A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes.

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a Km value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.

Evaluation of physical and psychological status of health care workers infected with COVID-19 during a hospital outbreak in Japan.

PURPOSE: COVID-19 causes physical and psychological impacts on health care workers (HCWs), especially when it occurs during an outbreak. As there are few reports on outcomes of HCWs infected with COVID-19 during a hospital outbreak, we investigated the physical and psychological impacts on HCWs infected with COVID-19 during an outbreak in our hospital. METHODS: During the outbreak in our hospital, 231 people were infected with COVID-19 including patients, HCWs and their families. Among them, 83 HCWs were enrolled in this study. Current quality of life (QOL) was assessed with the EuroQol-visual analogue scales (EQ-VAS), and motivation to keep on working was evaluated by a 10-point analogue scale. Physiological recovery rates including return to work (RTW) period were also analyzed. RESULTS: One nurse quit work due to anxiety regarding re-infection with COVID-19. The median period to RTW from the diagnosis was 14.0 (12.0-17.0) days. Motivation to keep on working was slightly reduced, and the EQ-VAS was 75.0 (65.0-83.6). There were no significant differences in QOL and motivation between male and female HCWs, nurses and other HCWs, treatment and non-treatment group, and supplemental and non-supplemental oxygen group. The most frequent persistent symptoms at 1,3 and 6 months after infection were anosmia followed by fatigue. CONCLUSION: Although QOL and motivation to keep on working were slightly reduced, only one HCW quit work. No severe persistent symptoms were observed, and the RTW period was relatively short.

Minor Suture Fusion is Associated With Chiari Malformation in Nonsyndromic Craniosynostosis.

Here, we focused on the association between minor suture fusion and Chiari malformation (CM) occurrence in nonsyndromic craniosynostosis (NSC), and evaluated how the minor suture affects the posterior cranial fossa by measuring the posterior fossa deflection angle (PFA). In this retrospective study, the clinical records of 137 patients who underwent surgery for NSC at Aichi Children's Health and Medical Center between April 2010 and May 2022 were analyzed. Clinical data from Aichi Developmental Disability Center Central Hospital was collected for 23 patients as the external validation set. Among the 137 patients, 123 were diagnosed with NSC and the remaining 14 with syndromic craniosynostosis. Of the 123 NSC patients, 23 patients presented with CM. Multivariate analysis showed that occipito-mastoid fusion was the only significant risk factor for CM ( P =0.0218). Within the NSC group, CM patients had a significantly increased PFA (6.33±8.10 deg) compared with those without CM (2.76±3.29 deg, P =0.0487). Nonsyndromic craniosynostosis patients with occipito-mastoid suture fusion had a significantly increased PFA (6.50±7.60 deg) compared with those without occipito-mastoid fusion (2.60±3.23 deg, P =0.0164). In the validation cohort, occipito-mastoid suture fusion was validated as an independent risk factor for CM in univariate analysis. Minor suture fusion may cause CM associated with NSC. Chiari malformation could develop due to an increased PFA due to minor suture fusion, which causes growth disturbance in the affected side and compensatory dilation in the contralateral side within the posterior cranial fossa.

Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for cabazitaxel-resistant prostate cancer.

BACKGROUND: Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines. METHODS: Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis. RESULTS: Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated. CONCLUSIONS: Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.

Albumin-bilirubin score for predicting neuropsychiatric symptoms in patients receiving ifosfamide-based chemotherapy.

WHAT IS KNOWN AND OBJECTIVE: Ifosfamide, an alkylating agent, is widely used in the treatment of malignant diseases. However, these treatments are often limited due to the incidence of neuropsychiatric symptoms such as delirium, seizures, hallucinations and agitation. In this study, we examined risk factors for neuropsychiatric symptoms in patients receiving ifosfamide-based chemotherapy. METHODS: The study cases were patients with cancer receiving ifosfamide-based chemotherapy between April 2007 and March 2018. Risk analysis for ifosfamide-related neuropsychiatric symptoms was determined by time-dependent Cox proportional hazard regression analysis. RESULTS AND DISCUSSION: Of 183 eligible patients, 32 patients (17.5%) experienced ifosfamide-related neuropsychiatric symptoms. Time-dependent Cox proportional hazard model showed that the albumin-bilirubin (ALBI) score was significantly correlated with the incidence of ifosfamide-related neuropsychiatric symptoms (hazard ratio [HR] =1.45, 95% confidence interval [CI] = 1.05-2.01, p = 0.025). Additionally, there were correlations between the predicted risk of neuropsychiatric symptoms and ifosfamide-dose per cycle (HR =0.51, 95% CI = 0.27-0.94, p = 0.030) and creatinine clearance (Ccr) (HR = 0.53, 95% CI = 0.28-1.00, p = 0.050). In contrast, neither serum albumin nor total bilirubin was a significant risk factor for neuropsychiatric symptoms. WHAT IS NEW AND CONCLUSION: These findings indicate that ALBI score may be a useful biomarker for predicting neuropsychiatric symptoms in patients receiving ifosfamide-based chemotherapy.

[Paraurethral Leiomyoma in a Female Patient who Had Clinical Symptoms with Increase of Tumor Volume: A Case Report].

A 34-year-old woman underwent total hysterectomy for management of uterine leiomyoma. At the same time, a paraurethral tumor (2 cm in size) was diagnosed based on magnetic resonance imaging (MRI). However, the patient was not treated for the tumor considering its small size. Eight years later, the patient was referred to our institution with a chief complaint of urethral bleeding. Computed tomography revealed a paraurethral mass at the same location, which was 13 cm in size. A percutaneous needle biopsy was performed and the tumor was diagnosed as leiomyoma. Tumor extirpation was performed and immunohistochemical analysis of the specimen demonstrated positive estrogen and progesterone receptors. Recurrence was not observed on MRI taken 6 months after the surgery. Paraurethral leiomyoma is rare, but relatively common in young women.

[Renal Cell Carcinoma in the Left Pelvic Kidney : A Case Report].

We report the case of a 61-year-old man who was incidentally diagnosed with a left pelvic ectopic kidney with renal tumor. Computed tomography showed a hypervascular tumor at the posterior surface of the ectopic kidney with five arterial and two venous supply vessels. On preoperative examination, this patient had respiratory dysfunction. For these reasons, an open radical nephrectomy was performed. Histological examination revealed a clear cell renal cell carcinoma, pT1aN0M0, G1, and a Fuhrman nuclear grading system grade of G2. No evidence of disease was observed 15 months after surgery.

Serum Exosomal Gamma-Glutamyltransferase Activity Increased in Patients with Renal Cell Carcinoma with Advanced Clinicopathological Features.

BACKGROUND: There has been no clinically useful diagnostic or prognostic biomarker for renal cell carcinoma (RCC). Serum γ-glutamyltransferase (GGT) activity has been reported to be a prognostic marker for several types of cancer including RCC. Exosomes or small extracellular vesicles present in body fluids have potential as a biomarker. We have recently demonstrated that GGT activity on exosomes isolated from serum is useful for the differential diagnosis of prostate cancer and benign prostate hyperplasia. In this study, we aimed to examine if serum exosomal GGT activity could be a marker for RCC. METHODS: We examined GGT1 expression and GGT activity in cell lysates and exosomes from culture medium of HK-2 proximal tubule epithelial and RCC cell lines. GGT activity was measured using a fluorescent probe for GGT, γ-glutamyl hydroxymethyl rhodamine green. Serum and serum exosomal GGT activities were measured in patients with RCC. GGT1 expression in RCC tissues was evaluated by immunohistochemical staining. RESULTS: GGT1 levels in exosomes from KMRC-1, OS-RC-2 and 786-O cells were elevated compared with those from HK-2 cells. In exosomes, GGT1 expression correlated with GGT activity determined using a fluorescent probe for GGT. In RCC patients, serum exosomal GGT activity was elevated in those with advanced stages (III/IV vs. I/II, p = 0.037) and those with microvascular invasion (with vs. without, p = 0.034). Immunohistochemical analysis showed that membranous GGT1 expression was increased in RCC with microvascular invasion. Notably, preoperative serum exosomal GGT activity could predict the likelihood of having microvascular invasion diagnosed by pathological examination of surgically resected specimens. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a clinically useful marker for advanced clinicopathological features of RCC patients, and its combined use with conventional diagnostic modalities may improve the diagnosis and treatment of patients.

Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

Predictive factors of rectal hemorrhage in patients with localized prostate cancer who underwent low-dose-rate brachytherapy.

BACKGROUND: This study aimed to evaluate the association between clinical covariates or the prescribed radiation dose for the prostate and rectal hemorrhage in patients with prostate cancer (PCa) who received iodine-125 low-dose-rate brachytherapy (LDR-BT group) or the combination of LDR-BT and external beam radiation therapy (CMT group). METHODS AND MATERIALS: In this retrospective study, we reviewed the clinical records of 298 consecutive PCa patients with clinical stage T1c/T2 who underwent LDR-BT between August 2004 and August 2016 at a single institution. The prescribed minimum peripheral doses were 145 Gy for the LDR-BT group and 104 Gy for the CMT group. The dosimetric parameters analyzed were minimal dose received by 90% of the prostate gland, biologically effective dose, and rectal volume receiving 100% (RV100) or 150% of the prescribed dose. The endpoint of this study was the onset of any-grade clinical rectal hemorrhage after treatment. RESULTS: The median follow-up period was 6.8 years. The 5-year overall survival rate was found to be 98.3%, and two patients (0.7%) reported biochemical recurrence during follow-up period. A total of 33 patients (11%) experienced rectal hemorrhage. However, ≥ grade 2 rectal hemorrhage occurred in eight patients (2.7%). On multivariate analysis, CMT, RV100 ≥ 0.66 mL, and hemorrhoids before treatment were identified as predictors of rectal hemorrhage after radiation therapy. CONCLUSIONS: Maximal reduction of the rectal dose seems very important to prevent serious rectal hemorrhage. In addition, we should consider the risk of rectal toxicities in patients with abnormalities in the rectal mucosa, especially hemorrhoids.

The Utility and Efficacy of Laparoscopic Radical Cystectomy in Patients with Muscle-Invasive Bladder Cancer at a Single Institution.

BACKGROUND: The aim of this study was to compare the surgical and oncological outcomes and complications of laparoscopic radical cystectomy (LRC) to those of open radical cystectomy (ORC) in patients with muscle-invasive bladder cancer (MIBC). METHODS: Our study focused on patients with histologically confirmed stage T2-T4a urothelial carcinoma of the bladder without distant metastases, who underwent LRC (LRC group) or ORC (ORC group). The primary endpoints in this study were the overall survival (OS) and recurrence-free survival (RFS) rates. RESULTS: In this study, 59 patients, 17 underwent LRC and 42 underwent ORC, were enrolled. The 2-year OS rate was 100% in the LRC group and 88.0% in the ORC group (p = 0.85). The 2-year RFS rate was 63.5% in the LRC group and 69.5% in the ORC group (p = 0.321). On multivariate analysis, the histological type, positive lymph node, and positive resection margin were significantly associated with the OS rates. CONCLUSIONS: This study suggested that LRC may achieve similar oncological outcomes and fewer perioperative complications and less blood loss compared to ORC. Therefore, LRC should be considered as one of the treatment options for patients with MIBC.

[Metachronous Pubic Bone Metastases from Synchronous Double Cancer with Prostate and Ascending Colon Cancers : A Case Report].

A 70-year-old man visited a private hospital with the chief complaint of right lower limb pain. Fluorodeoxyglucose-emission tomography (FDG-PET) showed abnormal uptake in the pubic bone, right femur, and ascending colon. The patient was referred to our hospital for further evaluation. The following tumor marker levels were found : prostate-specific antigen (PSA) 20.57 ng/ml, carcinoembryonic antigen (CEA) 108.5 ng/ml, carbohydrate antigen 19-9 (CA19-9) 1,002.1 U/ml. An open pubic bone biopsy was performed. The pathological diagnosis was metastatic adenocarcinoma from prostate cancer. Prostate and ascending colon cancers were clinically diagnosed as T2bN0M1b and T2N0M0, respectively. Laparoscopic colectomy was performed. Androgen deprivation therapy started immediately and the serum PSA level was maintained at <0.2 ng/ml during the follow-up period. However, the CEA and CA19-9 were higher than the normal level 2 years after the surgery. In addition, the FDG-PET revealed abnormal uptake in the pubic bone. Thus, a pubic bone biopsy was performed again. The histological diagnosis was metastatic adenocarcinoma from the ascending colon cancer. Although the patient received combination chemotherapy, he died of colon cancer.

[Retroperitoneal Dedifferentiated Liposarcoma which Could Be Diagnosed Using Dual Color in Situ Hybridization : A Case Report].

Ten years ago, a seventy-year-old female underwent extirpation of a left retroperitoneal tumor that was 58×36 mm in size. The pathological diagnosis was malignant peripheral nerve sheath tumor (MPNST) at that time. The patients visited our hospital with the chief complaint of back pain at ten years after surgery. Computer tomography (CT) showed recurrent tumors at the pancreas and the left kidney. Fine-needle aspiration biopsy was performed because of the possibility of pancreatic tumor. The pathological diagnosis was the recurrence of MPNST. The patient underwent extirpation of the recurrent tumors along with the pancreatic body and tail, transverse colon, spleen and left kidney. The definitive diagnosis was dedifferentiated liposarcoma with murine double minute 2 (MDM2) gene amplification and positive of p16Ink4 (p16). The previously resected tumor also revealed MDM2 gene amplification and positive of p16. Based on these results, our diagnosis in this case was recurrence of dedifferentiated liposarcoma. At 6 months after surgery, the patient had no local recurrence or distant metastases.

Risk factors and incidence of malignant neoplasms after kidney transplantation at a single institution in Japan.

BACKGROUND: The risk of malignant neoplasms increases in kidney transplantation (KT) recipients (KTRs). However, Japanese registry studies have not been reported since 2000. METHODS: We retrospectively reviewed the medical records of 346 patients who underwent KT at Gifu University Hospital, Japan since 2000. Patients were divided into two groups based on whether they developed malignancy after KT or not. The incidence, type of malignancy, risk factors, and prognosis for malignancy were examined. RESULTS: In this study, 22 de novo malignant neoplasms were identified in 20 KTRs (7.3%), with a median follow-up period of 8.2 years. Cumulative incidence of any malignant neoplasms was 1.1% within 1 year and 4.4% within 5 years. The 5-year overall survival (OS) rates were 71.8% in KTRs with malignant neoplasms and 98.6% in KTRs without malignant neoplasms. Uni- and multivariate analysis revealed that age at KT and acute rejection (AR) episode were significant predictors for incidence of malignancy. CONCLUSIONS: The development of malignant neoplasms was associated with poor OS and graft survival. We consider that appropriate screening and investigation of symptoms are important for KTRs, particularly for older KTRs at transplantation and those with AR episode.

Utility of robot-assisted radical cystectomy with intracorporeal urinary diversion for muscle-invasive bladder cancer.

Radical cystectomy remains the gold standard for treatment of muscle-invasive bladder cancer. Robot-assisted radical cystectomy has technical advantages over laparoscopic radical cystectomy and has emerged as an alternative to open radical cystectomy. Despite the advancements in robotic surgery, experience with total intracorporeal reconstruction of urinary diversion remains limited. Most surgeons have carried out the hybrid approach of robot-assisted radical cystectomy and extracorporeal reconstruction of urinary diversion, as intracorporeal reconstruction of urinary diversion remains technically challenging. However, intracorporeal reconstruction of urinary diversion might potentially proffer additional benefits, such as decreased fluid loss, reduction in estimated blood loss and a quicker return of bowel function. The adoption of intracorporeal ileal neobladder reconstruction has hitherto been limited to high-volume academic institutions. In the present review, we compare the totally intracorporeal robot-assisted radical cystectomy approach with open radical cystectomy and robot-assisted radical cystectomy + extracorporeal reconstruction of urinary diversion in muscle-invasive bladder cancer patients.

[Cardiac Metastasis in a Patient with Clear Cell Renal Cell Carcinoma who Underwent Multimodality Therapy for Multiple Metastases after Radical Nephrectomy].

The patient underwent laparoscopic left radical nephrectomy for clear cell renal cell carcinoma (ccRCC). After surgery, the patient had multiple lung metastases and underwent the combination therapy of radiofrequency ablation, interferon-alpha, and inteleukin-2. Thereafter, computed tomography showed multiple lymph node and brain metastases. The patient was administered targeted therapy and radiation. Eventually, the patient suddenly complained of dyspnea. An echocardiogram, coronary angiography and magnetic resonance imaging suggested acute heart failure and pericardial effusion due to a metastatic tumor in the cardiac anteroseptal and posterior wall. Nivolumab was administered for cardiac metastases. The patient has been in stable condition with no progression of cardiac metastases after the administration of nivolumab for 22 months.

GyrA and/or ParC alterations of Haemophilus influenzae strains isolated from the urethra of men with acute urethritis.

Of 73 clinical strains of Haemophilus influenzae isolated from the urethra of men with urogenital infections, we enrolled 6 strains (8.2%) with levofloxacin (LVFX) minimum inhibitory concentrations (MICs) of ≥0.03 µg/ml in this study. All the strains were isolated from non-gonococcal urethritis (NGU). We amplified the quinolone resistance-determining region of the gyrA gene and the analogous region of the parC gene from bacterial DNAs by PCR and sequenced the PCR products. Two strains with a LVFX MIC of 0.03 µg/ml had an amino acid change of Asp88 to Gly in GyrA. One with a LVFX MIC of 0.06 µg/ml had a change of Asp88 to Tyr in GyrA. Two with respective LVFX MICs of 0.12 and 0.25 µg/ml had a change of Ser84 to Leu in GyrA. One with a LVFX MIC of 1 µg/ml had changes of Ser84 to Leu in GyrA and of Ser84 to Ile in ParC. Multilocus sequence typing showed two strains with a change of Asp88 to Gly in GyrA had the same sequence type, but the others had sequence types different from each other. Single amino acid changes in GyrA alone or single changes in both GyrA and ParC could contribute to decreased susceptibility to fluoroquinolones in H. influenzae isolates from NGU. Most of the isolates with GyrA and/or ParC alterations would be multiclonal. The prevalence of such isolates would be relatively low, and they would still be susceptible to fluoroquinolones commonly prescribed for treatment of NGU.

Macrolide and fluoroquinolone resistance is uncommon in clinical strains of Chlamydia trachomatis.

We analyzed the 23S rRNA, gyrA and parC genes of Chlamydia trachomatis DNAs from men with urethritis and determined microbiological outcomes of an extended-release azithromycin (azithromycin-SR) regimen (2 g once daily for 1 day) and a sitafloxacin regimen (100 mg twice daily for 7 days) for chlamydial urethritis to clarify the macrolide and fluoroquinolone resistance status of clinical strains of C. trachomatis. We amplified the portions of 2 alleles of the 23S rRNA gene and the gyrA and parC genes from C. trachomatis DNAs in 284 first-voided urine specimens from men with chlamydial urethritis by PCR and sequenced their PCR products. We enrolled 369 men with chlamydial urethritis, comprising 314 and 55 treated with the azithromycin-SR regimen and the sitafloxacin regimen, respectively. Alleles 1 and/or 2 of the 23S rRNA gene were analyzed in 162 specimens. No mutations were found in the sequenced regions, including the central portion of domain V. The gyrA and parC genes were analyzed in 118 and 113 specimens, respectively. No amino acid changes were found within the quinolone resistance-determining region of the gyrA gene and in the sequenced region of the parC gene. The microbiological outcomes of the azithromycin-SR and sitafloxacin regimens were assessed in 176 and 30 men, respectively. The eradication rates were 96.0% (95% CI 93.1%-98.9%) for the azithromycin-SR regimen and 100% for the sitafloxacin regimen. Clinical strains of C. trachomatis with macrolide and/or fluoroquinolone resistance would be uncommon, and azithromycin or fluoroquinolone regimens could be recommended as treatments for chlamydial infections.

Exosomes expressing carbonic anhydrase 9 promote angiogenesis.

Exosomes or microvesicles that are secreted from cells are considered to play important roles in tumor microenvironment. Carbonic anhydrase 9 (CA9), which is induced by hypoxia-inducible factor 1 (HIF1) in response to hypoxia, is overexpressed in many types of cancer including renal cell carcinoma (RCC). We examined the expression level of CA9 in several RCC cell lines and found that the basal level of CA9 was much higher in OSRC-2 cells than in Caki-1, KMRC-1 and 786-O cells. Consistent with the intracellular expression levels, CA9 was abundantly detected in exosomes isolated by ultracentrifugation from OSRC-2 cells. Density gradient centrifugation of OSRC-2 and 786-O exosomes confirmed the co-presence of CA9 with exosomal markers. Upon hypoxia and treatment with CoCl2, a hypoxia mimic agent, the CA9 level in exosomes was increased for all cell lines. In order to examine the effects of CA9 exosomes on angiogenesis, we generated stably transfected HEK293 cells expressing CA9. Immunocytochemical staining demonstrated the uptake of CA9 exosomes by human umbilical vein endothelial cells (HUVEC). In vitro angiogenesis assays using HUVEC revealed that CA9 exosomes promoted migration and tube formation. Lastly, MMP2 expression was increased by treatment with CA9 exosomes in HUVEC. Taken together, our results suggest the possibility that CA9 exosomes released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression.