Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing

Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders.Medical Science

Mechanotransduction of substrate stiffness in endothelial cell collective migration

Endothelial damage during life-saving percutaneous angioplasty contributes to re-stenosis rates of nearly 20% within 5 years. Re-endothelialization, the collective endothelial cell migration over exposed extracellular matrix (ECM) and stent struts, can restore a continuous, functional endothelium. During atherosclerotic disease, vascular ECM becomes stiffer. ECM stiffness affects epithelial cell collective migration in other pathogenic contexts. However, substrate stiffness effects on endothelial cell collective migration have yet to be explored. We developed quantitative computational image processing algorithms for assessing collective migration. We then used these image analysis techniques to measure the effect of substrate stiffness on critical aspects of porcine aortic endothelial cell (PAEC) two-dimensional collective migration: (1) migration distance, (2) directedness, and (3) togetherness. PAEC were seeded on collagen-coated polyacrylamide hydrogels (4-50 kPa) in a 5 mm cloning ring and then allowed to migrate outwards. We found that migration distance increased with substrate stiffness and that there was a concomitant increase in PAEC alignment. We found that decreased togetherness on stiffer substrates led to enhanced proliferation at the migratory interface. We used the specific Rho kinase (ROCK) inhibitor Y27632 to show that ROCK-mediated contractility limited endothelial cell collective migration on soft substrates. We observed that PAEC secrete and remodel fibronectin on collagen-coated substrates. Interestingly, α5 integrin, but not fibronectin, was important for directed collective migration on stiff substrates. These findings provide insight into how substrate stiffness affects endothelial cell collective migration. This work will inform how the mechanical properties of tissue and tissue engineered construct could be designed to promote a functional endothelium.Ph.D., Biomedical Engineering -- Drexel University, 201

Erythropoietin signaling regulates heme biosynthesis

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)Massachusetts Institute of Technology. Poitras Center for Affective Disorders ResearchUnited States. Department of Energy (Computational Science Graduate Fellowship)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)New York Stem Cell FoundationPoitras FoundationSimons FoundationPaul G. Allen Family FoundationVallee FoundationTom HarrimanB. Metcalf

Antibiotic resistance and extended spectrum beta-lactamase production in fecal Escherichia coli strains isolated from human and cattle

Son bir ay içinde antibiyotik kullanmamış insanların dışkılarından izole edilen herbiri farklı bireye ait 140 Escherichia coli susu ile sığırların rektum mukozasından izole edilen 114 E.coli susunun 10 antibiyotiğe duyarlılığı disk difüzyon yöntemi ile, bu suşlarda genişlemiş spektrumlu beta-laktamaz (GSBL) üretimi fenotipik doğrulama testi ile belirlenmiştir. İnsan suşlannda ampisilin, ampisilin-sulbaktam, amoksisilin-klavulanik asit, piperasilin-tazobaktam, sefotaksim, siprofloksasin, trimetoprim-sulfametoksazol direnci sığır suşlarındakinden, sığır suşlannda amikasin direnci insan suşlanndakinden anlamlı derecede daha yüksek bulunmuştur. Seftazidim ve gentamisin direnci insan suşlannda daha yüksek bulunmuşsa da fark anlamlı değildir. İki grup susta da en yüksek direnç ampisilin için bulunmuştur. İnsan suşlannda direnç cinsiyete göre fark göstermemiş, ancak 13 yaş ve altındaki çocuklara ait suşlarda büyüklerden izole edilenlere göre ampisilin, ampisilin-sulbaktam, seftazidim, gentamisin ve trimetoprim-sulfametoksazol direnci anlamlı derecede yüksek bulunmuştur. İnsan suşlannda GSBL üretimi % 3 olarak saptanmış, sığır suşlannda GSBL üretimine rastlanmamıştır.A hundred forty E.coli strains isolated from stools of humans who did not use antibiotics in the last one month and 114 E.coli strains isolated from cattle's rectal mucosa were enrolled in this study. Each strain was from different individuals. Susceptibility of these strains to ten antibiotics were determined by disk diffusion test and extended spectrum beta-lactamase (ESBL) production by phenotypic confirmation test. The resistance to ampicillin, ampicillin-sulbactam, amoxicilin-clavulanic asid, piperacillin-tazobactam, sefotaxime, ciprofloxacin and trimethoprim-sulfamethoxazole in human E. coli strains were found significantly higher than the resistance of cattle E. coli strains and the resistance to amikacin in cattle E. coli strains was found significantly higher than the resistance of human E. coli strains. Although the resistance to ceftazidime and gentamicin in human E.coli strains were found higher, the differences was not statistically significant. The highest resistance was for ampicillin in both cattle and human strains. The differences in antibiotic resistance of human strains were insignificant in two genders. But the antibiotic resistance were significantly higher in the strains from children under 13 age than the strains from adults for ampicillin, ampicillin-sulbactam, trimetoprim-sulfamethoxazole, gentamisin and ceftazidime. While ESBL production from human strains was 3 %, there was no production in cattle strains

Heating Residences Using Solar Power

In this study, it was aimed to use the hot water produced using solar power in central heating systems in addition to places such as kitchen, bathroom and toilets, and a prototype was prepared. In the prototype, two 33.5x68.5 cm solar collectors, two boilers one of which is insulated (28 x 28 x 50 cm) and the other stripped (26.5 x 26.5 x 59 cm), a solar panel one radiator (40.5 x 60 cm), automated air relief cock, solenoid valve (12 V DC 14 W), two turbine motors (0.2 A, 220 V AC 40 W), one battery and two solar panels were used. The battery acts as a backup energy source in order for the water to be heated to the desired temperatures at nights, overcasts and times of precipitation, and it is charged using the energy from the solar panels. This is how the system may be used during winter months. As the electrical energy to run the system is taken from the sun, it contributes to minimisation of the increasing electricity costs and protection of the nature by reduction of air pollution. Proton IDE software was used to programme the PIC microcontroller. The software produced using this programme was placed on the PIC with Usburn programme using a Branner 8 PIC programmer

Solitary caecal diverticulitis as an unusual cause of a right iliac fossa mass: a case report

Inflammation of a solitary caecal diverticulum is an uncommon pathological condition. Preoperatively the condition is almost indistinguishable from appendicitis, and is often confused with carcinoma of the caecum during operation. The typical patient with this condition is male, Asian, and in the fourth decade of life. This case is unusual in that the patient was a 26-year-old Caucasian man

Good News as the News a Content Analysis of News Values

"İyi haberler de değerli haberler midir; yoksa yalnızca kötü haberler mi daha yüksek değere sahiptir?" Bu sorudan hareketle bu çalışmada günlük gazetelerde yayımlanan haberlerin duygu durumu irdelenmiştir. Türkçe literatürde bu alanda yapılmış ilk çalışma niteliğindeki araştırmada, yaygın günlük gazetelerden beşinin (Hürriyet, Posta, Sabah, Sözcü ve Zaman) belirli bir zaman kesiti içinde (6 Ocak - 13 Nisan 2014) örneklem alınan ilk sayfa haberlerinin duygu tonu ve duygu türü içerik analizine dayalı olarak çözümlenmiştir. Analizde duygu tonu bağlamında haberlerin pozitif, negatif ve nötr şeklindeki duygu çağrışımları; duygu türü bağlamında da endişe, ilgi duyma, korku, memnuniyet, merak, öfke ve şaşkınlık gibi öne çıkan duygular tanımlanmıştır. Bulgular, incelenen 668 haberin yarıdan fazlasında siyasetin konu edildiğini en fazla güncellik, önemlilik, ilgi düzeyi ve ilginçlik şeklindeki haber değerlerine önem verildiğini, baskın duygu tonunun nötr (%45,8) ve negatif (%41,6) olduğunu, buna karşılık pozitif tonlu haberlerin %12,6 düzeyinde kaldığını ortaya koymaktadır. Belirlenebilen en baskın üç duygu türü ise üzüntü, öfke/kızgınlık ve meraktır. Sonuç olarak iyi haberlerin kökü haberler kadar değerli olmadıkları söylenebilmektedir. Bu da eşik bekçilerine daha dengeli bir dağılıma özen göstermeleri yolunda tavsiyede bulunulmasını gerekli kılmaktadırThis study examined the mood of news on the basis of driving question "Do only the bad news have value or are the good news valuable, too?" In this study - which is the first of its kind on this topic in Turkish - front pages of five widely distributed newspapers (Hurriyet, Posta, Sabah, Sozcu and Zaman) have been analyzed using content analysis for a certain period of time (between January 6 and April 13, 2014) with regard to tone and type of feeling. The analysis identified positive, negative and neutral feeling associations with regards to feeling tone and worry, interest, fear, appreciation, curiosity, anger and surprise similar feelings with regards to types of feeling. Considering 668 news reports examined, more than half of the news reports are political news and the most frequent news values are timeliness, importance, level of interest and interestingness. As regards to feeling tone of the news, 45.8 % of news reports are coded as having neutral feeling tone while negative news are 41.6 % and positive news are only 12.6 %. The most common types of feelings are sadness, anger/fury and curiosity. The findings imply that gatekeepers must pay attention to balance of good and bad new