Profiling of ligand-receptor induced signalling- a novel protein chip technique

Cellular signalling pathways are the master controls of the biology of the cell, which includes cell communication, growth, death, and differentiation. The activities of these signalling proteins directly influence gene function by regulation of the signalling pathways that mediate cellular responses. Recent advanced techniques have given rise to a number of emerging tools for the analysis of cellular signalling that profile the proteome or the protein complement of the genome. However, these tools for signal profiling still face significant challenges such as sensitivity, specificity and be a high throughput method before they are widely adopted. Sensitivity issues are paramount in detecting signalling proteins that are normally in low amounts. Conventional protein chip technology promises to be a powerful tool for large scale high-throughput proteome profiling but there are still significant drawbacks. Here we report the development and application of a novel multiplexed and high-throughput platform for the quantitative profiling of activated intracellular sig nalling proteins subsequent to ligand-receptor induced signalling. This spatially addressable biochip platform will allow comprehensive mapping of interconnected signal pathways, through identification of key functional signalling proteins (ânodes’) in each pathway and quantifying their state of activity.Singapore-MIT Alliance (SMA

Signaling Biochip – Profiling ligand-receptor induced signalling

Intracellular communication controls most cellular functions, which includes cell growth, death, and differentiation. The current challenge in cell biology is to decipher the spatial-temporal interactions of these highly complex intracellular signaling pathways in mediating the various cellular responses. A number of emerging tools are available for the analyses of the proteome or the protein complement of the genome. However, these tools are generally not applicable for the profiling of molecules involved in the signaling pathways. The significant challenges include the lack of sensitivity, specificity and throughput. Conventional protein chip technology with capture moieties immobilized onto solid-surface, promises to be a powerful tool for large scale high-throughput proteome profiling. This approach has a number of drawbacks, among which the stability of the capture moieties in the given format pose a significant problem. Here we report the development and application of a novel multiplexed and high-throughput platform for the quantitative profiling of activated intracellular signalling proteins subsequent to ligand-receptor induced signalling. This spatially addressable biochip platform will allow comprehensive mapping of interconnected signal pathways, through identification of key functional signalling proteins (ânodes’) in each pathway and quantifying their state of activity.Singapore-MIT Alliance (SMA

Brain regional angiogenic potential at the neurovascular unit during normal aging

While mounting evidence indicates angiogenesis is impaired during normal aging, this has largely been investigated in peripheral organs, with much less effort focused on the aging brain. Given the strong regional specialization of the brain and increased vulnerability of certain brain regions to age-related deficits, the broad hypothesis of this dissertational research was cerebral angiogenic potential is regionally modified during normal aging. To test this, the expression of a broad group of angiogenesis-associated genes was assayed at the neurovascular unit (NVU) in discrete brain regions of normal aging mice by immunohistochemistry-guided laser capture microdissection (Immuno-LCM) coupled to quantitative real-time PCR (qRT-PCR). Complementary quantitative microvascular density/branching studies were performed as well. Effects of physical exercise on gene expression and microvascular density/branching were assayed to determine if such an intervention could reverse age-related regional cerebral angiogenic trends. Additionally, the response in gene expression to chronic mild hypoxia, which is normally a strong stimulus for angiogenesis in young healthy subjects, was further probed in the cortex to highlight any age-associated weaknesses in adaption to this stress. Normal aging was found to significantly impact resting expression of angiogenesis-associated genes at the NVU in a region-dependent manner—implying regional adaptation to the aging process. Physical exercise reversed some of these age-associated regional gene trends, as well as positively influenced cerebral capillary density and branching in a region-dependent way. Lastly, hypoxia revealed the angiogenic response to be weakened in aged brain, which displayed significantly muted changes in gene expression. Collectively, these results suggest a picture of heterogeneous changes in angiogenic capacity of the brain that accompany normal aging, and suggest a therapeutic benefit of physical exercise that acts at the NVU. Furthermore, a pilot study on the feasibility of coupling Immuno-LCM to a downstream mass spectrometric platform to study the brain microvascular endothelial proteome was also done, setting the stage for future analysis of brain regional angiogenic potential in the aged NVU, at the protein level.

Nivetha_poster.pdf

<p>Sexual size dimorphism is common across many species. Males being larger than females is the commonly observed pattern among mammals, and is explained as sexual selection favouring larger males. Some gliding mammals are known to exhibit the opposite pattern, with females larger than males. Functional advantages to large size (efficient gliding while pregnant and carrying young) have been shown to determine this pattern in flying squirrels. In this study, we examine whether female-biased dimorphism is consistent across gliding mammals belonging to different families (nine species in four families).</p

Thrombosis And Hemorrhage In Diabetic Retinopathy: A Perspective From An Inflammatory Standpoint

Retinal ischemia and hemorrhage are hallmarks of worsening diabetic retinopathy, which can lead to neovascularization, macular edema, and severe vision loss. Although diabetes alters expression of clotting factors and their activities, and increases retinal microthromboses, the effects of thrombotic processes on the pathogenesis of diabetic retinopathy are not fully understood. In addition to the roles of coagulation and fibrinolytic cascades in thrombosis and hemostasis, components in these systems also mediate multiple effects on the vasculature that promote inflammation. Plasma kallikrein, thrombin, and urokinase are increased in diabetic retinopathy, and exert proinflammatory effects that contribute to retinal vascular dysfunction. The accumulation and activation of these and additional coagulation factors in the vitreous due to hemorrhage and chronic retinal injury in the diabetic retina may contribute to worsening of retinal inflammation and capillary dysfunction, which lead to retinal ischemia and edema. Further understanding of the role for specific coagulation factors in diabetic retinopathy may suggest new therapeutic opportunities for this vision-threatening disease.WoSScopu

Removal of microplastic for a sustainable strategy by microbial biodegradation

Predictions indicate a concerning surge in plastic waste, expected to reach 380 million tonnes by 2040 from 188 million tonnes in 2016. Strategies to combat this could reduce yearly waste to 140 million tonnes, but this may not fully address existing pollution levels. Urgent action is needed to devise safe and effective methods for degrading microplastics. Use of microorganisms ranging from bacteria to algae to help in the biodegradation of these particles has been employed and researched upon extensively. Microorganisms belonging to the following genus: Bacillus, Actinobacteria, Pseudomonas, Aspergillus, Penicillium, Cyanobacteria and various species of microalgae have shown remarkable capabilities to degrade microplastic present in the environment. The main focus of this review is to highlight the potential microorganisms and organisms that can help with biodegradation of microplastics. The review also touches upon new biotechnological advancements like the use of genetically modified organisms to aid in the biodegradation of microplastics. It also highlights the gaps in research regarding the use of microorganisms and genetically modified organisms in a large scale and in combination with other degradation techniques in order to efficiently and safely degrade microplastics

Active Induction of Experimental Autoimmune Encephalomyelitis by MOG35-55 Peptide Immunization is Associated with Differential Responses in Separate Compartments of the Choroid Plexus

Background There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. Methods To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. Results The CP became swollen and displayed significant molecular changes in response to MOGCFA/ PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, proinflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTXtreatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. Conclusions MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease

Active induction of experimental autoimmune encephalomyelitis by MOG_35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus

<p>Abstract</p> <p>Background</p> <p>There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process.</p> <p>Methods</p> <p>To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and <it>in situ</it> expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG_35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an <it>Immune Panel</it> TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts.</p> <p>Results</p> <p>The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments.</p> <p>Conclusions</p> <p>MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.</p

Active Induction of Experimental Autoimmune Encephalomyelitis by MOG35-55 Peptide Immunization is Associated with Differential Responses in Separate Compartments of the Choroid Plexus

Background There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. Methods To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. Results The CP became swollen and displayed significant molecular changes in response to MOGCFA/ PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, proinflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTXtreatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. Conclusions MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease

Analysis of mouse brain microvascular endothelium using immuno-laser capture microdissection coupled to a hybrid linear ion trap with Fourier transform-mass spectrometry proteomics platform

The purpose of this study was to identify the protein profile of the mouse brain microvascular endothelium in situ. This involved coupling of a double-label, immuno-laser capture microdissection (LCM) process with LTQ-FT mass spectrometry to perform the in situ proteomic analysis. LCM was utilized to isolate cells from frozen mouse brain tissue sections. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and LC-MS analysis. Processed samples were subsequently analyzed using a linear IT coupled with a Fourier transform mass spectrometer (LTQ-FT MS). Overall, in this study, 881 proteins were identified from a specific cell category using immuno-guided LCM to probe these cell types along the entirety of the cerebral microvascular tree. The identification of sufficient numbers of proteins with high biological interest should allow us to study protein expression by specific cell types - as defined by certain cell markers - in complex tissues.7 page(s