This document describes the development and validation of a new LC/MS/MS method for quantifying four gangliosides (GM2, GM3, GD2, and GD3) in human plasma. Gangliosides are glycosphingolipids that play important roles in neurological function and disease. The method uses protein precipitation for sample extraction, UPLC separation, and MS/MS detection in MRM mode for high sensitivity and specificity. Product ion mass spectra were obtained for the gangliosides to select optimal MRM transitions for quantification. The method was validated for extraction recovery, calibration linearity, precision, and accuracy. This sensitive assay can be used to monitor ganglioside levels in plasma from normal and
26 ASBMB TODAY FEBRUARY 2021Discovering an old DoGs’ neMargaritoWhitt221
26 ASBMB TODAY FEBRUARY 2021
Discovering an old DoGs’
new trick
Heterotrimeric G proteins regulate
a variety of signaling pathways that
control cell development and influ-
ence cell morphology via actin/cyto-
skeleton remodeling. There are four
main families of G proteins: Gi/Go,
Gq, Gs and G12/13. Researchers long
have thought that Gs, unlike its family
members, is coupled specifically and
exclusively to adenylyl cyclases.
In a new study published in the
Journal of Biological Chemistry,
Alejandro Castillo–Kauil of the
Center for Research and Advanced
Studies of the National Polytechnic
Institute and collaborators challenge
this dogmatic view by identifying a
new Gs target. Using biochemical,
molecular biological and chemo-
genetic approaches, the researchers
demonstrated that the Gαs subfamily
of G proteins can regulate the activity
of Rho GTPases such as Rho guanine
nucleotide exchange factor, or Rho-
GEF. The interaction identified by the
group activates the small G protein
Cdc42 by Gs-coupled GPCRs, stimu-
lating a rearrangement of the cyto-
skeleton and inducing formation of
fingerlike protrusions called filopodia.
These results provide new insight
into G protein activity and define a
new role for RhoGEF coupling in G
protein function.
DOI: 10.1074/jbc.AC120.015204
A pathogen’s proteins target
mitochondria
The tick-borne pathogen Coxiella
burnetii causes Q fever, or query fever,
a rare flulike disease that can spread
to humans who inhale dust particles
contaminated by infected farm or
CONTINUED FROM PAGE 25
Noninvasive tool provides oral cancer prognosis
Oral squamous cell carcinoma, which affects about 34,000 people
in the U.S. each year, is found in the cells lining the lips and mouth.
Metastasis to the lymph nodes is a sign of disease progression and may
be accompanied by changes in proteolytic activity. During proteolysis,
enzymes cut up proteins into short fragments called peptides. Recent
work suggests that characterizing the sequence and abundance of these
molecules — a method dubbed peptidomics — might provide new in-
sight on cancer biology and in the clinic. In a recent paper in the journal
Molecular & Cellular Proteomics, Leandro Xavier Neves of the Brazil-
ian Biosciences National Laboratory and a team of Brazilian clinicians
and scientists describe their analysis of oral squamous cell carcinoma
patient saliva using peptidomics.
After extracting peptides from saliva samples, the research team ana-
lyzed and compared the peptide content in samples from patients with
and without metastasis to the lymph nodes. They found more than 1,000
uniquely expressed peptides in each group and an additional 1,628 pep-
tides expressed by both groups. A series of statistical analyses identified
77 peptides of particular interest; all of these peptides are overexpressed
in samples from patients with lymph node metastasis, which supports the
hypothesis that proteolytic activity increases ...
This thesis investigated using ellipsometry to analyze ligand binding to G-protein coupled receptors (GPCRs). GPCRs are important cell surface proteins linked to many diseases. Ellipsometry is an optical technique that can quantify ligand-receptor interactions by measuring changes at a surface when polarized light is reflected off. An A549 epithelial cell line expressing the CXCR4 GPCR and its ligand CXCL12 was used. Enzyme-linked immunosorbent assays were performed to determine optimal ligand and antibody concentrations. Baseline characterization of glass slides and binding experiments with ligand and antibody were conducted using ellipsometry. Psi-delta analysis of collected data showed trends in binding.
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
Key-words- CAD, PCSK9, SNP, Eam1104I, Polymorphism, West Bengal population
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal, which aims to develop coherent means to modify drug therapy, with respect to the patient's genotype, and to ensure maximum efficiency with minimal contrary effects.
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal and a peer-reviewed journal. Clinical Pharmacology & Toxicology is the all-encompassing and becoming an increasingly important discipline for the identification of disease targets and drug designing with their toxicological effects and means to eradicate diseases.
2009 JCEM Detection of growth hormone doping by gene expression profiling of ...Selina Sutton
Gene expression profiling of peripheral blood from athletes administered growth hormone (GH) for 8 weeks found:
1) GH increased circulating insulin-like growth factor I (IGF-I) levels approximately 2-fold in both men and women.
2) Microarray analysis detected small changes in gene expression with GH, with a maximum 2-fold increase or decrease. 353 genes were differentially expressed in women and 41 in men.
3) The effect of GH on gene expression was similar in magnitude to natural variation between individuals, making it an unlikely approach for detecting GH doping.
This document summarizes lysosomal storage diseases (LSDs), which are caused by defects in lysosomal proteins leading to substrate accumulation. LSDs include over 50 disorders categorized by substrate stored. While rare, treatment involves hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, chaperones, and gene therapy. Management requires a multidisciplinary team and monitoring for medical and psychosocial effects. The guideline provides diagnostic and treatment guidance for identifying LSDs through newborn screening.
This study identified a panel of 10 lipids in plasma that can predict the conversion from normal cognition to mild cognitive impairment or Alzheimer's disease within 2-3 years with over 90% accuracy. The panel distinguished a group of older adults who later developed cognitive impairment (converters) from those who remained normal. The lipids were lower in converters before their decline and may reflect early neurodegeneration. Validation in an independent sample confirmed the panel's ability to classify converters and controls with high sensitivity and specificity. The findings suggest blood-based lipid biomarkers could enable early detection of preclinical Alzheimer's disease.
26 ASBMB TODAY FEBRUARY 2021Discovering an old DoGs’ neMargaritoWhitt221
26 ASBMB TODAY FEBRUARY 2021
Discovering an old DoGs’
new trick
Heterotrimeric G proteins regulate
a variety of signaling pathways that
control cell development and influ-
ence cell morphology via actin/cyto-
skeleton remodeling. There are four
main families of G proteins: Gi/Go,
Gq, Gs and G12/13. Researchers long
have thought that Gs, unlike its family
members, is coupled specifically and
exclusively to adenylyl cyclases.
In a new study published in the
Journal of Biological Chemistry,
Alejandro Castillo–Kauil of the
Center for Research and Advanced
Studies of the National Polytechnic
Institute and collaborators challenge
this dogmatic view by identifying a
new Gs target. Using biochemical,
molecular biological and chemo-
genetic approaches, the researchers
demonstrated that the Gαs subfamily
of G proteins can regulate the activity
of Rho GTPases such as Rho guanine
nucleotide exchange factor, or Rho-
GEF. The interaction identified by the
group activates the small G protein
Cdc42 by Gs-coupled GPCRs, stimu-
lating a rearrangement of the cyto-
skeleton and inducing formation of
fingerlike protrusions called filopodia.
These results provide new insight
into G protein activity and define a
new role for RhoGEF coupling in G
protein function.
DOI: 10.1074/jbc.AC120.015204
A pathogen’s proteins target
mitochondria
The tick-borne pathogen Coxiella
burnetii causes Q fever, or query fever,
a rare flulike disease that can spread
to humans who inhale dust particles
contaminated by infected farm or
CONTINUED FROM PAGE 25
Noninvasive tool provides oral cancer prognosis
Oral squamous cell carcinoma, which affects about 34,000 people
in the U.S. each year, is found in the cells lining the lips and mouth.
Metastasis to the lymph nodes is a sign of disease progression and may
be accompanied by changes in proteolytic activity. During proteolysis,
enzymes cut up proteins into short fragments called peptides. Recent
work suggests that characterizing the sequence and abundance of these
molecules — a method dubbed peptidomics — might provide new in-
sight on cancer biology and in the clinic. In a recent paper in the journal
Molecular & Cellular Proteomics, Leandro Xavier Neves of the Brazil-
ian Biosciences National Laboratory and a team of Brazilian clinicians
and scientists describe their analysis of oral squamous cell carcinoma
patient saliva using peptidomics.
After extracting peptides from saliva samples, the research team ana-
lyzed and compared the peptide content in samples from patients with
and without metastasis to the lymph nodes. They found more than 1,000
uniquely expressed peptides in each group and an additional 1,628 pep-
tides expressed by both groups. A series of statistical analyses identified
77 peptides of particular interest; all of these peptides are overexpressed
in samples from patients with lymph node metastasis, which supports the
hypothesis that proteolytic activity increases ...
This thesis investigated using ellipsometry to analyze ligand binding to G-protein coupled receptors (GPCRs). GPCRs are important cell surface proteins linked to many diseases. Ellipsometry is an optical technique that can quantify ligand-receptor interactions by measuring changes at a surface when polarized light is reflected off. An A549 epithelial cell line expressing the CXCR4 GPCR and its ligand CXCL12 was used. Enzyme-linked immunosorbent assays were performed to determine optimal ligand and antibody concentrations. Baseline characterization of glass slides and binding experiments with ligand and antibody were conducted using ellipsometry. Psi-delta analysis of collected data showed trends in binding.
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
Key-words- CAD, PCSK9, SNP, Eam1104I, Polymorphism, West Bengal population
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal, which aims to develop coherent means to modify drug therapy, with respect to the patient's genotype, and to ensure maximum efficiency with minimal contrary effects.
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal and a peer-reviewed journal. Clinical Pharmacology & Toxicology is the all-encompassing and becoming an increasingly important discipline for the identification of disease targets and drug designing with their toxicological effects and means to eradicate diseases.
2009 JCEM Detection of growth hormone doping by gene expression profiling of ...Selina Sutton
Gene expression profiling of peripheral blood from athletes administered growth hormone (GH) for 8 weeks found:
1) GH increased circulating insulin-like growth factor I (IGF-I) levels approximately 2-fold in both men and women.
2) Microarray analysis detected small changes in gene expression with GH, with a maximum 2-fold increase or decrease. 353 genes were differentially expressed in women and 41 in men.
3) The effect of GH on gene expression was similar in magnitude to natural variation between individuals, making it an unlikely approach for detecting GH doping.
This document summarizes lysosomal storage diseases (LSDs), which are caused by defects in lysosomal proteins leading to substrate accumulation. LSDs include over 50 disorders categorized by substrate stored. While rare, treatment involves hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, chaperones, and gene therapy. Management requires a multidisciplinary team and monitoring for medical and psychosocial effects. The guideline provides diagnostic and treatment guidance for identifying LSDs through newborn screening.
This study identified a panel of 10 lipids in plasma that can predict the conversion from normal cognition to mild cognitive impairment or Alzheimer's disease within 2-3 years with over 90% accuracy. The panel distinguished a group of older adults who later developed cognitive impairment (converters) from those who remained normal. The lipids were lower in converters before their decline and may reflect early neurodegeneration. Validation in an independent sample confirmed the panel's ability to classify converters and controls with high sensitivity and specificity. The findings suggest blood-based lipid biomarkers could enable early detection of preclinical Alzheimer's disease.
Plasma phospholipids identify antecedent memory impairment in older adultsJosé Luis Moreno Garvayo
En este trabajo publicado en la revista Nature medicine el pasado mes de marzo, el equipo del Dr. Federoff plantea un novedoso enfoque que consiste en analizar un grupo de diez fosfolípidos para la detección de la enfermedad de Alzheimer antes de la manifestación clínica de los síntomas.
This document summarizes a study that used proteomics to identify serum biomarkers for Alzheimer's disease (AD) and mild cognitive impairment (MCI) by analyzing serum samples from patients selected based on their PiB-PET imaging scores. The researchers used isobaric tagging and liquid chromatography-tandem mass spectrometry to perform proteome profiling on serum from control, MCI, and AD patients. They identified 79 and 72 differentially expressed proteins in MCI and AD serum, respectively, compared to controls. Integrated analysis with brain tissue data identified three biomarker candidates related to proteolysis: PCSK9, F13A1, and DCD. Validation in independent serum samples confirmed elevated levels of these candidates in MCI
Jianying Xiao has over 16 years of experience in pharmaceutical research. She has expertise in in vivo and in vitro drug discovery techniques related to drug metabolism, infectious diseases, immunology, and cardio-metabolic disorders. She is proficient in various research techniques including animal handling, molecular biology, cell culture, and data analysis software. Jianying has worked at Merck & Co. for over 18 years, leading numerous projects that resulted in publications, patents, and awards. Her work has advanced drug programs from research through clinical trials.
Transcriptional signaling pathways inversely regulated in alzheimer's disease...Elsa von Licy
This document summarizes a study that analyzed gene expression data from patients with Alzheimer's disease and glioblastoma multiforme to identify signaling pathways that are inversely regulated between the two diseases. The study found that the ERK/MAPK pathway is upregulated in glioblastoma but downregulated in Alzheimer's disease. Additionally, the angiopoietin signaling pathway is upregulated in Alzheimer's disease but downregulated in glioblastoma. Conditioned media containing amyloid-beta peptide suppressed glioblastoma cell growth and ERK/MAPK signaling, suggesting amyloid-beta may contribute to the inverse relationship between the diseases by inhibiting glioblastoma signaling pathways.
Biomarkers of Systemic Lupus Erythematosus and Systemic Sclerosis diseases ac...Prof. Hesham N. Mustafa
Systemic Lupus Erythematosus (SLE) and systemic sclerosis (SSc) are systemic inflammatory autoimmune disorders characterized by a large spectrum of clinical and laboratory features. The aim of the present study was to investigate the possible use of serum level of soluble intercellular adhesion molecule-1(sICAM-1) and soluble interleukin-2 receptor (sIL-2Ra) as biomarkers for monitoring of SLE and SSc disease activity. Moreover, it aimed to compare the specificity and sensitivity as well as cut-off value of both biomarkers in a sample of Egyptian patients. 50 SLE patients, 30 SSc patients and 60 age and sex matched healthy controls were enrolled in our study. sICAM-1and sIL-2Ra were measured in serum samples obtained from all participants. In addition to Erythosedimentation rate (ESR), complete blood count (CBC), Antineuclearantibodies (ANA) estimation, disease activity of both diseases were also assessed. sICAM-1and sIL-2Ra levels were higher in SLE and SSc patients versus control. Both parameters are correlated with each other as well as the activity parameters. A cut-off levels of 455.59 (ng/ml) &2525935 (pg/ml) in both SLE & SSs respectively was observed with the highest specificity and sensitivity. It could be concluded that sICAM-1 and sIL-2Ra are noninvasive biomarkers for SLE and SSc that could play a pathophysiologic role in development and progression of both diseases. Moreover, sICAM-1 and sIL-2Ra are correlated with the disease activity at cut-off values of 455.59 (ng/ml) & 2525935(pg/ml) respectively.
Review (ca 2007) of Uremic Toxins Accumulating in Patients with Chronic and End Stage Renal Disease modified from a presentation I gave in Fellow's Grand rounds.
Relied heavily on publications from the EU Toxin Work Group Work, which provides more up to date information:
http://www.uremic-toxins.org/
Glypican and Biglycan in the Nuclei of Neurons and Glioma CellsYu Liang
This document describes a study that found glypican and biglycan, two proteoglycans, localized to the nuclei of neurons and glioma cells. The researchers identified nuclear localization signals in the amino acid sequences of both proteoglycans. They demonstrated that these signals were functional by showing nuclear localization of beta-galactosidase fusion proteins containing the sequences. Nuclear localization was reduced or abolished when the basic amino acids in the signals were mutated. Dynamic changes in glypican immunoreactivity in the nucleus of glioma cells were also observed during cell division and different phases of the cell cycle. The findings suggest glypican and biglycan may be involved in regulating cell division and survival by participating in nuclear processes in
This study investigated how tumor secreted factors alter the responses of human endothelial cells (HUVECs) to matrix stiffness during capillary formation. The researchers used synthetic hydrogels of varying stiffness to culture HUVECs with or without tumor cells. They found that while HUVECs prefer a substrate of intermediate stiffness for optimal capillary formation in the absence of tumor factors, stimulation by tumor secreted factors disrupts the HUVECs' mechanosensitive behavior. Additionally, the anti-angiogenic drug vandetanib was less effective at reducing capillary formation when HUVECs were activated by tumor factors, particularly on stiffer gels. This suggests tumor activation alters the mechanosensitivity and drug responsiveness
This document provides a biographical sketch for Phillip B. Hylemon including:
1) His education, positions held, and honors received including his work in bile acid metabolism and gut bacteria.
2) Contributions to science including discovering pathways of bile acid metabolism by gut bacteria and bile acid activation of cell signaling pathways.
3) Current research support including grants to study role of bile acids and gut bacteria in disease and regulation of hepatic sphingosine kinase by bile acids.
Personalized & Translational Medicine - KineMed, Inc. - Marc Hellerstein, MD,...KineMed, Inc.
The document discusses using measurements of causal pathways to improve predictability in disease outcomes and drug development. It outlines some key challenges, including the fundamental unpredictability of complex biological systems and high attrition rates in pharmaceutical development. The author proposes that measuring the activity of disease-driving processes, or "causal pathways", could help navigate this complexity and transform medicine development by providing a link between molecular targets and whole system outcomes. Examples of causal pathways for various diseases are given, and new kinetic technologies for measuring dynamic proteomes and metabolic water fluxes are described.
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 study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of these exons and certain polymorphisms were associated with T2DM. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
This study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of these exons and certain polymorphisms were associated with T2DM. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
Mucopolysaccharidosis type II MPS II or Hunter syndrome is Metabolism by lysosomal accumulation with a recessive inheritance pattern associated with the X chromosome. It is caused by lack of activity of the lysosomal enzyme iduronate 2 sulfatase, encoded by the IDS gene. Plasma iduronate 2 sulfatase enzymatic activity was measured and the IDS gene in genomic DNA was analyzed by automated direct sequencing. The enzyme activity was 1.2 mol l h reference value 2 mol l h and molecular analysis detected the mutation c.1403G A p.R468Q , confirming the diagnosis of MPS II. rice field. In conclusion, there are few groups dedicated to this disease family here in Mexico, highlighting the need to form an expert team of physicians and scientists dedicated to inborn errors of metabolism to stay up to date. Miss. Parimala L | Babu M "Hunter Syndrome: A Case Report" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-7 | Issue-1 , February 2023, URL: https://www.ijtsrd.com/papers/ijtsrd52814.pdf Paper URL: https://www.ijtsrd.com/medicine/other/52814/hunter-syndrome-a-case-report/miss-parimala-l
Princeton Thesis on Neuroeconomics: Ambition and Reward Greg Schundler
This study examined the association between the Taq A1 allele of the D2DR gene and behaviors in 160 Caucasian undergraduate students at Princeton University. The study found a 16.9% prevalence of the A1 allele, consistent with previous research. Unlike prior research, no associations were found between the A1 allele and addictive behaviors. Interestingly, the study found a negative trend between the A1 allele and alcohol addiction. Additionally, positive trends were found between the A1 allele and academic performance as well as artistic, musical and leadership abilities. The study suggests the A1 allele may confer beneficial behavioral phenotypes rather than only undesirable behaviors.
This study generated a transgenic knockin mouse model expressing the Pro32Pro33 variant of the integrin β3 subunit. Compared to wild-type mice, Pro32Pro33 mice showed decreased bleeding and clotting times, increased thrombosis risk, and enhanced platelet adhesion and spreading through increased integrin αIIbβ3 function. Under unstimulated conditions, Pro32Pro33 mice had elevated Src phosphorylation and talin interactions with the β3 cytoplasmic domain, priming the integrin for activation. Acute treatment with a Src inhibitor rescued the clotting phenotype in Pro32Pro33 mice to wild-type levels. These findings establish that the Pro32Pro33 variant modifies integrin αIIbβ3
This study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of exons 4 and 5 were identified in T2DM patients but not controls. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
This study aimed to analyze maternal serum peptides in the early second trimester to identify potential biomarkers for predicting gestational diabetes mellitus (GDM). Serum samples were collected from women at 16-18 weeks of pregnancy and later classified as having GDM or being healthy controls based on later glucose tolerance tests. Peptidomic analysis using liquid chromatography-tandem mass spectrometry identified 297 peptides from 228 proteins that were differentially expressed between GDM and control groups. These peptides may help predict GDM and allow for earlier intervention. The study provides the first validated peptidome profile of early second trimester serum to investigate biomarkers for predicting GDM.
Plasma phospholipids identify antecedent memory impairment in older adultsJosé Luis Moreno Garvayo
En este trabajo publicado en la revista Nature medicine el pasado mes de marzo, el equipo del Dr. Federoff plantea un novedoso enfoque que consiste en analizar un grupo de diez fosfolípidos para la detección de la enfermedad de Alzheimer antes de la manifestación clínica de los síntomas.
This document summarizes a study that used proteomics to identify serum biomarkers for Alzheimer's disease (AD) and mild cognitive impairment (MCI) by analyzing serum samples from patients selected based on their PiB-PET imaging scores. The researchers used isobaric tagging and liquid chromatography-tandem mass spectrometry to perform proteome profiling on serum from control, MCI, and AD patients. They identified 79 and 72 differentially expressed proteins in MCI and AD serum, respectively, compared to controls. Integrated analysis with brain tissue data identified three biomarker candidates related to proteolysis: PCSK9, F13A1, and DCD. Validation in independent serum samples confirmed elevated levels of these candidates in MCI
Jianying Xiao has over 16 years of experience in pharmaceutical research. She has expertise in in vivo and in vitro drug discovery techniques related to drug metabolism, infectious diseases, immunology, and cardio-metabolic disorders. She is proficient in various research techniques including animal handling, molecular biology, cell culture, and data analysis software. Jianying has worked at Merck & Co. for over 18 years, leading numerous projects that resulted in publications, patents, and awards. Her work has advanced drug programs from research through clinical trials.
Transcriptional signaling pathways inversely regulated in alzheimer's disease...Elsa von Licy
This document summarizes a study that analyzed gene expression data from patients with Alzheimer's disease and glioblastoma multiforme to identify signaling pathways that are inversely regulated between the two diseases. The study found that the ERK/MAPK pathway is upregulated in glioblastoma but downregulated in Alzheimer's disease. Additionally, the angiopoietin signaling pathway is upregulated in Alzheimer's disease but downregulated in glioblastoma. Conditioned media containing amyloid-beta peptide suppressed glioblastoma cell growth and ERK/MAPK signaling, suggesting amyloid-beta may contribute to the inverse relationship between the diseases by inhibiting glioblastoma signaling pathways.
Biomarkers of Systemic Lupus Erythematosus and Systemic Sclerosis diseases ac...Prof. Hesham N. Mustafa
Systemic Lupus Erythematosus (SLE) and systemic sclerosis (SSc) are systemic inflammatory autoimmune disorders characterized by a large spectrum of clinical and laboratory features. The aim of the present study was to investigate the possible use of serum level of soluble intercellular adhesion molecule-1(sICAM-1) and soluble interleukin-2 receptor (sIL-2Ra) as biomarkers for monitoring of SLE and SSc disease activity. Moreover, it aimed to compare the specificity and sensitivity as well as cut-off value of both biomarkers in a sample of Egyptian patients. 50 SLE patients, 30 SSc patients and 60 age and sex matched healthy controls were enrolled in our study. sICAM-1and sIL-2Ra were measured in serum samples obtained from all participants. In addition to Erythosedimentation rate (ESR), complete blood count (CBC), Antineuclearantibodies (ANA) estimation, disease activity of both diseases were also assessed. sICAM-1and sIL-2Ra levels were higher in SLE and SSc patients versus control. Both parameters are correlated with each other as well as the activity parameters. A cut-off levels of 455.59 (ng/ml) &2525935 (pg/ml) in both SLE & SSs respectively was observed with the highest specificity and sensitivity. It could be concluded that sICAM-1 and sIL-2Ra are noninvasive biomarkers for SLE and SSc that could play a pathophysiologic role in development and progression of both diseases. Moreover, sICAM-1 and sIL-2Ra are correlated with the disease activity at cut-off values of 455.59 (ng/ml) & 2525935(pg/ml) respectively.
Review (ca 2007) of Uremic Toxins Accumulating in Patients with Chronic and End Stage Renal Disease modified from a presentation I gave in Fellow's Grand rounds.
Relied heavily on publications from the EU Toxin Work Group Work, which provides more up to date information:
http://www.uremic-toxins.org/
Glypican and Biglycan in the Nuclei of Neurons and Glioma CellsYu Liang
This document describes a study that found glypican and biglycan, two proteoglycans, localized to the nuclei of neurons and glioma cells. The researchers identified nuclear localization signals in the amino acid sequences of both proteoglycans. They demonstrated that these signals were functional by showing nuclear localization of beta-galactosidase fusion proteins containing the sequences. Nuclear localization was reduced or abolished when the basic amino acids in the signals were mutated. Dynamic changes in glypican immunoreactivity in the nucleus of glioma cells were also observed during cell division and different phases of the cell cycle. The findings suggest glypican and biglycan may be involved in regulating cell division and survival by participating in nuclear processes in
This study investigated how tumor secreted factors alter the responses of human endothelial cells (HUVECs) to matrix stiffness during capillary formation. The researchers used synthetic hydrogels of varying stiffness to culture HUVECs with or without tumor cells. They found that while HUVECs prefer a substrate of intermediate stiffness for optimal capillary formation in the absence of tumor factors, stimulation by tumor secreted factors disrupts the HUVECs' mechanosensitive behavior. Additionally, the anti-angiogenic drug vandetanib was less effective at reducing capillary formation when HUVECs were activated by tumor factors, particularly on stiffer gels. This suggests tumor activation alters the mechanosensitivity and drug responsiveness
This document provides a biographical sketch for Phillip B. Hylemon including:
1) His education, positions held, and honors received including his work in bile acid metabolism and gut bacteria.
2) Contributions to science including discovering pathways of bile acid metabolism by gut bacteria and bile acid activation of cell signaling pathways.
3) Current research support including grants to study role of bile acids and gut bacteria in disease and regulation of hepatic sphingosine kinase by bile acids.
Personalized & Translational Medicine - KineMed, Inc. - Marc Hellerstein, MD,...KineMed, Inc.
The document discusses using measurements of causal pathways to improve predictability in disease outcomes and drug development. It outlines some key challenges, including the fundamental unpredictability of complex biological systems and high attrition rates in pharmaceutical development. The author proposes that measuring the activity of disease-driving processes, or "causal pathways", could help navigate this complexity and transform medicine development by providing a link between molecular targets and whole system outcomes. Examples of causal pathways for various diseases are given, and new kinetic technologies for measuring dynamic proteomes and metabolic water fluxes are described.
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 study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of these exons and certain polymorphisms were associated with T2DM. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
This study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of these exons and certain polymorphisms were associated with T2DM. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
Mucopolysaccharidosis type II MPS II or Hunter syndrome is Metabolism by lysosomal accumulation with a recessive inheritance pattern associated with the X chromosome. It is caused by lack of activity of the lysosomal enzyme iduronate 2 sulfatase, encoded by the IDS gene. Plasma iduronate 2 sulfatase enzymatic activity was measured and the IDS gene in genomic DNA was analyzed by automated direct sequencing. The enzyme activity was 1.2 mol l h reference value 2 mol l h and molecular analysis detected the mutation c.1403G A p.R468Q , confirming the diagnosis of MPS II. rice field. In conclusion, there are few groups dedicated to this disease family here in Mexico, highlighting the need to form an expert team of physicians and scientists dedicated to inborn errors of metabolism to stay up to date. Miss. Parimala L | Babu M "Hunter Syndrome: A Case Report" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-7 | Issue-1 , February 2023, URL: https://www.ijtsrd.com/papers/ijtsrd52814.pdf Paper URL: https://www.ijtsrd.com/medicine/other/52814/hunter-syndrome-a-case-report/miss-parimala-l
Princeton Thesis on Neuroeconomics: Ambition and Reward Greg Schundler
This study examined the association between the Taq A1 allele of the D2DR gene and behaviors in 160 Caucasian undergraduate students at Princeton University. The study found a 16.9% prevalence of the A1 allele, consistent with previous research. Unlike prior research, no associations were found between the A1 allele and addictive behaviors. Interestingly, the study found a negative trend between the A1 allele and alcohol addiction. Additionally, positive trends were found between the A1 allele and academic performance as well as artistic, musical and leadership abilities. The study suggests the A1 allele may confer beneficial behavioral phenotypes rather than only undesirable behaviors.
This study generated a transgenic knockin mouse model expressing the Pro32Pro33 variant of the integrin β3 subunit. Compared to wild-type mice, Pro32Pro33 mice showed decreased bleeding and clotting times, increased thrombosis risk, and enhanced platelet adhesion and spreading through increased integrin αIIbβ3 function. Under unstimulated conditions, Pro32Pro33 mice had elevated Src phosphorylation and talin interactions with the β3 cytoplasmic domain, priming the integrin for activation. Acute treatment with a Src inhibitor rescued the clotting phenotype in Pro32Pro33 mice to wild-type levels. These findings establish that the Pro32Pro33 variant modifies integrin αIIbβ3
This study investigated CD36 gene status in north Indian subjects with type 2 diabetes (T2DM) by screening for deletions of exons 3, 4 and 5 and certain polymorphisms. Blood samples were taken from 300 T2DM patients and 100 healthy controls. Biochemical parameters and DNA extraction were performed, and exons 3-5 were analyzed via PCR. Deletions of exons 4 and 5 were identified in T2DM patients but not controls. The study aims to further analyze genetic factors and deletions related to the CD36 gene in T2DM patients in north India.
This study aimed to analyze maternal serum peptides in the early second trimester to identify potential biomarkers for predicting gestational diabetes mellitus (GDM). Serum samples were collected from women at 16-18 weeks of pregnancy and later classified as having GDM or being healthy controls based on later glucose tolerance tests. Peptidomic analysis using liquid chromatography-tandem mass spectrometry identified 297 peptides from 228 proteins that were differentially expressed between GDM and control groups. These peptides may help predict GDM and allow for earlier intervention. The study provides the first validated peptidome profile of early second trimester serum to investigate biomarkers for predicting GDM.
This document provides an introduction to biostatistics. It discusses key concepts including descriptive statistics, inferential statistics, hypothesis testing, and sampling techniques. It outlines the role of biostatistics in various areas like clinical medicine, preventive medicine, health planning and evaluation, and medical research. Biostatistics helps manage uncertainties in medicine by providing statistical methods to analyze data, evaluate treatments and programs, and make inferences about populations. It is important for designing valid research studies and interpreting medical literature.
X-rays were discovered in 1895 by Wilhelm Röntgen. X-rays are produced when high velocity electrons hit a metal target, knocking electrons out of inner shells of atoms within the target. As outer electrons fall into these vacancies, characteristic X-rays are emitted with energies specific to the element. X-ray diffraction methods like Bragg's law are used to analyze crystal structures of materials by detecting the angles and intensities of diffracted X-ray beams. Common applications include determining structures of polymers, particle sizes, and states of materials.
This document provides an overview of systematic reviews and meta-analyses. It defines systematic reviews as reviews that use explicit and reproducible methods to minimize bias in identifying, selecting, and synthesizing studies. Key elements of systematic reviews include formulating a clear question, conducting a comprehensive search, applying objective selection criteria, critically appraising included studies, and synthesizing data using meta-analysis if appropriate. Heterogeneity and reporting biases are important considerations in analyzing and interpreting systematic review results. The document outlines the process for conducting high-quality systematic reviews and meta-analyses.
This document provides guidance on critically evaluating biomedical literature. It discusses key elements to examine such as the title, abstract, introduction, objectives, methods, study design, potential biases, statistical analysis, results, discussion and conclusion. The document emphasizes reviewing these sections systematically to determine the validity, relevance and applicability of a study's findings. Selecting high quality literature that could impact clinical practice is important. A thorough evaluation of all aspects of a scientific paper is required to make well-informed judgments.
This document summarizes different types of literature reviews that are commonly used in health research. It discusses 8 major types of reviews: literature review, critical review, scoping review, systematic review, meta-analysis, qualitative systematic review, realist review, and review of reviews. For each type of review, it provides a brief description, discusses their methodology using the Search, Appraisal, Synthesis, Analysis (SALSA) framework, and notes their strengths and weaknesses. The document aims to provide beginners with an overview of different review types and their implications for practice.
This document describes a method for quantitatively separating and estimating lipids in nervous tissue using thin-layer chromatography. The method involves extracting lipids from brain tissue using chloroform and methanol, applying the extract to a thin-layer chromatography plate, separating the lipids using a mobile phase, visualizing the lipids by spraying with sulfuric acid and scanning, and then quantifying the lipids by measuring the area under peaks on a densitometer trace. The method was found to accurately separate and quantify major brain lipids like sphingomyelin, cerebrosides, and phospholipids with standard errors of 2% or less for major lipids.
This document discusses the stepwise synthesis of glycolipids in the Golgi complex. It summarizes that glycolipid expression is highly regulated during development through transcriptional control of key glycosyltransferases. These transferases reside in the Golgi and depend on N-glycosylation and oligosaccharide processing for proper folding. Their N-terminal domain transports them to the Golgi where some associate to form multienzyme complexes. The precise organization and dynamics of this machinery in the Golgi is a potential target for fine-tuning glycolipid expression.
This document presents a targeted liquid chromatography-mass spectrometry workflow for comprehensive profiling of sphingolipids. The workflow improves upon previous methods by using (1) an optimized extraction procedure, (2) a segmented gradient to better separate isobaric species, and (3) parallel reaction monitoring of sphingolipid fragments to identify species. The workflow was tested on RAW 264.7 cells and quantified 61 sphingolipids over a wide dynamic range with low femtomole detection limits, making it a high-throughput tool for sphingolipidomics.
This study evaluates an internal standard cocktail developed for the LIPID MAPS Consortium for quantitative analysis of sphingolipids using mass spectrometry. The internal standard cocktail contains uncommon chain length analogs of sphingoid bases and sphingolipids that have similar ionization and fragmentation properties to the target analytes. Two mass spectrometers, a triple quadrupole and a quadrupole linear ion trap, were used to optimize the method and validate the internal standard cocktail. The internal standard cocktail and mass spectrometry methods were applied to analyze sphingolipids extracted from RAW264.7 mouse macrophage cells.
The document discusses infrared (IR) spectroscopy, which analyzes the vibrational frequencies of bonds in molecules. It describes the different types of molecular vibrations that can be observed in an IR spectrum, such as stretching and bending modes. Specific functional groups absorb at characteristic wavelengths - for example, O-H and N-H stretches appear around 3300 cm-1, while C=O stretches are around 1710 cm-1. The document outlines the theory behind IR spectroscopy and provides examples of IR spectra for various compounds like alkanes, alkenes, alcohols and amines to demonstrate the technique.
Hydrophilic interaction liquid chromatography (HILIC) is a powerful technique for separating polar compounds. The document reviews options for characterizing HILIC stationary phases and their applications. HILIC employs polar stationary phases like silica, amino, or cyano bonded phases. Retention depends on factors like the stationary phase properties, mobile phase composition, and analyte structure. HILIC has advantages over normal and reversed phase chromatography and is useful for separating compounds that are too polar for reversed phase methods.
This document provides an overview of the role of process validation in tablet manufacturing. It discusses that process validation is required to demonstrate that a manufacturing process is capable of consistently producing tablets that meet predetermined specifications and quality attributes. The key aspects of process validation covered include the types of validation, phases of validation, critical factors, and use of sample thieves. Process validation helps assure quality, reduce costs from rejects and failures, and ensures compliance with regulatory requirements. It is an important part of good manufacturing practices for pharmaceutical products.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
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Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
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The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
1. Accepted Manuscript
A new LC/MS/MS method for quantification of gangliosides in human plasma
Qianyang Huang, Xiang Zhou, Danting Liu, Baozhong Xin, Karen Cechner,
Heng Wang, Aimin Zhou
PII: S0003-2697(14)00112-2
DOI: http://dx.doi.org/10.1016/j.ab.2014.03.014
Reference: YABIO 11686
To appear in: Analytical Biochemistry
Received Date: 26 November 2013
Revised Date: 1 March 2014
Accepted Date: 19 March 2014
Please cite this article as: Q. Huang, X. Zhou, D. Liu, B. Xin, K. Cechner, H. Wang, A. Zhou, A new LC/MS/MS
method for quantification of gangliosides in human plasma, Analytical Biochemistry (2014), doi: http://dx.doi.org/
10.1016/j.ab.2014.03.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
2. A new LC/MS/MS method for quantification of gangliosides in human plasma
Qianyang Huanga
, Xiang Zhoua*
, Danting Liua
, Baozhong Xinc
, Karen Cechnerc
, Heng Wangc
,
and Aimin Zhoua,b*
a
Clinical Chemistry Program, Department of Chemistry, b
Center for Gene Regulation in
Health and Diseases, Cleveland State University Cleveland State University, 2121 Euclid
Avenue, Cleveland, Ohio, 44115, United States
c
DDC Clinic, Center for Special Needs Children, 14567 Madison Road, Middlefield, Ohio,
44062, United States
Running title: Development of a method for analyzing gangliosides
Keyword: Gangliosides, Mass spectrometry, GM3 synthase deficiency, neurological disorders
To whom correspondence should be addressed:
Dr. Aimin Zhou, Clinical Chemistry Program, Department of Chemistry, Cleveland State
University, Cleveland, OH 44115; Phone: 216-687-2416; Fax: 216-687-9298; e-mail:
a.zhou@csuohio.edu
Dr. Xiang Zhou, Department of Chemistry, Cleveland State University, Cleveland, OH
44115; Phone: 216-687-39226; Fax: 216-687-9298; e-mail: x.zhou34@csuohio.edu
3. Abstract
Gangliosides are a family of glycosphingolipids characterized by mono- or poly-sialic
acid-containing oligosaccharides linked through 1, 3- and 1, 4-β glycosidic bonds with subtle
differences in structure, which are abundantly presented in the central nervous system of
many living organisms. Their cellular surface expression and physiological malfunction are
believed to be pathologically implicated in considerable neurological disorders, including
Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental
retardation or physical stagnation deteriorates as the physiological profile of gangliosides
becomes progressively and distinctively abnormal during the development of these typical
neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography-tandem
mass spectrometry assay using standard addition calibration for determination of GM2, GM3,
GD2, and GD3 in human plasma has been developed and validated. The analytes and internal
standard were extracted from human plasma using a simple protein precipitation procedure.
Then the samples were analyzed by reverse phase UPLC/MS/MS interfaced to mass
spectrometry with electrospray ionization using a Multiple Reaction Monitoring (MRM)
mode to obtain superior sensitivity and specificity. This assay was validated for extraction
recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can
be applied to monitor ganglioside levels in plasma from normal people and
neurodegenerative patients.
4. 1. Introduction
Gangliosides are a subgroup of glycosphingolipids that present abundantly in the central
nervous system (1). They are generally consisted of mono- or poly-sialic acid-containing
hydrophilic oligosaccharide moiety linked with a hydrophobic ceramide portion that
comprises a sphingosine backbone and a fatty acid chain with a variable number of carbons.
Such compounds are predominately localized at the cellular membrane of neuronal and glial
cells with their ceramide portion embedded inward into the outer leaflet of the lipid bilayer
and the oligosaccharide moiety protrudes outward into the extracellular matrix (2). Up to date,
over 180 ganglioside species with characteristic carbohydrate moieties have been identified
in vertebrates. In addition to disparate linking arrangements among various monosaccharide
units in carbohydrate moiety, each species possesses multiple components with the ceramide
portion encompassing alterable chain-length of a fatty acid, which implies the great structural
heterogeneity of such molecules (3) (corresponding classifications and nomenclatures are
shown in Fig 1). The structural diversity empowers gangliosides the versatility for
biologically functioning as modulators of signaling receptors in the regulation of various
cellular events such as neurotrophy and neurotransmission by physiologically interacting with
regulatory proteins in the nervous system (4).
The exact physiological role of gangliosides in the brain is not fully understood yet, but they
are believed to be functioning in the regulation of receptor-mediated cell signaling pathways,
especially in cell differentiation, cell proliferation, and cell death (5-8). Such functions turn
out to be typically serving as integral components of cell surface microdomains (9) or lipid
5. rafts (10) along with proteins, sphingomyelins, and cholesterols in administrating signal
events affecting neural development and contributing to the pathogenesis of numerous
clinical diseases and disorders, including GM3 synthase deficiency (11-13), Tay-Sachs (14,
15) and Sandhoff diseases (16-18). GM3 synthase deficiency is a neurological disorder
recently identified among the Amish people in the United States, caused by mutation of the
GMs synthetase gene, resulting in the absence of GM3 and its downstream metabolites. The
clinical manifestation of these patients is similar to intractable seizures with profound mental
and physical retardation (11). Recent years studies have illustrated aberrant ganglioside
profiles in neurodegenerative syndromes, implying their potential contribution to the
development or progression of these chronic and incurable neurodegenerative disorders, such
as Parkinson’s (19, 20) and Alzheimer diseases (21).
In the past decades, measurement or profiling of gangliosides in animal tissues, cultured cells,
or cerebrospinal fluid (CSF) has been reported using thin layer chromatography (TLC)
coupled with densitometric (22) or immunochemical detection (23) high performance liquid
chromatography (HPLC) (24, 25), supercritical fluid chromatography (SFC) (26) and
enzyme-linked immuno-sorbent assay (ELISA) (27). These methods are subjected to such
limitations as large sample requirement, laborious sample preparation, time-consuming
measurement, and insufficient sensitivity and specificity. Recently, the advent of mass
spectrometry has given rise to the excellent quantitative analysis featured by promising
sensitivity and resolution for the determination of gangliosides. Gu et al. established a
method for simultaneous quantification of GM1 and GM2 gangliosides in human
6. cerebrospinal fluid using reverse phase LC/MS (28). Sorensen et al. reported a liquid
chromatographic approach interfaced with tandem mass spectrometry for the quantification
of gangliosides GD3 and GM3 in bovine milk and infant formula (29).
In the present work, we have developed and validated a UPLC/MS/MS method for the
measurement of four common ganglioside species including GM2, GM3, GD2, and GD3 in
human plasma. This assay employs a simple protein precipitation strategy for sample
extraction, UPLC for chromatographic separation, and tandem mass spectrometry in the
MRM mode for detection to achieve high sensitivity and specificity for rapid analysis. We
have successfully applied this method to measure the four species of gangliosides in human
plasma.
7. 2. Experimental
2.1 Materials
Ganglioside standards GM3 and GD3 were purchased from Avantilipids (Alabaster, AL).
GM2 and GD2 ganglioside standards were obtained from EMD Chemicals (Billerica, MA)
and Enzo Life Sciences (Farmingdale, NY), respectively. The internal standard (IS)
N-omega-CD3-Octadecanoyl monosialoganglioside GM3 (GM3-D3) was purchased from
Matreya LLC (Pleasant Gap, PA). HPLC grade methanol and isopropanol were obtained from
EMD Milipore (Billerica, MA). Deionized water was prepared through the Barnstead
Nano-PURE Water Purification System (Asheville, NC). Ammonium acetate was from VWR
(Bridgeport, NJ).
2.2 Human Plasma
The study was approved by the DDC Clinic Institutional Review Board, and written informed
consent was obtained from their legal guardians. Clinical information was collected when the
patients received medical services at DDC Clinic. Human plasma samples were stored at
-20℃ before analysis.
2.3 Stock and Work Solutions
Stock solutions of GM2, GM3, GD2, GD3, and IS GM3-D3 were prepared by dissolving the
original materials individually into 80% MeOH to obtain a concentration of 0.25 mg/ml for
each, and were stored at -20 ℃ before use. Three work solutions were prepared in 80%
8. MeOH from the stock solutions to obtain ganglioside concentrations as following: 3, 13.6,
0.125, 2 g/ml (WS-QC); 4.5, 20.4, 0.125, 3 g/ml (WS-1), and 18, 81.6, 0.5, 12 g/ml
(WS-2) for GM2, GM3, GD2, GD3, respectively. The IS stock solution also served as its
work solution, named as WS-IS. These work solutions were used for preparation of spiked
plasma samples as below.
2.4 Plasma Sample Preparation
For method validation, known-concentration samples were prepared by spiking ganglioside
standards into the analyte-free plasma from patients with GM3 Synthase Deficiency. In brief,
30 L analyte-free plasma was transferred into each of three 0.6-mL centrifuge vials (a, b,
and c) followed by individual addition of 10L WS-QC to obtain the plasma concentrations
of 1.00, 4.53, 0.04, and 0.67 g/ml for GM2, GM3, GD2, and GD3, respectively. Thereafter,
10 L of 80% methanol, WS-1, and WS-2 were spiked into the vials a, b, and c, respectively.
Afterwards, 10 L of WS-IS was spiked into each of the three samples, and this set of
samples were named as spiked analyte-free plasma (SAP), Standard Addition 1 (SA-1), and
Standard Addition 2 (SA-2), respectively.
For measurement of the gangliosides in unknown plasma samples from normal human
subjects, similar procedure as described above was followed except that WS-QC was not
added. The three samples in each set were named as unknown plasma sample (UPS), SA-1
and SA-2, respectively. All the plasma samples were extracted as described below prior to
LC/MS/MS analysis.
9. 2.5 Sample Extraction
Following the plasma sample preparation, 240 L of methanol was added into each of them
followed by vigorous vortexing. Then, the spiked samples were subjected to centrifugation at
14,000 G for 15 min at 4 ℃ for protein precipitation. The supernatants were transferred into
auto-sampler vials with micro-inserts for LC/MS analysis.
2.6 LC/MS Instrumentation
The UPLC system was composed of a DGU-20A3R degasser, two LC-30AD pumps, a
SIL-30AC autosampler, a CTO-10A column oven and a CBM-20A system controller from
Shimadzu (Columbia, MD). The UPLC system was interfaced to a Qtrap 5500 mass
spectrometer equipped with an electrospray ionization source and a built-in Valco switch
valve from AB SCIEX (Framingham, MA). The Multiple Reaction Monitoring (MRM) mode
was utilized for quantitation. Data acquisition and chromatographic peak integration were
conducted using Analysis 1.6.1 software package from AB SCIEX.
2.7 LC/MS/MS Procedure
After extraction, 30l of each sample was injected onto a Kinetex-C18 UPLC column (1.7
m, 50 mm × 2.1 mm; Phenomenex, Torrance, CA) guarded with a C18 Security Cartridge.
The mobile phase was a mixture of 2% MeOH plus 5 mM ammonium acetate (A) and
MeOH-Isopropanol (1:1) plus 5 mM ammonium acetate (B) using the gradient program
shown in Table 1. The LC flow at 0.3 mL/min was introduced to the waste within the first 2
minutes, and then switched to the mass spectrometer between 2.1 and 9 minutes while all the
10. monitored ganglioside components were gradually eluted out. Afterwards, the LC flow at 0.6
mL/min was directed to the waste again, during which the column was cleaned up and
re-equilibrated for next run. The column temperature was constantly controlled at 35 ℃
during the analysis.
2.8 Calibration Curve
Quantitation of the gangliosides in the spiked analyte-free plasma (SAP) and unknown
plasma samples (UPS) was implemented using standard addition calibration based on three
levels, including one to-be-measured sample with nil addition and two standard addition
calibrators (SA-1 and SA-2). For each measured ganglioside specimen, the total peak area
(TPA) was obtained by summing the individual peak area from every MRM transition
channel in the corresponding sample from a single injection using the equation below.
The three-level calibration curve for each ganglioside sample was then established by plotting
its normalized TPA by the internal standard as y-axis versus the plasma concentration of
standard addition as x-axis. The linear regression with a weighting factor of 1/y2
was
employed. The calculation of each ganglioside plasma concentration in a sample was
conducted using the corresponding equation obtained from the regression line as its intercept
with x-axis through extrapolation. Exemplified standard addition calibration curves for each
of the four measured gangliosides from the measurement of healthy individuals were shown
in Fig.2.
11. 3. Results and Discussion
3.1 Product Ion Mass Spectra
The representative product ion mass spectra of D18:1-20:0 ganglioside components for the
four ganglioside species from added standards were shown in Fig.3. Apparently,
fragmentation pattern relevance can be observed within GM series and GD series, including
the common product ions m/z = 274 and 292 for GM series, and m/z = 290 and 581 for GD
series. Owing to their relatively greater molecular masses, the singly-charged ions of
gangliosides are beyond the detection ranges of our mass spectrometer and many other
commercially available triple quadruple instruments. Therefore, the selection of
doubly-charged precursor ions and their appropriate daughter ions as MRM transition
channels has been necessitated for quantitation in this assay.
For GD series, two proton-donating carboxyl groups are attached to their sialic acid moieties
in the carbohydrate portion, the generation of doubly-charged ions had been observed to be
more favorable in the negative than that in a positive ionization mode. For GM series that
contain only one proton-donating carboxyl group, the signal responses from doubly-charged
precursor ions were practically undetectable in a negative ionization mode under various
conditions we studied during the method development. Since the positive mode was found to
be more effective in yielding doubly-charged precursor ions from GM2 and GM3, positive
ionization has been selected for determining GM series.
12. After evaluating different pairs of precursor → product ions for quantitation, it was revealed
that the following MRM transitions provided the optimal sensitivity and selectivity:
[GM2+2H+
]2+
→ 204, [GM3+2H+
]2+
→ 274, [GD2-2H+
]2-
→ 290, and [GD3-2H+
]2-
→ 290.
The fragmentation mechanisms from the precursor to the daughter ions have been interpreted
and elaborated in Fig 4.
3.2 Selection of MRM Transitions
As shown in Fig 1, each ganglioside species include multiple components that share the same
oligosaccharide moiety but differ in fatty acid chain length within their ceramide portion. For
accurate quantitation, all the major ganglioside components from each ganglioside species
with appreciable abundance in the plasma and commercial standards need to be detected in
the analysis. Therefore, we first carried out a screening study on predominant ganglioside
components of individual ganglioside species in both normal human plasma and commercial
standards using the predicted MRM channels through LC/MS/MS analysis. The selected
MRM transitions corresponding to all the significant ganglioside components observed by
such analysis for the quantitation of the four ganglioside species are listed in Table 2. In
addition to the quantitation MRM channels, quality assurance MRM channels with transitions
from their precursor ions to m/z 274, 292, 290, and 290 were also employed for GM2, GM3,
GD2, and GD3, respectively. Our experiments showed that the response ratio for each pair of
MRM channels was practically constant in standard-spiked solvent and unknown plasma
extracts, confirming the identity of the monitored components.
13. 3.3 HPLC Conditions
In view of multiple MRM transitions have been exploited to monitor each of the four
ganglioside species, a sufficient chromatographic separation between ganglioside molecules
and plasma interferences is essential to eliminate the potential ionization suppression
influence from the matrix for the acquisition of high quality quantification during the analysis.
Based on our preliminary studies during the method development, a C18 reverse-phase
UPLC column was found to provide the best resolution for multiple ganglioside components.
Under our optimized LC conditions, all the ganglioside components were eluted out and
spread within a retention time window from 3 to 9 minutes. In general, gangliosides could be
strongly retained by the C18 stationary phase due to the hydrophobic nature of their ceramide
portion. We observed that the use of Methanol-Isopropanol (1:1) as the solvent of mobile
phase B could elute the gangliosides from the C18 stationary phase more effectively than the
use of methanol only, and lead to shorter chromatographic running time and better peak
shapes. The MRM chromatograms for the major components of GM3 ganglioside are shown
in Fig.5.
It is noteworthy that the addition of 5 mM ammonium acetate in the mobile phases
considerably reinforced the peak intensities from all MRM channels in both positive and
negative ionization modes. This experimental phenomenon can be reasonably ascribed to
ammonium acetate’s ability to stabilize the PH at neural range, enabling both the
proton-donating interaction between ammonium group and gangliosides in the positive mode
and proton-accepting interaction between acetate group and gangliosides in the negative
14. mode to be undertaken for the promotion of ionizing efficiency during the electrospray
ionization. While ammonium formate or formic acid was added into the mobile phases, the
responses from positive ionization was enhanced to a certain extent, whereas substantial
signal suppression was occurred in the negative ionization, which was apparently attributable
to intensified proton-donating tendency of formic acid and ammonium formate as compared
with ammonium acetate.
3.4 Sample Extraction Procedure
The most commonly used method for the isolation of gangliosides from biomatrices is a
liquid-liquid partition strategy that first reported by Svennerholm and Fredman (30) for the
treatment of human brain samples, which has been used or modified later by other
investigators (29, 31, 32) for extracting gangliosides from milk and other matrices. In brief,
the workflow is constructed by the steps as following: (a) mixing the biomatrix with
chloroform/methanol/water (4:8:3) by vigorous shaking; (b) transferring the
ganglioside-enriched methanol/waster upper phase into a new container without disturbing
the middle and lower phase; (c) drying the crude extract with inert gas purging, and (d)
reconstituting the residues with methanol to a certain volume for quantitative analysis. To
compare the extraction recovery by using our simple protein-precipitation procedure and the
existed extraction method, we performed a series of parallel studies. In these assays,
analyte-free plasmas were spiked with the gangliosides followed by extraction with two
different extraction methods. As expected, our protein precipitation method was not only
easy-handling and time-saving, but also provided full recovery for all of the four ganglioside
15. species (Table 3). In contrast, the reported method generated insufficient recovery for GM2
and GM3 gangliosides. According to the literatures, protein-precipitation from plasma
samples was commonly conducted by the addition of 3 to 4-fold of methanol or acetonitrile,
followed by high speed centrifugation. However, the widely adopted condition generated
unsatisfactory recovery of gangliosides from plasma. Based on modest polarity of
gangliosides from their hydrophilic oligosaccharide moiety and hydrophobic ceramide
portion, we attempted to achieve the best extraction recovery through adjustment of the
polarity by increasing portion of methanol. Nearly 100% extraction recovery was achieved
for all of the four ganglioside species by the addition of 8-fold methanol into plasma for
protein precipitation.
3.5 Standard Addition Calibration
According to the FDA guidelines for bioanalytical method validation (33), the same type of
matrix as actual samples should be used for the preparation of calibrators and QCs during the
quantitative method development and validation whenever feasible concerning the potential
pronounced signal interference imposed by the complex matrices. For the determination of
exogenous compounds that do not present in intended matrices, the conventional standard
calibration is usually employed to generate calibration curves based on six or more levels of
analytes spiked into analyte-free matrices. However, for the measurement of endogenous
compounds that originally present in the sample biomatrices at significant concentrations, the
standard addition calibration has been widely used for the establishment of matrix-matched
calibration. Commonly, each standard calibration curve is constructed upon a set of 3-4
16. different concentrations prepared by addition (including a nil addition) of known amounts of
analyte standard into aliquots of a sample to be measured. Recent publications involving the
application of standard addition calibration include the quantification of bile acids (34), sialic
acids (35), gangliosides (29, 36), and other endogenous compounds (37, 38, 39) in various
biomatrices. In most of these studies, including determination of gangliosides in milk and
buttermilk, three or four points were used to construct the calibration curves.
As indicated earlier, one of our objectives for the development of this assay is to screen the
ganglioside levels in plasma from patients with GM3 synthase deficiency during clinical
treatments. We have chosen three-level standard addition upon such a consideration that these
patients are mostly children or even infants, and that they can only provide small amounts of
plasma for the study. As shown in Fig 2, the exemplified calibration curves had R2
values in a
range of 0.9998 - 1 for all four ganglioside species, demonstrating a good linear relationship
between the analyte response and concentration. For the method validation, we prepared
known-concentration samples by spiking ganglioside standards into aliquots of analyte-free
plasma from GSD patients. Analysis of such known samples has shown that our assay
employing the three-level standard addition offered satisfactory precision and accuracy for
the measurement of all the four ganglioside species in plasma, which is detailed below.
3.6 Precision and Accuracy
The precision and accuracy for both intra-assay and inter-assay were assessed by measuring
analyte-free plasma samples spiked with the ganglioside standards in triplicate. As shown in
Table 4, the CV % for intra- and inter-assay of all measured ganglioside species was within a
17. range from 0.4 to 6 % with the relative error below 9 %. Conclusively, the precision and
accuracy of our quantitative method well fulfill the 15 % error and variation tolerance as
required by the FDA guidelines for bioanalytical method validation (33).
3.7 Stability
The pre- and post- extraction stabilities of gangliosides in plasma under various storage
conditions were evaluated via the analysis of samples prepared from the analyte-free plasma
spiked with the gangliosides after undergoing the studied storage conditions, and the results
are listed in Table 5. The stability of “4-hour pre-extraction storage at room temperature” was
assessed by spiking gangliosides into the thawed analyte-free plasma, letting the spiked
samples stand at room temperature for 4 hours, and then analyzing the samples via LC/MS
immediately following the extraction procedure. The stability of “8-hour post-extraction
storage at room temperature” was evaluated by spiking gangliosides into analyte-free plasma,
extracting the spiked samples, letting the extracts stand at room temperature for 8 hours, and
then injecting them into LC/MS for analysis. The measured stability values for the four
ganglioside species range from 86 to 110 % under three different storage conditions,
including one month pre-extraction at -20 ℃, 4-hour pre-extraction at room temperature, and
8-hour post-extraction at room temperature. Apparently, the gangliosides were stable in the
plasma at the stages of frozen storage, sample treatment, and analysis.
3.8 Matrix Effect
The matrix effect for mass spectrometry detection of gangliosides was assessed through
18. comparable analysis of analyte-free plasma extracts spiked with ganglioside standards and
the pure solvent spiked with such standards at the same contractions in triplicate. It was found
that the matrix impurities substantially suppressed the ganglioside signals, and the
LC/MS/MS peak areas from the plasma extracts were only 61.3, 36.0, 68.3, and 49.0% in
comparison to the pure solvent for ganglioside GM2, GM3, GD2, and GD3, respectively. By
using the standard addition calibration, the matrix effect practically had no influence on the
measurement because the same matrix was utilized for the calibrators and the relevant
samples, which was proven by the high linearity of calibration, precision, and accuracy.
3.9 Method Application
The validated LC/MS/MS assay has been successfully applied to determine ganglioside
concentrations in human plasma from different specimens, including 20 normal human
subjects and 6 GSD patients. As shown in Table 6, the averaged ganglioside levels in the
plasma samples from 20 normal human subjects (include 12 males and 8 females) were 2.37,
9.72, 0.01, and 1.80 g/ml for GM2, GM3, GD2, and GD3, respectively. In contrast, the
analyte-free plasma from 6 patients with GSD had no gangliosides presented at the
measurable level, which was consistent with the pathological manifestation of the
neurodegenerative disorder. The averaged GM3 concentration in plasma samples from eight
healthy human subjects determined by a published LC/MS method (40) was 7.2 g/ml, which
was quite comparable to our result (9.72 g/ml).
19. 4. Conclusion
This paper detailed the development and validation of a sensitive LC/MS/MS method for the
determination of gangliosides GM2, GM3, GD2, and GD3 in human plasma. This method
utilized a simple protein-precipitation procedure for rapid extraction of the gangliosides from
the matrix, UPLC for high resolution chromatographic separation, and multiple MRM
transitions for sensitive and specific mass spectrometric detection, which had been
successfully applied to determine the level of gangliosides in plasma from normal human
subjects. Considering the quickness, sensitivity, and specificity of this assay, we believe that
it is also applicable to other potential biomatrices with some appropriate modifications.
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