The CDK Inhibitor SIAMESE Targets Both CDKA;1 and CDKB1 Complexes to Establish Endoreplication in Trichomes.

Endoreplication, also known as endoreduplication, is a modified cell cycle in which DNA is replicated without subsequent cell division. Endoreplication plays important roles in both normal plant development and in stress responses. The SIAMESE (SIM) gene of Arabidopsis (Arabidopsis thaliana) encodes a cyclin-dependent kinase (CDK) inhibitor that plays a central role in establishing endoreplication, and is the founding member of the SIAMESE-RELATED (SMR) family of plant-specific CDK inhibitor genes. However, there has been conflicting evidence regarding which specific cyclin/CDK complexes are inhibited by SIM in vivo. In this work, we use genetic evidence to show that SIM likely inhibits both CDKA;1- and CDKB1-containing CDK complexes in vivo, thus promoting endoreplication in developing Arabidopsis trichomes. We also show that SIM interacts with CYCA2;3, a binding partner of CDKB1;1, via SIM motif A, which we previously identified as a CDK-binding motif. By contrast, SIM motif C, which has been indicated as a cyclin binding motif in other contexts, appears to be relatively unimportant for interaction between SIM and CYCA2;3. Together with earlier results, our work suggests that SIM and other SMRs likely have a multivalent interaction with CYC/CDK complexes.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

Impact of radiology reports on timely tuberculosis diagnosis.

PURPOSE: As tuberculosis becomes less common in higher income countries, clinician familiarity with the disease is declining. Little is known about how chest radiograph interpretations affect tuberculosis care. We sought to determine how tuberculosis-related terminology in an initial chest radiograph reading impacted patient care. STUDY DESIGN: We examined a retrospective cohort of patients with pulmonary tuberculosis in North Carolina from 1 January 2011 to 31 December 2014. Tuberculosis-related terminology was categorised into four mutually exclusive categories. The primary outcomes of interest were the time from the chest radiograph to (1) obtaining the first sputum specimen for acid-fast smear and mycobacterial culture, and (2) initiation of antituberculous treatment. RESULTS: Of 550 available chest radiograph reports, 175 (31.8%) contained the word 'tuberculosis', 30 (5.5%) contained the word 'mycobacteria' or 'granulomatous', 43 (7.8%) contained the word 'cavity', and 301 (54.7%) had none of the above terms mentioned. Patients with the word 'tuberculosis' in the radiology report had a significantly shorter time to collection of the initial sputum specimen for acid-fast smear and mycobacterial culture (median 2 days) and to the start of antituberculous treatment (median 4 days) than patients with none of the keywords. Use of the term 'cavity' in the report was associated with a shorter time to initiation of antituberculous treatment (median 4 days) than if none of the keywords were used. CONCLUSION: Chest radiograph reports that contained keywords for pulmonary tuberculosis, such as 'tuberculosis' or 'cavity', were associated with less time to collection of sputum and antituberculous treatment.

Association Between Staff Experience and Effective Tuberculosis Contact Tracing in North Carolina, 2008-2009.

BACKGROUND: Effective investigation of tuberculosis (TB) contacts is essential for continued progress toward TB elimination. As the incidence of TB declines, staff experience will also decline. Little is known about the association between the experience level of public health TB staff and the quality of contact investigations. METHODS: Contact investigations involving fewer than 30 contacts during the period 2008-2009 were included in this analysis. Multivariable models were used to examine associations between staff TB experience (assessed by a standardized survey) and measures of contact investigation quality: time from case identification to contact identification and number of contacts identified per case investigated. RESULTS: A total of 501 cases and 3,230 contacts met the inclusion criteria. Data were stratified by the number of cases in the county and whether the case was smear-positive or smear-negative. For contacts of smear-positive cases, greater staff experience was associated with more rapid contact identification both in counties with high case counts (hazard ratio [HR] = 2.43; 95% CI, 1.79-3.31) and in counties with low case counts (HR = 1.142; 95% CI, 0.95-1.37). However, for smear-negative cases, staff in counties with low case counts identified contacts more slowly as years of experience increased (HR = 0.82; 95% CI, 0.62-1.07). For contacts of smear-negative cases, more contacts (relative risk [RR] = 1.20; 95% CI, 1.07-1.35) were identified per case in high case-count counties (more than 20 cases during 2008-2009). Conversely, in low case-count counties, fewer contacts were identified per case (RR = 0.94; 95% CI, 0.82-1.08); however, this finding was not significant. DISCUSSION: Speed of identification and number of contacts are imperfect surrogates for the most important outcome of contact investigations-that is, the rapid identification and treatment of infected contacts. CONCLUSION: More TB experience was associated with more rapid and thorough TB contact investigations. Retaining experienced staff and mentoring staff new to case management should be high priorities for TB control programs.

HIV-specific health care utilization and mortality among tuberculosis/HIV coinfected persons.

Persons coinfected with tuberculosis (TB) and HIV are at high risk of death, in part due to suboptimal utilization of HIV-specific health care. We sought to better understand HIV-associated health care utilization and mortality in a retrospective cohort of TB/HIV coinfected cases reported in North Carolina 1993-2003. In this cohort, HIV was newly diagnosed during TB presentation for 34.2% of coinfected patients. Patients had advanced HIV (median CD4 104 cells/mm(3)) at TB diagnosis. Of 260 patients previously known to be HIV positive, 32.3% had seen a physician for HIV care in the previous 6 months and only 18.5% were taking antiretrovirals when TB was diagnosed; 34.8% of patients started antiretrovirals during TB treatment. Twenty-seven (5%) patients died prior to starting TB treatment; of those who survived, 13.6% (70/515) died prior to completing TB treatment, and 42.7% (220/515) died during a median 1408 days of follow-up. CD4 count (relative risk [RR] 0.53 per 100 cell increase, 95% confidence interval [CI] 0.34, 1.02) and highly active antiretroviral therapy (HAART) use during TB therapy (RR 0.37, 95% CI 0.13, 1.02) were independently associated with decreased mortality, while age greater than 45 (RR 2.18, 95% CI 1.11, 4.29) was independently associated with increased mortality during TB treatment. We conclude that TB/HIV coinfected patients had low utilization rates of HIV-specific care prior to TB diagnosis. Many did not receive potentially lifesaving HIV treatment while on TB therapy, and mortality was high as a result. Interventions to enhance utilization of HIV-related health care and integration of TB and HIV services should be studied to improve outcomes.