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Hu Y.,Center Hospitalier Of Luniversite Of Montreal Chum | Lao L.,Center Hospitalier Of Luniversite Of Montreal Chum | Mao J.,Center Hospitalier Of Luniversite Of Montreal Chum | Jin W.,Center Hospitalier Of Luniversite Of Montreal Chum | And 8 more authors.
Nature Communications | Year: 2017

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways. © The Author(s) 2017.


Wang X.,Center Hospitalier Of Luniversite Of Montreal Crchum | Marcinkiewicz M.,Cytochem Inc. | Gatain Y.,McGill University | Bouchard M.,McGill University | And 7 more authors.
PLoS ONE | Year: 2013

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP+/- mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP+/- mice were in the normal range. The functions of MLF1-IP+/- T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation. © 2013 Wang et al.


Wang X.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Wu T.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Wu T.,Zhejiang University | Hu Y.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | And 6 more authors.
PLoS ONE | Year: 2012

Pno1 is a protein that plays a role in proteasome and ribosome neogenesis in yeast. So far, its functions in mammalian cells have not been investigated. To understand its function in mammals, we performed in situ hybridization analysis of Pno1 expression in different development stages and generated Pno1 gene knockout (KO) and transgenic (Tg) mice lineages. The results showed early lethality of homozygous Pno1 KO lineage caused, as demonstrated in parallel by ex vivo experiments, by arrest of embryo development before compaction stage. Though, heterozygous (HET) mice with 50% of normal Pno1 mRNA concentration were fertile and showed no obvious anomalies. The lymphoid organs of HET mice were normal in size, weight and cellularity, with normal T and B cell subpopulations. TCR-triggered activation and proliferation of HET T cells were normal. Proteasome activities in HET organs were uncompromised. Tg mice with actin promoter-driven Pno1 expression were also fertile, with no apparent anomalies, although they expressed 2-5-fold higher Pno1 mRNA levels. The lymphoid organs of Tg mice were of normal size, weight and cellularity with normal T and B cell sub-populations. TCR-triggered activation and proliferation of Tg T cells were normal. Tg organs and tissues presented normal proteasome activity as did their wild type counterparts. Tagged Pno1 over-expression in L cells and density gradient fractionation established that Pno1 existed in large complexes with sedimentation rates between 20S and 26S, bigger than mature 26S proteasomes. Pno1 in fractions did not coincide with 40S or 60S ribosome subunits. Our study indicates that Pno1 is essential for cellular functions, but only a small percentage of its normal level is sufficient, and excessive amounts are neither harmful nor useful. The nature of the large complexes it associates with remains to be identified, but it is certain that they are not mature proteasomes or ribosomes. © 2012 Wang et al.


Papapietro O.,McGill University | Teatero S.,McGill University | Thanabalasuriar A.,McGill University | Yuki K.E.,McGill University | And 11 more authors.
Nature Communications | Year: 2013

Citrobacter rodentium is a natural mouse pathogen widely used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhoea in susceptible strains. The physiological pathways as well as the genetic determinants leading to susceptibility have remained largely uncharacterized. Here we use a forward genetic approach to identify the R-spondin2 gene as a major determinant of susceptibility to C. rodentium infection. Robust induction of R-spondin2 expression during infection in susceptible mouse strains causes a potent Wnt-mediated proliferative response of colonic crypt cells, leading to the generation of an immature and poorly differentiated colonic epithelium with deficiencies in ion-transport components. Our data demonstrate a previously unknown role of R-spondins and Wnt signalling in susceptibility to infectious diarrhoea and identify R-spondin2 as a key molecular link between infection and intestinal homoeostasis. © 2013 Macmillan Publishers Limited. All rights reserved.


Shekarabi M.,University of Montréal | Lafreniere R.G.,University of Montréal | Gaudet R.,University of Montréal | Laganiere J.,University of Montréal | And 3 more authors.
PLoS ONE | Year: 2013

The With No lysine (K) family of serine/threonine kinase (WNK) defines a small family of kinases with significant roles in ion homeostasis. WNK1 has been shown to have different isoforms due to what seems to be largely tissue specific splicing. Here, we used two distinct in situ hybridization riboprobes on developing and adult mouse tissues to make a comparative analysis of Wnk1 and its sensory associated splice isoform, Wnk1/Hsn2. The hybridization signals in developing mouse tissues, which were prepared at embryonic day e10.5 and e12.5, revealed a homogenous expression profile with both probes. At e15.5 and in the newborn mouse, the two probes revealed different expression profiles with prominent signals in nervous system tissues and also other tissues such as kidney, thymus and testis. In adult mouse tissues, the two expression profiles appeared even more restricted to the nervous tissues, kidney, thymus and testis, with no detectable signal in the other tissues. Throughout the nervous system, sensory tissues, as well as in Cornu Ammonis 1 (CA1), CA2 and CA3 areas of the hippocampus, were strongly labeled with both probes. Hybridization signals were also strongly detected in Schwann and supporting satellite cells. Our results show that the expression profiles of Wnk1 isoforms change during the development, and that the expression of the Wnk1 splice variant containing the Hsn2 exon is prominent during developing and in adult mouse tissues, suggesting its important role in the development and maintenance of the nervous system. © 2013 Shekarabi et al.


Thomas A.,University of Montréal | Patterson N.H.,University of Montréal | Marcinkiewicz M.M.,Cytochem Inc. | Lazaris A.,McGill University | And 2 more authors.
Analytical Chemistry | Year: 2013

Imaging mass spectrometry (IMS) represents an innovative tool in the cancer research pipeline, which is increasingly being used in clinical and pharmaceutical applications. The unique properties of the technique, especially the amount of data generated, make the handling of data from multiple IMS acquisitions challenging. This work presents a histology-driven IMS approach aiming to identify discriminant lipid signatures from the simultaneous mining of IMS data sets from multiple samples. The feasibility of the developed workflow is evaluated on a set of three human colorectal cancer liver metastasis (CRCLM) tissue sections. Lipid IMS on tissue sections was performed using MALDI-TOF/TOF MS in both negative and positive ionization modes after 1,5-diaminonaphthalene matrix deposition by sublimation. The combination of both positive and negative acquisition results was performed during data mining to simplify the process and interrogate a larger lipidome into a single analysis. To reduce the complexity of the IMS data sets, a sub data set was generated by randomly selecting a fixed number of spectra from a histologically defined region of interest, resulting in a 10-fold data reduction. Principal component analysis confirmed that the molecular selectivity of the regions of interest is maintained after data reduction. Partial least-squares and heat map analyses demonstrated a selective signature of the CRCLM, revealing lipids that are significantly up- and down-regulated in the tumor region. This comprehensive approach is thus of interest for defining disease signatures directly from IMS data sets by the use of combinatory data mining, opening novel routes of investigation for addressing the demands of the clinical setting. © 2013 American Chemical Society.


Westlund K.N.,University of Kentucky | Zhang L.P.,University of Kentucky | Ma F.,University of Kentucky | Nesemeier R.,University of Kentucky | And 7 more authors.
Neuroscience | Year: 2014

Acute and chronic pain resulting from injury, surgery, or disease afflicts >100million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca2+ signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives. © 2013 IBRO.


PubMed | McGill University, Gza Hospitals St Augustinus, Cytochem Inc., University of Montréal and University of Geneva
Type: | Journal: Scientific reports | Year: 2016

In many cancers, the establishment of a patients future treatment regime often relies on histopathological assessment of tumor tissue specimens in order to determine the extent of the pathological response to a given therapy. However, histopathological assessment of pathological response remains subjective. Here we use MALDI mass spectrometry imaging to generate lipid signatures from colorectal cancer liver metastasis specimens resected from patients preoperatively treated with chemotherapy. Using these signatures we obtained a unique pathological response score that correlates with prognosis. In addition, we identify single lipid moieties that are overexpressed in different histopathological features of the tumor, which have potential as new biomarkers for assessing response to therapy. These data show that computational methods, focusing on the lipidome, can be used to determine prognostic markers for response to chemotherapy and may potentially improve risk assessment and patient care.


PubMed | AngioChem, University of Montréal and Cytochem Inc.
Type: | Journal: Analytical and bioanalytical chemistry | Year: 2016

Mucopolysaccharidosis type II (Hunters disease) mouse model (IdS-KO) was investigated by both imaging mass spectrometry (IMS) and immunohistochemistry (IHC) performed on the same tissue sections. For this purpose, IdS-KO mice brain sections were coated with sublimated 1,5-diaminonaphtalene and analyzed by high spatial resolution IMS (5m) and anti-GM3 IHC on the same tissue sections to characterize the ganglioside monosialated ganglioside (GM) deposits found in Hunters disease. IMS analysis have found that two species of GM3 and GM2 that are only different due to the length of their fatty acid residue (stearic or arachidic residue) were overexpressed in the IdS-KO mice compared to a control mouse. GM3 and GM2 were characterized by on-tissue exact mass and MS/MS compared to a GM3 standard. Realignment of both IMS and IHC data sets further confirmed the observed regioselective signal previously detected by providing direct correlation of the IMS image for the two GM3 overly expressed MS signals with the anti-GM3 IHC image. Furthermore, these regioselective GM MS signals were also found to have highly heterogeneous distributions within the GM3-IHC staining. Some deposits showed high content in GM3 and GM2 stearic species (r=0.74) and others had more abundant GM3 and GM2 arachidic species (r=0.76). Same-section analysis of Hunters disease mouse model by both high spatial resolution IMS and IHC provides a more in-depth analysis of the composition of the GM aggregates while providing spatial distribution of the observed molecular species. Graphical Abstract Ganglioside imaging mass spectrometry followed by immunohistochemistry performed on the same tissue section.


PubMed | University of Kentucky, Transposagen Biopharmaceuticals, Inc. and Cytochem Inc.
Type: | Journal: Neuroscience | Year: 2014

Acute and chronic pain resulting from injury, surgery, or disease afflicts >100 million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives.

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