Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli.

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.

Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.

Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Stromal-AR influences the growth of epithelial cells in the development of benign prostate hyperplasia.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, ß-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Preoperative Wire Localization of the Breast on the Day Before Surgery.

OBJECTIVE: To assess the feasibility and accuracy of preoperative wire localization performed one day prior to surgery and the relationship between the time interval following wire placement with migration distance within the time-window examined. METHODS: Two trials were performed with next-day mammography to assess migration. Trial 1 used a standard hooked wire (50 patients, 61 wires). Trial 2 employed a looped wire (50 patients, 59 wires). A third trial was subsequently performed (16 patients, 18 wires) using the looped wire without repeat mammograms. Complications were recorded. Comparative statistical analyses were performed between patients in Trial 1 and Trial 2. RESULTS: In Trials 1 and 2, no wires required readjustment on the day of surgery. Mean and maximum migration were less with the looped wire (range: 0-7 mm) compared to the hooked wire (range: 0-18 mm), allowing for the elimination of next-day mammograms in Trial 3. A Mann-Whitney U test showed no significant difference between the migration distances for the first two trials (P = 0.11). A Chi-square test showed no significant difference in the direction of the migration between the two trials (P = 0.15). There was no correlation between the time interval of localization and needle migration in the first two trials (r = -0.16, P = 0.22 and -0.12, P = 0.36). Specimen radiographs demonstrated the lesion/biopsy marker clip in all cases in all three trials. No infections or bleeding occurred. Two patients developed an allergic reaction to adhesive. CONCLUSION: Wire localization performed on the day before surgery is feasible, inexpensive, did not compromise accuracy, and successfully unlinked the radiologic and surgical procedures.

Use of Contrast-Enhanced MRI in Management of Discordant Core Biopsy Results.

OBJECTIVE. Evaluating concordance between core biopsy results and imaging findings is an integral component of breast intervention. Pathologic results deemed benign discordant reflect concern that a malignancy may have been incorrectly sampled. Standard of care currently is surgical excision, although a large percentage of these lesions will be benign at final pathologic analysis. The purpose of this study was to determine whether inclusion of contrast-enhanced MRI would optimize patient care. MATERIALS AND METHODS. Forty-five patients with 46 lesions were identified who underwent contrast-enhanced MRI after receiving discordant ultrasound or stereotactic biopsy results between 2012 and mid 2018. These findings were classified BI-RADS category 4 at diagnostic imaging. Disease-positive was defined as all malignancies and borderline lesions. RESULTS. Fourteen patients had suspicious MRI findings; 31 patients did not. Negative or benign MRI findings were validated by stability at imaging follow-up of at least 1 year in 27 patients (28 lesions) and at least 6 months in four patients. Eight of the total of 46 discordant lesions were ultimately malignant, a rate of 17.3%, an expected result for BI-RADS 4 lesions. Sensitivity, specificity, positive predictive value, and negative predictive value of MRI calculated in the group of 41 patients (42 lesions) with documented stability for at least 1 year were 100%, 93.3%, 85.7%, and 100%. The false-negative rate of MRI was 0%; the false-positive rate was 2 of 30 (6.7%). CONCLUSION. In the management of discordant benign core biopsy results, contrast-enhanced MRI facilitated successful triage of patients to surgery; 31 of the original 45 patients (68.9%) avoided surgery.

Preventing Ventilator-Associated Infections.

Mechanical ventilator use is fraught with risk of complications. Ventilator-associated pneumonia (VAP) is a common complication that prolongs stays on the ventilator and increases mortality and costs. The Centers for Disease Control and Prevention recommend the use of the term, ventilator-associated event. Prevention and/or interruption of cycle of inflammation, colonization of respiratory tract, and ventilator-associated tracheobronchitis are key to managing VAP. Modifying risk factors using a ventilator bundle is considered standard of care. The contentious factors and the lack of support for early tracheotomy, parenteral nutrition, and monitoring of gastric residuals are also addressed. Finally, the role of ventilator-associated tracheobronchitis in VAP is discussed.

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication.

To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1α associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1α/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.

Practices, Beliefs, and Perceived Barriers to Adolescent Human Immunodeficiency Virus Screening in the Emergency Department.

OBJECTIVE: Limited data exist regarding knowledge of and compliance to the Centers for Disease Control and Prevention's universal adolescent human immunodeficiency virus (HIV) screening recommendations. Our objective was to assess current guideline knowledge, practice, and perceived barriers to emergency department (ED)-based adolescent HIV screening. METHODS: We administered an anonymous Web-based cross-sectional survey from May 1, 2012, to June 30, 2012, to 1073 physicians from the American Academy of Pediatrics Section on Emergency Medicine LISTSERV. Survey participants were included if they (1) practiced as attending-level physicians, (2) practiced primarily in pediatric emergency medicine or general emergency medicine, and (3) provided clinical care for patients younger than the age of 21 years. The survey examined respondent demographics, knowledge, attitudes, beliefs, practices, and barriers to ED-based HIV screening. Standard descriptive statistics and comparative analyses were performed. RESULTS: A total of 220 responses were obtained; 29 responses were excluded and 191 responses were included in the study. Most of the participants were from urban, free-standing children's hospitals and had an annual ED volume of more than 61,000 patient visits. Respondent knowledge of the Centers for Disease Control and Prevention guidelines was low; less than 40% of the respondents identified correct consent requirements. Only 15.4% of the respondents reported screening for HIV more than 10 times for the prior 6 months. Most frequently cited barriers included concerns for privacy (67.4%), follow-up (67%), and cost-effectiveness (65.4%). Human immunodeficiency virus screening facilitators included availability of health educators (83%), established follow-up (74.7%), and rapid HIV tests (65.2%). CONCLUSIONS: Emergency department clinicians exhibit poor knowledge of adolescent HIV screening recommendations. Current universal screening practices remain low; barriers to screening are numerous. Future efforts should disseminate guideline knowledge, increase rapid HIV testing and health educator availability, as well as reduce adolescent-specific barriers.

Pediatric primary care provider practices, knowledge, and attitudes of human immunodeficiency virus screening among adolescents.

OBJECTIVES: To evaluate pediatric primary care provider (PCP) HIV screening practices, knowledge, and attitudes. STUDY DESIGN: Anonymous cross-sectional, internet-based survey of pediatric PCPs from 29 primary care practices. Survey items assessed current HIV screening practices and knowledge, attitudes, and perceived barriers towards screening. Provider demographics and practice characteristics were analyzed for associations with screening through logistic regression. RESULTS: Of 190 PCPs, there were 101 evaluable responses (response rate: 53.2%). PCPs reported a screening rate for HIV of 39.6% ("most" or "all of the time") during routine adolescent visits compared with violence (60.4%), substance abuse (92.1%), and depression (94.1%) (P < .001). Less than 10% of PCPs correctly answered questions related to Centers for Disease Control and Prevention and state HIV screening recommendations. Of 20 potential HIV screening barriers assessed, mean number of reported barriers was 4.8 (SD ± 2.9); with most concerns related to confidentiality, time for counseling, and follow-up. In a multivariable model, the only factor significantly associated with HIV screening "most" or "all of the time" during routine adolescent visits was urban practice site (aOR 9.8, 95% CI 2.9, 32.9). Provider type, sex, years since training, HIV screening guideline knowledge, and endorsing ≤5 barriers were not associated with HIV screening. CONCLUSIONS: Although providers practicing in urban areas were more likely to report screening adolescents for HIV than those in suburban areas, overall self-reported screening rates were low, and several barriers were identified commonly. Future interventions should target increasing providers' knowledge and addressing concerns about confidentiality, requirements and counseling time, and follow-up of results.