This document describes various in vitro models and methods that can be used to study hepatotoxicity, including hepatocyte cell cultures, assays to measure cell viability and metabolic activity (trypan blue dye exclusion test, MTT assay), staining to visualize lipid accumulation (Oil Red O), and techniques to examine gene and protein expression changes (RT-PCR, western blotting). Specifically, it discusses using these methods to establish models of non-alcoholic fatty liver disease (NAFLD) by treating hepatocyte cultures with fatty acids like palmitic and oleic acid, and models of drug-induced hepatotoxicity by treating with acetaminophen or amiodarone. Key readouts include lipid accumulation, apoptosis levels
This document summarizes various liver diseases and their etiologies. It discusses alcoholic liver disease, drug-induced liver injury, viral hepatitis infections from hepatitis B, C, and D viruses, autoimmune disorders like autoimmune hepatitis and primary biliary cirrhosis, genetic disorders, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. The liver's important functions are outlined. Causes, pathogenesis, clinical features, diagnosis, and treatment approaches are described for each disease.
An introduction to experimental epidemiology improvemed
This document provides an overview of experimental epidemiology methods. It discusses the key features and types of experimental epidemiology studies, including controlled field trials and community trials. Controlled field trials involve dividing healthy subjects into an exposed group that receives an active substance (like a vaccine) and an unexposed control group that receives a placebo. Community trials involve entire exposed and unexposed communities. Randomized controlled trials, which assign individual subjects randomly to intervention or control groups, are described as the most common experimental method but are covered in more depth separately. Overall, the document outlines the design and purpose of various experimental epidemiology study types.
Genotyping methods of nosocomial infections pathogenimprovemed
Nosocomial infections afflict around 2 million patients in the US each year, resulting in around 88,000 deaths and $4.5 billion in excess healthcare costs. Understanding the distribution and relatedness of pathogens that cause these infections is important for designing effective control methods. Historically, phenotypic characterization was used, but increasingly molecular or genotyping techniques are being used, including pulsed-field gel electrophoresis, multilocus sequence typing, and polymerase chain reaction-based methods. Studies have shown that integrating molecular typing into infection control programs can significantly reduce infection rates and healthcare costs.
Use of MALDI-TOF in the diagnosis of infectious diseasesimprovemed
MALDI-TOF MS has revolutionized clinical microbiology by drastically improving the time needed to identify bacterial cultures from over 24 hours to just a few minutes. Whereas the entire process from sampling to results previously took 2-3 days or more, new methods like MALDI-TOF MS and molecular technology have reduced this to just a few hours or one day. MALDI-TOF MS is a powerful, cost-effective, and easy to implement technique that provides rapid and reliable identification of bacteria and yeast from clinical samples at the genus and species level through analysis of their protein mass spectral signatures.
This document describes various in vitro models and methods that can be used to study hepatotoxicity, including hepatocyte cell cultures, assays to measure cell viability and metabolic activity (trypan blue dye exclusion test, MTT assay), staining to visualize lipid accumulation (Oil Red O), and techniques to examine gene and protein expression changes (RT-PCR, western blotting). Specifically, it discusses using these methods to establish models of non-alcoholic fatty liver disease (NAFLD) by treating hepatocyte cultures with fatty acids like palmitic and oleic acid, and models of drug-induced hepatotoxicity by treating with acetaminophen or amiodarone. Key readouts include lipid accumulation, apoptosis levels
This document summarizes various liver diseases and their etiologies. It discusses alcoholic liver disease, drug-induced liver injury, viral hepatitis infections from hepatitis B, C, and D viruses, autoimmune disorders like autoimmune hepatitis and primary biliary cirrhosis, genetic disorders, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. The liver's important functions are outlined. Causes, pathogenesis, clinical features, diagnosis, and treatment approaches are described for each disease.
An introduction to experimental epidemiology improvemed
This document provides an overview of experimental epidemiology methods. It discusses the key features and types of experimental epidemiology studies, including controlled field trials and community trials. Controlled field trials involve dividing healthy subjects into an exposed group that receives an active substance (like a vaccine) and an unexposed control group that receives a placebo. Community trials involve entire exposed and unexposed communities. Randomized controlled trials, which assign individual subjects randomly to intervention or control groups, are described as the most common experimental method but are covered in more depth separately. Overall, the document outlines the design and purpose of various experimental epidemiology study types.
Genotyping methods of nosocomial infections pathogenimprovemed
Nosocomial infections afflict around 2 million patients in the US each year, resulting in around 88,000 deaths and $4.5 billion in excess healthcare costs. Understanding the distribution and relatedness of pathogens that cause these infections is important for designing effective control methods. Historically, phenotypic characterization was used, but increasingly molecular or genotyping techniques are being used, including pulsed-field gel electrophoresis, multilocus sequence typing, and polymerase chain reaction-based methods. Studies have shown that integrating molecular typing into infection control programs can significantly reduce infection rates and healthcare costs.
Use of MALDI-TOF in the diagnosis of infectious diseasesimprovemed
MALDI-TOF MS has revolutionized clinical microbiology by drastically improving the time needed to identify bacterial cultures from over 24 hours to just a few minutes. Whereas the entire process from sampling to results previously took 2-3 days or more, new methods like MALDI-TOF MS and molecular technology have reduced this to just a few hours or one day. MALDI-TOF MS is a powerful, cost-effective, and easy to implement technique that provides rapid and reliable identification of bacteria and yeast from clinical samples at the genus and species level through analysis of their protein mass spectral signatures.
1. Molecular microbiology methods like PCR and hybridization have revolutionized clinical diagnostics by enabling fast and direct detection of pathogens from clinical samples.
2. PCR in particular has become a mainstay technique, allowing amplification of specific DNA sequences from small amounts of input DNA. Variations like real-time PCR, multiplex PCR, and broad-range PCR further expanded diagnostic capabilities.
3. Emerging technologies like DNA microarrays promise even greater multiplexing, with the ability to simultaneously genotype large genomic regions or measure expression of many genes, positioning them as promising future molecular diagnostic tools.
This document provides information about setting up and conducting experiments with isolated organs and tissue rings, including:
1. Describing the mechanical setup for a four-channel system bath for isolated organs.
2. Explaining the preparation of Krebs-Hanseleit solution and common drugs used.
3. Outlining typical experiment protocols, including stabilizing tissues, pre-contraction testing, and assessing endothelial function.
4. Noting that each experiment begins by preparing Krebs-Hanseleit solution and activating the system before surgery and setting rings in wells.
This document describes the components, work principles, and experimental protocols for using a pressure myograph system to study isolated blood vessels. The system allows measuring vessel diameter in response to drugs and stimuli while maintaining constant temperature. Experiments involve isolating small arteries from rats and attaching them to glass micropipettes in a chamber filled with physiological salt solution. Vessel diameter is recorded under varying pressures and drug exposures to study endothelial function and vasoactive mechanisms. Statistical analysis of diameter changes under different conditions uses repeated measures ANOVA to compare responses between experimental groups.
Notes for Measuring blood flow and reactivity of the blood vessels in the ski...improvemed
This document describes the laser Doppler flowmetry (LDF) method for measuring blood flow in the microcirculation of skin. Specifically, it discusses post-occlusive reactive hyperemia (PORH) testing using LDF to assess microvascular reactivity by inducing a brief occlusion of blood vessels. It also covers iontophoresis of acetylcholine and sodium nitroprusside combined with LDF to evaluate endothelium-dependent and independent vasodilation respectively. Standardization of methods like occlusion duration and probe placement is important for reproducibility. LDF provides a general index of microvascular function rather than direct flow measurements.
Notes for STAINING AND ANALYSIS of HISTOLOGICAL PREPARATIONSimprovemed
This document provides an overview of histological staining techniques. It discusses how histological preparations are stained using interactions between dyes, solvents, and tissue components. Different staining methods result in different colors that highlight various structures. A classic example is hematoxylin and eosin staining, where hematoxylin stains acidic components blue and eosin stains basic components pink. Specialized staining techniques also exist, such as immunohistochemistry. Proper staining selection depends on the tissue and research goals. Histological preparations are then analyzed under a microscope to study cell and tissue morphology.
Notes for Fixation of tissues and organs for educational and scientific purposesimprovemed
Fixation of tissues and organs is done to preserve them for scientific and educational purposes. Various chemical fixatives are used including formaldehyde, alcohols, and acids. Formaldehyde cross-links proteins to harden the tissue while maintaining the original structure. Several fixation protocols are used for different purposes, balancing preservation of color and long-term durability. Key steps include diffusion or injection of fixatives, followed by storage in preservative solutions. Proper fixation and storage are necessary to prevent degradation over time.
The document summarizes the process of preparing tissue samples for histological analysis, including fixation, dehydration, infiltration/embedding, sectioning, staining, and examination. Key steps involve fixing tissues to prevent degradation, dehydrating using increasing alcohol concentrations, infiltrating with paraffin wax or resin for structural support during sectioning, precisely cutting thin sections, mounting them to glass slides, staining, and examining under a microscope. The quality of prepared samples depends on carefully following each step of the preparation process.
Notes for The principle and performance of capillary electrophoresisimprovemed
This document provides an overview of capillary electrophoresis (CE). It begins by introducing CE and its advantages over other separation techniques. It then describes the basic theory behind CE, including electrophoretic mobility, electroosmotic flow, and how samples migrate through the capillary when an electric field is applied. The document details the key components of a CE instrument and various CE separation techniques such as capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary isoelectric focusing. It focuses on the principles and applications of CE.
Notes for The principle and performance of liquid chromatography–mass spectro...improvemed
This document provides an overview of liquid chromatography-mass spectrometry (LC-MS). It describes the basic components and functioning of an LC-MS system, including the liquid chromatograph and mass spectrometer connected by an interface. The document discusses various ionization sources like electrospray ionization and atmospheric pressure chemical ionization, as well as mass analyzers like quadrupoles and time-of-flight analyzers. It also covers detectors used in LC-MS like electron multipliers and photomultipliers. Overall, the document serves as a technical introduction to the principles and components of LC-MS.
This document provides an overview of basic cell culture techniques. It discusses the history of cell culture, defining primary and secondary cell cultures. It describes different types of cell lines and how cells grow as monolayers or in suspension. The document outlines the key equipment needed for a cell culture laboratory, including biosafety cabinets, CO2 incubators, centrifuges, microscopes, and supplies. It emphasizes the importance of aseptic technique to prevent microbial contamination when working with cell cultures.
This document discusses systems biology and its goals of understanding how biological molecules interact and systems function as a whole. It covers:
1) Systems biology uses large datasets from "omics" experiments and computational models to understand complex biological interactions beyond individual molecules.
2) Pioneering work used microarrays to measure thousands of genes in serum-stimulated cells, finding over 500 changed in proliferation.
3) The field aims to discover emergent system properties and functions not evident from separate parts, like switches that change cell behavior.
Systems biology for Medicine' is 'Experimental methods and the big datasetsimprovemed
This document discusses experimental methods used in systems biology to generate large datasets, including microarrays, sequencing-based methods, mass spectrometry, and liquid chromatography. It explains that systems biology studies must be quantitative and enable computational modeling. Key methods covered are microarrays, RNA-seq, ChIP-seq, whole-genome sequencing, whole-exome sequencing, proteomics using mass spectrometry, and combining liquid chromatography with mass spectrometry for lipidomics, metabolomics and glycomics. Sources of variation are also discussed for genomic and proteomic studies.
Systems biology for medical students/Systems medicineimprovemed
Systems biology takes a holistic approach to studying biological systems by considering all the interactions within a system and how they generate complex behaviors. Lecture 1 introduces key concepts in systems biology like how increasing levels of biological organization give rise to new system properties like robustness. Lecture 2 discusses experimental methods like genomics, proteomics, and metabolomics that generate large data sets for systems analysis. Lecture 3 covers mathematical and statistical tools for analyzing these data sets, such as using differential equations to model signaling networks. Lecture 4 provides examples of medical applications of systems biology in finding diagnostic markers, personalizing therapy, and predicting disease interactions from human disease networks, with the future of medicine taking a more predictive, preventive, and personalized approach
The document discusses several use cases for applying data mining and machine learning techniques in healthcare and biomedical research. Three examples are:
1) Early diagnosis of cancers like lung cancer and breast cancer through predictive modeling of patient data to detect cancers at earlier stages when survival rates are higher.
2) Predicting patient responses to drug therapies for cancers like breast cancer by combining different types of molecular profiling data using techniques like support vector machines and random forests.
3) Using imaging data and temporal analysis of metrics like medication purchases to better understand and predict chronic diseases like diabetes and associated health complications.
1. Improved Medical Education in Basic
Sciences
for Better Medical Practicing
ImproveMEd
Sistemska biologija za medicinu
II. Eksperimentalne metode i skupovi velikih podataka
2. Eksperimenti u sistemskoj biologiji ne moraju biti omički razmjeri kako bi zadovoljili kriterije biologije
sustava!
Razmotrite eksperimente usmjerene na podsustave kao što su npr. praćenje mRNA energetskog
metabolizma pod različitim režimom hranjenja, ili u više vremenskih točaka od početka hranjenja.
Koja se biologija sustava razlikuje od uobičajenih znanstvenih istraživanja:
1. Kvantitativni podaci - gdje, koliko i koliko brzo (dinamički, ovisnost o vremenu) će se entitet promijeniti
2. Računalni modeli - simulacije temeljene na preciznoj kvantifikaciji i vremenu
Još uvijek su potrebne pozitivne i negativne kontrole i najmanje 3 ponavljanja!
2. Hipoteze pokrenute studije koje
prate ciljani podskup molekula (ili ciljanih
organela) - biologija sustava malih
razmjera.
1. Hypothesis generating studies!
3. molekule:
• DNAs tehnologije s mikroračunalima i sekvenciranjem
• RNAs mRNA sequencing
• proteini Proteomika na bazi masene spektrometrije
• lipidi tekućinska kromatografija i masena spektrometrija
• metaboliti tekućinska kromatografija Masena spektrometrija
4. Prosječne populacijske tehnike
• Populacija stanica ili uzorak tkiva
≈ 1 milijun stanica ili više
• Prosječno u mnogim stanicama
Tehnike pojedinačnih stanica
• Jedan uzorak stanica
• Varijabilnost stanica-stanica
HepaRG stabilna stanična linijaTkivo jetre Sferoidi hepatocita
5. Mikropostrojima -
diferencijalni izraz
• Dominantna tehnika 2000-ih
• Uglavnom se koristi za mjerenje razine transkripta
• Druge namjene: genotipizacija, DNA mapiranje
(variranje broja kopija), metilacija DNA
• Microarray se sastoji od mjesta otisnutih različitim
oligonukleotidom
• Hibridizacija između fluorescentno obilježenog uzorka
(probe) i tiskanih oligonukleotida
• GEO baza podataka
https://www.ncbi.nlm.nih.gov/geo/info/qqtutorial.ht
ml
6. Array CGH usporedna genomska
hibridizacija
• molekularna citogenetska metoda /
kariotipizacija
• CNV relativan za ispitni uzorak
• Na temelju pretpostavke da se 2 uzorka blisko
povezanih osoba razlikuju (zdravi i bolesni) u
dobitku ili gubitku kromosoma ili kromosomske
regije
• Analiza velikog opsega tumor-specifičnih DNK
neuravnoteženih pregrada s rezolucijom od 5-10
megabaza
• OMIM baza podataka
https://www.ncbi.nlm.nih.gov/omim
7. Microarrays analiza metilacije -
epigenome
• Epigenetska regulacija ekspresije gena važna u razvoju, genetski otisak,
tumorogeneza ...
• Razina metilacije u genomu CpG na 30 000 do 500 000 CpG lokusa pokriva
cijeli genom
• Prvi korak je bisulfitna konverzija uzoraka - nemetilirana mjesta pretvaraju C u
U, dok metilirani gubitak metilacije
• Pretvorena DNA se dalje pojačava (i U se razmjenjuje za T)
• Fragmentirani i denaturirani oligonukleotidi su hibridizirani s dvije vrste alel
specifičnih zrnaca za svako mjesto
• Konačni nukleotid aniliranog alel specifičnog oligonukleotida se dalje proširuje
obilježenim dDNT
• Softver izračunava relativnu fluorescenciju svakog lokusa i razreda kao 0, 0,5 ili
1 (homozigotni nemetilirani, heterozigotni ili homozigotni metilirani)
• ENCODE
https://genome.ucsc.edu/encode/dataMatrix/encodeChipMatrixHuman.html
8. Tehnologije utemeljene na
sekvenciranju
• Počinje s izolacijom DNA ili RNA nakon čega
slijedi ili DNA fragmentacija ili sinteza cDNA
• Sljedeći korak je amplifikacija (fragmenti
klonova u vektor, transformiranje bakterija s
vektorima i amplifikacija)
• Paralelno sekvenciranje mnogih kratkih
dijelova DNA
• Sastavljanje susjednih fragmenata
9. Whole-genome sequencing (WGS)
vs. whole-exome sequencing (WES)
• WGS pokušava slijediti cijeli genom. Neke sekvence su
tehnički izazovne za sekvencu (telomere, centromeres,
visoki CG sadržaj ili ponavljajući lokusi) i propustile su
ih uobičajene platforme za sekvenciranje što rezultira
95-98% pokrivenosti genoma, ali na uniformiran način.
• WES slijed samo eksome (kodirajuće sekvence) ili 2%
genoma, pomoću svakog egzoma je sekvenca 30-100x
(visoka dubina). DNA-RNA hibridizacija koristi se za
odabir kodirajućih regija što uzrokuje prekomjernu
zastupljenost takozvanih "vrućih točaka" i nedovoljnu
reprezentaciju propuštenih varijanti. Brža, sitnija i
jednostavnija analiza podataka, ali niska ukupna
pokrivenost.
uniform
bias
10. Sekvenciranje exomea koristi korak
obogaćivanja za odabir ciljane DNA
• Mendelski poremećaji često narušavaju regije koje
kodiraju proteine
• Exom je dobar izvor varijanti rijetkih bolesti
• Mikroarije se mogu koristiti za izolaciju fragmenta
• WES daje visoku dubinu sekvenciranja
• Bamshat i sur. (2011) Pregled prirode Genetika
11. Trošak po genomu je pobijedio u utrci, a
WGS postaje poželjna metoda, posebno
za pregradnje specifične za tumor
Evolucija metoda sekvenciranja
• Prva generacija Sanger sekvenci; tehnologija
završetka lanca - ddNTPs koji se koriste za
prekidanje sinteze lanca dalje odvojene
kapilarnom elektroforezom (jedna traka u isto
vrijeme, spora, točna, skupa)
• Drugi (sljedeći) niz sekvenciranja -
sekvenciranje sintezom - uvođenje nano-
tehnologije - paralelno sekvenciranje i nema
potrebe za korak razdvajanja - granice u
dužini čitanja
• Sekvenciranje treće generacije - imobilizirana
polimeraza + fluorescentna dNTP + izvrsna
optika - detekcija ugradnje baze i modifikacija
baze
12. Podaci koji dolaze iz WGS-a ili WES-
a zahtijevaju provjeru preko velike
populacije! (više od 1000
pojedinaca)
Baza podataka o genotipovima i
fenotipovima (dbGaP)
https://www.ncbi.nlm.nih.gov/gap
Studije udruživanja u genomu
Foo et al. (2012) Nature Review Neurology
13. TranskriptomRNA-Seq
• Paralelno sekvenciranje mRNA, rRNA, miRNA ...
• Profil ekspresije gena
• Kvantitativna metoda idealna za eksperimente
biologije sustava
• Identifikacija alternativnih varijanti spajanja i nove
varijante transkripta
• GEO baza podataka
https://www.ncbi.nlm.nih.gov/geo/info/qqtutorial.htm
l
• Ciljne baze podataka skeniranja (miRNA)
• http://www.targetscan.org/vert_71/
Wang et al.(2009) Nature Reviews Genetics
14. RNA-seq postaje dominantna metoda
transkriptoma i zamjenjuje mikromreže
Wang et al.(2009) Nature
Reviews Genetics
15. ChIP-seq kromatinsko
imunoprecipitacijsko sekvenciranje
• Kombinira taloženje transkripcijskih faktora ili
drugih DNA vezujućih proteina (koristeći
specifična antitijela) ili histonske modifikacije
i duboko sekvenciranje ko-
imunoprecipitirane DNA
• Otkriva regulatorne sekvence, promotore,
pojačivače, prigušivače, razmaknice
• Funkcionalna organizacija genoma
16. Western blot je prva metoda prema
kvantifikaciji i identifikaciji više od jednog
proteina
• Polu-kvantitativna zbog nelinearne kinetike
iza enzimskog obilježavanja sekundarnih
antitijela i reakcije supstrata
• Također ima nelinearnu kinetiku u slučaju
kemiluminescentne reakcije ili ekspozicije X-
zraka
• LICOR je infracrvena fluorescencija koja daje
malo pozadine i fluorescentni signal je
linearno proporcionalan količini antitijela
• https://www.licor.com/bio/applications/quan
titative_western_blots/
17. Napredna i obrnuta fazna
proteinska matrica (FPPA i RPPA)
• Pokušaj transformacije Western blot metoda niske
propusnosti (8-16 traka za uzorke) u metodu visokog
protoka
• U verziji s prednje strane jedan se antibiotik nalazi na
slajdovima, a uzorci griva su ispitani na prisutnost
epitopa
• U obrnutoj verziji, mnoga antitijela (s visokom
specifičnošću) su uočena na slajdu i jedan uzorak je
ispitan za sva antitijela.
• Zahtijeva antitijela visoke specifičnosti, skupa.
18. Masovna spektrometrija
proteomics, lipidomics,
metabolomics
• Proteomika često koristi tandemsku masenu spektrometriju
(dvije masovne specifikacije izvedene paralelno)
• Kvantitativno zato što mjeri ukupnu razinu proteina
• Daje pojedinosti o post-translacijskim modifikacijama
• Ako se kombinira s imunoprecipitacijom daje informacije o
interakcijama proteina
• Uključuje mnoge korake: odvajanje, probavu, obogaćivanje,
ponavljanje razdvajanja, ionizaciju, filtriranje mase (MS1),
analizu fragmentacije i mase (MS2), identifikaciju,
kvantifikaciju
• Mnoge varijacije
19. Masovna spektrometrija
proteomics, lipidomics,
metabolomics
Završni korak temelji se na velikoj bazi
podataka poznatih peptida i bioinformatskih
potraga za šibicama.
Za lipidomiku i metabolomiku koriste se
različite verzije MS i različite baze podataka.
UniProtKB baza podataka
https://web.expasy.org/docs/swiss-
prot_guideline.html
20. Analiza proteomike dekodira razlike između MAPK puta u
normalnoj i tumorskoj stanici.
Choudhary & Mann (2010) Nature Reviews Molecular and Cell Biology
21. proteomics, lipidomics,
metabolomics
Završni korak temelji se na velikoj bazi
podataka poznatih peptida i bioinformatskih
potraga za šibicama.
Za lipidomiku i metabolomiku koriste se
različite verzije MS i različite baze podataka.
UniProtKB database
https://web.expasy.org/docs/swiss-
prot_guideline.html
22. Tekuća kromatografija (LC) i LC / MS
lipidomics, metabolomics
Palermo et al. (2017) Analytica Chimica Acta
Vrlo često se LC & MS kombiniraju i
koriste u slijedu. Također, lipidomska
i metabolomika mogu se vršiti u nizu
na istim uzorcima.
LC se koristi za dijeljenje uzorka u
frakcijama, kao tehnika razdvajanja,
dok se MS koristi za identifikaciju
molekula.
23. Eksperimenti biologije sustava prikupljaju
velike podatke koristeći metode visoke
propusnosti :
• DNAs Tehnologije temeljene na mikročipovima i sekvenciranju (NGS) exome / genome
• DNA + regulatory proteins ChIP-seq ReMap
• RNAs RNA-seq transcriptome
• Proteins MS proteome
• Lipids LC/MS lipidom
• Metabolites LC/MS metabolom