Probes for INS

Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

The classical motor center cerebellum is one of the most consistent structures of abnormality in autism spectrum disorders (ASD), and neuropeptide oxytocin is increasingly explored as a potential pharmacotherapy for ASD. However, whether oxytocin targets the cerebellum for therapeutic effects remains unclear. Here, we report a localization of oxytocin receptor (OXTR) in Purkinje cells (PCs) of cerebellar lobule Crus I, which is functionally connected with ASD-implicated circuits. OXTR activation neither affects firing activities, intrinsic excitability, and synaptic transmission of normal PCs nor improves abnormal intrinsic excitability and synaptic transmission of PCs in maternal immune activation (MIA) mouse model of autism. Furthermore, blockage of OXTR in Crus I in wild-type mice does not induce autistic-like social, stereotypic, cognitive, and anxiety-like behaviors. These results suggest that oxytocin signaling in Crus I PCs seems to be uninvolved in ASD pathophysiology, and contribute to understanding of targets and mechanisms of oxytocin in ASD treatment.

The nonapeptide system modulates numerous social behaviors through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin receptor (AVPR1A) in the brain. OXTRs and AVPR1As are widely distributed throughout the brain and binding densities exhibit substantial variation within and across species. Although OXTR and AVPR1A binding distributions have been mapped for several rodents, this system has yet to be characterized in the spiny mouse (Acomys cahirinus). Here we conducted receptor autoradiography and in situ hybridization to map distributions of OXTR and AVPR1A binding and Oxtr and Avpr1a mRNA expression throughout the basal forebrain and midbrain of male and female spiny mice. We found that nonapeptide receptor mRNA is diffuse throughout the forebrain and midbrain and does not always align with OXTR and AVPR1A binding. Analyses of sex differences in brain regions involved in social behavior and reward revealed that males exhibit higher OXTR binding densities in the lateral septum, bed nucleus of the stria terminalis, and anterior hypothalamus. However, no association with gonadal sex was observed for AVPR1A binding. Hierarchical clustering analysis further revealed that co-expression patterns of OXTR and AVPR1A binding across brain regions involved in social behavior and reward differ between males and females. These findings provide mapping distributions and sex differences in nonapeptide receptors in spiny mice. Spiny mice are an excellent organism for studying grouping behaviors such as cooperation and prosociality, and the nonapeptide receptor mapping here can inform the study of nonapeptide-mediated behavior in a highly social, large group-living rodent.

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.

Objective: To assess the clinical features and treatment outcomes in patients with primary ovarian squamous cell carcinoma (POSCC). Methods: Fifteen patients with primary ovarian squamous cell carcinoma diagnosed from January 2009 to December 2018 in Cancer Hospital of the University of Chinese Academy of Sciences were collected. The expression of p16, hMLH1, hMSH2, hMSH6 and PMS2 in POSCC was detected by immunohistochemistry, and the status of high-risk human papillomavirus (HPV) by RNAscope test. Results: Squamous cell carcinoma with different degrees of differentiation was found in 15 cases, including three cases with high differentiation and 12 cases with medium to low differentiation. There were four cases with in situ squamous cell carcinoma, four cases with teratoma, one case with endometrial carcinoma/atypical hyperplasia, and one case with endometriosis. p16 was expressed in five cases (5/15), indicating coexisting high-risk HPV infection. There was no high-risk HPV infection in the remaining 10 cases, and p16 staining was negative. There was no deficient mismatch repair protein in all cases. The overall survival time (P=0.038) and progression free survival (P=0.045) of patients with high-risk HPV infection were longer than those without HPV infection. Conclusions: POSCC is more commonly noted in postmenopausal women and often occurs unilaterally. Elevated serological indexes CA125 and SCC are the most common finding. Morphologically, the tumors show variable degrees of differentiation, but the current data suggest that the degree of differentiation cannot be used as an independent prognostic index. High-risk HPV infection may be associated with the occurrence of POSCC, and that the prognosis of POSCC patients with HPV infection is better than that of patients without infection.

Gastric-type cervical adenocarcinoma (GCA) is a rare and aggressive type of endocervical adenocarcinoma (ECA) with distinct histopathologic features and unfavorable treatment outcomes, but no genomic prognostic factor has been revealed. We aimed to systematically investigate the somatic alterations of GCA at genome-wide level and evaluate their prognostic value.We performed whole-exome sequencing (WES) on 25 pairs of tumor and matched normal samples to characterize the genomic features of Chinese patients with GCA and investigated their relations to histopathological characterizations and prognosis. The prognostic value of the genomic alterations was evaluated in a total of 58 GCA patients.Mutations were commonly observed in reported GCA-related driver genes, including TP53 (32%), CDKN2A (20%), SKT11 (20%), BRCA2 (12%), SMAD4 (12%), and ERBB2 (12%). Recurrent novel trunk mutations were also observed in PBRM1 (12%), FRMPD4 (12%), and NOP2 (8%) with high variant allele frequency. Moreover, enrichment of the APOBEC signature was attributed to frequent gain of somatic copy number alteration (SCNA) of APOBEC3B (20%), which perfectly matched the nuclear-positive staining of APOBEC3B through immunohistochemistry. In contrast, APOBEC3B alteration was absent in patients with conventional type of ECA (N = 52). Notably, positive APOBEC3B was consistently enriched in patients with favorable prognosis in both the discovery cohort and an additional 33 GCA patients, thus indicating a significant association with lower relapse risk of GCA independent of cancer stage (P = 0.02).Our results can aid understanding of the molecular basis of GCA in the Chinese population by providing genomic profiles and highlighting the potential prognostic value of APOBEC3B for GCA through routine clinical IHC.

Seasonal changes in food intake and adiposity in many animal species are triggered by changes in the photoperiod. These latter changes are faithfully transduced into a biochemical signal by melatonin secreted by the pineal gland. Seasonal variations, encoded by melatonin, are integrated by third ventricular tanycytes of the mediobasal hypothalamus through the detection of the thyroid-stimulating hormone (TSH) released from the pars tuberalis. The mediobasal hypothalamus is a critical brain region that maintains energy homeostasis by acting as an interface between the neural networks of the central nervous system and the periphery to control metabolic functions, including ingestive behavior, energy homeostasis, and reproduction. Among the cells involved in the regulation of energy balance and the blood-hypothalamus barrier (BHB) plasticity are tanycytes. Increasing evidence suggests that anterior pituitary hormones, specifically TSH, traditionally considered to have unitary functions in targeting single endocrine sites, display actions on multiple somatic tissues and central neurons. Notably, modulation of tanycytic TSH receptors seems critical for BHB plasticity in relation to energy homeostasis, but this needs to be proven.

Independent primary uterine and cervical adenocarcinoma are rare and difficult to identify their origins, which makes treatment decision difficult. A 46-year-old female with endometrioid carcinoma and adenocarcinoma, human papilloma virus (HPV)-associated of the uterine cervix was reported. The patient presented with increased menstrual flow, contact bleeding and watery leucorrhea for more than one year, and the imaging findings showed abnormal uterine morphology, irregular margins, and multiple abnormal signals in uterine cavity and myometrium, which suggested multiple leiomyomas of the uterus. The signal intensity in the right muscle layer was markedly enhanced, suggesting a smooth muscle tumor of uncertain malignant potential. A large number of cystic hypointensity was seen in the cervix, and multiple cysts were considered. The initial preoperative diagnosis was multiple leiomyoma of the uterus, and a hysterectomy operation was planned. During the operation, the uterus was sent for frozen sections. There was a mass in the endometrium of the fundus, with a soft grayish-red cut surface and a clear border with the myometrium, and there was a grayish-white nodule in the cervix with a hard grayish-white cut surface. The two masses were well demarcated from each other, and the distance between them was 30 mm. The result of the frozen sections indicated the malignant tumor of the endometrium, and the extended hysterectomy+pelvic lymphadenectomy+partial resection of the greater omentum was performed. After the operation, the paraffin sections were sent to the Department of Pathology of the Peking University Third Hospital for histochemistry, POLE gene sequencing and HPV RNAscope tests, and the final diagnosis was a synchronous endometrioid carcinoma (POLE-mutant according to the WHO classification) and an adenocarcinoma, HPV-associated of the uterine cervix. Now the patient had been treated with 2 cycles of chemotherapy and her condition was fine. Through the analysis of the histological, immunohistochemical and molecular detection results of this case, the importance of applying HPV RNAscope and TCGA molecular typing in the diagnosis of cervical adenocarcinomas and endometrial carcinomas was emphasized. At the same time, gynecologists should not blindly rely on intraoperative frozen sections, and should pay attention to preoperative pathological examination, and make appropriate operation methods according to the results in order to prevent passivity in the surgery.

Primary female urethral carcinoma (PUC-F) accounts for less than 1% of all genitourinary malignancies and comprises a histologically diverse group of tumors that are usually associated with poor prognosis. The carcinomas documented at this site include adenocarcinoma (clear cell adenocarcinoma, columnar cell carcinoma, and Skene gland adenocarcinoma), urothelial carcinoma (UCa), and squamous cell carcinoma (SCC). Recent studies have shown adenocarcinomas to be the most common type of primary urethral carcinoma in females. As most of the urethral carcinomas morphologically resemble carcinomas arising from surrounding pelvic organs or metastases, these should be ruled out before making the diagnosis of PUC-F. These tumors are currently staged according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. However, the AJCC system has limitations, including the staging of tumors involving the anterior wall of the urethra. Staging systems like the recently proposed histology-based female urethral carcinoma staging system (UCS) takes into account the unique histological landmarks of the female urethra to better stratify pT2 and pT3 tumors into prognostic groups, that correlate with clinical outcomes including recurrence rates, disease-specific survival and overall survival. Further larger multi-institutional cohorts are however required to validate the results of this staging system. There is very limited information regarding the molecular profiling of PUC-F. Thirty-one percent of clear cell adenocarcinomas have been reported to show PIK3CA alterations, whereas 15% of adenocarcinomas show PTEN mutations. Higher tumor mutational burden and PD-L1 staining have been reported in UCa and SCC. Although multimodality treatment is usually recommended in locally advanced and metastatic disease, the role of immunotherapy and targeted therapy is promising in select PUC-F cases.

The incorporation of HPV DNA testing into cervical screening programs has shown that many HPV-positive women are cytologically normal, with HPV-positivity fluctuating throughout life. Such results suggest that papillomaviruses may persist in a latent state after disease clearance, with sporadic recurrence. It appears that virus latency represents a narrow slot in a wider spectrum of subclinical and possibly productive infections. Clinical studies, and animal model infection studies, suggested a key role for host immune surveillance in maintaining such asymptomatic infections, and although infections may also be cleared, most studies have used the term 'clearance' to describe a situation where the presence of HPV DNA falls below the clinical detection level. Given our knowledge of papillomavirus immune evasion strategies and the restricted pattern of viral gene expression required for 'basal cell' persistence, the term 'apparent clearance' and 'subclinical persistence' of infection may better summarise our understanding. Subclinical infection also encompasses the lag phase, which occurs between infection and lesion development. This is dependent on infection titre, with multifocal infections developing more rapidly to disease. These concepts can usefully influence patient management where HPV-positivity occurs sometime after the onset of sexual activity, and where vertical transmission is suspected despite a lag period.