Understanding pluripotency by global and targeted quantification of chromatin-associated proteins

The maintenance of pluripotency in embryonic stem cells (ESC) is regulated by a network of chromatin-associated proteins coordinated by three master transcription factors Oct4, Sox2 and Nanog. To understand how different states of pluripotency are established, I developed three methods for studying chromatin-associated proteins globally, protein-targeted and locus-targeted in mouse ESC. Firstly, to study chromatin protein composition in a global manner, for the first time I developed in-vitro enzymatic labeling of chromatin by biotinylated nucleotides using Terminal deoxynucleotidyltransferase (TdT). As a result, more than 5000 proteins were significantly enriched in mouse ESCs in comparison to the negative control omitting the biotinylation step. In addition to the canonical chromatin-binding proteins, SICAP suggests chromatin association of some unexpected proteins such as Fgf4, which is a growth factor. This observation was further verified by immuno-staining. Secondly, I combined SICAP with chromatin immuno-precipitation (ChIP-SICAP) to identify proteins that interact with a target protein specifically on chromatin. Using endogenous Oct4, Sox2 and Nanog (OSN) as the targets of ChIP-SICAP, I identified about 400 proteins, as the overlap of the three assays. These 400 proteins include a large number of established interaction partners of the target proteins known to participate in the core pluripotency network (e.g. Rex1, Prdm14, Tcf3, Sall4, Esrrb, Tbx3, Stat3 etc). To reveal the co-localization sites with OSN, I selected Trim24. Interestingly, using ChIP-seq it turned out that Trim24 co-localizes with OSN on many super-enhancers of pluripotency. Thirdly, I developed a method to identify proteins bound to the Nanog promoter using biotinylated oligonucleotides. The specificity of the method, called targeted isolation of genomic regions (TIGR), was validated using qPCR and high-throughput sequencing. Hence, several proteins have been identified that are known to bind to, and regulate transcriptional activity of Nanog. Comparing the meta-stable and the ground-state of pluripotency, TIGR identified several nucleoporins that associate with the Nanog promoter preferentially in the ground-state of pluripotency. Using ChIP-qPCR I could validate the association of Nup98 to the Nanog promoter. Taken together, the data generated by the aforementioned methods expand the circuitry of pluripotency, and shed a new light on the differences between the ground-state and meta-stable state of pluripotency. Additionally, the newly developed methods are highly generalizable and independent of cell culture or genetic engineering so that they can be used for studying diverse biological systems

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.journal articl

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration

Enrichment of A Rare Subpopulation of miR-302-Expressing Glioma Cells by Serum Deprivation

Objective: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. Materials and Methods: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. Results: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Conclusion: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues

Effect of medicinal smokes on some nosocomial infection factors

زمینه و هدف: مقاومت دارویی امروزه یکی از مشکلات اساسی در درمان عفونت هاست و مقابله با این پدیده و استفاده از جایگزین های طبیعی اهمیت زیادی دارد. در این میان استفاده از دودهای طبی در درمان بسیاری از بیماری ها و از جمله بیماری های میکروبی و عفونی در ایران و بخصوص در استان چهارمحال و بختیاری از قدیم الایام رواج داشته که از جمله این دودها، دود حاصل از سوزاندن اسپند و سرگین می باشد. این مطالعه با هدف مقایسه خواص ضد میکروبی دود حاصل از دانه های اسپند و سرگین بر روی سودوموناس آئروژینوزا و استافیلوکوک اورئوس طراحی و انجام شد. روش بررسی: در این مطالعه تجربی آزمایشگاهی، گروه های دود اسپند و سرگین الاغ ماده به عنوان مورد و گروه های آنتی بیوتیک و دود کاه به عنوان شاهد در نظر گرفته شدند. سوش های استاندارد سودوموناس آئروژینوزا و استافیلوکوک اورئوس در محیط کشت های مناسب (مولر هینتون آگار، EMB و بلادآگار) کشت داده شدند. دیسک های بلانک آنتی بیوگرام دود دهی شده با سرگین، اسپند و کاه به طور جداگانه با کمک پنس استریل بر روی سطح پلیت های حاوی این سوش ها قرار داده شده و پس از 48 ساعت انکوباسیون در 37 درجه سانتی گراد از نظر وجود یا عدم وجود هاله رشد بررسی گردید. عمل دود دهی در محفظه های طراحی شده هر 20 دقیقه یکبار و حداکثر تا 24 بار تکرارگردید. یافته ها: هر دو گونه نسبت به دود کاه مقاوم بودند. استافیلوکوک اورئوس نسبت به دود سرگین و اسپند و سودوموناس آئروژینوزا نسبت به سرگین حساس بودند. در گروه شاهد نیز استافیلوکوک اورئوس نسبت به کلوگزاسیلین مقاوم بوده و سودوموناس آئروژینوزا تنها نسبت به اریترومایسین و سیپروفلوکساسین حساس بود. با افزایش زمان دود دهی، قطر هاله عدم رشد در موارد حساس بزرگتر شده و به عبارت دیگر اثرات ضد میکروبی دود افزایش می یافت. نتیجه گیری: با توجه به یافته های پژوهش و اثرات ضدمیکروبی دود سرگین بر روی میکروب های بسیار مقاومی مانند استافیلوکوک اورئوس و سودوموناس آئروژینوزا، لزوم انجام تحقیقات وسیع تر در مورد مواد موثره و خواص دود سرگین مطرح می گردد

Cell-specific Bioorthogonal Tagging of Glycoproteins

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function

The global burden of cancer attributable to risk factors, 2010-19 : a systematic analysis for the Global Burden of Disease Study 2019

Background Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. Methods The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk-outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. Findings Globally, in 2019, the risk factors included in this analysis accounted for 4.45 million (95% uncertainty interval 4.01-4.94) deaths and 105 million (95.0-116) DALYs for both sexes combined, representing 44.4% (41.3-48.4) of all cancer deaths and 42.0% (39.1-45.6) of all DALYs. There were 2.88 million (2.60-3.18) risk-attributable cancer deaths in males (50.6% [47.8-54.1] of all male cancer deaths) and 1.58 million (1.36-1.84) risk-attributable cancer deaths in females (36.3% [32.5-41.3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20.4% (12.6-28.4) and DALYs by 16.8% (8.8-25.0), with the greatest percentage increase in metabolic risks (34.7% [27.9-42.8] and 33.3% [25.8-42.0]). Interpretation The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.Peer reviewe

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

A Plausible Anti-Apoptotic Role of Up-Regulated OCT4B1 in Bladder Tumors

PURPOSE: To investigate and compare the expression of OCT4B1 between tumor and non-tumor bladder tissues. MATERIALS AND METHODS: We investigated the expression of OCT4B1 in 30 tumor and non-tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reaction approach and by carefully designing primers and probes specific for the amplification of the variant. RESULTS: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells. CONCLUSION: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings

Protease‐resistant streptavidin for interaction proteomics

Abstract Streptavidin‐mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin‐derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100‐fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow