The role of protein phosphatase 2A (PP2A) in the unfolded protein response (UPR) of plants.

Protein phosphatase 2A (PP2A) is a key regulator of plant growth and development, but its role in the endoplasmic reticulum (ER) stress response remains elusive. In this study, we investigated the function of PP2A under ER stress using loss-of-function mutants of ROOTS CURL of NAPHTHYLPHTHALAMIC ACID1 (RCN1), a regulatory A1 subunit isoform of Arabidopsis PP2A. RCN1 mutants (rcn1-1 and rcn1-2) exhibited reduced sensitivity to tunicamycin (TM), an inhibitor of N-linked glycosylation and inducer of unfolded protein response (UPR) gene expression, resulting in less severe effects compared to wild-type plants (Ws-2 and Col-0). TM negatively impacted PP2A activity in Col-0 plants but did not significantly affect rcn1-2 plants. Additionally, TM treatment did not influence the transcription levels of the PP2AA1(RCN1), 2, and 3 genes in Col-0 plants. Cantharidin, a PP2A inhibitor, exacerbated growth defects in rcn1 plants and alleviated TM-induced growth inhibition in Ws-2 and Col-0 plants. Furthermore, cantharidin treatment mitigated TM hypersensitivity in ire1a&b and bzip28&60 mutants. These findings suggest that PP2A activity is essential for an efficient UPR in Arabidopsis.

CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.

Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.

Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction.

TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

Inhibition of S6K1 enhances glucose deprivation-induced cell death via downregulation of anti-apoptotic proteins in MCF-7 breast cancer cells.

Nutrient-limiting conditions are frequently encountered by tumor cells in poorly vascularized microenvironments. These stress conditions may facilitate the selection of tumor cells with an inherent ability to decrease apoptotic potential. Therefore, selective targeting of tumor cells under glucose deprivation conditions may provide an effective alternative strategy for cancer therapy. In the present study, we investigated the effects of S6 kinase 1 (S6K1) inhibition on glucose deprivation-induced cell death and the underlying mechanisms in MCF-7 breast cancer cells. PF4708671, a selective inhibitor of S6K1, and knockdown of S6K1 with specific siRNA enhanced cell death induced under glucose deprivation conditions. Moreover, inhibition of S6K1 led to apoptosis in glucose-starved MCF-7 cells via downregulation of the anti-apoptotic proteins, Mcl-1 and survivin. Further experiments revealed that sorafenib, shown to be involved in Mcl-1 and survivin downregulation via mTOR/S6K1 inhibition significantly promotes cell death under glucose deprivation conditions. These findings collectively suggest that S6K1 plays an important role in tumor cell survival under stress conditions, and thus inhibition of S6K1 may be an effective strategy for sensitizing cells to glucose deprivation.

Effects of the molecular weight and the degree of deacetylation of chitosan oligosaccharides on antitumor activity.

Effects of the degree of deacetylation (DDA) and the molecular mass of chitosan oligosaccharides (CTS-OS), obtained from the enzymatic hydrolysis of high molecular weight chitosan (HMWC), on antitumor activity was explored. The DDA and molecular weights of CTS-OS were determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF MS) analysis. The CTS-OS were found to be a mixture of mainly dimers (18.8%), trimers (24.8%), tetramers (24.9%), pentamers (17.7%), hexamers (7.1%), heptamers (3.3%), and octamers (3.4%). The CTS-OS were further fractionated by gel-filtration chromatography into two major fractions: (1) COS, consisting of glucosamine (GlcN)(n), n = 3-5 with DDA 100%; and (2) HOS, consisting of (GlcN)(5) as the minimum residues and varying number of N-acetylglucosamine (GlcNAc)(n), n = 1-2 with DDA about 87.5% in random order. The cytotoxicities, expressed as the concentration needed for 50% cell death (CC(50)), of CTS-OS, COS, and HOS against PC3 (prostate cancer cell), A549 (lung cancer cell), and HepG2 (hepatoma cell), were determined to be 25 µg·mL(-1), 25 µg·mL(-1), and 50 µg·mL(-1), respectively. The HMWC was approximately 50% less effective than both CTS-OS and COS. These results demonstrate that the molecular weight and DDA of chitosan oligosaccharides are important factors for suppressing cancer cell growth.

The Effect of Mechanical Tongue Cleaning on Oral Malodor and Tongue Coating.

BACKGROUND: Mechanical tongue cleaning is an important oral hygiene procedure; it is known that a significant cause of volatile sulfur compounds (VSCs), a major component of bad breath, is due to the bacteria coating the tongue. This study was conducted to identify the effect of mechanical tongue cleaning on reducing bad breath and tongue coating. METHODS: Various mechanical tongue-cleaning methods were studied, including removing tongue coating using a toothbrush, removing tongue coating using a tongue scraper, and removing tongue coating using a toothbrush and a tongue scraper together. The results were as follows. RESULTS: First, the organic bad breath measurement value after cleaning the tongue significantly decreased in the group using only the toothbrush, the group using only the tongue scraper, and the group using both the toothbrush and the tongue scraper. However, there was no difference between the groups. Second, after cleaning the tongue, the measured values of the tongue coating in the values of WTCI (Winkel's tongue coating index) and Qray view were significantly reduced in all three groups, and there was no difference between the groups. Third, the gas measurement value in the oral cavity using a machine significantly decreased only the H2S value of the group using the tongue scraper immediately after the mechanical tongue cleaning. CONCLUSIONS: From these results, it can be confirmed that mechanical tongue cleaning is effective at reducing bad breath and tongue coating. However, in this study, there was no difference in the reduction effect according to the tools (groups) used for mechanical tongue cleaning. It can therefore be seen that wiping accurately from the rear of the tongue to the front is more effective at reducing bad breath and tongue coating.

Determination of Residual Triflumezopyrim Insecticide in Agricultural Products through a Modified QuEChERS Method.

A rapid and simple analytical method for triflumezopyrim, a new class of mesoionic insecticides and commercialized molecules from DuPont, was developed with a modified QuEChERS method. The pH adjustment was used to improve the extraction efficiency of acetonitrile solvent, and dispersive solid-phase extraction was employed for the clean-up process. The five selected food commodities were used to verify the present optimized method, which displayed good linearity with an excellent correlation coefficient (R2 = 0.9992-0.9998) in the 0.003-0.30 mg/kg calibration range. The method limits of detection (LOD) and quantification (LOQ) were determined to be a value of 0.003 and 0.01 mg/kg, respectively. The mean recovery for the triflumezopyrim was in the 89.7-104.3% range. The relative standard deviations were ≤9.8% for intra- (n = 5) and inter-day (n = 15) precisions at concentrations of 0.01, 0.1, and 0.5 mg/kg in the five representative samples. The matrix effect has been calculated to confirm the effect during ionization of the analyte in the UPLC-MS/MS. The matrix effects of the instrumental analysis showed that triflumezopyrim was less susceptible to matrices. The proposed analytical method in this study has effectively improved the accuracy, selectivity, and sensitivity for the determination of triflumezopyrim in agricultural commodities; therefore, it can serve as a reference method for the establishment of maximum residue limits (MRLs).

JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-kappaB activation and the JAK/STAT pathway.

JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic beta-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1beta and interferon (IFN)-gamma-induced beta-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor kappaB (NF-kappaB) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3(-/-) mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects beta-cells from cytokine toxicity through suppression of the NF-kappaB and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional beta-cell mass.

Dihydroavenanthramide D protects pancreatic beta-cells from cytokine and streptozotocin toxicity.

Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Although its anti-inflammatory, antiatherosclerotic, and antioxidant effects have been reported, the effect of DHAvD on type 1 diabetes is unknown. Therefore, in this study, the effect of DHAvD on cytokine- or streptozotocin-induced beta-cell damage was investigated. Treatment of RINm5F insulinoma cells or isolated islets with IL-1beta and IFN-gamma induced beta-cell damage through a NF-kappaB-dependent signaling pathway. DHAvD-pretreated RINm5F cells or islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced the inducible form of NO synthase expression, and decreased beta-cell destruction and the normal insulin secretion capacity. Furthermore, pretreatment with DHAvD blocked the development of type 1 diabetes in streptozotocin-treated mice. Prior injection with DHAvD maintained a normal range of plasma glucose and insulin, and retained immunoreactivity for insulin in the pancreas. These results suggest that DHAvD may be used to preserve functional beta-cell mass.

Serum response factor enhances liver metastasis of colorectal carcinoma via alteration of the E-cadherin/beta-catenin complex.

Serum response factor (SRF) is a transcription factor that controls cell growth, differentiation, and tumor progression as well as muscle development and function. Reduced expression of cell adhesion molecules has been reported to be associated with tumor metastasis. The aim of this study was to evaluate the expression and a role of SRF in liver metastasis of primary colorectal carcinomas. We examined the expression of SRF, E-cadherin, and beta-catenin by the use of immunochemical staining in 43 cases as a set of primary colorectal carcinomas and liver metastases. We also examined the role of SRF in colorectal carcinoma by overexpression of SRF in a colon cancer cell line. In metastatic carcinoma surgical samples, there was a marked increased expression of SRF as compared to expression in primary colorectal carcinoma surgical samples (P<0.05). E-cadherin expression was significantly decreased in metastatic liver carcinoma samples as compared to primary colorectal carcinoma samples (P<0.001). Frequent nuclear translocation of beta-catenin protein in primary and metastatic carcinoma cells was observed. Overexpression of SRF in SW480 cells resulted in a decreased expression of E-cadherin and an increased expression of non-phosphorylated nuclear beta-catenin. Overexpression of SRF in colorectal carcinoma cells enhanced cell motility and invasiveness. These results indicate that overexpression of SRF in colorectal carcinoma cells is associated with modulation of E-cadherin/beta-catenin expression and may play an important role in colorectal cancer metastasis.

Expression of the serum response factor in hepatocellular carcinoma: implications for epithelial-mesenchymal transition.

The acquisition of a migratory and invasive phenotype by cells of epithelial origin is associated with a gain of mesenchymal characteristics concomitant with a loss of the epithelial phenotype, a phenomenon referred to as epithelial-mesenchymal transition (EMT). Vimentin, a cytoplasmic intermediate filament, is characteristic of mesenchymal cells and is usually not expressed in epithelial cells. Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor invasion. Serum response factor (SRF) regulates transcription of immediate early genes and triggers proliferation, migration and differentiation in several types of cells. However, the role of SRF in hepatocellular carcinoma (HCC) has not been explored. The aims of this study were to evaluate the expression of SRF and to assess a functional role of SRF in HCC. First, we examined the expression of SRF in 55 human specimens of HCC and four different HCC cell lines, including a sarcomatoid HCC cell line. We also examined the role of SRF in HCC by transfection of an SRF expression plasmid into a HCC cell line. SRF was expressed in 13 of 55 cases of HCC. SRF was predominantly expressed in HCC cells, with intense labeling in the nucleus. No staining was observed in hepatocytes of normal and cirrhotic liver outside the tumor. SRF was significantly up-regulated in high grade HCC, especially in sarcomatoid HCC. Overexpression of SRF in hepatocellular carcinoma cells accelerates migration and invasion with subsequent acquisition of mesenchymal phenotype by expression of a mesenchymal marker (vimentin) and activation of immediate early genes. We propose for the first time that the expression of SRF in HCC cells associated with EMT may play an important role in HCC progression. Thus, functional antagonism of SRF will provide an additional therapeutic approach by controlling tumor cell invasion and metastasis.

Overexpression of discoidin domain receptor 1 increases the migration and invasion of hepatocellular carcinoma cells in association with matrix metalloproteinase.

The discoidin domain receptor (DDR) is a class of receptor tyrosine kinases that binds to several collagens. DDR1 is widely expressed in fast-growing invasive tumors of the breast, ovary, esophagus, brain and lung. However, there is little information on the expression of DDR1 in hepatocellular carcinoma (HCC) or its function in migration and invasion. Western blot analysis was performed to determine if four HCC cell lines (HLE, Huh-7, HepG2 and SH-J1) express DDR1. The HLE and Huh-7 cell lines were transfected with two isoforms of DDR1, DDR1a and DDR1b. Immunoprecipitation for DDR1 was then performed. Migration and invasion assays were carried out and the number of migrating cells was counted in 6 randomly selected fields per well under an optical microscope. Zymography was used to determine the level of the matrix metalloproteinase (MMP)-2 and -9 expression. DDR1 was expressed in all four cell lines. In the migration assay, the number of migrating cells was significantly higher in the DDR1a- or DDR1b-overexpressing HLE and Huh-7 cells, particularly after collagen type I stimulation (P<0.001). Collagen type I stimulation activated DDR1. In the invasion assay, there was a significantly higher number of invading cells in the DDR1a- or DDR1b-overexpressing HLE cells and DDR1a-overexpressing Huh-7 cells than in the control (P<0.01). The DDR1a- and DDR1b-overexpressing HLE cells showed a remarkable increase in the MMP-9 and -2 expression, particularly the active MMP-2. The DDR1a- and DDR1b-overexpressing Huh-7 cells showed a slight increase in the MMP-9 and -2 expression. The increased invasiveness of the HCC may be associated with the overexpression of either DDR1a or DDR1b mediated by MMP-2 and -9. Although this study provided one possible mechanism for the invasion of HCC cells, more studies are needed to understand the signal through which DDR1a and DDR1b act in invasion.

A peroxisome proliferator-activated receptor gamma antagonist induces vimentin cleavage and inhibits invasion in high-grade hepatocellular carcinoma.

Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor metastasis. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been intensively evaluated as a potential target for the inhibition of cell growth and metastasis in cancer cells. In the present study, we examined whether PPARgamma is a possible target molecule for the prevention of cell growth and invasion by treatment with agonists (troglitazone, rosiglitazone) and antagonists (T0070907, GW9662) in four different hepatocellular carcinoma (HCC) cell lines. We also evaluated the effects of the PPARgamma agonists and antagonists on tumor cell migration and invasion. The expression level of PPARgamma protein was higher in the sarcomatoid SH-J1 and poorly differentiated HLE cell lines than that in the well-differentiated HCC cell lines (HepG2 and Huh-7). Expression of vimentin was high in the SH-J1 HCC cell line and minimally detected in the HLE cell line. Treatment with low doses of the PPARgamma antagonists inhibited cell growth and colony formation of all four of the HCC cell lines. Vimentin in the high-grade HCC cells was cleaved by the treatment with the PPARgamma antagonists. Furthermore, treatment with the PPARgamma antagonists also strongly inhibited migration and invasion of the SH-J1 and HLE cells. However, treatment with low doses of the agonists had no effect on vimentin expression, migration, and invasion of the high-grade HCC cells but cell growth was inhibited by treatment with high concentrations of the agonists. Our results indicate that treatment with a PPARgamma antagonist may prevent cell growth and invasion of high-grade HCC cells. Our findings also suggest that PPARgamma antagonists inhibit cell growth and invasion through vimentin disarrangement in high-grade HCC.

[Expression of caveolin in hepatocellular carcinoma: association with unpaired artery formation and radiologic findings].

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide, and it is characterized by its high vascularity. Caveolin is the major structural protein in caveolae, which are small omega-shaped invaginations within the plasma membrane. Caveolin has been implicated in mitogenic signaling, oncogenesis and angiogenesis. The expression of caveolin-1 and -2 in HCC and its potential relationship with angiogenesis has not been examined. METHODS: Paraffin sections of 35 HCC specimens were immunostained with caveolin-1, caveolin-2, alpha-smooth muscle actin, and CD34 antibodies. In addition, the expression of caveolin-1 and -2 mRNA in HCC was examined. The relationship between the radiological findings and the number of unpaired arteries and microvessel density (MVD) was also investigated. RESULTS: Caveolin-1 and -2 were expressed in the sinusoidal endothelial cells in 20 out of 35, and 18 out of 35 HCC specimens, respectively. Caveolin-1 and -2 were also expressed in the smooth muscle cells of the unpaired arteries in 26 out of 35, and 18 out of 35 HCC specimens, respectively. Increased expression of caveolin-1 and -2 mRNA was detected in 26.7% and 33.3% of the tumor specimens, respectively, compared with the corresponding non-tumorous adjacent liver tissues. There was a significant correlation between expression of caveolin-1, -2 in the smooth muscle cells of unpaired arteries and the number of unpaired arteries. The number of unpaired arteries in HCCs was found to be associated with the degree of contrast enhancement in the arterial phase imaging. However, it did not correlate with the degree of MVD. CONCLUSIONS: These findings suggest that the expression of caveolin-1, -2 is associated with the formation of unpaired arteries in HCC. In addition, there is a correlation between the degree of contrast enhancement of the HCC in the arterial phase image and the number of unpaired arteries.

Prosthetic Valve Endocarditis caused by HACEK Organisms: a Case Reports and Systematic Review of the Literature.

HACEK is a rare cause of prosthetic valve endocarditis (PVE). We describe 42-year-old male patient who presented with Aggregatibacter aphrophilus PVE and cerebral infarct. A. aphrophilus was isolated from his blood cultures as the sole pathogen, which was confirmed by subsequent 16S rRNA sequencing. He was treated with valve replacement surgery and an 8 week course of pathogen-directed antibiotic therapy and followed for 20 months without recurrence.

Simple assessment of olfaction in patients with chronic rhinosinusitis.

CONCLUSION: The brief-smell identification test (B-SIT) can substitute for the butanol threshold test (BTT) in screening of anosmia and postoperative assessment of olfactory outcomes in patients with chronic rhinosinusitis (CRS). A time-effective test battery composed of B-SIT and the visual analog scale (VAS) can be implemented for simple olfactory assessment in any otolaryngology clinic. OBJECTIVES: Anosmia is a distinct clinical entity requiring special attention. Unpredictable olfactory outcomes after surgery make preoperative assessment more important. We compared the results of the BTT, B-SIT, and VAS to investigate whether B-SIT or VAS can substitute for BTT in screening of anosmia and postoperative follow-up. METHODS: We collected data on 68 CRS patients who had bilateral CRS and underwent endoscopic sinus surgery. Olfactory performance was graded using the BTT: normosmia, hyposmia, or anosmia. VAS and B-SIT were also performed. All tests were repeated 6 months after surgery. Postoperative improvement was defined by an increase of the BTT score ≥ 2. RESULTS: The B-SIT and VAS scores of the anomics were significantly lower than those of the normosmics. B-SIT discriminated anosmia with high specificity. Within the improvement group, postoperative increase of B-SIT/VAS score showed significance. However, neither the B-SIT nor the VAS differentiated between no change and deterioration of olfaction.

Do sinus computed tomography findings predict olfactory dysfunction and its postoperative recovery in chronic rhinosinusitis patients?

BACKGROUND: Olfactory dysfunction secondary to chronic rhinosinusitis (CRS) is a mixed disorder of conductive and sensorineural olfactory impairment. Although endoscopic sinus surgery has some beneficial effects on olfaction, the outcomes are challenging to predict. The aim of this study was to assess the olfactory outcomes after surgery, to investigate the correlation between the severity of regional computed tomography (CT) findings and olfactory performance, and to identify the predictors of postoperative outcomes based on unilateral olfactory threshold analysis. METHODS: This study included 167 CRS nostrils of 97 patients with/without polyps (68/99 nostrils) undergoing sinus surgery between January 2007 and December 2011. Olfactory function was evaluated using the butanol threshold test (BTT) before and 6 months after surgery. Clinical and nasal factors from sinus CT scan (sinuses, ostiomeatal complex, olfactory cleft [OC], nasal polyps, and unilateral Lund-Mackay CT score) were analyzed to correlate them with pre- and postoperative olfactory performances. RESULTS: Eighty-two percent of the CRS nostrils had anosmia or hyposmia. After surgery, 42% of them showed an improvement in BTT score. Despite improvement, most of the subjects remained with residual hyposmia. The BTT scores deteriorated after surgery in 23% of the total subjects. The disease severity of the OC, posterior ethmoid, and frontal sinus were the significant risk factors for CRS-related anosmia. The strongest risk factor for anosmia was totally obstructed OC (odds ratio [OR], 16.56; 95% CI, 4.31-63.71; p = 0.000). The nostrils with anosmia or partly opacified anterior ethmoid benefited from surgery with respect to olfaction. CONCLUSION: Our results can give support to the combined use of the butanol threshold and sinonasal CT findings in the evaluation of olfaction in CRS patients and help us counsel the patients about the likelihood of postoperative olfactory recovery.

Clinical Usefulness of PCR for Differential Diagnosis of Tuberculosis and Nontuberculous Mycobacterial Infection in Paraffin-Embedded Lung Tissues.

The need for isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased in recent years. Our aim was to determine the clinical usefulness of PCR for differential diagnosis of tuberculosis and nontuberculous mycobacterial infection in lung tissue that show chronic granulomatous inflammation. A total of 199 formalin-fixed, paraffin-embedded specimens, including 137 Mycobacterium tuberculosis (MTB), 17 NTM cases, and 45 other than mycobacterial cases were collected. We performed acid-fast staining, MTB and NTM nested PCRs, and MTB and NTM real-time PCRs. No histologic difference between MTB and NTM infections was observed. Sensitivity and specificity for detecting MTB were 70.1% and 95.1% by nested PCR, respectively, and 70.8% and 100.0% by real-time PCR, respectively. Sensitivity and specificity for detecting NTM were 52.9% and 96.15% by nested PCR, respectively, and 35.3% and 100.0% by real-time PCR, respectively. Mycobacteria were identified by acid-fast staining in 50 of 154 cases (32.5%). All 50 acid-fast staining-positive cases showed positive nested and real-time PCR results (n = 47 MTB PCR positive; n = 3 NTM PCR positive), and results agreed with final diagnosis. PCR will be useful for the rapid diagnosis of mycobacterial infection and differentiation of MTB from NTM in formalin-fixed, paraffin-embedded specimens, especially in acid-fast staining-positive specimens.

Characterization of CD45-/CD31+/CD105+ circulating cells in the peripheral blood of patients with gynecologic malignancies.

PURPOSE: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells. EXPERIMENTAL DESIGN: The frequency of representative CEC subsets (i.e., CD45(-)/CD31(+), CD45(-)/CD31(+)/CD146(+), CD45(-)/CD31(+)/CD105(+)) was analyzed in the peripheral blood of patients with gynecologic cancer (n = 56) and healthy volunteers (n = 44). CD45(-)/CD31(+) cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry. RESULTS: CD45(-)/CD31(+)/CD105(+) cells were significantly increased in the peripheral blood of patients with cancer, whereas evaluation of CD45(-)/CD31(+)/CD146(+) cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry analyses showed that these CD45(-)/CD31(+)/CD105(+) cells did not express vWF and CD146 but rather CD144. Furthermore, CD45(-)/CD31(+)/CD105(+) cells uniformly expressed the monocyte-specific markers CD14 and CD68. These results suggest that CD45(-)/CD31(+)/CD105(+) cells carry the characteristics of monocytes rather than endothelial cells. CONCLUSIONS: Our data indicate that CD45(-)/CD31(+)/CD105(+) circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis.

Sustained overexpression of Redd1 leads to Akt activation involved in cell survival.

Herein, we show that the constitutive overexpression of Redd1, a negative regulator of mTORC1, induces Akt activation in lung cancer cells. Akt phosphorylation was reduced to basal levels by Rictor siRNA, suggesting the involvement of mTORC2 in this process. Perifosine and PP242, selective inhibitors of Akt and mTORC1/2, respectively, efficiently suppressed the Akt phosphorylation that was induced by the sustained overexpression of Redd1 and increased the sensitivity of the cells to cisplatin. Therefore, the sustained overexpression of Redd1 leads to mTORC1 inhibition and to consequent Akt activation that is involved in cell survival. This finding highlights the importance of Akt activation as a therapeutic target to overcome resistance to chemotherapy.