Building a Professional Identity and an Academic Career Track in Translational Medicine

Biomedical scientists aim to contribute to further understanding of disease pathogenesis and to develop new diagnostic and therapeutic tools that relieve disease burden. Yet the majority of biomedical scientists do not develop their academic career or professional identity as “translational scientists,” and are not actively involved in the continuum from scientific concept to development of new strategies that change medical practice. The collaborative nature of translational medicine and the lengthy process of bringing innovative findings from bench to bedside conflict with established pathways of building a career in academia. This collaborative approach also poses a problem for evaluating individual contributions and progress. The traditional evaluation of scientific success measured by the impact and number of publications and grants scientists achieve is inadequate when the product is a team effort that may take decades to complete. Further, where scientists are trained to be independent thinkers and to establish unique scientific niches, translational medicine depends on combining individual insights and strengths for the greater good. Training programs that are specifically geared to prepare scientists for a career in translational medicine are not widespread. In addition, the legal, regulatory, scientific and clinical infrastructure and support required for translational research is often underdeveloped in academic institutions and funding organizations, further discouraging the development and success of translational scientists in the academic setting. In this perspective we discuss challenges and potential solutions that could allow for physicians, physician scientists and basic scientists to develop a professional identity and a fruitful career in translational medicine

Building a Professional Identity and an Academic Career Track in Translational Medicine

Biomedical scientists aim to contribute to further understanding of disease pathogenesis and to develop new diagnostic and therapeutic tools that relieve disease burden. Yet the majority of biomedical scientists do not develop their academic career or professional identity as “translational scientists,” and are not actively involved in the continuum from scientific concept to development of new strategies that change medical practice. The collaborative nature of translational medicine and the lengthy proce

Using Data-mining Techniques for the prediction of the severity of road crashes in Cartagena, Colombia

Objective: Analyze the road crashes in Cartagena (Colombia) and the factors associated with the collision and severity. The aim is to establish a set of rules for defining countermeasures to improve road safety. Methods: Data mining and machine learning techniques were used in 7894 traffic accidents from 2016 to 2017. The severity was determined between low (84%) and high (16%). Five classification algorithms to predict the accident severity were applied with WEKA Software (Waikato Environment for Knowledge Analysis). Including Decision Tree (DT-J48), Rule Induction (PART), Support Vector Machines (SVMs), Naïve Bayes (NB), and Multilayer Perceptron (MLP). The effectiveness of each algorithm was implemented using cross-validation with 10-fold. Decision rules were defined from the results of the different methods. Results: The methods applied are consistent and similar in the overall results of precision, accuracy, recall, and area under the ROC curve. Conclusions: 12 decision rules were defined based on the methods applied. The rules defined show motorcyclists, cyclists, including pedestrians, as the most vulnerable road users. Men and women motorcyclists between 20–39 years are prone in accidents with high severity. When a motorcycle or cyclist is not involved in the accident, the probable severity is low

Novel role for the transient receptor potential channel TRPM2 in prostate cancer cell proliferation

We have identified a novel function for a member of the transient receptor potential (TRP) protein super-family, TRPM2, in prostate cancer cell proliferation. TRPM2 encodes a non-selective cation-permeable ion channel. We found that selectively knocking down TRPM2 with the small interfering RNA technique inhibited the growth of prostate cancer cells but not of non-cancerous cells. The subcellular localization of this protein is also remarkably different between cancerous and non-cancerous cells. In BPH-1 (benign), TRPM2 protein is homogenously located near the plasma membrane and in the cytoplasm, whereas in the cancerous cells (PC-3 and DU-145), a significant amount of the TRPM2 protein is located in the nuclei in a clustered pattern. Furthermore, we have found that TRPM2 inhibited nuclear ADP-ribosylation in prostate cancer cells. However, TRPM2 knockdown-induced inhibition of proliferation is independent of the activity of poly(ADP-ribose) polymerases. We conclude that TRPM2 is essential for prostate cancer cell proliferation and may be a potential target for the selective treatment of prostate cancer

PAX4 Enhances Beta-Cell Differentiation of Human Embryonic Stem Cells

Background Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of β-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. Methods and Findings Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a β-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca2+ homeostasis of β-cells developing in embryoid bodies produced from such HESC. Cells expressing key β-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. Conclusion Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative β-cells. Our findings provide a novel foundation to study the mechanism of pancreatic β-cells differentiation during early human development and to help evaluate strategies for the generation of purified β-cells for future clinical applications

Conductance Ratios and Cellular Identity

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible

A Functional Nuclear Localization Sequence in the C. elegans TRPV Channel OCR-2

The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Cav1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated

The carboxy-terminal fragment of α1A calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4–20Q; SCA6: 20–33Q) in the carboxyl(C)-terminal cytoplasmic domain of the α1A voltage-dependent calcium channel (Cav2.1). Although a 75–85-kDa Cav2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Cav2.1 in human SCA6 brains. New antibodies against the Cav2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Cav2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus

Functional KV10.1 Channels Localize to the Inner Nuclear Membrane

Ectopically expressed human KV10.1 channels are relevant players in tumor biology. However, their function as ion channels at the plasma membrane does not totally explain their crucial role in tumors. Both in native and heterologous systems, it has been observed that a majority of KV10.1 channels remain at intracellular locations. In this study we investigated the localization and possible roles of perinuclear KV10.1. We show that KV10.1 is expressed at the inner nuclear membrane in both human and rat models; it co-purifies with established inner nuclear membrane markers, shows resistance to detergent extraction and restricted mobility, all of them typical features of proteins at the inner nuclear membrane. KV10.1 channels at the inner nuclear membrane are not all transported directly from the ER but rather have been exposed to the extracellular milieu. Patch clamp experiments on nuclei devoid of external nuclear membrane reveal the existence of channel activity compatible with KV10.1. We hypothesize that KV10.1 channels at the nuclear envelope might participate in the homeostasis of nuclear K+, or indirectly interact with heterochromatin, both factors known to affect gene expression

Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts