Background: In October 2010 an outbreak of staphylococcal food poisoning occurred in an elementary school with a kindergarten in Gorenjska. Methods: Using a questionnaire we performed a retrospective ...cohortanalytical epidemiological study. We calculated attack rate (AR) and relative risk for each food item. In patients we cultivated stools and vomit. We performed an onsite audit where the food samples, environmental samples, and swabs of the hands of the cooks were taken. We performed medical examination of those employed in the kitchen where specimens were taken for microbiological examination. Staphylococcal enterotoxins were detected with agglutination test SET RPLA (Oxoid) and genotypes determined by the pulsed-field gel electrophoresis (PFGE). Results: Out of 374 exposed 73 subjects got ill. Attack rate in the outbreak was 19,5 %. The most frequent symptoms were vomiting (87,7 %), stomach ache (75,3 %), diarrhea (64,2 %) and nausea ( 59,3 %). The highest relative risk (RR) were found for the meatloaf (RR= 24,2 (95 % CI 12,1-48,5; p<0,001)) and potato salad (RR= 19,4 (95 % CI 10,7 - 35,2; p<0,001)). Enterotoxin A producing S. aureus was isolated from hand of a cook, from potato salad and meatlof, from vomit and stools of patients. These strains had the same antibiotic sensitivity and were genetically closely related (96,3 %). Conclusions: In the article an optimal team approaches of a community epidemiologist and microbiologist in the occasion of outbreak are described. We confirmed epidemiologically related isolates and determined the importance of individual methods.
U ovom se radu raspravlja o nekim ograničenjima i pitanjima vezanim uz trenutačno popularnu proteomičku analizu, te o prednostima uporabe jednodimenzionalne elektroforetske analize proteoma u ...agronomiji. Jednodimenzionalna elektroforeza prednjači nad dvodimenzionalnom, jer je statistički točnija i ekonomičnija. Uzimanjem većeg broja uzoraka može se smanjiti pogreška u rezultatima, koja je posljedica prirodne varijabilnosti u agrosustavima.
Proteome analysis has emerged as a powerful tool to decipher (patho) physiological processes, resulting in the establishment of the field of clinical proteomics. One of the main goals is to discover ...biomarkers for diseases from tissues and body fluids. Due to the enormous complexity of the proteome, a separation step is required for mass spectrometry (MS)-based proteome analysis. In this review, the advantages and limitations of protein separation by two-dimensional gel electrophoresis, liquid chromatography, surface-enhanced laser desorption/ionization and capillary electrophoresis (CE) for proteomic analysis are described, focusing on CE-MS. CE-MS enables separation and detection of the small molecular weight proteome in biological fluids with high reproducibility and accuracy in one single processing step and in a short time. As sensitive and specific single biomarkers generally may not exist, a strategy to overcome this diagnostic void is shifting from single analyte detection to simultaneous analysis of multiple analytes that together form a disease-specific pattern. Such approaches, however, are accompanied with additional challenges, which we will outline in this review. Besides the choice of adequate technological platforms, a high level of standardization of proteomic measurements and data processing is also necessary to establish proteomic profiling. In this regard, demands concerning study design, choice of specimens, sample preparation, proteomic data mining, and clinical evaluation should be considered before performing a proteomic study.
Analiza proteoma je postala je moćno sredstvo za dešifrovanje (pato)fizioloških procesa, što je za rezultat imalo uspostavljanje oblasti kliničke proteomike. Jedan od glavnih ciljeva je otkrivanje biomarkera oboljenja iz tkiva i telesnih tečnosti. Zbog ogromne složenosti proteoma, pri proteomskoj analizi zasnovanoj na masenoj spektrometriji potrebno je izvršiti separaciju. U radu su opisane prednosti i ograničenja proteomske analize pri separaciji proteina putem dvodimenzionalne gel elektroforeze, tečne hromatografije, SELDI i kapilarne elektroforeze (KE), sa fokusom na KE-MS. KE-MS omogućava separaciju i detekciju proteoma male molekularne težine u biološkim tečnostima uz visoku reproducibilnost i preciznost u samo jednom koraku radnog postupka i za kratko vreme. Pošto pojedinačni senzitivni i specifični biomarkeri možda i ne postoje, strategija za premošćivanje te dijagnostičke praznine pomera se sa detekcije pojedinačnog analita na simultanu analizu više analita koji zajedno čine obrazac specifičan za dato oboljenje. Takvi pristupi, međutim, nose sa sobom dodatne izazove, koje ćemo predstaviti u ovom radu. Pored izbora odgovarajućih tehnoloških platformi, neophodan je visok nivo standardizacije proteomskih merenja i obrade podataka kako bi se vršilo profilisanje proteoma. U tom pogledu, zahtevi koji se tiču nacrta studija, izbora primeraka uzoraka, analize proteomskih podataka i kliničke evaluacije trebalo bi da budu razmotreni pre izvođenja proteomske studije.
Urine samples are easily attainable which makes them ideal substrates for biomarker research. Various techniques have been employed to unravel the urine proteome and identify disease biomarkers. Even ...though the presence of high abundance proteins in urine is not so pronounced as in the case of plasma, the presence of proteolytic products, many of which at low abundance, along with numerous frequently random chemical modifications, makes the analysis of urinary proteins challenging. To facilitate the detection of low abundance urinary proteins, in the study presented herein we applied two different electrophoretic techniques, preparative Lithium Dodecyl Sulfate (LDS)-PAGE in combination with 2-DE for urinary protein separation and enrichment. Our results indicate the effectiveness of this approach for the enrichment of low abundance and low molecular weight proteins and peptides in urine, and contribute towards the establishment of a urinary proteomic database. The application of this technique as a biomarker discovery tool faces several challenges: these include down-scaling of the technique, possible recompensation for the consequent expected decrease in protein resolution, by optimizing steps of the experimental workflow as well as getting a good understanding of the technical variability of the technique. Under these conditions, preparative electrophoresis can become an effective tool for clinical proteomics applications.
Uzorke urina je lako uzeti i oni su stoga idealan materijal za istraživanje biomarkera. Za ispitivanje proteoma urina i identifikaciju biomarkera oboljenja koriste se razne tehnike. Iako prisustvo proteina u urinu nije takao izraženo kao u plazmi, prisustvo proteolitičkih proizvoda, od kojih se mnogi nalaze u maloj količini, uz brojne, često nasumične hemijske modifikacije, predstavlja izazov za analizu proteina u urinu. Kako bi se olakšala detekcija retkih proteina u urinu, u ovoj studiji primenjene su dve različite elektroforetske tehnike, preparativna LDS-PAGE u kombinaciji sa 2-DE, u cilju separacije i obogaćivanja urinarnih proteina. Naši rezultati ukazuju na efikasnost takvog pristupa za obogaćivanje retkih proteina i peptida male molekularne mase u urinu, i doprinosi formiranju baze podataka o proteomima u urinu. Postoji nekoliko izazova za primenu te tehnike kao sredstva za otkrivanje biomarkera: smanjenje obimnosti tehnike, moguća rekompenzacija za posledični očekivani pad rezolucije proteina, usavršavanjem koraka u eksperimentalnom radnom postupku, kao i sticanje detaljnog uvida u tehničku varijabilnost tehnike. Pod tim uslovima, preparativna elektroforeza može postati efikasno sredstvo za aplikacije u kliničkoj proteomici.
Microplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemio-logical studies. MADGE is a highly flexible, cost effective, microplate compatible solution to high ...throughput electrophoresis needs. It enables several thousands to million gel lines per day for direct assay of single-base variation in different capacity laboratories. Variants of the standard 96-well MADGE include: 96-well stretch-MADGE, 192-well MADGE, 384-well MADGE, and 768-well MADGE. Melt-MADGE combines the temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning. Basic MADGE principles and procedures, preparation of MADGE gels, electrophoresis, visualization and analysis of these gels, as well as modifications of the basic 96-well MADGE will be discussed in detail. For the first time in our country, this revolutionary polyacrylamid electrophoresis was done in 1998. We shortly review our studies which used MADGE for high throughput genotyping of the apolipoprotein E. MADGE and melt-MADGE will have an important role in the future genetic research of complex diseases and especially in pharmacogenomics.
»Microplate array diagonal gel electrophoresis« (MADGE) primenjuje se u molekularno genetskim epidemiološkim studijama. MADGE je fleksibilna, jeftina metoda, kompatibilna sa formatom standardnih mikrotitarskih ploča, kojom se može vršiti elektroforeza nekoliko hiljada-miliona uzoraka dnevno kako u malim tako i u velikim laboratorijama. Postoji nekoliko modifikacija standardne MADGE metode sa 96 bunara i to su »stretch« MADGE, kao i MADGE metode u kojima se koristi veći broj bunara (192, 384 i 768). »Melt«-MADGE koristi se za utvrđivanje novih mutacija. U ovom radu biće detaljno prikazani osnovni principi MADGE, priprema gela, elektroforeza, vizuelizacija i analiza tih gelova, kao i modifikacije standardne MADGE metode sa 96 bunara. Ovaj revolucionarni pristup poliakrilamidnoj elektroforezi prvi put je u našoj zemlji primenjen 1998. godine. U radu će ukratko biti prikazani rezultati studija u kojima je za genotipizaciju apolipoproteina E korišćen MADGE. Treba očekivati da će MADGE i »melt«-MADGE imati značajnu ulogu u genetici poligenskih bolesti, posebno u farmakogenomici.
Protein Purification Janson, Jan-Christer
2011, 2011-03-03, 2012-01-03, Volume:
151
eBook
This thoroughly revised and updated third edition provides an overview of the principles and methodology of the most frequently used biochemical separation techniques for proteins used today. ...Balancing theory, procedures, and applications, it offers professionals and students in biochemistry, organic chemistry, and analytical chemistry quick access to a wide range of important techniques, including: Gel filtration and ion exchange chromatography Affinity and covalent chromatography Reversed phase and hydrophobic interaction chromatography Ligands from combinatorial libraries Electrophoresis in gels Immunoelectrophoresis and capillary electrophoresis Two-dimensional electrophoresis in proteomics Protein elution and blotting techniques Large-scale electrophoresis processes
For many years, Bence Jones proteinuria has been an important diagnostic marker for multiple myeloma. Relatively new serum tests for free kappa and free lambda light chains of immunoglobulins reflect ...the production of free light chains more accurately than urine tests. In this study, we examined the value of serum free light chains measurement in the diagnosis of some neoplastic diseases and the discrepance between the findings of serum protein electrophoresis and serum free light chains. Thirty one patients (f=19, m=12) were included in the study, most of them with blood malignant diseases. The results show that in six patients with normal gamma and beta electrophoresis fractions there are abnormal levels of free light chains and/or an abnormal κ/LD ratio. In 20 patients we found an abnormal κ/LD ratio, and in 21 patients we found an abnormal κ or LD level, or both. The obtained results show the important role of serum free light chains determination in identifying patients with monoclonal gammopathies.
Bence Jones proteinurija se već dugo upotrebljava kao važan marker u dijagnostici multiplog mijeloma. Relativno novi serumski testovi za određivanje slobodnih κ i LD lakih lanaca imunoglobulina bolje reflektuju produkciju lakih lanaca od urinskih testova. U ovoj studiji ispitivali smo značaj određivanja slobodnih lakih lanaca u serumu u sklopu dijagnostike pojedinih malignih bolesti, kao i diskrepancu između nalaza serumskih lakih lanaca i elektroforeze serumskih proteina. U studiju je uključen 31 pacijent (ž=19, m=12). Većina ovih pacijenata obolela je od neke od malignih bolesti krvi. Rezultati pokazuju da kod 6 pacijenata sa urednim vrednostima gama i beta frakcije elektroforeze serumskih proteina postoje abnormalne vrednosti serumskih lakih lanaca i/ili abnormalni κ/LD odnos. Ovaj odnos bio je poremećen kod 20 pacijenata, dok je kod 21 pacijenta bio povećan nivo ili κ ili LD slobodnog lakog lanca ili oba istovremeno. Dobijeni rezultati ukazuju na značajnu ulogu određivanja serumskih koncentracija slobodnih lakih lanaca imunoglobulina u dijagnostici monoklonskih gamapatija.
Proteins in clinical practice are analyzed as important parameters in the determination and treatment of different diseases. The scopes of the analyses are mainly concentrated in two levels - ...analyses of the complete protein profile, or determination of an isolated protein. In this work, despite of the use of conventional methods, mainly electrophoresis, new techniques have been implemented in protein analyses. Lab-on-a-chip is an electrophoretic technique that, when optimized, provides analyses of the total protein profile. When normal samples are compared to samples obtained from patients with different neurological diseases, characteristic patterns can be noted. Also, correlation and comparison can be made between the newly developed microchip electrophoresis method and the results obtained using the conventional techniques. When an analysis of a specific protein is necessary, mass spectrometry has proven to give best results, in both the se lectivity and specificity of analyses. It is believed that cystatin C is a potential biomarker in neurological diseases; therefore, the mass spectrometry method has been developed in order to obtain qualitative and quantitative analyses of biological fluids. Using the developed method of mass spectrometry immunoassay (MSIA), cystatin C was easily isolated and analyzed, obtaining complete analysis within minutes. The resulting mass spectra revealed various levels of cystatin C isoforms in serum and CSF samples.
U kliničkoj praksi, proteini se analiziraju kao parametri važni za određivanje i lečenje različitih bolesti. Postoje dva opsega analiza: analize kompletnog proteinskog profila ili određivanje izolovanog proteina. U ovom radu, pored korišćenja uobičajenih metoda, pre svega elektroforeze, u analizu proteina uključene su nove tehnike. Laboratorija na čipu je elektroforetska tehnika koja, optimizovana, omogućava analize ukupnog proteinskog profila. Kada se normalni uzorci uporede sa uzorcima dobijenim od pacijenata s različitim neurološkim oboljenjima, postaju uočljivi karakteristični obrasci. Takođe, moguće je uspostaviti korelaciju i poređenje između nedavno razvijenih metoda »mikročip« elektroforeze i rezultata dobijenih korišćenjem uobičajenih tehnika. Kada je neophodna analiza specifičnog proteina, masena spektrometrija je dala najbolje rezultate, što se tiče selektivnosti kao i specifičnosti analiza. Postoji uverenje da je cistatin C potencijalni biomarker za neurološka oboljenja, stoga je razvijen metod masene spektrometrije kako bi se postigle kvalitativne i kvantitativne analize bioloških tečnosti. Pomoću metode imunoeseja masenom spektrometrijom (MSIA), cistatin C je lako izolovan i analiziran a kompletna analiza trajala je nekoliko minuta. Maseni spektri koji su nastali kao rezultat otkrili su različite nivoe izoformi cistatina C u serumu i uzorcima cerebrospinalne tečnosti.
The stability of proteins is a subject of intense current interest. Aggregation, as a dominant degradation pathway for therapeutic proteins, may cause multiple adverse effects, including loss of ...efficacy and immunogenicity. In the present study, the formation of aggregates in lenograstim under physiological conditions was monitored. For this purpose, a simple and selective size-exclusion high-performance liquid chromatography method for detection and separation of aggregates from intact protein was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions to determine the nature of aggregate bond formation. Using both techniques, the presence of a low aggregate content attached via disulfide bonds was detected.
Stabilnost proteina vrlo je aktualna problematika. Agregacija, kao dominantni put razgradnje terapijskih proteina, može uzrokovati različite nuspojave, koje uključuju i gubitak učinkovitosti imunološkog sustava. U ovom radu praćeno je stvaranje agregata u lenograstimu pod fiziološkim uvjetima. Razvijena je jednostavna i selektivna tekućinska kromatografija visoke djelotvornost s isključenjem prema veličini za detekciju i razdvajanje agregata od intaktnog proteina. Pomoću natrij-dodecil sulfat poliakrilamidne gel elektroforeze u reducirajućim i nereducirajućim uvjetima utvrđen je način stvaranja agregata. Pomoću obje tehnike moguće je detektirati prisutnost malih količina agregata koji su spojeni disulfidnim vezama.
Detekcija apoptoze Žlender, Vilim
Arhiv za higijenu rada i toksikologiju,
06/2006, Volume:
57, Issue:
2
Journal Article
Peer reviewed
Open access
Apoptoza ili programirana smrt stanice ima vodeću ulogu u mnogim biološkim procesima. Njezino određivanje od velike je važnosti u različitim područjima moderne biologije, uključujući različite ...studije o embrionalnom razvoju, biologiji tumora te brojnim degenerativnim i imunodeficijentnim bolestima koje se povezuju s poremećajima u apoptozi. Mehanizam aktivacije i pokretanja apoptotskog procesa vrlo je kompleksan i prate ga specifične morfološke i biokemijske promjene stanica, koje često variraju ovisno o tipu stanica i specifičnim uvjetima okoline u kojoj rastu. Upravo zbog tog razloga bitno je upotrijebiti više različitih detekcijskih metoda koje će obuhvatiti različite stadije apoptoze kako bi se izbjegli lažno pozitivni ili lažno negativni rezultati. Svaka od danas poznatih i upotrebljavanih metoda ima svoje prednosti i nedostatke te ni za jednu od njih ne možemo reći da potpuno zadovoljava sve kriterije. U ovom radu prikazan je kratak pregled metoda koje se najčešće rabe pri detekciji apoptoza te osnovni morfološki i biokemijski procesi na kojima se te metode zasnivaju.
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