Publications by authors named "Shesh N Rai"

156 Publications

Evidence and magnitude of the effects of meteorological changes on SARS-CoV-2 transmission.

PLoS One 2021 17;16(2):e0246167. Epub 2021 Feb 17.

Christina Lee Brown Envirome Institute, University of Louisville School of Medicine, Louisville, Kentucky, United States of America.

Importance: Intensity and duration of the COVID-19 pandemic, and planning required to balance concerns of saving lives and avoiding economic collapse, could depend significantly on whether SARS-CoV-2 transmission is sensitive to seasonal changes.

Objective: Hypothesis is that increasing temperature results in reduced SARS CoV-2 transmission and may help slow the increase of cases over time.

Setting: Fifty representative Northern Hemisphere countries meeting specific criteria had sufficient COVID-19 case and meteorological data for analysis.

Methods: Regression was used to find the relationship between the log of number of COVID-19 cases and temperature over time in 50 representative countries. To summarize the day-day variability, and reduce dimensionality, we selected a robust measure, Coefficient of Time (CT), for each location. The resulting regression coefficients were then used in a multivariable regression against meteorological, country-level and demographic covariates.

Results: Median minimum daily temperature showed the strongest correlation with the reciprocal of CT (which can be considered as a rate associated with doubling time) for confirmed cases (adjusted R2 = 0.610, p = 1.45E-06). A similar correlation was found using median daily dewpoint, which was highly colinear with temperature, and therefore was not used in the analysis. The correlation between minimum median temperature and the rate of increase of the log of confirmed cases was 47% and 45% greater than for cases of death and recovered cases of COVID-19, respectively. This suggests the primary influence of temperature is on SARS-CoV-2 transmission more than COVID-19 morbidity. Based on the correlation between temperature and the rate of increase in COVID-19, it can be estimated that, between the range of 30 to 100 degrees Fahrenheit, a one degree increase is associated with a 1% decrease-and a one degree decrease could be associated with a 3.7% increase-in the rate of increase of the log of daily confirmed cases. This model of the effect of decreasing temperatures can only be verified over time as the pandemic proceeds through colder months.

Conclusions: The results suggest that boreal summer months are associated with slower rates of COVID-19 transmission, consistent with the behavior of a seasonal respiratory virus. Knowledge of COVID-19 seasonality could prove useful in local planning for phased reductions social interventions and help to prepare for the timing of possible pandemic resurgence during cooler months.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888632PMC
February 2021

Residential proximity to greenness mitigates the hemodynamic effects of ambient air pollution.

Am J Physiol Heart Circ Physiol 2021 Mar 8;320(3):H1102-H1111. Epub 2021 Jan 8.

Christina Lee Brown Envirome Institute, University of Louisville, Louisville, Kentucky.

Residential proximity to greenness is associated with a lower risk of cardiovascular disease (CVD) and all-cause mortality. However, it is unclear whether the beneficial effects of greenness are linked to a reduction in the effects of ambient air pollutants. We measured arterial stiffness in 73 participants with moderate to high CVD risk. Average levels of ambient PM and ozone were calculated from local monitoring stations. Residential greenness was estimated using satellite-derived normalized difference vegetation index (NDVI) for a 200-m and 1-km radius around each participant's home. Participants were 51% female, average age of 52 yr, and 79% had diagnosed hypertension. In multiple linear regression models, residential NDVI was negatively associated with augmentation index (-3.8% per 0.1 NDVI). Ambient levels of PM [per interquartile range (IQR) of 6.9 μg/m] were positively associated with augmentation pressure (3.1 mmHg), pulse pressure (5.9 mmHg), and aortic systolic pressure (8.1 mmHg). Ozone (per IQR of 0.03 ppm) was positively associated with augmentation index (5.5%), augmentation pressure (3.1 mmHg), and aortic systolic pressure (10 mmHg). In areas of low greenness, both PM and ozone were positively associated with pulse pressure. Additionally, ozone was positively associated with augmentation pressure and systolic blood pressure. However, in areas of high greenness, there was no significant association between indices of arterial stiffness with either PM or ozone. Residential proximity to greenness is associated with lower values of arterial stiffness. Residential greenness may mitigate the adverse effects of PM and ozone on arterial stiffness. Previous studies have linked proximity to green spaces with lower cardiovascular disease risk. However, the mechanisms underlying the salutary effects of green areas are not known. In our study of participants at risk of cardiovascular disease, we found that arterial stiffness was positively associated with short-term exposure to PM, PM, and ozone and inversely associated with greenness. The association between pollution and arterial stiffness was attenuated in areas of high greenness, suggesting that living green neighborhoods can lessen the adverse cardiovascular effects of air pollution.
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http://dx.doi.org/10.1152/ajpheart.00689.2020DOI Listing
March 2021

Age-related transcriptome changes in melanoma patients with tumor-positive sentinel lymph nodes.

Aging (Albany NY) 2020 12 29;12(24):24914-24939. Epub 2020 Dec 29.

The Hiram C. Polk, Jr., MD. Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40292, USA.

Age is an important factor for determining the outcome of melanoma patients. Sentinel lymph node (SLN) status is also a strong predictor of survival for melanoma. Paradoxically, older melanoma patients have a lower incidence of SLN metastasis but a higher mortality rate when compared with their younger counterparts. The mechanisms that underlie this phenomenon remain unknown. This study uses three independent datasets of RNA samples from patients with melanoma metastatic to the SLN to identify age-related transcriptome changes in SLNs and their association with outcome. Microarray was applied to the first dataset of 97 melanoma patients. NanoString was performed in the second dataset to identify the specific immune genes and pathways that are associated with recurrence in younger versus older patients. qRT-PCR analysis was used in the third dataset of 36 samples to validate the differentially expressed genes (DEGs) from microarray and NanoString. These analyses show that FOS, NR4A, and ITGB1 genes were significantly higher in older melanoma patients with positive SLNs. IRAK3- and Wnt10b-related genes are the major pathways associated with recurrent melanoma in younger and older patients with tumor-positive SLNs, respectively. This study aims to elucidate age-related differences in SLNs in the presence of nodal metastasis.
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http://dx.doi.org/10.18632/aging.202435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803563PMC
December 2020

Protocol to assess the efficacy of carnosine supplementation in mitigating the adverse cardiovascular responses to particulate matter (PM) exposure: the Nucleophilic Defense Against PM Toxicity (NEAT) trial.

BMJ Open 2020 12 28;10(12):e039118. Epub 2020 Dec 28.

Division of Environmental Medicine, Christina Lee Brown Envirome Institute, University of Louisville, Louisville, Kentucky, USA.

Introduction: Exposure to airborne particulate matter (PM) is associated with cardiovascular disease. These outcomes are believed to originate from pulmonary oxidative stress and the systemic delivery of oxidised biomolecules (eg, aldehydes) generated in the lungs. Carnosine is an endogenous di-peptide (β-alanine-L-histidine) which promotes physiological homeostasis in part by conjugating to and neutralising toxic aldehydes. We hypothesise that an increase of endogenous carnosine by dietary supplementation would mitigate the adverse cardiovascular outcomes associated with PM exposure in humans.

Methods And Analysis: To test this, we designed the Nucleophilic Defense Against PM Toxicity trial. This trial will enroll 240 participants over 2 years and determine if carnosine supplementation mitigates the adverse effects of PM inhalation. The participants will have low levels of endogenous carnosine to facilitate identification of supplementation-specific outcomes. At enrollment, we will measure several indices of inflammation, preclinical cardiovascular disease and physical function. Participants will be randomly allocated to carnosine or placebo groups and instructed to take their oral supplement for 12 weeks with two return clinical visits and repeated assessments during times of peak PM exposure (June-September) in Louisville, Kentucky, USA. Statistical modelling approaches will be used to assess the efficacy of carnosine supplementation in mitigating adverse outcomes.

Ethics And Dissemination: This study protocol has been approved by the Institutional Review Board at the University of Louisville. Results from this study will be disseminated at scientific conferences and in peer-reviewed publications. NCT03314987; Pre-results.
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http://dx.doi.org/10.1136/bmjopen-2020-039118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772308PMC
December 2020

Statistical Approach for Biologically Relevant Gene Selection from High-Throughput Gene Expression Data.

Entropy (Basel) 2020 Oct 25;22(11). Epub 2020 Oct 25.

Biostatistics and Bioinformatics Facility, JG Brown Cancer Center, University of Louisville, Louisville, KY 40292, USA.

Selection of biologically relevant genes from high-dimensional expression data is a key research problem in gene expression genomics. Most of the available gene selection methods are either based on relevancy or redundancy measure, which are usually adjudged through post selection classification accuracy. Through these methods the ranking of genes was conducted on a single high-dimensional expression data, which led to the selection of spuriously associated and redundant genes. Hence, we developed a statistical approach through combining a support vector machine with Maximum Relevance and Minimum Redundancy under a sound statistical setup for the selection of biologically relevant genes. Here, the genes were selected through statistical significance values and computed using a nonparametric test statistic under a bootstrap-based subject sampling model. Further, a systematic and rigorous evaluation of the proposed approach with nine existing competitive methods was carried on six different real crop gene expression datasets. This performance analysis was carried out under three comparison settings, i.e., subject classification, biological relevant criteria based on quantitative trait loci and gene ontology. Our analytical results showed that the proposed approach selects genes which are more biologically relevant as compared to the existing methods. Moreover, the proposed approach was also found to be better with respect to the competitive existing methods. The proposed statistical approach provides a framework for combining filter and wrapper methods of gene selection.
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http://dx.doi.org/10.3390/e22111205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712650PMC
October 2020

Fifteen Years of Gene Set Analysis for High-Throughput Genomic Data: A Review of Statistical Approaches and Future Challenges.

Entropy (Basel) 2020 Apr 10;22(4). Epub 2020 Apr 10.

School of Interdisciplinary and Graduate Studies, University of Louisville, Louisville, KY 40292, USA.

Over the last decade, gene set analysis has become the first choice for gaining insights into underlying complex biology of diseases through gene expression and gene association studies. It also reduces the complexity of statistical analysis and enhances the explanatory power of the obtained results. Although gene set analysis approaches are extensively used in gene expression and genome wide association data analysis, the statistical structure and steps common to these approaches have not yet been comprehensively discussed, which limits their utility. In this article, we provide a comprehensive overview, statistical structure and steps of gene set analysis approaches used for microarrays, RNA-sequencing and genome wide association data analysis. Further, we also classify the gene set analysis approaches and tools by the type of genomic study, null hypothesis, sampling model and nature of the test statistic, etc. Rather than reviewing the gene set analysis approaches individually, we provide the generation-wise evolution of such approaches for microarrays, RNA-sequencing and genome wide association studies and discuss their relative merits and limitations. Here, we identify the key biological and statistical challenges in current gene set analysis, which will be addressed by statisticians and biologists collectively in order to develop the next generation of gene set analysis approaches. Further, this study will serve as a catalog and provide guidelines to genome researchers and experimental biologists for choosing the proper gene set analysis approach based on several factors.
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http://dx.doi.org/10.3390/e22040427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516904PMC
April 2020

Circulating plasma microRNAs in colorectal neoplasia: A pilot study in assessing response to therapy.

Transl Oncol 2021 Jan 4;14(1):100962. Epub 2020 Dec 4.

Price Institute of Surgical Research, The Hiram C. Polk Jr. MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40292, USA. Electronic address:

Introduction: Current serological surveillance markers to monitor colorectal cancer (CRC) or colorectal advanced adenomas (CAA) are hampered by poor sensitivity and specificity. The aim of this study is to identify and validate a panel of plasma microRNAs which change in expression after resection of such lesions.

Methods: A prospectively maintained colorectal surgery database was queried for patients in whom both pre- and post-procedural serum samples had been obtained. An initial screening analysis of CRC and CAA patients (5 each) was conducted using screening cards for 380 miRNAs. Four identified miRNAs were combined with a previously described panel of 7 miRNAs that were diagnostically predictive of CRC and CAA. Differential miRNA expression was assessed using quantitative real-time polymerase chain reaction(qRT-PCR).

Results: Fifty patients were included (n = 27 CRC, n = 23 CAA). There was no difference in age, gender, or race profile of CRC patients compared to CAA patients. Six miRNA were significantly increased after CRC resection (miR-324, let7b, miR-454, miR-374a, miR-122, miR-19b, all p<0.05), while three miRNAs were significantly increased following CAA resection (miR-454, miR-374a, miR-122, all p<0.05). Three miRNA were increased in common for both (miR-454, miR-374a, miR-122).

Discussion: The expression of miRNAs associated with neoplasia (either CRC or CAA) was significantly increased following surgical resection or endoscopic removal of CRC or CAA. Future studies should focus on the evaluation of these miRNAs in CRC and CAA prognosis.
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http://dx.doi.org/10.1016/j.tranon.2020.100962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720092PMC
January 2021

Assessment of hospitalization rates for immune-related adverse events with immune checkpoint inhibitors.

J Oncol Pharm Pract 2020 Oct 25:1078155220968909. Epub 2020 Oct 25.

Department of Pharmacy, University of Louisville Hospital, Louisville, KY, USA.

Introduction: Immune checkpoint inhibitors (ICIs) have become the standard of care in many cancer types. As the number of patients receiving ICIs for various cancers continues to expand, patients and practitioners should be aware of potentially severe immune-related adverse events (irAEs). Despite reports of the incidence of grade 3/4 toxicities, the proportion of patients whose symptoms were clinically severe enough to warrant hospitalization for adverse event management is unknown.

Methods: This single center, retrospective, observational study was designed to determine the impact of irAEs on patients and the hospital. Patients who started ICIs from May 2016 through May 2019 for melanoma or lung cancer were included. The primary outcome was incidence of hospitalization for irAE. Secondary outcomes included median length of hospitalization, time to onset of irAE, rates of hospitalization for irAE per each checkpoint inhibitor regimen, organ system affected, progression free survival, and overall survival.

Results: Of 384 patients with melanoma or lung cancer, 27 (7%) were hospitalized at our institution for an irAE. The most common irAE leading to hospitalization was colitis for patients with melanoma and pneumonitis for patients with lung cancer. The median length of stay across all hospitalizations was 10 days. Twenty-five patients required the use of corticosteroids while hospitalized, while eight of these patients required second line irAE treatment. For the total patient population, 34.7% experienced a grade 1/2 irAE and 13.1% experienced a grade 3/4 irAE.

Conclusion: Our cohort of patients experienced similar rates irAEs as reported in clinical trials and published reports.
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http://dx.doi.org/10.1177/1078155220968909DOI Listing
October 2020

NHERF1 Loss Upregulates Enzymes of the Pentose Phosphate Pathway in Kidney Cortex.

Antioxidants (Basel) 2020 Sep 14;9(9). Epub 2020 Sep 14.

Department of Medicine, Division of Nephrology, University of Louisville, Louisville, KY 40202, USA.

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.
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http://dx.doi.org/10.3390/antiox9090862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554817PMC
September 2020

Statistical design of Phase II/III clinical trials for testing therapeutic interventions in COVID-19 patients.

BMC Med Res Methodol 2020 08 31;20(1):220. Epub 2020 Aug 31.

Department of Medicine, Christina Lee Brown Envirome Institute, University of Louisville, Louisville, KY, 40202, USA.

Background: Because of unknown features of the COVID-19 and the complexity of the population affected, standard clinical trial designs on treatments may not be optimal in such patients. We propose two independent clinical trials designs based on careful grouping of patient and outcome measures.

Methods: Using the World Health Organization ordinal scale on patient status, we classify treatable patients (Stages 3-7) into two risk groups. Patients in Stages 3, 4 and 5 are categorized as the intermediate-risk group, while patients in Stages 6 and 7 are categorized as the high-risk group. To ensure that an intervention, if deemed efficacious, is promptly made available to vulnerable patients, we propose a group sequential design incorporating four factors stratification, two interim analyses, and a toxicity monitoring rule for the intermediate-risk group. The primary response variable (binary variable) is based on the proportion of patients discharged from hospital by the 15 day. The goal is to detect a significant improvement in this response rate. For the high-risk group, we propose a group sequential design incorporating three factors stratification, and two interim analyses, with no toxicity monitoring. The primary response variable for this design is 30 day mortality, with the goal of detecting a meaningful reduction in mortality rate.

Results: Required sample size and toxicity boundaries are calculated for each scenario. Sample size requirements for designs with interim analyses are marginally greater than ones without. In addition, for both the intermediate-risk group and the high-risk group, the required sample size with two interim analyses is almost identical to analyses with just one interim analysis.

Conclusions: We recommend using a binary outcome with composite endpoints for patients in Stage 3, 4 or 5 with a power of 90% to detect an improvement of 20% in the response rate, and a 30 day mortality rate outcome for those in Stage 6 or 7 with a power of 90% to detect 15% (effect size) reduction in mortality rate. For the intermediate-risk group, two interim analyses for efficacy evaluation along with toxicity monitoring are encouraged. For the high-risk group, two interim analyses without toxicity monitoring is advised.
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http://dx.doi.org/10.1186/s12874-020-01101-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456751PMC
August 2020

Dioxin-like and non-dioxin-like PCBs differentially regulate the hepatic proteome and modify diet-induced nonalcoholic fatty liver disease severity.

Med Chem Res 2020 Jul 7;29:1247-1263. Epub 2020 Jun 7.

Department of Biochemistry & Molecular Genetics, School of Medicine, University of Louisville, Louisville, KY, 40202, USA.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with metabolic disruption and non-alcoholic fatty liver disease (NAFLD). Based on their ability to activate the aryl hydrocarbon receptor (AhR), PCBs are subdivided into two classes: dioxin-like (DL) and non-dioxin-like (NDL) PCBs. Previously, we demonstrated that NDL PCBs compromised the liver to promote more severe diet-induced NAFLD. Here, the hepatic effects and potential mechanisms (by untargeted liver proteomics) of DL PCBs, NDL PCBs or co-exposure to both in diet-induced NAFLD are investigated. Male C57Bl/6 mice were fed a 42% fat diet and exposed to vehicle control; Aroclor1260 (20 mg/kg, NDL PCB mixture); PCB126 (20 μg/kg, DL PCB congener); or a mixture of Aroclor1260 (20 mg/kg)+PCB126 (20 μg/kg) for 12 weeks. Each exposure was associated with a distinct hepatic proteome. Phenotypic and proteomic analyses revealed increased hepatic inflammation and phosphoprotein signaling disruption by Aroclor1260. PCB126 decreased hepatic inflammation and fibrosis at the molecular level; while altering cytoskeletal remodeling, metal homeostasis, and intermediary/xenobiotic metabolism. PCB126 attenuated Aroclor1260-induced hepatic inflammation but increased hepatic free fatty acids in the co-exposure group. Aroclor1260+PCB126 exposure was strongly associated with multiple epigenetic processes, and these could potentially explain the observed non-additive effects of the exposures on the hepatic proteome. Taken together, the results demonstrated that PCB exposures differentially regulated the hepatic proteome and the histologic severity of diet-induced NAFLD. Future research is warranted to determine the AhR-dependence of the observed effects including metal homeostasis and the epigenetic regulation of gene expression.
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http://dx.doi.org/10.1007/s00044-020-02581-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440142PMC
July 2020

Palbociclib treatment alters nucleotide biosynthesis and glutamine dependency in A549 cells.

Cancer Cell Int 2020 1;20:280. Epub 2020 Jul 1.

Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY USA.

Background: Aberrant activity of cell cycle proteins is one of the key somatic events in non-small cell lung cancer (NSCLC) pathogenesis. In most NSCLC cases, the retinoblastoma protein tumor suppressor (RB) becomes inactivated via constitutive phosphorylation by cyclin dependent kinase (CDK) 4/6, leading to uncontrolled cell proliferation. Palbociclib, a small molecule inhibitor of CDK4/6, has shown anti-tumor activity in vitro and in vivo, with recent studies demonstrating a functional role for palbociclib in reprogramming cellular metabolism. While palbociclib has shown efficacy in preclinical models of NSCLC, the metabolic consequences of CDK4/6 inhibition in this context are largely unknown.

Methods: In our study, we used a combination of stable isotope resolved metabolomics using [U-C]-glucose and multiple in vitro metabolic assays, to interrogate the metabolic perturbations induced by palbociclib in A549 lung adenocarcinoma cells. Specifically, we assessed changes in glycolytic activity, the pentose phosphate pathway (PPP), and glutamine utilization. We performed these studies following palbociclib treatment with simultaneous silencing of to define the pRB-dependent changes in metabolism.

Results: Our studies revealed palbociclib does not affect glycolytic activity in A549 cells but decreases glucose metabolism through the PPP. This is in part via reducing activity of glucose 6-phosphate dehydrogenase, the rate limiting enzyme in the PPP. Additionally, palbociclib enhances glutaminolysis to maintain mitochondrial respiration and sensitizes A549 cells to the glutaminase inhibitor, CB-839. Notably, the effects of palbociclib on both the PPP and glutamine utilization occur in an RB-dependent manner.

Conclusions: Together, our data define the metabolic impact of palbociclib treatment in A549 cells and may support the targeting CDK4/6 inhibition in combination with glutaminase inhibitors in NSCLC patients with RB-proficient tumors.
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http://dx.doi.org/10.1186/s12935-020-01357-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329430PMC
July 2020

Statistical Issues and Group Classification in Plasma MicroRNA Studies With Data Application.

Evol Bioinform Online 2020 14;16:1176934320913338. Epub 2020 Apr 14.

Price Institute of Surgical Research, Hiram C. Polk Jr. M.D. Department of Surgery, School of Medicine, University of Louisville, Louisville, KY, USA.

The analysis of plasma microRNAs (miRNAs) has been widely used as a method for finding potential biomarkers for human diseases, especially those with a link to cancer. Methods of analyzing plasma miRNA have been thoroughly discussed from sample extraction to data modeling. However, some issues exist within the process that have rarely been talked about. Rice et al. discussed some issues in plasma miRNA studies, such as the lack of standard methodology including the use of different cycle threshold, time to plasma extraction, among others. These issues can lead to inconsistent data, and thus impact the result and assay reproducibility. Other external issues, such as batch effect and operator effect, may also indirectly impact the statistical analysis. Here, we discuss issues in plasma miRNA studies from a statistical point of view. The interaction effect of different ways of calculating fold-change, the choice of housekeeping genes, and methods of normalization are among the issues we discuss, with data demonstrations. values are calculated and compared to determine the effect of those issues on statistical conclusions. Statistical methods such as analysis of variance and analysis of covariance are crucial in the analysis of miRNA but investigators are often confused about them; therefore, a brief explanation of these statistical methods is also included. In addition, 3-group classification is discussed, as it is often challenging, compared with 2-group classification.
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http://dx.doi.org/10.1177/1176934320913338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157974PMC
April 2020

Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing.

Chem Res Toxicol 2020 06 27;33(6):1403-1417. Epub 2020 Apr 27.

Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202, United States.

Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As)-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As can displace zinc (Zn) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As could displace Zn from ZRANB2, altering its structure, expression, and splicing function. As/Zn binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As (0 or 5 μM, 0-72 h; or 0-5 μM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As bound to, as well as displaced Zn from, each zfm. Also, Zn displaced As from As-bound zfm's acutely, albeit transiently. As exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As exposures, starting at 100 nm. We conclude that As exposure displaces Zn from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405655PMC
June 2020

Preoperative Opioid Prescription Is Associated With Major Complications in Patients With Crohn's Disease Undergoing Elective Ileocolic Resection.

Dis Colon Rectum 2020 08;63(8):1090-1101

Price Institute of Surgical Research, The Hiram C. Polk Jr., M.D., Department of Surgery, University of Louisville, Louisville, Kentucky.

Background: Opioid use has grown exponentially over the last decade. The effect of preoperative opioid prescription in patients with Crohn's disease undergoing surgery is unknown.

Objective: The purpose of this study was to identify whether preoperative opioid prescription is associated with adverse postoperative outcomes in Crohn's disease.

Design: This is a single-institution retrospective observational study.

Settings: This study was performed at an academic tertiary care center. Details of preoperative opioid prescription were collected from the Kentucky All-Schedule Prescription Electronic Reporting database and the electronic databases of bordering states.

Patients: Consecutive patients undergoing ileocolic resection for Crohn's disease from 2014 to 2018 were included.

Main Outcome Measures: The outcomes examined were major complications (Clavien-Dindo ≥3a), length of stay, and 30-day hospital readmission.

Results: Fifty one of 118 patients were prescribed opioids within 6 months preoperatively (range, 0-33,760 morphine milligram equivalents). Patients with preoperative opioid prescription compared with no preoperative opioid prescription required more daily opioids during hospital admission (p = 0.024). Nineteen patients had a major postoperative complication (preoperative opioid prescription: 26% (13/51) vs no preoperative opioid prescription: 9% (6/67)). On multivariable analysis, preoperative opioid prescription (OR = 2.994 (95% CI, 1.024-8.751); p = 0.045) was a significant risk factor for a major complication. Preoperative opioid prescription was associated with increased length of stay (p < 0.001) and was a risk factor for readmission (OR = 2.978 (95% CI, 1.075-8.246); p = 0.036). Twenty-four patients were readmitted. Using a cutoff for higher opioid prescription of 300 morphine milligram equivalents within 6 months preoperation (eg, 60 tablets of hydrocodone/acetaminophen 5/325), preoperative opioid prescription remained a risk factor for major postoperative complications (OR = 3.148 (95% CI, 1.110-8.928); p = 0.031).

Limitations: This was a retrospective study and could not assess nonprescribed opioid use.

Conclusions: Preoperative opioid prescription was a significant risk factor for adverse outcomes in patients with Crohn's disease undergoing elective ileocolic resection. See Video Abstract at http://links.lww.com/DCR/B113. LA PRESCRIPCIÓN PREOPERATORIA DE OPIOIDES SE ASOCIA CON COMPLICACIONES MAYORES EN PACIENTES CON ENFERMEDAD DE CROHN SOMETIDOS A RESECCIÓN ILEOCÓLICA ELECTIVA: El uso de opioides ha crecido exponencialmente en la última década. Se desconoce el efecto de la prescripción preoperatoria de opioides en pacientes con enfermedad de Crohn sometidos a cirugía.Identificar si la prescripción preoperatoria de opioides está asociada con resultados postoperatorios adversos en la enfermedad de Crohn.Este es un estudio observacional retrospectivo de una sola institución.Este estudio se realizó en un centro académico de atención terciaria. Los detalles de la prescripción preoperatoria de opiáceos se recopilaron de la base de datos de "Kentucky All-Schedule Prescription Electronic Reporting" y de las bases de datos electrónicas de los estados fronterizos.Pacientes consecutivos sometidos a resección ileocólica por enfermedad de Crohn entre 2014-2018.Los resultados examinados fueron complicaciones mayores (Clavien-Dindo ≥3a), duración de la estancia y el reingreso hospitalario de 30 días.A cincuenta y uno de 118 pacientes se le recetaron opioides dentro de los 6 meses preoperatorios (rango, 0 a 33,760 equivalentes de miligramos de morfina). Los pacientes con prescripción preoperatoria de opioides en comparación con ninguna prescripción preoperatoria de opioides requirieron más opioides diarios durante el ingreso hospitalario (p = 0,024). Diecinueve pacientes tuvieron una complicación postoperatoria importante (prescripción preoperatoria de opioides: 26% [13/51] frente a ninguna prescripción preoperatoria de opioides: 9% [6/67]). En el análisis multivariable, la prescripción de opioides preoperatorios (OR = 2.994, IC 95%: 1.024-8.751, p = 0.045) fueron factores de riesgo significativos para una complicación mayor. La prescripción preoperatoria de opioides se asoció con un aumento de la duración de la estadía (p <0.001) y fue un factor de riesgo para el reingreso (OR = 2.978, IC 95%: 1.075-8.246, p = 0.036). Veinticuatro pacientes fueron readmitidos. Utilizando un límite para una mayor prescripción de opioides de 300 miligramos equivalentes de morfina dentro de los 6 meses previos a la operación (p. Ej., 60 tabletas de hidrocodona / acetaminofén 5/325), la prescripción preoperatoria de opioides siguió siendo un factor de riesgo para complicaciones postoperatorias mayores (OR = 3.148 IC 95%: 1.110-8.928, p = 0.031).Este fue un estudio retrospectivo y no pudo evaluar el uso de opioides no prescritos.La prescripción preoperatoria de opioides fue un factor de riesgo significativo para los resultados adversos en pacientes con enfermedad de Crohn sometidos a resección ileocólica electiva. Consulte Video Resumen en http://links.lww.com/DCR/B113.
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http://dx.doi.org/10.1097/DCR.0000000000001571DOI Listing
August 2020

Association between residential greenness and exposure to volatile organic compounds.

Sci Total Environ 2020 Mar 23;707:135435. Epub 2019 Nov 23.

Christina Lee Brown Envirome Institute, University of Louisville, 302 E Muhammad Ali Blvd., Louisville, KY 40202, United States; Superfund Research Center, University of Louisville, 302 E Muhammad Ali Blvd., Louisville, KY 40202, United States; Diabetes and Obesity Center, University of Louisville, 580 S. Preston St., Louisville, KY 40202, United States. Electronic address:

Residential proximity to vegetation and plants is associated with many health benefits, including reduced risk of cardiovascular disease, diabetes and mental stress. Although the mechanisms by which proximity to greenness affects health remain unclear, plants have been shown to remove particulate air pollution. However, the association between residential-area vegetation and exposure to volatile organic chemicals (VOCs) has not been investigated. We recruited a cohort of 213 non-smoking individuals and estimated peak, cumulative, and contemporaneous greenery using satellite-derived normalized difference vegetation index (NDVI) near their residence. We found that the urinary metabolites of exposure to VOCs - acrolein, acrylamide, acrylonitrile, benzene, 1-bromopropane, propylene oxide were inversely associated (7-31% lower) with 0.1 higher peak NDVI values within 100 m radius of the participants' home. These associations were significant at radii ranging from 25 to 300 m. Strongest associations were observed within a 200 m radius, where VOC metabolites were 22% lower per 0.1 unit higher NDVI. Of the 18 measured urinary metabolites, 7 were positively associated with variation of greenness within a 200 m radius of homes. The percent of tree canopy and street trees around participants' residence were less strongly associated with metabolite levels. The associations between urinary VOC metabolites and residential NDVI values were stronger in winter than in summer, and in participants who were more educated, White, and those who lived close to areas of high traffic. These findings suggest high levels of residential greenness are associated with lower VOC exposure, particularly in winter.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294698PMC
March 2020

Exposure to airborne fine particulate matter is associated with impaired endothelial function and biomarkers of oxidative stress and inflammation.

Environ Res 2020 01 5;180:108890. Epub 2019 Nov 5.

Christina Lee Brown Envirome Institute, University of Louisville, Louisville, KY, 40292, USA. Electronic address:

Epidemiological evidence suggests that exposure to air pollution is a leading risk factor for cardiovascular disease (CVD). However, there is little direct evidence linking exposure to vascular dysfunction. We conducted a cross-sectional study of 100 participants, recruited from the University of Louisville Clinics. Endothelial function was assessed by calculating the reactive hyperemia index (RHI). Oxidative stress was indexed by measuring urinary levels of isoprostanes (n = 91). Inflammatory biomarkers were measured in the plasma (n = 80). Daily average PM levels were obtained from regional monitoring stations. Adjusted associations between PM levels and measured outcomes were tested using generalized linear models. The average age of participants was 48 years (44% male, 62% white); 52% had a diagnosis of hypertension, and 44% had type-2 diabetes. A 12.4% decrease in RHI was associated with 10 μg/m increase in PM (95% CI: 21.0, -2.7). The F-2 isoprostane metabolite showed a positive association of 28.4% (95% CI: 2.7, 60.3) per 10 μg/m increase in PM. Positive associations were observed with angiopoietin 1 (17.4%; 95% CI: 2.8, 33.8), vascular endothelial growth factor (10.4%; 95% CI: 0.6, 21.0), placental growth factor (31.7%; 95% CI: 12.2, 54.5), intracellular adhesion molecule-1 (24.6%; 95% CI: 1.6, 52.8), and matrix metalloproteinase-9 (30.3%; 95% CI: 8.0, 57.5) per 10 μg/m increase in PM. Additionally, a 10 μg/m increase in PM was associated with 15.9% decrease in vascular cell adhesion molecule-1 (95% CI: 28.3, -1.3). These findings suggest that exposure to PM is associated with impaired vascular function, which may result from oxidative stress and inflammation, thereby leading to a pro-atherogenic state.
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http://dx.doi.org/10.1016/j.envres.2019.108890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899204PMC
January 2020

Multi-group diagnostic classification of high-dimensional data using differential scanning calorimetry plasma thermograms.

PLoS One 2019 20;14(8):e0220765. Epub 2019 Aug 20.

Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.

The thermoanalytical technique differential scanning calorimetry (DSC) has been applied to characterize protein denaturation patterns (thermograms) in blood plasma samples and relate these to a subject's health status. The analysis and classification of thermograms is challenging because of the high-dimensionality of the dataset. There are various methods for group classification using high-dimensional data sets; however, the impact of using high-dimensional data sets for cancer classification has been poorly understood. In the present article, we proposed a statistical approach for data reduction and a parametric method (PM) for modeling of high-dimensional data sets for two- and three- group classification using DSC and demographic data. We compared the PM to the non-parametric classification method K-nearest neighbors (KNN) and the semi-parametric classification method KNN with dynamic time warping (DTW). We evaluated the performance of these methods for multiple two-group classifications: (i) normal versus cervical cancer, (ii) normal versus lung cancer, (iii) normal versus cancer (cervical + lung), (iv) lung cancer versus cervical cancer as well as for three-group classification: normal versus cervical cancer versus lung cancer. In general, performance for two-group classification was high whereas three-group classification was more challenging, with all three methods predicting normal samples more accurately than cancer samples. Moreover, specificity of the PM method was mostly higher or the same as KNN and DTW-KNN with lower sensitivity. The performance of KNN and DTW-KNN decreased with the inclusion of demographic data, whereas similar performance was observed for the PM which could be explained by the fact that the PM uses fewer parameters as compared to KNN and DTW-KNN methods and is thus less susceptible to the risk of overfitting. More importantly the accuracy of the PM can be increased by using a greater number of quantile data points and by the inclusion of additional demographic and clinical data, providing a substantial advantage over KNN and DTW-KNN methods.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220765PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701772PMC
March 2020

Adaptive Signature Design- review of the biomarker guided adaptive phase -III controlled design.

Contemp Clin Trials Commun 2019 Sep 16;15:100378. Epub 2019 May 16.

Department of Bioinformatics and Biostatistics, University of Louisville, Louisville, KY, USA.

Genomics having a profound impact on oncology drug development necessitates the use of genomic signatures for therapeutic strategy and emerging medicine proposals. Since its advent in the arena of clinical trials biomarker-related predictive methods for the identification and selection of patient subgroups, with optimal treatment response, are widely used. Genetic signatures which are accountable for the differential response to treatments are experimentally recognizable and analytically validated in phase II stage of clinical trials. The availability of robust and validated biomarkers in phase III is limited. Hence, the development of a clinical trial design without the availability of biomarker identity for treatment-sensitive patients becomes indispensable. Adaptive Signature Design (ASD) is a design procedure of developing and validating a predictive classifier (diagnostic testing strategy) when the signature of subjects responding differentially to treatment is remote in the context of the study. This review provides a detailed methodology and statistical background of this pioneering design developed by Freidlin and Simon (2005). In addition, it concentrates on the advances in ASD regarding statistical issues such as predictive assay identification, classification techniques, statistical methods, subgroup search, choice of differentially expressed genes, and multiplicity correction. The statistical methodology behind the design is explained with the intent of building the ground steps for future research approachable, especially for beginning researchers. Most of the existing research articles give a microcosmic view of the design and lack in describing the details behind the methodology. This study covers those details and marks the novelty of our research.
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http://dx.doi.org/10.1016/j.conctc.2019.100378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591770PMC
September 2019

Overexpression of hsa-miR-186 induces chromosomal instability in arsenic-exposed human keratinocytes.

Toxicol Appl Pharmacol 2019 09 6;378:114614. Epub 2019 Jun 6.

Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY 40292, USA. Electronic address:

The mechanism of arsenic-induced skin carcinogenesis is not yet fully understood. Chromosomal instability contributes to aneuploidy and is a driving force in carcinogenesis. Arsenic causes mitotic arrest and induces aneuploidy. hsa-miR-186 overexpression is associated with metastatic cancers as well as arsenic-induced squamous cell carcinoma and is reported to target several mitotic regulators. Decreased levels of these proteins can dysregulate chromatid segregation contributing to aneuploidy. This work investigates the potential aneuploidogenic role of hsa-miR-186 in arsenic carcinogenesis. Clones of immortalized human keratinocytes (HaCaT) stably transfected with a hsa-miR-186 expression or empty vector were isolated. Three clones with high and low hsa-miR-186 expression determined by RT-qPCR were selected for further analysis and cultured with 0 or 100 nM NaAsO for 8 weeks. Analysis of mitoses revealed that chromosome number and structural abnormalities increased in cells overexpressing hsa-miR-186 and were further increased by arsenite exposure. Double minutes were the dominant structural aberrations. The peak number of chromosomes also increased. Cells with >220 to >270 chromosomes appeared after 2 months in hsa-miR-186 overexpressing cells, indicating multiple rounds of endomitosis had occurred. The fraction of cells with increased chromosome number or structural abnormalities did not increase in passage matched control cells. Levels of selected target proteins were determined by western blot. Expression of BUB1, a predicted hsa-miR-186 target was suppressed in hsa-miR-186 overexpressing clones, but increased with arsenite exposure. CDC27 remained constant under all conditions. These results suggest that overexpression of miR-186 in arsenic exposed tissues likely induces aneuploidy contributing to arsenic-induced carcinogenesis.
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http://dx.doi.org/10.1016/j.taap.2019.114614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746570PMC
September 2019

Adherence to anti-estrogen therapy in women with hormone receptor-positive breast cancer utilizing bubble packaging: a pilot study.

Breast Cancer Res Treat 2019 Sep 6;177(2):395-399. Epub 2019 Jun 6.

Division of Hematology and Medical Oncology, Department of Medicine, James Graham Brown Cancer Center, University of Louisville, 529 S Jackson St, Louisville, KY, 40202, USA.

Purpose: This pilot study evaluated adherence to anti-estrogen therapy in women with hormone receptor-positive breast cancer utilizing bubble packaging.

Methods: This was a single-arm prospective investigational pilot study that enrolled 86 patients between August 2012 and April 2014. Descriptive statistics for patient age, race, insurance, stage, duration of treatment, and comorbidities were computed. All patients received routine prescriptions in a "bubble" pack or daily blister pack dispensed by one pharmacy. Participants were considered adherent if they had taken ≥ 80% of the dispensed drug. Disease-free survival (DFS) and overall survival (OS) data were obtained at 78 months.

Results: Fifty patients were included in the analysis. The overall adherence rate was 97%. None of the variables examined (race, age, insurance status, and stage) had an impact on adherence rate. Only duration of endocrine therapy had a marginal effect on adherence (p value = 0.06). The late cohort (duration of therapy 37-60 months) was least likely to be compliant at 89.53%. Our 5-year DFS was 94% and 5-year OS was 96%. There was no statistically significant difference in DFS and OS between patients with adherence rate > 90% and < 90%.

Conclusion: Adherence rate to bubble packaging was higher than that in historical studies. Although this is a single-arm pilot study, these data suggest bubble packaging of anti-estrogen may be a reasonable option to improve adherence in hormone receptor-positive breast cancer patients.
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http://dx.doi.org/10.1007/s10549-019-05308-7DOI Listing
September 2019

Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients.

Lab Chip 2019 06 3;19(11):1899-1915. Epub 2019 May 3.

Small Systems Laboratory, Department of Mechanical Engineering, Worcester Polytechnic Institute, Worcester, MA 01609, USA.

In this paper, we report the development of the nanotube-CTC-chip for isolation of tumor-derived epithelial cells (circulating tumor cells, CTCs) from peripheral blood, with high purity, by exploiting the physical mechanisms of preferential adherence of CTCs on a nanotube surface. The nanotube-CTC-chip is a new 76-element microarray technology that combines carbon nanotube surfaces with microarray batch manufacturing techniques for the capture and isolation of tumor-derived epithelial cells. Using a combination of red blood cell (RBC) lysis and preferential adherence, we demonstrate the capture and enrichment of CTCs with a 5-log reduction of contaminating WBCs. EpCAM negative MDA-MB-231/luciferase-2A-green fluorescent protein (GFP) cells were spiked in the blood of wild mice and enriched using an RBC lysis protocol. The enriched samples were then processed using the nanotube-CTC-chip for preferential CTC adherence on the nanosurface and counting the GFP cells yielded anywhere from 89% to 100% capture from the droplets. Electron microscopy (EM) studies showed focal adhesion with filaments from the cell body to the nanotube surface. We compared the nanotube preferential adherence to collagen adhesion matrix (CAM) scaffolding, reported as a viable strategy for CTC capture in patients. The CAM scaffolding on the device surface yielded 50% adherence with 100% tracking of cancer cells (adhered vs. non-adhered) versus carbon nanotubes with >90% adherence and 100% tracking for the same protocol. The nanotube-CTC-chip successfully captured CTCs in the peripheral blood of breast cancer patients (stage 1-4) with a range of 4-238 CTCs per 8.5 ml blood or 0.5-28 CTCs per ml. CTCs (based on CK8/18, Her2, EGFR) were successfully identified in 7/7 breast cancer patients, and no CTCs were captured in healthy controls (n = 2). CTC enumeration based on multiple markers using the nanotube-CTC-chip enables dynamic views of metastatic progression and could potentially have predictive capabilities for diagnosis and treatment response.
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http://dx.doi.org/10.1039/c9lc00274jDOI Listing
June 2019

Sample size calculations for the differential expression analysis of RNA-seq data using a negative binomial regression model.

Stat Appl Genet Mol Biol 2019 01 22;18(1). Epub 2019 Jan 22.

Department of Bioinformatics and Biostatistics, School of Public Health and Information Sciences, University of Louisville, Louisville, KY 40202, USA.

High throughput RNA sequencing (RNA-seq) technology is increasingly used in disease-related biomarker studies. A negative binomial distribution has become the popular choice for modeling read counts of genes in RNA-seq data due to over-dispersed read counts. In this study, we propose two explicit sample size calculation methods for RNA-seq data using a negative binomial regression model. To derive these new sample size formulas, the common dispersion parameter and the size factor as an offset via a natural logarithm link function are incorporated. A two-sided Wald test statistic derived from the coefficient parameter is used for testing a single gene at a nominal significance level 0.05 and multiple genes at a false discovery rate 0.05. The variance for the Wald test is computed from the variance-covariance matrix with the parameters estimated from the maximum likelihood estimates under the unrestricted and constrained scenarios. The performance and a side-by-side comparison of our new formulas with three existing methods with a Wald test, a likelihood ratio test or an exact test are evaluated via simulation studies. Since other methods are much computationally extensive, we recommend our M1 method for quick and direct estimation of sample sizes in an experimental design. Finally, we illustrate sample sizes estimation using an existing breast cancer RNA-seq data.
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http://dx.doi.org/10.1515/sagmb-2018-0021DOI Listing
January 2019

5-(3,3-Dimethyle-1-Triazeno) Imidazole-4-Carboxamide and Interleukin-2 Adjuvant Therapy in Resected High-Risk Primary and Regionally Metastatic Melanoma.

Am J Med Sci 2019 01 25;357(1):43-48. Epub 2018 Oct 25.

University of Louisville, Department of Medicine, Division of Medical Oncology/Hematology, Louisville, Kentucky; James Graham Brown Cancer Center, Louisville, Kentucky. Electronic address:

Background: In the precheckpoint inhibitor era, high-dose interferon was the only approved adjuvant therapy for high-risk melanoma. In this manuscript, we analyze the recurrence-free survival, overall survival and toxicity profile of adjuvant treatment with interleukin-2 (IL-2) and 5-(3,3-dimethyle-1-triazeno) imidazole-4-carboxamide (DTIC) for resected high-risk melanoma patients.

Methods: All patients with stage IIB, IIC or stage III melanoma who were treated with DTIC/IL-2 combination therapy at a single institution from 2000 to 2010 were identified from the University of Louisville Hospital medical record. Patients received 6 months of subcutaneous IL-2 (12 × 10 units days 1-4) and intravenous DTIC (750 mg/m day 1 of each cycle) every 28 days for 6 cycles. Individual medical records were accessed to collect the data.

Results: Of the 112 patients treated, all underwent surgical resection and then received adjuvant treatment. A total of 58.7% of the patients were male, 42.2% female; 99% were Caucasian. A total of 79 (72.5%) of the patients were alive at the time of analysis and 57 (47.7%) patients were currently event free. A total of 69 (63.3%) patients completed all 6 months of adjuvant combination treatment with 13.8% of the patients requiring IL-2 and 21.1% of the patients requiring DTIC dose reduction. Five year overall survival was 75.57% with recurrence-free survival of 53.05%.

Conclusions: For several decades, there has not been an ideal adjuvant treatment for patients with resected high risk melanoma. Our retrospective analysis suggests that combination therapy with DTIC/IL-2 is beneficial and relatively well tolerated as an alternative adjuvant treatment for patients with high-risk melanoma.
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http://dx.doi.org/10.1016/j.amjms.2018.10.009DOI Listing
January 2019

Interactive Web Tool for Standardizing Proteomics Workflow for Liquid Chromatography-Mass Spectrometry Data.

J Proteomics Bioinform 2019 23;12(4):85-88. Epub 2019 May 23.

Department of Bioinformatics & Biostatistics, University of Louisville, Louisville, Kentucky, United States of America.

Introduction: The proteomics experiments involve several steps and there are many choices available for each step in the workflow. Therefore, standardization of proteomics workflow is an essential task for design of proteomics experiments. However, there are challenges associated with the quantitative measurements based on liquid chromatography-mass spectrometry such as heterogeneity due to technical variability and missing values.

Methods: We introduce a web application, Proteomics Workflow Standardization Tool (PWST) to standardize the proteomics workflow. The tool will be helpful in deciding the most suitable choice for each step of the experimentation. This is based on identifying steps/choices with least variability such as comparing Coefficient of Variation (CV). We demonstrate the tool on data with categorical and continuous variables. We have used the special cases of general linear model, analysis of covariance and analysis of variance with fixed effects to study the effects due to various sources of variability. We have provided various options that will aid in finding the contribution of sum of squares for each variable and the CV. The user can analyze the data variability at protein and peptide level even in the presence of missing values.

Availability And Implementation: The source code for "PWST" is written in R and implemented as shiny web application that can be accessed freely from https://ulbbf.shinyapps.io/pwst/.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059686PMC
May 2019

Standardizing Proteomics Workflow for Liquid Chromatography-Mass Spectrometry: Technical and Statistical Considerations.

J Proteomics Bioinform 2019 4;12(3):48-55. Epub 2019 Apr 4.

Department of Bioinformatics & Biostatistics, University of Louisville, Louisville, Kentucky, United States of America.

Introduction: The quantitative measurements based on liquid chromatography (LC) coupled with mass spectrometry (MS) often suffer from the problem of missing values and data heterogeneity from technical variability. We considered a proteomics data set generated from human kidney biopsy material to investigate the technical effects of sample preparation and the quantitative MS.

Methods: We studied the effect of tissue storage methods (TSMs) and tissue extraction methods (TEMs) on data analysis. There are two TSMs: frozen (FR) and FFPE (formalin-fixed paraffin embedded); and three TEMs: MAX, TX followed by MAX and SDS followed by MAX. We assessed the impact of different strategies to analyze the data while considering heterogeneity and MVs. We have used analysis of variance (ANOVA) model to study the effects due to various sources of variability.

Results And Conclusion: We found that the FFPE TSM is better than the FR TSM. We also found that the one-step TEM (MAX) is better than those of two-steps TEMs. Furthermore, we found the imputation method is a better approach than excluding the proteins with MVs or using unbalanced design.
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http://dx.doi.org/10.35248/0974-276x.19.12.496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059694PMC
April 2019

Association Between Residential Greenness and Cardiovascular Disease Risk.

J Am Heart Assoc 2018 12;7(24):e009117

1 Envirome Institute University of Louisville Louisville KY.

Background Exposure to green vegetation has been linked to positive health, but the pathophysiological processes affected by exposure to vegetation remain unclear. To study the relationship between greenness and cardiovascular disease, we examined the association between residential greenness and biomarkers of cardiovascular injury and disease risk in susceptible individuals. Methods and Results In this cross-sectional study of 408 individuals recruited from a preventive cardiology clinic, we measured biomarkers of cardiovascular injury and risk in participant blood and urine. We estimated greenness from satellite-derived normalized difference vegetation index ( NDVI ) in zones with radii of 250 m and 1 km surrounding the participants' residences. We used generalized estimating equations to examine associations between greenness and cardiovascular disease biomarkers. We adjusted for residential clustering, demographic, clinical, and environmental variables. In fully adjusted models, contemporaneous NDVI within 250 m of participant residence was inversely associated with urinary levels of epinephrine (-6.9%; 95% confidence interval, -11.5, -2.0/0.1 NDVI ) and F2-isoprostane (-9.0%; 95% confidence interval, -15.1, -2.5/0.1 NDVI ). We found stronger associations between NDVI and urinary epinephrine in women, those not on β-blockers, and those who had not previously experienced a myocardial infarction. Of the 15 subtypes of circulating angiogenic cells examined, 11 were inversely associated (8.0-15.6% decrease/0.1 NDVI ), whereas 2 were positively associated (37.6-45.8% increase/0.1 NDVI ) with contemporaneous NDVI . Conclusions Independent of age, sex, race, smoking status, neighborhood deprivation, statin use, and roadway exposure, residential greenness is associated with lower levels of sympathetic activation, reduced oxidative stress, and higher angiogenic capacity.
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http://dx.doi.org/10.1161/JAHA.118.009117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405613PMC
December 2018

Impact of 21-Gene Expression Assay on Staging Estrogen Receptor-Positive HER2-Negative Breast Cancer.

Clin Breast Cancer 2019 02 29;19(1):e261-e269. Epub 2018 Oct 29.

Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, University of Louisville Hospital, Louisville, KY. Electronic address:

Purpose: The 8th edition of the American Joint Committee on Cancer (AJCC) breast cancer staging system requires histologic grade (GR), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and stage (assessed by the tumor, node, metastasis classification system). For T1-2 N0, ER/HER2- tumors, if the 21-gene expression assay is ordered and Oncotype DX (ODX) recurrence score (RS) is 0 to 10, the stage is IA. The purpose of this study was to determine the impact of the ODXRS on staging ER/HER2- tumors.

Materials And Methods: This is a retrospective review of ER/HER2- invasive breast cancer (BC) with ODXRS results from 2 institutions (n = 816) between 2006 and 2018. Stage based on the AJCC 7th and 8th editions, and stage using the 8th edition with and without ODXRS were compared. Significant associations among pathologic parameters and ODX risk groups were determined. Clinical histories were reviewed.

Results: Nearly half of the patients (43%) had a change in BC stage using the new staging system. Only 4 patients changed stage as a direct result of ODXRS. Influence of ODXRS on staging is limited to T2N0 tumors that are either GR 3 and strongly ER and PR or GR 1-2 and ER/PR-. Sixty-one percent of cases of recurrence (11/18) were downstaged using the new staging system.

Conclusion: ODXRS has little influence on staging, thus supporting the view of the AJCC 8th edition expert panel that ODX is not required for staging. Downstaging of more than half of cases of recurrence suggests that continued refinement of the staging system, as proposed by the expert panel, could be beneficial in this subgroup of patients.
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http://dx.doi.org/10.1016/j.clbc.2018.10.005DOI Listing
February 2019

Prevalence, patterns, and predictors of diarrhea: a spatial-temporal comprehensive evaluation in India.

BMC Public Health 2018 Nov 23;18(1):1288. Epub 2018 Nov 23.

Biostatistics Shared Facility, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA.

Background: Spatial analysis has been vital in mapping the spread of diseases and assisting in policy making. Targeting diarrhea transmission hotspots is one of the potential strategies for reducing diarrhea cases. This study aimed to examine the spatial-temporal variations and to identify the modifiable determinants of diarrhea while controlling for the spatial dependence in the data.

Methods: An ecological study on diarrhea data from DLHS-3 and NFHS- 4 in India. Moran's I and LISA were used to detect the spatial clustering of diarrhea cases and to test for clustering in the data. Spatial regression was used to identify the modifiable factors associated with the prevalence of diarrhea. The study comprised of the prevalence of diarrhea among the children below the age of five years (U-5 s) across different states in India. The determinants of diarrhea were obtained using spatial lag models. The software used were GeoDa 1.6.6 and QGIS 2.0.

Results: The presence of spatial autocorrelation in DLHS-3 and NFHS-4 (Moron's I = 0.577 and 0.369 respectively) enforces the usage of geographical properties while modeling the diarrhea data. The geographic clustering of high-prevalence districts was observed in the state of UP consistently. The spatial pattern of the percentage of children with diarrhea was persistently associated with the household with a sanitation facility (%) (p = 0.023 and p = 0.011). Compared to the diarrhea cases in the period 2007-2008, no much reduction was observed in the period 2015-2016. The prevalence of diarrhea and percentage of household with sanitation were ranging between 0.1-33.8% and 1.3-96.1% in the period 2007-2008 and 0.6-29.1% and 10.4-92.0% in the period 2015-2016 respectively. The least and highest prevalence of diarrhea being consistently from Assam and UP respectively.

Conclusion: Despite improvements in controlling spread of diarrheal disease, the burden remains high. Focus on widespread diarrheal disease control strategy by addressing the social determinants of health like basic sanitation is crucial to reduce the burden of diarrhea among U-5 s in India. The identification of hotspots will aid in the planning of control strategies for goal setting in the targeted regions.
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http://dx.doi.org/10.1186/s12889-018-6213-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251155PMC
November 2018

Circulating angiogenic stem cells in type 2 diabetes are associated with glycemic control and endothelial dysfunction.

PLoS One 2018 15;13(10):e0205851. Epub 2018 Oct 15.

Department of Medicine, Diabetes and Obesity Center, University of Louisville, Louisville, Kentucky, United States of America.

Circulating angiogenic cells (CACs) of various described phenotypes participate in the regeneration of the damaged endothelium, but the abundance of these cells is highly influenced by external cues including diabetes. It is not entirely clear which CAC populations are most reflective of endothelial function nor which are impacted by diabetes. To answer these questions, we enrolled a human cohort with variable CVD risk and determined relationships between stratified levels of CACs and indices of diabetes and vascular function. We also determined associations between CAC functional markers and diabetes and identified pro-angiogenic molecules which are impacted by diabetes. We found that subjects with low levels of CD34+/AC133+/CD31+/CD45dim cells (CAC-3) had a significantly higher incidence of diabetes (p = 0.004), higher HbA1c levels (p = 0.049) and higher CVD risk scores. Furthermore, there was an association between low CAC-3 levels and impaired vascular function (p = 0.023). These cells from diabetics had reduced levels of CXCR4 and VEGFR2, while diabetics had higher levels of certain cytokines and pro-angiogenic molecules. These results suggest that quantitative and functional defects of CD34+/AC133+/CD31+/CD45dim cells are associated with diabetes and vascular impairment and that this cell type may be a prognostic indicator of CVD and vascular dysfunction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205851PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188890PMC
April 2019