Pax6 regulates gene expression in the vertebrate lens through miR-204.

During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

Roles for Dicer1 in the patterning and differentiation of the optic cup neuroepithelium.

The embryonic ocular neuroepithilium generates a myriad of cell types, including the neuroretina, the pigmented epithelium, the ciliary and iris epithelia, and the iris smooth muscles. As in other regions of the developing nervous system, the generation of these various cell types requires a coordinated sequence of patterning, specification and differentiation events. We investigated the roles of microRNAs (miRNAs) in the development of optic cup (OC)-derived structures. We inactivated Dicer1, a key mediator of miRNA biosynthesis, within the OC in overlapping yet distinct spatiotemporal patterns. Ablation of Dicer1 in the inner layer of the OC resulted in patterning alteration, particularly at the most distal margins. Following loss of Dicer1, this region generated a cryptic population of cells with a mixed phenotype of neuronal and ciliary body (CB) progenitors. Notably, inactivation of Dicer1 in the retinal progenitors further resulted in abrogated neurogenesis, with prolongation of ganglion cell birth and arrested differentiation of other neuronal subtypes, including amacrine and photoreceptor cells. These alterations were accompanied by changes in the expression of Notch and Hedgehog signaling components, indicating the sensitivity of the pathways to miRNA activity. Moreover, this study revealed the requirement of miRNAs for morphogenesis of the iris and for the regulation of CB cell type proliferation and differentiation. Together, analysis of the three genetic models revealed novel, stage-dependent roles for miRNAs in the development of the ocular sub-organs, which are all essential for normal vision.

A novel urine cytology stain for the detection and monitoring of bladder cancer.

PURPOSE: CellDetect® is a unique platform technology comprising a proprietary plant extract and 3 dyes that enables color discrimination between malignant (red) and benign (green) cells based on specific metabolic alterations exclusive to the former. Preclinical studies and clinical trials demonstrated the applicability of the new technology in many cell culture lines and various cancers. We explored its performance characteristics in bladder cancer. MATERIALS AND METHODS: We performed an open label, 2-step study at tertiary medical centers. The study enrolled patients with newly diagnosed or a history of urothelial carcinoma. Step 1 involved staining archived biopsies. Slides were evaluated by 2 independent pathologists, who determined the concordance of the new staining technology with the hematoxylin and eosin based diagnosis. Step 2 included staining urine specimens with the new method and comparing findings to the patient final diagnosis and the results of standard urine cytology. RESULTS: A total of 58 archived biopsies were collected. The concordance of staining using the new platform technology with the hematoxylin and eosin based diagnosis was 100%. The new method applied to 44 urine smears showed 94% sensitivity and 89% specificity to detect urothelial carcinoma. Compared to standard urine cytology the new technology had overall superior sensitivity (94% vs 46%), particularly for low grade tumors (88% vs 17%, each p <0.005). There was no significant difference in specificity between the 2 staining techniques. CONCLUSIONS: Findings show the capability of CellDetect to accurately identify urothelial carcinoma. This indicates that the technology can be further developed to provide an alternative urine cytology test with diagnostic value that may have significant clinical benefits.

Reduced expression of Pax6 in lens and cornea of mutant mice leads to failure of chamber angle development and juvenile glaucoma.

Heterozygous mutations in PAX6 are causative for aniridia, a condition that is frequently associated with juvenile glaucoma. Defects in morphogenesis of the iridocorneal angle, such as lack of trabecular meshwork differentiation, absence of Schlemm's canal and blockage of the angle by iris tissue, have been described as likely causes for glaucoma, and comparable defects have been observed in heterozygous Pax6-deficient mice. Here, we employed Cre/loxP-mediated inactivation of a single Pax6 allele in either the lens/cornea or the distal optic cup to dissect in which tissues both alleles of Pax6 need to be expressed to control the development of the tissues in the iridocorneal angle. Somatic inactivation of one allele of Pax6 exclusively from epithelial cells of lens and cornea resulted in the disruption of trabecular meshwork and Schlemm's canal development as well as in an adhesion between iris periphery and cornea in juvenile eyes, which resulted in the complete closure of the iridocorneal angle in the adult eye. Structural changes in the iridocorneal angle presumably caused a continuous increase in intraocular pressure leading to degenerative changes in optic nerve axons and to glaucoma. In contrast, the inactivation of a single Pax6 allele in the distal optic cup did not cause obvious changes in iridocorneal angle formation. We conclude that the defects in iridocorneal angle formation are caused by non-autonomous mechanisms due to Pax6 haploinsufficiency in lens or corneal epithelial cells. Pax6 probably controls the expression of signaling molecules in lens cells that regulate the morphogenetic processes during iridocorneal angle formation.

Pax6 dosage requirements in iris and ciliary body differentiation.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.

Link between personality and response to THC exposure.

The effects of cannabis reported by users range from experiences of euphoria and anxiolytic effects to paranoia, anxiety, and increased risk of depression. Attempts to reconcile the apparent contradictions in user response have not been conclusive. Here, we utilized selectively-bred stress-resilient socially dominant (Dom) and stress-sensitive socially submissive (Sub) mice to elucidate this contradiction. Following short-term, repeated treatment with delta-9-tetrahydrocannabinol (THC) at two different doses (1.5 mg/kg and 15 mg/kg), Sub mice presented significant place-aversion in a Conditioned Place Preference paradigm at a high dose, whereas Dom mice displayed no place preference or aversion. Forced Swim test conducted after 6-week of washout period, revealed differential impact of the two THC doses depending upon behavioral pattern. Specifically, the low dose alleviated depressive-like behavior in Sub mice, while the high dose produced the opposite effect in Dom mice. Interestingly, corticosterone concentration in serum was elevated at the high dose regardless of the mice-population tested. We conclude here that differences in dominance behavior and stress vulnerability are involved in the regulation of cannabis response among users and should be considered when prescribing THC-containing medications to patients.

SIRT1/HERC4 Locus Associated With Bisphosphonate-Induced Osteonecrosis of the Jaw: An Exome-Wide Association Analysis.

Osteonecrosis of the jaw (ONJ) is a rare, but serious drug side effect, mainly associated with the use of intravenous (iv) bisphosphonates (BPs). The purpose of this study was to identify genetic variants associated with ONJ in patients of European ancestry treated with iv BPs using whole-exome sequencing (WES). The WES phase 1 included 44 multiple myeloma patients (22 ONJ cases and 22 controls) and WES phase 2 included 17 ONJ patients with solid tumors. Multivariable logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age, sex, and principal components for ancestry. Meta-analysis of WES phase 1 and 2 was performed to estimate the combined ORs. In silico analyses were then performed to identify expression quantitative loci (eQTL) single-nucleotide polymorphisms (SNPs) that are in high linkage disequilibrium (LD) with the top SNPs. The associations of the potentially functional SNPs were replicated and validated in an independent case-control study of 48 patients of European ancestry treated with iv BPs (19 ONJ cases and 29 controls). The top SNPs in the exome-wide association meta-analysis were two SNPs on chromosome 10: SIRT1 SNP rs7896005 and HERC4 SNP rs3758392 with identical OR of 0.07 (0.01-0.46; p = 3.83 × 10-5 ). In the in silico functional analyses, two promoter region SNPs (rs7894483 and rs3758391) were identified to be in high LD with the index SNPs and are eQTLs for SIRT1 gene in whole blood in the GTEx database. The ORs were 0.30 (0.10-0.88), 0.26 (0.12-0.55), and 0.26 (0.12-0.55) for the WES top SNP rs7896005 and two promoter SNPs rs7894483 and rs3758391, respectively, in the replication sample. In summary, we identified the SIRT1/HERC4 locus on chromosome 10 to be associated with iv BP-induced ONJ and two promoter SNPs that might be the potential genetic markers for this association. © 2017 The Authors.Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

A Novel Urine-Based Assay for Bladder Cancer Diagnosis: Multi-Institutional Validation Study.

BACKGROUND: CellDetect is a unique histochemical stain enabling color and morphological discrimination between malignant and benign cells based on differences in metabolic signature. OBJECTIVE: The objective of the present study was to validate the performance of this assay in a controlled, blinded, multicenter study. DESIGN, SETTING, AND PARTICIPANTS: The study, conducted in nine hospitals, included patients with documented history of bladder cancer, monitored for urothelial carcinoma (UCC) or scheduled for bladder cancer surgery. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cystoscopy and/or biopsy were used as a reference standard to determine sensitivity and specificity. Smears were stained by CellDetect and interpreted by two cytologists blinded to the patient's final diagnosis. The findings were compared with those of standard urine cytology and BTA stat. RESULTS AND LIMITATIONS: Two hundred and seventeen voided urine specimens were included. Ninety-six (44%) were positive by histology and 121 (56%) were negative by either cystoscopy or histology. The overall sensitivity of CellDetect was 84%. Notably, the sensitivity for detecting low-grade nonmuscle-invasive bladder cancer tumors was greater than this of BTA stat (78% vs 54%) and more than two-fold higher compared with standard cytology (33%, p ≤ 0.05). The specificity was 84% in patients undergoing routine surveillance by cystoscopy. At a median follow-up of 9 mo, 21% of the patients with positive CellDetect and negative reference standard developed UCC, which was significantly higher compared with the 5% of the true negative cases. Limitations include the lack of instrumental urine samples and the lack of patients with nongenitourinary cancers in the study population. CONCLUSIONS: This study validates the performance of CellDetect as a urine-based assay to identify UCC in patients with history of bladder cancer. The high sensitivity was maintained across all cancer grades and stages without compromising the assay specificity. Further studies are required to test whether this novel stain can be incorporated in routine bladder cancer surveillance as a noninvasive alternative to cystoscopy. PATIENT SUMMARY: Surveillance of bladder cancer requires frequent invasive procedures. In the present study, we validate the ability of a novel biomarker to accurately identify early-stage tumors in urine specimens for the noninvasive monitoring of patients with history of bladder cancer.