We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors—the local density of DnaA boxes and their affinities for DnaA—are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.

The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under-and overexpression of YabA. ChAP-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaA Y144C mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.

BackgroundThe sensitivities and specificities of interferon-gamma release assays (IGRAs) vary among different population studies, and the data on the routine use of IGRAs are limited. The aim of this study was to evaluate the role of QuantiFERON-TB Gold In-Tube (QFT-GIT) test in the diagnosis of active tuberculosis.MethodsWe conducted a prospective study, enrolling 77 patients with suspected pulmonary tuberculosis (TB), at a secondary care teaching hospital in Seoul.ResultsIn total, 12 (15.6%) patients showed indeterminate results due to positive control failure on the QFT-GIT test. Indeterminate results were significantly associated with the elderly, history of the intensive care unit stay, lymphocytopenia, especially low CD4 count, increased C-reactive protein and decreased protein levels. Of the 77 patients, 44 (57.1%) were diagnosed with active pulmonary tuberculosis, and the percentage of false negative results of the QFT-GIT was 36.4% (vs. 31.8% with TST). In the TB group with >65 years old (n=12), the proportions of the indeterminate (33.3% vs. 3.1%) and the false negative results (58.3% vs. 25.0%) of the QFT-GIT were significantly higher than in the younger TB group (n=32).ConclusionIndeterminate and false negative results of QFT-GIT test were not infrequent in tuberculosis, especially in the elderly. Care should be considered for the interpretation with the elderly, immunocompromised, chronic and severely diseased patients.

Adiponectin predominantly secreted from adipose tissue has exhibited potent anti-proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti-cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF-7) cancer cells. Gene ablation of FoxO3A prevented gAcrp-induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF-7 cells. Transfection with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp-induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of FoxO3A in HepG2 and MCF-7 cells. In addition, suppression of AMPK also blocked gAcrp-induced cell cycle arrest and further attenuated gAcrp-induced caspase-3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp-induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti-proliferative effect of adiponectin in cancer cells.

Although it is assumed that most patients with terminal cancer are reluctant to receive life-sustaining treatment, there is a paucity of evidence supporting this assumption. We retrospectively analyzed the advance directives of terminal cancer patients to determine the preferences of patients. Patients with cancer who had life expectancy of less than 6 months were admitted to a palliative care unit in Seoul Medical Center from March 2008 to February 2010. Among a total of 247 patients, advance directives were present in the medical records of 168 patients (68.0%). Most of the advance directives were written by the patients’ families (95.2%) and they stated that they did not want most of the invasive procedures. Patients with advanced cancer mostly requested that only minimally invasive treatments that eased suffering be performed.