Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters.

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

A high-content AlphaScreen™ identifies E6-specific small molecule inhibitors as potential therapeutics for HPV+ head and neck squamous cell carcinomas.

The incidence of human papillomavirus-positive head and neck squamous cell carcinoma (HPV+-HNSCC) has increased dramatically over the past decades due to an increase in infection of the oral mucosa by HPV. The etiology of HPV+-HNSCC is linked to expression of the HPV oncoprotein, E6, which influences tumor formation, growth and survival. E6 effects this oncogenic phenotype in part through inhibitory protein-protein interactions (PPIs) and accelerated degradation of proteins with tumor suppressor properties, such as p53 and caspase 8. Interfering with the binding between E6 and its cellular partners may therefore represent a reasonable pharmacological intervention in HPV+ tumors. In this study, we probed a small-molecule library using AlphaScreen™ technology to discover novel E6 inhibitors. Following a cascade of screens we identified and prioritized one hit compound. Structure activity relationship (SAR) studies of this lead uncovered an analog, 30-hydroxygambogic acid (GA-OH), that displayed improved activity. Further testing of this analog in a panel of HPV+ and HPV- cell lines showed good potency and a large window of selectivity as demonstrated by apoptosis induction and significant inhibition of cell growth, cell survival in HPV+ cells. In summary, GA-OH may serve as a starting point for the development of potent E6-specific inhibitors.

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.

Independent associations of urinary albumin-to-creatinine ratio and serum cystatin C with carotid intima-media thickness in community-living Taiwanese adults.

BACKGROUND: Renal function is a key factor of cardiovascular disease. Carotid intima-media thickness (IMT) has been widely used as a marker of early subclinical atherosclerosis. The determinants of cystatin C, a novel marker of renal function, have not been extensively studied in the Asian population. This study aimed to assess the determinants of cystatin C and explore whether carotid thickening was associated with urinary albumin-creatinine ratio and cystatin C in community-living Taiwanese adults. METHODS: A cross-sectional study was conducted on participants from Taichung City, Taiwan. All the participants underwent carotid ultrasonography. Carotid IMT-mean and IMT-maximum were derived. Kidney biomarkers were measured on the basis of urinary albumin-to-creatinine ratio (ACR) and cystatin C. Multiple linear regression analysis was used. RESULTS: A total of 1032 individuals were recruited, and 469 (45.44%) of them were men. An increased cystatin C level was significantly associated with older age, male gender, lack of physical activity, low HDL cholesterol, abdominal obesity, high hs-CRP, and high ACR. The multivariate-adjusted mean carotid IMT-mean and IMT-maximum values significantly increased by 80.49 and 195.23 µm for every one unit of increase in cystatin C level and by 0.07 and 0.14 µm for every one unit of increase in ACR, respectively (all p < 0.001 except ACR on IMT-maximum with p < 0.01). Lack of physical activity, low HDL, abdominal obesity, high hs-CRP, and high ACR were the determinants of cystatin C. CONCLUSION: Cystatin C and ACR were strongly and linearly associated with carotid thickening, a marker of subclinical atherosclerosis.

Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells.

Valproic acid (VPA) is a multifunctional medication used for the treatment of epilepsy, mania associated with bipolar disorder, and migraine. The pharmacological effects of VPA involve a variety of neurotransmitter and cell signaling systems, but the molecular mechanisms underlying its clinical efficacy is to date largely unknown. In this study, we used the isobaric tags for relative and absolute quantitation shotgun proteomic analysis to screen differentially expressed proteins in VPA-treated SH-SY5Y cells. We identified changes in the expression levels of multiple proteins involved in Alzheimer's disease, Parkinson's disease, chromatin remodeling, controlling gene expression via the vitamin D receptor, ribosome biogenesis, ubiquitin-mediated proteolysis, and the mitochondrial oxidative phosphorylation and electron transport chain. Our data indicate that VPA may modulate the differential expression of proteins involved in mitochondrial function and vitamin D receptor-mediated chromatin transcriptional regulation and proteins implicated in the pathogenesis of neurodegenerative diseases.

Smoking Paradox in Stroke Survivors?: Uncovering the Truth by Interpreting 2 Sets of Data.

Background and Purpose- The observation that smokers with stroke could have better outcome than nonsmokers led to the term "smoking paradox." The controversy of such a complex claim has not been fully settled, even though different case mix was noted. Analyses were conducted on 2 independent data sets to evaluate and determine whether such a paradox truly exists. Methods- Taiwan Stroke Registry with 88 925 stroke cases, and MJ cohort with 541 047 adults participating in a medical screening program with 1630 stroke deaths developed during 15 years of follow-up (1994-2008). Primary outcome for stroke registry was functional independence at 3 months by modified Rankin Scale score ≤2, for individuals classified by National Institutes of Health Stroke Scale score at admission. For MJ cohort, mortality risk by smoking status or by stroke history was assessed by hazard ratio. Results- A >11-year age difference in stroke incidence was found between smokers and nonsmokers, with a median age of 60.2 years for current smokers and 71.6 years for nonsmokers. For smokers, favorable outcome in mortality and in functional assessment in 3 months with modified Rankin Scale score ≤2 stratified by the National Institutes of Health Stroke Scale score was present but disappeared when age and sex were matched. Smokers without stroke history had a ≈2-fold increase in stroke deaths (2.05 for ischemic stroke and 1.53 for hemorrhagic stroke) but smokers with stroke history, 7.83-fold increase, overshadowing smoking risk. Quitting smoking at earlier age reversed or improved outcome. Conclusions- "The more you smoke, the earlier you stroke, and the longer sufferings you have to cope." Smokers had 2-fold mortality from stroke but endured stroke disability 11 years longer. Quitting early reduced or reversed the harms.

A nonsense mutant of the hepatitis B virus large S protein antagonizes multiple tumor suppressor pathways through c-Jun activation domain-binding protein1.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Previous studies have identified recurrent nonsense mutations in the HBV large S (LHBs) gene from the liver from HBV core antigen-positive HCC patients. These nonsense mutants have been shown to be oncogenic in mouse xenograft models using a mouse embryonic fibroblast cell line. Here, we expressed in a liver cell line Huh-7 a carboxy terminally truncated protein from a nonsense mutant of the LHBs gene, sW182* (stop codon at tryptophane-182). Although the sW182* protein appeared not to be very stable in the cultured liver cells, we confirmed that the protein can be highly expressed and retained for a prolonged period of time in the hepatocytes in the mouse liver, indicating its stable nature in the physiological condition. In the Huh-7 cells, the sW182* mutant downregulated tumor suppressors p53 and Smad4. This downregulation was reversed by a proteasome inhibitor MG132, implying the involvement of proteasome-based protein degradation in the observed regulation of the tumor suppressors. On the other hand, we found that c-Jun activation domain-binding protein 1 (Jab1) physically interacts with the sW182*, but not wild-type LHBs. RNA interference (RNAi) of Jab1 restored the levels of the downregulated p53 and Smad4. The sW182* mutant inhibited the promoter activity of downstream target genes of the tumor suppressors. Consistently, Jab1 RNAi reversed the inhibition. These results suggest that the LHBs nonsense mutant antagonizes the tumor suppressor pathways through Jab1 in the liver contributing to HCC development.

Multimodal imaging platform for optical virtual skin biopsy enabled by a fiber-based two-color ultrafast laser source.

We demonstrate multimodal label-free nonlinear optical microscopy in human skin enabled by a fiber-based two-color ultrafast source. Energetic femtosecond pulses at 775 nm and 1250 nm are simultaneously generated by an Er-fiber laser source employing frequency doubling and self-phase modulation enabled spectral selection. The integrated nonlinear optical microscope driven by such a two-color femtosecond source enables the excitation of endogenous two-photon excitation fluorescence, second-harmonic generation, and third-harmonic generation in human skin. Such a 3-channel imaging platform constitutes a powerful tool for clinical application and optical virtual skin biopsy.

Impaired mammary tumor formation and metastasis by the point mutation of a Smad3 linker phosphorylation site.

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-ß acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-ß signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways. Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles. The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21Cip1 and downregulation of c-Myc. The T179V KI also reduced cell migration and invasion in vitro. In the mouse xenograft models, the T179V KI markedly reduced the establishment of primary tumor in the mammary fat pad and the lung metastasis. Our results using gene editing indicate the cancer-promoting role of Smad3 T179 phosphorylation in the human TNBC cells. Our findings highly suggest that controlling this phosphorylation may have therapeutic potential for TNBC.

Combinatorial inhibition of PTPN12-regulated receptors leads to a broadly effective therapeutic strategy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year and is recalcitrant to targeted therapies. Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC. PTPN12 restrains several RTKs, suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRß, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRß and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRß and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.

Renal function is associated with 1-month and 1-year mortality in patients with ischemic stroke.

BACKGROUND AND AIMS: Renal dysfunction is a potent risk factor for cardiovascular diseases, including stroke. This study aimed to evaluate the impact of admission estimated glomerular filtration rate (eGFR) levels on short-term (1-month) and long-term (1-year) mortality in patients with acute ischemic stroke. METHODS: From the Taiwan Stroke Registry data, we classified ischemic stroke patients, identified from April 2006 to December 2015, into 5 groups by eGFR at admission: ≥ 90, 60-89, 30-59, 15-29, and <15 mL/min/1.73 m2 or on dialysis. Risks of 1-month mortality and 1-year mortality after ischemic stroke were investigated by the eGFR level. RESULTS: Among 52,732 ischemic stroke patients, 1480 died within one month. The 1-month mortality rate was over 5-fold greater in patients with eGFR <15 mL/min/1.73 m2 or dialysis than in patients with eGFR ≥90 mL/min/1.73 m2 (2.88 versus 0.56 per 1000 person-days). The adjusted hazard ratio (HR) of 1-month mortality increased from 1.31 (95% CI = 1.08-1.59) for patients with eGFR 60-89 mL/min/1.73 m2 to 2.33 (95% CI = 1.80-3.02) for patients with eGFR < 15 mL/min/1.73 m2 or on dialysis. 3226 patients died within one year. The adjusted HR of mortality increased from 1.38 (95% CI = 1.21-1.59) for patients with eGFR 60-89 mL/min/1.73 m2 to 2.60 (95% CI 2.18-3.10) for patients with eGFR < 15 mL/min/1.73 m2 or on dialysis, compared to patients with eGFR ≥ 90 mL/min/1.73 m2. CONCLUSIONS: After acute ischemic stroke, patients with reduced eGFR are at elevated risks of short-term and long-term deaths in a graded relationship.

Erratum: USP11 regulates PML stability to control Notch-induced malignancy in brain tumours.

This corrects the article DOI: 10.1038/ncomms4214.

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

BACKGROUND: Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. METHODS: Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. RESULTS: Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. CONCLUSIONS: Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

Sentinel lymph node biopsy for early oral cancers: Westmead Hospital experience.

BACKGROUND: Sentinel lymph node biopsy (SLNB) has become an alternative option to elective neck dissection (END) for early oral cavity squamous cell carcinoma (OCSCC) outside of Australia. We sought to assess the technical feasibility of SLNB and validate its accuracy against that of END in an Australian setting. METHODS: We performed a prospective cohort study consisting of 30 consecutive patients with cT1-2 N0 OCSCC referred to the Head and Neck Cancer Service, Westmead Hospital, Sydney, between 2011 and 2014. All patients underwent SLNB followed by immediate selective neck dissection (levels I-III). RESULTS: A total of 30 patients were diagnosed with an early clinically node-negative OCSCC (seven cT1 and 23 cT2), with the majority located on the oral tongue. A median of three (range: 1-14) sentinel nodes were identified on lymphoscintigraphy, and all sentinel nodes were successfully retrieved, with 50% having a pathologically positive sentinel node. No false-negative sentinel nodes were identified using selective neck dissection as the gold standard. The negative predictive value (NPV) of SLNB was 100%, with 40% having a sentinel node identified outside the field of planned neck dissection on lymphoscintigraphy. Of these, one patient had a positive sentinel node outside of the ipsilateral supraomohyoid neck dissection template. CONCLUSION: SLNB for early OCSCC is technically feasible in an Australian setting. It has a high NPV and can potentially identify at-risk lymphatic basins outside the traditional selective neck dissection levels even in well-lateralized lesions.

Effects of the traditional Chinese herb Astragalus membranaceus in patients with poststroke fatigue: A double-blind, randomized, controlled preliminary study.

BACKGROUND AND PURPOSE: Astragalus membranaceus (AM) is the first-choice herb for fatigue treatment in traditional Chinese medicine and the main herb used for stroke treatment in China and Taiwan. The purpose of this study was to evaluate the effect of AM on poststroke fatigue (PSF). MATERIALS AND METHODS: This study was designed as a double-blind, randomized, controlled preliminary study. Sixty-four patients with PSF were assigned to treatment group (TG; 31 patients), which received oral administration of AM (2.8g three times per day) for 28 days, and a control group (CG; 33 patients), which received a placebo. The primary outcome measures were the changes in the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and Brief Fatigue Index (BFI) scores RESULTS: A total of 61 patients (29 patients in the TG and 32 patients in the CG) completed the trial. The difference in BFI scores between Visit 2 and Visit 1 was -17.83±17.70 in the TG, which was greater than that in the CG (-8.03±9.95; p=0.01); additionally, the difference in BFI scores between Visit 3 and Visit 1 was -16.48±16.41 in the TG, which was also greater than that in the CG (-9.47±13.39; p=0.05). In the EORTC QLQ-C30, the difference in cognitive functioning scores between Visit 2 and Visit 1 was 14.37±13.89 in the TG, which was greater than that in the CG (3.65±19.74; p=0.02); additionally, the difference in these scores between Visit 3 and Visit 1 was 14.37±16.50 in the TG, which again was greater than that in the CG (6.25±19.74; p=0.04). The difference in social functioning scores between Visit 3 and Visit 1 was 9.77±15.12 in the TG, which was greater than that in the CG (-1.56±20.46; p=0.01). The difference in global quality of life (QOL) scores between Visit 2 and Visit 1 was 14.08±18.78 in the TG, which was also greater than that in the CG (1.56±18.14; p=0.003); moreover, the difference in these scores between Visit 3 and Visit 1 was 10.92±17.55 in the TG, and this was greater than that in the CG (1.82±15.8; p=0.05). CONCLUSION: AM can improve BFI scores; cognitive functioning, social functioning, and global QOL scores in the EORTC QLQ-C30. Our results suggest that physicians should pay close attention to the unmet medical needs of patients with PSF. AM is helpful for treating patients with PSF; however, additional studies with a larger sample and a longer period of investigation are required.

Amniotic membrane and adipose-derived stem cell co-culture system enhances bone regeneration in a rat periodontal defect model.

BACKGROUND/PURPOSE: Periodontal disease is a chronic inflammatory process that potentially leads to alveolar bone destruction and tooth loss. Tissue engineering combined with stem cell therapy is a potential effective treatment for periodontal bone loss. Amniotic membrane (AM) is a potential scaffold enriched with multiple growth factors. It has the effects of anti-inflammation, antiangiogenesis, and immunosuppression. Herein, we used adipose-derived stem cells (ADSCs) and an AM co-cultured system to study bone regeneration in a rat periodontal defect model in vivo. METHODS: Human ADSCs were isolated from the infrapatellar fat pad, and characterized by flow cytometry, reverse transcription-polymerase chain reaction, and multipotent differentiation assays. The co-culture system was applied in the periodontal two-wall osseous defect in a rat model, and computed tomography was used to measure the effect. RESULTS: Human ADSCs isolated from the infrapatellar fat pad showed spindle-like morphology. Flow cytometry results demonstrated that ADSCs expressed a high level of CD90 and CD105, but not CD31, CD34, and CD45. ADSCs strongly expressed stemness genes, including SOX2, OCT4, NANOG, and KLF4 on different passages. Furthermore, ADSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic cells. In the periodontal osseous defect rat model, ADSCs and the AM co-culture system significantly increased bone regeneration. CONCLUSION: This study provides the basis for using ADSCs with an AM co-culture system as stem cell therapy and scaffold transplantation in clinical periodontology.

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10.

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.

USP11 regulates PML stability to control Notch-induced malignancy in brain tumours.

The promyelocytic leukaemia (PML) protein controls multiple tumour suppressive functions and is downregulated in diverse types of human cancers through incompletely characterized post-translational mechanisms. Here we identify USP11 as a PML regulator by RNAi screening. USP11 deubiquitinates and stabilizes PML, thereby counteracting the functions of PML ubiquitin ligases RNF4 and the KLHL20-Cul3 (Cullin 3)-Roc1 complex. We find that USP11 is transcriptionally repressed through a Notch/Hey1-dependent mechanism, leading to PML destabilization. In human glioma, Hey1 upregulation correlates with USP11 and PML downregulation and with high-grade malignancy. The Notch/Hey1-induced downregulation of USP11 and PML not only confers multiple malignant characteristics of aggressive glioma, including proliferation, invasiveness and tumour growth in an orthotopic mouse model, but also potentiates self-renewal, tumour-forming capacity and therapeutic resistance of patient-derived glioma-initiating cells. Our study uncovers a PML degradation mechanism through Notch/Hey1-induced repression of the PML deubiquitinase USP11 and suggests an important role for this pathway in brain tumour pathogenesis.

The Nogo-C2/Nogo receptor complex regulates the morphogenesis of zebrafish lateral line primordium through modulating the expression of dkk1b, a Wnt signal inhibitor.

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.