Publications by authors named "Peter Lopez"

86 Publications

Evaluating the Effects of Cell Sorting on Gene Expression.

J Biomol Tech 2020 09;31(3):100-111

West Virginia University, Morgantown, West Virginia, USA.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, , gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.
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http://dx.doi.org/10.7171/jbt.20-3103-004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497499PMC
September 2020

Evaluating the Effects of Cell Sorting on Gene Expression.

J Biomol Tech 2020 Jun 22. Epub 2020 Jun 22.

West Virginia University, Morgantown, West Virginia, USA.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, , gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.
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http://dx.doi.org/10.7171/jbt.2020-3103-004DOI Listing
June 2020

Special Issue on Enhancement of Reproducibility and Rigor.

Cytometry A 2020 Feb;97(2):105-106

Cellular Technologies Bioconsulting, LLC, Cytometry, Bethesda, Maryland.

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http://dx.doi.org/10.1002/cyto.a.23972DOI Listing
February 2020

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments.

Cytometry A 2020 02 26;97(2):148-155. Epub 2019 Nov 26.

New York University School of Medicine, New York, New York.

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23940DOI Listing
February 2020

Survey on Scientific Shared Resource Rigor and Reproducibility.

J Biomol Tech 2019 09;30(3):36-44

University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (, those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.
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http://dx.doi.org/10.7171/jbt.19-3003-001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657953PMC
September 2019

Risk Factors for Dysphagia Following a Cervical Fusion in a Trauma Population.

Cureus 2018 Oct 24;10(10):e3489. Epub 2018 Oct 24.

Surgery, Ascension Providence Hospital, Michigan State University, College of Human Medicine, Southfield, USA.

Background Dysphagia following a cervical fusion is a known complication; however, this has not been examined in the trauma population. We sought to identify risk factors that can be optimized in this population. Methods We performed a retrospective chart review on consecutive trauma patients who underwent a cervical fusion from 2014 to 2017 at a single institution with multiple surgeons. We included patients more than 18-years-old who were admitted through the emergency department with a diagnosis of acute cervical injury and underwent a cervical fusion during the same admission. We excluded patients who remained intubated postoperatively or underwent a tracheostomy. The primary outcome was dysphagia as evaluated by a bedside swallow test on postoperative day one by the nursing staff. This was followed by a standardized assessment performed by a speech therapist on postoperative day two in some cases. Variables of interest included sex, age, mechanism of injury, surgical approach, cervical levels, and Charlson comorbidity index. Univariate analysis was also utilized. Results Sixty patients met the study criteria. Nineteen patients (31.7%) developed dysphagia postoperatively. Mechanical falls were the most common injury mechanism (80%) and most surgical procedures were performed on the subaxial cervical spine (68.3%). Comparing the dysphagia groups, there was no significant difference among the confounding variables. Patients with dysphagia had an increased length of stay (10.6 ± 6.7 vs. 7.4 ± 3.1, = 0.056) and were more likely to have had an anterior vs. posterior cervical fusion (63.2% vs. 34.1%, = 0.056). Conclusions We found no statistically significant risk factors leading to postoperative dysphagia. The objective of this pilot is to find the baseline dysphagia rate and the potential modifiable factors in this unique patient population undergoing cervical fusion procedures.
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http://dx.doi.org/10.7759/cureus.3489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314396PMC
October 2018

When is Thigh Pain a Problem?

J Emerg Med 2018 08 2;55(2):e53-e55. Epub 2018 Jun 2.

Department of Surgery, Providence and Providence Park Hospitals, Southfield, Michigan.

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http://dx.doi.org/10.1016/j.jemermed.2018.04.062DOI Listing
August 2018

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes.

Cell Rep 2018 01 28;22(4):876-884. Epub 2018 Jan 28.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology and Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.
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http://dx.doi.org/10.1016/j.celrep.2017.12.098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664440PMC
January 2018

A novel vector for transgenesis in the rat CNS.

Acta Neuropathol Commun 2017 Nov 21;5(1):84. Epub 2017 Nov 21.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, Sandler Neurosciences Center, 675 Nelson Rising Lane, San Francisco, CA, 94158, USA.

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175 ± 3 days post inoculation (dpi), one line, Tg2919, overexpressed RaPrP at 4.4-fold and exhibited a reduced incubation time of 149 ± 2 dpi. The second line, Tg2922, overexpressed RaPrP at 9.7-fold compared with WT animals and had an incubation time of 112 ± 0 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.
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http://dx.doi.org/10.1186/s40478-017-0484-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697436PMC
November 2017

Repeat Head CT? Not Necessary for Patients with a Negative Initial Head CT on Anticoagulation or Antiplatelet Therapy Suffering Low-Altitude Falls.

Am Surg 2017 May;83(5):429-435

Anticoagulation and antiplatelet (ACAP) medications are increasingly prescribed to patients at high risk for falls. Many trauma centers have developed protocols for obtaining repeat head CT (HCT) for patients with low-altitude falls on ACAP therapy. We assess the need for routine scheduled repeat HCT in this population. Prospective, observational analysis of all low-altitude fall (<6 feet) patients on ACAP therapy evaluated at a Level II trauma center. All low-altitude fall patients with visible or suspected head trauma received an initial HCT. Patients were admitted and repeat HCT was obtained 12 hours later or earlier if acute neurologic decline developed. Chi-squared, Fischer exact, t, and Wilcoxon rank-sum tests were used. Statistical significance was defined as P < 0.05. Total of 1501 patients enrolled suffering low-altitude falls with initial HCT. Among them 1379(91.2%) were negative and 122(8.1%) were initially positive for intracranial hemorrhage. Mean age was 79.9 ± 11.4 years, 61 per cent were female and 85 per cent had visible head trauma at presentation. One hundred ninety-nine were excluded secondary to not receiving repeat HCT. Of the 1180 patients with normal initial HCT who underwent repeat HCT, only 7 (0.51%) had delayed intracranial hemorrhage. None of these patients required surgery, major changes in medical management or suffered head trauma-related mortality; 69 per cent were taking aspirin (acetylsalicylic acid, ASA), 19 per cent warfarin, 17 per cent clopidogrel, 6 per cent other anticoagulants, and 11 per cent were on combination therapy. Repeat HCT for patients on any ACAP therapy after low-altitude fall with a negative initial HCT is not necessary. Thorough neurologic examination and close monitoring is as effective as obtaining a repeat HCT.
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May 2017