This document discusses non-invasive methods for diagnosing acute rejection in renal transplant patients. Currently, graft biopsy is the gold standard but it is invasive and detects rejection at a late stage. The presentation evaluates various potential biomarkers being studied through genomics, proteomics, and metabolomics approaches. Several individual studies are highlighted that found biomarkers like urinary MCP-1, VEGF, cytokines, and certain metabolites that showed potential for detecting early acute rejection non-invasively. Magnetic resonance imaging is also discussed as a non-invasive imaging technique being researched. In summary, the ideal non-invasive biomarker has yet to be identified but a combination of markers may provide a more realistic approach for different clinical scenarios.
Quantitative Analysis of 30 Drugs in Whole Blood by SPE and UHPLC-TOF-MSAnnex Publishers
Abstract
An Ultra-High Pressure Liquid Chromatography Time-of-Flight Mass Spectrometry (UHPLC-TOF-MS) method for quantitative analysis of 30 drugs in whole blood was developed and validated. The method was used for screening and quantification of common drugs and drugs of abuse in whole blood received from autopsy cases and living persons. The compounds included: alprazolam, amphetamine, benzoylecgonine, bromazepam, cathine, cathinone, chlordiazepoxide, cocaine, codeine, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, flunitrazepam, 7-aminoflunitrazepam, ketamine, ketobemidone, 3,4-Methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, methadone, morphine, 6-monoacetylmorphine, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, tramadol, O-desmethyltramadol, and zolpidem. Blood samples (200 μL) were subjected to Solid Phase Extraction (SPE). Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2 TOF-MS apparatus. Extraction recoveries ranged from 41% (7-aminoclonazepam) to 111% (ketamine) and matrix effects ranged from -13% (temazepam) to 50% (7-aminonitrazepam). For all compounds, a quadratic polynomial was applied for fitting the calibration curves. Lower Limits of Quantification (LOQ) ranged from 0.005 to 0.05 mg/kg. Satisfactory precisions below 15% and accuracies within 85-115% were obtained for all compounds at concentrations exceeding the LOQ. In conclusion, we present a validated UHPLC-TOF-MS method for simultaneous quantification of 30 drugs in whole blood with a run time of 15 min using 200 μL of whole blood.
Keywords: Drugs of abuse, UHPLC-TOF-MS, Whole blood, SPE, Quantification
Quantitative Analysis of 30 Drugs in Whole Blood by SPE and UHPLC-TOF-MSAnnex Publishers
Abstract
An Ultra-High Pressure Liquid Chromatography Time-of-Flight Mass Spectrometry (UHPLC-TOF-MS) method for quantitative analysis of 30 drugs in whole blood was developed and validated. The method was used for screening and quantification of common drugs and drugs of abuse in whole blood received from autopsy cases and living persons. The compounds included: alprazolam, amphetamine, benzoylecgonine, bromazepam, cathine, cathinone, chlordiazepoxide, cocaine, codeine, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, flunitrazepam, 7-aminoflunitrazepam, ketamine, ketobemidone, 3,4-Methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, methadone, morphine, 6-monoacetylmorphine, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, tramadol, O-desmethyltramadol, and zolpidem. Blood samples (200 μL) were subjected to Solid Phase Extraction (SPE). Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2 TOF-MS apparatus. Extraction recoveries ranged from 41% (7-aminoclonazepam) to 111% (ketamine) and matrix effects ranged from -13% (temazepam) to 50% (7-aminonitrazepam). For all compounds, a quadratic polynomial was applied for fitting the calibration curves. Lower Limits of Quantification (LOQ) ranged from 0.005 to 0.05 mg/kg. Satisfactory precisions below 15% and accuracies within 85-115% were obtained for all compounds at concentrations exceeding the LOQ. In conclusion, we present a validated UHPLC-TOF-MS method for simultaneous quantification of 30 drugs in whole blood with a run time of 15 min using 200 μL of whole blood.
Keywords: Drugs of abuse, UHPLC-TOF-MS, Whole blood, SPE, Quantification
The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence. It uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra.This method can obtain the peptide sequences without a protein database. It can be used for un-sequenced organisms, antibodies, peptides with posttranslational modifications, and endogenous peptides.
Application of proteomics for identification of abiotic stress tolerance in c...Vivek Zinzala
It is the study of “Proteome”.
The word "proteome" is a blend of "protein" and "genome”.
Large scale study of Proteins.
Particularly their structures and functions.
Study of full set of proteins in a cell type or tissue, and changes during various conditions
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence. It uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra.This method can obtain the peptide sequences without a protein database. It can be used for un-sequenced organisms, antibodies, peptides with posttranslational modifications, and endogenous peptides.
Application of proteomics for identification of abiotic stress tolerance in c...Vivek Zinzala
It is the study of “Proteome”.
The word "proteome" is a blend of "protein" and "genome”.
Large scale study of Proteins.
Particularly their structures and functions.
Study of full set of proteins in a cell type or tissue, and changes during various conditions
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
In this webinar Dr. Bertrand Rochat of Faculté de Biologie et de Médecine of the Centre Hospitalier Universitraire Vaudois (CHUV) at Lausanne discusses the paradigm shift to high resolution mass spectrometry (HRMS) in clinical research for quantitative analyses (sensitivity, selectivity, etc.). Quantifications in high resolution full scan or MS/MS mode will be compared with triple quadrupole MS. He will present Quan/Qual analysis with a study on the fate of an anti-cancer agent in human: with over 40 metabolites being identified and quantified; as well as metabolomics data underscoring the versatility of high resolution Orbitrap MS.
Anti-Mullerian hormone (AMH) is a glycoprotein, a member of the transforming growth factor-B super family. This hormone is a sensitive marker of ovarian reserve. The present study aims to measure the Anti-Mullerian hormone in thalassemic females receiving the regular blood transfusion as well as patients of chronic idiopathic thrombocgtopenic purpura and age and sex matched controls. Serum Anti-Mullerian hormone was measured by ELISA and Ferritin were measured by RIA. Clinical evaluation was done for all patients including anthropometric measurements, pubertal staging and history taking. Results of the study were analyzed by appropriate statistical methods. Obtained results revealed that the values of Body Mass Index as well as Anti-Mullerian were significantly higher in controls than thalassemics and chronic idiopathic thrombocytopenic purpura and there was a negative correlation between serum Ferritin and Anti-Mullerian hormone. Moreover, Anti-Mullerian hormone was significantly higher in patients receiving Desferal than in those receiving Deferriprone. Reduced Anti-Mullerian hormone in thalassemics as well as chronic ITP patients are considered an important indicator declines in ovarian function which entail modification in the therapeutic plans for thalassemic and chronic ITP patients.
dkNET Webinar: Leveraging Computational Strategies to Identify Type 1 Diabete...dkNET
dkNET New Investigator Pilot Program in Bioinformatics Awardee Webinar Series
Presenter: Wenting Wu, PhD. Research Assistant Professor, Center for Diabetes and Metabolic Diseases, Department of Medical and Molecular Genetics, Associate Director of Data and Analytics Core for Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine
Abstract
Type 1 diabetes (T1D) is an immune-mediated disease that results in insulin insufficiency and affects 0.3% of the population, including both children and adults. To support clinical trial efforts, there is an urgent need to develop reliable biomarkers capable of predicting T1D risk and guiding therapeutic interventions. Recently, whole blood bulk RNA sequencing has been used to guide T1D clinical trial design and assess response to disease modifying interventions. While the use of bulk RNA sequencing is cost-effective, these datasets provide limited information about cell specific gene expression changes. Here, we aimed to apply computational strategies to deconvolute cell type composition using cell specific gene expression references. Single-cell RNA sequencing (scRNA-seq) was conducted to profile peripheral blood mononuclear cells obtained from youth within recent T1D onset and age- and sex-matched controls and identified 31 distinct cell clusters. Using this pre-defined reference dataset, we ran computational algorithms CIBERSORTx and other deconvolution methods simultaneously to deconvolute cell proportions using public clinical trial data. We focused our initial analysis on data from the TN-20 Rituximab trial, which tested the anti-CD20 monoclonal antibody rituximab vs placebo in recent onset T1D. This talk will introduce recent advances of scRNA-seq techniques and computational deconvolution methods and demonstrate that how we apply different deconvolution approaches for secondary analysis of existing clinical trial data, in the purpose of linking cell specific immune signatures associated with drug responder status.
Upcoming webinars schedule: https://dknet.org/about/webinar
Robert P. Edwards, MD, Chair of OB/GYN/RS, Co-Director of Women's Cancer Program at University of Pittsburgh, offers information about the current state of immunotherapy for recurrent ovarian cancer patients.
L-arginine modulates T cell metabolism and enhances survival and anti-tumor A...Gul Muneer
L-arginine modulates T cell metabolism and enhances survival and anti-tumor Activity
How metabolic reprogramming of T cells impact T cells fate and function? Indeed, these cells enhance their metabolic throughput upon antigenic stimulation to ensure the production of sufficient biomass and energy for their proliferation, differentiation and effector functions. The naïve T cells, which utilize oxidative phosphorylation for energy supply but upon activation, these cells switch their metabolic pathways toward glycolysis. An important question arises here that whether differential metabolic reprogramming (i.e., use of oxidative phosphorylation versus glycolytic activity) directs the fate and function of a given cell? And whether this process shapes the immune response and individual cell survival? The researchers from Institute for Research in Biomedicine took advantage of high-resolution Mass Spectrometry to investigate dynamics of proteome and metabolome following activation of naïve T cells.
Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2QIAGEN
Advanced prostate cancer is highly heterogeneous but this inter-patient heterogeneity has until recently not been understood. We have through an international research effort dissected the molecular landscape of advanced castration resistant prostate, elucidating key molecular targets in this group of diseases. We have also shown that PARP inhibitors have antitumor activity against a significant proportion of these cancers, mainly in men whose cancers harbor DNA repair defects.
EVALUATION OF SERUM LEVELS OF FASTING LIPID PROFILE IN PRE-ECLAMPTIC WOMEN
Wuraola Serah Nnaemeka, Olisekodiaka, MJ, Onuegbu, AJ, Ezeugwunne, IP, Maduka, IG, Suru, SM , Johnkennedy Nnodim
IRO INTERNATIONAL JOURNAL OF MEDICAL AND APPLIED SCIENCES 2018, 1(1):20-23.
Similar to Non biopsy diagnosis of acute rejection of Renal allograft (20)
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
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We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journey
Non biopsy diagnosis of acute rejection of Renal allograft
1. Non biopsy diagnosis of
acute rejection
Presenter: Dr Bakshish Singh
Preceptor: Prof SK Agarwal
2. Introduction
Acute rejection key factor for long-term graft
function and survival in RT patients
Timely detection and treatment of rejection an
important goal.
The standard care with S.Creat measurements and
biopsy usually detects AR in an advanced stage
Hence the need for Non invasive markers that can
detect AR at an earlier stage.
2
3. Current Approach
Clinical signs
Decreased urine output
Graft tenderness
Hypertension
USG
Serial USG with echo enhancers
S creatinine
Greater than 15% rise suspicious
Biopsy
GOLD STANDARD
3
4. Graft biopsy
Graft biopsy currently
the GOLD STANDARD
for diagnosis of acute
rejection.
Criteria for AR defined
by consensus (Updated on
regular intervals)
Can pick up other causes
of graft dysfunction
Invasive
Subjective variability
May be non
representative
Timing of biopsy
Detects injury at a late
stage
4
5. Acute Rejection
Heterogenous
Involved structures
Mechanism
Clinical presentation
Severity
Time related
Immunosuppression
ALL THE ABOVE AFFECT THE BIOMARKERS
5
8. 8
Given the the heterogeneity and complexity of AR,
it is unlikely that one marker will fit all facets of
this process.
Different markers may detect incipient, fully
established and resolving stages of rejection.
Different rejection types (T-cell vs. antibody-
mediated, tubulo-interstitial vs. vascular) may
have distinct sets of markers.
Further, quantifiable markers could give additional
information about the severity of rejection
24. Why study proteins
Proteins, rather than nucleic
acids, mediate most of the
physiologic functions within the
cell.
Analysis of body fluids such as
urine can only be accomplished
by proteomics approaches
because nucleic acids play no
direct functional role in
extracellular fluids
Proteomics can be viewed as
being complementary to the
area of functional genomics
24
25. The common element of proteomics studies is
“Multiplexing”
The simultaneous study of multiple proteins rather than one
protein at a time (as in traditional biochemistry)
Most proteomics-based investigations focus on defined
subpopulations of proteins
Set of all proteins found in urine or blood plasma,
Set of proteins present in endosomes from collecting duct principal
cells
Set of all proteins that form complexes with the proximal tubule Na-H
exchanger NHE3
Set of all Na transporters expressed along the renal tubule
Set of all renal cortical proteins detectable in two-dimensional gels
25
28. Two broad areas
Mass spectrometry to
detect and identify
proteins and
Approaches using
arrays or ensembles of
binding molecules to
detect and identify
proteins using
antibodies as the
binding molecules
MS: Initial separation
technique by one of the
following followed by
identification
2 D Electrophoresis
Liquid chromatography
Surface-enhanced laser
desorption/ ionization
Capillary electrophoresis
Protein microarrays
28
30. Mass spectrometry
Two basic approaches using trypsin
digest
Peptide mass finger printing usually
employing matrix-assisted laser
desorption and ionization–time of- flight
(MALDI-TOF) mass spectrometers
Peptide sequencing using tandem mass
spectrometers
30
32. A. Diagram of a
quadrupole/time-of-flight
(Q-TOF) tandem mass
spectrometer. Peptide
ionization uses the
electrospray method B. Peptide sequencing using tandem mass spectrometry.
Y- series of peaks is derived from sequential elimination
of amino acids from the amino terminus of the peptide
by collision-induced dissociation. Differences in masses
between successive peaks correspond to residue masses
of individual amino acids. For example, the difference
between y12 and y11 peak is 115.03 Daltons
corresponding to aspartate (D). Difference between y11
and y10 is 113.08 Daltons corresponding to leucine (L).
32
38. Metabolomics
DEFINITION
Dresdale coined this term in the year 2000.
Also known as metabonomics or metabolic
profiling
High-throughput identification and
quantification of the small molecule (1,000 Da)
metabolites in the metabolome
Meatabolome
Collection of all small molecule metabolites
(endogenous or exogenous) that can be found in
a cell, organ or organism
38
39. This is possible due to
High-resolution mass spectrometry (MS) instruments for
precise mass determination
High-resolution, high-throughput nuclear magnetic
resonance (NMR) spectrometers for accelerated
compound identification
Capillary electrophoresis, high-pressure liquid
chromatography (HPLC), and ultra-high pressure liquid
chromatography systems for rapid compound separation
Software programs to rapidly process spectral or
chromatographic patterns
39
40. Approaches to metabolomics
Two Approaches
Chemometrics
compounds are not formally identified
spectral patterns and intensities are recorded, compared
and used to make diagnoses, identify phenotypes or draw
conclusions
Targeted profiling
compounds are formally identified and quantified
Being preferred these days
Human Metabolome Database available online
40
42. Most associated with generic metabolic processes
glycolysis
gluconeogenesis
lipid metabolism
Changes in relative concentrations of certain
‘universal’ metabolites such as glucose, citrate,
lactate, alpha-ketoglutarate and others can
reflect changes in
cell viability (apoptosis),
levels of oxygenation (anoxia, ischemia, oxidative stress)
local pH
general homeostasis
42
43. Methyl-histidine, creatine, taurine and glycine
reflect the extent of tissue repair or tissue
damage
Trimethylamine-N-oxide (TMAO) act as buffer to
stabilize serum proteins from the effects of
accumulated waste product
EACH PRODUCT TELLS A UNIQUE STORY
43
44. Transplanted, dysfunctional, rejected
kidneys show increased
Urine and serum concentrations of TMAO
Organic amines : trimethylamine & dimethylamine
Amino acids : glycine & alanine
Nephrotoxins : hippuric acid & uric acid in serum
Nitric oxide synthase inhibitors : phenylacetic acid &
dimethylarginine in the serum
Markers of Kreb cycle distress: lactate, acetate, succinate,
citrate and urea in the serum & urine
44
45. Rat models
Proximal straight
tubules injury (via the
toxin D-serine)
Increased lactate,
phenylalanine,
tryptophan, tyrosine
and valine
Straight tubule injury
Reduced levels of
methylsuccinic, sebacic
and xanthurenic acid
Proximal convoluted
tubule injury (via the
toxin gentamicin)
Elevated levels of urinary
glucose
Reduced levels of TMAO,
xanthurenic acid and
kynurenic acid
45
46. Papillary and medullary
injury (via
bromoethaneamide)
Increased urinary
concentrations of glutaric
acid, creatine and adipic
acid
Reduced levels of citrate,
succinate, oxoglutarate
and TMAO
Cortical damage
(induced via mercuric
chloride)
Increased urinary
glucose, alanine, valine,
lactate, hippurate
Decreased citrate,
succinate and
oxoglutarate
46
48. Metabolomics may well be the most useful
biomarkers for monitoring kidney function and
detecting adverse renal events.
This is because the kidney is specifically designed to
concentrate or filter small molecule metabolites and
small molecule toxins.
As a result, one would expect changes in metabolite
levels in blood or urine to be more detectable and
reflective of kidney function than subtle changes to
the kidney proteome or transcriptome
48
53. Initial Study : Rapid multi component analysis of
low molecular weight compounds in urine
The patterns of metabolic changes associated
with early renal allograft dysfunction
Increased urinary levels of trimethylamine-N-
oxide (TMAO), dimethylamine (DMA), lactate,
acetate, succinate, glycine and alanine during
episodes of graft dysfunction
53
54. 100 pts: AR (n=35) Stable graft fxn (n=65)
Urinary sediments by cytospin
Stained with anti-CD3, anti-CD64, anti-HLA-DR labeled monoclonal
antibodies.
Urinary expression of MCP-1 was assayed by ELISA
AR :
MCP-1 level was ten-fold higher
The number of CD3+ cells was over 5 times higher
The number of HLA-DR+ cells was 6 times higher
CD3+, HLA-DR+ and CD64+ cell counts strongly correlated with urine
excretion of MCP-1.
54
57. Urinary concentration of VEGF was determined by an ELISA
in 215 renal allograft recipients and 80 healthy controls
AR pts higher urinary excretion of VEGF
Urinary VEGF/creatinine ratio of 3.64 pg/mmol cut-off
point: Sensitivity and specificity for diagnosing acute
rejection were 85.1 and 74.8%, respectively
SRAR had significantly greater urinary VEGF concentration
than patients with SSAR
Urinary VEGF/creatinine ratio of 22.48 pg/mmol cut-off
point: sensitivity and specificity of the prediction to graft
loss after AR were 85.7% and 78.3%, respectively
57
60. 60
61 patients studied, 13 had no rejection
episodes, 8 had a proven acute
rejection, and 40 were excluded for
graft dysfunction causes
Mitogen induced peripheral blood
mononuclear cells tested for IL-2, IL-
4, IL-5, IL-6, IL-10, IL-15, IFN-g,
Perforin, Granzyme B, and Fas L using
(RT-PCR)
IL-4, IL-5, IL-6, IFN-g, Perforin, and
Granzyme B mRNA levels were
associated with AR
Using 2 markers 75% pt with AR
identified
80. Summary
Non invasive diagnosis of
acute rejection DESIRABLE
GOAL
Biomarker Development in
early stages
Ideal Biomarker still to be
found
Possibly combination markers
for different scenario more
realistic
Genomics, proteomics &
metabolomics complimentary
approach appears promising
80