Alicja Urbaniak has been selected to present in the “Guppy Tank” Translational Science Pitch Showcase at the American Society for Pharmacology and Experimental Therapeutics (ASPET) meeting.  Alicja is a postdoctoral fellow in Timothy Chamber‘s lab. Congratulations Alicja!

To conduct biomedical research, scientists must be able to identify, analyze and compare proteins in biological samples. This complex process requires facilities – known as proteomics cores – that house the specialized equipment and highly trained staff required for such a task.

The fourth annual Proteomics Facility Staff Symposium on Jan. 29-30 at UAMS brought together 30 proteomics core directors and staff members to learn how best to operate and maintain these facilities at their institutions.

All of the participants came from IDeA (Institutional Development Award) states and Puerto Rico, all of which have been identified by the National Institutes of Health as historically receiving less grant funding for biomedical research than other states.

“The first symposium was in 2017, and it has grown each year since. By getting together on a regular basis, we can learn from each other, share our successes and ensure we all have the support and knowledge to meet the needs of our researchers,” said Alan Tackett, Ph.D., associate director for basic science in the UAMS Winthrop P. Rockefeller Cancer Institute.

Tackett also serves as co-director of the IDeA National Resource for Quantitative Proteomics, a partnership between the Arkansas INBRE (IDeA Networks of Biomedical Research Excellence) and Oklahoma INBRE.

The national resource combines the strengths of the two INBREs to guide and assist other IDeA states where core facilities may be underfunded or lack resources.

“At UAMS, our discovery phase proteomic capabilities are very strong, while Oklahoma has a state-of-the-art, targeted validation proteomics program. Together we offer expertise and access to equipment that facilities in other IDeA states may lack,” Tackett said.

Mike Kinter, Ph.D., of the University of Oklahoma Health Sciences Center, serves as co-director of the national resource with Tackett.

The INBRE program supports research in public and private four-year colleges by building research capacity and raising awareness about career opportunities in biomedical research. It is supported by the National Institutes of Health (NIH) Institutional Development Award (IDeA), which was established to broaden the geographic distribution of NIH funding for biomedical and behavioral research.

Lawrence Cornett, Ph.D., professor in the UAMS College of Medicine Department of Physiology and Biophysics, serves as principal investigator and director of Arkansas INBRE.

Symposium participants took part in breakout sessions and heard speakers on administrative topics related to operating a proteomics core and establishing a rate structure, as well as information on topics such as sample preparation and data collection. Sessions were led by UAMS faculty Sam Mackintosh, Ph.D.; Rick Edmondson, Ph.D.; and Stephanie Byrum, Ph.D.

“We covered a wide range of topics to help core directors and staff develop and maintain programs that will succeed at their universities,” said Tackett, who also serves as a professor in the UAMS College of Medicine Department of Biochemistry and Molecular Biology.

The Proteomics Core at UAMS is one of several core facilities where technology, tools and collaborative services are pooled together and made available on a pay-per-use basis to investigators both within and outside of UAMS who may not otherwise have access to them in their individual labs.

In the Proteomics Core, staff use a process known as mass spectrometry to help researchers identify proteins. The core is a one-stop-shop, where staff not only process the samples but can help researchers design experiments and analyze results.

A Discovery-Phase Proteomics Faculty and Student Workshop is scheduled for Feb. 27-28 at UAMS and will emphasize new approaches that researchers can implement in their own laboratories and how to best use the resulting data to be more competitive for extramural funding.

The American Society for Microbioogy journal mSphere recently launched a new series of commentaries called “mSphere of Influence.” Young scientists were invited to write about how one or more important papers made a substantial impact on their own work and thinking. The paper “A CRISPR-based approach for proteomic analysis of a single genomic locus​” which was a collaborative effort between the labs of Kevin Raney and Alan Tackett was recognized by Lucy Glover in her commentary.

Brian Koss, a graduate student in the Biochemistry and Molecular Biology track, and Bobby McGehee, Dean of the Graduate School, were interviewed on KARK about graduate school at UAMS. Brian is a student in the lab of Alan Tackett.

Congratulations to Eugene Nyamugenda, who successfully defended his dissertation entitled “Effect of high-fat diet on the melanocortin-4 receptor (MC4R) neurons in the paraventricular nucleus of the hypothalmus”. Eugene is a student in Giulia Baldini’s laboratory.

Victoria Hwang, a senior at the Arkansas School for Mathematics, Sciences, & the Arts, has been named a top 300 Scholar in the 79^th Regeneron Science Talent Search. Victoria was selected for her work entitled “POLK^KO GBM-derived Cells Exhibit Increased Replication Catastrophe” in Dr. Robert Eoff’s laboratory.  Congratulations Victoria!

Mechanistic Insights into Chemoresistance Mediated by Oncogenic Viruses in Lymphomas.

Duah Alkam, a Ph.D. student in Mark Smeltzer‘s and Dave Ussery‘s laboratories was invited to present her work at the Nanopore Community Meeting in New York. A description of her presentation from Oxford Nanopore Technologies is below.

Duah Alkam – PCR-free transposon sequencing (TnSeq): Cas9/dCas9-mediated transposon enrichment

Duah Alkam (University of Arkansas for Medical Sciences) opened the Targeted Sequencing breakout session by introducing the goal of her team: to find therapeutic targets for the prominent bacterial pathogen Staphylococcus aureus. Staphylococcus bacteria are a leading cause of healthcare-associated infections, with Methicillin-resistant S. aureus (MRSA) causing over 80,000 severe infections and over 11,000 deaths per year.

Duah and the team take a whole-genome screening approach to the search for novel therapeutics via transposon sequencing (TnSeq), enabling analysis of the genes that affect the fitness of microorganisms in a particular condition. She described how, in a transposon library, each cell is mutagenized by a transposon – a DNA segment that inserts into a gene, resulting in dysfunction. The transposon library used by Duah and her colleagues is in a background of a clinically-relevant strain of S. aureus; Duah gave an overview of a typical workflow, in which the libraries are grown in different conditions, then enriched for the region of interest. The current protocol for TnSeq requires PCR amplification of the transposon-library junctions prior to sequencing; Duah noted that this method has been used in many papers. However, Duah explained, there’s a problem with this method: inherent bias is introduced through the PCR steps. Looking to overcome this, Duah asked: “can we enrich for the transposon via a PCR-free method?”. Here, Duah explained, the team turned to Oxford Nanopore and decided to explore PCR-free dCas9/Cas9-mediated transposon enrichment.

Duah outlined the four methods tested to investigate Cas9-mediated enrichment and nanopore sequencing of S. aureus TnSeq libraries; in each case, samples were prepared for sequencing without PCR using the Ligation Sequencing Kit and sequenced on the MinION device.

In the first condition, the TnSeq library was sequenced without any enrichment. This no-enrichment condition resulted in just 8,566, or 0.2% of the 5.1m reads representing transposons, as expected – transposons are “pretty rare in the library”. This confirmed the need for enrichment of the transposons.

In the second condition, CRISPR/Cas9 was used to induce a double-stranded cut targeting transposon insertion sites, then the library was sequenced. Duah outlined the CRISPR/Cas9-mediated enrichment method: firstly, the 5′ ends of all strands in the sample are dephosphorylated. Next, the Cas9 enzyme is directed to and cleaves DNA at transposon sites via RNAs including custom transposon-specific probes. This reveals phosphorylated ends, to which sequencing adapters can be ligated so that the enriched DNA can be sequenced. In this dataset, transposons were successfully enriched, representing 45% of reads in sequencing – “we were very excited when we saw this” – but sequencing yield was depleted, so that this was comprised of 3.7k reads.

To investigate whether yield could be improved, in the third condition, dCas9 – “dead” Cas9, an inactive form of the enzyme which does not cut the DNA – and biotinylated RNA probes were used, so that the biotinylated targets could be captured using Streptavidin beads then prepared for sequencing. Whilst this protocol resulted in only 0.8% transposon reads, sequencing output was higher, resulting in 94.6k reads.

Aiming to make the most of the enrichment from Cas9 and yield from dCas9, in the last condition, a combination of these methods were used. dCas9 and biotinylated probes were first used to capture the DNA, then active Cas9 was used to cleave the transposons at a single site prior to sequencing. Here, enrichment was lower than when using Cas9 alone, with 31% of reads representing transposons, but as sequencing output was higher, 19.4k reads were on-target.

Summarising, Duah concluded that she and her team had successfully devised and utilized a PCR-free method of transposon enrichment from a TnSeq S. aureus library, representing the first time this has been achieved without PCR. Furthermore, the use of dCas9 in pulldown prior to cleavage by Cas9 improved sequencing yield, enabling a greater number of transposon reads. They are now optimizing their protocol to further improve sequencing yield and recover greater numbers of insertions.

Congratulations to Tresor O. Mukiza, who successfully defended his dissertation entitled “Multiple DNA Sequence-Specific Protein-DNA Complexes Activate Meiotic Recombination Hotspots Via Chromatin Remodeling”. Tresor is a student in Wayne Wahls‘ laboratory.

Jacy Wagnon, who earned her Ph.D. in Dr. Wayne Wahls’ laboratory, has now obtained a tenure-track faculty position at The Ohio State University.  Doctor Wagnon is an Assistant Professor in the Department of Neuroscience, with research interests in epilepsy, neurodegenerative disorders, and genetics of neurological diseases.