Maximizing Black applicant matriculation in U.S. PA programs: associations between the number of submitted applications and likelihood of matriculation

Background: Physician Assistants (PA) are important members of the medical team, and increasing diversity in healthcare professionals has been consistently associated with improved health outcomes for underrepresented minority patients. In this study of a national cohort of PA program applicants, we investigated whether the number of programs a student applied to (Application Number, AN) was significantly associated with increased likelihood of matriculation into a PA program. Methods: We examined all applications (n = 27,282) to the 2017–2018 admissions cycle of the Central Application Service for Physician Assistants, which is utilized by over 90% of accredited PA programs in the US. As we a priori hypothesized that associations would be non-linear, we used natural cubic splines to estimate the associations between matriculation and AN, controlling for multiple metrics of academic achievement, experience, and applicant demographics. We subsequently used segmented regression analyses (modified poisson regression with robust error variance) to investigate log-linear associations above and below inflection points identified in the spline analyses. Additionally, we explored for effect modification by race/ethnicity. Results: The strongest associations were observed between application number 2–7, and a threshold effect was observed at \u3e 16 applications, beyond which there was no significant, incremental benefit in matriculation likelihood. Associations differed by race, particularly for application number 2–7, wherein the incremental benefit from each additional application was highest for Black applicants (Likelihood Ratio [LR]: 1.243, 95% CI: 1.136 to 1.360) vs non-Latinx White (LR: 1.098, 95% CI: 1.072 to 1.125), with no additional, incremental benefit beyond 7 program applications. For all other races, significant increased likelihoods of matriculation were observed until 16 program applications. Conclusions: These findings can help guide pre-PA advisors and PA programs, providing recommended thresholds to applicants on the most cost effective ways to increase their likelihood of admissions, and the PA profession as a whole by providing actionable information that can potentially increase Race/Ethnic diversity in the PA profession and, by extension, medical teams

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma

Differential impacts of particulate air pollution exposure on early and late stages of spermatogenesis

Background: Despite increasing evidence that particulate air pollution has adverse effects on human semen quality, few studies examine the impact of air pollution on clinically relevant thresholds used to diagnose male fertility problems. Furthermore, exposure is often assessed using average air pollution levels in a geographic area rather than individualized estimates. Finally, physiologically-informed exposure windows are inconsistent. Objectives: We sought to test the hypothesis that airborne particulate exposures during early-phase spermatogenesis will have a differential impact on spermatogenic formation compared to late-phase exposures, using an individualized model of exposure to particulate matter ≤ 2.5 µm and ≤ 10 µm (PM2.5 and PM10, respectively). Methods: From an original cohort of 183 couples, we conducted a retrospective analysis of 130 healthy males seeking to become parents, using spermatogenesis-relevant exposure windows of 77–34 days and 37–0 days prior to semen collection to encompass sperm development stages of mitosis/meiosis and spermiogenesis, respectively. Individualized residential exposure to PM2.5 and PM10 was estimated by selecting multiple air pollution sensors within the same geographic air basin as participants and employing inverse distance weighting to calculate mean daily exposure levels. We used multiple logistic regression to assess the association between pollution, temperature, and dichotomized World Health Organization semen parameters. Results: During the early phase of spermatogenesis, air pollution exposure is associated with 1.52 (95% CI: 1.04–2.32) times greater odds of < 30% normal heads per 1-unit increase in IQR for PM2.5. In the late phase of spermatogenesis, air pollution exposure is associated with 0.35 (95% CI: 0.10–0.74) times greater odds of semen concentration < 15 million/mL per 1-unit increase in IQR for PM2.5, and 0.28 (95% CI: 0.07–0.72) for PM10. Conclusion: Particulate exposure has a differential and more deleterious impact on sperm during early-phase spermatogenesis than late-phase

Prolyl-4-Hydroxylase 3 (PHD3) Expression Is Downregulated during Epithelial-to-Mesenchymal Transition

<div><p>Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O₂. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O₂ and the migratory and metastatic potential of tumor cells.</p></div

PHD3 expression in BxPC3 cells.

<p>BxPC3 cells stably transduced with retrovirus containing PHD3Wt (BxPC3-Wt), Vector (BxPC3-Vec) or anti-PHD3 shRNA (BxPC3-KD) were harvested for RNA and protein following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) PHD3 mRNA expression was determined by qRT-PCR in BxPC3-Vec and BxPC3-KD cells. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3-Vec Normoxia (lane 1). n = 3, error bars  = 1 S.D. p-value represents Student's 2-tailed, type 2 t-test comparison (B) Whole cell lysate from BxPC3-Wt, BxPC3-Vec and BxPC3-KD cells following 24 hours exposure to normoxia (N) or hypoxia (H) was resolved by SDS-PAGE and blotted for actin (top) and PHD3 (bottom). PHD3 band intensity was quantified relative to actin in each lane and then normalized to BxPC3-Vec (N). Relative band intensity is indicated below the figure. NS  =  non-specific band running just above PHD3 band. Data is representative of >3 biological replicates.</p

Flow cytometric analysis of E-cadherin in MDCK cells. (A) MDCK cells (parental population) were labeled with phalloidin (red) and anti-E-cadherin antibody (green) and visualized by confocal microscopy.

<p>Arrow indicates E-cadherin negative cells within the parental population. (B) Live MDCK cells (parental population) were labeled with primary anti-E-cadherin (red) or isotype-matched control antibody (blue), then with goat anti-mouse Alexa-fluor 488 (A488)-conjugated secondary antibody and analyzed by flow cytometry. (C) Flow chart for separation of mesenchymal and epithelial subpopulations of the MDCK parental cell line. (D) MDCK-L and MDCK-E3 cell lines were analyzed for surface E-cadherin expression by flow cytometry as done in (A). (E) MDCK-L and MDCK-E3 cells were subjected to normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours. mRNA was harvested and PHD3 and E-cadherin expression was quantified by qRT-PCR. All data points represent the average of 3 biological replicates. Quantification of mRNA is set relative to MDCK-E3 samples at normoxia. Error bars  = +/− 1 S.D.</p

PHD3 expression correlates with a mesenchymal-like morphology in pancreatic ductal adenocarcinoma cell lines.

<p>NHF-1 (Fibroblast) MiaPaca2, Panc1, CAPAN1 and BxPC3 cells were harvested for RNA and protein following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) Phase-contrast images at 10× magnification were taken of MiaPaca2 (mesenchymal-like) morphology and BxPC3 cells (differentiated, epithelial morphology) under normoxic conditions. (B) PHD3 mRNA expression was determined by qRT-PCR and graphed relative to BxPC3 in normoxia. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3-Vec Normoxia (lane 1). n = 3, error bars  = 1 S.D. (C) Whole cell lysate was resolved by SDS-PAGE and blotted for β-tubulin PHD3 and PHD2. N = normoxia, H = hypoxia (1% O₂).</p

PHD3 knockdown increases the migratory capacity of cells.

<p>BxPC3 cells stably transduced with retrovirus containing PHD3Wt (BxPC3-Wt), Vector (BxPC3-Vec) or anti-PHD3 shRNA (BxPC3-KD) were seeded at confluence in 60 mm tissue culture dishes and allowed to adhere for 24 hours. (A) Scratches were made using a 5 ml stripette and photographed at 0 hr and 24 hr under normoxia (21% O₂) or hypoxia (1% O₂). Arrows highlight the directional migration of cells (B) Migration speed for each cell line was normalized to BxPC3-Vec normoxia. Data is representative of 3 independent biological replicates and 8 scratches each. Error bars  = 1 S.D. p-value represents Student's 2-tailed, type 2 t-test comparison. * samples are significantly different p<.01 than all other samples. (C) Live cells were counted using trypan blue exclusion at the indicated time points. Lines represent best fit for the data.</p

SNAIL-induced EMT in MDCKE3 Cells.

<p>(A–D). Stable pQCXIP-SNAIL (hSNAIL) or pQCXIP-vector (Vector)-expressing MDCK-E3 cells were subjected to normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours. mRNA was harvested and subjected to qRT-PCR analysis for the indicated genes. All data points represent the average of 3 biological replicates. mRNA quantification is set relative to the Vector samples at normoxia. Error bars  = 1 S.D.</p

E-cadherin and PHD3 expression are inversely correlated with SNAIL expression.

<p>NHF-1 (fibroblast), MiaPaca2, Panc1, CAPAN1, and BxPC3 cells were harvested for RNA following 24 hours exposure to normoxia (21% O₂) or hypoxia (1% O₂). (A) E-cadherin and SNAIL mRNA expression was determined by qRT-PCR. All samples were normalized to 18S rRNA and graphed as expression relative to BxPC3 (lane 9). n = 3, error bars  = 1 S.D. (B) Hypoxic SNAIL mRNA expression was graphed relative to hypoxic PHD3 expression as was determined in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083021#pone-0083021-g004" target="_blank">Figure 4B</a>). ρ = Pearson's correlation coefficient. Line represents best fit for the data.</p