T-cell lymphocytes' aging clock: telomeres, telomerase and aging.

Aging is the decline of physiological capabilities required for life maintenance and reproduction over time. The human immune cells, including T-cells lymphocytes, undergo dramatic aging-related changes, including those related to telomeres and telomerase. It was demonstrated that telomeres and telomerase play crucial roles in T-cell differentiation, aging, and diseases, including a well-documented link between short telomeres and telomerase activation demonstrated in several T-cells malignancies. Herein, we provide a comprehensive review of the literature regarding T-cells' telomeres and telomerase in health and age related-diseases.

Training the next generation of Canadian Clinician-Scientists: charting a path to success.

Clinician-scientists are physicians with training in both clinical medicine and research that enables them to occupy a unique niche as specialists in basic and translational biomedical research. While there is widespread acknowledgement of the importance of clinician-scientists in today's landscape of evidence-based medical practice, training of clinician-scientists in Canada has been on the decline, with fewer opportunities to obtain funding. With the increasing length of training and lower financial compensation, fewer medical graduates are choosing to pursue such a career. MD-PhD programs, in which trainees receive both medical and research training, have the potential to be an important tool in training the next generation of clinician-scientists; however, MD-PhD trainees in Canada face barriers that include an increase in medical school tuition and a decrease in the amount of financial support. We examined the available data on MD-PhD training in Canada and identified a lack of oversight, a lack of funding and poor mentorship as barriers experienced by MD-PhD trainees. Specific recommendations are provided to begin the process of addressing these challenges, starting with the establishment of an overseeing national body that would track long-term outcome data for MD-PhD trainees. This national body could then function to implement best practices from individual programs across the country and to provide further mentorship and support for early-career physician-scientists. MD-PhD programs have the potential to address Canada's growing shortage of clinician-scientists, and strengthening MD-PhD programs will help to effect positive change.

Scientific Overview: CSCI-CITAC Annual General Meeting and Young Investigators' Forum 2015.

The 2015 Annual General Meeting of The Canadian Society of Clinician Investigators (CSCI) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was held in Toronto November 23-25, 2015, in conjunction with The University of Toronto Clinician Investigator Program Research Day. The theme for this year's meeting was "It takes a village" and the focus was the various support systems necessary to train a successful clinician scientist. The meeting featured an opening presentation by Dr. Vincent Dumez and workshops by Dr. Peter Nickerson, Dr. Jane Aubin, Dr. Kelly Warmington and Dr. Norman Rosenblum, and MD/PhD trainees Nardin Samuel, Kevin Wang and Kirill Zaslavsky. The keynote speakers were Dr. David Malkin (Hospital for Sick Children) who received the CSCI-RCPSC Henry Friesen Award, Dr. Brent Richards (McGill University) who received the Joe Doupe Award and Ernesto Shiffrin (Lady Davis Institute) who received the Distinguished Scientist Award. As always, the conference showcased outstanding scientific presentations from clinician investigator trainees from across the country at the Young Investigators' Forum. The research topics, which ranged from basic sciences to clinical medicine and translational work, are summarized in this review. Over 90 abstracts were presented at this year's meeting during two poster sessions, with several of the outstanding abstracts selected for oral presentations.

MYCN overexpression is associated with unbalanced copy number gain, altered nuclear location, and overexpression of chromosome arm 17q genes in neuroblastoma tumors and cell lines.

MYCN amplification and MYCN overexpression are poor prognostic factors in neuroblastoma. Tumors with unbalanced chromosome arm 17q gain are often associated with MYCN amplification; however, the relationship between chromosome 17 copy number status and MYCN expression is not known. We investigated the relationship between MYCN expression and chromosome 17 copy number, nuclear location, and gene expression. By performing dual-colored fluorescence in situ hybridization on 16 primary neuroblastomas, we found that those with unbalanced gain of 17q have high MYCN expression, those with no gain have medium expression, and those with numerical gain have low expression (P < 0.0001). We also found that the nuclear location of 17q correlates with chromosome 17 copy number status: copies in tumors with unbalanced gain and no gain of chromosome 17 occupy a more central location than those in tumors with balanced gain (P < 0.0001). We show that a more central nuclear location of 17q coincides with increased expression of genes found within this chromosome arm. To further understand the association between MYCN expression and chromosome 17, we overexpressed MYCN in two low-expressing MYCN cell lines, SHEP and GIMEN. We found that both cell lines had an unbalanced gain of chromosome 17q, a more central nuclear location of the region and increased expression of the 17q genes. Therefore, this study indicates, for the first time, a functional relationship between MYCN overexpression and the gain of 17q in neuroblastoma.

Measuring murine chromosome orientation in interphase nuclei.

The nuclear architecture of a cell may change as a result of various diseases, including cancer. A variety of nuclear features are, therefore, of interest to cell biologists. Recently, several studies have investigated the orientation of chromosomes in the interphase nucleus either visually or semi-automatically. In this article an automated method to measure this orientation is presented. The theoretical difference between performing these measurements in two and three dimensions is discussed and experimentally verified. The results computed from measurements of murine nuclei correspond with results from visual inspection. We found significant differences in the orientation of chromosome 11 between nuclei from a PreB cell line of BALB/c origin and primary B nuclei from congenic [T38HxBALB/c]N wild-type mice. Since our new automatic method concurs with both the visual and semi-automatic methods, we conclude that the automatic method can replace these methods in assessing chromosome orientation.

Distinct nuclear orientation patterns for mouse chromosome 11 in normal B lymphocytes.

BACKGROUND: Characterizing the nuclear orientation of chromosomes in the three-dimensional (3D) nucleus by multicolor banding (mBANDing) is a new approach towards understanding nuclear organization of chromosome territories. An mBANDing paint is composed of multiple overlapping subchromosomal probes that represent different regions of a single chromosome. In this study, we used it for the analysis of chromosome orientation in 3D interphase nuclei. We determined whether the nuclear orientation of the two chromosome 11 homologs was random or preferential, and if it was conserved between diploid mouse Pre B lymphocytes of BALB/c origin and primary B lymphocytes of congenic [T38HxBALB/c]N wild-type mice. The chromosome orientation was assessed visually and through a semi-automated quantitative analysis of the radial and angular orientation patterns observed in both B cell types. RESULTS: Our data indicate that there are different preferential patterns of chromosome 11 orientation, which are not significantly different between both mouse cell types (p > 0.05). In the most common case for both cell types, both copies of chromosome 11 were oriented in parallel with the nuclear border. The second most common pattern in both types of B lymphocytes was with one homolog of chromosome 11 positioned with its telomeric end towards the nuclear center and with its centromeric end towards the periphery, while the other chromosome 11 was found parallel with the nuclear border. In addition to these two most common orientations present in approximately 50% of nuclei from each cell type, other orientations were observed at lower frequencies. CONCLUSIONS: We conclude that there are probabilistic, non-random orientation patterns for mouse chromosome 11 in the mouse B lymphocytes we investigated (p < 0.0001).

Association among amyloid plaque, lipid, and creatine in hippocampus of TgCRND8 mouse model for Alzheimer disease.

Amyloid peptide (Aß) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30-50 µm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.

The Rising Challenge of Training Physician-Scientists: Recommendations From a Canadian National Consensus Conference.

Physician-scientists are individuals who actively participate in patient care, have undergone additional research training, and devote the majority of their time to research. Physician-scientists are traditionally the primary catalysts in bridging the translational gap-that is, the failure to link fundamental new knowledge in the pathobiology of disease with advances in health care and health policy in a timely manner. However, there has been a shift away from training physician-scientists, and financial support for the physician-scientist is diminishing globally, causing the translational gap to grow. Given its socialized health care system and cultural and geographic diversity, Canada can serve as a unique case study in understanding how to address this phenomenon as a national priority. To this end, a Canadian national consensus conference was convened to develop recommendations for training programs and early-career supports for physician-scientists. Five recommendations were generated: (1) Establish an independent, national council whose mandate is to provide pan-Canadian oversight of physician-scientist training programs; (2) develop capacity for funding and mentorship support for physician-scientists; (3) develop coherent networks across a broad range of clinician-scientists, including physician-scientists, to reflect the unique cultural and geographic diversity of Canada and to reflect the interdisciplinarity of health research; (4) ensure that medical school curricula integrate, as a core curriculum feature, an understanding of the scientific basis of health care, including research methodologies; and (5) ensure that the funding of the physician-scientist trainee is viewed as portable and distinct from the operational funding provided to the training program itself.

Role of non-government organizations in engaging medical students in research.

The continued decline in medical trainees entering the workforce as clinician-scientists has elevated the need to engage medical students in research. While past studies have shown early exposure to generate interest among medical students for research and academic careers, financial constraints have limited the number of such formal research training programs. In light of recent government budget cuts to support research training for medical students, non-government organizations (NGOs) may play a progressively larger role in supporting the development of clinician-scientists. Since 2005, the Mach-Gaensslen Foundation has sponsored 621 Canadian medical student research projects, which represents the largest longitudinal data set of Canadian medical students engaged in research. We present the results of the pre- and post-research studentship questionnaires, program evaluation survey and the 5-year and 10-year follow-up questionnaires of past recipients. This paper provides insight into the role of NGOs as stakeholders in the training of clinician-scientists and evaluates the impact of such programs on the attitudes and career trajectory of medical students. While the problem of too few physicians entering academic and research-oriented careers continues to grow, alternative-funding strategies from NGOs may prove to be an effective approach in developing and maintaining medical student interest in research.

Characteristics and outcomes of Canadian MD/PhD program graduates: a cross-sectional survey.

BACKGROUND: Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs. METHODS: We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015. Respondents were categorized into 2 groups according to whether they had or had not completed all training. RESULTS: Of the 186 eligible alumni of MD/PhD programs, 139 (74.7%) completed the survey. A total of 136/138 respondents (98.6%) had completed or were currently completing residency training, and 66/80 (82%) had completed at least 1 postgraduate fellowship. Most (58 [83%]) of the 70 respondents who had completed all training were appointed as faculty at academic institutions, and 37 (53%) had been principal investigators on at least 1 recent funded project. Among the 58 respondents appointed at academic institutions, 44/57 (77%) dedicated at least 20% of their time to research, and 25/57 (44%) dedicated at least 50% to research. During their combined degree, 102/136 respondents (75.0%) published 3 or more first-author papers, and 133/136 (97.8%) matched with their first choice of specialty. The median length of physician-scientist training was 13.5 years. Most respondents graduated with debt despite having been supported by Canadian Institutes of Health Research MD/PhD studentships. INTERPRETATION: Most Canadian MD/PhD program alumni pursued careers consistent with their physician-scientist training, which indicates that these programs are meeting their primary objective. Nevertheless, our findings highlight that a minority of these positions are research intensive; this finding warrants further study. Our data provide a baseline for future monitoring of the output of Canadian MD/PhD programs.

Identification of Neuroblastoma Subgroups Based on Three-Dimensional Telomere Organization.

Using 3D telomere quantitative fluorescence in situ hybridization, we determined the 3D telomere organization of 74 neuroblastoma tissue samples. Hierarchical cluster analysis of the measured telomere parameters identified three subgroups from our patient cohort. These subgroups have unique telomere profiles based on telomere length and nuclear architecture. Subgroups with higher levels of telomere dysfunction were comprised of tumors with greater numbers of telomeres, telomeric aggregates, and short telomeres (P<.0001). Tumors with greater telomere dysfunction were associated with unfavorable tumor characteristics (greater age at diagnosis, unfavorable histology, higher stage of disease, MYCN amplification, and higher MYCN expression) and poor prognostic risk (P<.001). Subgroups with greater telomere dysfunction also had higher intratumor heterogeneity. MYCN overexpression in two neuroblastoma cell lines with constitutively low MYCN expression induced changes in their telomere profile that were consistent with increased telomere dysfunction; this illustrates a functional relationship between MYCN and 3D telomere organization. This study demonstrates the ability to classify neuroblastomas based on the level of telomere dysfunction, which is a novel approach for this cancer.

Changes in Nuclear Orientation Patterns of Chromosome 11 during Mouse Plasmacytoma Development.

Studying changes in nuclear architecture is a unique approach toward the understanding of nuclear remodeling during tumor development. One aspect of nuclear architecture is the orientation of chromosomes in the three-dimensional nuclear space. We studied mouse chromosome 11 in lymphocytes of [T38HxBALB/c]N mice with a reciprocal translocation between chromosome X and 11 (T38HT(X;11)) exhibiting a long chromosome T(11;X) and a short chromosome T(X;11) and in fast-onset plasmacytomas (PCTs) induced in the same strain. We determined the three-dimensional orientation of chromosome 11 using a mouse chromosome 11 specific multicolor banding probe. We also examined the nuclear position of the small translocation chromosome T(X;11) which contains cytoband 11E2 and parts of E1. Chromosomes can point either with their centromeric or with their telomeric end toward the nuclear center or periphery, or their position is found in parallel to the nuclear border. In T38HT(X;11) nuclei, the most frequently observed orientation pattern was with both chromosomes 11 in parallel to the nuclear border ("PP"). PCT cells showed nuclei with two or more copies of chromosome 11. In PCTs, the most frequent orientation pattern was with one chromosome in parallel and the other pointing with its centromeric end toward the nuclear periphery ("CP"). There is a significant difference between the orientation patterns observed in T38HT(X;11) and in PCT nuclei (P < .0001).

c-MYC-induced genomic instability.

MYC dysregulation initiates a dynamic process of genomic instability that is linked to tumor initiation. Early studies using MYC-carrying retroviruses showed that these viruses were potent transforming agents. Cell culture models followed that addressed the role of MYC in transformation. With the advent of MYC transgenic mice, it became obvious that MYC deregulation alone was sufficient to initiate B-cell neoplasia in mice. More than 70% of all tumors have some form of c-MYC gene dysregulation, which affects gene regulation, microRNA expression profiles, large genomic amplifications, and the overall organization of the nucleus. These changes set the stage for the dynamic genomic rearrangements that are associated with cellular transformation.

Three-dimensional Nuclear Telomere Organization in Multiple Myeloma.

Multiple myeloma (MM) is preceded by monoclonal gammopathy of undetermined significance (MGUS). Up to date, it is difficult to predict an individual's time to disease progression and the treatment response. To examine whether the nuclear telomeric architecture will unravel some of these questions, we carried out. Three-dimensional (3D) telomere analysis on samples from patients diagnosed with MGUS and MM, as well as from patients who went into relapse. Telomere signal intensity, number of telomere aggregates, nuclear volume, and the overall nuclear telomere distribution (a/c ratio) were analyzed. The telomeric profiles allowed for the differentiation of the disease stages. The telomeric profiles of myeloma cells obtained from blood and bone marrow aspirates were identical. Based on this study, we discuss the use of 3D telomere profiling as a potential future tool for risk stratification and personalized treatment decisions.

Selected telomere length changes and aberrant three-dimensional nuclear telomere organization during fast-onset mouse plasmacytomas.

Mouse plasmacytoma (PCT) can develop within 45 days when induced by a v-abl/myc replication-deficient retrovirus. This fast-onset PCT development is always associated with trisomy of cytoband E2 of mouse chromosome 11 (11E2). Trisomy of 11E2 was identified as the sole aberration in all fast-onset mouse PCTs in [T38HxBALB/c]N congenic mice, with a reciprocal translocation between chromosome X and 11 (rcpT(X;11)) (Genes Cancer 2010;1:847-858). Using this mouse model, we have now examined the overall and individual telomere lengths in fast-onset PCTs compared with normal B cells using two-dimensional and three-dimensional quantitative fluorescent in situ hybridization of telomeres. We found fast-onset PCTs to have a significantly different three-dimensional telomere profile, compared with primary B cells of wild-type littermates with and without rcpT(X;11) (P < .0001 and P = .006, respectively). Our data also indicate for primary PCT cells, from the above mouse strain, that the translocation chromosome carrying 11E2 is the only chromosome with telomere lengthening (P = 4 x 10(-16)). This trend is not seen for T(X;11) in primary B cells of control [T38HxBALB/c]N mice with the rcpT(X;11). This finding supports the concept of individual telomere lengthening of chromosomes that are functionally important for the tumorigenic process.