Structural basis for DNA recognition and processing by UvrB.

DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.

Emerging therapies for the treatment of nonalcoholic steatohepatitis: A systematic review.

To describe the mechanism, efficacy, and safety of novel agents that have reached phase 3 clinical trials for the treatment of biopsy-proven nonalcoholic steatohepatitis (NASH). A literature search was conducted using the PRISMA guidelines of MEDLINE databases (1990 to October 2020) with the following MeSH terms: NASH, nonalcoholic liver disease, fatty liver, liver diseases, steatohepatitis, liver fibrosis; combined with obeticholic acid, FXR agonist, cenicriviroc, CCR5 receptor antagonist, elafibranor, PPAR, selonsertib, ASK-1 inhibitor, resmetirom, THR-ß receptor, arachidyl amido cholanoic acid (Aramchol™), and SCD-1 modulator. Results were verified via clinicaltrials.gov, Google Scholar, and Google. Articles were included if the medications of interest had ongoing or completed phase 3 trials in biopsy-proven NASH with outcomes directly related to NASH resolution. Eleven studies were identified involving obeticholic acid (OCA), elafibranor, cenicriviroc, Aramchol, and resmetirom. Two agents have reported data from phase 3 trials: OCA and elafibranor. OCA demonstrated safety and efficacy in NASH with a primary end point of improvement or NASH resolution; a new drug approval has been submitted. Elafibranor failed to show efficacy in NASH in the preliminary report from the RESOLVE-IT trial; however, the study is being extended to reassess outcomes. The remaining agents demonstrated positive results in phase 2b studies and have initiated phase 3 trials. With projections for increased prevalence of patients with NASH and the current lack of treatment options, novel agents with targeted mechanisms could potentially change the treatment landscape. The manufacturer of OCA is first to submit a new drug application for the treatment of NASH. These novel agents may fill a pharmacotherapy gap in patients with NASH and possibly prevent progression to advanced liver disease.

A multiyear comparison of flipped- vs. lecture-based teaching on student success in a pharmaceutical science class.

BACKGROUND AND PURPOSE: To gauge the potential effect of mode of content delivery on overall student success in a pharmaceutical sciences course in a doctor of pharmacy program. EDUCATIONAL ACTIVITY AND SETTING: Principles of Drug Action I (PDAI) is a first-year pharmaceutical science course typically taught by multiple faculty, and each utilizes their own approach to deliver course content. Over a seven year period, the course naturally separated into blocks. Block-1 was taught using a traditional lecture-based approach while Block-2 varied between either a lecture-based or a flipped-classroom format. Student success was evaluated by exam at the end of each block. FINDINGS: For the four years in which Block-2 was taught by lecture, the number of exam failures was similar to Block-1. For each of the three years Block-2 was taught via the flipped-classroom format, the number of exam failures was approximately half that of Block-1. While grades for the lecture-based Block-1 trended downward over the seven-year evaluation period, average exam grades overall were similar between Block-1 and Block-2 each year regardless of teaching modality. SUMMARY: Retrospective analysis of this novel blocked approach within PDAI provided a means of internally evaluating the potential effect of teaching format on overall student performance. The results described here support previous studies that indicate that the flipped-classroom approach can reduce course failures. These findings also show that flipped-classroom teaching may have a greater impact on improving learning in lower-performing students.

Rebooting a sports pharmacy advanced pharmacy practice experience: Utilizing medicinal chemistry as a foundational approach to get pharmacists back in the game.

BACKGROUND AND PURPOSE: To implement a sports pharmacy advanced pharmacy practice experience (SP-APPE) utilizing medicinal chemistry as a foundational approach. EDUCATIONAL ACTIVITY AND SETTING: A student-pharmacist and medicinal chemistry faculty member collaborated to reboot a SP-APPE. Approached from a medicinal chemistry perspective and tailored to the infrastructure of the university, three fourth-year student-pharmacists piloted the SP-APPE (fall 2017 to fall 2018). Various performance enhancing drugs (PEDs) and supplements were investigated. Student-pharmacists evaluated general knowledge of PEDs along with the perceived value of pharmacists in sports among student-athletes, athletic personnel, and pre-APPE students. FINDINGS: By demonstrating how legitimate medications were chemically similar to substances banned by sports governing agencies, comparing chemical structures/pharmacophores of PEDs to those of various prescription drugs, and comprehending athletes' misuse of PEDs, student-pharmacists educated student-athletes and athletic personnel on pertinent topics spanning pharmacy and sports (e.g., marijuana, medication sharing, deciphering supplement labels). Laboratory analytical methods that detect PEDs and educational points regarding potential adverse health risks posed by PED usage were also reviewed. Survey participants (approximately 75%, n = 134) perceived pharmacists as valuable to student-athletes and athletic personnel. Student-pharmacists indicated that medicinal chemistry knowledge was strengthened by completing the APPE and relevant to their pharmacy careers. SUMMARY: Based on medicinal chemistry principles, the SP-APPE provided a venue for student-pharmacists to interact with athletics, assist with drug information education, and identify drugs or supplements as chemically related to those banned by sports governing agencies. Confident in medicinal chemistry concepts, student-pharmacists translated their expertise to benefit the patient/student-athlete.

Cooperative damage recognition by UvrA and UvrB: identification of UvrA residues that mediate DNA binding.

Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway, it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acid residues in UvrA that provide binding energy to DNA. Based on conservation among approximately 300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3-37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA(-) strain of bacteria 10-100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA.

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution.

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.

A pharmacy student's role as a teaching assistant in an undergraduate medicinal chemistry course - Implementation, evaluation, and unexpected opportunities for educational outreach.

BACKGROUND AND PURPOSE: To describe 1) a pharmacy student's teaching assistant (TA) role in an undergraduate medicinal chemistry course, 2) an active learning module co-developed by the TA and instructor, and 3) the unexpected opportunities for pharmacy educational outreach that resulted from this collaboration. EDUCATIONAL ACTIVITY AND SETTING: Medicinal Chemistry (CHM3413) is an undergraduate course offered each fall at Palm Beach Atlantic University (PBA). As a TA for CHM3413, a pharmacy student from the Gregory School of Pharmacy (GSOP) at PBA co-developed and implemented an active learning module emphasizing foundational medicinal chemistry concepts as they pertain to performance enhancing drugs (PEDs). Surveys assessed undergraduate students' perceived knowledge of medicinal chemistry concepts, PEDs, and TA involvement. FINDINGS: Students' (total n = 60, three fall semesters) perceived confidence in knowledge of medicinal chemistry concepts and PEDs increased significantly (p < 0.001) after the TA's module. Nearly 93% of students acknowledged this was their first interaction with a TA at PBA, ~ 82% "agreed/strongly agreed" that the TA provided effective instruction, and ~ 62% "agreed/strongly agreed" that TA availability raised overall confidence in CHM3413. Unexpected "side-effects" of this collaboration included opportunities for the TA and instructor to discuss health risks associated with PED usage with student-athletes and coaches at PBA. DISCUSSION: This collaboration developed the pharmacy student's teaching skills and reinforced knowledge of foundational pharmaceutical science concepts for both the TA and undergraduate students. Unexpected "side-effects" that resulted from this collaboration included opportunities for the TA and instructor to discuss health risks associated with PED usage with student-athletes in PBA's athletic department. SUMMARY: Educational/interprofessional outreach opportunities resulted from a pharmacy student TA's involvement in an undergraduate medicinal chemistry course. An advanced pharmacy practice experience elective in sports pharmacy (based on Ambrose's model) begins Fall 2017.

'Close-fitting sleeves': DNA damage recognition by the UvrABC nuclease system.

DNA damage recognition represents a long-standing problem in the field of protein-DNA interactions. This article reviews our current knowledge of how damage recognition is achieved in bacterial nucleotide excision repair through the concerted action of the UvrA, UvrB, and UvrC proteins.

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding.

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.

Structural insights into the first incision reaction during nucleotide excision repair.

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.

Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling.

To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process. One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC. To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands. The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety. These analogs were incorporated into double-stranded DNA using DNA polymerase beta and functioned as both the damage site and the cross-linking reagent. The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (Delta2 and Deltabeta-hairpin), were assayed. Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.

Identification of residues within UvrB that are important for efficient DNA binding and damage processing.

The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.

Interactions between UvrA and UvrB: the role of UvrB's domain 2 in nucleotide excision repair.

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the pre-incision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA-UvrB interaction, which is a pre-requisite for all subsequent steps in nucleotide excision repair.