Biochemistry and Molecular Genetics

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Biochemistry and Molecular Genetics

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News Article | April 19, 2017
Site: phys.org

Published in Nature Communications, the study showed this posttranslational modification of the protein, centromere protein A (CENP-A), distinguishes it from a similar protein that is found in the rest of the chromosome. CENP-A is a type of histone, a protein with DNA wrapped around it, and specifies the location of the centromere in the nucleus. Lead author Daniel Foltz, '01 PhD, associate professor of Biochemistry and Molecular Genetics, and his team conducted functional analyses of the modifications on CENP-A, which they had previously identified. "It's interesting because this is a novel type of modifications on histones and because we can go in and really show what function is being mediated by the amino-terminal methylation, which has not been previously well-defined for this type of modification," Foltz said. The investigators discovered that CENP-A, when correctly methylated on its amino terminus, influences the recruitment of CCAN proteins, which are part of a large centromere complex of proteins. They showed that a subset of components of CCAN are dependent on this methylation. When the scientists blocked methylation from this process, they observed defects in chromosome segregation. "We really defined a different arm of recruitment for the CCAN proteins than has been understood before," Foltz said. Next, the scientists studied hallmarks of cancer cells, including defects in chromosome segregation and spindle polarity. A bipolar spindle is essential to equally segregate chromosomes into two separate cells during cell division. In cancer cells, defects in the spindle lead to chromosomes that are pulled in multiple directions and can result in chromosome breakage and genomic instability. The scientists found that reducing the amount of methylation of CENP-A drives this process. Foltz and the other investigators also found that in the absence of the tumor suppressor protein p53, loss of CENP-A methylation promotes more rapid tumor formation. Next, Foltz and his team want to study how tumor cells are using this pathway. "What we've done in this paper is engineer this defect into cells to see what the phenotype is," Foltz said. "The next question is what happens to CENP-A methylation in cancer cells, and when CENP-A is overexpressed, to what degree is it driving the genomic instability in cancers?" More information: Kizhakke M. Sathyan et al. α-amino trimethylation of CENP-A by NRMT is required for full recruitment of the centromere, Nature Communications (2017). DOI: 10.1038/ncomms14678


News Article | April 12, 2017
Site: www.cemag.us

Northwestern Medicine scientists have developed a novel testing platform to assess, in real time, the efficacy of nanomaterials in regulating gene expression. The findings, published in Proceedings of the National Academy of Sciences, could help to facilitate preclinical investigations and optimize nanotherapeutics for cancers before they reach clinical trials. Timothy Sita, a seventh-year MD/PhD student in the Medical Scientist Training Program, was the first author of the study, which looked at the platform in animal models. “This is an important step forward for the field,” said principal investigator Alexander Stegh, PhD, assistant professor of Neurology and of Medicine. “The very thorough optimization that we see in conventional drug development had been missing in the nanotech space, and we felt very strongly about changing this. The system that we developed here really allows us to support those efforts, and evaluate our nanoparticles in the most relevant models, in an in vivo setting.” Chad Mirkin, PhD, the George B. Rathmann Professor of Chemistry in the Weinberg College of Arts and Sciences and a professor of Medicine in the Division of Hematology/Oncology, was also a corresponding author of the paper. The scientists demonstrated the concept while using nanostructures called spherical nucleic acids (SNAs) to target a resistance factor gene in glioblastoma, an aggressive, incurable type of brain tumor. SNAs, first developed by Mirkin at Northwestern in 1996, consist of dense strands of RNA packed around a nanoparticle core. Because of their unique properties, SNAs are capable of both crossing the blood-brain barrier and entering into tumor cells, where they can directly target gene activity that encourages cancer growth. While these conjugates are a promising tool in the era of precision medicine, scientists previously lacked a quantitative method to assess how SNAs regulated gene activity in living organisms, which would provide new insights into how to optimize the therapies. “We’ve seen that these particles can basically target any cancer gene, but we didn’t know when they worked best or what dosing regimens to use,” Sita says. “As such, preclinical trials weren’t as successful as they could have been.” In the current study, the scientists showed that by using a type of non-invasive imaging on the mice, they could gauge in real time how the nanoparticles affected levels of an intratumoral target protein. “Now we can tweak these particles — play with the shape of the nanoparticle, or how much RNA we load onto the particle, for example — and then assess very quickly whether those changes are more effective or not,” Sita explains. “It’s a platform to help optimize the drugs in mice before they go to human trials, and make something that will translate better to the clinic.” While the method could be generalizable to investigating nanotherapeutics for many types of cancers, the study also has clinical implications unique to glioblastoma. The scientists developed nanoparticles to knock down O6-methylguanine-DNA methyltransferase (MGMT) — a protein which reduces the impact of chemotherapy — in mice with glioblastoma. Through the imaging platform, they discovered that mice had the lowest levels of the protein between 24 to 48 hours after receiving the nanoparticles, suggesting the optimal time to administer chemotherapy. “We showed a very significant reduction in tumor volume when we combined these particles with the chemotherapy,” Sita says. “By silencing this gene that’s causing resistance to the chemotherapy, we can have a much more profound response. That’s the key clinical angle.” Sita, who will be graduating in May, recently matched into a radiation oncology residency at the McGaw Medical Center of Northwestern University, where he intends to continue his research into glioblastoma therapies. Jasmine May, a fifth-year student in the Medical Scientist Training Program, was also a co-author of paper, along with Charles David James, PhD, professor of Neurological Surgery and of Biochemistry and Molecular Genetics, and other Northwestern scientists. Mirkin, Stegh, and James are also members of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. This research was supported by the Center for Cancer Nanotechnology Excellence initiative of the National Institutes of Health (NIH) under Award U54 CA151880, National Institute of Arthritis and Musculoskeletal and Skin Diseases Award R01AR060810, Defense Advanced Research Projects Agency Grant HR0011-13-2-0018, the John McNicholas Foundation and the American Cancer Society, National Cancer Institute (NCI)/NIH National Research Service Award Fellowship F30CA174058-01, a Ryan Fellowship, a National Science Foundation graduate research fellowship and a P.E.O. scholar award, the Northwestern University Flow Cytometry Facility, Northwestern University Center for Advanced Microscopy, and Cancer Center Support Grant NCI CA060553.


News Article | April 17, 2017
Site: www.eurekalert.org

CHARLOTTESVILLE, Va., April 13, 2017 - For Mazhar Adli, the little glowing dots dancing about on the computer screen are nothing less than the fulfillment of a dream. Those fluorescent dots, moving in real time, are set to illuminate our understanding of the human genome, cancer and other genetic diseases in a way never before possible. Adli, of the University of Virginia School of Medicine's Department of Biochemistry and Molecular Genetics, has developed a way to track genes inside living cells. He can set them aglow and watch them move in three dimensions, allowing him to map their positions much like star charts record the shifting heavens above. And just as the moon influences the tides, the position of genes influences the effects they have; thus, 3D maps of gene locations could lead scientists to a vastly more sophisticated appreciation of how our genes work and interact -- and how they affect our health. "This has been a dream for a long time," Adli said. "We are able to image basically any region in the genome that we want, in real time, in living cells. It works beautifully. ... With the traditional method, which is the gold standard, basically you will never be able to get this kind of data, because you have to kill the cells to get the imaging. But here we are doing it in live cells and in real time." DNA is often depicted as tidy strands stretched out in straight lines. But in reality, our DNA is clumped up inside the nuclei of our cells like cooked spaghetti. "We have two meters of DNA folded into a nucleus that is so tiny that 10,000 of them will fit onto the tip of a needle," Adli explained. "We know that DNA is not linear but forms these loops, these large, three-dimensional loops. We want to basically image those kind of interactions and get an idea of how the genome is organized in three-dimensional space, because that's functionally important." Thinking about DNA as a neat line, he noted, can create misconceptions about gene interactions. Two genes that are far apart in a linear diagram may actually be quite close when folded up inside the cell's nucleus, and that can affect what they do. He used a map analogy: "That's how we believe an element that appears to be in Los Angeles is regulating an element in Virginia - [when the DNA is folded up,] they're not actually that far apart." Adli's new approach, developed in conjunction with colleagues at UVA and the University of California, Berkeley, uses the CRISPR gene editing system that has proved a sensation in the science world. The researchers flag specific genomic regions with fluorescent proteins and then use CRISPR to do chromosome imaging. If they want, they can then use CRISPR to turn genes on and off, using the imaging approach to see what happens. The new method overcomes longstanding limitations of gene imaging. "We were told we would never be able to do this," Adli said. "There are some approaches that let you look at three-dimensional organization. But you do that experiment on hundreds of millions of cells, and you have to kill them to do it. Here, we can look at the single-cell level, and the cell is still alive, and we can take movies of what's happening inside." The business of growing cells just to kill them is both time consuming and a poor way to figure out what was happening with the DNA inside them, he said. It is like trying to figure out the rules of football by looking at blurry pictures of a game. Adli's new approach, on the other hand, lets him sit back and watch the plays unfold in real time. "It's a super exciting thing to be able to do," he said. Adli and his team have described their new method in an article in the scientific journal Nature Communications, making it available to scientists around the world. The paper was authored by Peiwu Qin, Mahmut Parlak, Cem Kuscu, Jigar Bandaria, Mustafa Mir, Karol Szlachta, Ritambhara Singh, Xavier Darzacq, Ahmet Yildiz and Adli. The work was supported by the V Foundation for Cancer Research; the UVA Cancer Center; the National Institutes of Health, grants U54-DK107980, U01-EB021236 and GM094522; the National Science Foundation; and the California Institute for Regenerative Medicine.


Elbarbary R.A.,University of Rochester | Miyoshi K.,University of Rochester | Myers J.R.,University of Rochester | Du P.,Rutgers University | And 3 more authors.
Science | Year: 2017

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis.We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2-loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation. © 2016 by the American Association for the Advancement of Science; all rights reserved.


MORRISVILLE, N.C., March 01, 2017 (GLOBE NEWSWIRE) -- Novan, Inc. (“the Company” or “Novan”) (NASDAQ:NOVN) today announced that preclinical data demonstrating the anti-viral effects of the Company’s nitric oxide-releasing drug candidates will be presented at the 31st International Papillomavirus Conference in Cape Town, South Africa. Thomas Broker, Ph.D., and Louise Chow, Ph.D., both of the University of Alabama at Birmingham, or UAB, are scheduled to present “Antiviral Efficacy of Nitric Oxide-Releasing Drug Candidates in Suppressing Productive Infection by HPV-18 in the Organotypic Epithelial Raft Culture Model System” on Thursday, Mar. 2. Drs. Broker and Chow are professors in the Department of Biochemistry and Molecular Genetics at UAB’s School of Medicine. The Broker-Chow research program has been focused on human papillomavirus, or HPV, for more than 33 years. “We are very excited by the continuing success of the investigational nitric oxide-releasing agents against HPV infections,” said Dr. Broker. “The data we are presenting showed significant inhibition of the high-risk HPV-18 genotype and reduction of the E6 viral protein in the raft culture model. We are quite pleased that the testing results from our laboratory have had a very constructive impact in elucidating some of the probable mechanisms of action and the affected cellular and viral pathways.” Published studies have demonstrated that the E6 protein impedes the body’s ability to recognize HPV-infected cells and disrupts the cells’ ability to repair DNA damage and prevent abnormal cellular replication. As a result, E6 is believed to be a primary driver of HPV-related cancers and other abnormal growths. “The findings generated in Broker-Chow’s human tissue model support the novel mechanism of action of Novan’s topical SB206 anti-viral development program,” said Nathan Stasko, Ph.D., President and Chief Executive Officer of Novan. “HPV affects millions of Americans, and we believe nitric oxide-releasing drug candidates have incredible potential not only for the topical treatment of genital, common and plantar warts caused by HPV, but also against cancers associated with high-risk HPV subtypes.” Top-line results from Novan’s Phase 2 clinical trial with SB206 for the treatment of genital warts caused by HPV were announced in November 2016. HPV refers to a large family of double-stranded DNA viruses that induce abnormal growths on the skin or mucosal surfaces. HPV affects nearly 80 million Americans, and an estimated 14 million new cases of the virus are reported each year, according to the Centers for Disease Control and Prevention, or CDC. There are over 100 subtypes of the virus, characterized as low-risk or high-risk based on their cancer-causing potential. The virus is typically transmitted via direct skin-to-skin contact through disruptions in the normal skin barrier. All warts are caused by HPV, including genital and perianal warts, common warts and plantar warts. Genital warts are among the world's most common sexually transmitted diseases. Genital warts are usually flesh-colored growths that can be raised, flat or cauliflower-shaped and are typically found on the surface of the external genitalia or in and around the anus. In males, they can appear on the surface of the penis and scrotum, and in females inside the vagina or on the cervix. Genital warts carry a substantial psychosocial burden due to the shame and embarrassment related to having a sexually transmitted disease as well as the inconvenience and discomfort of current treatment modalities. Current treatment options for genital warts consist of ablative procedures that cut, burn or freeze the warts but do not address the underlying viral infection, and there are no currently approved oral or topical prescription products indicated for the treatment of genital warts with a direct anti-viral mechanism of action. Novan, Inc. is a late-stage pharmaceutical company focused on redefining the standard of care in dermatology through the development and commercialization of innovative therapies using the Company’s nitric oxide-releasing platform. Nitric oxide plays a vital role in the natural immune system response against microbial pathogens and is a critical regulator of inflammation. Our ability to harness nitric oxide and its multiple mechanisms of action has enabled us to create a platform with the potential to generate differentiated, first-in-class product candidates. We are rapidly advancing programs in five dermatological conditions with significant unmet medical need. We believe that our ability to conveniently deploy nitric oxide on demand in topical formulations allows us the potential to significantly improve patient outcomes in a variety of skin diseases and positions us to be a commercially successful leader in the dermatology market. For more information, visit the Company’s website at www.Novan.com. This press release contains forward-looking statements including, but not limited to, statements related to pharmaceutical development of nitric oxide-releasing product candidates, expected performance of our product candidates, publication and presentation of our trial results in the medical community and future prospects of our business and our product candidates. Forward-looking statements are subject to a number of risks and uncertainties that could cause actual results to differ materially from our expectations, including, but not limited to, uncertainties and risks in the clinical development process, including, among others, length, expense, ability to enroll patients, reliance on third parties, and that results of earlier research and preclinical or clinical trials may not be predictive of results, conclusions or interpretations of later research or trials; the lengthy and unpredictable nature of the U.S. Food and Drug Administration’s drug approval process; whether we will be able to obtain additional funding when needed; and other risks and uncertainties described in our prospectus dated Sept. 20, 2016, filed with the Securities and Exchange Commission, or SEC, in our quarterly report filed with the SEC on Form 10-Q for the three months ended Sept. 30, 2016, and in any subsequent filings with the SEC. These forward-looking statements speak only as of the date of this press release, and Novan disclaims any intent or obligation to update these forward-looking statements to reflect events or circumstances after the date of such statements, except as may be required by law.


Kurosaki T.,University of Rochester | Li W.,Biochemistry and Molecular Genetics | Hoque M.,Biochemistry and Molecular Genetics | Popp M.W.-L.,University of Rochester | And 3 more authors.
Genes and Development | Year: 2014

Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptomewide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay. © 2014 Kurosaki et al.


Stern P.L.,Paterson Institute for Cancer Research | van der Burg S.H.,Leiden University | Hampson I.N.,University of Manchester | Broker T.R.,Biochemistry and Molecular Genetics | And 4 more authors.
Vaccine | Year: 2012

This chapter reviews the current treatment of chronic and neoplastic human papillomavirus (HPV)-associated conditions and the development of novel therapeutic approaches. Surgical excision of HPVassociated lower genital tract neoplasia is very successful but largely depends on secondary prevention programmes for identification of disease. Only high-risk HPV-driven chronic, pre-neoplastic lesions and some very early cancers cannot be successfully treated by surgical procedures alone. Chemoradiation therapy of cervical cancer contributes to the 66-79% cervical cancer survival at 5 years. Outlook for those patients with persistent or recurrent cervical cancer following treatment is very poor. Topical agents such as imiquimod (immune response modifier), cidofovir (inhibition of viral replication; induction apoptosis) or photodynamic therapy (direct damage of tumour and augmentation of anti-tumour immunity) have all shown some useful efficacy (~50-60%) in treatment of high grade vulvar intraepithelial neoplasia (VIN). Provider administered treatments of genital warts include cryotherapy, trichloracetic acid, or surgical removal which has the highest primary clearance rate. Patient applied therapies include podophyllotoxin and imiquimod. Recurrence after "successful" treatment is 30-40%. Further improvements could derive from a rational combination of current therapy with new drugs targeting molecular pathways mediated by HPV in cancer. Small molecule inhibitors targeting the DNA binding activities of HPV E1/E2 or the antiapoptotic consequences of E6/E7 oncogenes are in preclinical development. Proteasome and histone deacetylase inhibitors, which can enhance apoptosis in HPV positive tumour cells, are being tested in early clinical trials. Chronic high-risk HPV infection/neoplasia is characterised by systemic and/or local immune suppressive regulatory or escape factors. Recently two E6/E7 vaccines have shown some clinical efficacy in high grade VIN patients and this correlated with strong and broad systemic HPV-specific T cell response and modulation of key local immune factors. Treatments that can shift the balance of immune effectors locally in combination with vaccination are now being tested. © 2012 Elsevier Ltd. All rights reserved.


Zheng D.,Biochemistry and Molecular Genetics | Liu X.,Biochemistry and Molecular Genetics | Tian B.,Biochemistry and Molecular Genetics
RNA | Year: 2016

Sequencing of the 3′ end of poly(A)+ RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3′ region extraction and deep sequencing (3′READS), to address mispriming issues that often plague 3′ end sequencing. Here we report a new version, named 3′READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)+ RNA and to remove the bulk of the poly(A) tail, 3′READS+ generates RNA fragments with an optimal number of terminal A's that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3′READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (∼50%). 3′READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3′ end counting, especially when the amount of input total RNA is limited. © 2016 Zheng et al.


Silver B.,Biochemistry and Molecular Genetics | Zhu H.,Biochemistry and Molecular Genetics
Virologica Sinica | Year: 2014

Varicella zoster virus (VZV) is the causative agent of varicella (chicken pox) and herpes zoster (shingles). After primary infection, the virus remains latent in sensory ganglia, and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine (v-Oka) is highly attenuated in the skin, yet retains its neurovirulence and may reactivate and damage sensory neurons. The reactivation is sometimes associated with postherpetic neuralgia (PHN), a severe pain along the affected sensory nerves that can linger for years, even after the herpetic rash resolves. In addition to the older population that develops a secondary infection resulting in herpes zoster, childhood breakthrough herpes zoster affects a small population of vaccinated children. There is a great need for a neuro-attenuated vaccine that would prevent not only the varicella manifestation, but, more importantly, any establishment of latency, and therefore herpes zoster. The development of a genetically-defined live-attenuated VZV vaccine that prevents neuronal and latent infection, in addition to primary varicella, is imperative for eventual eradication of VZV, and, if fully understood, has vast implications for many related herpesviruses and other viruses with similar pathogenic mechanisms. © 2014, Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg.


Lea M.A.,Biochemistry and Molecular Genetics
Journal of Cellular Biochemistry | Year: 2015

Flavonols comprise a group of flavonoid molecules that are widely distributes in fruits and vegetables. There is epidemiological data to suggest that consumption of flavonols can be accompanied by decreased cancer incidence. The anti-oxidant activity of flavonols may have an important role in preventing carcinogenesis. Therapeutic potential of flavonols is indicated by their growth inhibitory action accompanied by a decrease in several hallmarks of cancer such as resistance to apoptosis. Multiple mechanisms of action have been reported for the action of flavonols on cancer cells. Particular emphasis has been directed to inhibitory effects on several protein kinases and on the potential for prooxidant effects. The diversity of actions presents a problem in trying to elucidate primary and secondary effects but it may be a strength of the therapeutic potential of flavonols that it renders development of resistance more difficult for cancer cells. Cancer chemotherapy is usually characterized by the use of drug combinations. Some additive or synergistic combinations have been identified for flavonols and this is an area of ongoing investigation. As with other polyphenolic molecules there have been questions of cellular uptake and bioavailability. Several investigations have been and are being conducted to modify the structures of flavonols with the goal of increasing bioavailability. At present many investigators are sufficiently encouraged by past observations that they are responding to the challenge to optimize the dietary and therapeutic use of flavonols in cancer prevention and treatment. J. Cell. Biochem. 116: 1190-1194, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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