3,528 research outputs found
Energy profile and secondary structure impact shRNA efficacy
<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a cellular mechanism in which a short/small double stranded RNA induces the degradation of its sequence specific target mRNA, leading to specific gene silencing. Since its discovery, RNAi has become a powerful biological technique for gene function studies and drug discovery. The very first requirement of applying RNAi is to design functional small interfering RNA (siRNA) that can uniquely induce the degradation of the targeted mRNA. It has been shown that many functional synthetic siRNAs share some common characteristics, such as GC content limitation and free energy preferences at both terminals, etc.</p> <p>Results</p> <p>Our three-phase algorithm was developed to design siRNA on a whole-genome scale based on those identified characteristics of functional siRNA. When this algorithm was applied to design short hairpin RNA (shRNA), the validated success rate of shRNAs was over 70%, which was almost double the rate reported for TRC library. This indicates that the designs of siRNA and shRNA may share the same concerns. Further analysis of the shRNA dataset of 444 designs reveals that the high free energy states of the two terminals have the largest positive impact on the shRNA efficacy. Enforcing these energy characteristics of both terminals can further improve the shRNA design success rate to 83.1%. We also found that functional shRNAs have less probability for their 3' terminals to be involved in mRNA secondary structure formation.</p> <p>Conclusion</p> <p>Functional shRNAs prefer high free energy states at both terminals. High free energy states of the two terminals were found to be the largest positive impact factor on shRNA efficacy. In addition, the accessibility of the 3' terminal is another key factor to shRNA efficacy.</p
Computational Design of Artificial RNA Molecules For Gene Regulation
This volume provides an overview of RNA bioinformatics methodologies, including basic strategies to predict secondary and tertiary structures, and novel algorithms based on massive RNA sequencing. Interest in RNA bioinformatics has rapidly increased thanks to the recent high-throughput sequencing technologies allowing scientists to investigate complete transcriptomes at single nucleotide resolution. Adopting advanced computational technics, scientists are now able to conduct more in-depth studies and present them to you in this book. Written in the highly successful Methods of Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and equipment, step-by-step, readily reproducible bioinformatics protocols, and key tips to avoid known pitfalls.Authoritative and practical, RNA Bioinformatics seeks to aid scientists in the further study of bioinformatics and computational biology of RNA
A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.
Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases
The lncRNA landscape of breast cancer reveals a role for DSCAM-AS1 in breast cancer progression.
Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer
A gene related study with a review of current osteoporosis medications and a comparison between two kinds of BMP-2
ํ์๋
ผ๋ฌธ(๋ฐ์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :์๊ณผ๋ํ ์ํ๊ณผ,2019. 8. ์ด์ฌํ.Osteoporosis is caused by an imbalance between bone formation and bone resorption that results in low bone mass and deteriorated bone microstructure and finally elevates the risk of low-trauma fracture. For developing new therapies for managing osteoporosis, this study compromised 3 stages as follows: 1, the bone formation efficacy of recombinant human bone morphogenetic protein 2 (rhBMP-2) was investigated since bone substitute is necessary for osteoporosis patients; 2, the efficacy of currently available medications was analyzed via meta-analysis; 3, the impact brought by the mutation in Drg2 in bone homeostasis was researched. Their methods and results were as follow. In the first part, we compared the osteoinductivity of Escherichia coli rhBMP-2 (ErhBMP-2) with Chinese hamster ovary cell-derived rhBMP-2 (CrhBMP-2) with human mesenchymal stem cells and rat calvarial defect. In the second part, we systematically reviewed the effect of current osteoporosis medications on preventing secondary osteoporotic vertebral and non-vertebral fractures from randomized controlled studies and synthesized their result via meta-analysis. In the third part, we compared the transcription level of DRG2 in osteoporosis and non-osteoporosis subjects, and furtherly fabricated Drg2 knockout mice and analyzed the difference in bone phenotype of wild type and Drg2 knockout mice. At the end of this part, we investigated the possible mechanisms and signals Drg2 involved in osteoblastic differentiation. The results from the first part showed ErhBMP-2 could have comparable osteoinductivity with Chinese hamster ovary cell-derived BMP-2 while using the demineralized bone matrix as the carrier. In the second part, we found the medications could have a consistent effect on osteoporosis patients, regardless of their fracture history. And in the third part, we found osteoporosis patients had higher expression level of Drg2 and knocking out of Drg2 in mice significantly improved bone mass and mineral density even if mice were ovariectomized. The bone marrow-derived macrophage in Drg2 knockout mice showed lower osteoclastogenesis while the bone marrow mesenchymal stem cell concurrently showed higher osteoblastogenesis than wild type mice. Furtherly, inhibition of Drg2 expression in mouse MC3T3-E1 cells elevated its osteogenicity via canonical and non-canonical BMP pathway. In summary, we found the ErhBMP-2 might have the potential of being used as an anabolic agent for osteoporosis fracture; currently available medications could have a significant effect on preventing secondary osteoporotic fracture; and Drg2 as an important regulator in bone remodeling, which suggested Drg2 inhibitor could be a potential anabolic for treating osteoporosis.๊ณจ๋ค๊ณต์ฆ์ ๊ณจ๋ ๊ฐ์์ ๊ณจ ๋ฏธ์ธ๊ตฌ์กฐ ์ด์์ ์ผ๊ธฐํ๋ ์งํ์ผ๋ก ๊ณจํ์ฑ๊ณผ ๊ณจํก์๊ฐ ๋ถ๊ท ํ์ ์ํด ๋ฐ์ํ๋ฉฐ, ์ ์์ ๊ณจ์ ์ ์ํ์ ์ฆ๊ฐ์ํค๋ ์งํ์ด๋ค. ์๋ก์ด ๊ณจ๋ค๊ณต์ฆ ์น๋ฃ๋ฒ์ ๊ฐ๋ฐํ๊ธฐ ์ํ์ฌ ๋ค์ 3๋จ๊ณ์ ์ฐ๊ตฌ๋ฅผ ์งํํ์๋ค. ๊ณจ๋ค๊ณต์ฆ ํ์๋ ๊ณจ๋์ฒด์ ๊ฐ ํ์ํ๊ธฐ ๋๋ฌธ์ ์ฌ์กฐํฉ ๊ณจํ์ฑ๋จ๋ฐฑ์ง ์ 2ํ(rhBMP-2) ์ ๊ณจํ์ฑ ํจ๋ฅ ์ฐ๊ตฌ, ํ์ฌ ์ฌ์ฉ๋๋ ๊ณจ๋ค๊ณต์ฆ ์น๋ฃ์ ์ 2์ฐจ ๊ณจ์ ์๋ฐฉ ํจ๋ฅ์ ๊ดํ ๋ฉํ ๋ถ์ ์ฐ๊ตฌ์ ํจ๊ป, developmentally regulated GTP binding protein 2 (Drg2) ์ ๊ณจ ํญ์์ฑ์ ๋ฏธ์น๋ ์ํฅ์ ์ฐ๊ตฌํ์๋ค. ์ฒซ๋ฒ์งธ ์ฐ๊ตฌ์์๋, ๋์ฅ๊ท ์ ๋ ๊ณจํ์ฑ ๋จ๋ฐฑ์ง ์ 2ํ(ErhBMP-2)๊ณผ ๋๋ฌผ์ธํฌ ์ ๋ ๊ณจ ํ์ฑ ๋จ๋ฐฑ์ง ์ 2ํ(CrhBMP-2)์ ๊ณจ์ ๋์ฑ์ ์ธ๊ฐ ๊ฐ์ฝ์ค๊ธฐ์ธํฌ ๋ฐ ๋ซ๋ ๋๊ฐ๊ณจ ๊ฒฐ์๋ชจ๋ธ์์ ๋น๊ตํ์๊ณ ๋๋ฒ์งธ ์ฐ๊ตฌ์์๋, ๊ธฐ์กด ๊ณจ๋ค๊ณต์ฆ ์น๋ฃ์ ๊ฐ ๊ณจ๋ค๊ณต์ฆ์ฑ ์ฒ์ถ ๋ฐ ๋น์ฒ์ถ ๊ณจ์ ์ ์๋ฐฉํ๋ ํจ๊ณผ์ ๋ํ์ฌ ๋ฉํ๋ถ์์ ์ํํ์์ผ๋ฉฐ ์ธ๋ฒ์งธ ์ฐ๊ตฌ์์๋, ๊ณจ๋ค๊ณต์ฆ์ด ์๋ ํ์๊ตฐ๊ณผ ์ ์ ๋์กฐ๊ตฐ์ ๊ณจ์ ์ ๋ ๊ฐ์ฝ์ค๊ธฐ์ธํฌ ์์ DRG2์ ๋ฐํ์ ๋น๊ตํ์๊ณ , Drg2 ๊ฒฐ์ ๋ง์ฐ์ค๋ฅผ ์ ์ํ์ฌ ๋์กฐ๊ตฐ๊ณผ ๊ณจ ํํํ์ ์ฐจ์ด๋ฅผ ๋ถ์ํ์๋ค. ๋ํ Drg2 ๊ฐ ์กฐ๊ณจ์ธํฌ์ ๋ถํ์ ๊ด์ฌํ๋ ๊ธฐ์ ๊ณผ ์ ํธ์ ๋ฌ ์ฐ๊ตฌ๋ฅผ ์ํํ์๋ค. ์ฒซ๋ฒ์งธ ์ฐ๊ตฌ์์ ErhBMP-2๊ฐ ํํ๊ณจ๊ธฐ์ง์ ๋ด์ฒด๋ก ์ฌ์ฉํ ๋ CrhBMP-2์ ์ ์ฌํ ๊ณจ ์ ๋๋ฅ๋ ฅ์ ๊ฐ์ง ์๋ ์์์ ํ์ธํ์์ผ๋ฉฐ ๋๋ฒ์งธ ์ฐ๊ตฌ์์๋ ๊ณจ๋ค๊ณต์ฆ ํ์์ ๊ณจ์ ๋ณ๋ ฅ๊ณผ ๋ฌด๊ดํ๊ฒ ์ฝ๋ฌผ์น๋ฃ๊ฐ ์ง์์ ์ธ ์ํฅ์ด ์์ ์ ์๋ค๋ ๊ฒ์ ์ ์ ์์์ต๋๋ค. ์ธ๋ฒ์งธ ์ฐ๊ตฌ์์ ๊ณจ๋ค๊ณต์ฆ ํ์๋ Drg2 mRNA ๋ฐํ ์ ๋๊ฐ ์ ์ ๋์กฐ๊ตฐ๋ณด๋ค ๋ ๋์๊ณ , Drg2 ๊ฒฐ์๋ง์ฐ์ค๋ ๋์์ ์ ์ ์ ์ํํ์์ ๋ ๊ณจ๋ ๋ณดํธ ํจ๊ณผ๊ฐ ๊ด์ฐฐ๋์๋ค. Drg2 ๊ฒฐ์ ๋ง์ฐ์ค์์ ์ป์ ๊ณจ์์ ๋ ๋์์ธํฌ๋ฅผ ๋์กฐ๊ตฐ๊ณผ ๋น๊ตํ์ ๋, ํ๊ณจ์ธํฌ ๋ถํ๋ ฅ์ด ๋ฎ์์ผ๋ฉฐ ๊ณจ์์ ๋ ์ค๊ธฐ์ธํฌ์ ์กฐ๊ณจ์ธํฌ ๋ถํ๋ ฅ์ ๋์๋ค. ๋ํ ๋ง์ฐ์ค MC3T3-E1 ์ธํฌ์์์ Drg2 ๋ฐํ์ ์ต์ ์ํค๋ฉด ์ ์ ๋ฐ ๋น์ ์ BMP ๊ฒฝ๋ก๋ฅผ ํตํ์ฌ ์กฐ๊ณจ์ธํฌ์ ๋ถํ๊ฐ ์ฆ๊ฐํ์๋ค. ๊ฒฐ๋ก ์ ์ผ๋ก, ๋ณธ ์ฐ๊ตฌ์์๋ ErhBMP-2๊ฐ ๊ณจ๋ค๊ณต์ฆ์ฑ ๊ณจ์ ์์ ๋ํ์ ์ ๋ก์ ์ฌ์ฉ ๋ ์ ์๋ ๊ฐ๋ฅ์ฑ๊ณผ Drg2 ์ ์ ์๊ฐ ๊ณจ ์ฌํ์ฑ์ ์์ด ์ค์ํ ์กฐ์ ์ธ์์์ ํ์ธํ์๋ค.Abstract I
Table of Contents: IV
List of Tables VI
List of Figures VII
Introduction 1
Methods 5
Osteoinductive treatment of human mesenchymal stem cells 5
ALP staining and and ALP activity assay 5
Calcium staining and assay 6
Real-time PCR 7
Rat calvarial defect model 7
Micro-CT evaluation 8
Hematoxylin and eosin staining 9
Search for studies 10
Selection of studies 10
Data extraction and risk of bias 11
Data analysis and quality of evidence 12
Extraction of mesenchymal stem cells from human 13
Fabrication of DRG2 knock out mouse 13
Genomic typing and gender determination 14
Primary culture of BMMCs and bone marrow MSC 14
TRAP staining 15
shRNA transfection of MC3T3-E1 cell 15
Inducing osteoblastic differentiation in MC3T3-E1 cells 16
Western blot 16
Semiquantitative RT-PCR 17
Feeding and maintaining 17
Serum P1NP and CTX measurement 18
Ovariectomy 18
Calcein labeling 19
Statistics 19
Results 20
ALP assay 20
ALP staining 20
Calcium assay 20
Alizarin red staining 21
Real-time PCR 21
Animal experiments 21
Characteristics of included studies and risk of bias 22
Comparison with control group 24
Comparison between interventions 29
Higher DRG2 expression correlates with lower BMD 30
Knocking out of Drg2 affects mice postnatal bone formation 30
Inhibition of DRG2 improves bone architecture and BMD even in ovariectomized mice 31
Results of the GO enrichment and KEGG pathway analysis 33
Inhibiting the expression of DRG2 inhibits the osteoclastic differentiation of BMMCs and elevates osteoblastic differentiation of bone marrow MSCs 33
Inhibiting the expression of DRG2 elevates osteogenicity of MC3T3-E1 cells 34
Inhibition of DRG2 elevates OB differentiation via canonical and non-canonical BMP signaling 35
Discussion 37
The first section 38
The second section 41
The third section 46
References 50
Figures 61
Table 90
Supplementary material 104
๋
ผ ๋ฌธ ์ด ๋ก 123
Acknowledgments 126Docto
High affinity binding of H3K14ac through collaboration of bromodomains 2, 4 and 5 is critical for the molecular and tumor suppressor functions of PBRM1.
Polybromo-1 (PBRM1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains (BDs), which are specialized structures that recognize acetyl-lysine residues. While BD2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BDs collaborate with BD2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM1 is also unknown. We discovered that full-length PBRM1, but not its individual BDs, strongly binds H3K14ac. BDs 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD4 was found to be required for H3K14ac recognition, the conserved acetyl-lysine binding site of BD4 was not. Furthermore, simultaneous point mutations in BDs 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM1 to relocalize to the cytoplasm. In contrast, tumor-derived point mutations in BD2 alone lowered PBRM1\u27s affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM1 variants containing point mutations in BDs 2, 4, and 5 or BD2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BDs 2, 4, and 5 of PBRM1 collaborate to generate high affinity to H3K14ac and tether PBRM1 to chromatin. Mutations in BD2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions
Identification of Allele-Specific RNAi Effectors Targeting Genetic Forms of Parkinson's Disease
Parkinson's disease (PD) is a progressive neurological disorder affecting an estimated 5โ10 million people worldwide. Recent evidence has implicated several genes that directly cause or increase susceptibility to PD. As well as advancing understanding of the genetic aetiology of PD these findings suggest new ways to modify the disease course, in some cases through genetic manipulation. Here we generated a โwalk-throughโ series of RNA Pol III-expressed shRNAs targeting both the ฮฑ-synuclein A30P and LRRK2 G2019S PD-associated mutations. Allele-specific discrimination of the ฮฑ-synuclein A30P mutation was achieved with alignments at position 10, 13 and 14 in two model systems, including a heterozygous model mimicking the disease setting, whilst 5โฒRACE was used to confirm stated alignments. Discrimination of the most common PD-linked LRRK2 G2019S mutation was assessed in hemizygous dual-luciferase assays and showed that alignment of the mutation opposite position 4 of the antisense species produced robust discrimination of alleles at all time points studied. Discrimination at this position was subsequently confirmed using siRNAs, where up to 10-fold discrimination was seen. The results suggest that RNAi-mediated silencing of PD-associated autosomal dominant genes could be a novel therapeutic approach for the treatment of the relevant clinical cases of PD in future
High-throughput next-generation sequencing technologies foster new cutting-edge computing techniques in bioinformatics
The advent of high-throughput next generation sequencing technologies have fostered enormous potential applications of supercomputing techniques in genome sequencing, epi-genetics, metagenomics, personalized medicine, discovery of non-coding RNAs and protein-binding sites. To this end, the 2008 International Conference on Bioinformatics and Computational Biology (Biocomp) โ 2008 World Congress on Computer Science, Computer Engineering and Applied Computing (Worldcomp) was designed to promote synergistic inter/multidisciplinary research and education in response to the current research trends and advances. The conference attracted more than two thousand scientists, medical doctors, engineers, professors and students gathered at Las Vegas, Nevada, USA during July 14โ17 and received great success. Supported by International Society of Intelligent Biological Medicine (ISIBM), International Journal of Computational Biology and Drug Design (IJCBDD), International Journal of Functional Informatics and Personalized Medicine (IJFIPM) and the leading research laboratories from Harvard, M.I.T., Purdue, UIUC, UCLA, Georgia Tech, UT Austin, U. of Minnesota, U. of Iowa etc, the conference received thousands of research papers. Each submitted paper was reviewed by at least three reviewers and accepted papers were required to satisfy reviewers' comments. Finally, the review board and the committee decided to select only 19 high-quality research papers for inclusion in this supplement to BMC Genomics based on the peer reviews only. The conference committee was very grateful for the Plenary Keynote Lectures given by: Dr. Brian D. Athey (University of Michigan Medical School), Dr. Vladimir N. Uversky (Indiana University School of Medicine), Dr. David A. Patterson (Member of United States National Academy of Sciences and National Academy of Engineering, University of California at Berkeley) and Anousheh Ansari (Prodea Systems, Space Ambassador). The theme of the conference to promote synergistic research and education has been achieved successfully
From molecular targets to antitumor immunity
This thesis aimed at the development of strategies that could contribute to the evaluation of the clinical potential of new anticancer therapies. The work was divided into two main sections comprising the development of a therapeutic approach to target cancer genetic vulnerabilities and the development of 3D tumor models incorporating cues from the stromal and immune microenvironments. (...
- โฆ