72 research outputs found
Practical, ethical and regulatory considerations for the evolving medical and research genomics landscape.
We are entering a fascinating and uncertain period of medical history, as today’s DNA sequencing
technology has the potential to help each of us direct our care and predict our future based on knowledge of
our own individual inherited and acquired genetics. However, from a global and local economic
perspective, these are lean years, and this adds a significant degree of uncertainty to the immediate future
of this enterprise. It is therefore incumbent upon us as a community to show that personalized genomic
medicine will not just be a luxury or a burdensome cost center, but that it truly has the potential to save
both lives and health care expenses via data-driven management, early disease detection/screening and
more efficacious pharmaceutical delivery. To do this, we need to determine how to move forward towards
expanded clinical use of this technology in a manner both rapid and economical, while ensuring the
integrity of the process and the safety and well-being of patients and research participants. Here, we
discuss some of the ethical, regulatory and practical considerations that are emerging in the field of
genomic medicine. We also propose that many of the cost and safety issues we are facing can be mitigated
through expanded reliance on existing clinical regulatory frameworks and the implementation of worksharing
strategies designed to leverage the strengths of our genomics centers and clinical interpretive
teams
Dihydropyrimidine dehydrogenase deficiency as a cause of fatal 5-Fluorouracil toxicity
5-Fluorouracil (5-FU), in combination with other cytotoxic drugs, is commonly used to treat a variety of cancers. Dihydropyrimidine dehydrogenase (DPD) catalyzes the first catabolic step of the 5-FU degradation pathway, converting 80% of 5-FU to its inactive metabolite. Approximately 0.3% of the population demonstrate complete DPD deficiency, translating to extreme toxicity of 5-FU. Here we present a case of a patient who had a fatal outcome after treatment with 5-FU who was found to have an unknown DPD deficiency discovered at autopsy
Loop Groups and Discrete KdV Equations
A study is presented of fully discretized lattice equations associated with
the KdV hierarchy. Loop group methods give a systematic way of constructing
discretizations of the equations in the hierarchy. The lattice KdV system of
Nijhoff et al. arises from the lowest order discretization of the trivial,
lowest order equation in the hierarchy, b_t=b_x. Two new discretizations are
also given, the lowest order discretization of the first nontrivial equation in
the hierarchy, and a "second order" discretization of b_t=b_x. The former,
which is given the name "full lattice KdV" has the (potential) KdV equation as
a standard continuum limit. For each discretization a Backlund transformation
is given and soliton content analyzed. The full lattice KdV system has, like
KdV itself, solitons of all speeds, whereas both other discretizations studied
have a limited range of speeds, being discretizations of an equation with
solutions only of a fixed speed.Comment: LaTeX, 23 pages, 1 figur
Dihydropyrimidine dehydrogenase deficiency as a cause of fatal 5-Fluorouracil toxicity
5-Fluorouracil (5-FU), in combination with other cytotoxic drugs, is commonly used to treat a variety of cancers. Dihydropyrimidine dehydrogenase (DPD) catalyzes the first catabolic step of the 5-FU degradation pathway, converting 80% of 5-FU to its inactive metabolite. Approximately 0.3% of the population demonstrate complete DPD deficiency, translating to extreme toxicity of 5-FU. Here we present a case of a patient who had a fatal outcome after treatment with 5-FU who was found to have an unknown DPD deficiency discovered at autopsy
Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo
Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection
Shotgun Proteomics Identifies Serum Fibronectin as a Candidate Diagnostic Biomarker for Inclusion in Future Multiplex Tests for Ectopic Pregnancy
Ectopic pregnancy (EP) is difficult to diagnose early and accurately. Women often present at emergency departments in early pregnancy with a 'pregnancy of unknown location' (PUL), and diagnosis and exclusion of EP is challenging due to a lack of reliable biomarkers. The objective of this study was to identify novel diagnostic biomarkers for EP. Shotgun proteomics, incorporating combinatorial-ligand library pre-fractionation, was used to interrogate pooled sera (n = 40) from women undergoing surgery for EP, termination of viable intrauterine pregnancy and management of non-viable intrauterine pregnancy. Western blot was used to validate results in individual sera. ELISAs were developed to interrogate sera from women with PUL (n = 120). Sera were collected at time of first symptomatic presentation and categorized according to pregnancy outcome. The main outcome measures were differences between groups and area under the receiver operating curve (ROC). Proteomics identified six biomarker candidates. Western blot detected significant differences in levels of two of these candidates. ELISA of sera from second cohort revealed that these differences were only significant for one of these candidates, fibronectin. ROC analysis of ability of fibronectin to discriminate EP from other pregnancy outcomes suggested that fibronectin has diagnostic potential (ROC 0.6439; 95% CI 0.5090 to 0.7788; P>0.05), becoming significant when 'ambiguous' medically managed PUL excluded from analysis (ROC 0.6538; 95% CI 0.5158 to 0.7918; P<0.05). Fibronectin may make a useful adjunct to future multiplex EP diagnostic tests
A Linear Framework for Time-Scale Separation in Nonlinear Biochemical Systems
Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level
An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge
There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
RESULTS:
A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.
CONCLUSIONS:
The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups
31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two
Background
The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd.
Methods
We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background.
Results
First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001).
Conclusions
In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival
A Molecular Dissection of the Ventromedial Hypothalamic Nucleus
The ventromedial hypothalamic nucleus (VMH) plays an important role in the regulation of food intake, glucose metabolism, and body weight. However, in contrast to other hypothalamic nuclei that are also known to regulate energy homeostasis, there is a paucity of nucleus-specific marker genes for the VMH that can be used to label its constituent neurons. This represents a significant impediment to the application of molecular approaches for analyzing VMH circuitry and function. Thus, we conducted a microarray screen in order to identify VMH-specific genes that could be used to label populations of VMH neurons. Laser-capture microdissection was used to isolate RNA from the VMH and from two adjacent hypothalamic nuclei known to play a role in energy balance, the arcuate (ARC) and dorsomedial hypothalamic nucleus (DMH). Amplified RNA from these three nuclei were intercompared to identify genes with VMH-enriched expression. The top 12 VMH marker gene candidates were screened by real-time PCR, and three genes (Cerebellin 1, PACAP, and a novel gene we characterized, LBH2) were examined by in situ hybridization for further validation and examination of their subnuclear expression profile. One of the VMH markers, steroidogenic factor 1 (SF-1), is an orphan nuclear receptor with few known target genes. As this transcription factor is responsible for proper developmental formation of the VMH and also for normal energy homeostasis, we endeavored to determine whether any of the VMH marker genes may be regulated by SF- 1. The expression of 4 markers was significantly altered in VMH neurons of SF-1 2 knockout mice, and this result was confirmed by an in situ hybridization study of cerebellin 1 expression in brain-specific SF-1 knockout mice. One of the VMH markers was a previous undescribed gene that we further characterized and named LBH2 owing to its similarity to a presumed transcription factor called limb bud and heart (LBH). To further examine the expression of this gene in the VMH, and to begin to describe VMH neuronal populations, we created LBH2-GFP BAC transgenic mouse line expressing GFP under the control of the LBH2 promoter. These mice were validated for correct expression of the transgene and examined for overlap with other populations of interest in the VMH, including estrogen receptor-alpha neurons and leptin-responsive neurons. Finally, we used a TK+ strain of PRV Bartha2001 to trace neuronal inputs from AgRP neurons in the ARC using AgRP-cre mice. Using this technique, it could be determined that the dorsomedial and intermediate VMH sends significant outputs to this population of ARC neurons. This technique, together with the marker genes discovered, may now be used to identify and catalog individual VMH neuronal subsets that project to these neurons. In conclusion, we have discovered a set of marker genes for the VMH using lasercapture microdissection coupled with cDNA microarray analysis. This combination of techniques represents a powerful approach for the identification of genes enriched in specific, anatomically-defined brain regions. The discovery of multiple genes regulated by SF-1 also suggests this technique may be useful for identifying nucleus-specific transcriptional networks. The VMH-enriched genes identified here, in conjunction with LBH2-GFP mice and other transgenic animals, will provide a basis for a full 3 characterization of VMH neurons, and will prove greatly useful for future neuroanatomic and transgenic-based studies of this important nucleus
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