Probes for INS

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.

In situ follicular neoplasia (ISFN) is usually an occasional incidental finding in lymph nodes by BCL2 immunohistochemistry, and its true scale is unknown. We have identified 6 cases of follicular lymphoma (FL) with a history of solid neoplasm 4-16 years ago, from which ISFN was identified widely in the surgically cleared lymph nodes (LNs). Using clone-specific PCR and BaseScope in situ hybridisation with primers or probes specific to the VDJ or BCL2-IGHJ junction sequence, we confirmed the clonal identity among different ISFNs and overt-FL in each of the 4 cases successfully investigated. Mutation analyses of overt-FL by targeted next-generation sequencing identified multiple potential pathogenic changes involving CREBBP, EZH2, KMT2D, TNFRS14 and STAT6. Further investigations of these mutations in paired ISFNs using Fluidigm PCR and Illumina sequencing showed the presence of the FL associated mutations in early lesions for 2 of the 6 cases investigated (CREBBP and KMT2D in one case and STAT6 in the other), with one case displaying stepwise accumulation of its observed mutations. Remarkably, there were considerable divergences in BCL2 variants among different ISFN involved lymph nodes in all 4 cases successfully investigated, indicating ongoing intraclonal diversification by somatic hypermutation machinery. Our findings demonstrate widespread distribution of ISFN lesions, further implicating their dynamic nature with the neoplastic cells undergoing active trafficking and clonal evolution. This article is protected by

Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment.We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically.In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without.Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.

Background The heterogeneity of salivary gland neoplasms, within and between histologic types, presents a major diagnostic challenge. Mucoepidermoid carcinoma (MEC), the most common salivary gland cancer in adults, children, and adolescents, is associated with the presence of a novel CRTC1-MAML2 fusion gene. The translocation can be detected by fluorescence in situ hybridization or reverse transcription polymerase chain reaction but without information regarding transcript level, identification of the cell type(s) harboring the translocation and histologic architecture is not preserved. This study describes, for the first time, a novel in situ chromogenic assay, BaseScope, to detect CRTC1-MAML2 translocation expression. Objective Design a novel BaseScope probe targeting the novel exon-exon junction in the CRTC1-MAML2 fusion transcript, determine expression levels of the transcript, and identify specific cell types harboring the translocation. Methods Formalin-fixed paraffin-embedded tissues and known fusion-positive and -negative human MEC cells were subjected to the assay. Results The CRTC1-MAML2 RNA transcript was detected in known fusion-positive cells but not in fusion-negative cells. In MEC tissues distinct fusion events, in the form of punctate red dots, were detected in all tumor grades and all cell types. Interestingly, the translocation was specifically identified in tumor cells that had direct contact with tumor stroma or nerve invasion. No positive staining was seen in normal tissue or surrounding stroma and no unique morphological features were noted in negative cases. Conclusions The BaseScope assay accurately detects the CRTC1-MAML2 fusion translocation and thus provides an alternative chromogenic technique, for easy use in routine clinical labs, to aid accurate diagnosis of MEC.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

Women with atypical hyperplasia (AH) or well-differentiated early-stage endometrioid endometrial carcinoma (EEC) who wish to retain fertility and/or with comorbidities precluding surgery, are treated with progestin. Clinically approved predictive biomarkers for progestin therapy remain an unmet need. The objectives of this study were to document the overall response rate (ORR) of levonorgestrel intrauterine device (LNG-IUD) treatment, and determine the association of FGFR2b and FGFR2c expression with treatment outcome. BaseScope RNA ISH assay was utilized to detect expression of FGFR2b and FGFR2c mRNA in the diagnostic biopsies of 89 women (40 AH and 49 EEC) treated with LNG-IUD. Detailed clinical follow-up was available for 69 women which revealed an overall response rate (ORR) of 44% (30/69) with a higher ORR seen in AH (64%) compared to EEC (23%). The recurrence rate in women who initially responded to LNG-IUD was 10/30 (33.3%). RNA ISH was successful in 72 patients and showed FGFR2c expression in 12/72 (16.7%) samples. In the 59 women with detailed clinical follow-up and RNA-ISH data, women with tumours expressing FGFR2c were 5-times more likely to have treatment failure in both univariable (HR 5.08, p < 0.0001) and multivariable (HR 4.5, p < 0.002) Cox regression analyses. In conclusion, FGFR2c expression appears to be strongly associated with progestin treatment failure, albeit the ORR is lower in this cohort than previously reported. Future work to validate these findings in an independent multi-institutional cohort is needed.

The limited ability for axonal repair after spinal cord injury underlies long-term functional impairment. DLK (MAP3K12) is an evolutionarily conserved MAP3K implicated in neuronal injury signaling from C. elegans to mammals. However, whether DLK or its close homologue LZK (MAP3K13) regulates axonal repair in the mammalian spinal cord remains unknown. Here we assess the role of endogenous DLK and LZK in the regeneration and compensatory sprouting of corticospinal tract (CST) axons in mice of both sexes with genetic analyses in a regeneration competent background provided by PTEN deletion. We found that inducible neuronal deletion of both DLK and LZK, but not either kinase alone, abolishes PTEN deletion-induced regeneration and sprouting of CST axons, and reduces naturally-occurring axon sprouting after injury. Thus, DLK/LZK-mediated injury signaling operates not only in injured neurons to regulate regeneration, but also unexpectedly in uninjured neurons to regulate sprouting. Deleting DLK and LZK does not interfere with PTEN/mTOR signaling, indicating that injury signaling and regenerative competence are independently controlled. Together with our previous study implicating LZK in astrocytic reactivity and scar formation, these data illustrate the multicellular function of this pair of MAP3Ks in both neurons and glia in the injury response of the mammalian spinal cord.SIGNIFICANCE STATEMENT:Functional recovery after spinal cord injury is limited due to a lack of axonal repair in the mammalian central nervous system (CNS). DLK and LZK are two closely related protein kinases that have emerged as regulators of neuronal responses to injury. However, their role in axon repair in the mammalian spinal cord has not been described. Here we show that DLK and LZK together play critical roles in axonal repair in the mammalian spinal cord, validating them as potential targets to promote repair and recovery after spinal cord injury. In addition to regulating axonal regeneration from injured pathways, they also regulate compensatory axonal growth from uninjured pathways, indicating a more pervasive role in CNS repair than originally anticipated.

Endometrial carcinoma (EC) is the most common gynecological malignancy and fibroblast growth factor receptor 2 (FGFR2) is a frequently dysregulated receptor tyrosine kinase. FGFR2b and FGFR2c are the two main splice isoforms of FGFR2 and are normally localized in epithelial and mesenchymal cells, respectively. Previously, we demonstrated that FGFR2c mRNA expression was associated with aggressive tumor characteristics, shorter progression-free survival (PFS), and disease-specific survival (DSS) in endometrioid ECs (EECs). The objectives of this study were to investigate the spatial expression of FGFR2b in normal and hyperplasia with and without atypia of human endometrium and to assess the prognostic significance of FGFR2b expression in EC. FGFR2b and FGFR2c mRNA expression was evaluated in normal (proliferative [n = 10], secretory [n = 15], and atrophic [n = 10] endometrium), hyperplasia with and without atypia (n = 19) as well as two patient cohorts of EC samples (discovery [n = 78] and Vancouver [n = 460]) using isoform-specific BaseScope RNA in situ hybridization assays. Tumors were categorized based on FGFR2 isoform expression (one, both, or neither) and categories were correlated with clinicopathologic markers, molecular subtypes, and clinical outcomes. The FGFR2b splice isoform was exclusively expressed in the epithelial compartment of normal endometrium and hyperplasia without atypia. We observed FGFR2c expression at the basalis layer of glands in 33% (3/9) of hyperplasia with atypia. In patients with EEC, FGFR2b+/FGFR2c- expression was found in 48% of the discovery cohort and 35% of the validation Vancouver cohort. In univariate analyses, tumors with FGFR2b+/FGFR2c- expression had longer PFS (hazard ratio [HR] 0.265; 95% CI 0.145-0.423; log-rank p < 0.019) and DSS (HR 0.31; 95% CI 0.149-0.622; log-rank p < 0.001) compared to tumors with FGFR2b-/FGFR2c+ expression in the large EEC Vancouver cohort. In multivariable Cox regression analyses, tumors with FGFR2b+/FGFR2c- expression were significantly associated with longer DSS (HR 0.37; 95% CI 0.153-0.872; log-rank p < 0.023) compared to FGFR2b-/FGFR2c+ tumors. In conclusion, FGFR2b+/FGFR2c- expression is associated with favorable clinicopathologic markers and clinical outcomes suggesting that FGFR2b could play a role in tailoring the management of EEC patients in the clinic if these findings are confirmed in an independent cohort.

There is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC).We conducted whole-transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression.In this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27-JNK/P38 mitogen-activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27-JNK/P38 MAPK signaling and promoted tumor progression.Our results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27-JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Circular RNAs (circRNAs) are endogenous single-stranded RNAs characterized by covalently closed loop structures with neither 5′ to 3′ polarity nor poly(A) tails. They are generated most commonly from back-splicing of protein-coding exons. CircRNAs have a tissue-specific distribution and are evolutionarily conserved, and many circRNAs play important biological functions by combining with microRNAs and proteins to regulate protein functions and their own translation. Numerous studies have shown that circRNAs are enriched in the central nervous system (CNS) and play an important role in the development and maintenance of homeostasis. Correspondingly, they also play an important role in the occurrence and progression of CNS diseases. In this review, we highlight the current state of circRNA biogenesis, properties, function and the crucial roles they play in the CNS.