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.

Recent analyses of genome data indicate that members of the cyanobacterial order Pleurocapsales show tremendous potential for natural product discovery. However, only a few compounds have been reported from this order. Here, we report the isolation of hyellamide (1), a glycosylated N-acyl tyrosine-derived eneamide, from the pleurocapsalean cyanobacterium Hyella patelloides LEGE 07179. The putative biosynthetic gene cluster for 1 was identified in the genome of the producing organism and a biosynthetic proposal is presented. This work sheds light on the chemistry of the Pleurocapsales and expands the chemical repertoire of cyanobacterial natural products to include N-acyl tyrosine-derived molecules.

Cathepsin B has been shown to contribute to deficits in traumatic brain injury (TBI), an important risk factor for Alzheimers disease (AD). Cathepsin B is elevated in TBI and AD patients, as well as in animal models of these conditions. Knockout of the cathepsin B gene results in amelioration of TBI-induced motor dysfunction and improvement of AD memory deficit in mice. The mechanism of cathepsin B pathogenesis in these brain disorders has been hypothesized to involve its translocation to the cytosol from its normal lysosomal location. This study, therefore, evaluated brain cytosolic cathepsin B activity in the controlled cortical impact (CCI) mouse model of TBI. CCI-TBI resulted in motor deficits demonstrated by the rotarod assay, brain tissue lesions, and disorganization of the hippocampus. Significantly, CCI-TBI increased cytosolic cathepsin B activity in the brain cortex in the ipsilateral brain hemisphere that received the CCI-TBI injury, with a concomitant decrease in the lysosomal fraction. Cathepsin B activity was monitored using the substrate Z-Nle-Lys-Arg-AMC which specifically detects cathepsin B activity but not other cysteine proteases. The normal lysosomal distribution of cathepsin B was observed by its discrete localization in brain cortical cells. CCI-TBI resulted in a more diffuse cellular distribution of cathepsin B consistent with translocation to the cytosol. Further studies utilized the novel neutral pH-selective inhibitor, Z-Arg-Lys-AOMK, that specifically inhibits cathepsin B at neutral pH 7.2 of the cytosol but not at acidic pH 4.6 of lysosomes. Daily administration of Z-Arg-Lys-AOMK (ip), beginning 1 day before CCI-TBI, resulted in the reduction of the increased cytosolic cathepsin B activity induced by CCI-TBI. The inhibitor also reduced cathepsin B activities in homogenates of the brain cortex and hippocampus which were increased by CCI-TBI. Furthermore, the Z-Arg-Lys-AOMK inhibitor resulted in the reduction of motor function deficit resulting from CCI-TBI. These findings demonstrate the activation of cytosolic cathepsin B activity in CCI-TBI mouse brain injury.

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.

With diabetes reaching epidemic proportions globally, it is imperative to increase the number of providers equipped to screen, educate, and help patients achieve glycemic control. This study evaluated the long-term results of student pharmacists attending a first-year Diabetes Self-Care Education Program (DSEP) by measuring knowledge retention, confidence, and clinical applicability of skills learned over time. The DSEP, integrated into the early pharmacy curriculum, is a 9-h training program made up of interactive lectures, glucose monitoring assignments, and active-learning workshops. Following DSEP training, two cohorts of first-year student pharmacists were surveyed annually for 3 and 5 years to assess knowledge retention, confidence, and clinical use of the DSEP content in their practice sites. By the year 1 follow-up survey, the response rate from the pre-survey period for cohort 1 was 88% and 78% for cohort 2; over time, the response rate decreased. For the long-term follow-up surveys, cohort 1 (5 years) and cohort 2 (3 years) demonstrated overall significantly improved sustained knowledge of diabetes (48% higher average test score, p < 0.001), perceived confidence, and clinical ability (over 60% and 76% increases from baseline, p < 0.001). Within 12 months of completing the DSEP, about two-thirds of students applied their training to assist patients with diabetes and their caregivers. Long-term, participants in both cohorts reported educating and screening over 22,000 patients with diabetes and caregivers in multiple clinical settings over 3 years and 5 years, respectively, following DSEP training. The positive impact of improved knowledge, confidence, and clinical applicability of the DSEP training obtained by student pharmacists was sustained for 3 or more years, impacting thousands of patients with diabetes and caregivers. Considering the growing global diabetes epidemic, pharmacy schools around the world should consider implementing an early DSEP program.

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.

INTRODUCTION: This study compared the calculated vancomycin area under the curve (AUC0-24) using trapezoidal and non-trapezoidal first-order pharmacokinetic equations. METHODS: This retrospective observational study included adult patients with documented MRSA bacteremia who received ≥ 48 h of intravenous vancomycin and had two consecutive serum levels after the first dose. AUC0-24 was calculated using trapezoidal and non-trapezoidal equations. Correlation and agreement between methods were assessed using Pearsons correlation coefficient (r) and Bland-Altman plots. Significant predictors (p < 0.05) from simple linear regression were included in a multiple linear regression model to evaluate their impact on AUC0-24 for both methods. RESULTS: Fifty-two patients were included. The median age was 63 years (interquartile range [IQR]: 50-73), and the median vancomycin clearance was 4 l/h (IQR: 2-6). Median vancomycin AUC0-24 was 399 mg∙h/l (IQR: 257-674) for the trapezoidal method and 572 mg∙h/l (IQR: 466-807) for the non-trapezoidal method. There was a strong correlation between the methods (r = 0.87 [95% CI, 0.79-1]; P < 0.01), but Bland-Altman analysis showed poor agreement, with a bias of - 198 mg∙h/l and 95% limits of agreement from - 482 to 86 mg∙h/l. In multiple linear regression, total daily dose and vancomycin clearance were independent predictors of AUC0-24 for both methods, with a stronger impact on non-trapezoidal AUC0-24 (adjusted R2 = 0.70) than trapezoidal AUC0-24 (adjusted R2 = 0.59). CONCLUSIONS: Trapezoidal and non-trapezoidal equations are not interchangeable for estimating vancomycin AUC0-24. The trapezoidal method consistently results in lower AUC0-24 estimates than the non-trapezoidal method.