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Biochemical and Structural Characterization of Divergent Members of the Protein Kinase Family
Get PDFThe protein kinase family of enzymes catalyzes the transfer of the γ phosphate from an ATP molecule onto protein substrates. Using bioinformatic approaches, proteins with barely recognizable similarity to the known kinases can be found. Divergent catalytic motifs and sequence insertions allow such enzymes to catalyze unexpected reactions. In this work, I characterize two processes catalyzed by distant members of the protein kinase family: SidJ-catalyzed Glutamylation, and NiRAN-catalyzed RNA capping.
A Legionella pneumophila effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. SidJ becomes activated upon translocation into the host cell and uses cellular ATP and glutamic acid to inactivate another bacterial effector family, the SidE all-in-one ubiquitin ligases. By determining cryo-EM structures of the SidJ-SidE reaction intermediates, I show that SidJ uses its kinase-like active site to adenylate an active site Glu in SidE, forming a stable acyl adenylate intermediate complex. Subsequently, a secondary active site formed by unique insertions in the kinase catalytic loop binds the reaction intermediate, and positions glutamate for peptide bond formation. A similar reaction is catalyzed by SdjA, a SidJ paralog in the L. pneumophila genome. I show that SdjA has distinct substrate specificity from that of SidJ and identify structural motifs responsible for the difference.
SARS-CoV-2 is a positive-sense single-stranded RNA virus, the causative agent of the COVID 19 pandemic. The RNA genome of SARS-CoV-2 is protected by a 5′ cap structure, allowing the virus to avoid the cellular immune response, and enables translation of its genes. Although the enzymatic machinery decorating the cap with methyl groups was characterized, how the core cap structure is made was not known. I found, along with another student Gina Park, that the virus uses a kinase-like NiRAN domain attached to its RNA-dependent RNA polymerase (nsp12) to transfer its transcripts onto the N-terminus of the viral nsp9 protein in a RNAylation reaction. The NiRAN domain then transfers the RNA chain from the RNA-nsp9 species onto a GDP molecule, forming the core cap structure, GpppA. I characterized the reaction by performing cryo-EM analysis of the replication/transcription complex with nsp9 bound to the NiRAN active site
Impaired Control of Chronic Herpesvirus Infection in Tissue Resident Macrophages During Coinfection with Intestinal Parasite and Other Cell-intrinsic Herpesvirus Interactions
Get PDFGammaherpesviruses are chronic pathogens that infect lymphocytes, such as B cells, dendritic cells, and macrophages. Reactivation of gammaherpesviruses is thought to induce lymphomas and can be caused by a variety of signals, such as HDAC inhibitors. While some of these signals are known, it is still unclear whether there are cell-specific reactivation mechanisms, and if so, which signals cause them. Further, human gammaherpesviruses are endemic in areas that also have a high prevalence of helminth infections. Coinfection with parasites has been shown to induce murine gammaherpesvirus-68 (MHV68) reactivation in vivo, suggesting that coinfection with chronic pathogens is one mechanism herpesviruses can use to reactivate. To examine cell-specific MHV68 reactivation, we utilized conditional knockout mice to remove IL-4 receptor signaling on B cells or macrophages. We found that IL-4 signaling in combination with interferon-γ blockade reactivates MHV68 from latently infected macrophages, but not B cells. We continued our work on cell-specific reactivation signals by examining MHV68 infection in tissue resident peritoneal macrophages, called large peritoneal macrophages (LPMs), during coinfection with a parasite, Heligmosomoides polygyrus (HP). We found that coinfection with HP increases the number of MHV68-infected LPMs during acute replication and latency. Further, coinfected mice had increased ex vivo reactivation of MHV68. By using dietary and genetic depletion of the LPMs, we found that HP-induced expansion of the LPMs was necessary for the increased infection and reactivation. Overall, this work demonstrates a new role for LPMs during viral infection and highlights cell-specific mechanisms gammaherpesviruses use to reactivate from latency
Kidney Allograft Outcomes of Dual-Organ Heart Kidney Transplant Recipients Following Donation after Circulatory Determination of Death
Get PDFThe 63rd Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, January 28, 2025; 3-6 p.m.; D1.700 Lecture Hall)BACKGROUND: Dual Organ Heart Kidney (HK) transplants following donation after circulatory determination of death (DCD) are increasing. DCD donation has expanded the donor pool and reduced transplant wait times for critically ill recipients. Whether kidney allograft outcomes, including the need for dialysis and/or kidney transplant, differ between DCD and DBD HK recipients has not been well studied.
HYPOTHESIS: Kidney allograft outcomes in dual-organ heart-kidney DCD transplants will be non-inferior to DBD donors.
METHODS: Leveraging the UNOS database, we identified adults (≥ 18 years old) who were listed for dual-organ HK and transplanted after 10/18/2018. Baseline recipient and donor characteristics were compared between recipients of DBD and DCD donors. The primary outcome of interest was the composite of non-receipt of kidney allograft, acute or chronic dialysis, and kidney transplant. Univariate and multivariable logistic regression were used to identify risk factors for this composite outcome.
RESULTS: From October 2018 to June 2024, there were 2178 dual-organ HK recipients, with 2014 receiving allografts from DBD donors and 162 from DCD donors. Compared to DBD donors, DCD donors had significantly lower BUN and creatinine (Cr). DCD recipients were significantly older, listed at lower transplant status, and less likely to be on dialysis, on inotropes, or mechanical support with IABP before transplant. After a median follow-up of 1.2 years, there was no significant difference in the primary composite outcome between both groups. Donor age and terminal Cr were associated with the primary endpoint. Recipient risk factors included age, BMI, baseline Cr, total bilirubin (mg/dL), bridge to transplant with LVAD or VA-ECMO, and IV antibiotics at the time of transplant. These associations persisted in multivariable modeling. There was no difference in post-transplant survival at 1 year.
CONCLUSION: Kidney allograft outcomes are similar in DBD and DCD dual-organ HK recipients. Risk factors for unfavorable kidney allograft outcomes in both DBD and DCD recipients include donor and recipient baseline kidney function and recipient peri-operative hemodynamic status. These findings further support DCD in expanding the donor pool for multiorgan HK candidates.Southwestern Medical Foundatio
Transforming Single Ventricle Care: Early Successes of the Complex Biventricular Repair Program
Get PDFThe 63rd Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, January 28, 2025; 3-6 p.m.; D1.700 Lecture Hall)BACKGROUND: The Complex Biventricular Repair Program at Children's Health, Dallas, was launched in 2023 to assess patients with single ventricle heart disease for potential biventricular repair. This program aims to alleviate the limitations associated with single ventricle palliation (Norwood, Glenn, and Fontan) through ventricular recruitment and biventricular conversion.
HYPOTHESIS: Complex biventricular repair can provide a viable alternative to procedures that establish a single ventricle system, improving outcomes for patients with challenging cardiac anatomies and borderline ventricles.
METHODS: The program includes a comprehensive evaluation of patients with single ventricle heart disease, beginning with a baseline assessment of anatomy using transthoracic echocardiography and cardiac magnetic resonance imaging. Virtual and physical 3D models are generated to visualize the anatomy and plan surgical repairs. Based on each patient's needs, an individualized plan is developed, incorporating recruitment strategies to optimize ventricular size and function and considering surgical interventions, including both staged and direct biventricular conversions.
RESULTS: Since its inception, the program has evaluated 35 patients, performing 46 biventricular repair operations. Among 11 patients with complex anatomy, 14 operations were performed, with 0% mortality, 0% transplants, 0% ECMO, and 18% pacemaker implantation. For 11 patients with borderline left ventricles, 18 operations resulted in 0% mortality, 0% transplants, 0% ECMO, and 11% pacemaker implantation. In 13 patients with borderline right ventricles, 14 operations led to 0% mortality, 0% transplants, 14% ECMO, and 8% pacemaker implantation. Overall, median hospital stay was 10 days, and no patients experienced death, transplant, or biventricular takedowns. In total, 35 patients are expected to achieve a biventricular circulation without need for long-term single ventricle palliation.
CONCLUSION: The Complex Biventricular Repair Program has demonstrated promising results in treating patients with single ventricle heart disease. The program's approach has effectively reduced the reliance on the Fontan procedure, offering a new pathway for improved patient outcomes.Southwestern Medical Foundatio
Value Based Care in Transplantation: Is There Value in Volume? An Assessment of Liver Transplant Practices in the United States
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.BACKGROUND: Liver transplantation has increased in volume and provides substantial survival benefit. Value-based practice has become a central theme in many surgical fields but has not been well studied in liver transplantation. Given an increasing focus on optimizing outcomes with containing costs, defining value in liver transplantation warrants investigation.
OBJECTIVE: We hypothesize value in liver transplantation has improved over time, and centers that perform more transplants (i.e. high volume centers), deliver more value.
METHODS: MELD era adult (n=75,988) and pediatric (n=5,770) liver transplant recipients were identified using UNOS STARfile data and compared across time periods (Period A: 2/2002-1/2007, B: 2/2007-1/2013, C: 2/2013-1/2019). Status 1, multiorgan, living donor, lost to follow-up, retransplants, and recipients dying during index post-transplant hospitalization were excluded. Liver centers were divided into volume tertiles for each period (small, medium, large) based upon the cumulative number of transplants completed at each center during each analysis period, and pediatric recipients were further stratified by age (0-4, 5-11, 12-18 years). Value for the index transplant episode was defined as % graft survival ≥ 1 year divided by mean post-transplant length-of-stay (LOS). The statistical analyses were performed using Stata 16/MP4 (StataCorp LP, College Station, TX), and p values < 0.05 (2-tailed) were considered statistically significant. Nearest-neighbor Mahalanobis metric matching was used to account for confounding when assessing the impact of center volume on value.
RESULTS: Adult centers increased value over time due to ubiquitous improvement in 1-year graft survival. However, large centers demonstrated the most significant value change (large +17% vs small +7.0%, p<0.001) due to a -8.5% reduction in large centers LOS from Period A to C, while small centers LOS remained unchanged (-0.1%). Large centers delivered higher value despite more complex care: older recipients (54.8±10.3 vs 53.0±11.4 years p<0.001), fewer MELD exceptions (34.0% vs 38.2%, p<0.001), higher rates of candidate portal vein thrombosis (10.1% vs 8.5%, p<0.001) and prior abdominal surgery (43.4% vs 37.4%, p<0.001), and more marginal donor utilization (DRI 1.45±0.38 vs 1.36±0.33, p<0.001). Mahalanobis matching demonstrated that compared to small centers, large centers progressively shortened recipient LOS per transplant in each Period (A: -0.36 days, p=0.437) (B: -2.14 days, p<0.001) (C: -2.49 days, p<0.001). In pediatric transplantation, compared to small centers, large centers delivered better outcomes (1-year graft survival 93.7% vs 89.4%, p=0.017) without increased resource utilization (LOS 20.8±15.6 days vs 19.6±17.0, p=0.281) during the 17-year study period. Mahalanobois-matched cohorts demonstrated a volume-value relationship (higher value care with better outcomes and decreased resource utilization) in the 0-4 age group, but not in older recipients. The 0-4 age group comprised the largest proportion of Status 1B patients (21.8%, p<0.001) and the highest utilization rate of partial liver allografts (40.9%, p<0.001).
CONCLUSION: There is value in liver transplant volume. In the adult population, value was observed with volume, driven by lower resource utilization. In the pediatric population, value was observed with volume specifically in very young (0 - 4 y/o) patients, driven by better outcomes. High volume centers scale more rapidly to deliver value and drive resource utilization reduction without compromising graft survival, and adoption of value-based practices from large centers may allow optimization of health care delivery for this costly procedure. Further detailed study of the factors at large volume centers that enable greater healthcare value delivery is warranted
Gout 2025
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern’s Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Identifying Metabolic Liabilities in Hürthle Cell Thyroid Carcinoma
Get PDFA metabolic hallmark of cancer identified by Warburg is the increased consumption of glucose and secretion of lactate, even in the presence of oxygen. Although many tumors exhibit increased glycolytic activity, most forms of cancer rely on mitochondrial respiration for tumor growth. Hürthle cell carcinoma of the thyroid (HTC) represents an outlier wherein HTC tumors enrich for mitochondrial DNA mutations that are predicted to impair complex I of the electron transport chain (ETC). Consistent with these mutations, our work shows that HTC models harboring mitochondrial DNA-encoded defects in complex I of the mitochondrial electron transport chain exhibit impaired respiration and alterations in central carbon metabolism. We investigated whether the metabolic adaptations to complex I deficiency constitute HTC-specific vulnerabilities that could be therapeutically targeted. Our initial studies demonstrated that aspartate biosynthesis, a key output of respiration in proliferation cells, was altered in complex I-deficient HTC cells. Genetic disruption of alternative aspartate biosynthetic pathways selectively impaired HTC cell growth in culture; however, inhibition of these pathways did not impair HTC growth in vivo. We utilized proliferation-based CRISPR-Cas9 pooled screening to identify additional HTC-specific metabolic liabilities and found that glycolytic enzymes are selectively essential in complex I-mutant HTC cells. The respiratory defects and shifts to glycolytic metabolism found in complex I-deficient HTC cells enforce a reliance on fermentation that is targetable with small molecule inhibitors of lactate dehydrogenase (LDH) both in cultured cells and in vivo models. Using forward genetic screens, we identified mechanisms of resistance to LDH inhibitor treatment including inhibitor-specific mutations and upregulation of LDH isoforms. Combined, this work demonstrates that complex I loss exposes fermentation as a therapeutic target in HTC and has implications for other tumors bearing mutations that irreversibly damage mitochondrial respiration
The Role of Dispositional Optimism in Interdisciplinary Chronic Pain Management
Get PDFBACKGROUND: Millions of US adults suffer from chronic pain, affecting their quality of life and costing hundreds of billions of dollars in lost productivity annually. Interdisciplinary chronic pain management programs are backed by decades of research. However, unworkable insurance policies and high attrition rates have undermined their effectiveness and generalizability.
OBJECTIVE: Optimism has been associated with adaptive coping. Although prior research has examined the association between optimism and chronic pain, the impact of optimism in interdisciplinary treatment merits additional exploration. This study examined the role of dispositional optimism in interdisciplinary chronic pain management, including treatment completion.
METHODS: A total of 178 participants were enrolled in the interdisciplinary program at the McDermott Center for Pain Management. Measures related to chronic pain and psychological functioning were gathered at treatment intake, midpoint (2 weeks), and endpoint (4 weeks).
ANALYSES: Binomial logistic and Poisson regressions were utilized to predict program completion and attendance, respectively, using dispositional optimism, with covariates of age, opioid use, and employment. Additional attempts were made to examine the mediating effect of attendance on the association between dispositional optimism and chronic pain outcomes.
RESULTS: Participants who were older, employed, and taking opioid medication at intake were more likely to attend more sessions, whereas those who were employed and using opioid medication at intake were more likely to complete the program. Surprisingly, dispositional optimism neither predicted treatment completion nor was associated with attendance. Mediation analyses were also inconclusive.
DISCUSSION: It is possible that optimists' active role in illness prevention may have equally driven them to seek treatment or reduce their perceived need for treatment. Additionally, some optimistic participants may have practiced emotion-focused coping (e.g., acceptance), while others may have relied on problem-focused coping (e.g., seeking support in treatment), leading to inconclusive results. Participants who used opioids at intake had increased likelihood of program completion and attendance, possibly due to increased motivation after learning the deleterious effects of long-term opioid use. Participants who were employed had a higher likelihood of completion. Conversely, any cause of unemployment could have contributed to dropout (e.g., lack of transportation, physical disability, intensity of pain, cognitive difficulties, etc.)
RNA Exonuclease Xrn1 Regulates TORC1 and Autophagy in Response to SAM Availability
Get PDFDuring methionine deprivation, yeast cells experience loss of S-adenosyl methionine (SAM), resulting in globally reduced histone methylation levels. Investigations into the location of the enduring histone methylation initiated our studies into Y' elements, subtelomeric noncoding RNAs that we have found are highly expressed under this condition. Their accumulation following loss of methionine is caused by reduced degradation by the conserved 5'-3' exonuclease Xrn1, rather than increased transcription during this condition. The finding that Xrn1 has altered activity under methionine deprivation led to investigate how Xrn1 may sense the change in nutrient availability.
Autophagy is a conserved process of cellular self-digestion that promotes survival during nutrient stress. In yeast, methionine starvation is sufficient to induce autophagy. One pathway of autophagy induction is governed by the SEACIT complex, which regulates TORC1 activity in response to amino acids through the Rag GTPases Gtr1 and Gtr2. However, the precise mechanism by which SEACIT senses amino acids and regulates TORC1 signaling remains incompletely understood. We identified Xrn1 as a surprising and novel regulator of TORC1 activity in response to methionine starvation. This role of Xrn1 is dependent on its catalytic activity, but not on degradation of any specific class of mRNAs. Instead, Xrn1 modulates the nucleotide-binding state of the Gtr1/2 complex, which is key for its interaction with and activation of TORC1. This work identifies a critical role for Xrn1 in nutrient sensing and growth control that extends beyond its canonical housekeeping function in RNA degradation and indicates an avenue for RNA metabolism to function in amino acid signaling into TORC1
It's a Girl!
Get PDFThe excitement or being a first daughter, ridicule of being a little girl, and fear of being a woman