Probes for INS

Transthyretin (TTR) distributes thyroxine in the cerebrospinal fluid of mammals. Choroid plexus epithelial cells produce and secrete TTR, and were long recognized as the only CNS source of TTR. However, research over the last years has reported neuronal-specific expression as well, but without a clear function. Recently, we found Ttr transcripts in cells of the adult mouse subventricular zone (SVZ), the largest neural stem cell (NSC) region, but the protein was undetectable. We therefore investigated in more detail what role TTR might play in the SVZ, and when. We mapped temporal-spatial Ttr expression by re-analysing publicly available single-cell RNA-Seq data obtained from dissected mouse SVZs at E14-E17-P2-P7-P20-P61. We observed a peak in Ttr expression in NSCs, neural progenitors and differentiating cells at postnatal day 7 (P7). That is one week prior to when thyroxine serum levels peak and T3 activates SVZ-NSCs that start generating neurons and glia at a constant rate. RNAscope on P7 brain sections confirmed that few Ttr transcripts are present in a many SVZ-progenitors, oligodendrocyte precursors and neuroblasts. Unexpectedly though, no protein was detectable using commercially available antibodies, signal amplification and appropriate controls. This might suggest TTR is rapidly secreted to affect nearby cells. To test this hypothesis, we prepared neurospheres from dissected SVZ-progenitors at P7. After 7 days of proliferation, cells were dissociated, and allowed to differentiate for 1 or 5 days. In parallel with controls, we treated them once at day 0 of differentiation with a low (2.5 µg/ml) or a high dose (25 µg/ml) of human recombinant TTR, or with 5 nM T3. Low TTR doses reduced cell mitosis at day 1, as did T3. After 5 days, we counted a 30% lower proportion of differentiated neuroblasts with the highest TTR dose. That proportion had dropped 3-fold in the presence of T3. Proportions of oligodendroglia after 5 days of differentiation were only significantly higher in T3 conditions. As a result, the neuron/glia balance shifted in favour of oligodendrogenesis under T3, and borderline-significantly following high TTR doses. Altogether, the murine SVZ represents a novel region containing cells that express Ttr, with a peak at P7, despite seeming absence of the protein itself, precluding deducing its exact role. Single-cell RNA-Seq on treated neurospheres could reveal how exogenous TTR affects intracellular pathways, and whether its action is TH-dependent or not. This can help unravelling the pathophysiology of familial amyloid polyneuropathy, in which misfolded TTR proteins cause neurodegeneration.

Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice.Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group).We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05).Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF1 and CRF2), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF1, the role of CRF2 in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF2, interacting with a C terminal fragment of CRFBP, CRFBP(10kD), may have a role in the modulation of neuronal activity. However, the mechanism by which CRF interacts with CRFBP(10kD) and CRF2 has not been fully elucidated due to the lack of useful chemical tools to probe CRFBP.We miniaturized a cell-based assay, where CRFBP(10kD) is fused as a chimera with CRF2, and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF2 complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF2 in the absence of CRFBP(10kD), and toward CRF1 in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF1+ neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF1 promoter, and 2) the CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in dopamine neurons in the ventral tegmental area (VTA).We found that CRFBP(10kD) potentiates CRF-intracellular Ca2+ release specifically via CRF2, indicating that CRFBP may possess excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP, CRFBP(27kD). We identified novel small molecule CRFBP-CRF2 NAMs that do not alter the CRF1-mediated effects of exogenous CRF but blunt CRF-induced potentiation of NMDAR-mediated synaptic transmission in dopamine neurons in the VTA, an effect mediated by CRF2 and CRFBP.These results provide the first evidence of specific roles for CRF2 and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF2 NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.

Introduction. During the COVID-19 pandemic, the question regarding an effect of related infection on the body of pregnant women and the fetoplacental complex has emerged, with many aspects of this issue still being unknown. At the moment, it has been proven that in some cases the course of COVID-19 can be accompanied by severe systemic inflammatory reaction leading to hypercoagulable state.Aim: to search for evidence of a direct and/or indirect effect of SARS-CoV-2 infection on human placenta structure.Materials and Methods. Taking into account the goal, this review was compiled according to the type of a narrative review of publications on a topic of interest. A search for English-language publications dated of 01.12.2019 till 01.12.2021 in PubMed/MEDLINE, Cochrane, Web of Science databases was made. The search queries included the following keywords: combinations of «coronavirus» and «infection during pregnancy», «placental structure» and «2019-nCoV», «COVID-19 and pregnancy», «SARSCoV-2 and pregnancy». In the process of writing the article, in order to improve the reader's understanding of the essence of debated issue, there was a need to discuss some of the results with literary sources published earlier 2019 that were not directly related to the topic of the new coronavirus infection (there are 6 such sources). We analyzed full-text publications, both reports on original research and meta-analyses on relevant topics. In total, 351 full-text publications met the query criteria, of which 54 were selected as meeting the objectives of the study. The select reports were discussed by the co-authors, duplicates were excluded and 34 of them were included in this review. In those that were excluded from the review, information about the clinical course of pregnancy and its outcome during novel coronavirus infection prevailed, or isolated cases of studying insignificant placental structural changes were discussed. Studies with a small number of observations were selected only in the case of the uniqueness of the published data, the absence of scientific papers where similar studies would have been conducted in larger sample.Results. Pregnancy complicated by COVID-19 may be accompanied by placental structural changes, which represent both a manifestation of compensatory-adaptive reactions and a consequence of the damaging effect to the placenta due to infectious process. In case of late (in the III trimester) disease in pregnant woman with mild COVID-19, placental disorders are predominantly of compensatory-adaptive nature, specific cytological signs of viral cell damage are uncharacteristic. During COVID-19 infection, chronic histiocytic intervillositis and syncytiotrophoblast necrosis occur more often than in average population, and adverse fetal outcomes are characterized by additional marked increase in intervillous fibrinoid deposition. Before COVID-19 pandemic, chronic histiocytic intervillositis was described in about 6 out of 10,000 placentas (0.6 %) in II and III trimesters.Conclusion. The high frequency of chronic histiocytic intervillositis, both in the placenta of paired women with live-born infants infected prenatally due to maternal virus transmission, and in the placentas of stillborn infected infants, allows us to cautiously assume that such placental structural changes are more characteristic for damage by SARS-CoV-2 rather than other infectious agents. It is necessary to study a relationship between placental structural changes occurred at different gestation ages, as well as clinical course and outcome of pregnancy during COVID-19.

Introduction: Four randomized controlled trials studying fecal microbiota transplantation (FMT) in active ulcerative colitis (UC) patients showed variable success rates. The efficacy of FMT appears to be influenced by various factors including donor- and procedure-specific characteristics. Aim: We hypothesized that the outcome of FMT in patients with active UC could be improved by donor preselection on microbiota level, by using a strict anaerobic approach, and by repeated FMT administration. Methods: The RESTORE-UC trial (NCT03110289) was a national, multi-centric double-blind, sham-controlled randomized trial. Active UC patients (Total Mayo score 4-10 with endoscopic sub-score > or = 2) were randomly allocated (1:1) to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT by permutated blocks (2- 4) and stratified for weight, concomitant steroid use, and therapy refractoriness. S-FMTs were selected after a rigorous screening excluding samples with Bacteroides 2 enterotype, high abundances of Fusobacterium, Escherichia coli and Veillonella and the lowest microbial loads (Q1). A futility analysis after 66% (n=72) of inclusions was planned per protocol including a modified intention-to-treat (mITT) analysis using non-responder imputation (NRI) for patients receiving at least one FMT. The primary endpoint was steroid-free clinical remission (Total Mayo ≤ 2, with no subscore >1) at week 8. Secondary outcomes included steroid-free PRO-2 remission (Combined Mayo subscores of ≤1 for rectal bleeding plus stool frequency) and response (≥3 points or/and ≥50% reduction from baseline in combined Mayo subscores for rectal bleeding plus stool frequency) and steroid-free endoscopic remission (Mayo endoscopic subscore ≤1) and response (Mayo endoscopy subscore ≤1 and ≥1 point reduction from baseline). Results: Between March 2017-2021, 72 patients signed the ICF and 66 were randomly allocated to S-FMT (n=30) or A-FMT (N=36) and received at least one FMT. Both study arms were matched for baseline characteristics, yet a trend (p= 0,07) towards higher concomitant biological use in the S-FMT arm was observed. A remarkably high proportion of patients were previously exposed to biologicals (58.3% and 60.0% for the A-FMT and S-FMT group respectively). In the S-FMT and the A-FMT respectively 4 and 5 patients terminated the trial early due to worsening of colitis (4 in both arms) or FMT enema intolerance (1 A-FMT). They were included in the mITT analysis using NRI, showing after 66% of intended inclusions, the primary endpoint was reached in 3/30 (mITT with NRI 10.0%) S-FMT and 5/31 (13.9%) patients randomized to A-FMT (p=0.72). As the predefined minimum difference of 5% between both treatment arms was not attained, the study was stopped due to futility. Steroid-free PRO-2 remission was achieved in 7/30 (23,3%) patients on S-FMT and 10/36 (27,8%) on A-FMT (p= 0,78). Steroid-free PRO-2 response was attained by respectively 9/30 (30,0%) patients in the S-FMT arm and 12/36 (33,3%) patients in the A-FMT arm (p= 0,80). Steroid-free endoscopic response and remission were noted in 5/30 (16,7%) assigned to the S-FMT arm compared with 7/36 (19,4%) allocated to the A-FMT arm (p= 1.0). Of note, no patients on concomitant biologicals reached the primary endpoint, and there were 2 serious adverse events in the A-FMT arm: dysuria requiring hospitalization and worsening of UC requiring colectomy. Conclusions: In this double-blind sham-controlled trial comparing repeated administrations of anaerobic-prepared S-FMT with A-FMT in patients with active UC, no significant difference in steroid-free remission rates at week 8 were observed. The FMT procedure was generally well tolerated, and no new safety signals were observed.

Introduction: Four randomized controlled trials studying fecal microbiota transplantation (FMT) in active ulcerative colitis (UC) patients showed variable success rates. The efficacy of FMT appears to be influenced by various factors including donor- and procedure-specific characteristics. Aim: We hypothesized that the outcome of FMT in patients with active UC could be improved by donor preselection on microbiota level, by using a strict anaerobic approach, and by repeated FMT administration. Methods: The RESTORE-UC trial (NCT03110289) was a national, multi-centric double-blind, sham-controlled randomized trial. Active UC patients (Total Mayo score 4-10 with endoscopic sub-score > or = 2) were randomly allocated (1:1) to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT by permutated blocks (2- 4) and stratified for weight, concomitant steroid use, and therapy refractoriness. S-FMTs were selected after a rigorous screening excluding samples with Bacteroides 2 enterotype, high abundances of Fusobacterium, Escherichia coli and Veillonella and the lowest microbial loads (Q1). A futility analysis after 66% (n=72) of inclusions was planned per protocol including a modified intention-to-treat (mITT) analysis using non-responder imputation (NRI) for patients receiving at least one FMT. The primary endpoint was steroid-free clinical remission (Total Mayo ≤ 2, with no subscore >1) at week 8. Secondary outcomes included steroid-free PRO-2 remission (Combined Mayo subscores of ≤1 for rectal bleeding plus stool frequency) and response (≥3 points or/and ≥50% reduction from baseline in combined Mayo subscores for rectal bleeding plus stool frequency) and steroid-free endoscopic remission (Mayo endoscopic subscore ≤1) and response (Mayo endoscopy subscore ≤1 and ≥1 point reduction from baseline). Results: Between March 2017-2021, 72 patients signed the ICF and 66 were randomly allocated to S-FMT (n=30) or A-FMT (N=36) and received at least one FMT. Both study arms were matched for baseline characteristics, yet a trend (p= 0,07) towards higher concomitant biological use in the S-FMT arm was observed. A remarkably high proportion of patients were previously exposed to biologicals (58.3% and 60.0% for the A-FMT and S-FMT group respectively). In the S-FMT and the A-FMT respectively 4 and 5 patients terminated the trial early due to worsening of colitis (4 in both arms) or FMT enema intolerance (1 A-FMT). They were included in the mITT analysis using NRI, showing after 66% of intended inclusions, the primary endpoint was reached in 3/30 (mITT with NRI 10.0%) S-FMT and 5/31 (13.9%) patients randomized to A-FMT (p=0.72). As the predefined minimum difference of 5% between both treatment arms was not attained, the study was stopped due to futility. Steroid-free PRO-2 remission was achieved in 7/30 (23,3%) patients on S-FMT and 10/36 (27,8%) on A-FMT (p= 0,78). Steroid-free PRO-2 response was attained by respectively 9/30 (30,0%) patients in the S-FMT arm and 12/36 (33,3%) patients in the A-FMT arm (p= 0,80). Steroid-free endoscopic response and remission were noted in 5/30 (16,7%) assigned to the S-FMT arm compared with 7/36 (19,4%) allocated to the A-FMT arm (p= 1.0). Of note, no patients on concomitant biologicals reached the primary endpoint, and there were 2 serious adverse events in the A-FMT arm: dysuria requiring hospitalization and worsening of UC requiring colectomy. Conclusions: In this double-blind sham-controlled trial comparing repeated administrations of anaerobic-prepared S-FMT with A-FMT in patients with active UC, no significant difference in steroid-free remission rates at week 8 were observed. The FMT procedure was generally well tolerated, and no new safety signals were observed.

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Xuming Zhu is first author on ‘ FZD2 regulates limb development by mediating β-catenin-dependent and -independent Wnt signaling pathways’, published in DMM. Xuming is an instructor in the lab of Sarah E. Millar at Icahn School of Medicine at Mount Sinai, New York, NY, USA, investigating the molecular mechanisms that underlie the development of appendages, epithelial homeostasis and diseases.

Adenoid cystic carcinoma (ACC) is one of the most common primary salivary gland cancers. ACC has several benign and malignant mimics amongst salivary gland neoplasms. An accurate diagnosis of ACC is essential for optimal management of the patients and their follow-up. Upregulation of MYB has been described in 85-90% of ACC, but not in other salivary gland neoplasms. In ACC, MYB upregulation can occur as a result of a genetic rearrangement t(6;9)(q22-23;p23-24), MYB copy number variation (CNV), or enhancer hijacking of MYB. All mechanisms of MYB upregulation result in increased RNA transcription that can be detected using RNA in situ hybridisation (ISH) methods. In this study, utilising 138 primary salivary gland neoplasms including 78 ACC, we evaluate the diagnostic utility of MYB RNA ISH for distinguishing ACC from other primary salivary gland neoplasms with a prominent cribriform architecture including pleomorphic adenoma, basal cell adenoma, basal cell adenocarcinoma, epithelial myoepithelial carcinoma, and polymorphous adenocarcinoma. Fluorescent in situ hybridisation and next generation sequencing were also performed to evaluate the sensitivity and specificity of RNA ISH for detecting increased MYB RNA when MYB gene alterations were present. Detection of MYB RNA has 92.3% sensitivity and 98.2% specificity for a diagnosis of ACC amongst salivary gland neoplasms. The sensitivity of MYB RNA detection by ISH (92.3%) is significantly higher than that of the FISH MYB break-apart probe (42%) for ACC. Next generation sequencing did not demonstrate MYB alterations in cases that lacked MYB RNA overexpression indicating high sensitivity of MYB RNA ISH for detecting MYB gene alterations. The possibility that the sensitivity may be higher in clinical practice with contemporary samples as compared with older retrospective tissue samples with RNA degradation is not entirely excluded. In addition to the high sensitivity and specificity, MYB RNA testing can be performed using standard IHC platforms and protocols and evaluated using brightfield microscopy making it a time and cost-efficient diagnostic tool in routine clinical practice.

Severe debilitating pain is the most common complication and reason for hospitalization for individuals with sickle cell disease (SCD), a genetic blood disorder that affects 100,000 people in the US and over 3 million worldwide. Despite this, the biological basis of chronic SCD pain is not fully understood. Using transgenic SCD mice and fecal material transplant paradigms, we determined that gastrointestinal tract contents drive persistent SCD pain. Mechanical allodynia was temporarily alleviated in SCD mice following fecal transplant from wildtype animals. In contrast, wildtype mice developed mechanical and cold allodynia following fecal transplant from SCD animals. To identify gut bacterial species and metabolites responsible for SCD pain, we completed 16s rRNA sequencing and metabolomic screening respectively on transplant recipient feces. Bilirubin, a product of heme breakdown, was significantly elevated in the feces of SCD mice and mice that received SCD fecal transplants, as well as in the plasma of individuals with SCD. Oral administration of bilirubin induced mechanical allodynia in wildtype mice that depended on vagus nerve signaling. Using whole cell patch clamp recordings, we demonstrated that bilirubin directly activates vagal afferents and increases afferent excitability. Ongoing experiments are investigating the specific receptors through which bilirubin alters neuronal activity as drugs targeting these proteins may prove effective analgesics for SCD pain. In summary, these experiments are the first to demonstrate that sickle cell gut contents drive chronic widespread pain in this disease, and furthermore, are the first to identify gut metabolites that should be targeted for chronic SCD pain management. National Institutes of Health: K99HL155791(KS), R01HL142657(AB), R01NS070711 CS).

Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder that with no effective treatments. Several microRNA (miRNA) are commonly dysregulated in CRPS patient and tibia fracture model of CRPS (TFM) mice, including miR-25 which is associated with positive treatment outcomes in patients. Interestingly, these miRNAs are predicted to target several genes critical to resident memory T cell (Trm) function. We hypothesize that miRNA dysregulation contributes to the pathology of CRPS through regulation of skin Trm development and maintenance. Therapeutic strategies blocking Trm development or maintenance may be beneficial in treating this disease. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Animals were treated with therapeutic agents after development of TFM and monitored for behavioral outcomes and T cell populations of collected tissues were analyzed at different time points by flow cytometry. There was an inverse correlation of miR-25 and CD69 in blood samples from CRPS patients compared to controls. TFM hindlimb skin shows increased epidermal CD8+ and CD4+ Trm, dermal CD4+ Trm. Epidermal CD8+ Trm, dermal CD4+ Trm are marked by increases in CD103+CD49a+ populations, and along with splenic CD8+ Tem show increased CD122+ cells. Therapeutic studies are ongoing. miRNA signatures in CRPS patients and TFM mice show common alterations which are capable of regulating CD69, a core Trm marker. TFM hindlimb skin shows increased pathological Trm populations and treatments targeting Trm development and maintenance may be beneficial in treating CRPS. 1RF1NS130481-01.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.