The Role Of Chemerin In Helicobacter Pylori Induced Gastric Pathogenesis

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2016Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2016Helicobacter pylori (H. pylori), insan gastik epitelinde kolonize olan, gram negatif, spiral-şekilli, mikroaerofilik, kamçılı bir bakteridir. Bu bakteri, kronik gastrit, gastrik adenokarsinom, peptik ülserler ve gastrik lenfomalar gibi gastrik hastalıkların gelişimiyle olan bağlantısından dolayı Tip I karsinojen olarak sınıflandırılmaktadır. Dünya nüfusunun %50’si H. pylori ile enfekte olduğu halde, enfeksiyonların büyük bir çoğunluğu, patojenin güçlü doğal ve edinsel bağışıklık cevaplarından kaçınabilme yeteneğinden ötürü, semptomlara veya gastrointestinal hastalıklara yol açmamaktadır. Bakteriler nadiren tamamen yok edilir, kolonizasyon genel olarak ömür boyu sürer, ve enfeksiyon hem doğal, hem de edinsel bağışıklı cevaplarını içerir. Kemerin (chemerin), katelisidin/sistatin ailesinden bir proteindir ve G proteinine bağlı reseptör CMKLR1 ( kemokin benzeri reseptör 1)’in ana ligandıdır. Epitel hücreler, karaciğer, dalak, lenf düğümleri vb. dahil birçok dokuda mevcuttur. Bu şekilde, makrofajlar, plazmositoid dendritik hücreler ve NK hücreleri gibi CMKLR1 ifade eden lökosit popülasyonlarının enflamasyon bölgesine kemotaksisini başlatır. Plazmada her yerde, sürekli, nanomolar konsantrasyonlarda 143 aa prokemerin olarak bulunan protein, serin ve sistein proteazlarının yanı sıra karboksipeptidazlar tarafından proteolitik kesime uğrayarak, farklı formlar halinde aktive olur. Kemerin, ortamdaki uyarıcıya ve incelenen hastalığa göre, hem pro-enflamatuvar, hem de anti-enflamatuvar cevaplara sebep olabilmektedir. Eritematöz, romatoid artrit, psöriyazis (sedef hastalığı), Crohn hastalığı gibi enflamatuvar hastalıklarda, birçok kobay ve insan çalışmalarında da gözlemlendiği gibi, enflamasyon gelişiminde artan kemerin seviyelerinin hastalıklarla ilişkisi gösterilmiştir. CMKLR1 nakavt farelerde, EAE’de merkezi sinir sistemi (MSS) enflamasyonunun ve sigara içmeye bağlı kronik obstrüktif akciğer hastalığı (KOAH) modelinde pulmoner enflamasyonun azaldığı gösterilmiştir. Bu şekilde, kemerinin pro-enflamatuvar rolü ortaya konmuştur. Fakat, LPS ile akciğer hasarlı modelde ve pro-enflamatuvar markerlerin ifadesi azaltılmış akut viral pönomi modelinde, CMKLR1 nakavt farelerde ve kemerinin baskılandığı durumlarda, kemerinin anti-enflamatuvar rolü de gözlemlenmiştir. Makrofajlar, dışarıdan gelen uyarılara adapte olmak için yerel enflamatuvar durumlarını şekillendirebilen, uyarana ve yerel mikroçevreye göre polarize olabilen, plastik ve heterojenik bir hücre grubudur. Makrofajlar, klasik yollardan aktive olan- M1 tipi-makrofajlara ve alternatif yollardan aktive olan –M2 tipi-makrofaj alt gruplarına polarize olabilmektedir. M1 tipi makrofajlar, granülosit makrofaj uyarıcı faktör (GM-CSF), lipopolisakkarit (LPS) ve IFN-γ tarafından üretilen doğal bağışıklık cevapta görülen, pro-enflamatuvar efektör hücrelerdir ve TNF-α, IL-1β, IL-6, IL-12/ IL-23 gibi pro-enflamatuvar sitokinler salgılarlar. M1 tipi makrofajların antimikrobiyal fonksiyonları, L-argininden nitrik oksit ve çok miktarda NO üreten, indüklenebilir niktik oksit sentazın (iNOS) ifadesinin artmasına bağlıdır. M2 tipi makrofajlar ise, IL-4/IL-13 veya M-CSF ile üretilen, immün baskılama ve doku tamirinde görev alan, IL-10 ve IL-1RA salgılayıp, düşük seviyelerde IL-12/ IL-23 ifade eden hücre gruplarıdır. Bu hücreler, NO üretmezler. M2 tipi makrofajlar, sitokin ifade profillerine göre kendi içlerinde M2a, M2b, ve M2c olmak üzere alt gruplara ayrılırlar. H. pylori'nin makrofajlardaki etkileri ile ilgili birkaç çalışma mevcuttur. İlk çalışmalarda, H. pylori'nin makrofajlarda IL-6, IL-8, TNF-α, IL-1-β gibi pro-enflamatuvar sitokinlerle birlikte indüklenebilir NO sentetaz (iNOS) enziminin ekspresyonunu indüklediği gösterilmiştir. H. pylori pozitif bireylerden alınan gastrik biyopsi örneklerinde alternatif olarak aktive olan M2 makrofajlar gözlenmiştir. Ayrıca, H. pylori enfeksiyonu durumunda insan monositleri, IL-1β, IL-6, IL-10 ve IL-12p40 (kısmen IL-23 olarak salgılanan) salgılarken IL-12p70 sitokini salgılamamıştır. Bununla beraber, doğal immün sistem hücreleri tarafından salgılanan sitokinler, makrofajların ve dendritik hücrelerin antijen sunması ve mikroçevredeki değişimler, H. pylori'ye karşı sonradan kazanılmış immün cevabı aktive eder. İnsanlarda H. pylori'ye karşı makrofaj aracılı pro-enflamatuvar Th1 ve Th17 immün cevap hakimdir. Kemerin/kemerin reseptör sinyalleşmesi, artan integrin kümelenmesi yoluyla (VLA-5 ve VLA-4) kemotaksise ve makrofajların hücre dışı matriks proteinlerine ve endotelyal hücrelere adhezyonuna aracılık eder. Farelerde kemerinin ChemR23-bağımlı anti-enflamatuvar ve koruyucu etkileri gözlenmiştir. İnsanlarda ise LPS, TNF-a and IFN-g uyarımı, kronik ve sistemik enflamasyonla ilişkili olan monositlerde ve makrofajlarda ChemR23 ekspresyonunu artırmıştır. Ayrıca, makrofajlar farklı kemerin izoformlarına cevap olarak pro- ve anti-enflamatuvar yanıt oluşturabilir. Kemerinin DSS ile indüklenen kolit modelinde in vivo and in vitro koşullarda artan pro-enflamatuvar sitokin profilinin yardımıyla M2 makrofaj polarizasyonunu baskıladığı gözlenmiştir. Bir başka çalışmada M1 makrofajlarda belirgin IL-10 ekspresyonu ile birlikte artan kemerin ekspresyonun gözlenmesi kemerinin M1 makrofajlardaki enflamatuvar etkisini açıklamaktadır. Belirgin kemerin ekspresyonunun, makrofaj öncüllerinin 10-24 haftalık fetal bağırsakta sürekli olarak toplanmasında görev alan ve yeni doğanlarda konak savunma mekanizmasını ve mukozal bağışıklığı artıran intestinal epitel hücreler (IECler) tarafından gerçekleştirildiği gösterilmiştir. Fakat, olgun IEC'lerin aksine sadece fetal IEC'ler kemerin üretir. Makrofajlarda H. pylori'nin etkilerini araştıran birçok çalışma değerli görüşler ortaya koymuştur. Ayrıca, kemerin ve makrofajlar arasındaki ilişkiyi araştıran çalışmalar da mevcuttur. Fakat, H. pylori ile indüklenen patogenezde kemerinin makrofaj polarizasyonuna etkisini araştıran çalışmalar henüz yoktur. H. pylori'nin insanda potansiyel olarak M1 ve M2 tipi makrofajları indükleyebildiği bilinirken; kemerinin bu ilişkiyi nasıl etkileyebildiği bilinmemektedir. Dolayısıyla bu çalışmada, kemerin ve H. pylori'nin monositik hücre hattı olan THP-1 hücreleri üzerindeki etki ve bu hücrelerin sitokin profillerine göre polarizasyon durumları arasındaki farklılıklar araştırılmıştır. Literatürden epiteliyal hücrelerin kemerin salgıladıkları bilinmektedir. Kemerinin gastrik patolojideki etkisini gözlemlemek için, ilk olarak 3 gastrik epiteliyal hücre hattında (KATO III, AGS ve MKN45) kemerin ekspresyonu araştırılmıştır. Klasik PCR sonuçları kemerinin bu hücreler tarafından eksprese edildiğini göstermiştir. Daha sonra, H. pylori'nin bu hücre hatlarında kemerin ekspresyonuna etkisi incelenmiştir. Bunu gerçekleştirmek için, KATO III, AGS ve MKN45 hücreleri yabani tip G27 suş sonikatı ve bu suşun ΔcagA mutant sonikatı ile olmak üzere 2 ayrı H. pylori sonikatı ile muamele edilmiştir. H. pylori virulans faktörü CagA'nın (sitotoksin-ilişkili gen-A) gastrik kötü huylu tümörlerle ilişkisi gösterildiğinden, bu çalışma için CagA virulans faktörü seçilmiştir. Kemerin ekspresyonu, KATO III and AGS hücre hatlarında yabani tip G27 ve ΔcagA mutant sonikatları ile 6 ve 24 saat muamele edildiğinde önemli ölçüde artmıştır. Bundan sonra ise kemerin ifadesi azalmıştır. Ancak MKN45 hücre hattında anlamlı bir ifade gözlenmemiştir. Bu nedenle diğer deneylere KATO III and AGS hücre hatları ile devam edilmiştir. Daha sonra, H. pylori muamelesi ile mRNA ekspresyonunun yanı sıra protein ifadesinin de artıp artmadığı kontrol edilmek istenmiştir. Tekrarlı western blot optimizasyon deneyleri sonucunda, herhangi bir hücre hattının muamele edilen ve edilmeyen gruplarında kemerin bandı görülmemiştir. Bu sonuçların nedeninin protenin olmaması veya antikorun proteine yetersiz bağlanması olduğunun kararlaştırılması amacıyla, insan kemerini klonlanmış ve HEK293T hücre hattına transfekte edilmiştir. Tüm örnekler kullanılarak western blot gerçekleştirilmiştir ve kemerin sadece insan kemerin genini içeren plasmid ile transfekte edilen HEK293T nin tüm hücre ekstraktında tespit edilmiştir. Sonuç olarak, H. pylori muamelesinden sonra kemerin artışı mRNA seviyesinde tespit edilirken, protein seviyesinde görülememiştir. Daha sonra, H. pylori pozitif akut gastrit ve kronik ülser hastalarında da kemerin ekspresyonunu incelenmiştir ve patoloji göstermeyen enfekte olmamış hastalarla karşılaştırılmıştır. Real-time PCR deneyler, ülser hastalarında kemerinin anlamlı ölçüde fazla bulunduğunu göstermiştir. Gastrit hastalarının numunelerinde, kemerin mRNA ifadesinin, kontrol grubuyla anlamlı farklılıklar göstermediğine dikkat edilmelidir. Bu veriler, akut gastrit ve kronik ülserli H. pylori pozitif hastaların ve H. pylori negatif sağlıklı bireylerin gastrik dokuları üzerinde yürütülen immünohistokimya analizlerinden elde edilen sonuçlar tarafından desteklenmektedir. Daha sonra, kemerinin makrofajların polarizasyon durumu üzerindeki etkilerini incelenmiştir. Bu amaçla, monositik bir hücre hattı olan THP-1 kullanılmış ve monositlerin makrofajlara farklılaşmasını indüklemek için PMA (phorbol 12-myristate 13-acetate) ile muamele edilmiştir. Farklılaşan makrofajların M1 and M2 alt tiplerine polarize olması için kemerin varlığında ve yokluğunda sırasıyla LPS ve rekombinant IL-4 gibi farklı uyaranlar eklenmiştir. Monositlerin makrofajlara farklılaştığını göstermek için makrofajlara özgü belirteçlerin varlığına bakılmıştır. Aynı zamanda, klasik PZR ile sitokin seviyeleri ölçülmüştür. IL-1β ve IL-6 pro-enflamatuvar sitokinleri M1 tipi makrofajların belirteci olarak kullanılmıştır. Her ikisinin de LPS ile muamele edilen örneklerde anlamlı ölçüde arttığı gözlemlenmiştir. Kemerin, LPS ile uygulandığı zaman, yalnızca ilk setteki LPS ile muamele edilen örnekler ile karşılaştırıldığında IL-1β’nın anlamlı ölçüde yüksek ekspresyonu ve IL-6’nın düşük ekspresyonu gözlenmiştir. Başlangıçta, kemerinin M1 tipi makrofajlara polarizasyonunda bir etkisinin olabileceği düşünülmüştür, ancak tekrarlanan üç deney sadece LPS ile veya kemerin ile muamele edilen örneklerde IL-1β ve IL6 ekspresyonunda bir değişim olmadığını göstermiştir. Bu da, kemerinin M1 tipi hücrelere polarizasyona bir etkisinin olma olasılığını ortadan kaldırmıştır. M2 polarizasyonunu tespit etmek için IL-10 ekspresyonu değerlendirilmiştir. Kontrol grubu ile karşılaştırıldığında, IL-4 muamele edilen gruplarda IL-10’un ekspresyonu artış göstermiştir ve kemerinin IL-4 ile birlikte uygulanması bu etkiyi ortadan kaldırmıştır. Bu da, Lin vd. (2014) tarafından yapılan kemerinin M2 ile ilişkili genlerin ekspresyonunu azalttığı çalışmayı desteklemektedir. Sonuç olarak, yapılan çalışmada, H. pylori’nin makrofajlar üzerindeki etkisini değiştirip değiştirmediğini kontrol etmek için, kemerin varlığında ve yokluğunda, H. pylori’nin makrofaj polarizasyonu üzerideki etkisi incelenmiştir. THP-1 makrofajları kemerinin varlığı ve yokluğunda yabani tip G27 ve ΔcagA mutant sonikatları ile 24 saat muamele edilmiştir. Sonikat muamelesi makrofajları yüksek IL-1β ve IL6 üreten, pro-enflamatuvar profilindeki M1 alt tipine polarize ederken IL-10’u da arttırmıştır. M1 makrofajlarının bakteriyal uyarımına cevap olarak IL-10 ürettiği bilinmektedir, bu durum ayrıca laboratuvarımızdaki diğer bir çalışmada H. felis ile uyarılan bir fare modelinde de gözlenmiştir (yayımlanmamış veri). Ancak, H. pylori sonikatlar ile birlikte kemerin muamele edildiğinde IL-1β, IL6 ve IL-10 ekspresyonunda hiçbir değişiklik gözlenmemiştir. Bu nedenle, sonuçlarımız, H. pylori ile indüklenen M1 makrofaj polarizasyonunda kemerinin bir rolü olmayabileceğini önermektedir, ancak M2 polarizasyonunun kemerin muamelesi ile H. pylori’den bağımsız şekilde engellendiği gözlemlenmiştir.Helicobacter pylori (H. pylori) is a gram negative, spiral –shaped, microaerophilic, flagellated bacterium which colonize in the human gastric epithelium and is classified as type I carcinogen for association with the development of gastric malignancies such as chronic gastritis, gastric adenocarcinoma, peptic ulcers and gastric lymphoma. Although 50% of the world’s population is infected with Helicobacter pylori, the majority of infections do not lead to symptoms or gastrointestinal disease due to the pathogen’s unique ability to avoid strong innate and adaptive immune responses. The bacteria are rarely eliminated, colonization usually persists throughout life, and infection involves both innate and adaptive immune responses. Chemerin is a protein from cathelicidine/cystatin family and the major ligand of G protein coupled receptor CMKLR1 (chemokine like receptor 1). It is present in various tissues including epithelial cells, liver, spleen, lymph nodes etc. and promotes chemotaxis of leukocyte populations that express CMKLR1, such as macrophages, plasmacytoid dendritic cells and natural killer (NK) cells to site of inflammation. It is ubiquitously found in plasma as 143 amino acids prochemerin at nanomolar concentrations and becomes activated into various isoforms through proteolytic cleavage by a myriad of serine and cysteine proteases as well as carboxypeptidases. Chemerin promotes both pro- and anti-inflammatory immune response based on the stimuli provided and the disease being investigated. Inflammatory diseases, such as erythematosus, rheumatoid arthritis, psoriasis, Crohn’s disease showed association of elevated chemerin in inflammation development which is also observed in several mice and human studies. CMKLR1 knockout mice showed reduced CNS inflammation in EAE and pulmonary inflammation in COPD (cigarette smoke- induced chronic obstructive pulmonary disease) model, suggesting a pro-inflammatory role of chemerin. However, anti-inflammatory response was also observed when chemerin was discarded and in CMKLR1 knockout mice in LPS-induced lung injury and acute viral pneumonia with reduced expression of pro-inflammatory markers. Macrophages are plastic and heterogenic group of cells, which can polarize according to stimuli and the local microenvironment and shape the local inflammatory status to adapt to outside stimuli. Macrophages can be polarized into the classically activated -M1 type- macrophages and the alternatively activated -M2 type- macrophage subsets. M1 type macrophages are the pro-inflammatory effector cells in innate immune response produced by granulocyte macrophage stimulating factor (GM-CSF), lipopolysaccharide (LPS) and IFN-γ and secrete pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-12/ IL-23. The antimicrobial functions of M1 macrophages are linked to up-regulation of inducible nitric oxide synthase (iNOS) that generates nitric oxide from L-arginine and substantial production of NO. M2 type macrophages work in immunosuppression and tissue repair, produced by IL-4/IL-13 or M-CSF and secrete IL-10 and IL-1RA and low expression of IL-12/ IL-23. They do not produce NO. M2 macrophages can be further divided into subsets based on their cytokine expression profiles; M2a, M2b, and M2c. There are several studies regarding effects of H. pylori on macrophages. In early studies, it has been shown that H. pylori induces the expression of inducible NO synthetase (iNOS) from macrophages along with pro- inflammatory cytokines such as IL-6, IL-8, TNF-α, IL-1-β. However, alternatively activated M2 macrophages were detected in human gastric biopsy specimens from H. pylori positive individuals. Also, upon H. pylori infection, human monocytes secreted IL-1β, IL-6, IL-10, and IL-12p40 (partially secreted as IL-23), but not IL-12p70. Furthermore, cytokines secreted from innate immune cells, antigen presentation from macrophages and dendritic cells and changes in the microenvironment, also activates the adaptive immune response against H. pylori. Macrophage mediated pro-inflammatory Th1 and Th17 response was observed as the pre-dominant adaptive response against H. pylori in humans. Chemerin/chemerin receptor signaling mediates chemotaxis and adhesion of macrophages to extracellular matrix proteins and endothelial cells through increased promotion of integrins clustering (VLA-5 and VLA-4). ChemR23-dependent anti- inflammatory and protective effects of chemerin was found in mice, whereas in humans LPS, TNF-alpha and IFN-gamma stimulation increased ChemR23 expression in monocytes and macrophages which is associated with chronic and systemic inflammation. Macrophages may also exert pro- or anti-inflammatory response to different chemerin isoforms. Chemerin was observed to suppress M2 macrophage polarization with increased pro-inflammatory cytokine profile in DSS induced colitis model in vivo and in vitro. Another study found increased chemerin expression on M1 macrophages with distinct IL-10 expression, explaining the inflammatory effect of chemerin on M1 macrophages. Marked expression of chemerin was shown to be produced by intestinal epithelial cells (IECS) which is involved in the continuous recruitment of macrophage precursors to 10-24 week fetal intestine and boosted the host defence mechanism and mucosal immunity in neonates. However, only fetal IECs but not mature IECs, produces chemerin. Many studies work on the effects of H. pylori on macrophages and gave valuable insights, there are studies that investigated the inter-relation between chemerin and macrophages as well. However, no studies have yet investigated the effect of chemerin on the polarization status of macrophages in H. pylori induced pathogenesis. It is known from the literature that H. pylori could potentially induce both M1 and M2 macrophages in human but how/whether chemerin could effect this inter-relation is not known. Therefore, in this study, we investigated the effect of both chemerin and H. pylori on the monocytic cell line THP-1 and observe the differences on its polarization status according to cytokine profiles. It was known from the literature that epithelial cells secrete chemerin. In order to observe the effect of chemerin on gastric immunopathology, it’s expression was first investigated on three gastric epithelial cell lines; KATO III, AGS and MKN45. Conventional PCR results revealed expression of chemerin in different levels by these cell lines. Next we evaluated the effect of H. pylori on chemerin expression by these cell lines. In order to do that, KATO III. AGS and MKN45 cells were treated with two H. pylori sonicates, the wild type G27 strain sonicate and its ΔcagA mutant sonicate. The H. pylori virulence factor Cag A (cytotoxin- associated gene-A) was chosen since its association with development of gastric malignancies have been reported. Chemerin expression was signifantly increased in KATO III and AGS cell line when they were treated with wild type G27 and ΔcagA mutant sonicate for 6 and 24 hours. After 24 hours, the expression level of chemerin reduced. No significant expression was observed on MKN45 cell line. Therefore, the following experiments were performed on KATO III and AGS cell lines. Next, we assess if chemerin protein expression was also increased along with the mRNA expression upon H. pylori treatment. Repeated attempts of western blot optimization experiments failed to show any expected band of chemerin in any treated or untreated groups of any cell line. To clarify if the lack of detection is real or due to inefficient antibody binding to chemerin, human chemerin was cloned and transfected into HEK293T cell line. Western blot was performed using various samples from different cell lines with different treatments and chemerin-expressing HEK293T cells. Chemerin was only detected in the whole cell extract of chemerin-transfected HEK293T cell lines but not in other samples. As a conclusion, we could detect elavated chemerin in mRNA levels upon H. pylori treatment but not in protein level. Next, we investigated expression of chemerin in gastric biopsy specimens of H. pylori positive patients of acute gastritis and chronic ulcer and compared it to uninfected patients without pathology. Real-time PCR experiments revealed presence of significantly higher levels of chemerin in ulcer patients. Of note, chemerin mRNA expression in samples from patients with gastritis was not significantly different when compared to control group. These data was supported by the results obtained from immunohistochemistry analysis performed on gastric tissues of H. pylori postive patients of acute gastritis, chronic ulcer and H. pylori negative healthy individiuals. Chemerin was shown to be highly expressed in gastric tissues of ulcer patients. We investigated effects of chemerin on the polarization status of macrophages. For that, a monocytic cell line THP-1 was used and PMA (phorbol 12-myristate 13-acetate) was administered to induce differentiation of monocytes into macrophages. The differentiated macrophages were then exposed to different stimuli such as LPS and recombinant IL-4 to polarize them into M1 and M2 subtypes respectively, with or without the presence of chemerin. The presence of macrophage specific markers were used to show the differentiation of monocytes into macrophages. Also, cytokine levels were measured by conventional PCR. Two pro-inflammatory cytokines namely IL-1β and IL-6 were used as a marker for M1 macrophages. Both of them were significantly increased in LPS- treated samples. When chemerin was administered with LPS, a significantly high expression of IL-1β and low expression of IL-6 observed when compared to LPS- treated sample only in the first set. Initially it was thought chemerin might have a role in the polarization of M1 macrophages but three repeated experiments showed no difference in the expression of IL-1β and

Screening of GPCR drugs for repurposing in breast cancer

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC

Copy Move and Splicing Image Forgery Detection using CNN

The boom of digital images coupled with the development of approachable image manipulation software has made image tampering easier than ever. As a result, there is massive increase in number of forged or falsified images that represent incorrect or false information. Hence, the issue of image forgery has become a major concern and it must be addressed with appropriate solution. Throughout the years, various computer vision and deep learning solutions have emerged with a purpose to detect forgery in case of digital images. This paper presents a novel approach to detect copy move and splicing image forgery using a Convolutional Neural Network (CNN) with three different models i.e. ELA (Error Level Analysis), VGG16 and VGG19. The proposed method applies the pre-processing technique to obtain the images at a particular compression rate. These images are then utilized to train the model and further the images are classified as authentic or forged. The paper also presents the experimental results of the proposed method and performance evaluation in terms of accuracy

Left Ventricular Noncompaction Is Associated with Valvular Regurgitation and a Variety of Arrhythmias

Left ventricular noncompaction (LVNC) is a type of cardiomyopathy characterized anatomically by prominent ventricular trabeculation and deep intertrabecular recesses. The mortality associated with LVNC ranges from 5% to 47%. The etiology of LVNC is yet to be fully understood, although decades have passed since its recognition as a clinical entity globally. Furthermore, critical questions, i.e., whether LVNC represents an acquired pathology or has a congenital origin and whether the reduced contractile function in LVNC patients is a cause or consequence of noncompaction, remain to be addressed. In this study, to answer some of these questions, we analyzed the clinical features of LVNC patients. Out of 9582 subjects screened for abnormal cardiac functions, 45 exhibit the characteristics of LVNC, and 1 presents right ventricular noncompaction (RVNC). We found that 40 patients show valvular regurgitation, 39 manifest reduced systolic contractions, and 46 out of the 46 present different forms of arrhythmias that are not restricted to be caused by the noncompact myocardium. This retrospective examination of LVNC patients reveals some novel findings: LVNC is associated with regurgitation in most patients and arrhythmias in all patients. The thickness ratio of the trabecular layer to compact layer negatively correlates with fractional shortening, and reduced contractility might result from LVNC. This study adds evidence to support a congenital origin of LVNC that might benefit the diagnosis and subsequent characterization of LVNC patients

DataSheet1_Screening of GPCR drugs for repurposing in breast cancer.PDF

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

Table4_Screening of GPCR drugs for repurposing in breast cancer.XLSX

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

Table3_Screening of GPCR drugs for repurposing in breast cancer.XLSX

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

Table1_Screening of GPCR drugs for repurposing in breast cancer.XLSX

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

DataSheet3_Screening of GPCR drugs for repurposing in breast cancer.PDF

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p

Table2_Screening of GPCR drugs for repurposing in breast cancer.XLSX

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.</p