Cost-effectiveness of dehydrated human amnion/chorion membrane allografts in lower extremity diabetic ulcer treatment.

OBJECTIVE: To evaluate the cost-effectiveness and budget impact of using standard care (no advanced treatment, NAT) compared with an advanced treatment (AT), dehydrated human amnion/chorion membrane (DHACM), when following parameters for use (FPFU) in treating lower extremity diabetic ulcers (LEDUs). METHOD: We analysed a retrospective cohort of Medicare patients (2015-2019) to generate four propensity-matched cohorts of LEDU episodes. Outcomes for DHACM and NAT, such as amputations, and healthcare utilisation were tracked from claims codes, analysed and used to build a hybrid economic model, combining a one-year decision tree and a four-year Markov model. The budget impact was evaluated in the difference in per member per month spending following completion of the decision tree. Likewise, the cost-effectiveness was analysed before and after the Markov model at a willingness to pay (WTP) threshold of $100,000 per quality adjusted life year (QALY). The analysis was conducted from the healthcare sector perspective. RESULTS: There were 10,900,127 patients with a diagnosis of diabetes, of whom 1,213,614 had an LEDU. Propensity-matched Group 1 was generated from the 19,910 episodes that received AT. Only 9.2% of episodes were FPFU and DHACM was identified as the most widely used AT product among Medicare episodes. Propensity-matched Group 4 was limited by the 590 episodes that used DHACM FPFU. Episodes treated with DHACM FPFU had statistically fewer amputations and healthcare utilisation. In year one, DHACM FPFU provided an additional 0.013 QALYs, while saving $3,670 per patient. At a WTP of $100,000 per QALY, the five-year net monetary benefit was $5003. CONCLUSION: The findings of this study showed that DHACM FPFU reduced costs and improved clinical benefits compared with NAT for LEDU Medicare patients. DHACM FPFU provided better clinical outcomes than NAT by reducing major amputations, ED visits, inpatient admissions and readmissions. These clinical gains were achieved at a lower cost, in years 1-5, and were likely to be cost-effective at any WTP threshold. Adoption of best practices identified in this retrospective analysis is expected to generate clinically significant decreases in amputations and hospital utilisation while saving money.

The xenobiotic sensing pregnane X receptor regulates tissue damage and inflammation triggered by C difficile toxins.

Clostridioides difficile (formerly Clostridium difficile; C difficile), the leading cause of nosocomial antibiotic-associated colitis and diarrhea in the industrialized world, triggers colonic disease through the release two toxins, toxin A (TcdA) and toxin B (TcdB), glucosyltransferases that modulate monomeric G-protein function and alter cytoskeletal function. The initial degree of the host immune response to C difficile and its pathogenic toxins is a common indicator of disease severity and infection recurrence. Thus, targeting the intestinal inflammatory response during infection could significantly decrease disease morbidity and mortality. In the current study, we sought to interrogate the influence of the pregnane X receptor (PXR), a modulator of xenobiotic and detoxification responses, which can sense and respond to microbial metabolites and modulates inflammatory activity, during exposure to TcdA and TcdB. Following intrarectal exposure to TcdA/B, PXR-deficient mice (Nr1i2-/- ) exhibited reduced survival, an effect that was associated with increased levels of innate immune cell influx. This exacerbated response was associated with a twofold increase in the expression of Tlr4. Furthermore, while broad-spectrum antibiotic treatment (to deplete the intestinal microbiota) did not alter the responses in Nr1i2-/- mice, blocking TLR4 signaling significantly reduced TcdA/B-induced disease severity and immune responses in these mice. Lastly, to assess the therapeutic potential of targeting the PXR, we activated the PXR with pregnenolone 16α-carbonitrile (PCN) in wild-type mice, which greatly reduced the severity of TcdA/B-induced damage and intestinal inflammation. Taken together, these data suggest that the PXR plays a role in the host's response to TcdA/B and may provide a novel target to dampen the inflammatory tissue damage in C difficile infections.

Mixing divalent ionic liquids: effects of charge and side-chains.

We have prepared novel divalent ionic liquids (ILs) based on the bis(trifluoromethylsulfonyl)imide anion where two charged imidazolium groups in the cations are either directly bound to each other or linked by a single atom. We assessed the influence of the side-chain functionality and divalency on their physical properties and on the thermodynamics of mixing. The results indicate that shortening the spacer of a divalent IL reduces its thermal stability and increases its viscosity. Mixtures of divalent and monovalent ILs show small but significant deviations from ideality upon mixing. These deviations appear to depend primarily on the (mis)match of the nature and length of the cation side-chain. The non-ideality imposed by mixing ILs with different side-chains appears to be enhanced by the increase in formal charge of the cations in the mixture.

Observed impact of skin substitutes in lower extremity diabetic ulcers: lessons from the Medicare Database (2015-2018).

OBJECTIVE: To evaluate large propensity-matched cohorts to assess outcomes in patients receiving advanced treatment (AT) with skin substitutes for lower extremity diabetic ulcers (LEDUs) versus no AT (NAT) for the management of LEDUs. METHOD: The Medicare Limited Dataset (1 October 2015 through 2 October 2018) were used to retrospectively analyse people receiving care for a LEDU treated with AT or NAT (propensity-matched Group 1). Analysis included major and minor amputations, emergency department (ED) visits and hospital readmissions. In addition, AT following parameters for use (FPFU) was compared with AT not FPFU (propensity-matched Group 2). A paired t-test was used for comparisons of the two groups. For comparisons of three groups, the Kruskal-Wallis test was used. A Bonferroni correction was performed when multiple comparisons were calculated. RESULTS: There were 9,738,760 patients with a diagnosis of diabetes, of whom 909,813 had a LEDU. In propensity-matched Group 1 (12,676 episodes per cohort), AT patients had statistically fewer minor amputations (p=0.0367), major amputations (p<0.0001), ED visits (p<0.0001), and readmissions (p<0.0001) compared with NAT patients. In propensity-matched Group 2 (1131 episodes per cohort), AT FPFU patients had fewer minor amputations (p=0.002) than those in the AT not FPFU group. CONCLUSION: AT for the management of LEDUs was associated with significant reductions in major and minor amputation, ED use, and hospital readmission compared with LEDUs managed with NAT. Clinics should implement AT in accordance with the highlighted parameters for use to improve outcomes and reduce costs.

An Unintentional Discovery of a Fluorogenic DNA Probe for Ribonuclease†I.

Ribonuclease I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well-known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNase I probe RFA13-1 isolated through in vitro selection with the crude extracellular mixture from Clostridium difficile contaminated with Klebsiella aerogenes as a selection target. Characterization of RFA13-1 reveals that it exhibits high sensitivity to Escherichia coli RNase I with a detection limit of 1.39 pm. Furthermore, RFA13-1 also shows high specificity for RNase I produced only in select bacteria from the Enterobacteriaceae family. As a result, this probe offers a simple tool for RNase I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.

Pregnane X Receptor Activation Triggers Rapid ATP Release in Primed Macrophages That Mediates NLRP3 Inflammasome Activation.

The pregnane X receptor (PXR) is a ligand-activated nuclear receptor that acts as a xenobiotic sensor, responding to compounds of foreign origin, including pharmaceutical compounds, environmental contaminants, and natural products, to induce transcriptional events that regulate drug detoxification and efflux pathways. As such, the PXR is thought to play a key role in protecting the host from xenobiotic exposure. More recently, the PXR has been reported to regulate the expression of innate immune receptors in the intestine and modulate inflammasome activation in the vasculature. In the current study, we report that activation of the PXR in primed macrophages triggers caspase-1 activation and interleukin-1ß release. Mechanistically, we show that this response is nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3-dependent and is driven by the rapid efflux of ATP and P2X purinoceptor 7 activation following PXR stimulation, an event that involves pannexin-1 gating, and is sensitive to inhibition of Src-family kinases. Our findings identify a mechanism whereby the PXR drives innate immune signaling, providing a potential link between xenobiotic exposure and the induction of innate inflammatory responses.

Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine.

Small noncoding microRNAs act as post-transcriptional regulators of gene expression involved in diverse biologic functions. Pregnane X receptor (PXR, NR1I2), a member of the superfamily of nuclear receptors, is a transcription factor governing the transport and biotransformation of various drugs and other chemicals. In the present study, we identified a specific microRNA (miR) involved in regulating the expression and functionality of human PXR (hPXR). According to bioinformatics analysis employing three commonly used algorithms (TargetScan, miRanda, and DIANA-microT-CDS), miR-18a-5p was predicted to be the top candidate microRNA regulator of hPXR. Consequently, this microRNA was selected for detailed experimental investigation. As shown in cell-based dual-luciferase reporter gene assays, functional interaction occurred between miR-18a-5p and the microRNA recognition element of miR-18a-5p in the 3'-untranslated region of hPXR mRNA. Transfection of LS180 human colorectal adenocarcinoma cells with an miR-18a-5p mimic decreased hPXR mRNA and protein expression, whereas transfection of LS180 cells with an miR-18a-5p inhibitor increased hPXR mRNA and protein expression. The decrease in hPXR expression by the miR-18a-5p mimic was associated with a reduction in the extent of hPXR target gene (CYP3A4) induction by rifampin and rilpivirine. Treatment of untransfected LS180 cells with either of these hPXR agonists decreased endogenous expression of miR-18a-5p, and this preceded the onset of CYP3A4 induction. In conclusion, miR-18a-5p is a negative regulator of hPXR expression and the hPXR agonists rifampin and rilpivirine are chemical suppressors of miR-18a-5p expression.

Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 µM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

3-Hydroxyflavone and structural analogues differentially activate pregnane X receptor: Implication for inflammatory bowel disease.

Pregnane X receptor (PXR; NR1I2) is a member of the superfamily of nuclear receptors that regulates the expression of genes involved in various biological processes, including drug transport and biotransformation. In the present study, we investigated the effect of 3-hydroxyflavone and its structurally-related analogues on PXR activity. 3-Hydroxyflavone, galangin, kaempferol, querceetin, isorhamnetin, and tamarixetin, but not but not datiscetin, morin, myricetin, or syringetin, activated mouse PXR, as assessed in a cell-based reporter gene assay. By comparison, 3-hydroxyflavone activated rat PXR, whereas 3-hydroxyflavone, galangin, quercetin, isorhamnetin, and tamarixetin activated human PXR (hPXR). A time-resolved fluorescence resonance energy transfer competitive ligand-binding assay showed binding to the ligand-binding domain of hPXR by 3-hydroxyflavone, galangin, quercetin, isorhamnetin, and tamarixetin. 3-Hydroxyflavone and galangin, but not quercetin, isorhamnetin, or tamarixetin, recruited steroid receptor coactivator (SRC)-1, SRC-2, and SRC-3 to hPXR. In LS180 human colon adenocarcinoma cells, 3-hydroxyflavone, quercetin, and tamarixetin increased CYP3A4, CYP3A5, and ABCB1 mRNA expression, whereas galangin and isorhamnetin increased CYP3A4 and ABCB1 but not CYP3A5 mRNA expression. Datiscetin, kaempferol, morin, myricetin, and syringetin did not attenuate the extent of hPXR activation by rifampicin, suggesting they are not hPXR antagonists. Overall, flavonols activate PXR in an analogue-specific and species-dependent manner. Substitution at the C2' or C5' position of 3-hydroxyflavone with a hydroxyl or methoxy group rendered it incapable of activating hPXR. Understanding the structure-activity relationship of flavonols in hPXR activation may facilitate nutraceutical development efforts in the treatment of PXR-associated intestinal diseases, such as inflammatory bowel disease.

Evaluation of in situ generated valproyl 1-O-ß-acyl glucuronide in valproic acid toxicity in sandwich-cultured rat hepatocytes.

Acyl glucuronides are reactive electrophilic metabolites implicated in the toxicity of carboxylic acid drugs. Valproyl 1-O-ß-acyl glucuronide (VPA-G), which is a major metabolite of valproic acid (VPA), has been linked to the development of oxidative stress in VPA-treated rats. However, relatively little is known about the toxicity of in situ generated VPA-G and its contribution to VPA hepatotoxicity. Therefore, we investigated the effects of modulating the in situ formation of VPA-G on lactate dehydrogenase (LDH) release (a marker of necrosis), BODIPY 558/568 C12 accumulation (a marker of steatosis), and cellular glutathione (GSH) content in VPA-treated sandwich-cultured rat hepatocytes. VPA increased LDH release and BODIPY 558/568 C12 accumulation, whereas it had little or no effect on total GSH content. Among the various uridine 5'-diphospho-glucuronosyltransferase inducers evaluated, ß-naphthoflavone produced the greatest increase in VPA-G formation. This was accompanied by an attenuation of the increase in BODIPY 558/568 C12 accumulation, but did not affect the change in LDH release or total GSH content in VPA-treated hepatocytes. Inhibition of in situ formation of VPA-G by borneol was not accompanied by substantive changes in the effects of VPA on any of the toxicity markers. In a comparative study, in situ generated diclofenac glucuronide was not toxic to rat hepatocytes, as assessed using the same chemical modulators, thereby demonstrating the utility of the sandwich-cultured rat hepatocyte model. Overall, in situ generated VPA-G was not toxic to sandwich-cultured rat hepatocytes, suggesting that VPA glucuronidation per se is not expected to be a contributing mechanism for VPA hepatotoxicity.

Fetal bovine serum and human constitutive androstane receptor: evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system.

The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular>dialyzed>CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1-10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition.

Peroneal Pathology in the Athlete.

The peroneal tendons play a critical role in stabilizing the foot and ankle especially in athletes with high demands on lateral ankle strength. A complete understanding of the anatomy of the lateral ankle as well as a careful physical examination is imperative to diagnosing peroneal pathology, which is commonly misdiagnosed and can lead to chronic pain and inability to perform high level sport. Although low-demand patients do well with a conservative approach, most high-demand athletes will benefit from surgical intervention.

Nanobiotechnological basis of an oxygen carrier with enhanced carbonic anhydrase for CO2 transport and enhanced catalase and superoxide dismutase for antioxidant function.

This is a mini review on the biotechnological aspects of the most extensively developed hemoglobin-based oxygen carriers The emphasis is on the most recent Polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase (PolyHb-CAT-SOD-CA), which is a nanobiotechnological complex that is being investigated and scaled up with the potential for clinical use as nanobiotherapeutics. Hemoglobin, a tetramer, is an excellent oxygen carrier. However, in the body it is converted into toxic dimers. Diacid or glutaraldehyde can crosslink hemoglobin into polyhemoglobin (PolyHb) and prevent its breakdown into toxic dimers. This has been developed and tested in clinical trials. A bovine polyhemoglobin has been approved for routine clinical use for surgical procedures in South Africa and Russia. Clinical trials with human PolyHb in hemorrhagic shock were effective but with a very slight increase in non-fatal myocardial ischemia. This could be due to a number of reasons. For those conditions with ischemia-reperfusion, one would need an oxygen carrier with antioxidant properties. One approach to remedy this is with prepared polyhemoglobin-catalase-superoxide dismutase (PolyHb-CAT-SOD). Another reason is an increase in intracellular pCO2. We therefore added an enhanced level of carbonic anhydrase to prepare a PolyHb-CAT-SOD-CA. The result is an oxygen carrier with enhanced Carbonic Anhydrase for CO2 transport and enhanced Catalase and Superoxide Dismutase for antioxidant functions. Detailed efficacy and safety studies have led to the industrial scale up towards clinical trial. In the meantime, oxygen carriers are being investigated around the world for use in ex vivo biotechnological fluid for organ preservation for transplantation, with one already approved in France.

Superparamagnetic Artificial Cells PLGA-Fe3O4 Micro/Nanocapsules for Cancer Targeted Delivery.

Artificial cells have been extensively used in many fields, such as nanomedicine, biotherapy, blood substitutes, drug delivery, enzyme/gene therapy, cancer therapy, and the COVID-19 vaccine. The unique properties of superparamagnetic Fe3O4 nanoparticles have contributed to increased interest in using superparamagnetic artificial cells (PLGA-Fe3O4 micro/nanocapsules) for targeted therapy. In this review, the preparation methods of Fe3O4 NPs and superparamagnetic artificial cell PLGA-drug-Fe3O4 micro/nanocapsules are discussed. This review also focuses on the recent progress of superparamagnetic PLGA-drug-Fe3O4 micro/nanocapsules as targeted therapeutics. We shall concentrate on the use of superparamagnetic artificial cells in the form of PLGA-drug-Fe3O4 nanocapsules for magnetic hyperthermia/photothermal therapy and cancer therapies, including lung breast cancer and glioblastoma.

Prospective Multicenter Diagnostic Performance of Technologist-Performed Screening Breast Ultrasound After Tomosynthesis in Women With Dense Breasts (the DBTUST).

PURPOSE: To assess diagnostic performance of digital breast tomosynthesis (DBT) alone or combined with technologist-performed handheld screening ultrasound (US) in women with dense breasts. METHODS: In an institutional review board-approved, Health Insurance Portability and Accountability Act-compliant multicenter protocol in western Pennsylvania, 6,179 women consented to three rounds of annual screening, interpreted by two radiologist observers, and had appropriate follow-up. Primary analysis was based on first observer results. RESULTS: Mean participant age was 54.8 years (range, 40-75 years). Across 17,552 screens, there were 126 cancer events in 125 women (7.2/1,000; 95% CI, 5.9 to 8.4). In year 1, DBT-alone cancer yield was 5.0/1,000, and of DBT+US, 6.3/1,000, difference 1.3/1,000 (95% CI, 0.3 to 2.1; P = .005). In years 2 + 3, DBT cancer yield was 4.9/1,000, and of DBT+US, 5.9/1,000, difference 1.0/1,000 (95% CI, 0.4 to 1.5; P < .001). False-positive rate increased from 7.0% for DBT in year 1 to 11.5% for DBT+US and from 5.9% for DBT in year 2 + 3 to 9.7% for DBT+US (P < .001 for both). Nine cancers were seen only by double reading DBT and one by double reading US. Ten interval cancers (0.6/1,000 [95% CI, 0.2 to 0.9]) were identified. Despite reduction in specificity, addition of US improved receiver operating characteristic curves, with area under receiver operating characteristic curve increasing from 0.83 for DBT alone to 0.92 for DBT+US in year 1 (P = .01), with smaller improvements in subsequent years. Of 6,179 women, across all 3 years, 172/6,179 (2.8%) unique women had a false-positive biopsy because of DBT as did another 230/6,179 (3.7%) women because of US (P < .001). CONCLUSION: Overall added cancer detection rate of US screening after DBT was modest at 19/17,552 (1.1/1,000; CI, 0.5- to 1.6) screens but potentially overcomes substantial increases in false-positive recalls and benign biopsies.

Selective agonism of human pregnane X receptor by individual ginkgolides.

Ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J are structurally related terpene trilactones present in Ginkgo biloba extract. Pregnane X receptor (PXR), glucocorticoid receptor (GR), and constitutive androstane receptor (CAR) regulate the expression of genes involved in diverse biological functions. In the present study, we investigated the effects of individual ginkgolides as single chemical entities on the function of human PXR (hPXR), human GR (hGR), and human CAR (hCAR). In cell-based reporter gene assays, none of the ginkgolides activated hGR or hCAR (wild-type and its SV23, SV24, and SV25 splice variants). Concentration-response experiments showed that ginkgolide A and ginkgolide B activated hPXR and rat PXR to a greater extent than ginkgolide C, whereas ginkgolide J had no effect. As determined by a time-resolved fluorescence resonance energy transfer competitive binding assay, ginkgolide A and ginkgolide B, but not ginkgolide C or ginkgolide J, were shown to bind to the ligand-binding domain of hPXR, consistent with molecular docking data. Compared with tetraethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate (SR12813) (a known agonist of hPXR), ginkgolide A and ginkgolide B were considerably less potent in binding to hPXR. These two ginkgolides recruited steroid receptor coactivator-1 to hPXR and increased hPXR target gene (CYP3A4) expression, as assessed by a mammalian two-hybrid assay and real-time polymerase chain reaction, respectively. In conclusion, the individual ginkgolides regulate the function of nuclear receptors in a receptor-selective and chemical-dependent manner. This study identifies ginkgolide A and ginkgolide B as naturally occurring agonists of hPXR and provides mechanistic insight into the structure-activity relationship in ligand activation of hPXR.

Species-dependent and receptor-selective action of bilobalide on the function of constitutive androstane receptor and pregnane X receptor.

Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6ß-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes.

Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by ß-oxidation. Another biotransformation pathway involves ß-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2',7'-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C12), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes.

From artificial red blood cells, oxygen carriers, and oxygen therapeutics to artificial cells, nanomedicine, and beyond.

The first experimental artificial red blood cells have all three major functions of red blood cells (rbc). However, the first practical one is a simple polyhemoglobin (PolyHb) that only has an oxygen-carrying function. This is now in routine clinical use in South Africa and Russia. An oxygen carrier with antioxidant functions, PolyHb-catalase-superoxide dismutase, can fulfill two of the three functions of rbc. Even more complete is one with all three functions of rbc in the form of PolyHb-catalase-superoxide dismutase-carbonic anhydrase. The most advanced ones are nanodimension artificial rbc with either PEG-lipid membrane or PEG-PLA polymer membrane. Extensions into oxygen therapeutics include a PolyHb-tyrosinase that suppresses the growth of melanoma in a mice model. Another is a PolyHb-fibrinogen that is an oxygen carrier with platelet-like function. Research has now extended well beyond the original research on artificial rbc into many areas of artificial cells. These include nanoparticles, nanotubules, lipid vesicles, liposomes, polymer-tethered lipid vesicles, polymersomes, microcapsules, bioencapsulation, nanocapules, macroencapsulation, synthetic cells, and others. These are being used in nanotechnology, nanomedicine, regenerative medicine, enzyme/gene therapy, cell/stem cell therapy, biotechnology, drug delivery, hemoperfusion, nanosensers, and even by some groups in agriculture, industry, aquatic culture, nanocomputers, and nanorobotics.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase: a novel biotechnology-based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA) is a therapeutic antioxidant that also transports both oxygen and carbon dioxide. This is formed by crosslinking Hb with SOD, CAT, and CA using glutaraldehyde. Crosslinking stroma-free Hb from red blood cell (RBC) reduces CA activity to 55%. Addition of more CA resulted in a preparation with the same CA activity as RBC. PolyHb in the complex acts as a buffer to prevent large pH changes as carbon dioxide is converted to carbonic acid. We then prepare and optimize a novel PolyHb-SOD-CAT-CA, a therapeutic antioxidant that also transports both oxygen and carbon dioxide.