Skaggs School of Pharmacy and Pharmaceutical Sciences (SSPPS)

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia/hypercapnia (IHC), affects predominantly obese individuals, and increases atherosclerosis risk. Since we and others have implicated gut microbiota and metabolites in atherogenesis, we dissected their contributions to OSA-induced atherosclerosis. Atherosclerotic lesions were compared between conventionally-reared specific pathogen free (SPF) and germ-free (GF) Apoe-/- mice following a high fat high cholesterol diet (HFHC), with and without IHC conditions. The fecal microbiota and metabolome were profiled using 16S rRNA gene amplicon sequencing and untargeted tandem mass spectrometry (LC-MS/MS) respectively. Phenotypic data showed that HFHC significantly increased atherosclerosis as compared to regular chow (RC) in both aorta and pulmonary artery (PA) of SPF mice. IHC exacerbated lesions in addition to HFHC. Differential abundance analysis of gut microbiota identified an enrichment of Akkermansiaceae and a depletion of Muribaculaceae (formerly S24-7) family members in the HFHC-IHC group. LC-MS/MS showed a dysregulation of bile acid profiles with taurocholic acid, taurodeoxycholic acid, and 12-ketodeoxycholic acid enriched in the HFHC-IHC group, long-chain N-acyl amides, and phosphatidylcholines. Interestingly, GF Apoe-/- mice markedly reduced atherosclerotic formation relative to SPF Apoe-/- mice in the aorta under HFHC/IHC conditions. In contrast, microbial colonization did not show a significant impact on the atherosclerotic progression in PA. In summary, this research demonstrated that (1) IHC acts cooperatively with HFHC to induce atherosclerosis; (2) gut microbiota modulate atherogenesis, induced by HFHC/IHC, in the aorta not in PA; (3) different analytical methods suggest that a specific imbalance between Akkermansiaceae and Muribaculaceae bacterial families mediate OSA-induced atherosclerosis; and (4) derived bile acids, such as deoxycholic acid and lithocholic acid, regulate atherosclerosis in OSA. The knowledge obtained provides novel insights into the potential therapeutic approaches to prevent and treat OSA-induced atherosclerosis.

Neglected tropical diseases such as Chagas disease, human African trypanosomiasis, leishmaniasis, and schistosomiasis have a significant global health impact in predominantly developing countries, although these diseases are spreading due to increased international travel and population migration. Drug repurposing with a focus on increasing antiparasitic potency and drug-like properties is a cost-effective and efficient route to the development of new therapies. Here we identify compounds that have potent activity against Trypanosoma cruzi and Leishmania donovani, and the latter were progressed into the murine model of infection. Despite the potent in vitro activity, there was no effect on parasitemia, necessitating further work to improve the pharmacokinetic properties of this series. Nonetheless, valuable insights have been obtained into the structure-activity and structure-property relationships of this compound series.

Untargeted metabolomics is frequently performed on human fecal samples in conjunction with sequencing to unravel the gut microbiome functionality. As sample collection efforts are rapidly expanding, with individuals often collecting specimens at home, metabolomics experiments should adapt to accommodate the safety and needs of bulk off-site collections and improve high throughput. Here, we show that a 95% ethanol, safe to be shipped and handled, extraction part of the Matrix Method pipeline recovers comparable amounts of metabolites as a validated 50% methanol extraction, preserving metabolic profile differences between investigated subjects. Additionally, we show that the fecal metabolome remains relatively stable when stored in 95% ethanol for up to 1 week at room temperature. Finally, we suggest a metabolomics data analysis workflow based on robust centered log ratio transformation, which removes the variance introduced by possible different sample weights and concentrations, allowing for reliable and integration-ready untargeted metabolomics experiments in gut microbiome research.

Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4CSA ubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR.

The configurational analysis of amino acids (AAs) in natural product peptides, often containing nonproteinogenic AAs, is mostly carried out by the venerable Marfeys method using a chiral derivatizing agent (CDA) 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA)─Marfeys reagent─which undergoes SNAr reaction of the 1° amino group. The resulting AA-DAA derivatives are mostly well-separated by reversed-phase HPLC, but some DAA derivatives resist resolution. Here, we report the synthesis and characterization of two CDAs: l-FDTA (4) in which the l-alanine-derived auxiliary is replaced by l-tryptophanamide and (S)-FDNE (3) where the auxiliary is S-(6-methoxynaphth-2-yl)-1-ethylamine. Side-by-side comparisons of the two reagents were carried out by AA derivatization and reversed-phase HPLC analysis with variables such as organic solvent, additives, and the ionic strength of the mobile phase. l-DTA derivatives of l- and d-AAs were found to show superior HPLC performance and an improvement in resolutions. When incorporated into the mobile phase, the ammonium ion (NH4+, 0-100 mM) showed a dramatic influence on differential retention times [ΔtR = ΔtRd - ΔtRl] of several key AAs. We attributed the effect to π-cation interactions between the indole ring of DTA and the NH4+ counterion in the analyte, a hypothesis supported by 1H NMR titrations and DFT calculations.

BACKGROUND: Limited data exist on bictegravir pharmacokinetics in pregnancy among persons with HIV (PWH) and infant washout. SETTING: Nonrandomized, open-label, multicenter phase-IV prospective study of bictegravir pharmacokinetics and safety in pregnant PWH and their infants. METHODS: Steady-state 24-hour pharmacokinetic sampling of oral bictegravir 50 mg once daily (a component of fixed-dose combination bictegravir/emtricitabine/tenofovir alafenamide) during the second and third trimesters and postpartum was performed. Cord blood and infant washout samples were collected. Total and free bictegravir concentrations were measured by validated liquid chromatography with tandem mass spectrometry methods. Within-participant geometric mean ratios (GMR) with 90% confidence intervals (CI) were calculated to compare pharmacokinetics between second and third trimester versus postpartum. Infant HIV testing results were obtained. RESULTS: Twenty-seven maternal-infant pairs were enrolled. Bictegravir area under the concentration-time curve from time 0 through 24 hours post-dose was 46% lower in the second trimester (n = 12; P = 0.002; GMR 0.54; 90% CI: 0.43 to 0.69) and 52% lower in the third trimester (n = 24; P < 0.0001; GMR 0.48; 90% CI: 0.43 to 0.55), compared with postpartum. C 24 concentrations were above the estimated bictegravir protein-adjusted 95% effective concentration of 0.162 μg/mL. The median ratio of cord-to-maternal blood concentration was 1.38 (n = 17; quartiles: 1.17-1.63). Median T 1/2 for infant bictegravir washout was 33.2 hours (quartiles: 25.7-45.9) with a C max of 2.06 μg/mL (quartiles: 1.37-2.72). Overall, 88%-92% of participants maintained suppression <40 copies/mL throughout pregnancy and postpartum. All available infant HIV testing results were negative. The safety profile for pregnant PWH and infants was acceptable. CONCLUSIONS: Bictegravir exposure was lower during pregnancy compared with postpartum, yet C 24 concentrations were greater than the bictegravir protein-adjusted 95% effective concentration.

A structurally novel metabolite, fatuamide A (1), was discovered from a laboratory cultured strain of the marine cyanobacterium Leptolyngbya sp., collected from Fagaitua Bay, American Samoa. A bioassay-guided approach using NCI-H460 human lung cancer cells directed the isolation of fatuamide A, which was obtained from the most cytotoxic fraction. The planar structure of fatuamide A was elucidated by integrated NMR and MS/MS analysis, and a combination of bioinformatic and computational approaches was used to deduce the absolute configuration at its eight stereocenters. A putative hybrid PKS/NRPS biosynthetic gene cluster responsible for fatuamide A production was identified from the sequenced genomic DNA of the cultured cyanobacterium. The biosynthetic gene cluster possessed elements that suggested fatuamide A binds metals, and this metallophore property was demonstrated by native metabolomics and indicated a preference for binding copper. The producing strain was found to be highly resistant to toxicity from elevated copper concentrations in culture media.

BACKGROUND: Opioid users across federal poverty levels have varying healthcare consumption, which could influence public health policies to address the opioid crisis. To better understand this relationship, we evaluated the associations between federal poverty level (FPL) with healthcare costs and utilizations among adult opioid users in the United States (US). METHODS: A serial cross-sectional study using pooled data (2008-2019) from the Medical Expenditure Panel Survey (MEPS) was used to evaluate the association between FPL with healthcare expenditures among a representative sample of the US adult population with > = 1 opioid prescription. FPL was defined as Poor/Near Poor-Income, Low-Income, Middle-Income, and High-Income. Healthcare expenditures included costs and resource utilization. Survey weights were applied to generate standard errors for the representative sample of the US population. Generalized linear models were constructed to evaluate the association between FPL and healthcare expenditures adjusting for confounders. FPL groups were stratified by insurance coverage, frequency of opioid prescriptions filled, and pain level to evaluate their impact on healthcare expenditures. RESULTS: Total weighted sample was 27,289,263 respondents; 21.6% in Poor/Near Poor-Income, 14.9% in Low-Income, 28.6% in Middle-Income, and 34.9% in High-Income groups. The average annual increase in total healthcare costs for the Poor/Near Poor-Income group was $451 (95% CI: $142-$761), $275 (95% CI: $48-$502) for the Low-Income group, $640 (95% CI: $447-$834) for the Middle-Income group, and $618 (95% CI: $360-$877) for the High-Income group. Between-group comparisons yielded significant increases in average annual total healthcare costs for Middle- and High-Income groups versus Low-Income group; significant increases in average annual emergency room costs between Middle- versus Low-Income groups, and significant increases in average annual inpatient costs between Middle-Income versus Poor/Near Poor- and Low-Income groups. Stratified analyses yielded several significant increases in average annual costs and expenditures. However, no differences were reported for respondents who were uninsured across FPL groups. CONCLUSIONS: Respondents across FPL groups consumed healthcare at various rates, particularly when stratified by insurance coverage, frequency of opioid prescriptions filled, and pain level. FPL plays an important role in healthcare consumption, but further research is needed to understand these mechanisms and their impact on the opioid crisis.

Context: The growing field of palliative care emphasizes the need for high-quality research, yet the contributions of nonphysician palliative care specialists to studying patient outcomes remain underexplored. Methods: This national observational study aims to identify barriers to conducting research among chaplains, nurses, pharmacists, and social workers in palliative care settings. An anonymous online survey was conducted across various disciplines. Results: A total of 173 participants reveal significant differences in research engagement and perceived barriers, with chaplains and pharmacists more likely to have research requirements and training, respectively, but face obstacles including insufficient time and article preparation. Nurse practitioners and social workers reported lower research involvement and higher perceived barriers in study design and mentorship. Conclusion: The study highlights the need for tailored interventions, including targeted training, mentorship, and financial support to enhance research engagement among nonphysician palliative care specialists. Addressing these barriers through institutional support and interprofessional collaboration is crucial for advancing palliative care research and practice.

BACKGROUND: For extended-release drug formulations, effective half-life (t 1/2eff ) is a relevant pharmacokinetic parameter to inform dosing strategies and time to reach steady state. Tacrolimus, an immunosuppressant commonly used for the prophylaxis of organ rejection in transplant patients, is available as both immediate- and extended-release formulations. To the best of our knowledge, the t 1/2eff of tacrolimus from these different formulations has not yet been assessed. The objective of this study was to characterize the t 1/2eff and terminal half-life (t 1/2z ) of an extended-release once-daily tacrolimus formulation (LCPT) and twice-daily immediate-release tacrolimus (IR-Tac). METHODS: A noncompartmental analysis of pharmacokinetic data obtained from a phase 2 study in de novo kidney transplant recipients receiving either LCPT or IR-Tac was conducted. Intensive blood sampling was performed on days 1, 7, and 14, and tacrolimus whole blood concentrations were measured using a validated liquid chromatography with tandem mass spectrometry method. T 1/2eff was estimated using within-participant accumulation ratios. T 1/2z was estimated by linear regression of the terminal phase of the concentration versus time profile. RESULTS: The median accumulation ratios of LCPT and IR-Tac on day 14 were 3.18 and 2.06, respectively.The median (interquartile range; IQR) t 1/2eff for LCPT at day 14 of dosing was 48.4 (37.4-77.9) hours, whereas the t 1/2z was 20.3 (17.6-22.9) hours. For IR-Tac, the median (IQR) t 1/2eff and t 1/2z on day 14 were 12.5 (8.8-23.0) hours and 12.2 (9.2-15.7) hours, respectively. CONCLUSIONS: Consistent with its prolonged release of tacrolimus, LCPT demonstrated a higher accumulation ratio and a longer t 1/2eff compared with IR-Tac. These findings underscore the pharmacokinetic differences between different drug formulations of the same moiety and may help inform dose adjustments for LCPT in kidney transplantation.