Translation Of Basic Science Discoveries Research Articles

A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

A Spontaneous Melanoma Mouse Model Applicable for a Longitudinal Chemotherapy and Immunotherapy Study

Mouse models that reflect human disorders provide invaluable tools for the translation of basic science discoveries to clinical therapies. However, many of these invivo therapeutic studies are short term and do not accurately mimic patient conditions. In this study, we used a fully immunocompetent, transgenic mouse model, TGS, in which the spontaneous development of metastatic melanoma is driven by the ectopic expression of a normal neuronal receptor, mGluR1, as a model to assess longitudinal treatment response (up to 8 months) with an inhibitor of glutamatergic signaling, troriluzole, which is a prodrug of riluzole, plus an antibody against PD-1, an immune checkpoint inhibitor. Our results reveal a sex-biased treatment response that led to improved survival in troriluzole and/or anti-PD-1-treated male mice that correlated with differential CD8+ T cells and CD11b+ myeloid cell populations in the tumor-stromal interface, supporting the notion that this model is a responsive and tractable system for evaluating therapeutic regimens for melanoma in an immunocompetent setting.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

Microsurgical reconstruction affects the outcome in a translational mouse model for Achilles tendon healing.

SummaryBackgroundAnimal models are one of the first steps in translation of basic science findings to clinical practice. For tendon healing research, transgenic mouse models are important to advance therapeutic strategies. However, the small size of the structures complicates surgical approaches, histological assessment, and biomechanical testing. In addition, available models are not standardized and difficult to compare. How surgery itself affects the healing outcome has not been investigated yet. The focus of the study was to develop a procedure that includes a transection and microsurgical reconstruction of the Achilles tendon but, unlike other models, preserves the sciatic nerve. We wanted to examine how distinct parts of the technique influenced healing.MethodsFor this animal model study, we used 96 wild-type male C57BL/6 mice aged 8–12 weeks. We evaluated different suture techniques and macroscopically confirmed the optimal combination of suture material and technique to minimize tendon gap formation. A key element is the detailed, step-by-step illustration of the surgery. In addition, we assessed histological (Herovici and Alcian blue staining) outcome parameters at 1–16 weeks postoperatively. Microcomputed tomography (micro-CT) was performed to measure the bone volume of heterotopic ossifications (HOs). Biomechanical analyses were carried out using a viscoelastic protocol on the biomechanical testing machine LM1.ResultsA modified 4-strand suture combined with a cerclage for immobilization without transection of the sciatic nerve reliably eliminated gap formation. The maximal dorsal extension of the hindlimb at the upper ankle joint from the equinus position (limited by the immobilization cerclage) increased over time postoperatively (operation: 28.8 ± 2.2°; 1 week: 54 ± 36°; 6 weeks: 80 ± 11.7°; 16 weeks: 96 ± 15.8°, p > 0.05). Histological staining revealed a maturation of collagen fibres within 6 weeks, whereas masses of cartilage were visible throughout the healing period. Micro-CT scans detected the development of HOs starting at 4 weeks and further progression at 6 and 16 weeks (bone volume, 4 weeks: 0.07604 ± 0.05286 mm3; 6 weeks: 0.50682 ± 0.68841 mm3; 16 weeks: 2.36027 ± 0.85202 mm3, p > 0.001). In-depth micro-CT analysis of the different surgical elements revealed that an injury of the tendon is a key factor for the development of HOs. Immobilization alone does not trigger HOs. Biomechanical properties of repaired tendons were greatly altered and remained inferior 6 weeks after surgery.ConclusionWith this study, we demonstrated that the microsurgical technique greatly influences the short- and longer-term healing outcome. When the sciatic nerve is preserved, the best surgical reconstruction of the tendon defect is achieved by a 4-strand core suture in combination with a tibiofibular cerclage for postoperative immobilization. The cerclage promotes a gradual increase in the range of motion of the upper ankle joint, comparable with an early mobilization rehabilitation protocol. HO, as a key mechanism for poor tendon healing, is progressive and can be monitored early in the model.The translational potential of this articleThe study enhances the understanding of model dependent factors of healing. The described reconstruction technique provides a reproducible and translational rodent model for future Achilles tendon healing research. In combination with transgenic strains, it can be facilitated to advance therapeutic strategies to improve the clinical results of tendon injuries.

BIOMARKERS OF SENESCENT CELL BURDEN

Senescent cells drive aging. Preclinical studies suggest that targeted elimination of senescent cells offers a unique therapeutic approach to counter numerous chronic diseases and geriatric syndromes. To foster the translation of basic science discoveries to clinical application, we have sought to identify circulating biomarkers that reflect systemic senescent cell burden. We first analyzed the secretome of multiple senescent human cell-types and developed a candidate panel of proteins that could be reliably measured in human blood. Multiple proteins demonstrated significant associations with chronological age in a community-based cohort of adults aged 20-to-90 years. Impressively, in two distinct surgical cohorts (severe aortic stenosis and ovarian cancer), candidate protein concentrations were associated with biological age indices, including frailty and adverse outcomes. Our data suggest senescence biomarkers may have utility for clinical practice as indicators of risk, and for clinical research as surrogate endpoints in trials of interventions targeting senescent cells.

Technology Transfer: From the Research Bench to Commercialization: Part 1: Intellectual Property Rights—Basics of Patents and Copyrights

SummaryProgress in medicine hinges on the successful translation of basic science discoveries into new medical devices, diagnostics, and therapeutics. “Technology transfer” is the process by which new innovations flow from the basic research bench to commercial entities and then to public use. In academic institutions, intellectual property rights do not usually fall automatically to the individual inventor per se, but most often are the property of the institution. Technology transfer offices are tasked with seeing to it that such intellectual property rights are properly managed and commercialized. This 2-part series explores the technology transfer process from invention to commercialization. Part 1 reviews basic aspects of intellectual property rights, primarily patents and copyrights. Part 2 will discuss the ways in which inventions become commercialized through startup companies and licensing arrangements with industry players.

Barriers to the Preclinical Development of Therapeutics that Target Aging Mechanisms.

Through the progress of basic science research, fundamental mechanisms that contribute to age-related decline are being described with increasing depth and detail. Although these efforts have identified new drug targets and compounds that extend life span in model organisms, clinical trials of therapeutics that target aging processes remain scarce. Progress in aging research is hindered by barriers associated with the translation of basic science discoveries into the clinic. This report summarizes discussions held at a 2014 Geroscience Network retreat focused on identifying hurdles that currently impede the preclinical development of drugs targeting fundamental aging processes. From these discussions, it was evident that aging researchers have varied perceptions of the ideal preclinical pipeline. To forge a clear and cohesive path forward, several areas of controversy must first be resolved and new tools developed. Here, we focus on five key issues in preclinical drug development (drug discovery, lead compound development, translational preclinical biomarkers, funding, and integration between researchers and clinicians), expanding upon discussions held at the Geroscience Retreat and suggesting areas for further research. By bringing these findings to the attention of the aging research community, we hope to lay the foundation for a concerted preclinical drug development pipeline.

Optimising Translational Research Opportunities: A Systematic Review and Narrative Synthesis of Basic and Clinician Scientists' Perspectives of Factors Which Enable or Hinder Translational Research.

IntroductionTranslational research is central to international health policy, research and funding initiatives. Despite increasing use of the term, the translation of basic science discoveries into clinical practice is not straightforward. This systematic search and narrative synthesis aimed to examine factors enabling or hindering translational research from the perspective of basic and clinician scientists, a key stakeholder group in translational research, and to draw policy-relevant implications for organisations seeking to optimise translational research opportunities.Methods and ResultsWe searched SCOPUS and Web of Science from inception until April 2015 for papers reporting scientists’ views of the factors they perceive as enabling or hindering the conduct of translational research. We screened 8,295 papers from electronic database searches and 20 papers from hand searches and citation tracking, identifying 26 studies of qualitative, quantitative or mixed method designs. We used a narrative synthesis approach and identified the following themes: 1) differing concepts of translational research 2) research processes as a barrier to translational research; 3) perceived cultural divide between research and clinical care; 4) interdisciplinary collaboration as enabling translation research, but dependent on the quality of prior and current social relationships; 5) translational research as entrepreneurial science. Across all five themes, factors enabling or hindering translational research were largely shaped by wider social, organisational, and structural factors.ConclusionTo optimise translational research, policy could consider refining translational research models to better reflect scientists’ experiences, fostering greater collaboration and buy in from all types of scientists. Organisations could foster cultural change, ensuring that organisational practices and systems keep pace with the change in knowledge production brought about by the translational research agenda.

Alpha-1 Antitrypsin Investigations Using Animal Models of Emphysema.

Animal models of disease help accelerate the translation of basic science discoveries to the bedside, because they permit experimental interrogation of mechanisms at relatively high throughput, while accounting for the complexity of an intact organism. From the groundbreaking observation of emphysema-like alveolar destruction after direct instillation of elastase in the lungs to the more clinically relevant model of airspace enlargement induced by chronic exposure to cigarette smoke, animal models have advanced our understanding of alpha-1 antitrypsin (AAT) function. Experimental in vivo models that, at least in part, replicate clinical human phenotypes facilitate the translation of mechanistic findings into individuals with chronic obstructive pulmonary disease and with AAT deficiency. In addition, unexpected findings of alveolar enlargement in various transgenic mice have led to novel hypotheses of emphysema development. Previous challenges in manipulating the AAT genes in mice can now be overcome with new transgenic approaches that will likely advance our understanding of functions of this essential, lung-protective serine protease inhibitor (serpin).

Challenges to the translation of basic science findings to atrial fibrillation therapies.

Faculty of Health, Medicine & Life Sciences, Department of Cardiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands Institute of Pharmacology, West German Heart & Vascular Center, University Duisburg-Essen, Essen, Germany *Author for correspondence: Tel.: +49 201 733 3477; dobromir.dobrev@uk-essen.de

The value of basic research insights into atrial fibrillation mechanisms as a guide to therapeutic innovation: a critical analysis.

Atrial fibrillation (AF) is an extremely common clinical problem associated with increased morbidity and mortality. Current antiarrhythmic options include pharmacological, ablation, and surgical therapies, and have significantly improved clinical outcomes. However, their efficacy remains suboptimal, and their use is limited by a variety of potentially serious adverse effects. There is a clear need for improved therapeutic options. Several decades of research have substantially expanded our understanding of the basic mechanisms of AF. Ectopic firing and re-entrant activity have been identified as the predominant mechanisms for arrhythmia initiation and maintenance. However, it has become clear that the clinical factors predisposing to AF and the cellular and molecular mechanisms involved are extremely complex. Moreover, all AF-promoting and maintaining mechanisms are dynamically regulated and subject to remodelling caused by both AF and cardiovascular disease. Accordingly, the initial presentation and clinical progression of AF patients are enormously heterogeneous. An understanding of arrhythmia mechanisms is widely assumed to be the basis of therapeutic innovation, but while this assumption seems self-evident, we are not aware of any papers that have critically examined the practical contributions of basic research into AF mechanisms to arrhythmia management. Here, we review recent insights into the basic mechanisms of AF, critically analyse the role of basic research insights in the development of presently used anti-AF therapeutic options and assess the potential value of contemporary experimental discoveries for future therapeutic innovation. Finally, we highlight some of the important challenges to the translation of basic science findings to clinical application.

Expanding D&I science capacity and activity within NIH Clinical and Translational Science Award (CTSA) Programs: guidance and successful models from national leaders

The NIH CTSA program (Clinical and Translational Science Awards) supports over 60 research centers working to accelerate scientific discovery, development and implementation to improve health. The CTSA initiative was launched in 2006 to accelerate progress along the research pipeline by overcoming the roadblocks identified by the Institute of Medicine's Clinical Research Roundtable (CRR)-and discussed in numerous additional reports published in the U.S. and internationally. Although the CRR and its international counterparts identified two broad categories of roadblocks, the first impeding the translation of basic science discoveries into effective clinical and public health interventions and the second impeding appropriate implementation-in-practice and societal benefits of these interventions, to date most CTSA activity has focused largely on the first translational roadblock. The presentations included in this panel represent national leaders in the establishment of effective programs to support and expand CTSA activity related to the second translational roadblock, barriers to effective dissemination, implementation and achievement of population health benefit of NIH-funded research. The presentations describe the components, activities and impacts of CTSA-based D&I science programs established at the University of California at San Francisco, University of North Carolina at Chapel Hill, and Washington University at St. Louis. The panel will also describe the ongoing development of a new program at the University of California at Los Angeles (guided by the three programs featured in the panel) and will feature comments by key national research leaders addressing the value and role of these national leaders in stimulating and guiding similar efforts across other CTSAs and similar research institutions.

Found in Translation: Training the Next Generation of Translational Neuroscientists

Integrated, multidisciplinary programs for training physicians and scientists in translational science have become essential in efforts to meet the demand for more effective translation of basic science discoveries into new clinical applications.

Form and Function of the intervertebral Disc: Human Organ Culture and Histological Stain Comparison Study

Introduction Multiple therapies for treating intervertebral disc (IVD) degeneration are under development and range from injectable biologics to implantable devices. Treatments that show promise in small animal and cell culture systems often fail in human trials. There is a lack of models that are comparable to human IVDs, and this is a major obstacle preventing effective translation of basic science findings to human treatment. Human IVDs remodel and accumulate damage over years of life and disease, and anatomical features of vertebrae and IVDs are likely to influence clinical outcomes. Organ culture of IVDs from human autopsy provides a model that represents the spectrum of IVD degeneration. Prior studies demonstrated that whole human IVD organ culture is possible, but required organ donation and described removal of vertebral endplates which can lead to swelling and matrix loss.1 The objectives of this study are to determine the feasibility of whole human IVD organ culture using human autopsy specimens and to highlight important anatomical features using a range of tinctorial stains. Materials and Methods Bioreactor Design An organ culture chamber for use with human IVDs was designed, based on a previously described bovine IVD system.2 IVDs from human autopsy are isolated, placed between two permeable platens of the chamber, sealed and loaded to prevent swelling (Fig. A). Human Autopsy IVD Isolation Six human spines were collected from autopsy services with approval and X-rayed to assess degenerative level and establish feasibility for organ culture. IVDs that retained the vertebral and cartilaginous endplates were harvested and vertebral endplates were cleaned as previously described.2 Culture Conditions IVDs from 3 different autopsy donors (average age 36 ± 24) were loaded for 7, 14, and 21 days in organ culture at static (0.2MPa) or diurnal (0.1-0.2MPa) conditions. All cultures occurred in 37°C and 5% CO2 using DMEM (high glucose, 10%FBS, 1% Pen/Strep, 0.2% ascorbic acid, and primocin) with media changes every 3-4 days. To assess transport through the endplates, 500 µM Texas Red labeled dextran (MW=3 kDa) was added to the media. Following culture, tissue was taken for cell viability (cryosections stained with MTT for live cells and DAPI for nuclei) and histology. Tissue Histology The 21 day organ culture specimens and additional human autopsy IVDs ( n = 6) were isolated for tinctorial staining. Tissue was fixed, embedded in plastic (PMA), and 5µm sagittal sections were used for histology (Fig. B). Multiple stains were compared: Safranin-O, Toluidine Blue, Picrosirius Red & Alcian Blue,3 modified Gomori's Tri-chrome, and XFAST.4 Annulus fibrosus (AF), nucleus pulposus (NP), endplate, Schmorl's node, and transitional regions were compared. Results Human IVDs from autopsy were successfully cultured for up to 21 days. At the culture period completion, evidence of dextran transport was observed at the interface of the cartilaginous endplate and AF. Effective transport was confirmed with the high cell viability that was maintained in both NP and AF through day 21. Tinctorial staining after 21 day organ culture highlighted many structural elements of human IVDs (Fig. C). A distinctive Schmorl's node region stained with XFAST revealed a protrusion disrupting the organization of chondrocytes and matrix in the cartilaginous endplate and intercalated blue and orange staining in the matrix surrounding cell clusters (Fig. D). Localized variations in proteoglycan staining were apparent from both XFAST and Safranin-O (Fig. E) staining including diminished staining in cartilage endplates. Color gradients were more obvious with XFAST than Safranin-O. The tri-chrome stain (Fig. F) highlighted the disrupted structure at the endplate to NP interface and surrounding the entire Schmorl's node, however it was difficult to discern proteoglycan rich and collagen rich regions. Picrosirius red and Alcian blue staining (Fig. G) highlighted the collagenous structure of the cartilaginous endplate and in pericellular regions around cell clusters, although proteoglycan staining was not well differentiated in this region with this stain. Fig. (A) Human autopsy IVD organ culture chamber setup. (B) IVD sections including vertebral endplate were prepared and cultured. Following culture, sagittal sections were used for histology. (C) Representative sagittal section of the IVD (47 years/o) with XFAST staining (scale=5,000µm, 2.5X). The Schmorl's node region of interest (scale = 1,000µm, 10X) was used to compare tinctorial stains: (D) XFAST; (E) Safranin-O; (F) modified Gomori's tri-chrome; (G) Picrosirius red & Alcian blue. Conclusion Key structural features in human IVDs are important in replicating clinical conditions; therefore the development of a relevant human organ culture model serves as an important tool to screen therapeutics and repair strategies to treat IVD degeneration. The presence of dextran at the interface of the endplate and AF, and high cell viability in the AF and NP after 21 days in culture demonstrated the feasibility of human organ culture using IVDs from autopsy. Tinctorial stain comparisons of sagittal IVD sections highlighted the complex structure of human IVDs, which are difficult to replicate in animal model systems. The use of IVDs from autopsy provides a viable source for ex vivo experiments to assess mechanisms of pathophysiology and to evaluate therapies for IVD degeneration in a highly clinically relevant model system. Acknowledgments Funded by NIH Grant R01 AR057397. We thank Dr. T. Naidich & D. Shiovitz for their assitance. Disclosure of Interest None declared References Gawri R, Mwale F, Ouellet J, et al. Development of an organ culture system for long-term survival of the intact human intervertebral disc. Spine 2011;36(22):1835–1842 Illien-Jünger S, Gantenbein-Ritter B, Grad S, et al. The combined effects of limited nutrition and high-frequency loading on intervertebral discs with endplates. Spine 2010;35(19):1744–1752 Gruber HE, Ingram J, Hanley EN Jr. An improved staining method for intervertebral disc tissue. Biotech Histochem 2002;77(2):81–83 Leung VY, Chan WC, Hung SC, Cheung KM, Chan D. Matrix remodeling during intervertebral disc growth and degeneration detected by multichromatic FAST staining. J Histochem Cytochem 2009;57(3):249–256

Mentoring Interdisciplinary Research Teams for the Study of Sex and Gender Differences in Health and Disease

Initiatives to hasten the translation of basic science discoveries to clinical care have necessitated the development of new approaches to interdisciplinary collaboration and training of future investigators. This has been nowhere more important than in the study of sex differences with implications for extension into areas of gender medicine. Clearly, gaining better understanding of the role that sex and gender play in health and disease is essential to the implementation of truly individualized medicine. This case report will describe our experiences in developing the Mayo Clinic Building Interdisciplinary Research Programs in Women's Health (BIRCWH) program, an interdisciplinary research and training program in women's health and sex and gender differences. We identify both our successes and the barriers we have encountered in order that others who are developing similar programs might benefit from our experiences.

P68: Laser Doppler perfusion imaging for non-invasive assessment of vascular responses in living mice

Background Methods for the assessment of vascular functions in mice are largely limited to ex vivo organ bath methods particularly the aortic ring assay. We aimed at establishing an easy to perform, sensitive, and valid method for the non-invasive measurement of vascular responses to pharmacological and physiological stimuli in vivo using laser Doppler perfusion imaging (LDPI). Methods Male C57Bl/6, 8–10 week old mice were anesthetized with isoflurane and tissue perfusion of the hindlimb was assessed by LDPI (Perimed, Stockholm, Sweden). The changes in tissue perfusion in response to vasodilators and vasoconstrictors was assessed by acute intraperitoneal (i.p) application of nitroglycerin, intravenous (i.v.) application of acethylcholine, and i.p application of epinephrine. Reactive hyperemic blood flow was induced by occluding the vessels of the hindlimb using an inflatable cuff until the perfusion signal reached the zero level. After 1–5 min of ischemia the cuff pressure was released. We determined intra-individual variability by measuring the same mouse on three consecutive days ( n  = 4) and inter-individual variability by comparing the coefficient of variation (CV) of 11 mice. The effect of acute and chronic inhibition of nitric oxide synthase (NOS) or cyclooxygenase (COX) on the hyperemic response was achieved by administration of l -NNA or indometacin i.p or for 6 days in drinking water ad libitum, respectively. Results Application of nitroglycerin increased the perfusion of the hindlimb within approximately 100 s by 127% (191 ± 36 vs. 435 ± 23 perfusion units, PU), while epinephrine (209 ± 59 vs. 90 ± 18 PU:) resulted in a maximum decrease of 57% of the mean perfusion ( n  = 5). I.v. administration of adenosine elevated the perfusion by 41% (224 ± 34 vs. 316 ± 43 PU). Reactive hyperemia progressively increased after 1, 3 and 5 min ischemia with the maximal response achieved after 5 min ischemia. Time to maximum reperfusion and the ratio of maximum/baseline perfusion values (perfusion reserve) showed the smallest variation, while the area under the perfusion curve exhibited a CV of 40–50%. Generally, intra-individual variability was lower as compared to inter-individual variability. Chronic oral treatment with l -NNA significantly prolonged the time to maximal perfusion (36 ± 5 vs. 77 ± 18 s), while acute NOS inhibition ( l -NNA i.P) reduced the perfusion reserve (2.5 ± 0.1 vs. 1.8 ± 0.0). In addition, chronic inhibition of COX resulted in a 16 s longer reperfusion (48 ± 5 vs. 64 ± 5 s) but had no effect on the other parameters. Conclusion Taken together, LDPI can be used as a valid and reproducible method for measuring vascular responses to physiological and pharmacological stimuli in the hindlimb of living mice. This may be a valuable experimental model that is similar to methodologies applied in humans enabling a translation of basic science findings. Disclosure Nothing to disclose.

Emerging menu options for gonadal reincarnation

For nearly a decade now, amidst the promises and purviews of regenerative medicine, the prospects for rehabilitation or replacement of tattered organs have resonated with those unwilling to accept the fate of their aging bodies. Your personal candidate for tissue replacement is often dictated by your genetics and some combination of luck–good or bad–that challenges your organs’ abilities to withstand the test of time after years of use and abuse. Regenerative medicine is fast becoming a reality, and will likely debut on a commercial level as tractable tissue replacements for joints, blood vessels, and bladders, which are less complex, histologically-speaking. But anxious moments accrue for those whose replacement needs include the likes of hearts, brains, kidneys, and livers. What about your gonads? The search for a fountain of youth when it comes to preserving or extending your reproductive ability reduces to a most fundamental biological principle: use it or lose it! Gonads carry the burden of performing two basic tasks during our reproductive lifespan and they do so on a continuous or discontinuous schedule. For males, the call of puberty enlightens the testes such that hormonal rhythms sustain the continuous process of spermatogenesis, yielding sufficient numbers of spermatozoa to meet the regular demands of a fully-loaded ejaculate. For females, on the other hand, the strategy of putting all the eggs in one basket (sic two ovaries) at birth leads to the discontinuous provision of a single mature oocyte at ovulation, the tell-tale outcome of periodic fluctuations in a hormonal conversation that took place between the brain and gonads. In both cases, germ plasm wastage is the norm, not the exception, with the vast majority of germ cells vanishing into an abyss not unlike the one that so many aspiring actors find themselves resolved to on the road to Broadway or Hollywood. Fortunately (or not), the one-on-one encounter of sperm and egg happens against all odds, accounting for our growing numbers on this planet. The less fortunate among us seek medical intervention for infertility treatment when and where available. Enter the world of fertility preservation. 2012 was indeed a remarkable year. The prospects for manipulating gonadal physiology and germ cell wastage were measurably advanced by a series of landmark papers, which spawned a journalistic feeding frenzy that captured the global attention of health specialists and the public at large. Fittingly, this was best evidenced by recognizing the breakthroughs of the 2012 Nobel Laureates in Physiology and Medicine, Sir John Gurdon and Professor Shinya Yamanaka. The notion that a nucleus from a differentiated cell could be coaxed back into a naive or pluripotent state (a notion that became known as reprogramming) transitioned from an eclectic observation on amphibian oocytes in the 1960s to iconic status after the demonstration of induced pluripotent stem cell (iPSCs) production early in the 21st century. To the matter of fertility preservation, a continuing special focus for JARG, three papers lead off this issue which set the stage for what is emerging as a central role for human ARTs in the field of regenerative medicine. Gonadotoxic therapies for management of cancer and hematological disorders in children pose one of the most serious threats to future gonadal function and understandably test physicians seeking to retain some measure of reproductive fitness for their patients. From Kutluk Oktay’s group comes a report on male and female children who were offered gonadal tissue cryopreservation during the course of various medically-indicated procedures. The findings illustrate how a successful team effort can be realized to minimize patient costs and risks, and afford an opportunity for restoration of gonadal function in their anticipated reproductive years of life. From Denmark comes an important study by Mikkel Rosendahl and colleagues in which cryopreserved ovarian tissues were transplanted into patients following gonadotoxic cancer treatments and biomarkers of functional restoration were apparent in many of the patients so treated. This work adds momentum to the importance of gonadal tissue cryopreservation for subsequent transplantation despite lingering concerns over reintroduction of disease and a recognized need to develop better methods and approaches for long term storage of ovarian or testicular biopsy material. Towards this end, we feature the paper by Sandra Sanfilippo and collaborators from several institutions who have tailored a novel slow-freeze formulation for ovarian tissue that alters both cryoprotectant and antioxidant components. In addition to evaluating follicular viability, the study uncovers important parameters of vascular endothelium and stromal compartments in thawed ovarian tissues that were subjected to an extended culture period, a measure of tissue functionality that had not been previously exploited and one that will likely set the stage for future investigations on tissue integrity prior to transplantation. Collectively, these papers emphasize the importance and applicability of gonadal reincarnation for patients who have the opportunity to undergo tissue cryopreservation with existing technology. It is fair then to ask whether other technologies loom on the horizon for recharging gonads that have endured complete or partial loss of germ plasm? The matter of stem cells and gametes will be featured in the February issue of JARG but, most certainly, a crowning accomplishment reported late in 2012 has brought to the surface an intermingling of reproductive and regenerative medicine. From Kyoto University in Japan arrives the publication by Hayashi and colleagues (“Offspring from oocytes derived from in vitro primordial Germ cell-like cells in mice”, Science, 4 October 2012 / Page 1 / 10.1126/science.1226889) describing closure of the circle of life between stem cells and female germ cells in the mouse, a repeat performance of their 2011 paper in Cell (doi:10.1016/j.cell.2011.06.052) when this was achieved in the male. The lessons obtained from this work are manifold. It is a technological tour de force, a roadmap for using developmental biology principles in experimental design, and a declaration for the expanding role that ARTs play in the field of regenerative medicine. The bottom line is that mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), converted into primordial germ cell-like cells when combined with age-appropriate ovarian somatic cells, assumed the characteristics of card-carrying oocytes. Proof of the pudding came in the form of demonstrating that oocytes retrieved from reconstituted ovaries transplanted into a “host” ovarian environment were capable of undergoing in vitro maturation and IVF, and produced viable offspring after embryo transfer. What distinguishes this work from the many other attempts to generate oocytes from various kinds of stem cells is the directness of the approach and its overall efficiency. Efforts to repeat this work are ongoing, and the implications it raises in terms of teasing apart the mechanistic attributes that confer reprogramming of ES or iPS lineages into the female germ line will surely raise our level of understanding of the biology of the oocyte in particular and the ovary more broadly. Satisfying our curiosity about the underpinnings of germ cell mystique is but one likely outcome from the studies described in Tsutomu Saitou’s work, as recently discussed by Hayashi et al.,( Fertility and Sterility, 2012; 97:1250–9). And, it would follow that some of the factors underlying infertility in men and women could be modeled through such an approach using stem cells of either embryonic or iPSC varieties from humans and other animals. What remains unknown, however, is the extent to which advances of this kind will be brought bear in a clinical setting. With the remarkable strides of the past year as a backdrop, there is much to anticipate in 2013 as the field of reproductive medicine contextualizes these and forthcoming discoveries with respect to new menu options in the human ART clinic, and the ethical and legal implications that will arise in the translation of basic science discoveries into clinical practice. I urge our readership to prepare for a roller coaster ride into the future. And finally, note that this excursion will be facilitated by a number of additions to our Editorial Board who we are pleased will be joining the JARG family. Welcome to our new Associate Editors Clarisa Gracia, Lawrence C. Layman, and Elizabeth McGee, and new board members Paul Brezina, Sherman Silber, Anil Dubey, Daniel DeMatos, Hananel Holzer, Karla Hutt, and Armand Zini. Best wishes for a healthy and productive 2013!

A Comprehensive Career-Success Model for Physician–Scientists

With today's focus on the translation of basic science discoveries into clinical practice, the demand for physician-scientists is growing. Yet, physicians have always found it challenging to juggle the demands of clinical care with the time required to perform research. The Research on Careers Workgroup of the Institute for Clinical Research Education at the University of Pittsburgh developed a comprehensive model for career success that would address, and allow for the evaluation of, the personal factors, organizational factors, and their interplay that contribute to career success. With this model, leaders of training programs could identify early opportunities for intervening with potential physician-scientists to ensure career success. Through an iterative process described in this article, the authors identified and examined potential models for career success from the literature, added other elements determined to be significant, and developed a comprehensive model to assess factors associated with career success for physician-scientists. The authors also present examples of ways in which this model can be adapted and applied to specific situations to assess the effects of different factors on career success.

Clinical advances in hemophilia management

Hemophilia is an excellent example in medicine where clinical translation of basic science discoveries has transformed the gloomy outlook of the disease. This review provides an overview of clinical advances in hemophilia management with a specific focus on the molecular heterogeneity of the disease and progress in management of patients with inhibitors. Novel therapeutics and the emerging ethical issues in the field of hemophilia are also discussed.