Human REV3L: Expression and Protein Interaction Studies

Human REV3L: Expression and Protein Interaction StudiesGregory N. Gan, Ph.D.University of Pittsburgh, 2007REV3L is a specialized DNA polymerase essential for DNA damage-induced mutagenesis and for the ability of cells to tolerate DNA damage. Our understanding of REV3L biochemistry stems predominantly from studies done with the budding yeast homolog, Rev3. Yeast DNA polymerase zeta consists of two proteins, Rev3, the catalytic subunit, and Rev7, an accessory factor which enhances the activity of Rev3, in vitro. Yeast Rev1 acts as a scaffold by associating with Polymerase zeta and enhances its translesion bypass activity on a mismatch primer template. Because of the large size of the mammalian REV3L cDNA (10.6 kbp) and protein (353 kDa), work in this field has focused solely on functional genetic studies associated with disruption or knockdown of the gene. Loss of REV3L causes embryonic lethality in mice and leads to progressive chromosomal instability in Rev3L disrupted cell lines. In the developing mouse embryo, Rev3L transcript is found in all tissues. However, its expression pattern at the cellular level in the adult mouse has not been examined. Determining the protein interactions of REV3L will provide a better understanding of how the protein functions at the molecular level. In addition, elucidating how Rev3L is expressed and regulated in mammalian cells will indicate what role it may play in tissues of the adult organism and why it is essential for life. In order to study human REV3L biochemistry, this project focused on cloning, expressing, purifying and detecting full-length human REV3L protein. Human REV3L was hypothesized to interact with REV1 and/or REV7 based on knowledge about te yeast homologs. Furthermore, using a REV3L lacZ expression mouse model, the expression of REV3L in mouse organs containing proliferative cells was characterized. It was hypothesized that organs with highly proliferative tissue require REV3L. First, the results of immunoprecipitation studies demonstrated that full-length human REV3L interacts with REV1, but does not interact with REV7 in a DNA damage independent fashion. Preliminary analysis of deletion mutants indicates that the C-terminal domain of REV1 is required for the protein-protein interaction. Secondly, REV1 and REV3L are ubiquitinated in a DNA damage independent fashion and this covalent modification is not required for REV1-REV3L interaction. Finally, REV3L expression in mice is highest in testis, cardiac tissue and the smooth musculature of lung and intestines and low in lymphoid tissues. These sites of expression suggest that REV3L may be important for highly oxidative tissue compared to proliferative tissue. In summary, this dissertation provides insight on human REV3L's protein-protein interaction with REV1 and REV7; their post-translational modification, and tissue-specific expression pattern in the adult mouse

Exploring the function of myeloid cells in promoting metastasis in head and neck cancer

Head and neck cancer (HNC) is a challenging disease that lacks effective treatment, particularly in the cases that spread locoregionally and metastasize distantly, dramatically reducing patient survival rates. Expanding the understanding of the mechanisms of the metastatic cascade is critical for creating more effective therapeutics that improve outcomes for HNC patients. A true grasp of cancer metastasis requires the consideration of all cell types that contribute to the inflammatory HNC microenvironment as drivers of this process. More emphasis now is being placed on exploring the roles of the different immune cells in cancer control, tumorigenesis and metastasis. Myeloid cells are the most numerous immune cell types in the body, and they are actively recruited and reprogrammed by tumor cells to behave in a variety of ways. These cells are remarkably diverse in phenotype and function, and the part they play in tumor spread greatly differs based on the cell type. This review will focus on summarizing the roles of macrophages, neutrophils, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs) in driving HNC metastasis by examining the current knowledge base and offering potential new routes through which to target and treat this deadly process

Microfluidic affinity selection of active SARS-CoV-2 virus particles

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus’s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip’s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent

On-chip picosecond pulse detection and generation using graphene photoconductive switches

We report on the use of graphene for room temperature on-chip detection and generation of pulsed terahertz (THz) frequency radiation, exploiting the fast carrier dynamics of light-generated hot carriers, and compare our results with conventional low-temperature-grown gallium arsenide (LT-GaAs) photoconductive (PC) switches. Coupling of picosecond-duration pulses from a biased graphene PC switch into Goubau line waveguides is also demonstrated. A Drude transport model based on the transient photoconductance of graphene is used to describe the mechanism for both detection and generation of THz radiation

Functional Dicer Is Necessary for Appropriate Specification of Radial Glia during Early Development of Mouse Telencephalon

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon

Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition

Polycomb repressive complex 2 (PRC2) is a key regulator of epigenetic states catalyzing histone H3 lysine 27 trimethylation (H3K27me3), a repressive chromatin mark. PRC2 composition is conserved from humans to plants, but the function of PRC2 during the early stage of plant life is unclear beyond the fact that it is required for the development of endosperm, a nutritive tissue that supports embryo growth. Circumventing the requirement of PRC2 in endosperm allowed us to generate viable homozygous null mutants for FERTILIZATION INDEPENDENT ENDOSPERM (FIE), which is the single Arabidopsis homolog of Extra Sex Combs, an indispensable component of Drosophila and mammalian PRC2. Here we show that H3K27me3 deposition is abolished genome-wide in fie mutants demonstrating the essential function of PRC2 in placing this mark in plants as in animals. In contrast to animals, we find that PRC2 function is not required for initial body plan formation in Arabidopsis. Rather, our results show that fie mutant seeds exhibit enhanced dormancy and germination defects, indicating a deficiency in terminating the embryonic phase. After germination, fie mutant seedlings switch to generative development that is not sustained, giving rise to neoplastic, callus-like structures. Further genome-wide studies showed that only a fraction of PRC2 targets are transcriptionally activated in fie seedlings and that this activation is accompanied in only a few cases with deposition of H3K4me3, a mark associated with gene activity and considered to act antagonistically to H3K27me3. Up-regulated PRC2 target genes were found to act at different hierarchical levels from transcriptional master regulators to a wide range of downstream targets. Collectively, our findings demonstrate that PRC2-mediated regulation represents a robust system controlling developmental phase transitions, not only from vegetative phase to flowering but also especially from embryonic phase to the seedling stage

Selective Serotonin Reuptake Inhibitor Use Is Associated with Right Ventricular Structure and Function: The MESA-Right Ventricle Study

PURPOSE:Serotonin and the serotonin transporter have been implicated in the development of pulmonary hypertension (PH). Selective serotonin reuptake inhibitors (SSRIs) may have a role in PH treatment, but the effects of SSRI use on right ventricular (RV) structure and function are unknown. We hypothesized that SSRI use would be associated with RV morphology in a large cohort without cardiovascular disease (N = 4114). METHODS:SSRI use was determined by medication inventory during the Multi-Ethnic Study of Atherosclerosis baseline examination. RV measures were assessed via cardiac magnetic resonance imaging. The cross-sectional relationship between SSRI use and each RV measure was assessed using multivariable linear regression; analyses for RV mass and end-diastolic volume (RVEDV) were stratified by sex. RESULTS:After adjustment for multiple covariates including depression and left ventricular measures, SSRI use was associated with larger RV stroke volume (RVSV) (2.75 mL, 95% confidence interval [CI] 0.48-5.02 mL, p = 0.02). Among men only, SSRI use was associated with greater RV mass (1.08 g, 95% CI 0.19-1.97 g, p = 0.02) and larger RVEDV (7.71 mL, 95% 3.02-12.40 mL, p = 0.001). SSRI use may have been associated with larger RVEDV among women and larger RV end-systolic volume in both sexes. CONCLUSIONS:SSRI use was associated with higher RVSV in cardiovascular disease-free individuals and, among men, greater RV mass and larger RVEDV. The effects of SSRI use in patients with (or at risk for) RV dysfunction and the role of sex in modifying this relationship warrant further study

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Competence in Endoscopic Ultrasound and Endoscopic Retrograde Cholangiopancreatography, From Training Through Independent Practice.

BACKGROUND & AIMS: It is unclear whether participation in competency-based fellowship programs for endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP) results in high-quality care in independent practice. We measured quality indicator (QI) adherence during the first year of independent practice among physicians who completed endoscopic training with a systematic assessment of competence. METHODS: We performed a prospective multicenter cohort study of invited participants from 62 training programs. In phase 1, 24 advanced endoscopy trainees (AETs), from 20 programs, were assessed using a validated competence assessment tool. We used a comprehensive data collection and reporting system to create learning curves using cumulative sum analysis that were shared with AETs and trainers quarterly. In phase 2, participating AETs entered data into a database pertaining to every EUS and ERCP examination during their first year of independent practice, anchored by key QIs. RESULTS: By the end of training, most AETs had achieved overall technical competence (EUS 91.7%, ERCP 73.9%) and cognitive competence (EUS 91.7%, ERCP 94.1%). In phase 2 of the study, 22 AETs (91.6%) participated and completed a median of 136 EUS examinations per AET and 116 ERCP examinations per AET. Most AETs met the performance thresholds for QIs in EUS (including 94.4% diagnostic rate of adequate samples and 83.8% diagnostic yield of malignancy in pancreatic masses) and ERCP (94.9% overall cannulation rate). CONCLUSIONS: In this prospective multicenter study, we found that although competence cannot be confirmed for all AETs at the end of training, most meet QI thresholds for EUS and ERCP at the end of their first year of independent practice. This finding affirms the effectiveness of training programs. Clinicaltrials.gov ID NCT02509416

Competence in Endoscopic Ultrasound and Endoscopic Retrograde Cholangiopancreatography, From Training Through Independent Practice.

BACKGROUND & AIMS: It is unclear whether participation in competency-based fellowship programs for endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP) results in high-quality care in independent practice. We measured quality indicator (QI) adherence during the first year of independent practice among physicians who completed endoscopic training with a systematic assessment of competence. METHODS: We performed a prospective multicenter cohort study of invited participants from 62 training programs. In phase 1, 24 advanced endoscopy trainees (AETs), from 20 programs, were assessed using a validated competence assessment tool. We used a comprehensive data collection and reporting system to create learning curves using cumulative sum analysis that were shared with AETs and trainers quarterly. In phase 2, participating AETs entered data into a database pertaining to every EUS and ERCP examination during their first year of independent practice, anchored by key QIs. RESULTS: By the end of training, most AETs had achieved overall technical competence (EUS 91.7%, ERCP 73.9%) and cognitive competence (EUS 91.7%, ERCP 94.1%). In phase 2 of the study, 22 AETs (91.6%) participated and completed a median of 136 EUS examinations per AET and 116 ERCP examinations per AET. Most AETs met the performance thresholds for QIs in EUS (including 94.4% diagnostic rate of adequate samples and 83.8% diagnostic yield of malignancy in pancreatic masses) and ERCP (94.9% overall cannulation rate). CONCLUSIONS: In this prospective multicenter study, we found that although competence cannot be confirmed for all AETs at the end of training, most meet QI thresholds for EUS and ERCP at the end of their first year of independent practice. This finding affirms the effectiveness of training programs. Clinicaltrials.gov ID NCT02509416