Molecular Genetic and Epigenetic Characterization of Tumor Suppressor Rbl2/p130 in Human Breast Cancer Tumor suppressor retinoblastoma like protein-2 (Rbl2/p130) regulate G1/S transition through its binding with basal transcription factors (E2F4 and E2F5). Activities of Rbl2 protein are regulated through phosphorylation and acetylation in a cell cycle dependent manner. Deregulated expression of Rbl2 and E2F4/5 may lead to neoplastic alterations. The involvement of Rbl2 and E2F4/5 genes in the development and progression of breast cancer among local Pakistani population was investigated. Various genetic and epigenetic mechanisms were probed to explore their potential involvement in gene expressions and tumor growth formation. Rbl2/p130 gene was comprehensively screened for mutations in all exons through single strand conformational polymorphism (SSCP) analysis followed by direct sequencing of suspected samples. Total 200 breast cancer women with mean age of ~45 years were participated in this study. Out of which 126 were at stage I (SI), 40 at stage II (SII) and 34 at stage III (SIII) of breast cancer. Moreover, 138 patients were from invasive ductal carcinoma (IDC), 48 from Invasive lobular carcinoma (ILC) and 14 from ductal carcinoma in situ (DCIS). Among these, 79 mutations in blood and 87 mutations in tissues were detected. Out of these, 12 mutations were previously reported whereas the remaining were found novel. Rbl2 gene was found heavily mutated in exons 19-22 encompassing pocket and carboxyl terminal domain of protein. Among total 28 mutations in exon-21, an A>G transition in codon 1083 that codes for an acetylatable lysine present in the bipartite nuclear localization signals (NLS) 1081PSKRLR1086, was observed. This lysine was mutated to an arginine in 102 (51%) blood and 118 (59%) tissue samples, which suggests that impairment in acetylation status of Rbl2 proteins as well as alterations in nuclear localization might be a key event in tumor initiation and progression. Rbl2/p130 gene was found down regulated while E2F4 and E2F5 genes were upregulated in all study cohorts devised on the basis of age, disease severity and tissue origin. Deregulated expression of these genes was investigated in connection to xi their promoter methylation status using methylation specific PCR (MSP) of bisulphite converted genomic DNA. Rbl2 promoter was found hyper-methylated while promoters of E2F4/5 genes were hypo-methylated in patient‟s samples, suggesting a potential role of promoter methylation in breast carcinogenesis. The CpG positions (-1, +3, +15 and +75) in Rbl2 promoter were specifically found methylated, however positions (-8, +9, +21, +28, +47 and +52) were un-methylated. Rbl2 promoter methylation was found positively associated with various disease stages. Dynamic positioning of nucleosomes is pivotal in determining levels of gene expression especially on or around transcription start site (TSS) of a gene. Nucleosome position around TSS of Rbl2/p130 was determined using micrococcal nuclease (MNAse) digestion assay and ChIP-PCR. Region between -145 -to- +140 around TSS was scanned for 6 positions (P1=-137 -to- +69; P2=-90 -to- +69; P3=-33 -to- +140; RX=-137 -to- +7; F1=-54 -to- +99; F2=-11 -to- +140). About 66% breast tumors and 26.6% ANCT samples were positive for P1. The difference was found statistically significant (p < 0.0001) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~9 times more probable in tumor samples, whereas P2, P3, RX, F1 and F2 were not that suitable for nucleosome positioning. These results indicate that nucleosomes are present slightly downstream of TSS under normal physiological circumstances, that upon breast carcinogenesis slides 55 bases upstream of the TSS, aligning position +1 at the center of nucleosome, hence hindering access to the transcriptional machinery. Breast tumor samples were found methylated at H3K4 to a lesser extent and these modifications were not found associated with Rbl2 proteins, although in precipitates where Rbl2 protein as enriched small fractions of K4 methylation were observed. Conversely, H3K9 tri-methylation as well as Suv39 H2 methyltransferase were found positively associated with Rbl2 proteins, which suggests a putative role of Rbl2 proteins in creating a pre-apoptotic environment in tumor tissues. Our results, highlights a cumulative impact of alterations in genetic and epigenetic mechanisms that culminate in neoplastic transformation. It is obvious from these findings that a cell cycle regulator like Rbl2 can act as a transducer molecule that provide platform for various other molecules to manipulate physiological environment of the cell. With this, we hypothesize that activities of Rbl2/p130 proteins are not limited to tumor suppressor only, rather it has the potential to regulate other physiological and biochemical activities of the cell.
حکیم محمد صادق سیالکوٹی ؒکی اردو سیرت ’’جمالِ مصطفیﷺ‘‘کا تعارف وجائزہ
ڈاکٹر محمد انصر جاوید گھمن
اردو سیرت کی کتاب "جمالِ مصطفیﷺ"مولاناحکیم محمدصاد ق سیالکوٹی رحمہ اللہ کی ایک مشہور تصنیف ہے ۔ یہ کتاب نعمانی کتب خانہ حق سٹریٹ اردوبازار، لاہورسے چھپی ہے ۔اس کتاب کا سرورق بہت زیادہ خوبصورتی سے مزین کیاگیاہے، جس کی ابتدااللہ تعالیٰ کے ارشادِ پاک "اور ہم نے آپﷺ کو سب جہانوں کے لیے رحمت بناکربھیجاہے۔"کے ساتھ درج ذیل شعر سے کی گئی ہے:
سیلاب رنگ و نور طلوع سحر میں ہے تابندہ کہکشاں تیر گر سفر میں ہے
حکیم صاحب اپنی کتاب کا تعارف ان الفاظ میں کرواتے ہیں:
"اس کتاب میں آرامِ جاں، سکون ایمان، کاشفِ سرکن فکان، دلدارِ مسکان، غم خوارِ عاصیاں، ممدوحِ قُدْسَیاں، سرخیلِ نوریاں، رحمتِ عالمیاں، سید الکونین، سید الثقلین، حبیبِ خدا، اشرف الانبیاء، شافعِ روزِ جزاء، حضرت محمد مصطفیﷺکا حسن صحیح معنوں میں اپنی راعنائیوں کے ساتھ جلوہ بار ہے۔"
مولانا حکیم محمد صادق سیالکوٹی رحمہ اللہ نے حسنِ صورت بھی بیان کیاہے اور حسنِ سیرت بھی بیان کیاہے۔ حکیم صاحب نے مندرجہ بالا القابات جو بیان کیے ہیں در حقیقت یہ رسول اللہﷺ کی سیرت کے مختلف پہلو ہیں جو حکیم صاحب نے بڑے حکیمانہ انداز میں بیان فرمائے ہیں۔ پھرحکیم صاحب نبیﷺ کی سیرت کو اشعارکی صورت میں بیان کرتےہیں اور آفتابِ نبوت میں آفتابِ کردار کو نمایاں کرتے ہیں۔جن میں سے چند حسب ذیل ہیں:
Tujuan Penelitian yaitu mendesain Villa Terapung di Pulau Kera yang mengambil unsur budaya lokal dengan pendekatan Arsitektur Vernakuler agar dapat memenuhi berbagai aspek seperti dimanfaatkan untuk tujuan penelitian, ilmu pengetahuan, pendidikan, menunjang budidaya, pariwisata dan rekreasi alam. Jenis penelitian yang digunakan pada perencanan dan perancangan vila terapung di Kawasan Pulau Kera Propinsi NusaTenggara Timur adalah penelitian kualitatif deskriptif yang mampu mendeskripsikan serta menganalisa kegiatan atau data yang bersifat alamiah. Variabel penelitian Perencanaan dan Perancangan Villa Terapung Di Pulau Kera Dengan Pendekatan Arsitektur Vernakular Timor Nusa Tenggara Timur terdiri dari Parameter dan Indikator yang telah ditentukan berdasarkan pada kajian yang dilakukan. Teknik pengumpulan data penelitian dilakukan untuk memperoleh data yang relevan maka beberapa teknik pengumpulan data yang dilakukan yaitu observasi, studi literatur, studi banding. Teknik analisis data yang digunakan untuk adalah analisis kualitatif model interaktif yang diajukan oleh Milles dan Huberman. Hasil penelitian ini diharapkan dapat membantu meningkatkan daya tarik wisatawan terhadap Propinsi Nusa Tenggara Timur, Kabupaten Kupang khususnya Kawasan Pulau Kera dan menjadi bahan masukan dan pertimbangan bagi pihak-pihak yang memerlukan.
A new facile and effortless method has been introduced for the fabrication of l-cysteine capped nickel nanoparticles (Ni NPs) in an organic solvent (ethylene glycol) under microwave irradiation with the aim to employ such nanoparticles as catalysts in remediation/reduction of pollutants for environmental or analytical purposes. Appropriate amounts of NaOH, Na 2 CO 3 and l-cysteine were significant for the formation of Ni NPs. The surface interaction of l-cysteine with Ni NPs was monitored by UV-Vis spectrometry and Fourier transform infrared (FTIR) spectroscopy while size and shape of as-synthesized Ni NPs were judged by transmission electron microscopy (TEM). These studies confirmed the interaction of biomolecules with the surface of Ni NPs via the -S- linkage to form spherical Ni NPs. The Ni NPs were recovered and reused four times for the reduction of fresh 4-nitrophenol (4-NPh) with 100-98% efficiency that exhibit negligible catalytic poisoning with excellent economic output. Further these Ni NPs were also used to investigate their catalytic efficiency to reduce hexavalent chromium i.e. Cr(VI) to trivalent chromium i.e. Cr(III) in aqueous system. We observed complete reduction of Cr(VI) in only five minutes by the use of 0.5 mg quantity of l-cysteine derived Ni NPs as catalysts. Synthesis of nickel nanowires (Ni NWs) by a simple chemical approach and their use as highly active and recyclable catalysts for conversion of isopropyl alcohol (IPA) to acetone by the transfer hydrogenation reaction was carried out in an aqueous medium. The Ni NWs were obtained by reducing Ni 2+ ions with hydrazine xiiimonohydrate as the reducing agent and capped by l-methionine (amino acid) molecules. The basic pH, high concentration of reducing agent and higher molar ratio of Ni/l-methionine were necessary for synthesis of Ni NWs. UV-Vis spectroscopy, FTIR spectroscopy and scanning electron microscopy (SEM) were used for characterization of Ni NWs. The catalytic test was performed in the presence of the rich hydrogen source NaBH 4 , which helps in the conversion of IPA to acetone. The effects of concentration of IPA, concentration of NaBH 4 , reaction time and amount of Ni NWs were monitored to investigate the efficiency of catalysts. The study also describes synthesis of highly active and ordered structures of nickel nanocatalysts by a green and economically viable approach. The study reveals efficient catalytic activity for the degradation of a number of toxic and lethal organic dyes such as Eosin-B (EB), Rose bengal (RB), Ereochrome black-T (ECBT) and Methylene blue (MB). The stable colloidal dispersions of ordered nickel nanostructures (Ni NSs) arrays were prepared via a modified hydrazine reduction route with unique and controllable morphologies in a lyotropic liquid crystalline medium using a nonionic surfactant (Triton X-100). Characterization studies and optimization of various parameters for preparation of these nanoscale nickel structures, surface binding interactions, size and morphologies of the fabricated Ni NSs were carried out using UV-Vis spectroscopy, FTIR spectroscopy, X-ray diffraction (XRD) and SEM analysis. We introduced a simple and primitive seed-mediated growth approach for fabrication of well dispersed l-threonine derived nickel nanoparticles (Ni NPs) using xivnickel chloride as the precursor in an aqueous medium via a modified borohydride reduction method. L-threonine molecules served to tune the nanoscale composites. Appropriate amounts of NaOH/HCl were added to adjust the pH range of the solution to the range 2.6-11.3, however basic pH 8.5 was found to favor the formation of spherical shapes and achieve well dispersed Ni NPs as shown in TEM micrographs. Freshly prepared Ni NPs covered mean nanoscale dimensions of 5.06 nm for bigger nanospheres and 1.68 nm of smaller NPs, determined from atomic force microscopy (AFM) and TEM data. Microscopy studies reveal that bigger Ni NPs consist of small individual nano-composites with fine crystal structures. The nanoparticles thus prepared were exploited to check their catalytic activity. Congo red (CR) dye was used as a model reagent to monitor catalytic degradation. Experiments highlighted no or very little reduction of dye in the absence of Ni NPs. Conversely the addition of only 0.2 mg of nano-catalysts (Ni NPs) produced 100% conversion/degradation efficiency within a fraction of a minute; the present study also showed recovery and reuse of the same catalysts which performed with no loss of activity even after several cycles of reuse.