A 50-year-old female with no significant medical history initially presented to an urgent care center with symptoms of acute onset abdominal pain, nausea, and emesis. Chest and abdominal X-ray revealed free air under the diaphragm, prompting immediate transfer to the emergency department. Continued abdominal tenderness and pain were concerning for perforated viscus. The patient was transferred to the operating room, and diagnostic laparoscopy was performed. Inflammation and contamination were discovered in the right side of the abdomen and pelvis secondary to a small bowel (SB) perforation. Segmental SB resection revealed a perforated diverticulum. Pathological examination confirmed a diagnosis of gastrointestinal stromal tumor (GIST) at the perforated segment. On postoperative day 5, the patient was discharged home, and at 30-month follow-up, the patient continued to do well. Although rare, SB diverticula are commonly false (i.e., pseudodiverticula). The concomitant presence of a GIST in a true SB diverticulum presenting with perforation has not yet been reported.

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Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome is encoded by the nuclear genome; which includes additional oxidative phosphorylation subunits, as well as the proteins necessary for mtDNA replication, expression and stability. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible protein post‐translational modifications of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. To address this gap in knowledge, this research focuses on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow for the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, threonine 313 and serine 197. To mimic the behavior of phosphorylation on these three sites, individual amino acids were mutated to glutamate, an amino acid that mimics the size and charge of the phosphoryl group. WT and phosphomimetic TFB2M were expressed in E. coli and purified. The purified proteins were analyzed for their ability to bind mtDNA through a pull‐down assay and fluorescence polarization was used to determine the binding dissociation constants for the light strand promoter. Interactions between TFB2M phosphomimics and POLRMT were also assessed. The data support the hypothesis that the phosphorylation at these sites may decrease the ability of TFB2M to bind to mtDNA. In vitro transcription assays were performed to determine the impact of these modifications on TFB2M’s role in transcription initiation. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function. Support or Funding Information This material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.

Mitochondrial DNA (mtDNA) encodes 13 of the protein subunits of the oxidative phosphorylation pathway, the pathway that produces the majority of ATP in eukaryotic cells. Mitochondrial dysfunction is associated with neurodegenerative disorders, cancer, inherited mitochondrial diseases, and other pathologies, making mitochondrial biochemistry an important area of investigation. However, there lacks an understanding of key elements of this biochemistry, including the mechanisms that regulate mtDNA transcription. mtDNA is known to be complexed to various proteins in structures known as nucleoids. Some nucleoid proteins are key members of the mtDNA transcription machinery; these include the mitochondrial RNA polymerase (POLRMT) and mitochondrial ribosomal protein L12 (MRPL12), which maintains POLRMT stability and promotes mtDNA transcription. We hypothesize that reversible post‐translational modifications (PTMs), such as phosphorylation of threonine, serine, or tyrosine residues or acetylation of lysine residues within these and other nucleoid proteins, may play a role in regulating mtDNA transcription. The objective of our study is to determine the effects of MRPL12 and POLRMT post‐translational modifications on the activities of these proteins in the context of mtDNA transcription in order to assess the roles of such modifications in regulating this process. To test this, we performed mutagenesis PCR on MRPL12 and POLRMT genes within bacterial expression vectors to replace the sequence encoding an amino acid at a PTM site with a sequence encoding a modified or unmodified amino acid mimic (T, S, Y → E/A, phosphorylated/dephosphorylated mimic; K → Q/R, acetylated/deacetylated mimic). We optimized bacterial expression and protein purification conditions and purified wild‐type and mutant MRPL12 proteins, as well as wild‐type POLRMT. We performed preliminary in vitro transcription assays to assess POLRMT functionality and found that our protein successfully transcribed short mtDNA templates. Going forward, we will perform protein‐protein binding assays to determine the effects of MRPL12 modifications on MRPL12's ability to bind POLRMT, as well as POLRMT mutagenesis and more in vitro transcription assays to measure the effects of MRPL12 and POLRMT modifications on mtDNA transcription.Support or Funding InformationThis research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845, with additional funding from the Arnold and Mabel Beckman Foundation Beckman Scholars Award and the ASBMB Undergraduate Research award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial DNA (mtDNA) encodes for 13 protein components required for oxidative phosphorylation (OXPHOS), the primary cellular energy production pathway. While the critical roles of mitochondria in metabolism and cellular function are well established, the mechanisms regulating expression of mtDNA‐encoded genes are poorly understood. Defects in the regulation of mtDNA transcription are implicated in numerous pathologies such as neurodegenerative disorders and cancers. mtDNA is complexed to proteins in structures known as nucleoids, of which some proteins play critical roles in transcriptional regulation. Our objective is to determine whether reversible post‐translational modifications (PTMs) of nucleoid proteins, including lysine acetylation and serine/threonine/tyrosine phosphorylation, regulate mtDNA transcription. Our central hypothesis is that PTMs alter the function of these proteins and provide a means of regulating mitochondrial gene expression. We highlight our work on the characterization of mitochondrial transcription factor B2 (TFB2M), focusing on the mutagenesis, bacterial expression, purification, and analysis. Site‐directed mutagenesis was used to alter the amino acids known to be post‐translationally modified to amino acids mimicking either the modified or unmodified state. Purified mutants and wild type TFB2M were analyzed in a mtDNA binding assay to determine the effects of PTMs on TFB2M function. Initial data suggest that TFB2M relies on modified or unmodified states of key PTM sites to regulate its ability to bind to mtDNA. Characterization of these sites is critical for determining TFB2M's role in mtDNA transcriptional regulation. Future work involves additional screening of TFB2M mutants, optimization of the mtDNA binding assay for analysis of other nucleoid proteins, and in vitro transcription assays to assess mutant protein functionality on mtDNA transcription.Support or Funding InformationThis research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.