groups receiving additional sodium supplements. Acute manipulation of daily sodium intake does alter renal function and specific hepatic CYP isoforms and should be considered when using these rat models.

PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum.

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.