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https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/SUCU5X.

Experimental design, materials and methods
Round fatigue specimens with uniform gage section and the dimensions and geometry, shown in Fig. 1, were designed according to ASTM standard E606/E606M-12 [2]. The specimens were fabricated from 12.7mm diameter round bars of Ti–6Al–4V ELI, received in a mill-annealed condition (annealed for 1h at 1300°F). All fatigue experiments were conducted under strain-controlled condition at room temperature, utilizing an MTS extensometer model 634.31F-25, a servohydraulic test frame with 100kN load cell capacity, and a sinusoidal waveform input. Tests were performed using strain amplitudes that ranged from 0.0015 to 0.012mm/mm. Influence of strain rate effects was minimized by adjusting the test frequency, ranging from 0.5 to 5Hz, for each test condition to maintain a relatively consistent average strain rate for all tests. In addition, for each prescribed test condition, duplicate tests were conducted to validate the collected data and ensure the repeatability of experiments. Further details on the experimental program are presented in the following subsections according to the type of loading utilized.

Disclaimer

Acknowledgements
This material is based upon work supported by the U.S. Army TACOM Life Cycle Command under Contract no. W56HZV-08-C-0236. Any opinions, findings and conclusions or recommendations expressed in this Tubastatin A HCl material are those of the authors and do not necessarily reflect the views of the U.S. Army TACOM Life Cycle Command. This work was done while Patricio E. Carrion and Nima Shamsaei were at Mississippi State University.

Data
The experimental recordings reported in this paper were collected during the tests of concrete ties reinforced with multiple bars [1]. Cross-sections of the ties and surface characteristics of the reinforcement bars are presented in Fig. 1 with main reinforcement characteristics described in Table 1. The tests were performed in the Research Laboratory of Innovative Building Structures at Vilnius Gediminas Technical University (Lithuania). The test setup is shown in Fig. 2. Crack spacing characteristics and crack development schemes are presented in Tables 2 and 3, respectively. The Supplementary material consists of the database of average strains of reinforcement and concrete surface of the ties.

Design of the tests, materials, and methods

Funding sources
This work is part of a Ph.D. thesis of Arvydas Rimkus and was funded by the Research Council of Lithuania (Research Project MIP–050/2014).

Data
The data collected in the article are related to the research article entitled Retrofit strategies towards Net Zero Energy Educational Buildings: a case study at the University of the Basque Country (Ref. 0378–7788)[1]. The dataset presented in nares paper correspond to the indoor temperature and relative humidity of a seminar room located on the second floor of the Architecture Faculty in San Sebastian (Spain). The inputs correspond to a typical spring week: from 25th April to 1st May 2016, being the heating system off. In the following Tables 1 and 2 data for the day with the maximum values (26th April) and the day Tubastatin A HCl with the minimum values (28th April) are presented.
The average outdoor temperature on 26th April was 11.8°C and the average relative humidity 73.3% (Table 1). The maximum outdoor temperature was 14.2°C and was registered at 14:00. The minimum outdoor temperature was 9.4°C and was registered at 06:00. The average indoor temperature on 26th April was 22.8°C and the average relative humidity 43.55%. The maximum indoor temperature was 24.4°C and was registered at 12:00 by the Sensor 3 (located at the back rows). The minimum indoor temperature was 21.4°C and was registered from 02:00 to 08:00 by the Sensor 2 (located on the façade). According to occupation, 19 students were registered at 10:00 and 11 at 19:00. Location of the sensors is shown in Fig. 1.

Por otra parte la tica del cuidado

Por otra parte, la ética del cuidado critica algunos de los valores que el feminismo liberal abandera: el feminismo liberal está moldeado a partir de valores propiamente masculinos, como son la individualidad, l-ascorbic acid la universalidad y la autonomía. Como señalé antes, la ética del cuidado enfatiza una perspectiva interpersonal, contextual y de dependencia mutua, que son los valores morales centrales que una ética debe promover. Según esta teoría, el feminismo liberal ha sido demasiado entusiasta en abrazar algunos valores masculinos, pero particularmente su concepción del yo como el de un individuo autónomo y racional. En lugar de verlo de ese modo, la ética del cuidado propondría un concepto del yo basado en la afectividad, más que en la racionalidad. Asimismo, mientras que buena parte del feminismo enfatiza un ideal moral de igualdad entre hombres y mujeres, la ética del cuidado, en cambio, enfatiza las diferencias en nuestras perspectivas morales —y en este sentido, mientras el primero formaría parte del llamado feminismo de la igualdad, el segundo, del de la diferencia—. Sin embargo, todos estos conceptos que la ética del cuidado critica del feminismo liberal —autonomía, individualidad, racionalidad, igualdad, etc.— son los conceptos que están en la l-ascorbic acid del concepto moderno de derechos humanos. Es aquí donde quiero centrarme para hacer el balance entre estas dos teorías éticas: en sus distintas posiciones frente al concepto de derechos y su utilidad para la ética.
Parece inevitable que una teoría moral particularista, que rechaza el empleo de criterios generales y abstractos, entre en conflicto con una teoría universalista, que precisamente apela a ese tipo de criterios. El discurso de los derechos morales está basado en una pretensión de universalidad que, según el particularismo, no ayuda a entender la moralidad de situaciones concretas. Podemos decir que el discurso de los derechos morales (o de los derechos humanos universales como aspiraciones morales) no forma parte del vocabulario de la ética del cuidado. Antes mencioné la cercanía teórica de la ética del cuidado con la ética de las virtudes, y creo que el escepticismo de esta segunda teoría con respecto a los derechos también es compartido por la ética del cuidado. Para ambas teorías el discurso sobre virtud y cuidado pueden sustituir el discurso sobre derechos morales. Para las éticas radicales de la virtud y del cuidado, la ética puede, y debe, deshacerse de estos conceptos y de los principios generales que los justifican, que no son más que ficciones generalizadas, simulacros de universalidad y racionalidad que solo encubren intereses particulares y arbitrarios.
Así, Rosalind Hursthouse, una teórica de la virtud, muestra este escepticismo sobre el discurso de los derechos en su análisis en torno a la moralidad del aborto. El ejercicio de los derechos no garantiza que esa acción sea moralmente buena. Los derechos no nos ayudan a principle of independent assortment determinar la corrección o incorrección moral de un aborto.
Apelar a derechos morales no nos dice nada sobre la moralidad del acto de interrumpir un embarazo. Lo realmente relevante para determinar la moralidad de un aborto son las actitudes, las respuestas emocionales y las virtudes, especialmente aquellas relacionadas con el cuidado, que una mujer manifieste en esa circunstancia. ¿Podríamos entonces tal vez, en la ética, prescindir del discurso de los derechos morales a favor de un discurso basado en las virtudes, y particularmente en los afectos, las relaciones interpersonales y el cuidado? ¿Podemos hacerlo, por ejemplo, para el caso del aborto? Mi respuesta es que no: la ética no debe prescindir del discurso de los derechos morales, sobre todo en lo que se refiere al tema del aborto. Pero esto hay que argumentarlo.
Sin duda, el discurso de los derechos no nos da respuesta a todas nuestras inquietudes morales ni debe ser el concepto central de la moralidad. Tal vez incluso, como dicen algunos comunitaristas y teóricos que subrayan el aspecto comunitario y afectivo de las relaciones humanas, las relaciones morales no deben pensarse como constituidas básicamente por derechos y por relaciones basadas en derechos entre individuos atomistamente concebidos. Eso parece ir en la dirección correcta: así como hay más, por ejemplo, en una relación matrimonial que una mera relación contractual de derechos y obligaciones, hay más en la evaluación moral del aborto que simplemente un asunto de derechos, como indica Hursthouse. El mero ejercicio de los derechos no garantiza la corrección moral de una acción —ni de un aborto en particular—. La relación que una mujer tiene consigo misma, con el embrión en gestación y con la sociedad, así como los aspectos afectivos que subraya la ética del cuidado, también deben tomarse en cuenta para apreciar la complejidad moral de la interrupción de un embarazo en particular. Es cierto, como dice Jeremy Waldron, que “la estructura de los derechos no es constitutiva de la vida social, sino que debe entenderse como una posición de reserva y de seguridad en caso de que otros elementos constitutivos de la relación social se rompieran” (Waldron 1993: 374). En otras palabras, los afectos y las relaciones interpersonales que constituyen la vida social y que son los elementos fundamentales de la vida moral, como vimos, no son solamente algo cambiante y relativo a contextos particulares, sino que son relaciones que fácilmente pueden fallar o en las que las cosas pueden salir mal. Para esos casos están los derechos. ¿A qué me refiero? Volvamos al asunto del aborto.

br Gender difference hypotheses br

Gender difference hypotheses

Discussion
According to the explained difference among ADS syllabusses in Section 5.3, the effect of IQ on expert design skills is better than that on novices (the P-value for IQ and ADS-4 is 0.07 and is close tobeingcorrelated). Moreover, when the complexity of a project and design constraints increase, and the degree of being ill-defined in a design problem decreases, the influence of IQ on design skills becomes evident. However, the item (designer experience, complexity and functionality of design project, and a design problem being less ill-defined) that has a larger effect on this correlation cannot be indentified because no continual decrease in P-value occurs from ADS-1 to ADS-5 (because of both ADS-2 and an IQ P-value of 0.56).
Testing the second AL 8697 about the effect of gender difference on IQ and design scores shows that no significant difference is observed in the IQ and design scores of males and females in this study compared with other predictors (i.e., spatial ability) that indicate that males are better than females (Newcombe et al., 1983). The implication is that if spatial ability is regarded as an indicator of design abilities, then spatial ability contradicts the results in terms of the absence of a significant difference between males and females in the aspect of design scores.
The correlation between IQ scores and the total GPA is also measured in this study (P-value=0.217, Pearson correlation=−0.15, no correlation). The results indicate the lack of a significant difference between the average of design scores and students’ GPA.
Furthermore, the threshold theory of creativity–intelligence about intelligence design for an IQ above 120 is measured. For students with an IQ above 120, the correlation between IQ and the mean ADS is the Pearson correlation=−0.073 and the P-value=0.79, which indicates the lack of a significant relationship, and threshold theory about creativity–intelligence is not confirmed in intelligence design.

Conclusion
Other concerns that emerged were about creativity versus intelligence tests. Meanwhile, researchers have emphasized the role of creativity in design, in which the broad concept of creativity induced difficulties in understanding the exact role of creativity in design. Moreover, the measurability of creativity and creativity tests is under debate. Certain intelligence innovation tests (Squalli and Wilson, 2014) that can be used for future studies are available.
Based on different effective design factors and the outcomes (mental, cognitive, social interaction, collaboration, personality, problem-solving skills) of these factors, every designed predictive test should consider all aspects or might be combinations of cognitive and personality tests. In terms of success in designing a reliable test or questionnaire, Cross’ (1999) theory can validate that design is a special and separate type of intelligence.

Acknowledgement

Introduction
The main aim of this paper is to analyze the results from a post-occupancy evaluation (POE) study of a restored industrial space: a former textile factory converted into a contemporary art and design gallery. The building is located in the city of Puebla, Mexico. The importance of preserving industrial spaces, in this case a textile factory, is discussed in relation to its new use. The research questions are as follows: (1) How convenient is it to provide a new use for an old and abandoned building? (2) Is the building appropriate and comfortable for its current use?(Figures 1 and 2).
This paper addresses the principles of the 1987 Declaration of Puebla City as a World Heritage City and the connotation of the conservation of the industrial space with the Angelopolis mega project and its relationship with the contemporary art and design gallery in Puebla, Mexico. This document is structured with the following the sections: historical background of the building and the area, analysis of the concept of Industrial Heritage, research methodology, analysis of the conversion of “La Violeta” textile factory into an art gallery, description of the building, and the results of the POE of a converted industrial space into a contemporary art and design gallery called “Angeles Espinosa Yglesias”.

Recommended treatment for intracranial mesenchymal chondrosarcoma is radical surgical excision

Recommended treatment for intracranial mesenchymal chondrosarcoma is radical surgical excision if possible. Preoperative embolization has been suggested because the tumor is highly vascular. Recurrence is common after resection, however. Some authors therefore recommend adjuvant radiation therapy to prevent recurrence, but it is difficult to tell if this mode of adjuvant therapy is effective for the purpose. Because of the few cases with metastases, adjuvant chemotherapy has also been recommended regardless of treatment for the primary tumor. Again, the small number of cases prohibits drawing any conclusions about such treatment.

Conclusions

Introduction

Case report
A man who was 20 years of age was admitted to the hospital due to sudden onset of left chest pain and dyspnea for several hours. A chest radiograph demonstrated a large pleural effusion or hemothorax over the left lobe (Fig. 1), and a chest tube was inserted that drained approximately 1000ml of blood. The patient was sent to the intensive care unit for close observation, and then a chest computed tomography (CT) scan was taken once the vital signs were stable 2 days later; the scan revealed a broad-based bony excrescence over the right scapular body and right 7th rib and another pin-like bony excrescence over the left 10th rib with indentation of the left renal leukotriene receptor antagonist (Figs. 2 and 3). Furthermore, a renal echogram showed multiple exophytic bony excrescences over the right anterior rib with indentation of the anterior liver surface. There was another long bony spur, approximately 3–4cm in length, with indentation of the left renal capsule and no penetration into the left renal parenchyma.
During a left 9th to 10th intercostal space posterolateral thoracotomy, an osteochondromatous spur was found on the anterior arc of the left 10th rib near the costochondral junction. It measured 2cm in length and presented with a thorn-shaped tip, which was not covered by the cap of cartilage from its surface and eroded the diaphragm. The affected diaphragm was inflamed by a laceration. There was no active bleeding during the operation. The exostosis and the abnormal segment of the rib were excised (Fig. 4). The postoperative course was uneventful; there were no complications such as diaphragmatic rupture or recurrent bleeding, and the patient was discharged 7 days after the operation. Pathologic examination of the resected specimen (Fig. 5) revealed an outgrowth of mature bone covered by a well-differentiated cartilaginous cap that surrounded a subperiosteal bony projection. The cells in the cartilaginous cap resembled those of normal hyaline cartilage. The pathologic diagnosis was costal osteochondroma.

Discussion
HME represents a small percentage of thoracic cage tumors. Except for an inward-facing exostosis in the thoracic cage, most solitary HMEs are discovered before adulthood and usually are nontender, painless, slow-growing masses. However, when an exostosis is either large or occurs at certain critical anatomic sites, it can lead to symptoms with potential complications. An exostosis becomes symptomatic on account of mechanical irritation to soft tissues, and involvement of a rib can rarely lead to pleural irritation and hemothorax. Few cases of hemothorax complicating a rib exostosis, however, have been reported. The complication may be associated with injury to the pleura, diaphragm, heart or lung. Shearing of the pleura or the diaphragm by the sharp margins of the intrathoracic exostosis is potentially lethal. Diaphragmatic rupture, bowel obstruction, pericardial effusion, repetitive chest infections and loculated empyema have also been reported. In our case, shearing of the diaphragm by the sharp margins of the intrathoracic exostosis causing hemothorax should be considered.
To the best of our knowledge, with regard to thorn-like exostoses without coverage by a cap of cartilage, only four cases have been reported in past and the only known adult case as we reported. The diagnosis was suggested by CT scan and confirmed by surgery with pathologic finding.

Laparoscopy would be an effective treatment method

Laparoscopy would be an effective treatment method in patients with internal hernia; however, some factors are considered contraindicative to laparoscopic surgery: dilated small-bowel diameter >4 cm, history of severe adhesions, bowel ischemia, inadequate field of view, complicated pathology, such as malignancy, and inflammatory bowel disease. We performed emergent laparotomy rather than laparoscopy due to significant dilatation of the small bowel (diameter >6 cm) and ischemic change in the CT image.

Introduction
A horseshoe kidney is the most common renal fusion anomaly and occurs in 0.2–0.3% of the population. Urothelial carcinoma (UC) of the upper urinary tract in a horseshoe kidney is extremely rare. According to our review of relevant literature, only a few studies have reported UC in a horseshoe kidney managed through a laparoscopic heminephroureterectomy. Herein, we present a case of UC in a horseshoe kidney managed through this technique.

Case Report
A 74-year-old man presented with complaints of gross hematuria ongoing for 10 months. Retrograde pyelography showed a filling defect in the left renal pelvis. Ureteroscopy revealed a papillary tumor in the left renal pelvis, and biopsy confirmed UC. Computed tomography (CT) angiography revealed a horseshoe kidney with four renal gingerol supplying the left moiety of the horseshoe kidney (Figure 1). Moreover, 99mTc-MAG3 renal scintigraphy revealed a renal function of 72.1% and 27.9% for the right and left parts, respectively. After receiving a careful explanation of the treatment risks and benefits, the patient chose to undergo a laparoscopic heminephroureterectomy.
Under general anesthesia, the patient was placed in a modified flank position, with the left side elevated 30°. A 2-cm incision was made over the paramedian line at the level of the umbilicus. The peritoneal cavity was entered and a 12-mm trocar was inserted. Pneumoperitoneum was created using carbon dioxide. Under direct laparoscopic vision, a 12-mm trocar was inserted on the ipsilateral midclavicular line, 3 cm below the costal margin and another 12-mm trocar was placed over the left Gibson incision line. Transperitoneal nephroureterectomy was performed. The renal arteries were ligated using Hem-O-Lok clips (Weck Surgical Instruments, Teleflex Medical, Durham, NC). Endo-GIA staplers (Covidien Inc, Mansfield, MA) were used to divide the renal vein and isthmus (Figure 2). Subsequently, open bladder cuff excision was performed through the Gibson wound. The surgical time was 210 minutes, and 200 mL of blood was lost. The patient resumed oral intake on Postoperative Day 1, and no perioperative complication was reported. He was discharged on Postoperative Day 4. Pathology revealed a pT3N0 UC in the left renal pelvis. No recurrent malignancy was observed over a 6-month follow-up period.

Discussion
Annually, UC affects 20 people/100,000 population, and 7–8% of UCs are located in the upper urinary tract. In a review gingerol of tumors in horseshoe kidneys, renal cell carcinoma, UC, and Wilm\’s tumor accounted for 50%, 25%, and 25% of all cases, respectively. UC occurs more frequently in horseshoe kidneys because of the greater susceptibility to urinary stasis, renal lithiasis, and urinary tract infection. Nephroureterectomy with bladder cuff resection is the standard treatment for UCs of the upper urinary tract. Two main challenges when conducting a laparoscopic heminephroureterectomy in a horseshoe kidney are managing the complex renal arteries and performing an isthmusectomy. Conducting a preoperative imaging study is crucial for identifying complex vascular anatomy, understanding the extent of tumor invasion, and facilitating optimal surgical planning. In 2001, Lee et al advocated three-dimensional multi-slice helical CT as the preferred imaging modality for clarifying tumors, vascular anatomy, and the collecting system of a horseshoe kidney. In a review of 209 cases with fused ectopia and a horseshoe kidney, CT was observed to be superior to magnetic resonance imaging and angiography in detecting arterial vessels. In addition to the complex vasculature supplying each kidney, considerable variations may exist in the blood supply to the isthmus of a horseshoe kidney. After the supplying renal vessels have been ligated and transected, the isthmus margin can be clearly observed. This can also be achieved through preoperative embolization. Complete mobilization of the kidney should be achieved before isthmusectomy, which can be performed using Endo GIA staplers, parenchymal sutures, electrocautery, or harmonic scalpels, depending on the preference of the surgeon and thickness of the renal isthmus. Possible major complications of a laparoscopic nephrectomy include bleeding requiring conversion (1.4%), splenic injury (1.4%), a fragmented tumor (0.35%), kidney fracture during retrieval (0.35%), hypotension in the recovery room with re-exploration (0.35%), pneumothorax (0.35%), pulmonary embolus (0.35%), a Mallory–Weiss tear (0.35%), a bleeding duodenal ulcer (0.35%), and acute renal failure (0.35%). Khan et al reviewed 21 case reports (on a total of 25 patients) of laparoscopic nephrectomy for horseshoe kidney. They reported that perioperative surgical complications included paralytic ileus (4%), sick sinus syndrome (4%), a colonic serosal tear (4%), skin separation (4%), urinoma (4%), anejaculation (4%), and neuralgia (4%). Additional large-scale studies are required in order to elucidate the actual complication rate of nephrectomy for horseshoe kidneys.

To develop an effective cell based therapy against

To develop an effective cell-based therapy against neurodegenerative disorders of the CNS, efficient recruitment of the cells into the CNS is essential. Previous findings suggest that particular bone marrow derived cells are able to cross the blood–brain barrier (Lebson et al., 2010; Simard and Rivest, 2004). To evaluate the migration of the iPS-ML into the CNS, we examined the effect of intravenous, intraperitoneal, and intracerebroventricular injection of iPS-ML into 5XFAD mice. To our disappointment, the iPS-ML injected via these routes did not efficiently infiltrate into Rocilinostat parenchyma and failed to reduce the amyloid burden. A possible reason of the failure to efficiently migrate in brain tissue may be the lack of CC chemokine receptor-2 (CCR2) expression in the iPS-ML (data not shown). To analyze the in vivo effect of iPS-ML/NEP2, we directly administered iPS-ML into the brain. To this end, we stereotaxically inserted microinjection tubes into the hippocampus of 5XFAD mice and transplanted iPS-ML through this tube. The hippocampus plays a major role in cognitive dysfunction of AD, and the 5XFAD hippocampus is one of the regions of the brain where Aβ plaques accumulate. iPS-ML transplanted by this procedure migrated to the brain parenchyma adjacent to the area of the tube insertion (Figs. 5B, C).
In vivo, transplantation of iPS-ML/NEP2 into the hippocampus of 3–4-month old 5XFAD/scid mice significantly diminished the levels of soluble Aβ1–42 in the brain ISF compared to the control Ringer\’s solution injection (Fig. 6). The reduction of Aβ was not significant when non-modified iPS-ML were transplanted. Therefore, the reduction of Aβ was caused by the secretion of NEP2 from the iPS-ML, but not phagocytosis of Aβ by the iPS-ML. Our intrahippocampus transplantation of iPS-ML demonstrated short-term and focal remote effects of the IPS-ML; only where the cells were transplanted. Furthermore we could not examine the therapeutic effect of cognitive function, because the mice were weakened by probe implantation. Future studies will be aimed at exploring whether iPS-ML are effective in preventing cognitive decline and neuronal damage in other AD models. In addition, to develop this technique as a therapy for AD, delivery of iPS-ML into the brain by systemic administration is necessary.
To examine the chromosomal alteration of iPS-ML, iPS-ML cultured for 6weeks after the introduction of proliferating factors were subjected to karyotype analysis. As shown in Supplemental Fig. S1, some karyotype abnormalities were detected in this analysis. For application of iPS-ML to clinical cellular therapy, we should resolve the issue of genetic instability of iPS-ML. To generate iPS-ML, our current method uses cMYC, BMI1 plus MDM2 to induce proliferation of iPS-MC. Among the introduced factors, MDM2 is involved in degradation of p53 protein as the E3 ubiquitin ligase (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). Forced expression of MDM2 in iPS-ML may cause complete loss of p53 function and result in the genetic instability of iPS-ML. Although co-introduction of MDM2 enhanced the proliferation rate of iPS-ML, this factor is not absolutely necessary for the establishment of iPS-ML, as previously reported (Haruta et al., 2013; Koba et al., 2013). Omission of MDM2 in the generation of iPS-ML may be one way to improve the genetic stability of iPS-ML.

Acknowledgments
The plasmids used for preparing recombinant lentivirus, pCSII-EF, pCMV-VSV-G-RSV-Rev, and pCAG-HIVgp were kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center). cDNAs for human BMI1 and EZH2 were provided by RIKEN BRC which is participating in National Bio-Resources Project of the MEXT, Japan. This work was supported in part by a Grant-in-Aid No. 23659158 from MEXT, Japan, a Research Grant for Intractable Diseases from Ministry of Health and Welfare, Japan, and a grant from Japan Science and Technology Agency (JST).

The primary determinant of the target specificity of an

The primary determinant of the target specificity of an individual designer nuclease is the length of its DNA recognition site, which is different in ZFNs, TALENs and the CRISPR/Cas9 system. Also, the GC content substantially influences the target specificity. In theory, a 16bp recognition site should already be statistically unique in the human genome. In reality, less than 50% of all 16-mer combinations are in fact unique and recognition sequences of at least 18bp should be preferred to achieve hybridisation to a distinct locus (Li et al., 2015; Liu et al., 2008). In general, TALENs can be expected to bind fewer off-target sequences than ZFNs and CRISPR/Cas9, as the target specificity relies on a relatively long recognition motif of 30–36bp in length which is rarely found in genomes. In addition, ZFNs and TALENs may be safer than the CRISPR/Cas9 system as they only act as a dimer, so that a pair of potential off-target sites has to be located within a range of 100bp to bring both FokI nucleases close enough for activation.
For the CRISPR/Cas9 system with its 20bp recognition sequence significant off-target mutagenesis in different human cell types could be observed, indicating that the Cas9 nuclease can be active even at sites with multiple mismatched SRT1720 pairs if they were not properly designed (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013). In case of demonstrated off-target activity, the respective imperfect target sequences were always followed by a suitable PAM (Hsu et al., 2013), and PAM-proximal mismatches were less tolerated than PAM-distal counterparts (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013) suggesting for a 5–8bp seed region. However, the overall specificity was largely determined by the complete guide sequence including the guide RNA architecture and dosage (Hsu et al., 2013; Pattanayak et al., 2013). In contrast to various studies showing substantial off-target damage at related sites when using common CRISPR/Cas9 systems, another study contradicted these findings since whole genome sequencing of hPSC clones revealed low incidence of off-target mutations after CRISPR/Cas9 mediated targeting (Veres et al., 2014). It is noteworthy, however, that insertions or deletions (INDELs) larger than 16bp are barely detectable using current Next-Generation Sequencing (NGS) technologies and might have been overseen in recent studies. It is likely that much more off-target effects, which occurred in subpopulations of the targeted cells, would be detectable in the targeted polyclonal cell population, if more accurate sequencing systems than the applied “sequencing-by-synthesis” technology (error rates per read between 10%, Illumina GA IIx datasheet, and 1% for HiSeq 2000 (Liu et al., 2012)) would be used. Furthermore, off-target mutations with frequencies below 1% or 0.1% would only be detected by sequencing of 150 or 1500 single cell clones, respectively (Tsai and Joung, 2014).
Very recently, two studies allowed detailed insights in the regulation of Cas9 target searching and proofreading mechanisms before double strand break induction (Knight et al., 2015; Sternberg et al., 2015). Thus, the target search mechanism involves rapid three-dimensional diffusion dynamics of Cas9 (Knight et al., 2015) and a final checkpoint beyond initial PAM recognition and RNA–DNA base pairing in terms of conformational change controlling DNA cleavage (Sternberg et al., 2015). This understanding will help to further limit off-target activity. So far, for the reduction of off-target effects in CRISPR/Cas9 targeting experiments, truncated gRNAs of 17–18nt length or “enhanced specificity” SpCas9 variants (Fu et al., 2014; Slaymaker et al., 2016) can be applied without decreasing on-target efficiency. Another approach to improve the specificity of the monomeric Cas9 nuclease, is the application of paired Cas9 nickases to generate DNA DSBs. One nickase produces a single strand break only, and similar to ZFNs and TALENs the targeted activity of two nickases leads to a double strand break that allows engineering the respective site via NHEJ or HR. Since it is extremely unlikely that a pair of nickases show an off-target activity at both complementary strands of one genomic site, and because off-target single strand breaks are repaired with high fidelity by the base-excision repair pathway instead of error prone NHEJ, the risk of off-site effects at genomic sites where only one of the guide RNAs binds is dramatically reduced (Mali et al., 2013; Ran et al., 2013; Shen et al., 2014). Similar to TALENs (Mussolino et al., 2011), also obligate heterodimeric FokI variants have been combined with the CRISPR system, which likewise improves its specificity (Tsai et al., 2014). In view of the recent contradictory studies and the ongoing development of improved CRISPR/Cas9 systems further studies are clearly required to obtain a reliable comparative estimation of the off-target efficacy of the different types of designer nucleases. Certainly, reduction of off-target effects is one major issue, especially for in vivo application. However, to date it is still unclear if off-target effects will have a real impact on cell function and cancerogenity since the enormous heterogeneity of the human genome makes prediction difficult and biological read-outs are missing.

We next used the high

We next used the high-content imaging to further define hypertrophic pathway interactions in hESC-CMs and hiPSC-CMs and to compare this with data defining the phosphorylation effects of PE treatment. We have developed 2D and 3D imaging processes to analyze hypertrophic phenotype of our Cyanine5.5 alkyne using high-content analysis, and we used these while extending our original panel of kinase inhibitors (Földes et al., 2011) to cover many pivotal kinase targets. This is an important advance on our previous experiments, where we used a limited panel, because few inhibitors are completely specific. In Figures 6C and S5, the abscissa titles show that many inhibitors have multiple targets; bioinformatic analysis was therefore necessary to deconvolve the pathways clusters involved. Although the positive effect of PE on hypertrophic markers was not evident in hiPSC-CMs, we were surprised to see negative effects of kinase inhibitors on a number of parameters. These included the original p38-MAPK, ERK1/2, and mTOR inhibitors that were effective in hESC-CMs (in both this and our previous study; Földes et al., 2011). Some inhibitors, such as those for GSK3β and EGFRK, were also able to increase cell size and area, suggesting that there had been tonic suppression of cell growth by these pathways. We also noted a number of clear differences between hESC-CM and hiPSC-CM responses, such as an increase of cell volume in hiPSC-CMs, but not hESC-CMs, with inhibitors of EGFRK or the STAT activator JAK-2. This suggests a greater tonic suppression of ADRA1B hypertrophic signaling in the hiPSC-CM, which would again contribute to the poor responses to PE.
Detection of PE-dependent phosphorylation of the kinases themselves with a proteome profiling array showed that Src family kinases, a nonreceptor tyrosine kinase group implicated in controlling G protein-coupled receptor trafficking and effects on cell proliferation and cytoskeletal rearrangement, were markedly activated in hESC-CMs and hiPSC-CMs. The Ingenuity Pathway Analysis identified an EGFR/Src/GSK3β/STAT3 network modulated by ADRA1B that could drive the hypertrophic process. STAT3 is an established ADRA1B downstream target (Han et al., 2008), and the finding is in line with previous reports of activation of Src-dependent pathways by ADRA1 and crosstalk between EGF signaling and ADRA1 in cardiac hypertrophy (Li et al., 2011; Zitron et al., 2008; Asakura et al., 2002). Supporting this schema, a STAT3 inhibitor was able to reduce both basal and PE-stimulated cell area in the hESC-CMs as well as basal in hiPSC-CMs. Also, a STAT activator (IL-6) caused a marked increase in PE-stimulated cell area in the hiPSC-CMs. PE was able to induce STAT3 movement to the nucleus, but it was only able to increase cell area in the presence of IL-6. The simultaneous activation of EGFRK/GSK3β by PE via STAT3 may have restrained the final cell size change following translocation. A smaller panel of inhibitors, based on the array above, was used in combination to identify the antihypertrophic pathways most active in the hiPSC-CMs. This confirmed the EGFRK/GSK3β combination, together with CamKII, as optimal to increase cell area.
We conclude that the main difference in hiPSC-CMs that accounts for the defective response to α1AR stimulation is the suppression of growth by tonic antihypertrophic pathways (Figure 7), including EGFRK, GSK3β, and CamKII. The limitations of this study are that this screen is not fully comprehensive, and so we may have missed other elements and combinations that could have further rescued hypertrophy in hiPSC-CMs. Nor can we say that this will be predictive of every hiPSC-CM line, since we have not identified the genetic or epigenetic change produced by the reprogramming process, which may have triggered the different balancing of hypertrophic/antihypertrophic pathways. We further note that even in hESC-CMs, where α1AR-mediated cell size increases are clear, there has been a differentiation-induced shift in the αAR subtype. These data send an important message that superficial similarities in phenotype between cardiomyocytes derived from hESCs or hiPSCs, and parallels to adult cardiomyocytes, may mask complex differences in signaling. This has implications for their use in drug discovery, where targets identified using pluripotent stem cell derivative may not ultimately act in the same way in adult human cardiac cells.

br Introduction Alkaline phosphatase ALP is a ubiquitous membrane bound

Introduction
Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Examination of expression of ALP at intracellular and extracellular level is a widely-used procedure in both clinical practice and basic research. In the clinic, changes of normal ALP level in plasma is a routine diagnostic marker for various pathological processes (Sharma et al., 2014). At the basic research level, ALP quantification is frequently used in the study of cancer physiology and for evaluation of pluripotency in stem cells. Intracellular expression of ALP is very high in induced pluripotent (iPS) and embryonic stem cells (ES) and therefore can be used as unique and unambiguous biomarker of stem cells (Martins et al., 2014; Stefkova et al., 2015). In cancer, ALP expression inversely correlates with disease severity in advanced colon cancer and positively correlates with the ability of cells to differentiate (Shin et al., 2015).
Multiplexing is common in dual luciferase reporter assays, where transfection of Renilla and Firefly luciferase reporter plasmids are performed in batch and then read sequentially (Liu et al., 2009). A combination of two detection methods, such as luminescence and fluorescence, is also possible although frequently there is a loss of sensitivity as optimal conditions for one platform may not be compatible with the other and requires either expression of fluorescent biomarkers like Green Fluorescent Protein (GFP) or live staining of cells with a fluorescent dye, such as Calcein AM.

Materials and methods

Results
In order to examine whether CDP could be effectively used as a reagent for detection of ALP in a multiplexed assay with metabolic proliferation reagents in-vitro, we prepared calf intestinal ALP into 1xCutSmart Reaction Buffer and then diluted in LB with serial dilutions to simulate conditions of cell lysis. Following CDP addition, we saw a linear luminescence signal in all range of concentrations: from 0.017nM to 20nM (data not shown). To further evaluate the compatibility of the ALP reaction with metabolic detection reagents, we added LB, CTG buffer, or ATPlite buffer. While addition of LB had no effect on the CDP signal, both CTG and PE GLPG0634 markedly quenched the CDP signal (Fig. 1, A). Notably, 6 other buffers used for cell viability detection were purchased from other companies (see Materials and methods) and had no effect on CDP signal (data not shown). Quenching of the CDP signal by subsequent addition of a viability reagent is useful as the secondary luminescent, reading from the viability reagent will not be convoluted by luminescence from two different enzymes in the same homogenous reaction.
CTG is a reagent used GLPG0634 to determine the number of viable cells in culture based on quantification of the total ATP correlating with cell number. Since ATP and ALP are both presented in cell lysates, it was important to exclude the possibility that one of the agents impacts the luminescent signal of the other. We measured ALP activity between and 1000nM by CDP reagent in the presence (10, 100, or 1000nM) or absence of ATP, and observed no effect of ATP on ALP signal (Fig. 1, B). Conversely, there was no effect of ALP on ATP signal when measured in the presence of ALP in 7 different concentrations (0 to 241nM) using CTG (Fig. 1, C).
Elevated ALP expression is one of the ubiquitous markers of embryonic stem cells and pluripotent stem cells (Stefkova et al., 2015), and therefore a potential application for CDP/CTG methods is the determination of the cellular “stemness” in a complex culture of cells from multiple lineages. Different numbers of either induced pluripotent stem cells (iPSC) or mouse embryonic fibroblasts (MEF) cells were plated and CDP/CTG signals were measured at the next day. Signals from both CDP and CTG increased with cell number (Fig. 2, A, B) when examining iPS cells. In contrast, we observed almost no CDP signal in MEF cells, while CTG signal was correlated directly with cell number. Further, the sequential addition of the metabolic CTG reagent requires compatibility of the protocols and reagents in the same reaction well. Therefore, we compared different methods of sequential addition of CTG reagents following the CDP reaction. Cells were lysed by the CDP-compatible LB protocol and then either LB or CDP reagents were applied, followed by the addition of CTG. The original manufacturer\’s CTG protocol recommends aspiration of growth medium and direct addition to cells of CTG into remaining medium. When applied sequentially in cells, prior reactions of CDP did not adversely affect the CTG signal (Fig. 2, C), with similar results when CTG was applied directly without aspiration of media. Taken together the results suggest the ability of CTG usage after CDP in same well without affecting of experimental parameters.

In the dermis the glial P NGFR positive BC

In the dermis, the glial P75NGFR-positive BC derivatives also include a neurogenic and gliogenic stem-like cell population. Multipotent stem-like cell populations have been described previously in the adult trunk skin, associated with the glial and melanocyte lineages and derived from the NC (Wong et al., 2006) or associated with hair follicle dermal Dabrafenib and derived from the mesoderm (Biernaskie et al., 2009; Jinno et al., 2010). Our results indicate that the BC-derived population constitutes the major, but not single, component of skin stem-like cells detected in these culture conditions, as they represent approximately 80% of the sphere population at late passage. Our work is consistent with recent observations indicating that human adult skin stem cells with neurogenic potential express P75NGFR and can be ascribed to the Schwann cell lineage (Etxaniz et al., 2014). Together, our results establish the precise origin of the large majority of the stem-like cells in the dermis and provide a unique and specific genetic tool for their identification, further study, and manipulation.
BC derivatives with self-renewal and neurogenic potential also have been identified in the mouse embryonic DRGs (Hjerling-Leffler et al., 2005; Li et al., 2007). In vitro, these stem-like cells re-express NC markers and differentiate efficiently into peripheral sensory neurons (Hjerling-Leffler et al., 2005). Recently, stem/progenitor cells were identified in the adult DRG (Vidal et al., 2015). However, their differentiation might be more restricted as, after transplantation into the lesioned spinal cord, they exclusively generated Schwann cells. It will be interesting to determine whether this population derives from BC cells.

Experimental Procedures

Author Contributions

Acknowledgments

Introduction
The generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is dependent on our ability to accurately recapitulate the intricate steps of early embryonic hematopoietic development in the differentiation culture. The mammalian embryonic hematopoietic system consists of at least three distinct programs—primitive, erythromyeloid progenitor (EMP)/yolk sac definitive, and intraembryonic definitive—that differ in their potential as well as spatial and temporal patterns of development (McGrath et al., 2011; Medvinsky and Dzierzak, 1996; Palis et al., 1999). Primitive hematopoiesis is the first to develop, initiates in the yolk sac at embryonic day 7.0 (E7.0), and produces a restricted repertoire of hematopoietic cells consisting of primitive erythrocytes that express embryonic forms of globin and macrophages. Shortly after the onset of primitive hematopoiesis, the yolk sac generates a cohort of erythroid, myeloid, and megakaryocytic progenitors that constitute a distinct phase of embryonic hematopoiesis known as the erythromyeloid progenitor program, also referred to as the yolk sac definitive program (McGrath et al., 2011). This program appears to be positioned developmentally between primitive and definitive hematopoiesis. In the mouse, it generates erythroid cells that express adult and embryonic globins but does not give rise to lymphoid lineage cells or hematopoietic stem cells (McGrath et al., 2011; Palis et al., 1999). HSCs are generated during the intraembryonic-definitive stage of hematopoiesis that is specified following the emergence of the two yolk sac programs at different sites within the embryonic arterial vasculature, the most well characterized being the developing aorta, gonad, and mesonephros (AGM) (Medvinsky and Dzierzak, 1996). In addition to HSC and lymphoid potential, murine definitive hematopoiesis is distinguished from the two earlier programs by the fact that the erythroid lineage expresses exclusively adult forms of globin. Although both the EMP and intraembryonic-definitive hematopoietic programs are considered to be “definitive” due to their ability to generate erythroid precursors, to distinguish between them in this study, we use the terms EMP for the yolk-sac-derived progenitors and definitive for those derived from intraembryonic sites.