Modeling MyD88 Deficiency In Vitro Provides New Insights in Its Function.

Inherited defects in MyD88 and IRAK4, two regulators in Toll-like receptor (TLR) signaling, are clinically highly relevant, but still incompletely understood. MyD88- and IRAK4-deficient patients are exceedingly susceptible to a narrow spectrum of pathogens, with ∼50% lethality in the first years of life. To better understand the underlying molecular and cellular characteristics that determine disease progression, we aimed at modeling the cellular response to pathogens in vitro. To this end, we determined the immunophenotype of monocytes and macrophages derived from MyD88- and IRAK4-deficient patients. We recognized that macrophages derived from both patients were particularly poorly activated by streptococci, indicating that both signaling intermediates are essential for the immune response to facultative pathogens. To characterize this defect in more detail, we generated induced pluripotent stem cells (iPSCs) of fibroblasts derived from an MyD88-deficient patient. The underlying genetic defect was corrected using Sleeping Beauty transposon vectors encoding either the long (L) or the short (S) MYD88 isoform, respectively. Macrophages derived from these iPSC lines (iMacs) expressed typical macrophage markers, stably produced either MyD88 isoform, and showed robust phagocytic activity. Notably, iMacs expressing MyD88-L, but not MyD88-S, exhibited similar responses to external stimuli, including cytokine release patterns, as compared to genetically normal iMacs. Thus, the two MyD88 isoforms assume distinct functions in signaling. In conclusion, iPSC technology, in combination with efficient myeloid differentiation protocols, provides a valuable and inexhaustible source of macrophages, which can be used for disease modeling. Moreover, iPSC-derived macrophages may eventually aid in stabilizing MyD88-deficient patients during pyogenic infections.

Reprogramming of Ovarian Granulosa Cells by YAP1 Leads to Development of High-Grade Cancer with Mesenchymal Lineage and Serous Features.

Understanding the cell-of-origin of ovarian high grade serous cancer (HGSC) is the prerequisite for efficient prevention and early diagnosis of this most lethal gynecological cancer. Recently, a mesenchymal type of ovarian HGSC with the poorest prognosis among ovarian cancers was identified by both TCGA and AOCS studies. The cell-of-origin of this subtype of ovarian cancer is unknown. While pursuing studies to understand the role of the Hippo pathway in ovarian granulosa cell physiology and pathology, we unexpectedly found that the Yes-associated protein 1 (YAP1), the major effector of the Hippo signaling pathway, induced dedifferentiation and reprogramming of the ovarian granulosa cells, a unique type of ovarian follicular cells with mesenchymal lineage and high plasticity, leading to the development of high grade ovarian cancer with serous features. Our research results unveil a potential cell-of-origin for a subtype of HGSC with mesenchymal features.

Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Although the role of the Hippo signaling pathway in development and tumorigenesis has been extensively studied in multiple organs, its role in ovarian follicle development remains largely unknown. Here, we report that Yes-Associated Protein 1 (YAP1), the major effector of Hippo signaling, is spatiotemporally expressed in ovarian granulosa cells and plays a critical role in the regulation of follicle development. We found that the active form of YAP1 (nuclear YAP1) was predominantly expressed in proliferative granulosa cells, whereas the inactive form of YAP1 (cytoplasmic YAP1) was mainly detected in luteal cells (terminally differentiated granulosa cells). Pharmacological inhibition of YAP1 activity disrupted mouse ovarian follicle development in vitro and in vivo. Foxl2 promoter-driven knockout of Yap1 in ovarian granulosa cells resulted in increased apoptosis of granulosa cells, decreased number of corpora lutea, reduced ovarian size, and subfertility in transgenic mice. However, Cyp19a1 promoter-driven knockout of Yap1 in differentiated granulosa cells of preovulatory follicles and luteal cells of corpora lutea had no effect on ovarian morphology and fertility. Mechanistic studies demonstrated that YAP1 interacted with epidermal growth factor receptor and TGF-ß signaling pathways to regulate granulosa cell proliferation, differentiation, and survival. Results from this study identify YAP1 as a critical regulator of granulosa cell proliferation and differentiation. Balanced expression and activation of YAP1 is essential for follicle development and successful reproduction. YAP1 is a promising target for treatment of subfertility associated with abnormal granulosa cell function.-Lv, X., He, C., Huang, C., Wang, H., Hua, G., Wang, Z., Zhou, J., Chen, X., Ma, B., Timm, B. K., Maclin, V., Dong, J., Rueda, B. R., Davis, J. S., Wang, C. Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

A multicentre evaluation of the Elecsys® anti-Müllerian hormone immunoassay for prediction of antral follicle count.

RESEARCH QUESTION: What concentration of anti-Müllerian hormone (AMH) corresponds to an antral follicle count (AFC) >15 for determination of ovarian reserve? DESIGN: A prospective study conducted at 13 US fertility clinics in women aged 21-44 years who presented for AFC evaluation by transvaginal ultrasound. Serum samples were collected at the time of AFC evaluation (menstrual cycle day 2-4). AMH concentrations were measured by the Elecsys® AMH immunoassay; oestradiol and follicle-stimulating hormone (FSH) concentrations were also measured. The serum AMH cut-off able to detect AFC >15 with high sensitivity was determined (derivation cohort). Clinical performance of the AMH assay at the derived cut-off was evaluated (validation cohort). Receiver operating characteristic (ROC) analyses were also performed. RESULTS: In the derivation cohort (n = 306), an optimal serum AMH cut-off value of 1.77 ng/ml was determined to correspond to AFC >15 with 89.63% sensitivity and 69.01% specificity, using the Elecsys AMH assay. In the validation cohort (n = 856), this 1.77 ng/ml cut-off could identify women with an AFC >15 with a sensitivity of 88.34% and a specificity of 68.29%; corresponding positive predictive and negative predictive values were 75.19% and 84.34%, respectively. ROC analyses demonstrated that AMH performed better than oestradiol or FSH in predicting AFC, with area under the curves of 85.7%, 57.1% and 69.7%, respectively, in the validation cohort. CONCLUSION: The Elecsys AMH immunoassay provides a robust and fully automated method to measure serum AMH levels. Women with AMH values below the cut-off of 1.77 ng/ml are unlikely to have AFC >15.

TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells.

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.

Função pulmonar após cirurgia de revascularização do miocárdiocom e sem circulação extracorpórea / Pulmonary function following on and off-pump coronary artery bypass grafting

Objetivos: avaliar e comparar a função pulmonar de pacientes submetidos à cirurgia de revascularização do miocárdio com e sem o uso de circulação extracorpórea. Métodos: a amostra foi composta por 40 pacientes submetidos à cirurgia de revascularização do miocárdio, classificados em dois grupos: com circulação extracorpórea (grupo CCEC ? 20 pacientes) ou sem circulação extracorpórea (grupo SCEC ? 20 pacientes). Registros espirométricos da capacidade vital forçada (CVF) e do volume expiratório forçado no primeiro segundo (VEF1) foram obtidos no período pré-operatório (considerado basal) e no primeiro, terceiro e quinto dias do período pós-operatório. Resultados: obser-vou-se que no geral os valores de CVF e VEF1 diminuíram no primeiro pós-operatório em relação ao basal (diferença média = 1,8±1,0, p<0,001 e 1,3±1,0, p<0,001, respectivamente), tendo recuperação parcial no terceiro e no quinto pós-operatórios, sem retornar aos valores iniciais (diferença média 1,2±1,1, p<0,001 e 0,9±0,9, p<0,001, respectivamente). Após controle para os valores basais, não foram observadas diferenças significativas entre os grupos CCEC e SCEC quanto à CVF e ao VEF1 no quinto dia pós-operatório. Conclusões: a cirurgia de revascularização do miocárdio associou-se a um decréscimo significante na função pulmonar em todos os pacientes, havendo recuperação parcial da CVF e do VEF1 no quinto pós-operatório para os dois grupos, porém sem retorno aos valores basais. Não se observou associação estatisticamente significativa entre realização de circulação extracorpórea e função pulmonar no quinto dia pós-operatório.

Aims: To evaluate and compare the pulmonary function of patients submitted to myocardial revascularization surgery, with and without the use of extracorporeal circulation. Methods: The sample comprised 40 patients submitted to myocardial revascularization surgery, classified into two groups, depending on whether extracorporeal circulation was used (wECC ? 20 patients) or not (nECC ? 20 patients). Spirometric recordings of forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1) were obtained during the preoperative period (considered baseline) and on the first, third and fifth days postoperatively. Results: In general, the values of FVC and FEV1 diminished on the first day postoperatively compared to the baseline (mean difference = 1.8±1.0, p<0.001 and 1.3±1.0, p<0.001, respectively), with partial recovery on the third and fifth day postoperatively, with no return to the initial values (mean difference = 1.2±1.1, p<0.001 and 0.9±0.9, p<0.001, respectively). After controlling for baseline values, no significant differences were observed between the wECC and nECC groups as to FVC and FEV1 on the 5th day postoperatively. Conclusions: Myocardial revascularization surgery was associated to a significant decrease in pulmonary function in all patients, with partial recovery of FVC and FEV1 on the fifth day postoperatively for all groups, with no return to baseline values. No statistically significant association was found between extracorporeal circulation and pulmonary function on the fifth day postoperatively.

Improving the interaction of Myc-interfering peptides with Myc using molecular dynamics simulations.

Previously, a Myc-interfering peptide (Mip) was identified for the targeted inactivation of the Myc:Max complex by the combination of rational design and an in vivo protein-fragment complementation assay. In the subsequent work presented here, molecular dynamics simulations and free energy calculations based on the molecular mechanics GBSA method were performed to define the contribution of the different amino acids in the Myc:Mip coiled coil domain, and compared to wild-type Myc:Max. For further optimization of the Myc interference, point mutations were introduced into Mip and analyzed, from which two showed much higher binding affinities in the computational studies in good agreement with the experiment. These mutants with very high potential for inactivation of Myc can now be used as starting point for further optimizations based on the computational as well as experimental protocols presented here.

Telemedicine in the work site: a study of feasibility, and patient and provider satisfaction.

We examined the use of telemedicine for improving access to care in a work-site clinic. A prospective study of 100 patients was conducted over a four-month period in a work site that housed 700 employees. Sinusitis (10 visits), upper respiratory tract infections (9 visits), otitis media (9 visits), hypertension (9 visits) and back pain (8 visits) were the most common reasons for the visits. In 99 visits, clinicians were of the opinion that the telemedicine visit felt similar to a face-to-face visit. For most of the visits (67), patients strongly agreed or agreed that telemedicine had a positive effect on their relationship with the health-care provider. The otoscope, microscope and stethoscope telemedicine peripherals were important in aiding diagnosis (and ruling out other causes) in about 55% of the visits (upper respiratory tract infection, sinusitis, otitis media, cough, sore throat, nevi, rhinitis and ear wax related concerns). The ability for the patient to watch their ENT examination and see any associated abnormalities was appreciated by many patients. Physicians, nurses and patients were capable of using the technology with little training.

Use of Physcomitrella patens actin 5' regions for high transgene expression: importance of 5' introns.

We have isolated four actin (Act) genes from Physcomitrella patens and used their corresponding 5' regions for recombinant expression of the human vascular endothelial growth factor (rhVEGF121) in transiently transformed Physcomitrella protoplasts and in stable transformed lines. In the transient system, we found up to 11-fold activity of the corresponding 5' regions as compared with that of the plant constitutive 35S promoter. Moreover, the use of an optimised expression vector in which the human VEGF signal peptide was exchanged with a plant signal peptide resulted in an additional 7-fold increase in secreted rhVEGF. We found that the 5' introns of PpAct1, PpAct5 and PpAct7 are essential for high expression. The enhancing mechanisms of the introns, however, seem to be different since in the case of PpAct1, the expression level is stimulated only in the presence of the endogenous promoter, whereas the 5' introns of PpAct5 and PpAct7 stimulate expression also in combination with the 35S promoter. Beyond this, the isolated 5' regions are shown to be useful for high expression levels in transgenic moss lines with values of secreted rhVEGF up to 96 microg g(-1) dry weight.