Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of ß-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

Extended-spectrum ß-lactamase-producing Plesiomonas shigelloides isolated from the stool of a Japanese traveler returning from Rwanda: A case report.

A 21-year-old previously healthy Japanese woman visited an outpatient clinic because of abdominal pain, watery diarrhea, vomiting, and mild fever that had started on the previous day. She traveled to rural and urban areas of Rwanda and returned to Japan 3 days before. Stool culture yielded the Plesiomonas shigelloides strain TMCH301018, against which minimum inhibitory concentrations of cefotaxime and cefotaxime-clavulanate were 128 and ≤0.12/4 µg/mL, respectively. The strain had the blaCTX-M-27 gene and an IncA/C replicon-type plasmid. Moreover, a transformant produced by introduction of an IncA/C plasmid extracted from TMCH301018 into Escherichia coli DH5α was positive for the blaCTX-M-27 gene and fulfilled the criteria of extended-spectrum ß-lactamase (ESBL) production described by the Clinical and Laboratory Standards Institute, indicating that TMCH301018 produced ESBL of CTX-M-27 and the ESBL-encoding gene was located on an IncA/C plasmid. Pathogenicity of TMCH301018 for the patient's complaints was uncertain because a molecular assay detected other enteropathogens in the stool specimen and the symptoms improved within 2 days with administration of oral ciprofloxacin, to which TMCH301018 was not susceptible. To our knowledge, this is the first report describing the isolation of ESBL-producing P. shigelloides.

Estrogen induces genomic instability in high-risk HPV-infected cervix and promotes the carcinogenesis of cervical adenocarcinoma.

High-risk human papillomavirus (HPV) infection is the major cause of cervical cancer. However, the factors that modulate the process from infection to carcinogenesis are poorly understood. Although cervical cancer is clinically considered an estrogen-independent tumor, the role of estrogen in cervical cancer, particularly cervical adenocarcinoma, remains controversial. In this study, we showed that estrogen/GPR30 signaling induced genomic instability, which leads to carcinogenesis in high-risk HPV-infected endocervical columnar cell lines. The expression of estrogen receptors in a normal cervix was confirmed through immunohistochemical analysis which showed that G protein-coupled receptor 30 (GPR30) was predominantly expressed in endocervical glands and estrogen receptor-α (ERα) was expressed at higher levels in the squamous epithelium than in the cervical gland. E2 increased the proliferation of cervical cell lines, particularly normal endocervical columnar and adenocarcinoma cells via GPR30 rather than ERα, and increased the accumulation of DNA double-strand breaks (DSBs) in high-risk HPV-E6-expressing cells. The increase in DSBs was caused by the impairment of Rad 51 and accumulation of topoisomerase-2-DNA complexes under HPV-E6 expression. In addition, chromosomal aberrations increased in cells with E2-induced DSB accumulation. Collectively, we conclude that E2 exposure in high-risk HPV-infected cervical cells increases DSBs, leading to genomic instability and thus carcinogenesis via GPR30.

Prospective study of three saliva qualitative antigen testing kits for the detection of SARS-CoV-2 among mainly symptomatic patients in Japan.

INTRODUCTION: Rapid qualitative antigen testing has been widely used for the laboratory diagnosis of COVID-19 with nasopharyngeal samples. Saliva samples have been used as alternative samples, but the analytical performance of those samples for qualitative antigen testing has not been sufficiently evaluated. METHODS: A prospective observational study evaluated the analytical performance of three In Vitro Diagnostics (IVD) approved COVID-19 rapid antigen detection kits for saliva between June 2022 and July 2022 in Japan using real-time reverse transcription polymerase chain reaction (RT-qPCR) as a reference. A nasopharyngeal sample and a saliva sample were simultaneously obtained, and RT-qPCR was performed. RESULTS: In total, saliva samples and nasopharyngeal samples were collected from 471 individuals (RT-qPCR-positive, n = 145) for the analysis. Of these, 96.6% were symptomatic. The median copy numbers were 1.7 × 106 copies/mL for saliva samples and 1.2 × 108 copies/mL for nasopharyngeal samples (p < 0.001). Compared with the reference, the sensitivity and specificity were 44.8% and 99.7% for ImunoAce SARS-CoV-2 Saliva, 57.2% and 99.1% for Espline SARS-CoV-2 N, and 60.0% and 99.1% for QuickChaser Auto SARS-CoV-2, respectively. The sensitivities of all antigen testing kit were 100% for saliva samples with a high viral load (>107 copies/mL), whereas the sensitivities were <70% for high-viral-load nasopharyngeal samples (>107 copies/mL). CONCLUSION: COVID-19 rapid antigen detection kits with saliva showed high specificity, but the sensitivity varied among kits, and were also insufficient for the detection of symptomatic COVID-19 patients.

Analytical performance of the rapid qualitative antigen kit for the detection of SARS-CoV-2 during widespread circulation of the Omicron variant.

INTRODUCTION: Rapid qualitative antigen testing is essential in the clinical management of COVID-19. However, most evaluations of antigen tests have been performed before the emergence of the Omicron variant. METHODS: This prospective observational study evaluated QuickNavi-COVID19 Ag, a rapid antigen detection test between December 2021 and February 2022 in Japan, using real-time reverse transcription (RT)-PCR as a reference. Two nasopharyngeal samples were simultaneously collected for antigen testing and for RT-PCR. Variant analysis of the SARS-CoV-2 genomic sequencing was also performed. RESULTS: In total, nasopharyngeal samples were collected from 1073 participants (417 positive; 919 symptomatic; 154 asymptomatic) for analysis. Compared with those of RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 94.2% (95% CI: 91.6%-96.3%), 99.5% (95% CI: 98.7%-99.9%), 99.2% (95% CI: 97.8%-99.8%), and 96.5% (95% CI: 94.8%-97.7%), respectively. The sensitivity among symptomatic individuals was 94.3% (95% CI: 91.5%-96.4%). Overall, 85.9% of sequences were classified as Omicron sublineage BA.1, 12.4% were Omicron sublineage BA.2, and 1.6% were Delta B.1.617.2. (Delta variant). Most of the samples (87.1%) had Ct values of <25, and the sensitivity was 47.4% for low viral load samples (Ct ≥ 30); a similar trend has been observed in both symptomatic and asymptomatic groups. CONCLUSIONS: The QuickNavi-COVID19 Ag test showed sufficient diagnostic performance for the detection of the SARS-CoV-2 Omicron sublineages BA.1 and BA.2 from nasopharyngeal samples. However, the current study was mainly performed in symptomatic patients and the results are not sufficiently applicable for asymptomatic patients.

Diagnostic performance of a novel digital immunoassay (RapidTesta SARS-CoV-2): A prospective observational study with nasopharyngeal samples.

INTRODUCTION: Digital immunoassays are generally regarded as superior tests for the detection of infectious disease pathogens, but there have been insufficient data concerning SARS-CoV-2 immunoassays. METHODS: We prospectively evaluated a novel digital immunoassay (RapidTesta SARS-CoV-2). Two nasopharyngeal samples were simultaneously collected for antigen tests and Real-time RT-PCR. RESULTS: During the study period, 1127 nasopharyngeal samples (symptomatic patients: 802, asymptomatic patients: 325) were evaluated. For digital immunoassay antigen tests, the sensitivity was 78.3% (95% CI: 67.3%-87.1%) and the specificity was 97.6% (95% CI: 96.5%-98.5%). When technicians visually analyzed the antigen test results, the sensitivity was 71.6% (95% CI: 59.9%-81.5%) and the specificity was 99.2% (95% CI: 98.5%-99.7%). Among symptomatic patients, the sensitivity was 89.4% (95% CI; 76.9%-96.5%) with digital immunoassay antigen tests, and 85.1% (95% CI; 71.7%-93.8%) with visually analyzed the antigen test, respectively. CONCLUSIONS: The sensitivity of digital immunoassay antigen tests was superior to that of visually analyzed antigen tests, but the rate of false-positive results increased with the introduction of a digital immunoassay device.

Evaluation and clinical implications of the time to a positive results of antigen testing for SARS-CoV-2.

INTRODUCTION: Antigen tests for severe acute respiratory coronavirus 2 sometimes show positive lines earlier than their specified read time, although the implication of getting the results at earlier time is not well understood. METHODS: We prospectively collected additional nasopharyngeal samples from patients who had already tested positive for SARS-CoV-2 by reverse transcription PCR. The swab was used for an antigen test, QuickNavi™-COVID19 Ag, and the time periods to get positive results were measured. RESULTS: In 84 of 96 (87.5%) analyzed cases, the results of QuickNavi™-COVID19 Ag were positive. The time to obtain positive results was 15.0 seconds in median (inter quartile range: 12.0-33.3, range 11-736) and was extended in samples with higher cycle thresholds (p < 0.001). Positive lines appeared within a minute in 85.7% of cases and within 5 min in 96.4%. CONCLUSION: QuickNavi™-COVID19 Ag immediately showed positive results in most cases, and the time to a positive reaction may have indicated the viral load.

A prospective clinical evaluation of the diagnostic accuracy of the SARS-CoV-2 rapid antigen test using anterior nasal samples.

INTRODUCTION: The diagnostic accuracy of antigen testing of anterior nasal (AN) samples for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has not been evaluated in the Japanese population. This study assessed the diagnostic accuracy of the Roche SARS-CoV-2 rapid antigen test (rapid antigen test) using AN samples. METHODS: Two AN samples and one nasopharyngeal (NP) sample were collected from individuals undergoing screening for SARS-CoV-2 infection. The results of the rapid antigen test and the reverse-transcription polymerase chain reaction (RT-PCR) test using AN samples were compared to those of RT-PCR tests using NP samples. RESULTS: Samples were collected from 800 participants, 95 and 110 of whom tested positive for SARS-CoV-2 on RT-PCR tests of AN and NP samples, respectively. The overall sensitivity/specificity of the AN rapid antigen test and AN RT-PCR were 72.7%/100% and 86.4%/100%, respectively. In symptomatic cases, the sensitivities of the AN rapid antigen test and AN RT-PCR were 84.7% and 94.9%, respectively. In asymptomatic cases, the sensitivities of the AN rapid antigen test and AN RT-PCR were 58.8% and 76.5%, respectively. The sensitivity of the AN rapid antigen test was over 80% in cases with cycle threshold (Ct) values < 25; it significantly decreased with an increase in the Ct values (p < 0.001). CONCLUSION: The rapid antigen test with AN samples had a favorable sensitivity, especially in symptomatic cases or in cases with Ct values < 25. It gave no false-positive results. Compared with AN-RT PCR, the AN rapid antigen test had a modestly lower sensitivity in asymptomatic cases.

A prospective evaluation of diagnostic performance of a combo rapid antigen test QuickNavi-Flu+COVID19 Ag.

INTRODUCTION: Since respiratory sample collection is an uncomfortable experience, simultaneous detection of pathogens with a single swab is preferable. We prospectively evaluated the clinical performance of a newly developed antigen test QuickNavi-Flu+COVID19 Ag (Denka Co., Ltd., Tokyo, Japan) which can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the same time with a single testing device. METHODS: We included those who were suspected of contracting coronavirus disease 2019 (COVID-19) and were referred to a PCR center at Ibaraki prefecture in Japan, between August 2, 2021 to September 13, 2021, when the variant carrying L452R spike mutation of SARS-CoV-2 were prevalent. Additional nasopharyngeal samples and anterior nasal samples were obtained for the antigen test and were compared with a reference real-time reverse transcription PCR (RT-PCR) using nasopharyngeal samples. RESULTS: In total, 1510 nasopharyngeal samples and 862 anterior nasal samples were evaluated. During the study period, influenza viruses were not detected by QuickNavi-Flu+COVID19 Ag and reference real-time RT-PCR. For SARS-CoV-2 detection in nasopharyngeal samples, the sensitivity and specificity of the antigen test were 80.9% and 99.8%, respectively. The sensitivity and specificity using anterior nasal samples were 67.8% and 100%, respectively. In symptomatic cases, the sensitivities increased to 88.3% with nasopharyngeal samples and 73.7% with anterior nasal samples. There were three cases of discrepant results between the antigen test and the real-time RT-PCR. All of them were positive with the antigen test but negative with the real-time RT-PCR in SARS-CoV-2 detection. CONCLUSION: A combo kit, QuickNavi-Flu+COVID19 Ag, showed an acceptable sensitivity and sufficient specificity for SARS-CoV-2 detection, especially using nasopharyngeal sample collected from symptomatic patients.

Clinical evaluation of the rapid nucleic acid amplification point-of-care test (Smart Gene SARS-CoV-2) in the analysis of nasopharyngeal and anterior nasal samples.

INTRODUCTION: Smart Gene is a point-of-care (POC)-type automated molecular testing platform that can be performed with 1 min of hands-on-time. Smart Gene SARS-CoV-2 is a newly developed Smart Gene molecular assay for the detection of SARS-CoV-2. The analytical and clinical performance of Smart Gene SARS-CoV-2 has not been evaluated. METHODS: Nasopharyngeal and anterior nasal samples were prospectively collected from subjects referred to the local PCR center from March 25 to July 5, 2021. Two swabs were simultaneously obtained for the Smart Gene SARS-CoV-2 assay and the reference real-time RT-PCR assay, and the results of Smart Gene SARS-CoV-2 were compared to the reference real-time RT-PCR assay. RESULTS: Among a total of 1150 samples, 68 of 791 nasopharyngeal samples and 51 of 359 anterior nasal samples were positive for SARS-CoV-2 in the reference real-time RT-PCR assay. In the testing of nasopharyngeal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 99.2% (95% confidence interval [CI]: 98.4-99.7%), 94.1% (95% CI: 85.6-98.4%) and 99.7% (95% CI: 99.0-100%), respectively. For anterior nasal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 98.9% (95% CI: 97.2-99.7%), 98.0% (95% CI: 89.6-100%) and 99.0% (95% CI: 97.2-99.8%), respectively. In total, 5 samples were positive in the reference real-time RT-PCR assay and negative in the Smart Gene SARS-CoV-2 assay, whereas 5 samples were negative in the reference real-time RT-PCR assay and positive in the Smart Gene SARS-CoV-2 assay. CONCLUSION: Smart Gene SARS-CoV-2 showed sufficient analytical performance for the detection of SARS-CoV-2 in nasopharyngeal and anterior nasal samples.

Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy.

Cancer immunotherapy has already shown significant improvements by combining different antibodies specific for distinct immune checkpoints, such as Ipilimumab and Nivolumab. Here, we tested combinatorial treatments of immunomodulatory antibodies, previously generated in our laboratory, for their effects on hPBMC activation, either upon stimulation with SEB or in co-cultures with tumor cells by cytokine secretion assays. We found that some of them showed additive or synergistic effects, and on the basis of these observations, we constructed, for the first time, four novel bispecific tribodies (TR), made up of a Fab derived from one anti-IC mAb and two scFvs derived from another mAb targeting a different IC. All four TRs cotargeting either programmed cell death protein 1 (PD-1) and Lymphocyte Activating 3 (LAG-3) or programmed death-ligand 1 (PD-L1) and LAG-3 retained binding affinity for their targets and the antagonistic effects of their parental mAbs, but some of them also showed an increased ability to induce lymphocyte activation and increased in vitro cytotoxicity against tumor cells compared to parental antibodies used either alone or in combinatorial treatments. Furthermore, none of the tribodies showed significant increased cytotoxicity on human cardiomyocytes. Considering that the tribody format reduces production costs (as only one construct provides the inhibitory effects of two antibodies), has an intermediate molecular size (100 kDa) which is well suited for both tumor penetration and an acceptable half-life, we think that these novel immunomodulatory TRBs have the potential to become precious tools for therapeutic applications, particularly in monotherapy-resistant cancer patients.

Prospective analytical performance evaluation of the QuickNavi™-COVID19 Ag for asymptomatic individuals.

INTRODUCTION: Antigen testing may help screen for and detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in asymptomatic individuals. However, limited data regarding the diagnostic performance of antigen tests for this group are available. METHODS: We used clinical samples to prospectively evaluate the analytical and clinical performance of the antigen test QuickNavi™-COVID19 Ag. This study was conducted at a PCR center between October 7, 2020 and January 9, 2021. Two nasopharyngeal samples per patient were obtained with flocked swabs; one was used for the antigen test, and the other for real-time reverse transcription PCR (RT-PCR). The diagnostic performance of the antigen test was compared between asymptomatic and symptomatic patients, and the RT-PCR results were used as a reference. RESULTS: Among the 1934 collected samples, 188 (9.7%) demonstrated detection of SARS-CoV-2 by real-time RT-PCR; 76 (40.4%) of these 188 samples were from asymptomatic individuals, and over half of the total samples were asymptomatic (1073; 55.5%). The sensitivity of the antigen test was significantly lower for the asymptomatic group than for symptomatic patients (67.1% vs. 89.3%, respectively, p < 0.001). The specificity was 100% for both groups, and no false positives were observed among all 1934 samples. The median cycle threshold value for the asymptomatic group was significantly higher than that of the symptomatic group (24 vs. 20, p < 0.001). CONCLUSIONS: The QuickNavi™-COVID19 Ag showed lower sensitivity for the asymptomatic group than for symptomatic patients. However, its specificity was consistently high, and no false positives were found in this study.

The evaluation of a newly developed antigen test (QuickNavi™-COVID19 Ag) for SARS-CoV-2: A prospective observational study in Japan.

INTRODUCTION: Several antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed worldwide, but their clinical utility has not been well established. In this study, we evaluated the analytical and clinical performance of QuickNavi™-COVID19 Ag, a newly developed antigen test in Japan. METHODS: This prospective observational study was conducted at a PCR center between October 7 and December 5, 2020. The included patients were referred from a local public health center and 89 primary care facilities. We simultaneously obtained two nasopharyngeal samples with flocked swabs; one was used for the antigen test and the other for real-time reverse transcription PCR (RT-PCR). Using the results of real-time RT-PCR as a reference, the performance of the antigen test was evaluated. RESULTS: A total of 1186 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 105 (8.9%). Of these 105 patients, 33 (31.4%) were asymptomatic. The antigen test provided a 98.8% (95% confidence interval [CI]: 98.0%-99.4%) concordance rate with real-time RT-PCR, along with a sensitivity of 86.7% (95% CI: 78.6%-92.5%) and a specificity of 100% (95% CI: 99.7%-100%). False-negatives were observed in 14 patients, 8 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). In symptomatic patients, the sensitivity was 91.7% (95% CI: 82.7%-96.9%). CONCLUSION: QuickNavi™-COVID19 Ag showed high specificity and sufficient sensitivity for the detection of SARS-CoV-2. This test is a promising potential diagnostic modality especially in symptomatic patients.

The evaluation of a novel digital immunochromatographic assay with silver amplification to detect SARS-CoV-2.

INTRODUCTION: Rapid antigen tests are convenient for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they have lower sensitivities than nucleic acid amplification tests. In this study, we evaluated the diagnostic performance of Quick Chaser® Auto SARS-CoV-2, a novel digital immunochromatographic assay that is expected to have higher sensitivity than conventional antigen tests. METHODS: A prospective observational study was conducted between February 8 and March 24, 2021. We simultaneously obtained two nasopharyngeal samples, one for evaluation with the QuickChaser® Auto SARS-CoV-2 antigen test and the other for assessment with reverse transcription PCR (RT-PCR), considered the gold-standard reference test. The limit of detection (LOD) of the new antigen test was compared with those of four other commercially available rapid antigen tests. RESULTS: A total of 1401 samples were analyzed. SARS-CoV-2 was detected by reference RT-PCR in 83 (5.9%) samples, of which 36 (43.4%) were collected from symptomatic patients. The sensitivity, specificity, positive predictive value, and negative predictive value were 74.7% (95% confidence interval (CI): 64.0-83.6%), 99.8% (95% CI: 99.5-100%), 96.9% (95% CI: 89.2-99.6%), and 98.4% (95% CI: 97.6-99.0%), respectively. When limited to samples with a cycle threshold (Ct) < 30 or those from symptomatic patients, the sensitivity increased to 98.3% and 88.9%, respectively. The QuickChaser® Auto SARS-CoV-2 detected 34-120 copies/test, which indicated greater sensitivity than the other rapid antigen tests. CONCLUSIONS: QuickChaser® Auto SARS-CoV-2 showed sufficient sensitivity and specificity in clinical samples of symptomatic patients. The sensitivity was comparable to RT-PCR in samples with Ct < 30.

Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor microRNA replacement therapy in ovarian cancer.

MicroRNA (miRNA) plays a pivotal role in cancer biology. Therefore, tumor suppressor (TS) miRNAs are an attractive target for cancer therapy. However, clinical trials have failed due to the difficulties in miRNA delivery, warranting the development of a novel drug delivery system (DDS). Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. The aim of this study was to explore the possibility of exosomes as a carrier for miRNA replacement therapy for ovarian cancer (OC). First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA, and the synthesized miR-199a-3p was loaded into exosomes by electroporation. Treatment with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) drastically increased miR-199a-3p expression level in OC cell lines (CaOV3; 8592-, SKOV3; 67188-, and OVCAR3; 2280-fold). miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cell proliferation and invasion. In a xenograft study, miR-199a-3p-Exo also drastically inhibited peritoneal dissemination in OC mice model, and diminished c-Met expression, ERK phosphorylation, and MMP2 expression in tumors. These results suggest that miRNA replacement therapy using exosomes shows promise for treatment of OC. Given that omental fibroblasts can be obtained from most OC patients, patient-derived exosomes can be utilized as a DDS for future molecular-targeted therapies.

[Lung Cancer Complicated with Empyema Due to Intratumoral Abscess;Report of a Case].

A 73-year-old man was admitted to our hospital because of fever. A chest computed tomography (CT) scan revealed a 10-cm-sized irregular mass with air-fluid level in the left lower lobe, and sputum cytology revealed squamous cell carcinoma, which was diagnosed as a giant squamous cell carcinoma with intratumoral abscess. Later, the empyema developed and drainage was performed for empyema and intratumoral abscess. Two weeks later, left lower lobectomy and lymph node dissection were performed by posterolateral thoracotomy. After surgery, there were no serious complications or relapse of empyema.

Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor.

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.

The novel small-molecule antagonist of PAC1 receptor attenuates formalin-induced inflammatory pain behaviors in mice.

We recently developed PA-8, a novel small-molecule antagonist of PACAP type 1 (PAC1) receptor. In the present study, we examined whether PA-8 was effective against formalin-induced inflammatory pain in mice. Both intrathecal and oral administration of PA-8 resulted in the dose-dependent attenuation of the second phase of formalin-induced nociceptive responses. PA-8 also inhibited c-fos upregulation in the ipsilateral dorsal horn of the spinal cord. The results suggested that PACAP-PAC1 receptor signaling system in the spinal cord were primarily involved in the transmission of inflammatory pain, and PA-8 could be useful for the development of novel analgesics for treating inflammatory pain.

Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells.

BACKGROUND: microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. METHODS: Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. RESULTS: The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. CONCLUSIONS: Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

Optimization and in vivo evaluation of pyrazolopyridines as a potent and selective PI3Kδ inhibitor.

Chemical optimization of pyrazolopyridine 1, focused on cellular potency, isoform selectivity and microsomal stability, led to the discovery of the potent, selective and orally available PI3Kδ inhibitor 5d. On the basis of its desirable potency, selectivity and pharmacokinetic profiles, 5d was tested in the trinitrophenylated aminoethylcarboxymethyl-Ficoll (TNP-Ficoll)-induced antibody production model, and showed higher antibody inhibition than a 4-fold oral dose of the starting compound 1. These excellent results suggest that 5d is a potential candidate for further studies in the treatment of autoimmune diseases and leukocyte malignancies.