Matrine inhibits invasion and migration of gallbladder cancer via regulating the PI3K/AKT signaling pathway.

Gallbladder cancer (GBC) is a common malignant cancer in the biliary system, which poses a serious threat to human health. It is urgent to explore ideal drugs for the treatment of GBC. Matrine is the main active ingredient of Sophora flavescentis, with a wide range of biological activities encompassing anti-inflammatory, antiviral, immunomodulatory, and anti-tumor. However, the underlying mechanism by which Matrine treats GBC is still unclear. The purpose of this study is to investigate the anti-tumor effects of Matrine on GBC in vivo and in vitro and to clarify the potential regulatory mechanisms. Here, we found that Matrine had a significant killing effect on GBC through CCK8 and flow cytometry, including arrest of cell cycle, inhibition of GBC cell, and induction of apoptosis. Further in vivo studies confirmed the inhibitory effect of Matrine on tumor growth in NOZ xenografted nude mouse. At the same time, Matrine also significantly suppressed the migration and invasion of GBC cells through scratch and Transwell experiments. In addition, by detecting the mRNA and protein levels of epithelial-mesenchymal transition (EMT) and matrix metalloproteinases, Matrine furtherly substantiated the inhibitory role on invasion and migration of GBC. From a mechanistic perspective, network pharmacology analysis suggests that the potential targets of Matrine in the treatment of GBC are enriched in the PI3K/AKT signaling pathway. Subsequently, Matrine effectively decreased the abundance of p-PI3K and p-AKT protein in vivo and in vitro. More importantly, PI3K activator (740 Y-P) antagonized the anti-tumor effect of Matrine, while PI3K inhibitor (LY294002) increased the sensitivity of Matrine for GBC. Based on the above findings, we conclude that Matrine inhibits the invasion and migration of GBC by regulating PI3K/AKT signaling pathway. Our results indicate the crucial role and regulatory mechanism of Matrine in suppressing the growth of GBC, which provides a theoretical basis for Matrine to be a candidate drug for the treatment and research of GBC.

Perovskite CoSn(OH)6 nanocubes with tuned d-band states towards enhanced oxygen evolution reactions.

The CoSn(OH)6 perovskite hydroxide is a structure stable and inexpensive electrocatalyst for the oxygen evolution reaction (OER). However, the OER activity of CoSn(OH)6 is still unfavorable due to its limited active sites. In this work, an Fe3+ doping strategy is used to optimize the d-band state of the CoSn(OH)6 perovskite hydroxide. The CoSn(OH)6 catalyst with slightly Fe3+ doped nanocubes is synthesized by a facile hydrothermal method. Structure characterization shows that Fe3+ ions are incorporated into the crystal structure of CoSn(OH)6. Owing to the regulation of the electronic structure, CoSn(OH)6-Fe1.8% exhibits an OER overpotential of 289 mV at a current density of 10 mA cm-2 in OER electrochemical tests. In situ Raman spectroscopy shows that no obvious re-construction occurred during the OER for both CoSn(OH)6 and CoSn(OH)6-Fe1.8%. DFT calculations show that the introduction of Fe3+ into CoSn(OH)6 can shift the d-band center to a relatively high position, thus promoting the OER intermediates' adsorption ability. Further DFT calculations suggest that incorporation of an appropriate amount of Fe3+ into CoSn(OH)6 significantly reduces the rate-determining Gibbs free energy during the OER. This work offers valuable insights into tuning the d-band center of perovskite hydroxide materials for efficient OER applications.

Dihydrotanshinone I inhibits gallbladder cancer growth by targeting the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation.

BACKGROUND: Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear. PURPOSE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms. METHODS: The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice. RESULTS: Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells. CONCLUSION: Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.

Linking New Alleles at the Oscillator Loci to Flowering and Expansion of Asian Rice.

The central oscillator is believed to be the key mechanism by which plants adapt to new environments. However, impacts from hybridization, the natural environment, and human selection have rarely been assessed on the oscillator of a crop. Here, from clearly identified alleles at oscillator loci (OsCCA1/LHY, OsPRR95, OsPRR37, OsPRR59, and OsPRR1) in ten diverse genomes of Oryza sativa, additional accessions, and functional analysis, we show that rice's oscillator was rebuilt primarily by new alleles from recombining parental sequences and subsequent 5' or/and coding mutations. New alleles may exhibit altered transcript levels from that of a parental allele and are transcribed variably among genetic backgrounds and natural environments in RIL lines. Plants carrying more expressed OsCCA1_a and less transcribed OsPRR1_e flower early in the paddy field. 5' mutations are instrumental in varied transcription, as shown by EMSA tests on one deletion at the 5' region of highly transcribed OsPRR1_a. Compared to relatively balanced mutations at oscillator loci of Arabidopsis thaliana, 5' mutations of OsPRR37 (and OsCCA1 to a less degree) were under negative selection while those of OsPRR1 alleles were under strong positive selection. Together, range expansion of Asian rice can be elucidated by human selection on OsPRR1 alleles via local flowering time-yield relationships.

Genetic Authentication of the Medicinal Plant Portulaca oleracea Using a Quick, Precise, and Sensitive Isothermal DNA Amplification Assay.

Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named "loop-mediated isothermal amplification (LAMP)" was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.

Development of a specific and sensitive diagnostic PCR for rapid molecular authentication of the medicinal plant Portulaca oleracea.

Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future.

A study on etiology of incontinence in double knockout mouse model.

INTRODUCTION AND OBJECTIVE: Stress urinary incontinence is of concern in both pediatric and adult population. Double mutant GLI family zinc finger Gli2+/-; Gli3Δ699/+ murine model of stress incontinence has been recently developed as a reliable model which does not require surgical manipulation to create incontinence and is shown to survive to adulthood. The aim of this study was to establish the etiology of incontinence in the double mutant Gli2+/-; Gli3Δ699/+ mice. STUDY DESIGN: We used 13 cluster of differentiation 1 (CD-1) mice (7-9 weeks) for demonstration of histology of the bladder and urethra. There were 3 Wild Gli2+/- females, 2 Wild Gli2+/- males, 4 Gli2+/-;Gli3Δ699/+ females and 4 Gli2+/-;Gli3Δ699/+ males. The Wild Gli2+/- mice served as the control group and Gli2+/-;Gli3Δ699/+ mice served as the test group. Additionally, eight 16.5 days mice (2 each of Wild Gli2+/- females, Wild Gli2+/- males, double knockout (DKO) Gli2+/-;Gli3Δ699/+ females and Gli2+/-;Gli3Δ699/+ males) were used to assess the histology of the spinal cord. The gross appearance of bladder and urethra was studied using ink injection assays. Immunohistochemistry was done for smooth muscle actin and cytokeratin. RESULTS: Gross and histologic appearance confirmed the previously reported widening of bladder outlet and hypoplasia of smooth muscles in female urethra and also established them in the male urethra of Gli2+/-;Gli3Δ699/+ mice compared to Gli2+/- mice. The double knockout mice were smaller than the Gli2 mice (5.2 vs 6.1 cm, p = 0.002). Immunohistochemistry demonstrated epithelial hyperplasia and smooth muscle hypoplasia. Additionally, there was prostatic hypoplasia in the Gli2+/-;Gli3Δ699/+ male mice. The spinal cord length for body size appeared comparable between the Gli2+/- and Gli2+/-;Gli3Δ699/+ mice but histological evaluation revealed abnormal development of the caudal end of the vertebral body with premature termination of the spinal cord (Figure). DISCUSSION: The histological changes in the bladder neck and urethra were consistent to those previously reported. While previous report described the findings in female mice only, we confirmed that these findings are also present in males as well as prostatic hypoplasia, a possible additional factor leading to stress incontinence. The most important finding in the present study however, was the detection of premature termination of spinal cord in the DKO Gli2+/-; Gli3Δ699/+ mice which has not been reported previously and is likely a major contributor to incontinence in this model. CONCLUSION: The incontinence in male as well as female Gli2+/-; Gli3Δ699/+ mice is due to both myogenic and neurogenic involvement. These double knockout mice are a valuable model of stress incontinence related to neurogenic bladder due to low outlet resistance.

Complete plastome sequence of Lonicera gynochlamydea Hemsl. (Caprifoliaceae).

We report and characterize the complete plastome of Lonicera gynochlamydea Hemsl. L. gynochlamydea is a shrub, belonging to the family Caprifoliaceae. Our results show that the length of the complete plastome is 154,643 bp, including 131 genes consisting of 84 protein-coding genes, 39 tRNA genes, and eight rRNA genes. The plastome exhibits the typical quadripartite structure and gene content of angiosperms, composed of two inverted repeats (IRs) regions of 23,846 bp, a large single-copy (LSC) region of 88,298 bp, and a small single-copy (SSC) region of 18,653 bp. The total G/C content in L. gynochlamydea plastome is 38.4%. The complete plastome sequence of L. gynochlamydea will make contributions to the conservation genetics of this species as well as to phylogenetic studies in Caprifoliaceae.

Association between periodic limb movements during sleep and neuroimaging features of cerebral small vessel disease: A preliminary cross-sectional study.

Evidence on the relationship between periodic limb movements during sleep (PLMS) and cerebral small vessel disease is lacking. This study aimed to assess the association between the PLMS index and the neuroimaging features of cerebral small vessel disease on magnetic resonance imaging. Consecutive patients diagnosed with cerebral small vessel disease were enrolled. Data on the clinical characteristics, polysomnography, and brain magnetic resonance imaging were collected. The Accubrain software was used to calculate automatically the volume of white matter hyperintensities, the number of lacunar infarctions, and cerebral microbleeds. The severity of white matter hyperintensities, enlarged basal ganglia perivascular spaces, and the total cerebral small vessel disease scores were also rated visually using semiquantitative scales. The severity of PLMS was measured using the PLMS index, and the patients were divided into two groups using an established cut-off value of ≥15 per hour. Logistic regression was used to examine the association between PLMS and the neuroimaging features of cerebral small vessel disease. In total, 37 patients were included in the final analyses. The mean age was 66.49 ± 11.31 years, and 73.0% were males. The mean PLMS index was 19.30 ± 10.18. In univariate analyses, it was found that patients with cerebral small vessel disease with a PLMS index ≥15 had increased enlarged basal ganglia perivascular spaces (OR 6.136, 95%CI 1.101-34.214) and increased total cerebral small vessel disease scores (OR 6.0, 95%CI 1.253-28.742). Only the association between the PLMS index and the total cerebral small vessel disease burden score remained statistically significant after adjusting for age, sex, and the presence of moderate to severe obstructive sleep apnea syndrome. In conclusion, an elevated PLMS index is likely to be associated with a greater cerebral small vessel disease burden. PLMS might be a novel potential marker of cerebral small vessel disease.

Ectopic expression of Irx3 and Irx5 in the paraventricular nucleus of the hypothalamus contributes to defects in Sim1 haploinsufficiency.

The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing neurons critical for feeding regulation. Sim1 haploinsufficiency results in hyperphagic obesity with disruption of PVH neurons, yet the molecular profiles of PVH neurons and the mechanism underlying the defects of Sim1 haploinsufficiency are not well understood. By single-cell RNA sequencing, we identified two major populations of Sim1+ PVH neurons, which are differentially affected by Sim1 haploinsufficiency. The Iroquois homeobox genes Irx3 and Irx5 have been implicated in the hypothalamic control of energy homeostasis. We found that Irx3 and Irx5 are ectopically expressed in the Sim1+ PVH cells of Sim1+/− mice. By reducing their dosage and PVH-specific deletion of Irx3, we demonstrate that misexpression of Irx3 and Irx5 contributes to the defects of Sim1+/− mice. Our results illustrate abnormal hypothalamic activities of Irx3 and Irx5 as a central mechanism disrupting PVH development and feeding regulation in Sim1 haploinsufficiency.

Irx3 and Irx5 in Ins2-Cre+ cells regulate hypothalamic postnatal neurogenesis and leptin response.

Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.

Enhanced Photoelectrochemical Water Oxidation Performance in Bilayer TiO2 /α-Fe2 O3 Nanorod Arrays Photoanode with Cu : NiOx as Hole Transport Layer and Co-Pi as Cocatalyst.

Efficient charge transfer and excellent surface water oxidation kinetics are key factors in determining the photoelectrochemical (PEC) water splitting performance in photoelectrodes. Herein, a bilayer TiO2 /α-Fe2 O3 nanorod (NR) arrays photoanode was prepared with deposited Cu-doped NiOx (Cu : NiOx ) hole transport layer (HTL) and Co-Pi oxygen evolution reaction (OER) cocatalyst for PEC water oxidation. The hierarchical TiO2 /α-Fe2 O3 composite obtained by a secondary hydrothermal process exhibited an inapparent bilayer structure by embedding the underlayer TiO2 NR arrays at the bottom part of the post-grown α-Fe2 O3 NR arrays. The underlayer TiO2 NRs acted as an effective shuttling pathway for transferring photoelectrons generated in the upper hematite light absorber layer. A p-type inter-Cu : NiOx HTL was introduced to form a build-in p-n electric field between Cu : NiOx and α-Fe2 O3 NRs, which improved the hole extraction from α-Fe2 O3 to Co-Pi OER catalyst. As expected, the as-engineered TiO2 /α-Fe2 O3 /Cu : NiOx /Co-Pi photoanode displayed an excellent photocurrent density of 2.43 mA cm-2 at 1.23 V versus the reversible hydrogen electrode (VRHE ), up to 4.05 and 2.23 times greater than those of the bare α-Fe2 O3 (0.60 mA cm-2 ) and TiO2 /α-Fe2 O3 , respectively. The results demonstrate that the bottom-up engineering of electron-hole transport channels and cocatalyst modification is an attractive maneuver to enhance the PEC water oxidation activity in hematite and other photoanodes.

The effects of the seat cushion contour and the sitting posture on surface pressure distribution and comfort during seated work.

OBJECTIVES: The purpose of this paper is to investigate the effects of the seat cushion contour and the sitting posture on the seat pan interface pressure distribution and subjective comfort perception. MATERIAL AND METHODS: Overall, 16 volunteers typed a text passage on a laptop while seated, by assuming 3 kinds of common sitting postures (forward, relaxed and upright) in 4 seat cushion configurations: chair only, and chair with 1 of 3 supplementary cushions. Pressure data and cushion comfort ratings were collected in the experiment. RESULTS: It was found that the sitting posture and the seat cushion contour had different impacts on surface pressure. The seat cushion contour had an impact on pressure parameters and pressure distribution on the seat pan, while the sitting posture affected the location of peak pressure on the seat pan. The correlation analysis revealed that the subjective comfort rating was significantly correlated with average pressure (AP) and mean peak pressure (MPP). CONCLUSIONS: The conclusion was that the cushion contour had a greater effect on seat pan interface pressure parameters than the sitting posture. Notably, AP and MPP can be indicators for assessing seat cushion comfort in a short-term perspective. Int J Occup Med Environ Health. 2020;33(5):675-89.

Emergence of Zika virus infection in China.

Currently, Zika virus (ZIKV) is spreading across the world and no ZIKV infection cases have ever been reported in China. Here, we aimed to determine whether ZIKV infection exists in China. Blood samples of 273 healthy individuals were collected from Nanning City, Guangxi Province, China in March 2019. We found that 9.5% (26/273) and 1.8% (5/273) of healthy persons were positive to ZIKV total antibody (IgG and/or IgM) IgM antibody, respectively. All ZIKV positive plasma samples were negative to Dengue virus and West Nile virus. Among the ZIKV antibody positive plasma samples, 65.4% (17/26) exhibited neutralizing activity to ZIKV. Followed up studies showed that none had clinical symptoms of ZIKV infection and oversea experience. Together, our study indicates that endemic ZIKV infections emerge in China, which not only suggested that ZIKV posed a potential threat to public health in China, but also expand the ZIKV epidemic areas in East and Southeast Asia.

Hospital and healthcare insurance system record-based epidemiological study of myasthenia gravis in southern and northern China.

OBJECTIVE: This is the first cross-region epidemiological study of myasthenia gravis (MG) in China. We estimated the incidence, prevalence, and medical costs of MG in southern China and explored the differences between the southern and northern Chinese populations. METHODS: We collected and analyzed records from 20 hospitals in the southern city, Guangzhou, 13 hospitals in the northern city, Harbin, and two healthcare insurance systems: job based and residence based in Guangzhou during 2000-2017. RESULTS: (1) The estimated annual incidence of MG was 1.55-3.66 per 100,000, and the estimated prevalence of MG was 2.19-11.07 per 100,000 in southern China based on insurance records. (2) The proportion of hospitalized MG patients in the south-based hospital records was three times as high as that in the north-based hospital records. (3) Female MG prevalence was significantly higher than male MG prevalence in Guangzhou, while the similar gender difference in Harbin was not statistically significant due to higher variation in earlier years. (4) The average expense was $35-42 for each outpatient service and $2526-2673 for each hospitalization expense in the south. (5) Contrary to the increase of insurance-based estimate of MG prevalence, the proportion of hospitalized MG patients did not increase over the years, suggesting rising awareness and utilization of health insurance. CONCLUSIONS: The southern MG population had a significantly higher prevalence and a lower response threshold to medication than the northern MG population. These results are calling for further investigations on the genetic, cultural, and environmental variations of the Chinese MG populations between north and south.

GLI2 Modulated by SUFU and SPOP Induces Intestinal Stem Cell Niche Signals in Development and Tumorigenesis.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis.

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation. Simultaneous loss of Spop, an E3 ubiquitin ligase targeting Gli2, restores robust Gli2 activation and induces rapid MB formation in Sufu knockout background. We also demonstrated a tumor-promoting role of Sufu in Smo-activated MB (∼60% of human SHH MB) by maintaining robust Gli activity. Having established Gli2 activation as a key driver of SHH MB, we report a comprehensive analysis of its targetome. Furthermore, we identified Atoh1 as a target and molecular accomplice of Gli2 that activates core SHH MB signature genes in a synergistic manner. Overall, our work establishes the dual role of SUFU in SHH MB and provides mechanistic insights into transcriptional regulation underlying Gli2-mediated SHH MB tumorigenesis.

A genetic female mouse model with congenital genitourinary anomalies and adult stages of urinary incontinence.

AIMS: To characterize the urinary incontinence observed in adult Gli2+/- ; Gli3Δ699/+ female mice and identify the defects underlying the condition. METHODS: Gli2+/- and Gli3Δ699/+ mice were crossed to generate: wild-type, mutant Gli2 (Gli2+/- ), mutant Gli3 (Gli3Δ699/+ ), and double mutant (Gli2+/- ; Gli3Δ699/+ ) female mice, verified via Polymerase Chain Reactions. Bladder functional studies including cystometrogram (CMG), leak point pressure (LPP), and voiding testing were performed on adult female mice. Female bladders and urethras were also analyzed via ink injection and histological assays. RESULTS: CMG tracing showed no signal corresponding to the filling of the Gli2+/- ; Gli3Δ699/+ bladders. LPP were significantly reduced in Gli2+/- ; Gli3Δ699/+ mice compared to wild-type mice. CMG studies revealed a decrease in peak micturition pressure values in Gli2+/- ; Gli3Δ699/+ mice compared with all other groups. No significant differences between mutant and wild-type mice were detected in urinary output. Histological analyses revealed Gli2+/- ; Gli3Δ699/+ mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONS: Gli2+/- ; Gli3Δ699/+ adult female mice display persistent urinary incontinence due to the malformation of the bladder outlet and urethra. This presents a consistent and reliable genetic mouse model for female urinary incontinence and alludes to the key role of genetic factors involved in the condition.

Adult Gli2+/-;Gli3Δ699/+ Male and Female Mice Display a Spectrum of Genital Malformation.

Disorders of sexual development (DSD) encompass a broad spectrum of urogenital malformations and are amongst the most common congenital birth defects. Although key genetic factors such as the hedgehog (Hh) family have been identified, a unifying postnatally viable model displaying the spectrum of male and female urogenital malformations has not yet been reported. Since human cases are diagnosed and treated at various stages postnatally, equivalent mouse models enabling analysis at similar stages are of significant interest. Additionally, all non-Hh based genetic models investigating DSD display normal females, leaving female urogenital development largely unknown. Here, we generated compound mutant mice, Gli2+/-;Gli3Δ699/+, which exhibit a spectrum of urogenital malformations in both males and females upon birth, and also carried them well into adulthood. Analysis of embryonic day (E)18.5 and adult mice revealed shortened anogenital distance (AGD), open ventral urethral groove, incomplete fusion of scrotal sac, abnormal penile size and structure, and incomplete testicular descent with hypoplasia in male mice, whereas female mutant mice displayed reduced AGD, urinary incontinence, and a number of uterine anomalies such as vaginal duplication. Male and female fertility was also investigated via breeding cages, and it was identified that male mice were infertile while females were unable to deliver despite becoming impregnated. We propose that Gli2+/-;Gli3Δ699/+ mice can serve as a genetic mouse model for common DSD such as cryptorchidism, hypospadias, and incomplete fusion of the scrotal sac in males, and a spectrum of uterine and vaginal abnormalities along with urinary incontinence in females, which could prove essential in revealing new insights into their equivalent diseases in humans.