This document summarizes an experiment that investigated the effects of granulocyte colony stimulating factor (GCSF) on rats that experienced stroke induced by focal cerebral ischemia. Western blot assays were used to analyze the molecular expression of caspase 12 and glucose regulated protein 78 (GRP78) in GCSF treated and untreated rats, as indicators of cell death. The results found decreased expression of these markers in the core region of treated rats' brains but increased expression in the penumbra region, though the results were not statistically significant. Future research with larger sample sizes is needed to better understand the effects of GCSF on apoptotic pathways following stroke.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
Poster - Gordon Research Conference 2008Donna Johnson
1. The study assessed how mutating regulatory elements (REs) of the GADD45a gene affected its induction in response to genotoxic stress.
2. It found that mutating the p53RE or WT1RE, or using a cell line with mutant p53, lessened the induction of GADD45a in response to DNA damaging chemicals.
3. The results suggest that for maximum induction of GADD45a, both functioning p53 and the intact p53RE are required to bind to regulatory elements in the GADD45a promoter region.
This study developed a novel methylation-specific high resolution melt (MS-HRM) assay to validate locus-specific DNA methylation profiles identified in previous epigenome-wide association studies. Using this new assay, the researchers detected significant hypomethylation of a CpG site (cg05575921) in the AHRR gene in adult smokers compared to non-smokers, consistent with prior studies. Hypomethylation at this site correlated with increased AHRR gene expression, and this effect was more prominent in monocytes than other white blood cells. The MS-HRM assay represents a useful and inexpensive tool for analyzing methylation differences identified in genome-wide studies.
- Ventral hippocampal corticosterone (vHipp CORT) enhances accumbal dopamine output in drug-naive rats, peaking 55 minutes post-infusion, suggesting a mechanism by which stress could enhance reward. However, in amphetamine withdrawal rats, vHipp CORT immediately reduces accumbal dopamine output, peaking at 20 and 80 minutes post-infusion, suggesting stress could contribute to dysphoric states and relapse.
- The findings support an opponent-process theory of addiction where blunted dopamine and enhanced CORT stress responses drive continued drug use. Receptor mechanisms are being tested to explain the differential dopamine responses to vHipp CORT in controls versus withdrawal.
This document describes research into the roles of prostaglandin receptors EP2 and EP4 in bone formation. The researchers aimed to clone and express the rat EP2 and EP4 receptors in cells to establish an assay for measuring cyclic AMP levels following ligand binding. Initial colorimetric assays were unsuccessful, but a quantitative enzymatic assay indicated varying transfection success rates depending on DNA-Lipofectamine ratios. Future work will determine optimal transfection conditions and test other prostaglandins to investigate potential anabolic treatments for osteoporosis.
This document describes the development of a cell-based assay to detect neutralizing antibodies against the drug alemtuzumab, which is used to treat multiple sclerosis. Researchers generated a stable CHO cell line expressing human CD52 and used it to detect anti-alemtuzumab antibodies in serum from an MS patient treated with alemtuzumab. The assay involves measuring inhibition of alemtuzumab-Alexa 488 binding to the CHO-CD52 cells by antibodies in patient serum. This assay provides a quantitative method for routine screening of serum from patients treated with alemtuzumab to detect neutralizing antibodies.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
Poster - Gordon Research Conference 2008Donna Johnson
1. The study assessed how mutating regulatory elements (REs) of the GADD45a gene affected its induction in response to genotoxic stress.
2. It found that mutating the p53RE or WT1RE, or using a cell line with mutant p53, lessened the induction of GADD45a in response to DNA damaging chemicals.
3. The results suggest that for maximum induction of GADD45a, both functioning p53 and the intact p53RE are required to bind to regulatory elements in the GADD45a promoter region.
This study developed a novel methylation-specific high resolution melt (MS-HRM) assay to validate locus-specific DNA methylation profiles identified in previous epigenome-wide association studies. Using this new assay, the researchers detected significant hypomethylation of a CpG site (cg05575921) in the AHRR gene in adult smokers compared to non-smokers, consistent with prior studies. Hypomethylation at this site correlated with increased AHRR gene expression, and this effect was more prominent in monocytes than other white blood cells. The MS-HRM assay represents a useful and inexpensive tool for analyzing methylation differences identified in genome-wide studies.
- Ventral hippocampal corticosterone (vHipp CORT) enhances accumbal dopamine output in drug-naive rats, peaking 55 minutes post-infusion, suggesting a mechanism by which stress could enhance reward. However, in amphetamine withdrawal rats, vHipp CORT immediately reduces accumbal dopamine output, peaking at 20 and 80 minutes post-infusion, suggesting stress could contribute to dysphoric states and relapse.
- The findings support an opponent-process theory of addiction where blunted dopamine and enhanced CORT stress responses drive continued drug use. Receptor mechanisms are being tested to explain the differential dopamine responses to vHipp CORT in controls versus withdrawal.
This document describes research into the roles of prostaglandin receptors EP2 and EP4 in bone formation. The researchers aimed to clone and express the rat EP2 and EP4 receptors in cells to establish an assay for measuring cyclic AMP levels following ligand binding. Initial colorimetric assays were unsuccessful, but a quantitative enzymatic assay indicated varying transfection success rates depending on DNA-Lipofectamine ratios. Future work will determine optimal transfection conditions and test other prostaglandins to investigate potential anabolic treatments for osteoporosis.
This document describes the development of a cell-based assay to detect neutralizing antibodies against the drug alemtuzumab, which is used to treat multiple sclerosis. Researchers generated a stable CHO cell line expressing human CD52 and used it to detect anti-alemtuzumab antibodies in serum from an MS patient treated with alemtuzumab. The assay involves measuring inhibition of alemtuzumab-Alexa 488 binding to the CHO-CD52 cells by antibodies in patient serum. This assay provides a quantitative method for routine screening of serum from patients treated with alemtuzumab to detect neutralizing antibodies.
C3G interacts with and inhibits the function of β-catenin. C3G forms a complex with β-catenin through its central proline-rich domain. Overexpression of C3G inhibits β-catenin/TCF transcriptional activity and destabilizes β-catenin protein levels by promoting its degradation. β-catenin activation leads to reduced expression of C3G, indicating reciprocal negative regulation between C3G and β-catenin. C3G exerts these effects on β-catenin independently of its guanine nucleotide exchange factor activity or GSK3β phosphorylation of β-catenin.
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
antiapoptotic effect of Ach (research paper by Marta sloniecka)Prajjwal Rajput
1) The study investigated the antiapoptotic effect of acetylcholine (Ach) in Fas-mediated apoptosis of human keratocytes. Ach treatment decreased Fas-induced apoptosis through activation of muscarinic acetylcholine receptors.
2) Ach reduced the activities of caspase-8 and caspase-9, key initiator caspases of apoptosis. This effect was mediated by mAchRs and partially by nAchRs.
3) Ach's antiapoptotic mechanism involved downregulating FasL-induced NF-κB gene expression and increasing levels of the antiapoptotic protein Bcl-2 by phosphorylating the proapoptotic protein Bad. This inhibited the
Participation of the gabaergic system on the glutamateankit
This study investigated the participation of the GABAergic system in regulating glutamate release from frontal cortex synaptosomes in a rat model of experimental autoimmune encephalomyelitis (EAE), which mimics multiple sclerosis in humans. The results showed reduced GABAergic inhibition of glutamate release and impaired GABA regulation of synapsin I phosphorylation in synaptosomes from EAE rats compared to controls. Specifically, GABA had no effect on glutamate release or synapsin I phosphorylation in EAE rats, suggesting alterations in the GABAergic system may contribute to cortical pathology in EAE.
The invention of sprycel from benchtop to bedside (Gengcheng Jack Yang)Jack Gengcheng YANG
1) The document describes the discovery and optimization of dasatinib (BMS-354825), a potent pan-Src kinase inhibitor. Initial screening identified a thiazole-based hit that inhibited Lck kinase. Extensive SAR explored substituents on the thiazole ring as well as replacements for the amide linker.
2) Replacing the amide with a 2-aminopyrimidine improved potency against Lck and cellular assays. Further appending a polar side chain to this analog enhanced aqueous solubility and potency, yielding dasatinib.
3) Dasatinib was found to be a highly potent, selective, ATP-competitive inhibitor of Src family kinases and
Expression of N-terminal seven amino acids peptide of fibrin (β–peptide) on...Aref Farokhi Fard
This document summarizes a research project on expressing the N-terminal seven amino acid peptide of fibrin (β-peptide) on the surface of M13KO7 phage. The goal is to develop a targeted drug delivery system for fibrinolytic drugs to treat vascular accidents like heart attacks and strokes. The β-peptide sequence was identified from the fibrinogen beta chain protein and used as the antigen. After cloning the β-peptide gene into the phage genome and transforming bacteria, recombinant phages were produced and the expression of the β-peptide on their surface was confirmed through PCR, sequencing and SDS-PAGE gel electrophoresis. This targeted drug delivery system could help increase the efficiency of fibrinolytic drugs while reducing side effects like hemorrh
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
1) The study investigated whether a catalytically inactive tPA variant (tPA-S481A) could prevent impairment of cerebral autoregulation and histopathology after traumatic brain injury (TBI) in piglets.
2) tPA-S481A prevented pial artery vasoconstriction and blunted dilation impairment during hypotension after TBI.
3) tPA-S481A also prevented neuronal cell loss in the hippocampus that normally occurs after TBI.
This study investigated the effects of various anabolic steroids and progesterone on progesterone production and STARD1 mRNA levels in human ovarian cells. The results showed that:
1) Dihydrotestosterone increased basal progesterone levels at some time points and increased STARD1 mRNA levels in the presence of a cAMP stimulant.
2) Progesterone increased STARD1 mRNA levels in the presence of cAMP at some time points.
3) Mesterolone and trenbolone increased basal progesterone levels but did not affect levels stimulated by cAMP, and had mixed effects on STARD1 mRNA levels.
4) Superdrol had little effect on progesterone levels but tended to reduce cAMP-stim
J Biomol Screen-2013-Maillard-868-78 (dragged)Hyunsun Park
This document describes the development of a new label-free LC/MS/MS assay for detecting caspase inhibitors. Previous fluorescence-based caspase assays were found to be susceptible to artifacts from compounds that interfere with the fluorescent readout. Only one series of published non-peptidic caspase inhibitors, the isatins, showed genuine inhibition in the improved fluorescence assays. However, even the isatin compounds showed differing potencies between the fluorescence and new label-free LC/MS/MS assays, highlighting the need for orthogonal assays to validate caspase inhibitors given the target's susceptibility to artifactual inhibition. The new label-free LC/MS/MS assay provides caspase activity measurements that agree with fluorescence assays for reference inhibitors, but detects differing
Genome sequencing uncovers MS phenocopies in primary progressive multiple scl...Sherman Jia
This document summarizes a study that performed whole-genome sequencing on patients with primary progressive multiple sclerosis (PPMS) and controls. The key findings were:
1) Some PPMS patients carried rare mutations in genes associated with neurological disorders that resemble MS, called phenocopies.
2) PPMS patients were enriched for rare mutations in genes that cause spastic paraplegias, a type of motor neuron disorder. This enrichment was unique to PPMS and not seen in relapsing-remitting MS.
3) Additional research is needed to understand how both rare and common genetic variations contribute to MS pathogenesis. The findings suggest PPMS may have features of both a complex genetic disease and monogenic
Yearly Clinical Review of a Patient’s WGS Results Leads to a New Gene Candida...Golden Helix
The rapid evolution of genetic disease understanding and expanding genetic databases behooves clinician specialists to regularly review the status of clinical genetic test panels, reevaluating the status of identified variants. With the increasing use of WES and WGS sequencing, these results likely also need regular review.
We present a four-generation family affected by sudden cardiac arrests, ventricular tachycardia and (within the last 2 generations) documented left ventricular non-compaction. An affected member underwent a 38-gene pan-cardiomyopathy clinical genetic panel with no identified pathogenic variants. Therefore, affected members from three surviving generations (*) agreed to undergo genetic testing by whole genome sequencing in 2016. Analysis with Golden Helix SVS identified 69 shared, rare (MAF≤0.00005) coding variants in 40 genes, but none were identified to be associated with cardiac phenotypes.
At clinical follow-up of the youngest family member in 2019, we elected to review our WGS results again. A recent review of the genetics and genomics of dilated cardiomyopathy now identifies the SRA1 gene as being potentially involved in this phenotype, although no human cases have yet been reported. Upon review of this families previous WGS sequencing using SVS, we identified a SRA1 c.328_329 ins GAC (p.Val110delinsGlyLeu) rare shared variant. This variant is only present in 2 of 142724 alleles in the gnomAD database and inserts an additional amino acid into a partially conserved two amino acid region within an otherwise highly conserved protein region.
Friedrichs and colleagues previously identified SRA1 as one of 3 genes within a 600kb haploblock associated with cardiomyopathy in three independent Caucasian populations. In zebrafish, sra1 morphants display severe pericardial edema. The human SRA1 region also encodes a long noncoding RNA, lnc-SRA1-2, that is modulated in heart failure, attenuates hypoxia-induced injury in experimental cardiomyocytes through PPARγ/NF-κB signalling, and promotes the activation of cardiac myofibroblasts. SRA1 knockdown in C2C12 mouse myoblast cells prevented proper muscle gene expression and cell differentiation.
Cardiac genetics assessments by whole genome or whole exome sequencing also need regular review, which can be easily performed by Golden Helix SVS software. Links to new and frequently updated gene tracks and other genetic databases facilitate these reviews.
1) The study investigated the effects of IRL-1620, an ETB receptor agonist, on beta amyloid (Aβ)-induced cognitive impairment and oxidative stress in non-diabetic and diabetic rats.
2) IRL-1620 treatment significantly reduced oxidative stress markers increased by Aβ and improved spatial memory impairment caused by Aβ in both non-diabetic and diabetic rats.
3) The results suggest that stimulation of ETB receptors by IRL-1620 provides neuroprotection against Aβ-induced cognitive impairment and oxidative stress.
This document summarizes a study that identified the drug tropisetron as a potential therapeutic for Alzheimer's disease (AD) through its ability to increase the ratio of sAPPα to Aβ peptides in the brain. Tropisetron was found to bind to and act on the α7 nicotinic acetylcholine receptor, 5-HT3 serotonin receptor, and amyloid precursor protein (APP). In a mouse model of AD, tropisetron treatment improved memory and normalized aberrant APP processing at a dose equivalent to the current human dose used to treat nausea. The results suggest tropisetron may be suitable for clinical trials in AD and mild cognitive impairment due to its multi-target effects and established safety profile
Neurodegenrative disorders by Dr Rbalaraman.pptxRBalaraman4
1. Neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and stroke result from neuronal death in the brain. The mechanisms of neuronal death include protein deposition, excitotoxicity, oxidative stress, and apoptosis.
2. While dead neurons cannot regenerate in the adult brain, pharmacological treatments can help prevent neuronal loss or compensate for it. Cholinesterase inhibitors are used for Alzheimer's disease and levodopa is used for Parkinson's disease.
3. Research is exploring new drug targets to stop the molecular pathways leading to neuronal death in these disorders, with the goal of developing disease-modifying treatments in the future.
This document contains summaries of 4 poster presentations from a conference on Alzheimer's disease and dementia research.
The first poster discusses findings that the ER stress transcription factor XBP1s protects against amyloid-beta neurotoxicity in a fruit fly model of Alzheimer's and in human neuroblastoma cells. XBP1s prevents the accumulation of free calcium in the cytosol, explaining its neuroprotective effects.
The second poster reports that amyloid-beta oligomers impair neuronal function and morphology in rat and mouse neuronal cultures and hippocampal slices through a mechanism linked to NMDA receptor signaling and activation of the protein Jacob.
The third poster examines the role of O-GlcNAc glycosylation in hippocampal synaptic
1) Neural stem cells cultured in vitro with epidermal growth factor and basic fibroblast growth factor proliferated and formed neurospheres. 2) Addition of PPARγ agonists like ciglitazone and 15d-PGJ2 inhibited neural stem cell proliferation in a dose-dependent manner. 3) Interestingly, neural stem cells cultured with PPARγ agonists showed increased expression of oligodendrocyte markers and a reduction in the neural stem cell marker nestin, indicating promotion of oligodendrocyte differentiation.
1. The document summarizes a study that screened three single nucleotide polymorphisms (SNPs) in the MDR1 gene in four non-tribal populations in Kerala, India.
2. The study found diversity in allele and genotype frequencies between populations, with some similarities to other world populations. It also found linkage disequilibrium between two of the SNPs.
3. The results provide background population data that could help with case-control studies of adverse drug reactions by accounting for population stratification. The four populations studied showed little genetic difference and can be considered relatively homogenous.
This study investigated the role of neuronal apoptosis in volumetric changes of the hippocampus in diabetes mellitus type 1 rats. The key findings were:
1. The volume of the dentate gyrus and CA3 region was reduced in diabetic and vitamin C-treated rats compared to controls, indicating volume reduction can occur independently of neuronal loss.
2. The number of apoptotic neurons in the dentate gyrus and CA3 was significantly higher in diabetic rats compared to other groups, showing neuronal apoptosis is increased by diabetes.
3. A response index using the ratio of dentate gyrus to CA3 volumes and neuronal densities provided a predictive model, with the curves meeting at a critical point of 0
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
C3G interacts with and inhibits the function of β-catenin. C3G forms a complex with β-catenin through its central proline-rich domain. Overexpression of C3G inhibits β-catenin/TCF transcriptional activity and destabilizes β-catenin protein levels by promoting its degradation. β-catenin activation leads to reduced expression of C3G, indicating reciprocal negative regulation between C3G and β-catenin. C3G exerts these effects on β-catenin independently of its guanine nucleotide exchange factor activity or GSK3β phosphorylation of β-catenin.
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
antiapoptotic effect of Ach (research paper by Marta sloniecka)Prajjwal Rajput
1) The study investigated the antiapoptotic effect of acetylcholine (Ach) in Fas-mediated apoptosis of human keratocytes. Ach treatment decreased Fas-induced apoptosis through activation of muscarinic acetylcholine receptors.
2) Ach reduced the activities of caspase-8 and caspase-9, key initiator caspases of apoptosis. This effect was mediated by mAchRs and partially by nAchRs.
3) Ach's antiapoptotic mechanism involved downregulating FasL-induced NF-κB gene expression and increasing levels of the antiapoptotic protein Bcl-2 by phosphorylating the proapoptotic protein Bad. This inhibited the
Participation of the gabaergic system on the glutamateankit
This study investigated the participation of the GABAergic system in regulating glutamate release from frontal cortex synaptosomes in a rat model of experimental autoimmune encephalomyelitis (EAE), which mimics multiple sclerosis in humans. The results showed reduced GABAergic inhibition of glutamate release and impaired GABA regulation of synapsin I phosphorylation in synaptosomes from EAE rats compared to controls. Specifically, GABA had no effect on glutamate release or synapsin I phosphorylation in EAE rats, suggesting alterations in the GABAergic system may contribute to cortical pathology in EAE.
The invention of sprycel from benchtop to bedside (Gengcheng Jack Yang)Jack Gengcheng YANG
1) The document describes the discovery and optimization of dasatinib (BMS-354825), a potent pan-Src kinase inhibitor. Initial screening identified a thiazole-based hit that inhibited Lck kinase. Extensive SAR explored substituents on the thiazole ring as well as replacements for the amide linker.
2) Replacing the amide with a 2-aminopyrimidine improved potency against Lck and cellular assays. Further appending a polar side chain to this analog enhanced aqueous solubility and potency, yielding dasatinib.
3) Dasatinib was found to be a highly potent, selective, ATP-competitive inhibitor of Src family kinases and
Expression of N-terminal seven amino acids peptide of fibrin (β–peptide) on...Aref Farokhi Fard
This document summarizes a research project on expressing the N-terminal seven amino acid peptide of fibrin (β-peptide) on the surface of M13KO7 phage. The goal is to develop a targeted drug delivery system for fibrinolytic drugs to treat vascular accidents like heart attacks and strokes. The β-peptide sequence was identified from the fibrinogen beta chain protein and used as the antigen. After cloning the β-peptide gene into the phage genome and transforming bacteria, recombinant phages were produced and the expression of the β-peptide on their surface was confirmed through PCR, sequencing and SDS-PAGE gel electrophoresis. This targeted drug delivery system could help increase the efficiency of fibrinolytic drugs while reducing side effects like hemorrh
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
1) The study investigated whether a catalytically inactive tPA variant (tPA-S481A) could prevent impairment of cerebral autoregulation and histopathology after traumatic brain injury (TBI) in piglets.
2) tPA-S481A prevented pial artery vasoconstriction and blunted dilation impairment during hypotension after TBI.
3) tPA-S481A also prevented neuronal cell loss in the hippocampus that normally occurs after TBI.
This study investigated the effects of various anabolic steroids and progesterone on progesterone production and STARD1 mRNA levels in human ovarian cells. The results showed that:
1) Dihydrotestosterone increased basal progesterone levels at some time points and increased STARD1 mRNA levels in the presence of a cAMP stimulant.
2) Progesterone increased STARD1 mRNA levels in the presence of cAMP at some time points.
3) Mesterolone and trenbolone increased basal progesterone levels but did not affect levels stimulated by cAMP, and had mixed effects on STARD1 mRNA levels.
4) Superdrol had little effect on progesterone levels but tended to reduce cAMP-stim
J Biomol Screen-2013-Maillard-868-78 (dragged)Hyunsun Park
This document describes the development of a new label-free LC/MS/MS assay for detecting caspase inhibitors. Previous fluorescence-based caspase assays were found to be susceptible to artifacts from compounds that interfere with the fluorescent readout. Only one series of published non-peptidic caspase inhibitors, the isatins, showed genuine inhibition in the improved fluorescence assays. However, even the isatin compounds showed differing potencies between the fluorescence and new label-free LC/MS/MS assays, highlighting the need for orthogonal assays to validate caspase inhibitors given the target's susceptibility to artifactual inhibition. The new label-free LC/MS/MS assay provides caspase activity measurements that agree with fluorescence assays for reference inhibitors, but detects differing
Genome sequencing uncovers MS phenocopies in primary progressive multiple scl...Sherman Jia
This document summarizes a study that performed whole-genome sequencing on patients with primary progressive multiple sclerosis (PPMS) and controls. The key findings were:
1) Some PPMS patients carried rare mutations in genes associated with neurological disorders that resemble MS, called phenocopies.
2) PPMS patients were enriched for rare mutations in genes that cause spastic paraplegias, a type of motor neuron disorder. This enrichment was unique to PPMS and not seen in relapsing-remitting MS.
3) Additional research is needed to understand how both rare and common genetic variations contribute to MS pathogenesis. The findings suggest PPMS may have features of both a complex genetic disease and monogenic
Yearly Clinical Review of a Patient’s WGS Results Leads to a New Gene Candida...Golden Helix
The rapid evolution of genetic disease understanding and expanding genetic databases behooves clinician specialists to regularly review the status of clinical genetic test panels, reevaluating the status of identified variants. With the increasing use of WES and WGS sequencing, these results likely also need regular review.
We present a four-generation family affected by sudden cardiac arrests, ventricular tachycardia and (within the last 2 generations) documented left ventricular non-compaction. An affected member underwent a 38-gene pan-cardiomyopathy clinical genetic panel with no identified pathogenic variants. Therefore, affected members from three surviving generations (*) agreed to undergo genetic testing by whole genome sequencing in 2016. Analysis with Golden Helix SVS identified 69 shared, rare (MAF≤0.00005) coding variants in 40 genes, but none were identified to be associated with cardiac phenotypes.
At clinical follow-up of the youngest family member in 2019, we elected to review our WGS results again. A recent review of the genetics and genomics of dilated cardiomyopathy now identifies the SRA1 gene as being potentially involved in this phenotype, although no human cases have yet been reported. Upon review of this families previous WGS sequencing using SVS, we identified a SRA1 c.328_329 ins GAC (p.Val110delinsGlyLeu) rare shared variant. This variant is only present in 2 of 142724 alleles in the gnomAD database and inserts an additional amino acid into a partially conserved two amino acid region within an otherwise highly conserved protein region.
Friedrichs and colleagues previously identified SRA1 as one of 3 genes within a 600kb haploblock associated with cardiomyopathy in three independent Caucasian populations. In zebrafish, sra1 morphants display severe pericardial edema. The human SRA1 region also encodes a long noncoding RNA, lnc-SRA1-2, that is modulated in heart failure, attenuates hypoxia-induced injury in experimental cardiomyocytes through PPARγ/NF-κB signalling, and promotes the activation of cardiac myofibroblasts. SRA1 knockdown in C2C12 mouse myoblast cells prevented proper muscle gene expression and cell differentiation.
Cardiac genetics assessments by whole genome or whole exome sequencing also need regular review, which can be easily performed by Golden Helix SVS software. Links to new and frequently updated gene tracks and other genetic databases facilitate these reviews.
1) The study investigated the effects of IRL-1620, an ETB receptor agonist, on beta amyloid (Aβ)-induced cognitive impairment and oxidative stress in non-diabetic and diabetic rats.
2) IRL-1620 treatment significantly reduced oxidative stress markers increased by Aβ and improved spatial memory impairment caused by Aβ in both non-diabetic and diabetic rats.
3) The results suggest that stimulation of ETB receptors by IRL-1620 provides neuroprotection against Aβ-induced cognitive impairment and oxidative stress.
This document summarizes a study that identified the drug tropisetron as a potential therapeutic for Alzheimer's disease (AD) through its ability to increase the ratio of sAPPα to Aβ peptides in the brain. Tropisetron was found to bind to and act on the α7 nicotinic acetylcholine receptor, 5-HT3 serotonin receptor, and amyloid precursor protein (APP). In a mouse model of AD, tropisetron treatment improved memory and normalized aberrant APP processing at a dose equivalent to the current human dose used to treat nausea. The results suggest tropisetron may be suitable for clinical trials in AD and mild cognitive impairment due to its multi-target effects and established safety profile
Neurodegenrative disorders by Dr Rbalaraman.pptxRBalaraman4
1. Neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and stroke result from neuronal death in the brain. The mechanisms of neuronal death include protein deposition, excitotoxicity, oxidative stress, and apoptosis.
2. While dead neurons cannot regenerate in the adult brain, pharmacological treatments can help prevent neuronal loss or compensate for it. Cholinesterase inhibitors are used for Alzheimer's disease and levodopa is used for Parkinson's disease.
3. Research is exploring new drug targets to stop the molecular pathways leading to neuronal death in these disorders, with the goal of developing disease-modifying treatments in the future.
This document contains summaries of 4 poster presentations from a conference on Alzheimer's disease and dementia research.
The first poster discusses findings that the ER stress transcription factor XBP1s protects against amyloid-beta neurotoxicity in a fruit fly model of Alzheimer's and in human neuroblastoma cells. XBP1s prevents the accumulation of free calcium in the cytosol, explaining its neuroprotective effects.
The second poster reports that amyloid-beta oligomers impair neuronal function and morphology in rat and mouse neuronal cultures and hippocampal slices through a mechanism linked to NMDA receptor signaling and activation of the protein Jacob.
The third poster examines the role of O-GlcNAc glycosylation in hippocampal synaptic
1) Neural stem cells cultured in vitro with epidermal growth factor and basic fibroblast growth factor proliferated and formed neurospheres. 2) Addition of PPARγ agonists like ciglitazone and 15d-PGJ2 inhibited neural stem cell proliferation in a dose-dependent manner. 3) Interestingly, neural stem cells cultured with PPARγ agonists showed increased expression of oligodendrocyte markers and a reduction in the neural stem cell marker nestin, indicating promotion of oligodendrocyte differentiation.
1. The document summarizes a study that screened three single nucleotide polymorphisms (SNPs) in the MDR1 gene in four non-tribal populations in Kerala, India.
2. The study found diversity in allele and genotype frequencies between populations, with some similarities to other world populations. It also found linkage disequilibrium between two of the SNPs.
3. The results provide background population data that could help with case-control studies of adverse drug reactions by accounting for population stratification. The four populations studied showed little genetic difference and can be considered relatively homogenous.
This study investigated the role of neuronal apoptosis in volumetric changes of the hippocampus in diabetes mellitus type 1 rats. The key findings were:
1. The volume of the dentate gyrus and CA3 region was reduced in diabetic and vitamin C-treated rats compared to controls, indicating volume reduction can occur independently of neuronal loss.
2. The number of apoptotic neurons in the dentate gyrus and CA3 was significantly higher in diabetic rats compared to other groups, showing neuronal apoptosis is increased by diabetes.
3. A response index using the ratio of dentate gyrus to CA3 volumes and neuronal densities provided a predictive model, with the curves meeting at a critical point of 0
This document reports on a study that found adult hippocampal neural stem and progenitor cells (NSPCs) secrete significant amounts of vascular endothelial growth factor (VEGF), which plays an important role in maintaining the adult neurogenic niche. The study showed that NSPCs in the adult hippocampus express VEGF both in vivo and in vitro. Inducible knockout of VEGF specifically in NSPCs led to a 20-30% reduction in total hippocampal VEGF levels and impaired stem cell maintenance over time, demonstrating the functional relevance of NSPC-derived VEGF. These findings reveal that NSPCs act as a previously unknown secretory cell type that helps regulate the neurogenic niche through VEGF secretion.
Effects of atorvastatin and streptozocin on immunohistochemical markers in hi...Ram Sahu
This study investigated the effects of atorvastatin on immunohistochemical markers in the hippocampus of rats with a streptozocin-induced model of Alzheimer's disease. The results showed that streptozocin increased glial fibrillary acidic protein and neuronal nitric oxide synthase in the hippocampus compared to controls. Atorvastatin treatment at 20 mg/kg reduced glial fibrillary acidic protein levels compared to streptozocin alone. Atorvastatin at doses of 5, 10, and 20 mg/kg increased glutathione reductase expression compared to streptozocin alone. Atorvastatin at 10 and 20 mg/kg also reduced neuronal nitric oxide synthase levels compared to streptozocin alone.
This study investigated how immune signaling and cell stress gene expression changes in the hippocampus and cerebellum of a rat model for glutamate excitotoxicity neurodegeneration. RNA was extracted from these brain regions in mutant and normal rats at different ages. Gene expression analysis found that genes involved in cytokine signaling were downregulated while cell stress genes were upregulated in mutant rats, suggesting altered immune regulation contributes to neurodegeneration. Specifically, genes related to glutamate metabolism and clusterin expression changed over time and differed between brain regions in ways that could enhance excitotoxic cell damage and death. The results provide insight into how the immune system may initiate neurodegeneration during glutamate excitotoxicity.
Marie-Laure Rives Allostery Functional Selectivity KOR Columbia University Marie-Laure Rives, PhD
The document discusses G protein-coupled receptors (GPCRs) and their modulation by signaling molecules and allosteric modulators. It focuses on the dopamine D2 and adenosine A2A receptors, describing how their heterodimerization was detected ex vivo in the striatum using proximity ligation assays. The document also discusses the therapeutic potential of kappa opioid receptor ligands and developing functionally selective ligands that activate signaling pathways for therapeutic effects but not adverse effects. Specifically, it describes developing G protein-biased kappa agonists like 6'GNTI that do not recruit arrestin in order to achieve pain relief without dysphoric effects.
This study investigated the effects of inhibiting glucocerebrosidase (GCase) activity on the behavior of A53T-α-synuclein transgenic mice, a model of synucleinopathies like Parkinson's disease. The inhibition of GCase with CBE exacerbated memory deficits and fine motor impairments already present in the transgenic mice. Specifically, CBE treatment further diminished nest-building ability, pole descent time, and fear memory in the mice. These findings suggest that reduced GCase activity can worsen behavioral symptoms and validate the relevance of GCase to synucleinopathy development and severity. Future work will include biochemical and histological analysis to support these behavioral results and evaluate if gene therapy to boost
This study investigated the use of quantum dots (qdots) to label and visualize two endogenous synaptic proteins, GABAA-1 receptors and glutamate transporters (VGLUT1), in the rat cerebellum. Qdots allowed for the clear visualization of these proteins in very small presynaptic structures like parallel fiber varicosities that are below the diffraction limit of conventional microscopy. Specifically, qdots formed clusters around interneurons and isolated clusters on interneuron dendrites, revealing the presence of GABAA receptors. They also labeled sub-micrometer parallel fibers and 1-2 micrometer presynaptic varicosities containing VGLUT1. While double labeling with two qdot colors was attempted, some receptor sites remained unlabeled
1. The document discusses delivering AAV2 gene therapy for Parkinson's disease through convection-enhanced delivery (CED), which uses a syringe pump to generate pressure and spread the vector throughout the targeted striatal area.
2. It examines delivering different doses of AAV2-hAADC, ranging from 6 to 500 units per hemisphere, to animal models and evaluating clinical responses and vector distribution up to 6 months later using various analysis methods.
3. The highest dosage group showed large discrepancies and no antibody response was detected, suggesting a potential delivery problem rather than immunogenicity issues.
Objective: To study the effects of resveratrol in neuronal structures in traumatic brain injury (TBI).
Study Design: Thirty rats were categorized as (1) control group (n=10), saline solution administered i.p. for 14 days, (2) TBI group (n=10), trauma induced by weight-drop model on brain, and (3) TBI+Resveratrol group (n=10), 15 minutes after injury the rats were given resveratrol (10 μmoL/kg/i.p.) for 14 days. At the end of the experiment the cerebellum was excised for routine paraffin tissue protocol. Blood samples were tested for serum biochemical markers (MDA, SOD, CAT, and GSH-x).
Results: SOD, GPx, and CAT values were lowest in the TBI group. MDA and histological scores of dilations in vessels, inflammation, degeneration in neurons, apoptosis in microglia, ADAMTS8, and GFAP expressions were highest in the TBI group. Sections of the control group showed normal cerebellar histology. The trauma group showed degenerated ganglion layer, pyknotic and apoptotic Purkinje cell nuclei. Vascular thrombus was seen in the substantia alba and substantia grisea. In the Trauma+Resveratrol group, most pa- thologies observed in the TBI group were improved. In the control group, GFAP protein was expressed in granular cells, axons, dendrites, Purkinje cells, and microglia cells. In the trauma group, increased GFAP expression was observed in glial processes, neurons, and Purkinje cells. In the Trauma+Resveratrol group, GFAP was expressed in molecular layer and glial processes. In the control group, ADAMTS-4 activity was observed in granulosa layer, glial cells, and Purkinje cells. In the trauma group, ADAMTS-4 expression was positive in Purkinje cells and glial cells. In the Trauma+ Resveratrol group, ADAMTS-4 was expressed in Purkinje cells, granular cells, and glial cells.
Conclusion: GFAP and ADAMTS-4 proteins may be involved in regeneration of damaged astroglial cells and other glial cells, Purkinje cells, and synaptic extensions. We suggest that antioxidative drugs such as resveratrol may be alternative target agents in neurological disease.
Keywords: ADAMTS-4, brain, cerebellum, GFAP, rat, resveratrol, traumatic brain injury
2. INTRODUCTION: GCSF
Granulocyte Colony Stimulating Factor (GCSF) is a growth factor
and cytokine that stimulates the proliferation and mobilization of
hematopoietic stem cells
Its actions are mediated by binding to specific GCSF receptors
(GCSFRs)
Although its main clinical use has been to counteract neutropenia,
recent studies have reported that GCSFRs are located on the
brain leading to neuroprotective effects of the drug
By decreasing the rate of neuronal death many central nervous
system (CNS) disorders may be counteracted against
3. INTRODUCTION: CEREBRAL ISCHEMIA
Focal cerebral ischemia (stroke) is a CNS disorder in which the
cerebral blood vessels are blocked, resulting in an inadequate
blood flow to the brain and subsequent oxygen (hypoxic) –
glucose deprived environment
In this state the brain cannot meet metabolic demands and
neuronal death will eventually be initiated via apoptosis
The resulting ischemic infarct (area of dead cell tissue) includes a
central core (an area of severe ischemia) and a surrounding
penumbra, which is vulnerable if the cell death is not arrested
White = core
Red = penumbra
4. INTRODUCTION: APOPTOTIC
PATHWAYS
The endoplasmic reticulum (ER) is responsible for the proper folding
and sorting of proteins
During an ischemic insult the ER becomes stressed and will try to
restore normal function by deactivating protein translation signaling
pathways while activating other pathways that increase the
production of chaperone molecules that facilitates protein folding
If it is unsuccessful in rectifying its stressed state and restoring
normal function, the ER will initiate an apoptotic cascade
Caspase 12 is an ER-membrane associated protein and one of the
initiators of the caspase-mediated apoptotic pathway
Glucose regulated protein 78 (GRP78) is a chaperon molecule
associated with the ER
5. INTRODUCTION: EXPERIMENT
The molecular expression of GRP78 and Caspase 12 were
compared in GCSF treated and untreated rats
Both GRP78 and Caspase 12 are excellent indicators of cell
death
6. HYPOTHESIS
If the molecular expression of GRP78 and Caspase 12 are
measured in GCSF treated and untreated rats after focal cerebral
ischemia, treated rats will have lower expression of the selected
indicators and therefore lower cell death in both the core and
penumbra of the brain
7. METHODS: WESTERN BLOT
Frozen homogenates/lysates of protein samples (from two rats to
increase diversity with sample population) were used to perform
western blot assays
Briefly, protein homogenates/lysates were boiled for 5 minutes and
centrifuged
Each lane was loaded with 50 μg of protein and subjected to sodium
dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on
12% gels and then electrophoretically transferred to nitrocellulose
membranes
Once protein transfers were completed, the nitrocellulose
membranes were blocked in 5% nonfat dry milk in TBST (20 mmol/L
Tris base, pH 7.6, 137 mmol/L NaCl, and 0.05% Tween 20) for 1 hour
at room temperature and rinsed in TBS-T (50 mM Tris, 170 mM NaCl,
0.2% [vol/vol] Tween 20; pH 7.5)
8. METHODS: WESTERN BLOT
They were then incubated with primary antibodies (anti-caspase
12, anti-Grp78 and anti-GAPDH, all at 1:1000 dilution) and
incubated in TBST and the corresponding primary antibody
(1:1000) overnight at 4°C
The membranes were subsequently washed in TBST and
incubated for 1.5 hours at room temperature with the same
corresponding secondary antibody in a dilution of 1:3000 with
TBST
Immunoreactive bands were visualized in the linear range with
enhanced chemoluminescence
Films were scanned and the optical density was determined with
ImageJ
9. METHODS: ANALYSIS
All statistical data shown was expressed as the mean optical
density for each treatment
A one-way ANOVA test was used to compare means between
groups and determine statistical significance
Differences of P<0.05 were considered statistically significant
10. RESULTS: CASPASE 12
This figure shows the mean normalized optical density values
determined by ImageJ for Caspase 12
GAPDH was used as the internal control to normalize values
Click to next slide for
more explanation
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF
11. RESULTS: CASPASE 12
The data demonstrates a relatively large decrease in expression
between the MCAO and MCAO+GCSF groups in the core and an
increase between groups in the penumbra
Caspase 12: 40 kDa
1 2 3 4
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF
12. RESULTS: CASPASE 12
ANOVA Tests were run for the results of both the core and
penumbra
Since both had p values > .05, results were determined to be
statistically insignificant
Core
Penumbra
13. RESULTS: CASPASE 12
A T-Test was then run which also proved results to be statistically
insignificant because the p value > .05
14. RESULTS: GRP78
This figure displays the mean normalized optical density values
determined by ImageJ for GRP78
GAPDH was used as the internal control to normalize values
Click to next slide for
more explanation
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF
15. RESULTS: GRP78
The data demonstrates a decrease in expression between the
MCAO and MCAO+GCSF groups in the core and an increase in
the penumbra
GRP78: 72-80 kDa
1 2 3 4
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF
16. RESULTS: SUMMARY
With both Caspase 12 and GRP78, there was a decrease in
expression in the treated rats in the core, but an increase in
expression in the treated rats in the penumbra
17. DISCUSSION: CASPASE 12
RESULTS
In response to excess calcium in the ER lumen, an apoptotic
cascade is initiated involving Caspase 12 with the final death
marker prior to apoptosis being Caspase 3
Consistent with previous studies, the core GCSF-treated rats
showed reduced Caspase 12 expression
The penumbra GCSF-treated rats showed increased Caspase 12
expression in the penumbra in complete disagreement with
previous studies
18. DISCUSSION: GRP78 RESULTS
In the absence of ER stress, the chaperone protein GRP78
protects cells from apoptosis by binding to IRE1, PERK, ATF6,
and Caspase 14
GRP78 normally inhibits the activation of these intracellular
cytoplasmic kinases but when the ER is stressed, GRP78
dissociates from the cytoplasmic molecules to sequester the extra
calcium
Similar to the results with Caspase 12, the core-GCSF treated
groups decreased GRP78 expression, but in the penumbra the
opposite effect occurred.
19. DISCUSSION: STATISTICS
There was no statistical significance between any of the treatment
groups analyzed
A possible reason for this result is the relatively small sample size of
data used in this experiment compared to similar studies
Also there was a relatively high level of variation within each group,
which may have contributed to some of the observed discrepancies
in the data analysis
Another statistical discrepancy may have come from GAPDH, which
was used to normalize OD values
As demonstrated in Figure 1, the expression of GAPDH in the MCAO
group was significantly smaller than any other group making the
normalized value of the MCAO group relatively smaller
20. FUTURE RESEARCH
In future research, additional Western Blots with Caspase 12 and
GRP78 would be run in order to compile more data and create
more reliable results
As this research only investigated pro-death markers (Caspase
12) and ER stress indicators (GRP78), it would be interesting to
assay BCl2, a pro-survival marker in future research
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