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Several therapeutic agents are available for metastatic castration-resistant prostate cancer (CRPC). However, prognosis is still not well developed. The Gleason score (GS) is a prognostic factor available for patients with metastatic CRPC. GSs ranging from 6 to 10 and GSs ≥8 are usually categorized as single prognostic factors. In this study, we evaluated the prognosis of high-GS metastatic CRPC in Japanese men.Overall, 105 patients with metastatic CRPC with a GS ≥8 were retrospectively analyzed. Multivariate analyses of patient age, GS, and Eastern Cooperative Oncology Group performance status (ECOG-PS) were performed using Cox proportional hazards analysis to predict overall survival (OS).GS 8 had all Gleason patterns of 4+4. Thirty patients (28.6%) had GS of 8, and 75 (71.4%) had GS of 9 or 10. As a first-line treatment for metastatic CRPC, 42 patients (40%) received abiraterone, 35 (33.3%) received enzalutamide, and 26 (24.8%) received docetaxel. The 5-year OS in patients with GS of 8 was 65.0% [95% confidence interval (CI): 43.07-86.82%], while the 5-year OS in patients with GS of 9 or 10 was 37.0% (95% CI: 24.41-56.11%). There was a significant difference in OS between the GS 8 and GS 9-10 groups (log-rank test, P=0.038). Multivariate analysis showed that GS and ECOG-PS were significant prognostic factors for OS.Patients with metastatic CRPC with GS 9-10 had poor prognoses, suggesting the need for additional treatment options.

Recently, combination therapy including immune checkpoint inhibition (ICI) has proven to be effective as first-line therapy for patients with metastatic renal cell carcinoma. Although the first-line combination therapies with ICI have shown clinical benefit, a number of patients require second-line treatment. We report a 60-year-old man with metastatic renal cell carcinoma who was treated with pazopanib soon after nivolumab plus ipilimumab combination therapy. He experienced Grade 3 disseminated intravascular coagulation (DIC). We suspect that this was caused by an interaction between pazopanib and nivolumab even though ICI therapy was discontinued. He was treated with thrombomodulin and platelet transfusion and recovered from DIC. Treatment with pazopanib was subsequently restarted. No evidence of DIC was observed thereafter. This severe adverse reaction may have been induced by an interaction between activated proinflammatory immune cells and cytokines from an exacerbated inflammatory state and pazopanib. This report highlights the need to perform careful monitoring of patients who receive molecular targeted therapy after ICI-based immunotherapy.

Hiroharu Kamioka,1 Yasunori Mori,2 Takahiko Horiuchi,3 Takahito Hayashi,4 Koichiro Ohmura,5 Satoru Yamaguchi,6 Mitsutoshi Kato7 1Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan; 2Mie Prefecture Health and Environment Research Institute, Yokkaichi, Mie, Japan; 3Department of Internal Medicine, Kyushu University Beppu Hospital, Beppu, Oita, Japan; 4Department of Legal Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; 5Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine, Kyoto, Japan; 6Department of Oriental Medicine, Saitama Medical University, Saitama, Japan; 7Kato Clinic of Internal Medicine, Tokyo, JapanCorrespondence: Hiroharu KamiokaFaculty of Regional Environment Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-Ku, Tokyo 156-8502, JapanTel/Fax +81 35477 2587Email h1kamiok@nodai.ac.jpPurpose: To clarify the relationship between daily hot water bathing (HWB) at home and glycemic control in middle-aged and elderly ambulatory patients with type 2 diabetes mellitus (T2DM).Methods: We defined hemoglobin A1c (HbA1c) as the main outcome. We set 7.0% based on the mean value of the dependent variable as the cut-off point for analysis. Frequency of HWB was an explanatory variable. A two-sample t-test was used to compare between groups with continuous variables. Multiple logistic regression analysis was performed for frequency, adjusted age, sex, BMI, T2DM duration (Model 1), and other confounding factors (Model 2). Odds ratio (OR) and 95% confidence interval (95% CI) were calculated.Results: Among 838 patients, there was a significant difference (p< 0.001) in age between males (n=528, 62.8± 8.7 years) and females (n=310, 65.0± 8.1 years). In Model 1, compared with participants who used HWB more than seven times a week, those with poorly controlled HbA1c were significantly associated with low frequency of HWB: four to six times a week (OR 1.32, 95% CI 0.87– 1.99) and less than three times a week (OR 1.43, 95% CI 0.98– 2.10); p-value for overall trend was 0.041. In Model 2, p-value for overall trend was 0.138.Conclusion: A higher frequency of HWB was moderately associated with a decreased risk of poor glycemic control in middle-aged and elderly ambulatory patients with T2DM.Keywords: hot water bathing, type 2 diabetes, hemoglobin A1c, middle-aged and elderly ambulatory patients

Significance Mammalian central synapses of diverse functions contribute to highly complex brain organization, but the molecular basis of synaptic diversity remains open. This is because current synapse proteomics are restricted to the “average” composition of abundant synaptic proteins. Here, we demonstrate a subcellular proteomic workflow that can identify and quantify the deep proteome of synaptic vesicles, including previously missing proteins present in a small percentage of central synapses. This synaptic vesicle proteome revealed many proteins of physiological and pathological relevance, particularly in the low-abundance range, thus providing a resource for future investigations on diversified synaptic functions and neuronal dysfunctions., Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Gut microbes are intricately involved in maintaining normal physiology and homeostasis and have become a major area of study to determine their relevance in human disease. In cancer, alterations to gut microbiota has been associated to resistance to chemo- and immuno-therapy, whereas supplementation with specific taxa improves antitumor treatment responses. Mouse models have been essential to study disease biology and drug discovery. However, mice are fundamentally quite different from humans, thus, questions have arisen regarding their utility as pertinent tools to study microbial influences and their impact on human disease. To address these questions, we have performed a cross-species comparative analysis of the fecal microbiota from a human cohort of prostate cancer patients and a preclinical mouse model of prostate cancer. Microbiota composition was determined by 16s RNA gene sequencing on stool samples from tumor-bearing prostate-specific conditional Pten-knockout and disease-free wildtype mice and 16s RNA gene sequencing data from a human cohort of patients with suspected prostate cancer was used. Human cases were assigned to no cancer and cancer groups based on diagnosis from prostate biopsy and as low-risk (negative biopsy of Gleason grade Citation Format: Chisato Wakamori, Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Kazuko Sakai, Makoto Matsushita, Eri Banno, Yasunori Mori, Masahiro Nozawa, Mitsuhisa Nishimoto, Kazuhiro Yoshimura, Norio Nonomura, Kazuto Nishio, Hirotsugu Uemura. Integrative gut microbiome analysis of human and mouse prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3044.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature immune cells that expand during cancer and can induce potent immune suppression. These cells play critical roles in supporting tumor growth and progression and their induction has been associated with the inactivation of PTEN. To date, few therapies targeting these cells are available which is partly due to their increasingly complex nature and context-dependent functions. Moreover, there is no clear definition to identify these cells. Enhancing our understanding of this cell population will aid in developing more effective cancer therapies. Here, we used gene expression profiling, flow cytometry, and quantitative immunohistochemistry to characterize tumor-infiltrating and circulating MDSCs in prostate carcinogenesis and progression in transgenic mouse models of prostate cancer driven by the conditional inactivation of Pten and Trp53. Transcriptomic profiling revealed enrichment of various myeloid-derived cell signatures in the early stages of prostate cancer including MDSCs, macrophages, and neutrophils. Flow cytometric analysis confirmed the increased infiltration of CD11b+/Ly6G+ (polymorphonuclear MDSCs (pMDSCs)) and CD11b+/Ly6C+ (monocytic MDSCs (mMDSCs)) cells in prostate tumors. The temporal dynamics of tumor-infiltrating myeloid cells were further profiled and showed that pMDSC and mMDSC abundances were 1.6 and 5.7-fold higher, respectively, in older mice with late-stage locally invasive adenocarcinomas (AdCa) compared to younger mice with early-stage prostatic intraepithelial neoplasia (PIN). In both instances, pMDSCs comprised most of the leukocyte infiltrate, however, mMDSCs tended to be positively correlated with tumor burden in AdCa but not PIN. In peripheral blood, CD44+/CD62L+ immature neutrophils were associated with high tumor burden in aged mice. Treatments targeting the androgen receptor (AR) signaling further exacerbated pMDSC infiltration in tumors and was associated with resistance to second androgen receptor-targeted agent (ARTA) therapy in a model of castration resistant prostate cancer. Our findings indicate that MDSCs are a prominent population in Pten-null prostate cancer and are implicated with resistance to prostate cancer treatment. MDSCs constitute a potentially targetable population and these models serve as viable platforms to further investigate their biological activity and further evaluate novel MDSC-directed therapies. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Eri Banno, Shogo Adomi, Mitsuhisa Nishimoto, Takafumi Minami, Yasunori Mori, Kazutoshi Fujita, Masahiro Nozawa Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2877.

Studies suggest that certain immune-mediated diseases may be associated with cancer risk beyond local organs. Prolonged systemic inflammation from inflammatory bowel diseases such as ulcerative colitis (UC) and Crohn’s disease (CD) pose a greater risk to colorectal cancer (CRC) and accumulating data suggests that prostate cancer (PCa) risk is higher in patients with UC but not CD and that patients with PCa may be at a greater risk for CRC. A better understanding of the biological interactions between these conditions could have important implications for cancer screening, detection, and prevention in high-risk populations. However, data to establish the relationships and relative contributions of these diseases are lacking. Here, we explored these relationships using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and DSS/azoxymethane (AOM)-induced CRC (DSS/AOM-CRC) in young and aged healthy wildtype mice and transgenic mice harboring Pten-deficient PCa. In the DSS/AOM-CRC model, aged PCa-bearing conditional Pten-knockout (KO) mice experienced poorer overall survival compared to age-matched wildtype (WT) without PCa because of CRC. DSS/AOM-CRC incidence was not affected by the presence of PCa, however tumor burden was higher in KO mice compared to WT and in KO mice, DSS/AOM-CRC accelerated PCa growth and progression. Prostates from WT DSS/AOM-CRC mice showed higher incidence of hyperplasia and greater myeloid cell infiltration compared to healthy controls. Systemic changes associated with DSS/AOM-CRC in both WT and KO mice included splenomegaly, lymphadenopathy, and immune cell composition. The impact of DSS-UC was investigated conditional Pten/Trp53-double knockout (DKO) mice with DDS only treatments starting at an age prior to the development of PCa. In this setting, cycling DSS led to the development of UC after 10 weeks of treatment and accelerated prostate tumor development characterized by high grade prostatic intraepithelial neoplasia (PIN) grade dysplasia and increased cell proliferation. Prostate tumors from mice with DSS-UC exhibited an altered tumor immune microenvironment characterized by a higher degree of inflammatory cell infiltrate. Changes in immune cell composition were also observed in peripheral blood and secondary lymphoid organs. These preclinical models recapitulated disease relationships observed in humans and provide a platform to further investigate biological mechanisms and therapeutic interventions. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Shogo Adomi, Eri Banno, Yasunori Mori, Takafumi Minami, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Exploring the relationships between prostate cancer, colorectal cancer, and ulcerative colitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 640.

The World Health Organisation (WHO) recommends that the concentration of radon in water should be no more than 100 kBq m−3 (100 BqL−1) and the Codex Alimentarius Commission states that the limit of quantification (LOQ) of a method should be no more than one-fifth of this value. In this study, a degassing method with an RAD7 device was used to measure radon concentrations in water, compared to a liquid scintillation counter (LSC) method used as the reference, to investigate whether the numerical value of the LOQ of this method was more than 1/5 (20 kBq m–3) of 100 kBq m–3. The degassing method with leak prevention was shown to reach a target value of 20 kBq m−3 or less under a relative humidity of 6% or lower in the chamber of the RAD7 device. Accordingly, the RAD7 degassing method with leak prevention can be used to accurately measure radon concentrations in water within the guidance level set out by the WHO.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertories were ‘delayed’, not disrupted, in gnrh3-/- and npff-/- males, while the double knockout showed normal behaviors. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in the motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which balanced release of co-existing peptides is important for the neuromodulatory function of TN-GnRH neurons in the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to external environments.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.

BACKGROUND: Inflammatory cytokines and immature myeloid derived suppressor cells (MDSCs), which increase during cancer progression, could lead to a neutrophil increase and lymphocyte reduction. Thus, the neutrophil-lymphocyte ratio (NLR) was used to predict survival of patients suffering from urological cancers including upper urinary tract carcinoma. We further determined whether the NLR during the first cycle of first-line chemotherapy could predict cancer specific survival. METHODS: We recruited patients with locally advanced or metastatic upper urinary tract urothelial carcinoma (UTUC) who received chemotherapy between January 2014 and July 2019. We investigated the impact of various clinical variables, including age, sex, performance status, and estimated creatinine clearance (CCr), and NLR before and after the first cycle of the first-line chemotherapy on prognosis. RESULTS: A total of 41 patients were included in our study. Cancer specific survival of the patients with lower NLR was significantly better than that of the patients with higher NLR measured after the first cycle of the first-line chemotherapy (log-rank test P=0.005, median 29.2 vs. 11.9 months, respectively). Cox proportional regression analysis showed that higher NLR after the first cycle of the first-line chemotherapy was a significant predictor of cancer specific survival. CONCLUSIONS: The NLR after the first cycle of the first-line chemotherapy could be an indication for patients with locally advanced or metastatic UTUC to maintain their first-line chemotherapy treatment.

A 67-year-old man underwent open radical left nephrectomy for left renal cell carcinoma [pT4N0M1 (right lower lobe of lung)] and thoracoscopic partial right lung resection for lung metastasis. The patient subsequently developed a solitary lung metastasis at 10 months and then at 26 months postoperatively. He underwent partial lung resection on each occasion. During the 28 months postoperatively, he was found to have a 12 mm middle mediastinal lymph node metastasis and a 30 mm splenic metastasis, which gradually increased in size. Three months after discovery, sunitinib was initiated at 37.5 mg 2 weeks on/1 week off. Twelve days later, the patient presented with complaints of fever. A gas-producing splenic abscess was diagnosed and he was admitted on the same day. His condition improved with antibiotics and splenic drainage. On day 35 of hospitalization, he underwent laparoscopic splenectomy. The patient's postoperative clinical course was uneventful and he was discharged 7 days after the surgery.

Replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained transmitter release during repetitive stimulation. Kinetics of replenishment and available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability is unknown. Here, using comprehensive optical imaging for various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, we demonstrate that part of the recycling pool bearing the endosomal Q–SNARE Syntaxin 7 (Stx7) is preferentially mobilized for release during high–frequency repetitive stimulation. Recruitment of the SV pool marked with the Stx7–reporter requires high intra–terminal Ca2+ concentrations and actin polymerization. Furthermore, disruption of Stx7 function by overexpressing the N–terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 is essential for adaptation of synapses to respond high-frequency repetitive stimulation.

Introduction: We investigated whether the change in the neutrophil lymphocyte ratio (NLR) from the first to the last repeat prostate biopsy (ΔNLR) could be the diagnostic tool or not for prostate cancer (PCa) detection. Materials and Methods: We retrospectively evaluated medical records of men who had undergone repeat prostate biopsy. The investigated parameters were white blood cell, neutrophil, lymphocyte counts, NLR at the last prostate biopsy, ΔNLR, prostate-specific antigen (PSA), PSA density (PSAD), and PSA velocity. Exclusion criteria were the presence of cancers other than prostate origin, medication, and diseases which induce the change of NLR. Results: A total of 301 men who had undergone repeat prostate biopsy were selected for this study. After applying exclusion criteria, 223 patients were included. Of these patients, 94 were diagnosed with PCa (Group I) and 129 with no malignancy (Group II). Only a single patient had metastasis. On evaluating the area under the receiver operating characteristic curve of all study parameters, ΔNLR was the most accurate marker, followed by PSAD and then NLR measured at the last biopsy. Conclusions: ΔNLR was the most accurate marker to improve the total predictive value in repeat prostate biopsy for diagnosing PCa.

Ca2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca2+] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/H+ antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca2+ homeostasis and the modulation of H+ gradient in SVs.

The purpose of this study was to clarify the relationship of daily hot water bathing at home (DHW) and hot water spa bathing (HSPA) with the number of underlying diseases in middle-aged and elderly ambulatory patients. We defined the number of underlying diseases as the main outcome and dependent (criterion) variable. The frequency and time of DHW and the frequency of HSPA were set as explanatory variables. Multiple logistic regression analysis was performed for each frequency and time, adjusted age and sex. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated. Among the 1261 patients who participated, there was no significant difference in age between males (n = 508, 72.8 ± 6.8 years) and females (n = 753, 73.5 ± 6.9 years). There was also no significant age difference between males (number of diseases: 2.7 ± 2.0 pts.) and females (number of diseases: 2.7 ± 2.1 pts.) in the occurrence of underlying diseases. Frequency and time of DHW were not associated with the occurrence of underlying diseases. However, compared with participants who utilized hot water spa at least once a week, the occurrence of underlying diseases was significantly associated with bathing frequency: one to three times per month (OR 2.72, 95% CI 1.63–4.52); twice or five times a year (OR 1.92, 95% CI 1.25–2.94). In conclusion, lower frequency of HSPA was significantly associated with increased risk of the occurrence of underlying diseases in middle-aged and elderly ambulatory patients. However, the relationship between proactive use of hot water spa and patients’ mental and physical support should be clarified by well-designed cohort studies. The present study was registered as UMIN000033018 by the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) in Japan (refer: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000037643 ).

Targeting gene expression to a particular subset of neurons helps study the cellular function of the nervous system. Although neuron-specific promoters, such as the synapsin I promoter and the α-CaMKII promoter, are known to exhibit selectivity for excitatory glutamatergic neurons in vivo, the cell type-specificity of these promoters has not been thoroughly tested in culture preparations. Here, by using hippocampal culture preparation from the VGAT-Venus transgenic mice, we examined the ability of five putative promoter sequences of glutamatergic-selective markers including synapsin I, α-CaMKII, the vesicular glutamate transporter 1 (VGLUT1), Dock10 and Prox1. Among these, a genomic fragment containing a 2.1 kb segment upstream of the translation start site (TSS) of the VGLUT1 implemented in a lentiviral vector with the Tet-Off inducible system achieved the highest preferential gene expression in glutamatergic neurons. Analysis of various lengths of the VGLUT1 promoter regions identified a segment between −2.1 kb and −1.4 kb from the TSS as a responsible element for the glutamatergic selectivity. Consistently, expression of channelrhodopsin under this promoter sequence allowed for selective light-evoked activation of excitatory neurons. Thus, the lentiviral system carrying the VGLUT1 promoter fragment can be used to effectively target exogenous gene expression to excitatory glutamatergic neurons in cultures.

Efficient retrieval of the synaptic vesicle (SV) membrane from the presynaptic plasma membrane, a process called endocytosis, is crucial for the fidelity of neurotransmission, particularly during sustained neural activity. Although multiple modes of endocytosis have been identified, it is clear that the efficient retrieval of the major SV cargos into newly formed SVs during any of these modes is fundamental for synaptic transmission. It is currently believed that SVs are eventually reformed via a clathrin-dependent pathway. Various adaptor proteins recognize SV cargos and link them to clathrin, ensuring the efficient retrieval of the cargos into newly formed SVs. Here, we summarize our current knowledge of the molecular signatures within individual SV cargos that underlie efficient retrieval into SV membranes, as well as discuss possible contributions of the mechanisms under physiological conditions.

Epidemiological data has shown that dietary practices can greatly influence cancer rates. Men in East Asian countries, men have significantly lower prostate cancer rates compared to their counterparts the US and Europe. Soybeans are a versatile and rich source of protein and its products constitute a rich portion of Asian diets. Recent interest in healthy eating has expanded the consumption of soy products which also provide a rich source of naturally occurring isoflavones and 17β-estradiol. In this study, we used roasted soybean flour (kinako), which contains high levels isoflavones glycosides and estradiol, as dietary soy source to determine the influence of isoflavones rich diets on prostate cancer. Six-week old conditional Pten/Trp53 double knockout mice were randomized and fed plain AIN-93M (Control) diets or a diets supplemented with kinako ad libitum. Concentrations of kinako were adjusted to for daily intakes of aglycone isoflavones (genistein, daidzein, and glycetein) of 400 (LDI) and 800 (HDI) mg. Mice were sacrificed at 16 and 20 weeks (n=6 mice/group) or maintained for survival assessment (n=8 mice/group). Dietary intake of kinako-supplemented diets did not influence the onset of prostatic intraepithelial neoplasia or tumor burden at the early stages. However, tumors from mice fed the HDI diet experienced reduced tumor proliferation rates. Moreover, mice fed LDI and HDI diets showed reduced androgen receptor (AR) protein expression levels as well as mRNA levels for the AR target genes Fkbp5, Nk3x3.1 and Timp4. Interestingly, mice on the LDI diet, but not the HDI, experienced longer times to disease progression (median times 264, 299 and 250 days for Control, LDI and HDI, respectively, P=0.663), tumor doubling (median times 14, 27 and 14 days for Control, LDI and HDI, respectively, P=0.083), cumulative survival (median times 292, 348 and 320 days for Control, LDI and HDI, respectively, P=0.199), and overall survival times (median times 28, 43 and 35 days for Control, LDI and HDI, respectively, P=0.324). The metastatic incidence was 33%, 14% and 14% for Control, LDI and HDI groups, respectively, P=0.631. We also investigated whether dietary intervention with kinako would impact previously stablished tumors. For this we fed kinako supplemented diets to conditional Pten-knockout mice with established tumors but no changes were observed in tumor burden, proliferation, apoptosis and AR activity. Together our data shows that long-term continuous ingestion of a diet rich in isoflavones may be necessary in order suppress tumor growth. Interestingly, this protective effect appears to be lost with high-doses of the dietary isoflavones. Further studies will need to be performed in order to decipher complex dynamic interplay between survival pathways isoflavones chemoprevention. Citation Format: Yasunori Mori, Marco A. De Velasco, Yurie Kura, Eri Banno, Naomi Ando, Noriko Sato, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Dietary isoflavone decreases prostate cancer progression and improves survival in conditional Pten/Trp53-deficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1613.

Exciting breakthroughs in tumor immunology have led to the discovery and development of several promising immunotherapeutic strategies for cancer patients. Thus far, immune checkpoint inhibitors have been the most encouraging for solid tumors, however, treatment responses are observed in only a subset of patients and these appear to be primarily dependent on a tumor’s baseline immune profile. Additionally, tumors respond differently to immunotherapy and criteria to assess treatment responses are still being refined. We have identified a set of immune-related genes and developed a scoring system to profile the tumor’s immune status and assess immunological responses. In this study, we used a qRT-PCR-based approach to assess this panel and determine baseline tumor immune-profiles in an immunocompetent mouse model of Pten-null prostate cancer. We also assessed and compared tumor immune responses following androgen withdrawal (via surgical castration) and anti-PD-L1 immune checkpoint blockade. A total of 96 genes were selected for this focused panel which consisted of cell-type, immuno-responsive and housekeeping control genes. Core modules were designated based on functional gene associations which included antigen presentation, tumor inflammation, effector cells, immunomodulatory cells, immunosuppressive signaling and immune checkpoints. Core modules were further subdivided in to appropriately relevant sub-modules. A score for each submodule was calculated and a weighed score was then assigned for each core module. Tumor immunophenotypes based on core and sub-core immune scores (IS) were corroborated with immunohistochemical and/or flow cytometric analyses. Mouse castration-naïve prostate tumors exhibited low scores indicating a ‘”cold tumor” phenotype with little variation between individual mice. Androgen withdrawal induced cancer cell death in these tumors that in turn promoted immune cell infiltration. Core and sub-core IS of individual mice identified varied signatures reminiscent of immune excluded and inflamed “hot tumor” phenotypes. Short-term treatment with anti-PD-L1 blockade (clone D265A, mouse/IgG1 kappa) elicited an increased immune response signature in tumors from surgically castrated mice, but not in tumors from intact mice. Notably, discernable differences were noted among individual responders supporting the notion that responses to immune checkpoint may indeed be dependent on baseline tumor immune-profiles. In summary, androgen withdrawal appears to promote a “hotter” immunological phenotype in this model of prostate cancer, that is increased further by PD-L1 inhibition. Moreover, this investigation provides proof of concept evidence for a qRT-PCR-based biomarker approach for immuno-oncology. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sato, Naomi Ando, Kazuko Sakai, Yasunori Mori, Barry R. Davies, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. A real-time PCR-based approach to quantitatively assess tumor immune profiles and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4996.

We screened for a Rab39-specific effector by performing a yeast two-hybrid assay with GTP-locked Rab39A/B as the bait and identified UACA (uveal autoantigen with coiled-coil domains and ankyrin repeats) as a specific Rab39A/B-binding protein. Deletion analysis revealed that a C-terminal coiled-coil domain of UACA functions as a GTP-dependent Rab39-binding domain. shRNA-mediated knockdown of endogenous Rab39A or UACA in mouse neuroblastoma Neuro2A cells resulted in a change in retinoic acid-induced neurite morphology from a multipolar morphology to a bipolar morphology. Taken together, these findings indicate that UACA functions as a Rab39A effector in the retinoic acid-induced differentiation of Neuro2A cells.

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca(2+)) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca(2+)-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca(2+)-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca(2+)-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca(2+)-dependent exocytosis.

Relaxin-3 is a recently discovered member of the insulin superfamily and is a ligand for three orphan G-protein-coupled receptors: GPCR135, GPCR142 and LGR7 (leucine-rich repeat-containing G-protein-coupled receptor 7). GPCR135 mRNA is expressed in the pancreas, however, it is not known, whether the peptide affects pancreatic islet function. Reverse transcriptase (RT)-PCR and radioreceptor assay have shown that the relaxin-3 receptor (GPCR135) is expressed in pancreatic islets and rat insulinoma, but LGR7 is not expressed. Moreover, relaxin-3 has been revealed to inhibit the secretion of insulin from pancreatic islets. However, we can not detect relaxin-3 in small intestine and pancreas. These results suggest a novel role of relaxin-3 in the regulation of insulin release.

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

Neurons are functionally and morphologically compartmentalized into axons and dendrites, and the localization of specific proteins within these compartments is critical to the proper formation of neuronal networks, which includes neurite morphogenesis and synapse formation. The small GTPase Rab17 is specifically localized in dendrites and is not found in axons, and it regulates the dendrite morphogenesis and postsynaptic development of mouse hippocampal neurons. However, the spatiotemporal regulation of Rab17 is poorly understood. We recently identified Rabex-5, originally described as a Rab5-guanine nucleotide exchange factor (GEF), as a physiological Rab17-GEF that promotes translocation of Rab17 from the cell body to the dendrites of developing hippocampal neurons. Knockdown of Rab17 in mouse hippocampal neurons resulted in reductions in dendrite growth, branch numbers, filopodium density, and active synapse numbers. Knockdown of Rab17-GEF Rabex-5 in hippocampal neurons resulted in decreased targeting of Rab17 to the dendrites, which led to a reduction in dendrite growth. In this chapter we describe the assay procedures for analyzing Rab17 and Rabex-5 in cultured mouse hippocampal neurons, and we particularly focus on the measurement of total dendrite (or axon) length and total dendrite (or axon) branch numbers, filopodium density, number of active synapses, and dendritic Rab17 signals.