Derivation of cochlear cells from pathological or isogenic human iPSCs for modeling hereditary hearing loss

Alström Syndrome (AS) is a human autosomal recessive genetic disorder characterized by numerous clinical symptoms including deafness. AS is caused by mutations in the ALMS1 gene encoding for ALMS1 protein expressed at the basal body and implicated in ciliogenesis, cell cycle and proliferation (Jagger et al., 2011; Zulato et al., 2011 & Shenje et al., 2014). We are interesting in understanding the unknown mechanisms involving this protein in the genetic deafness of AS patients. To develop a model as closer as possible to the human pathology, we are using human induced pluripotent stem cells (hiPSCs) generated from fibroblasts of healthy and AS patients. Using a stepwise protocol, we demonstrated that healthy hiPSCs (waiting for isogenic hiPSCs) can generate a population of cells with gene and protein expression patterns consistent with the ones of otic progenitor cells (OSCs). At this differentiation stage, we observed some proliferation and apoptotic defects between healthy and AS cells. When human OSCs are co-cultured with mouse feeder cells, they are able to differentiate into hair cells (HCs). We successfully differentiated AS hiPSCs generated from AS patients into HCs. We are currently confirming gene expression pattern and testing HCs functionality.  To exclude patient linked epigenetics and differentiation defects, we are correcting the genomic mutation in the AS hiPSCs to generate isogenic hiPSCs using the CRIPSR/Cas9 system. Thanks to the isogenic hiPSCs we will be able to confirm that these defects are well due to the ALMS1 mutation.Derivation of cochlear cells from pathological or isogenic human iPSCs for modeling hereditary hearing los

YY1 coopère avec AP-2 pour stimuler l'expression du gène ERBB2 dans les cellules de cancer du sein

peer reviewedOverexpression of the ERBB2 oncogene is observed in about 30% of breast cancers and is generally correlated with a poor prognosis. Previous results from our and other laboratories indicated that elevated transcriptional activity contributes significantly to the overexpression of ERBB2 mRNA in mammary adenocarcinoma cell lines. Activator protein 2 (AP-2) transcription factors account for this overexpression through two recognition sequences located 215 and 500 bp upstream from the transcription start site. Furthermore, AP-2 transcription factors are highly expressed in cancer cell lines overexpressing ERBB2. In this report, we examined the cooperative effect of Yin Yang 1 (YY1) on AP-2-induced activation of ERBB2 promoter activity. We detected high levels of YY1 transcription factor in mammary cancer cell lines. Notably, we showed that YY1 enhances AP-2alpha transcriptional activation of the ERBB2 promoter through an AP-2 site both in HepG2 and in HCT116 cells, whereas a carboxyl-terminal-truncated form of YY1 cannot. Moreover, we demonstrated the interaction between endogenous AP-2 and YY1 factors in the BT-474 mammary adenocarcinoma cell line. In addition, inhibition of endogenous YY1 protein by an antisense decreased the transcription of an AP-2-responsive ERBB2 reporter plasmid in BT-474 breast cancer cells. Finally, we detected in vivo AP-2 and YY1 occupancy of the ERBB2 proximal promoter in chromatin immunoprecipitation assays. Our data thus provide evidence that YY1 cooperates with AP-2 to stimulate ERBB2 promoter activity through the AP-2 binding sites

Caspase-8-dependent HER-2 cleavage in response to tumor necrosis factor alpha stimulation is counteracted by nuclear factor kappa B through c-FLIP-L expression

The oncoprotein HER-2/neu is a prosurvival factor, and its overexpression has been correlated with poor prognosis in patients with breast cancer. We report that HER-2 is a new substrate for caspase-8 and that tumor necrosis factor alpha (TNF-alpha) stimulation leads to an early caspase-8-dependent HER-2 cleavage in MCF7 A/Z breast adenocarcinoma cells defective for nuclear factor kappaB (NFkappaB) activation. We show that the antiapoptotic transcription factor NFkappaB counteracts this cleavage through induction of the caspase-8 inhibitor c-FLIP. Our results also demonstrate that this HER-2 cleavage contributes to the TNF-alpha-induced apoptosis pathway because ectopic expression of an uncleavable HER-2 protects NFkappaB-defective cells against TNF-alpha-mediated cell death. Therefore, we propose an original model in which NFkappaB exerts a new antiapoptotic function by counteracting TNF-alpha-triggered cleavage of the HER-2 survival factor

Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels

Application of Simple Smart Logic for Waterflooding Reservoir Management

A simple smart logic for controlling inflow control valves (ICV) in waterflooding reservoir management is implemented and analyzed, with the final objective of improving the long term financial return of a petroleum reservoir. Such a control is based in a reactive simple logic that responds to the watercut measured in the ICV. Basically, when the watercut increases, the ICV is set to close proportionally. For comparison purposes, four strategies are presented: base case scenario with conventional control, the best completion configuration found by trial-and-error, the reactive control, and a deterministic optimal control based on Nonlinear Gradient Method with adjoint-gradient formulation is shown for comparison purposes. Finally, all four strategies are tested again in different reservoir realizations in order to mimic the geological uncertainties. Two different synthetic reservoir models were studied. First, a simple cube with a five-spot well configuration, in which the permeability field has a horizontal pattern defined by lognormal distributions. The second model is a benchmark proposed by the Dutch university, TU delft, with 101 channelized permeability fields representing river patterns. For the first model, no significant relative gain is found neither in the variable control nor in the optimal control. Manly because of the high homogeneity of the reservoir models. Therefore, no intelligent completion is recommended. On the other hand, for the second and more complex case, the results indicate an expressive relative gain in the use of simple reactive logic. Besides, this type of control achieves results nearly as good as the optimal control. The test in different realizations, however, shows that reservoir characterization is still a key part of any attempt to improve production. Although the variable reactive control is semi-independent, with action being taken based on measurements, some parameters need a priori model to be tuned

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Role of SoxE genes in the development of the auditory portion of the inner ear

The transcription factors of the SoxE family, including Sox8, Sox9 and Sox10 play important roles in diverse developmental processes and particularly in the development of the auditory portion of the inner ear, the cochlea. Among these regulatory genes, Sox9 has been shown to be important in otic vesicle formation during early embryonic development. Indeed, Sox9 expression is detected as soon as embryonic day E9, in the forming otic placode. As the organ of Corti begins to differentiate, Sox9 is progressively restricted to supporting cells and its expression is absent from hair cells, responsible for sound transduction. This expression profile prompted us to evaluate the role of this gene in cochlear cell terminal differentiation. We therefore analysed the role of Sox9 in cell specification using both gain- and loss-of function approaches. We demonstrated, by ex vivo electroporation of mouse embryonic cochlea that Sox9 strongly inhibits hair cell fate even when it is forced by ectopic expression of Atoh1, a potent inducer of hair cell differentiation. On the contrary, suppression of Sox9 in sensory progenitors did not affect cell fate. We also showed that Sox9 is upregulated by Notch activation in mouse embryonic cochlea and partially contributes to Notch inhibition of hair cell fate. Loss of Notch signaling in cells that are committed to become hair cells could thus be responsible for Sox9 suppression in these cells. In parallel, we investigated the molecular mechanisms underlying Sox9 inhibitory effect on hair cell fate in UB/OC1 cell line (derived from mouse embryonic otocyst) and more specifically its relationship with other important factors for cell specification. We observed an inhibition of Atoh1 transcriptional activity upon Sox9 overexpression, concomitant with an increase in the expression levels of Hey1 and HeyL factors. These proteins are well-known effectors of the Notch cascade and act by inhibiting Atoh1 activity. Taken together, our data suggest that Sox9 potentiates Notch pathway by upregulating its effectors Hey1 and HeyL and that Sox9 misexpression in differentiating hair cells inhibits the activity of the major hair cell fate inducer Atoh1, thereby preventing sensory cell terminal differentiation