Preclinical Laboratories Research Articles

Challenges in current inhalable tobacco toxicity assessment models: A narrative review.

Emerging tobacco products such as electronic nicotine delivery systems (ENDS) and heated tobacco products (HTPs) have a dynamic landscape and are becoming widely popular as they claim to offer a low-risk alternative to conventional smoking. Most pre-clinical laboratories currently exploit in vitro, ex vivo, and in vivo experimental models to assess toxicological outcomes as well as to develop risk-estimation models. While most laboratories have produced a wide range of cell culture and mouse model data utilizing current smoke/aerosol generators and standardized puffing profiles, much variation still exists between research studies, hindering the generation of usable data appropriate for the standardization of these tobacco products. In this review, we discuss current state-of-the-art in vitro and in vivo models and their challenges, as well as insights into risk estimation of novel products and recommendations for toxicological parameters for reporting, allowing comparability of the research studies between laboratories, resulting in usable data for regulation of these products before approval by regulatory authorities.

Creating a Teaching Technician Team for Support of Veterinary Student Training Laboratories

Veterinary technicians play valuable roles in most veterinary colleges and have particular strengths in coaching new learners. We moved to a teaching technician ‘float team’ model to provide teaching support to core pre-clinical laboratories in our DVM (Doctor of Veterinary Medicine) curriculum. The teaching technician team model has enabled us to better utilize staff skill sets and training, created efficiencies and expanded support provided to core laboratory courses, provided skill development and advancement opportunities for team members, and improved our ability to recognize and mentor our teaching staff. This model also allowed us to effectively adjust to the rapidly changing structure and increased numbers of laboratories brought on by the COVID-19 pandemic without increasing numbers of staff involved in laboratory teaching.

Should attendance for preclinical simulation and clinical education be mandatory?

Mandatory attendance, particularly in didactic settings, is a highly debated topic in higher education, including dental education. Within dental education, a large portion of education occurs in preclinical laboratories and clinical environments. There is little to no research on attendance in these settings in dental schools. This point/counterpoint paper examines the pros and cons of mandatory attendance in these highly specialized educational settings. With the backdrop of the COVID-19 pandemic that began in March 2020 and continues to impact dental education at the time of publication, this topic has become even more relevant. Viewpoint 1 claims that attendance should be mandatory because a greater exposure to preclinical and clinical environments helps foster better clinical hand skills, critical thinking, decision-making, problem-solving skills, and an overall sense of professional identity. It goes on further to suggest that there may be a link between attendance and performance in exams and that attendance is part of the dental school's responsibility. Viewpoint 2 argues that the rationale for attendance is complex, and that creating learning environments that are psychologically safe will incentivize students to attend, even without mandatory attendance policies. Furthermore, it explains that technological advances have allowed dental schools to think creatively about asynchronous learning, which by its very nature does not require attendance at a given time. The authors of both viewpoints conclude that the preclinical and clinical education and experience are critical dental education and that dental school leaders should focus on improving the quality of these experiences.

Pre-Clinical Common Data Elements for Traumatic Brain Injury Research: Progress and Use Cases.

Traumatic brain injury (TBI) is an extremely complex condition due to heterogeneity in injury mechanism, underlying conditions, and secondary injury. Pre-clinical and clinical researchers face challenges with reproducibility that negatively impact translation and therapeutic development for improved TBI patient outcomes. To address this challenge, TBI Pre-clinical Working Groups expanded upon previous efforts and developed common data elements (CDEs) to describe the most frequently used experimental parameters. The working groups created 913 CDEs to describe study metadata, animal characteristics, animal history, injury models, and behavioral tests. Use cases applied a set of commonly used CDEs to address and evaluate the degree of missing data resulting from combining legacy data from different laboratories for two different outcome measures (Morris water maze [MWM]; RotorRod/Rotarod). Data were cleaned and harmonized to Form Structures containing the relevant CDEs and subjected to missing value analysis. For the MWM dataset (358 animals from five studies, 44 CDEs), 50% of the CDEs contained at least one missing value, while for the Rotarod dataset (97 animals from three studies, 48 CDEs), over 60% of CDEs contained at least one missing value. Overall, 35% of values were missing across the MWM dataset, and 33% of values were missing for the Rotarod dataset, demonstrating both the feasibility and the challenge of combining legacy datasets using CDEs. The CDEs and the associated forms created here are available to the broader pre-clinical research community to promote consistent and comprehensive data acquisition, as well as to facilitate data sharing and formation of data repositories. In addition to addressing the challenge of standardization in TBI pre-clinical studies, this effort is intended to bring attention to the discrepancies in assessment and outcome metrics among pre-clinical laboratories and ultimately accelerate translation to clinical research.

Development and application of a decision tree to determine appropriate handling of gene therapies

Background & Aim Executives at the Food and Drug Administration (FDA) state that by the year 2025, there will be 10 to 20 cell and gene therapy (GT) product approvals per year. Current infrastructure is not sufficient to accommodate the spike in GT at most healthcare facilities. Furthermore, few healthcare providers (HCP) are trained to assess the risk of GT, or to implement safe handling strategies. Containment of GT, from entry to exit, requires a customized approach dictated by the construct of the vector. The risk of hazardous exposure to a novel therapy is amplified when containment procedures are undefined. Uncertainty regarding GT handling is due to a lack of relevant contemporary information and training. The outdated guidance's are drafted for pre-clinical laboratories, and many HCP are forced to extrapolate them to alternate settings. Recent papers have attempted to bridge the gap, but there is much work to be done with GT risk level identification, infrastructure establishment, and policy development. This presentation aims to describe a decision tree that sends the HCP on a rational path toward safe handling, by taking precautions that match the risk level of the GT vector. Methods, Results & Conclusion The method to eliminate vector exposure is by careful consideration of its replication status, integration ability, carcinogenicity, and toxicity. Vectors that are replication deficient, do not integrate into the host genome, and are otherwise not hazardous, are routed toward minimal handling precautions on the decision tree. However, if any of these factors, or a combination of them are present in a vector, the routing process dictates a more cautious approach. Importantly, influences such as capsid size and seroconversion are being added to the current body of knowledge to form the decision tree. Escalated handling precautions involves moving from Biosafety Level 1 (BSL1) to Biosafety Level 2 (BSL2) or 3. Two vectors, one using Adeno-associated virus (AAV), and the other incorporating the Herpes Simplex Virus (HSV-1) are routed through the decision tree. Because AAV is non-replicating, does not integrate into the host genome, and is non-toxic, it is reasonable to infer that handling can be performed in a BSL1 environment. However, since HSV-1 is replication competent and integrates into the host genome, BSL2 is a prudent recommendation. The decision tree provides a safety tool for HCP tasked with GT handling. Its application depends on modern infrastructure, focused training, and clear policy.

The Impact of Background Music in Reducing Stress During Preclinical Dental Laboratories.

Objective:The reported high level of stress among dental students and its consequences, considering the known benefits of background music (BM). This study was designed to evaluate students’ level of stress and impact of BM on their efficiency of work, performance, and learning ability during preclinical laboratory.Materials and Methods:In this cross-sectional questionnaire study, 61 preclinical students from the College of Dentistry, Ajman university-Fujairah campus who met the defined inclusion criteria participated. After a semester of the normal laboratory without BM, various genres of BM were introduced in the laboratory. Psychological stress assessment was done using the perceived stress scale. Students were asked to fill up a written feedback questionnaire at the end of 6 weeks of BM. Descriptive statistics and Chi-square test were used to study the statistical relationships among groups.Results:About 100% male and female students were found to be highly stressed due to exams and tests, 98% were stressed due to lack of time in the laboratory. About 82.5% females and 76% of males sample population expressed passion for music and also reported BM to be helpful to reduce stress in the preclinical laboratory and also to increase their concentration level.Conclusion:A strong positive relation has been shown in the reduction of stress with the BM in the preclinical laboratory.

From bedside to bench and back again: translational studies of mechanical unloading of the left ventricle to promote recovery after acute myocardial infarction.

Heart failure is a major cause of global morbidity and mortality. Acute myocardial infarction (AMI) is a primary cause of heart failure due in large part to residual myocardial damage despite timely reperfusion therapy. Since the 1970’s, multiple preclinical laboratories have tested whether reducing myocardial oxygen demand with a mechanical support pump can reduce infarct size in AMI. In the past decade, this hypothesis has been studied using contemporary circulatory support pumps. We will review the most recent series of preclinical studies in the field which led to the recently completed Door to Unload ST-segment Elevation Myocardial Infarction (DTU-STEMI) safety and feasibility pilot trial.

Quantification of Mouse Renal Perfusion Using Arterial Spin Labeled MRI at 1 T

Quantitative measurement of renal perfusion in murine models provides important information on the organ physiology and disease states. The 1-T desktop magnetic resonance imaging has a small footprint and a self-contained fringe field. This resultant flexibility in siting makes the system ideal for preclinical imaging research. Our objective was to evaluate the capability of the 1-T desktop magnetic resonance imaging to measure mouse renal perfusion without the administration of exogenous contrast agents. We implemented a flow-sensitive alternating inversion recovery (FAIR)-based arterial spin labeling sequence with a mouse volume coil on a 1-T desktop magnetic resonance scanner. The validity of the implementation was tested by comparing obtained renal perfusion results with literature values for normal mice and challenging the technique with mice treated with furosemide, a blood vessel vasoconstrictor drug. The measured cortical and medullary perfusions were quantified to be 402 ± 95 and 184 ± 52 mL/100 g/min, respectively, in agreement with literature values. The ratio of cortical to medullary renal blood flow was between 2 and 3 and was independent of the mouse weight. As expected, upon furosemide injection, a decrease (~50%) in cortical perfusion was observed in the mice population, at 1 hour post injection compared to baseline (P < 0.0001), which returned to baseline after 24 hours (P = 0.68). We reported the successful application of FAIR-based arterial spin labeling for noncontrast perfusion measurement of mouse kidneys using a 1-T desktop scanner. The easy implementation of FAIR sequence on a 1-T desktop scanner offers the potential for longitudinal perfusion studies in limited access areas such as behind the barrier in mouse facilities and in multimodality preclinical imaging laboratories without the administration of exogenous contrast agents.

Guidance for Efficient Small Animal Imaging Quality Control

Routine quality control is a critical aspect of properly maintaining high-performance small animal imaging instrumentation. A robust quality control program helps produce more reliable data both for academic purposes and as proof of system performance for contract imaging work. For preclinical imaging laboratories, the combination of costs and available resources often limits their ability to produce efficient and effective quality control programs. This work presents a series of simplified quality control procedures that are accessible to a wide range of preclinical imaging laboratories. Our intent is to provide minimum guidelines for routine quality control that can assist preclinical imaging specialists in setting up an appropriate quality control program for their facility.

Preclinical tests in Brazil

Preclinical tests in Brazil

Due diligence in the characterization of matrix effects in a total IL-13 Singulex™ method.

After obtaining her PhD in Cellular and Molecular biology from the University of Nevada, Reno, Stephanie has spent the last 15 years in the field of bioanalysis. She has held positions in academia, biotech, contract research and large pharma where she has managed ligand binding assay (discovery to Phase IIb clinical) and flow cytometry (preclinical) laboratories as well as taken the lead on implementing new/emergent technologies. Currently Stephanie leads Pfizer's Regulated Bioanalysis Ligand Binding Assay group, focusing on early clinical biomarker support. Interleukin (IL)-13, a Th2 cytokine, drives a range of physiological responses associated with the induction of allergic airway diseases and inflammatory bowel diseases. Analysis of IL-13 as a biomarker has provided insight into its role in disease mechanisms and progression. Serum IL-13 concentrations are often too low to be measured by standard enzyme-linked immunosorbent assay techniques, necessitating the implementation of a highly sensitive assay. Previously, the validation of a Singulex™ Erenna(®) assay for the quantitation of IL-13 was reported. Herein we describe refinement of this validation; defining the impact of matrix interference on the lower limit of quantification, adding spiked matrix QC samples, and extending endogenous IL-13 stability. A fit-for-purpose validation was conducted and the assay was used to support a Phase II clinical trial.

Sunitinib LC–MS/MS Assay in Mouse Plasma and Brain Tissue: Application in CNS Distribution Studies

Sunitinib malate is a multi-targeted tyrosine-kinase inhibitor, currently in clinical trials for glioma. Previously developed methods for preclinical studies in species such as mice have either employed high-performance liquid chromatography (HPLC) or did not describe a detailed analytical method, which could be employed by other preclinical laboratories. In this paper, we have developed and validated a simple, sensitive high-performance liquid chromatography tandem mass-spectrometric method (LC-MS/MS) for the determination of sunitinib concentration in mouse plasma and brain tissue homogenate using dasatinib-free base as the internal standard. A single step liquid-liquid extraction method was used for both the matrices. Since sunitinib exhibits light-induced E/Z isomerism, all sample preparation was done in light-protected conditions. Separation was performed on a ZORBAX Eclipse XDB C18 column 4.6 × 50 mm, 1.8 μm. The mobile phase consisted of 20 mM ammonium formate (with 0.1 % formic acid): acetonitrile (70:30, v/v) pumped isocratically at a flow rate of 0.25 mL min-1 with a total run-time of 13 min. The retention times of sunitinib and dasatinib were 7.8 and 5.5 min, respectively. The calibration curve was linear over the range from 1.95 to 500 ng mL-1 in both plasma and brain tissue homogenate with 1.95 ng mL-1 as the lower limit of quantification (LLOQ) for both the matrices. Inter- and intra-day accuracy and precision was <15 % for low QC, med QC and high QC and <20 % for LLOQ. The method was applied to a pharmacokinetic study in FVB wild-type mice to determine the plasma and brain concentrations after a single oral sunitinib malate dose of 20 mg kg-1.

Use of optical imaging to progress novel therapeutics to the clinic

There is an undisputed need for employment and improvement of robust technology for real-time analyses of therapeutic delivery and responses in clinical translation of gene and cell therapies. Over the past decade, optical imaging has become the in vivo imaging modality of choice for many preclinical laboratories due to its efficiency, practicality and affordability, while more recently, the clinical potential for this technology is becoming apparent. This review provides an update on the current state of the art in in vivo optical imaging and discusses this rapidly improving technology in the context of it representing a translation enabler or indeed a future clinical imaging modality in its own right.

From mice to women and back again: Causalities and clues for Chlamydia-induced tubal ectopic pregnancy

To provide an overview of knockout mouse models that have pathological tubal phenotypes after Chlamydia muridarum infection, discuss factors and pathological processes that contribute to inflammation, summarize data on tubal transport and progression of tubal implantation from studies in humans and animal models, and highlight research questions in the field. A search of the relevant literature using PubMed and other online tools. University-based preclinical and clinical research laboratories. Women with tubal ectopic pregnancy after Chlamydia trachomatis infection. None. Critical review of the literature. Chlamydia trachomatis infection poses a major threat to human reproduction. Biological and epidemiological evidence suggests that progression of Chlamydia infection causes intense and persistent inflammation, injury, and scarring in the fallopian tube, leading to a substantially increased risk of ectopic pregnancy and infertility. The main targets of Chlamydia infection are epithelial cells lining the mucosal surface, which play a central role in host immune responses and pathophysiology. Tubal phenotypes at the cellular level in mutant mice appear to reflect alterations in the balance between inflammatory mediator and factor deficiency. While studies in mice infected with Chlamydia muridarum have provided insight into potential inflammatory mediators linked to fallopian tube pathology, it is unclear how inflammation induced by Chlamydia infection prevents or retards normal tubal transport and causes embryo implantation in the fallopian tube. Given the similarities in the tubal physiology of humans and rodents, knockout mouse models can be used to study certain aspects of tubal functions, such as gamete transport and early embryo implantation. Elucidation of the exact molecular mechanisms of immune and inflammatory responses caused by Chlamydia infection in human fallopian tubal cells in vitro and understanding how Chlamydia infection affects tubal transport and implantation in animal studies in vivo may explain how Chlamydia trachomatis infection drives inflammation and develops the tubal pathology in women with tubal ectopic pregnancy.

An investigation into fragmentation of hEGF in triple quadrupole mass spectrometry and its quantitative application to human plasma

A growing number of peptides are being used today as biomarkers and therapeutic drugs. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS), in providing a highly specific and reliable technique, is likely to have a substantial impact on peptide quantification. However, most of the applications developed in research laboratories have relied on capillary liquid chromatography, nanospray interfaces, and ion trap or other types of mass spectrometers, which are not commonly found in preclinical and clinical laboratories. Triple quadrupole mass spectrometers are not often considered for quantitative analysis of peptides, despite the fact that they are currently an increasingly common tool in preclinical and clinical laboratories. However, sensitive peptide analyses have been reported, as in the demonstration of structurally distinctive immonium ions that were formed in abundance. Thus, we have recently proposed that use of immonium ions might provide a potential pathway for the quantitative analysis of oligo-peptides with intra-chain disulfide bonds. In the present report, the capability of these ions in LC/MS/MS toward peptide determination was further explored using a polypeptide, human epidermal growth factor (hEGF). hEGF is much larger and more complex than the oligo-peptides previously investigated in our lab. Both the experiments and theoretical calculations were carried out. The results indicate that polypeptides with intra-chain disulfide bonds can increase fragmentation efficiency and raise the number of immonium product ions produced, on the condition that at least one of their intra-loops is sensitive to abrupt bond cleavage. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of hEGF in human plasma. Good linearity and accuracy were achieved.

Growth of an Industry: How U.S. Scientists and Clinicians Have Enabled Stem Cell Tourism

Direct consumer marketing for scientifically unproven “stem cell therapies” has accelerated in parallel with the pace of science-based progress in preclinical research laboratories. Murdoch and Sco...

Tracking problems and possible solutions in the quantitative determination of small molecule drugs and metabolites in biological fluids using liquid chromatography–mass spectrometry

During the last decade, quantification of low molecular weight molecules using liquid chromatography–tandem mass spectrometry in biological fluids has become a common procedure in many preclinical and clinical laboratories. This overview highlights a number of issues involving “small molecule drugs”, bioanalytical liquid chromatography–tandem mass spectrometry, which are frequently encountered during assay development. In addition, possible solutions to these issues are proposed with examples in some of the case studies. Topics such as chromatographic peak shape, carry-over, cross-talk, standard curve non-linearity, internal standard selection, matrix effect, and metabolite interference are presented. Since plasma is one of the most widely adopted biological fluid in drug discovery and development, the focus of this discussion will be limited to plasma analysis. This article is not intended to be a comprehensive overview and readers are encouraged to refer to the citations herein.

New Drugs for the Treatment of Advanced-Stage Diffuse Large Cell Lymphomas

The rapid pace of discovering new signaling pathways that influence the growth and survival of different types of cancer cells has produced a daunting array of potentially new, "drugable" targets for the treatment of cancer. This has been particularly true for the lymphomas. The empiric observation that many kinds of "novel targeted" drugs appear to exhibit relatively selective patterns of activity is testament to the concept that the lymphomas represent an incredibly diverse group of human cancers. Targeting one pathway in a subtype of lymphoma may not be universally effective against all subtypes of lymphoma, even closely related ones. These observations suggest that pharmacologically driven "target identification" plays an important role in the continued development of new therapeutic agents, and underscores the need for more research into the biologic basis for different subtypes of lymphoma. Over the past 10 years, there has been an explosion of new drugs making their way through preclinical laboratories and early clinical studies. These experiences have taught us some frustrating lessons. Sometimes, the strongest biologic rationale is associated with the poorest clinical results. Sometimes, where no rationale exists, golden therapeutic opportunities emerge. In either case, both experiences typically end up teaching us something about the disease we didn't historically appreciate. In this review, we will take a close look at some of the more intriguing targets in aggressive large B-cell lymphomas, all of which now have small molecules that can profoundly affect their activity, activity we hope will lead to new treatment opportunities. We will focus on the biologic rationale for targeting novel pathways regulated by Bcl-6, Bcl-2, BLysS, and APRIL for example. In addition, we will review how new concepts in structural biology and chemical design can help produce new generation compounds with novel activity, as is the case for pralatrexate and targeting the reduced folate carrier-type-1. Clearly, given the breadth and depth of information emerging on these and other relevant biologic pathways, we are limited to discussing only a few illustrative examples, which should in no way detract from the importance of other critical signaling and survival pathways now being exploited in the treatment of lymphoma.

The External RNA Controls Consortium: a progress report

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Mechanisms and the current state of deep brain stimulation in neuropsychiatry.

Deep brain stimulation (DBS) is established as a therapy for movement disorders, and it is an investigational treatment in other neurologic conditions. DBS precisely targets neuroanatomical targets deep within the brain that are proposed to be centrally involved in the pathophysiology of some neuropsychiatric illnesses. DBS is nonablative, offering the advantages of reversibility and adjustability. This might permit therapeutic effectiveness to be enhanced or side effects to be minimized. Preclinical and clinical studies have shown effects of DBS locally, at the stimulation target, and at a distance, via actions on fibers of passage or across synapses. Although its mechanisms of action are not fully elucidated, several effects have been proposed to underlie the therapeutic effects of DBS in movement disorders, and potentially in other conditions as well. The mechanisms of action of DBS are the focus of active investigation in a number of clinical and preclinical laboratories. As in severe movement disorders, DBS may offer a degree of hope for patients with intractable neuropsychiatric illness. It is already clear that research intended to realize this potential will require a very considerable commitment of resources, energy, and time across disciplines including psychiatry, neurosurgery neurology, neuropsychology, bioengineering, and bioethics. These investigations should proceed cautiously.