Probes for INS

Over the past decade, considerable efforts have been made to accelerate pathophysiological understanding of fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) with brain banks at the forefront. In addition to exploratory disease mechanisms, brain banks have aided our understanding with regard to clinical diagnosis, genetics and cell biology. Across neurodegenerative disorders, the impact of brain tissue in ALS research has yet to be quantified. This review aims to outline (i) how postmortem tissues from brain banks have influenced our understanding of ALS over the last 15 years, (ii) correlate the location of dedicated brain banks with the geographical prevalence of ALS, (iii) identify the frequency of features reported from postmortem studies and (iv) propose common reporting standards for materials obtained from dedicated brain banks. A systematic review was conducted using PubMed and Web of Science databases using key words. From a total of 1439 articles, 73 articles were included in the final review, following PRISMA guidelines. Following a thematic analysis, articles were categorised into five themes; clinico-pathological (13), genetic (20), transactive response DNA binding protein 43 (TDP-43) pathology (12), non-TDP-43 neuronal pathology (nine) and extraneuronal pathology (19). Research primarily focused on the genetics of ALS, followed by protein pathology. About 63% of the brain banks were in the United States of America and United Kingdom. The location of brain banks overall aligned with the incidence of ALS worldwide with 88% of brain banks situated in Europe and North America. An overwhelming lack of consistency in reporting and replicability was observed, strengthening the need for a standardised reporting system. Overall, postmortem material from brain banks generated substantial new knowledge in areas of genetics and proteomics and supports their ongoing role as an important research tool.

Emerging literature suggests that experiences of discrimination negatively influence health and well-being. It is unfortunately common for people living with HIV (PLWH) to be stigmatized and discriminated against because of their HIV status and other marginalized identities (e.g., ethnicity/race, sexual identity and orientation). To date, little research has specifically examined discrimination in PWLH and its associations with pain and other pain-relevant factors such as mood and sleep. The purpose of this ongoing study was to preliminarily analyze associations among daily experiences of discrimination, pain severity and interference, depressive symptoms, and sleep in PLWH. Participants included 24 PLWH recruited from a local HIV treatment center. Participants completed The Everyday Discrimination Scale (TEDS) followed by the Brief Pain Inventory - Short Form (BPI-SF), the Insomnia Severity Index (ISI), and the Center for Epidemiologic Studies - Depression Scale (CES-D). Initial findings tentatively suggest that more frequent daily experiences of discrimination may be significantly associated with greater pain interference on the BPI-SF (p = .030) and greater severity of insomnia symptoms on the ISI (p = .059). However, it appears that daily experiences of discrimination may not be meaningfully associated with pain severity on the BPI-SF (p = .401) or depressive symptoms on the CES-D (p = .235). Our findings highlight the potentially deleterious effects of daily discrimination experiences on pain and sleep in in PLWH. As this ongoing study recruits a larger sample of PLWH, data will need to be reanalyzed to better determine the durability of these preliminary findings. However, there is potential that findings from this study may assist in elucidating causal pathways linking discrimination to pain and pain relevant health behaviors like sleep in PLWH. Grant support from The Impact of Insomnia on Pain, Physical Function, and Inflammation in HIV (3R01HL147603-03S1).

To evaluate the application of RNAscope in the clinical diagnostic field compared to the current 'gold standard' methods employed for testing gene expression levels, including immunohistochemistry (IHC), quantitative real time PCR (qPCR), and quantitative reverse transcriptase PCR (qRT-PCR), and to detect genes, including DNA in situ hybridisation (DNA ISH).This systematic review searched CINAHL, Medline, Embase and Web of Science databases for studies that were conducted after 2012 and that compared RNAscope with one or more of the 'gold standard' techniques in human samples. QUADAS-2 test was used for the evaluation of the articles' risk of bias. The results were reviewed narratively and analysed qualitatively.A total of 27 articles (all retrospective studies) were obtained and reviewed. The 27 articles showed a range of low to middle risk of bias scores, as assessed by QUADAS-2 test. 26 articles studied RNAscope within cancer samples. RNAscope was compared to different techniques throughout the included studies (IHC, qPCR, qRT-PCR and DNA ISH). The results confirmed that RNAscope is a highly sensitive and specific method that has a high concordance rate (CR) with qPCR, qRT-PCR, and DNA ISH (81.8-100%). However, the CR with IHC was lower than expected (58.7-95.3%), which is mostly due to the different products that each technique measures (RNA vs. protein).This is the first systematic review to be conducted on the use of RNAscope in the clinical diagnostic field. RNAscope was found to be a reliable and robust method that could complement gold standard techniques currently used in clinical diagnostics to measure gene expression levels or for gene detection. However, there were not enough data to suggest that RNAscope could stand alone in the clinical diagnostic setting, indicating further prospective studies to validate diagnostic accuracy values, in keeping with relevant regulations, followed by cost evaluation are required.

Benign prostatic hyperplasia (BPH) is a progressive expansion of peri-urethral prostate tissue common in aging men. Patients with enlarged prostates are treated with 5-alpha reductase inhibitors (5ARIs) to shrink prostate volume by blocking the conversion of testosterone to dihydrotestosterone (DHT). A reduction in DHT levels can elicit atrophy and apoptosis of prostate secretory luminal cells, which results in a favorable clinical response characterized by improved lower urinary tract symptoms. However, the histologic response to 5ARI treatment is often heterogeneous across prostate acini and lower urinary tract symptoms can persist to require surgical intervention. We used two spatial profiling approaches to characterize gene expression changes across histologically normal and atrophied regions in prostates from 5ARI-treated men. Objective transcriptomic profiling using the Visium spatial gene expression platform showed that 5ARI-induced atrophy of prostate luminal cells correlated with reduced androgen receptor signaling and increased expression of urethral club cell genes including LTF, PIGR, OLFM4, SCGB1A1 and SCGB3A1. Prostate luminal cells within atrophied acini adapted to decreased DHT conditions by increasing NF-κB signaling and anti-apoptotic BCL2 expression, which may explain their survival. Using GeoMx digital spatial profiling with a probe set to assess ~18,000 RNA targets, we confirmed that atrophied acini expressing SCGB3A1 displayed higher levels of club cell markers compared to histologically normal acini with NKX3-1 expression. In addition, club-like cells within regions of 5ARI-induced atrophy closely resembled true club cells from the prostatic urethra. A comparison of histologically normal regions from 5ARI-treated men and histologically normal regions from untreated men revealed few transcriptional differences. Taken together, our results describe a heterogeneous response to 5ARI treatment where cells in atrophied acini undergo an adaptation from a prostate secretory luminal to a club cell-like state in response to 5ARI treatment. This article is protected by

Epilepsy-Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS, however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 knockout (KO) mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity.Significant StatementEpilepsy-Aphasia Syndromes are at the severe end of a spectrum of cognitive-behavioral symptoms that are seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the knockout of the EAS risk gene Cnksr2 We show these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools.

Porcine astrovirus type 3 (PoAstV3) is an emerging virus in the family Astroviridae that has been recently associated with polioencephalomyelitis/encephalitis. Herein, we describe the experimental oral and intravenous inoculation of an infectious central nervous system (CNS) tissue homogenate containing PoAstV3 to cesarean-derived, colostrum-deprived pigs, and the subsequent development of clinical signs, histologic lesions, specific humoral immune response, and detection of viral particles by electron microscopy (EM) and viral RNA by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) and in situ hybridization (ISH). IgG against a portion of the PoAstV3 ORF2 capsid was first detected at 7 days post-inoculation (DPI) in 2 of 4 inoculated animals and in all inoculated animals by 14 DPI. At 21 and 28 DPI, 2 of 4 inoculated animals developed ataxia, tetraparesis, and/or lateral recumbency. All inoculated animals had histologic lesions in the CNS including perivascular lymphoplasmacytic cuffs, multifocal areas of gliosis with neuronal necrosis, satellitosis, and radiculoneuritis, and PoAstV3 RNA as detected by RT-qPCR within multiple anatomic regions of the CNS. Consistent viral structures were within the soma of a spinal cord neuron in the single pig examined by EM. Of note, PoAstV3 was not only detected by ISH in neurons of the cerebrum and spinal cord but also neurons of the dorsal root ganglion and nerve roots consistent with viral dissemination via axonal transport. This is the first study reproducing CNS disease with a porcine astrovirus strain consistent with natural infection, suggesting that pigs may serve as an animal model to study the pathogenesis of neurotropic astroviruses.

The mammalian organ of Corti is a highly specialized sensory organ of the cochlea with a fine-grained pattern that is essential for auditory function. The sensory epithelium, the organ of Corti consists of a single row of inner hair cells and three rows of outer hair cells that are intercalated by support cells in a mosaic pattern. Previous studies show that the Wnt pathway regulates proliferation, promotes medial compartment formation in the cochlea, differentiation of the mechanosensory hair cells and axon guidance of Type II afferent neurons. WNT ligand expressions are highly dynamic throughout development but are insufficient to explain the roles of the Wnt pathway. We address a potential way for how WNTs specify the medial compartment by characterizing the expression of Porcupine (PORCN), an O-acyltransferase that is required for WNT secretion. We show PORCN expression across embryonic ages (E)12.5 - E14.5, E16.5, and postnatal day (P)1. Our results showed enriched PORCN in the medial domains during early stages of development, indicating that WNTs have a stronger influence on patterning of the medial compartment. PORCN was rapidly downregulated after E14.5, following the onset of sensory cell differentiation; residual expression remained in some hair cells and supporting cells. On E14.5 and E16.5, we also examined the spatial expression of Gsk3β, an inhibitor of canonical Wnt signaling to determine its potential role in radial patterning of the cochlea. Gsk3β was broadly expressed across the radial axis of the epithelium; therefore, unlikely to control WNT-mediated medial specification. In conclusion, the spatial expression of PORCN enriches WNT secretion from the medial domains of the cochlea to influence the specification of cell fates in the medial sensory domain.

Histone proteins undergo various modifications that alter chromatin structure, including addition of methyl groups. Enhancer of homolog 2 (EZH2), is a histone methyltransferase that methylates lysine residue 27, and thereby, suppresses gene expression. EZH2 plays integral role in the uterus and other reproductive organs. We have previously shown that conditional deletion of uterine EZH2 results in increased proliferation of luminal and glandular epithelial cells, and RNAseq analyses reveal several uterine transcriptomic changes in Ezh2 conditional (c) knockout (KO) mice that can affect estrogen signaling pathways. To pinpoint the origin of such gene expression changes, we used the recently developed spatial transcriptomics (ST) method with the hypotheses that Ezh2cKO mice would predominantly demonstrate changes in epithelial cells and/or ablation of this gene would disrupt normal epithelial/stromal gene expression patterns. Uteri were collected from ovariectomized adult WT and Ezh2cKO mice and analyzed by ST. Asb4, Cxcl14, Dio2, and Igfbp5 were increased, Sult1d1, Mt3, and Lcn2 were reduced in Ezh2cKO uterine epithelium vs. WT epithelium. For Ezh2cKO uterine stroma, differentially expressed key hub genes included Cald1, Fbln1, Myh11, Acta2, and Tagln. Conditional loss of uterine Ezh2 also appears to shift the balance of gene expression profiles in epithelial vs. stromal tissue toward uterine epithelial cell and gland development and proliferation, consistent with uterine gland hyperplasia in these mice. Current findings provide further insight into how EZH2 may selectively affect uterine epithelial and stromal compartments. Additionally, these transcriptome data might provide the mechanistic understanding and valuable biomarkers for human endometrial disorders with epigenetic underpinnings.

Background Oral squamous cell carcinoma (OSCC) is an aggressive, highly immunosuppressive cancer with a high mortality rate. Interactions between programmed cell death protein 1 (PD-1; on T cells) and programmed death ligand 1 (PD-L1; on tumor cells) within the tumor microenvironment facilitates T-lymphocyte exhaustion. Regulatory T cells (Treg) are a distinct lymphocyte population, expressing the transcription factor forkhead homeobox protein-3 (FoxP3), which downregulates immune responses in OSCC. PD-L1+ tumor cells and FoxP3+ Treg expression in OSCC has been associated with poor prognosis. This research investigates the expression of PD-L1+ cells and Tregs in control, dysplastic, and OSCC tissues. Objective To investigate and compare the expression of PD-L1+ tumor cells and FoxP3+ Tregs in nondysplastic tisssue, dysplastic tissue, and OSCC using immunohistochemistry. Methods Immunohistochemistry was performed on formalin-fixed, paraffin-embedded, archival tissues. Qualitative and quantitative analyses of positively stained cells were undertaken and the dysplastic (n = 20) and OSCC groups (n = 20) were compared against the non-dysplastic control group (n = 20), using image analysis Results A higher proportion score and immunoreactive score for PD-L1+ and FoxP3+ Tregs was found in OSCC and dysplastic groups when compared to the nondysplastic control group (P < .05). There was no significant difference between the OSCC and dysplastic tissues. Conclusions Significantly more PD-L1+ cells and Tregs were detected in dysplastic and OSCC tissues. An increase in PD-L1 and FoxP3 expression may serve as an indicator of progression from normal to a potentially malignant lesion.

Background: Preclinical studies indicate opioid administration evokes pro-inflammatory responses in both the periphery and brain. Opioid-induced pro-inflammatory responses influence both appetitive and dysphoric addiction processes and thus, may influence the development of opioid use disorder (OUD) and/or perpetuate continued opioid use among OUD patients. Herein, we investigated the neuroimmune effects of morphine administration using Positron Emission Tomography (PET) imaging with [11C]PBR28, a radiotracer that binds to the 18kDa translocator protein (TSPO), a marker sensitive to immune stimuli. Methods: Healthy individuals with prior medical opioid exposure (N¼4; 3M; 2 ‘high-affinity’ binders; Age¼30yrs [range¼26-38]; BMI¼26.5 [range¼24-30]) completed two Cold Pressor tasks and [11C]PBR28 PET scans (120min acquisition) in one day: one before and one after intramuscular morphine (0.07mg/kg). Arterial blood was acquired to measure the metabolite-corrected input function. Total volume of distribution (VT), i.e., TSPO availability, was estimated in 10 brain regions using multilinear analysis-1 (MA-1; t*¼30). Morphine’s effect on regional [11C]PBR28 VT was evaluated using a repeated-measures analysis of variance with rs6971 genotype as a fixed factor. Results: Morphine increased TSPO availability by 28%-39% across regions, F(1,2)¼9.56, p¼.09, partial h2¼0.83, ‘very large’ effect. Morphine increased hand withdrawal latency on the Cold Pressor task, F(1,2)¼3.98, p¼.18, partial h2¼0.66, ‘very large’ effect. Conclusions: Preliminary findings suggest that an analgesic morphine dose (4.69-5.95mg) induced a whole-brain neuroimmune response in healthy adults, the first such evidence in people. If confirmed, our findings suggest a plausible role for the neuroimmune system in the development of OUD. Future studies are needed to investigate opioid-neuroimmune relationships in OUD patients.

Chronic low back pain (cLBP) prevalence increases with advancing age and is a leading contributor to mobility disability among older adults. Opioids are commonly prescribed treatments to reduce pain related symptoms. The rise in opioid use and misuse can enhance a variety of issues in the adult population; such as, lack of mobility and decrease in overall health and wellbeing. Few studies have examined the impact of opioid use on mobility in older adults with cLBP. Therefore, we sought to examine the relationship between self-reported opioid use and self-reported mobility. cLBP participants (n = 140) completed a series of questionnaires regarding pain intensity, interference, and disability including demographics, clinical pain assessment, and the Brief Pain Inventory-Short form. Pearson's chi-square tests, and regression-based analyses were conducted using SPSS version 26.0. Among cLBP participants, those who self-reported opioid use were more likely to have greater self-reported difficulty climbing stairs (χ2 = 16.6, p < .05), walking for fifteen minutes (χ2 = 17.7, p < .05), performing chores (χ2 = 15.4, p < .05), and running errands (χ2 = 10.7, p < .05). Among the older cLBP participants above the age of 54 (n = 38), half used an opioid (n = 19) at some point of time as a form of cLBP pain treatment. Among older adults, opioid use was significantly associated with poorer self-reported outcomes for climbing stairs (Wald χ2(1) = 5.9, p < .05), walking (Wald χ2(1) = 7.4, p < .05), and performing chores (Wald χ2(1) = 7.5, p < .05). Opioid use predicts poorer self-reported mobility among adults and older adults with cLBP. Results inform associations between pain treatment and mobility in aging populations. Future research should seek to understand the influence of opioids on objective performance measures in cLBP. This work was supported by Examining Racial And SocioEconomic Disparities in cLBP; ERASED; R01MD010441.

Background and aims In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signalling. The Bone Morphogenetic Protein (BMP) pathway has a physiological, pro-differentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signalling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signalling landscape, and investigated the impact and therapeutic potential, of pathway manipulation in homeostasis and regeneration. Methods A novel mouse model was generated to assess the effect of autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signalling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. Results In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival and prevented dedifferentiation. Following ulceration, physiological attenuation of BMP signalling arose through upregulation of the secreted antagonist, Grem1, from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation following Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory, but functionally sub-maximal, as regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1 respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming, despite a convergent impact of YAP/TAZ on cell fate in remodelled wounds. Conclusions BMP signalling prevents epithelial de-differentiation, and pathway attenuation, through stromal Grem1 upregulation, was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally sub-maximal, raising the possibility of therapeutic pathway manipulation in Inflammatory Bowel Disease.