Functional analysis of mutant and wild-type Drosophila origin recognition complex

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.     

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.     

Spirituality and medicine

INTRODUCTION: Governing bodies for medical education recommend that spirituality and medicine be incorporated into training. AIM: To pilot a workshop on spirituality and medicine on a convenience sample of preclinical medical students and internal medicine residents and determine whether content was relevant to learners at different levels, whether preliminary evaluation was promising, and to generate hypotheses for future research. SETTING: Private medical school and university primary care internal medicine residency program, both in the Northeast. CURRICULUM DESCRIPTION: The authors designed and implemented a required 2-hour workshop for all second-year medical students and a separate required 1.5-hour workshop for all primary care internal medicine house staff. The workshops used multiple educational strategies including lecture, discussion, and role-play to address educational objectives. PROGRAM EVALUATION: Learners completed optional, anonymous pre and postworkshop surveys with six 5-point Likert-rated statements and space to cite the most useful part of the curriculum and their remaining questions. One hundred and thirty-seven learners participated and 100 completed both surveys. Medical students and residents had increased (all P< or =.002): agreement regarding the appropriateness of inquiring about spiritual and religious beliefs in the medical encounter, their perceived competence in taking a spiritual history, and their perceived knowledge of available pastoral care resources. Medical students, but not residents, had an increase in their perceived comfort in working with hospital chaplains. DISCUSSION: A brief pilot workshop on spirituality and medicine had a modest effect in improving attitudes and perceived competence of both medical students and residents.