Tumour tissue-derived small extracellular vesicles reflect molecular subtypes of bladder cancer.

mRNA-based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size-exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1 g of tissue, incubated with media for 30 min. FFPE tumour tissue and tumour tissue-derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV-related bladder cancer-specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26-1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue-derived sEVs may reflect more tissue- or disease-specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy-based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer-specific biomarkers in large cohorts.

Melanocortin agonism in a social context selectively activates nucleus accumbens in an oxytocin-dependent manner.

Social deficits are debilitating features of many psychiatric disorders, including autism. While time-intensive behavioral therapy is moderately effective, there are no pharmacological interventions for social deficits in autism. Many studies have attempted to treat social deficits using the neuropeptide oxytocin for its powerful neuromodulatory abilities and influence on social behaviors and cognition. However, clinical trials utilizing supplementation paradigms in which exogenous oxytocin is chronically administered independent of context have failed. An alternative treatment paradigm suggests pharmacologically activating the endogenous oxytocin system during behavioral therapy to enhance the efficacy of therapy by facilitating social learning. To this end, melanocortin receptor agonists like Melanotan II (MTII), which induces central oxytocin release and accelerates formation of partner preference, a form of social learning, in prairie voles, are promising pharmacological tools. To model pharmacological activation of the endogenous oxytocin system during behavioral therapy, we administered MTII prior to social interactions between male and female voles. We assessed its effect on oxytocin-dependent activity in brain regions subserving social learning using Fos expression as a proxy for neuronal activation. In non-social contexts, MTII only activated hypothalamic paraventricular nucleus, a primary site of oxytocin synthesis. However, during social interactions, MTII selectively increased oxytocin-dependent activation of nucleus accumbens, a site critical for social learning. These results suggest a mechanism for the MTII-induced acceleration of partner preference formation observed in previous studies. Moreover, they are consistent with the hypothesis that pharmacologically activating the endogenous oxytocin system with a melanocortin agonist during behavioral therapy has potential to facilitate social learning.

Natural variation in oxytocin receptor signaling causes widespread changes in brain transcription: a link to the natural killer gene complex.

Oxytocin (OXT) is a highly conserved neuropeptide that modulates social cognition, and variation in its receptor gene (Oxtr) is associated with divergent social phenotypes. The cellular mechanisms connecting Oxtr genotype to social phenotype remain obscure. We exploit an association between Oxtr polymorphisms and striatal-specific OXTR density in prairie voles to investigate how OXTR signaling influences the brain transcriptome. We discover widespread, OXTR signaling-dependent transcriptomic changes. Interestingly, OXTR signaling robustly modulates gene expression of C-type lectin-like receptors (CTLRs) in the natural killer gene complex, a genomic region associated with immune function. CTLRs are positioned to control microglial synaptic pruning; a process important for shaping neural circuits. Similar relationships between OXTR RNA and CTLR gene expression were found in human striatum. These data suggest a potential molecular mechanism by which variation in OXTR signaling due to genetic background and/or life-long social experiences, including nurturing/neglect, may affect circuit connectivity and social behavior.

Sugar signals from oral glucose transporters elicit cephalic-phase insulin release in mice.

Cephalic-phase insulin release (CPIR) occurs before blood glucose increases after a meal. Although glucose is the most plausible cue to induce CPIR, peripheral sensory systems involved are not fully elucidated. We therefore examined roles of sweet sensing by a T1R3-dependent taste receptor and sugar sensing by oral glucose transporters in the oropharyngeal region in inducing CPIR. Spontaneous oral ingestion of glucose significantly increased plasma insulin 5 min later in wild-type (C57BL/6) and T1R3-knockout mice, but intragastric infusion did not. Oral treatment of glucose transporter inhibitors phlorizin and phloretin significantly reduced CPIR after spontaneous oral ingestion. In addition, a rapid increase in plasma insulin was significantly smaller in WT mice with spontaneous oral ingestion of nonmetabolizable glucose analog than in WT mice with spontaneous oral ingestion of glucose. Taken together, the T1R3-dependent receptor is not required for CPIR, but oral glucose transporters greatly contribute to induction of CPIR by sugars.

Oxytocin receptors are widely distributed in the prairie vole (Microtus ochrogaster) brain: Relation to social behavior, genetic polymorphisms, and the dopamine system.

Oxytocin regulates social behavior via direct modulation of neurons, regulation of neural network activity, and interaction with other neurotransmitter systems. The behavioral effects of oxytocin signaling are determined by the species-specific distribution of brain oxytocin receptors. The socially monogamous prairie vole has been a useful model organism for elucidating the role of oxytocin in social behaviors, including pair bonding, response to social loss, and consoling. However, there has been no comprehensive mapping of oxytocin receptor-expressing cells throughout the prairie vole brain. Here, we employed a highly sensitive in situ hybridization, RNAscope, to construct an exhaustive, brain-wide map of oxytocin receptor mRNA-expressing cells. We found that oxytocin receptor mRNA expression was widespread and diffused throughout the brain, with specific areas displaying a particularly robust expression. Comparing receptor binding with mRNA revealed that regions of the hippocampus and substantia nigra contained oxytocin receptor protein but lacked mRNA, indicating that oxytocin receptors can be transported to distal neuronal processes, consistent with presynaptic oxytocin receptor functions. In the nucleus accumbens, a region involved in oxytocin-dependent social bonding, oxytocin receptor mRNA expression was detected in both the D1 and D2 dopamine receptor-expressing subtypes of cells. Furthermore, natural genetic polymorphisms robustly influenced oxytocin receptor expression in both D1 and D2 receptor cell types in the nucleus accumbens. Collectively, our findings further elucidate the extent to which oxytocin signaling is capable of influencing brain-wide neural activity, responses to social stimuli, and social behavior. KEY POINTS: Oxytocin receptor mRNA is diffusely expressed throughout the brain, with strong expression concentrated in certain areas involved in social behavior. Oxytocin receptor mRNA expression and protein localization are misaligned in some areas, indicating that the receptor protein may be transported to distal processes. In the nucleus accumbens, oxytocin receptors are expressed on cells expressing both D1 and D2 dopamine receptor subtypes, and the majority of variation in oxytocin receptor expression between animals is attributable to polymorphisms in the oxytocin receptor gene.

Helping behavior in prairie voles: A model of empathy and the importance of oxytocin.

Several studies suggest that rodents show empathic responses and helping behavior toward others. We examined whether prairie voles would help conspecifics who were soaked in water by opening a door to a safe area. Door-opening latency decreased as task sessions progressed. Female and male voles stayed close to the soaked voles' side at equal rates and opened the door with similar latencies. When the conspecific was not soaked in water, the door-opening latency did not decrease. This suggests that the distress of the conspecific is necessary for learning to open the door and that the door-opening performed by prairie voles corresponds to helping behavior. Additionally, we examined the helping behavior in prairie voles in which oxytocin receptors were genetically knocked out. Oxytocin receptor knockout voles demonstrated less learning of the door-opening behavior and less interest in soaked conspecifics. This suggests that oxytocin is important for the emergence of helping behavior.

CRISPR/Cas9-Mediated Genetic Engineering to Generate a Disease Model Prairie Vole, Based on Species-Optimized Assisted Reproductive Technology.

Social and prosocial behaviors, including communication, social bonding, and affiliation, parental behaviors, and empathy are key features of a highly social mammalian species. However, the neuronal mechanism in the brain underlying these behaviors remains unclear because of limited information on the social and prosocial behavioral levels in rodent models generally used in behavioral neuroscience studies.The rodent species, prairie vole (Microtus ochrogaster), is one of the nontraditional animal models with several advantages in experimental science over other rodent models, such as mice or rats. Additionally, it demonstrates characteristics advantageous in the study of social and prosocial behaviors, such as monogamous pair bonding behavior, biparental care, and consoling behavior toward partners stressed by aversive foot shock stimulus. Recent studies of prairie voles have highlighted the importance of oxytocin (OXT) and oxytocin receptor (OXTR)-mediated mechanisms in the regulation of these behaviors.Recently, we established assisted reproductive technologies for prairie voles, and successfully and efficiently generated an OXTR gene knockout (KO) prairie vole using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9 ), a powerful genome editing tool with artificially developed single-strand guide RNAs (sgRNA) and Cas9 endonucleases.Herein, we describe the method for CRISPR /Cas9-mediated generation of OXTR KO prairie vole. This OXTR KO prairie vole can be a valuable tool to understand their unique social and prosocial behaviors and elucidate how the oxytocin system influences or modulates these behaviors in the brain.

The accuracy of prostate cancer diagnosis in biopsy-naive patients using combined magnetic resonance imaging and transrectal ultrasound fusion-targeted prostate biopsy.

BACKGROUND: This study aimed to estimate whether multiparametric magnetic resonance imaging (mpMRI)-transrectal ultrasound (TRUS) fusion biopsy (FUS-TB) increases the detection rates of clinically significant prostate cancer (csPCa) compared with TRUS-guided systematic biopsy (TRUS-GB). METHODS: This retrospective study focused on patients who underwent mpMRI before prostate biopsy (PB) with Prostate Imaging Reporting and Data System version 2 (PI-RADS v2) scores ≥3 and prostate-specific antigen (PSA) level between 2.5 and 20 ng/mL. Before FUS-TB, the biopsy needle position was checked virtually using three-dimensional mapping. After confirming the position of the target within the prostate, biopsy needle was inserted and PB was performed. Suspicious lesions were generally targeted with 2 to 4 cores. Subsequently, 10-12 cores were biopsied for TRUS-GB. The primary endpoint was the PCa detection rate (PCDR) for patients with PCa who underwent combined FUS-TB and TRUS-GB. RESULTS: According to PI-RADS v2, 76.7% of the patients with PI-RADS v2 score ≥3 were diagnosed with PCa. The PCDRs in patients with PI-RADS v2 score of 4 or 5 were significantly higher than those in patients with PI-RADS v2 score of 3 (3 vs. 4, P<0.001; 3 vs. 5, P<0.001; 4 vs. 5, P=0.073). According to PCDR, the detection rates of PCa and csPCa in the FUS-TB were significantly higher than that in the TRUS-GB. CONCLUSIONS: Following detection of suspicious tumor lesions on mpMRI, FUS-TB use detects a higher number of PCa cases compared with TRUS-GB.

Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies.

We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.

Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro.

Renal cell carcinoma (RCC) accounts for over 400,000 new cases and 175,000 deaths annually. Diagnostic RCC biomarkers may prevent overtreatment in patients with early disease. Extracellular vesicles (EVs) are a promising source of RCC biomarkers because EVs carry proteins and messenger RNA (mRNA) among other biomolecules. We aimed to identify biomarkers and assess biological functions of EV cargo from clear cell RCC (ccRCC), papillary RCC (pRCC), and benign kidney cell lines. EVs were enriched from conditioned cell media by size exclusion chromatography. The EV proteome was assessed using Tandem Mass Tag mass spectrometry (TMT-MS) and NanoString nCounter technology was used to profile 770 cancer-related mRNA present in EVs. The heterogeneity of protein and mRNA abundance and identification highlighted the heterogeneity of EV cargo, even between cell lines of a similar pathological group (e.g., ccRCC or pRCC). Overall, 1726 proteins were quantified across all EV samples, including 181 proteins that were detected in all samples. In the targeted profiling of mRNA by NanoString, 461 mRNAs were detected in EVs from at least one cell line, including 159 that were present in EVs from all cell lines. In addition to a shared EV cargo signature, pRCC, ccRCC, and/or benign renal cell lines also showed unique signatures. Using this multi-omics approach, we identified 34 protein candidate pRCC EV biomarkers and 20 protein and 8 mRNA candidate ccRCC EV biomarkers for clinical validation.

Leptomeningeal Carcinomatosis in Urothelial Carcinoma of the Urinary Bladder: A Report of a Patient with a Fulminant Course Who Died of Cancer after Definitive Therapies.

A 45-year-old Japanese man visited a community hospital with the chief complaint of asymptomatic macrohematuria. He was diagnosed with muscle-invasive bladder cancer (MIBC), and he received intra-arterial chemotherapy followed by radiation therapy at another institution. Twenty-eight months after chemoradiotherapy, magnetic resonance imaging (MRI) revealed MIBC recurrence. After neoadjuvant chemotherapy, robot-assisted radical cystectomy was performed. Pathological examination indicated high-grade urothelial carcinoma with lymphovascular invasion, a positive surgical margin, and skip lesions of cancer cells in the perivesical adipose tissue. Three months after surgery, he was brought to our hospital in an ambulance with the chief complaint of rotatory vertigo and was speaking inarticulately. Head and whole spine MRI revealed meningeal metastasis along both the vestibulocochlear nerves and cauda equina. Analysis of the cerebrospinal fluid revealed malignant cells. The patient was diagnosed with leptomeningeal carcinomatosis originating from the MIBC. He received whole-brain radiotherapy followed by the administration of pembrolizumab. Unfortunately, the patient's condition quickly deteriorated, and he died of cancer 4 months after surgery.

High-Throughput Simultaneous mRNA Profiling Using nCounter Technology Demonstrates That Extracellular Vesicles Contain Different mRNA Transcripts Than Their Parental Prostate Cancer Cells.

Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.

Investigation of Oxtr-expressing Neurons Projecting to Nucleus Accumbens using Oxtr-ires-Cre Knock-in prairie Voles (Microtus ochrogaster).

Social bonds such as parent-infant attachment or pair bonds can be critical for mental and physical well-being. The monogamous prairie vole (Microtus ochrogaster) has proven useful for examining the neural substrates regulating social behaviors, including social bonding. Oxytocin (OXT) and oxytocin receptor (OXTR) play critical roles in alloparental care, pair bonding and consoling behavior in prairie voles. While OXTR in a few regions, such as the nucleus accumbnes (NAcc), prefrontal cortex (PFC) and anterior cingulate cortex (ACC), have been implicated in regulating these behaviors, the extent to which other OXT sensitive areas modulate social behaviors has not been investigated. The NAcc is a central hub for modulating OXTR dependent social behaviors. To identify neurons expressing Oxtr in prairie vole brain, we generated gene knock-in voles expressing Cre recombinase in tandem with Oxtr (Oxtr-ires-Cre) using CRISPR/Cas9 genome editing. We confirmed Oxtr and Cre mRNA co-localization in NAcc, validating this model. Next, we identified putative Oxtr-expressing neurons projecting to NAcc by infusing retrograde CRE-dependent EGFP AAV into NAcc and visualizing fluorescence. We found enhanced green fluorescent protein (EGFP) positive neurons in anterior olfactory nucleus, PFC, ACC, insular cortex (IC), paraventricular thalamus (PVT), basolateral amygdala (BLA), and posteromedial and posterolateral cortical amygdaloid area (PMCo, PLCo). The ACC to NAcc OXTR projection may represent a species-specific circuit since Oxtr-expressing neurons in the ACC of mice were reported not to project to the NAcc. This is the first delineation of Oxtr-expressing neural circuits in the prairie vole, and demonstrates the utility of this novel genetically modified organism for characterizing OXTR circuits involved in social behaviors.

Single administration of resveratrol improves social behavior in adult mouse models of autism spectrum disorder.

Resveratrol (RSV) is a natural polyphenol present in grapes, the skin of peanuts, and several other plants with many health benefits. Autism spectrum disorder (ASD) is a neurodevelopmental disorder that may be linked to neural and synaptic development impairments. The present study aimed to analyze the preventive effects of RSV on the development of ASD-like behavior, using oxytocin receptor gene knockout (Oxtr-KO) and valproic acid-induced ASD (VPA-ASD) model mice. Genetic deficiencies in Oxtr are suggested to be involved in ASD etiology. Twenty-four hours after a single RSV injection to the Oxtr-KO mice, the social impairments caused by OXTR deficiency were ameliorated. RSV also improved social impairments in the VPA-ASD mice. Administration of RSV up-regulated silent information regulator 1 (Sirt1) gene and early growth response factor 3 (Egr3) gene expressions in the amygdala of the Oxtr-KO mice. Our data suggest that RSV may have therapeutic effects on ASD with multiple targets.

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.

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.

Effect of Posttransplant Diabetes Mellitus on Graft Loss After Living-Donor Kidney Transplant at a Single Institution.

BACKGROUND: This study aimed to evaluate predictive factors for graft loss in patients who received kidney transplantation (KT) from living kidney donors (LKDs) at a single institute in Japan. METHODS: Our study focused on patients with end-stage renal disease who underwent KT from LKDs and were followed up for at least 1 year after surgery. The primary end point was graft survival (GS). GS after KT was analyzed using the Kaplan-Meier method. GS according to subgroup classification was analyzed using the log-rank test. A multivariate analysis was performed using a Cox proportional hazard model. RESULTS: The median follow-up period was 105.5 months after KT. The 5- and 10-year GS rates were 97.8% and 96.0% in KT recipients (KTRs) without posttransplant diabetes mellitus (PTDM) and 89.9% and 63.2% in those with PTDM, respectively. The rate of graft loss was significantly higher in KTRs with PTDM than in those without PTDM (P < .001). Of the KTRs whose diabetes mellitus (DM) was cured after KT, those who underwent dialysis because of diabetic nephropathy had no graft loss. In the multivariate analysis, the serum creatinine level at 1 month after KT, PTDM, and human leukocyte antigen mismatches were significantly associated with graft loss after KT. CONCLUSIONS: In this study, the rate of graft loss in KTRs with PTDM was significantly higher than that of KTRs without PTDM. However, among KTRs whose DM was cured after KT, those who underwent dialysis because of diabetic nephropathy had no graft loss.

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density-gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co-separate contaminants when the non-EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non-EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Oxytocin induced labor causes region and sex-specific transient oligodendrocyte cell death in neonatal mouse brain.

AIM: Previous reports showed associations between oxytocin induced labor and mental disorders in offspring. However, those reports are restricted in epidemiological analyses and its mechanism remains unclear. In this study, we hypothesized that induced labor directly causes brain damage in newborns and results in the development of mental disorders. Therefore we aimed to investigate this hypothesis with animal model. METHODS: The animal model of induced labor was established by subcutaneous oxytocin administration to term-pregnant C57BL/6J mice. We investigated the neonatal brain damage with evaluating immediate early gene expression (c-Fos, c-Jun and JunB) by quantitative polymerase reaction and TdT-mediated dUTP nick end labeling staining. To investigate the injured brain cell types, we performed double-immunostaining with TdT-mediated dUTP nick end labeling staining and each brain component specific protein, such as Oligo2, NeuN, GFAP and Iba1. RESULTS: Brain damage during induced labor led to cell death in specific brain regions, which are implicated in mental disorders, in only male offspring at P0. Furthermore, oligodendrocyte precursors were selectively vulnerable compared to the other cell types. This oligodendrocyte-specific impairment during the perinatal period led to an increased numbers of Olig2-positive cells at P5. Expression levels of oxytocin and Oxtr in the fetal brain were not affected by the oxytocin administered to mothers during induced labor. CONCLUSION: Oligodendrocyte cell death in specific brain regions, which was unrelated to the oxytocin itself, was caused by induced labor in only male offspring. This may be an underlying mechanism explaining the human epidemiological data suggesting an association between induced labor and mental disorders.

[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.