Flaviviruses such as dengue encode a protease that is essential for viral replication. The protease functions by cleaving well-conserved positions in the viral polyprotein. In addition to the viral polyprotein, the dengue protease cleaves at least one host protein involved in immune response. This raises the question, what other host proteins are targeted and cleaved? Here we present a new computational method for identifying putative host protein targets of the dengue virus protease. Our method relies on biochemical and secondary structure features at the known cleavage sites in the viral polyprotein in a two-stage classification process to identify putative cleavage targets. The accuracy of our predictions scaled inversely with evolutionary distance when we applied it to the known cleavage sites of several other flaviviruses—a good indication of the validity of our predictions. Ultimately, our classifier identified 257 human protein sites possessing both a similar target motif and accessible local structure. These proteins are promising candidates for further investigation. As the number of viral sequences expands, our method could be adopted to predict host targets of other flaviviruses.

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Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.

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The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.

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Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known.

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A protocol can help with the tricky conversations essential to responsible research conduct, says Mary A. Allen.

“Either this is sloppiness or misconduct, and either way I don’t think this is a lab I want to be in anymore.” I was terrified as I spoke these words to my first graduate-school adviser in November 2004. Members of my laboratory had seen suspicious data in grant proposals, and the discovery was causing me unmanageable stress. A month later, two colleagues went to the chair of our department, and an investigation began. From the outside, deciding to have that hard conversation was a small part of a gruelling ordeal involving many people. Our lab eventually shut down, and our principal investigator was found to have falsified data on grant applications.

For me, personally, that conversation is the result of one of the most important things that I have ever done: making the decision to have a difficult discussion when something needs to change. That’s why I’ve developed a way to help others to do so.

Now I am a principal investigator co-running a lab. Our website has a light-hearted Tolkienesque map. It shows the Isle of RNA modification, the Ivory Tower, a Sea of Data — and a career track running through an ethical swamp. Many ask why this is included amid the puns and in-jokes, but I ask why it wouldn’t be. Almost every scientist I know has been through at least one ethical morass in their career.

Partly because of my past experience, I teach the responsible conduct of research (RCR) courses required of many trainees who receive government funding. Most of the mandated topics, such as responsible authorship and publication, focus on compliance — following the rules. That is necessary. But it is not sufficient. A responsible researcher needs to be able to navigate conflicts and tricky situations.

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Mary Allen holds up a valentine sent to her from a childhood friend. It sits in her cubicle where she is hard at work tearing apart genomic data looking for patterns. This friend, who has Down syndrome, is part of the reason that Allen, a postdoctoral researcher in  at the , became interested in studying aneuploidy. Aneuploidy means that cells have too many, or too few, of one or more chromosomes. In the case of Down syndrome, there is an extra copy of chromosome 21. Allen is exploring what makes people with this extra chromosome survivors.

“Down syndrome is actually not all that survivable,” says Allen. “Only 25 percent of embryos with three copies of chromosome 21 survive to live birth. These people who are surviving and living long lives have something in their DNA—from their genetic background—that is helping them.”

Down syndrome is the most commonly occurring chromosomal condition and more than 400,000 people in the United States are currently living with it. Allen is right about them being survivors. According to the , life expectancy for people with the syndrome has increased dramatically from 25 years in 1983 to 60 years now, due in part to better educational programs, health care and support from families and communities.

Allen is taking genetic sequencing data from people with Down syndrome and their parents to understand how that extra copy of chromosome 21 puts this population at higher risk for health issues such as heart defects, thyroid conditions, leukemia, , and respiratory and hearing problems. She is also trying to understand why they are at lower risk for heart attack, stroke, and solid tumor cancers. Allen isn’t out to find a cure for Down syndrome. Her goal is to find what in their DNA is helping these survivors, and how can we design targeted molecular therapy to help them have better lives.

“Once you have had a friend with Down syndrome, stopping the occurrence of the syndrome isn’t on the table,” says Allen. “They are just such great people.”

Allen recently was awarded a Sie Foundation Postdoctoral Fellowship to continue her Down syndrome research for the next two years. This fellowship was created under the Anna and John J. Sie Endowment Fund for the BioFrontiers Institute, which is targeted specifically at funding research to prevent the cognitive and medical ill effects associated with the extra chromosome 21. The fellowship is offered as a collaboration between BioFrontiers and the  at the University of ŷ���ڱ���Ƶ, Anschutz Medical Campus.

The BioFrontiers Institute also awarded Sie Fellowships to Geertruida Josien Levenga of ŷ���ڱ���Ƶ-Boulder’s Institute of Behavioral Genetics and to  of ŷ���ڱ���Ƶ-Boulder’s Department of Molecular, Cellular and Developmental Biology. Dr. Levenga is a neuroscientist whose research holds promise for ameliorating the seizures that afflict so many individuals with Down syndrome. Dr. Garrido-Lecca will test the hypothesis that alteration of microRNA levels in individuals with Down syndrome contributes to some of their health challenges.

Dr. Allen sees the new fellowship as welcome news for her work. Research funding for Down syndrome has always been extremely low. The National Institutes of Health in 2012 allocated only $50 in research funding per person living with the condition, versus $270 for Fragile X research, $329 for multiple sclerosis research and $2,867 for cystic fibrosis research. Individuals with Down syndrome have special health needs, like heart conditions and decreased immunity, which can be helped by further research. In addition, since Alzheimer’s disease, leukemia, low muscle tone and weight gain are seen at a high incidence in people with Down syndrome, researching the syndrome may lead to treatments for these associated disorders in the broader population.

“Research on the smaller ear canals of people with Down syndrome is now helping people who suffer from deafness and other auditory disorders,” says Allen. “Unlocking the cellular processes behind one disorder can help us with so many others.”

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