1,720,991 research outputs found
Antibody biotinylation and its application in flow cytometry
No full textProtutijela nalaze široku primjenu u medicini i biotehnološkim istraživanjima zbog sposobnosti specifičnog vezanja na različite molekule (antigene). Mogu se koristiti u detekciji specifičnih antigena za dijagnozu bolesti, u terapiji imunodeficijencija, raka ili zaraznih bolesti te u raznim biomedicinskim istraživačkim i dijagnostičkim tehnikama: Western blot, imunohistokemija, protočna citometrija, itd. U kliničkoj se praksi sve više radi na stvaranju monoklonskih protutijela koja su mnogostruko učinkovitija i specifičnija od standardnih poliklonskih antiseruma. Kako bi se olakšala njihova detekcija u protočnoj citometriji, protutijela se obilježavaju fluorokromima ili biotinom. U ovom radu biotinom je obilježeno protutijelo anti-m04.17 koje prepoznaje m04 protein mišjeg citomegalovirusa (MCMV) te je ispitana kvaliteta i primjenjivost ovako obilježenog protutijela za detekciju MCMV-om inficiranih stanica u protočnoj citometriji. Dobiveni rezultati pokazali su da je biotinilacija uspješno provedena, da nije uzrokovala uništenje paratopa antitijela te da je protutijelo anti-m04 specifično za m04 protein MCMV-a.Antibodies have a wide use in medicine and biotechnological research due to their ability to bind various molecules (antigens) with high specificity. They can be used for detecting specific antigens for diagnosis of diseases, treatment of some immune deficiencies, cancer, infectious diseases and in different biomedical techniques: Western blotting, immunohistochemistry, flow citometry, etc. Monoclonal antibodies are of special interest for clinical practice since they are more efficient and have higher specificity compared to standard polyclonal antiserums. Antibodies can be labeled with fluorochromes or biotin to make it easier for detection in flow citometry. In this work antibody anti-m04.17 that recognizes m04 protein of mouse cytomegalovirus (MCMV) was labeled with biotin. After labeling, the quality and applicability of such labeled antibody for detection of cells infected with MCMC in flow citometry was tested. The results have shown that biotinylation was succesfull and did not cause the destruction of antibody's paratope and that the antibody anti-m04 is specific for m04 protein of MCMV
The effect of trypsin and EDTA solutions on the stability of surface markers of MEF, B12, RAW264.7 and DC 2.4 cells at different time points
No full textJedan od prvih koraka prilikom analize adherentnih stanica na protočnom citometru je njihovo odljepljivanje od podloge i dobivanje stanične suspenzije, međutim ovaj postupak može utjecati na kompoziciju i integritet molekula ispoljenih na samoj površini stanice. Kako bismo to izbjegli, odlučili smo ispitati utjecaj otopina tripsin i EDTA na izražaj određenih proteina, s obzirom na vrijeme inkubacije. Proteini čija razina je praćena su molekule MHC I, m04 te CD80 na čiji izražaj utječe mišji citomegalovirus (MCMV) kako bi izbjegao prepoznavanje od stanica imunosnog sustava, a njihova regulacija i ekspresija predmet su intenzivnog istraživanja u Centru za proteomiku. Razine navedenih molekula proučavane su na stanicama MEF, B12, DC 2.4 i Raw264.7 s i bez infekcije MCMV-om. Dobiveni rezultati analizom na protočnom citometru pokazali su da je izražaj MHC I i m04 značajno smanjen samo na inficiranim stanicama RAW264.7 nakon tretmana otopinom EDTA 10 minuta, a razina CD80 na neinficiranim i inficiranim stanicama DC2.4 tretmanom s otopinom tripsin 10 minuta. Dobiveni rezultati ukazuju na to da je bitno na vrijeme zaustaviti djelovanje otopina tripsin i EDTA kako bi se dobili točni i pouzdani rezultati u budućim eksperimentima. Osim toga, usporedila se i razina navedenih molekula na stanicama te je pokazana veća razina MHC I i m04 na stanicama RAW264.7 i DC 2.4 što ukazuje na to da je izražaj virusom izmijenjenih MHC I na njihovoj površini vjerojatno veći nego na stanicama MEF i B12. Kakav utjecaj na imunosni sustav ima ovaj fenomen treba još ispitati.One of the first steps in the analysis of adherent cells on a flow cytometer is their detachment from the substrate and obtaining a cell suspension, however this procedure can affect the composition and integrity of the molecules expressed on the cell surface. To avoid this, we decided to examine the effect of trypsin and EDTA solutions on the expression of certain proteins, given the incubation time. Proteins whose levels were monitored are the molecules MHC I, m04 and CD80, the expression of which is influenced by murine cytomegalovirus (MCMV) to avoid recognition by immune system cells, and their regulation and expression is the subject of intensive research at the Center for Proteomics. Levels of these molecules were studied on MEF, B12, DC 2.4, and Raw264.7 cells with and without MCMV infection. The results obtained by flow cytometer analysis showed that the expression of MHC I and m04 was significantly reduced only on infected RAW264.7 cells after treatment with EDTA solution for 10 minutes, and CD80 levels on uninfected and infected DC2.4 cells after treatment with trypsin solution for 10 minutes. The obtained results indicate that it is important to stop the action of trypsin and EDTA solutions on time in order to obtain accurate and reliable results in future experiments. In addition, the level of these molecules on the cells was compared and a higher level of MHC I and m04 was shown on RAW264.7 and DC 2.4 cells, which indicates that the expression of virus altered MHC I on their surface is probably higher than on MEF and B12 cells. What effect this phenomenon has on the immune system remains to be investigated
Murin citomegalovyrus protein gp34/m04 as a new marker for detection of virally infected cells
No full textCilj ovog rada bio je ispitati može li protutijelo m04.17, koje prepoznaje protein m04 mišjeg citomegalovirusa (MCMV-a), služiti kao marker infekcije stanica. Analiza specifičnosti vezanja protutijela anti-m04.17, proizvedenog u laboratoriju Centra za proteomiku, provedena je površinskim i unutarstaničnim obilježavanjem stanica inficiranih različitim sojevima MCMV-a 24 i 48 sati po infekciji.
Protein m04 ispoljava se rano po infekciji te se nakuplja u stanicama tijekom infekcije u značajnim količinama. Prema dobivenim rezultatima, pokazalo se da je protutijelo m04.17 specifično obilježilo samo stanice zaražene virusom MCMV koji sadrži gen m04 (WT i Δm138). Osim toga, pokazalo se da proteina m04 ima mnogo više u unutrašnjosti stanice, nego na površini te da proteina m04 ima značajno više 48 sati po infekciji u usporedbi sa 24 sata po infekciji.
Iz svega navedenog proizlazi da se protutijelo m04.17 može koristiti kao marker inficiranih stanica metodom koja će ga detektirati i u unutrašnjosti i na površini stanice.The aim of this work was to investigate whether the antibody m04.17, which recognizes protein m04 of mouse cytomegalovirus (MCMV), can be used as a marker of MCMV-infected cells. Analysis of the binding specificity of anti-m04.17 antibody, produced in the laboratory Center for proteomics, was performed by surface and intracellular labeling of the cells infected with various MCMV strains 24 and 48 hours after infection.
Protein m04 is expressed early after infection and accumulates in the cell during infection in significant quantities. In this work, I have shown that the antibody m04.17 specifically labels only the cells that were infected with MCMV containing the gene m04 (WT and Δm138). In addition, intracellular levels of protein m04 were significantly higher than the cell surface fraction. I could also observe increased staining with anti-m04 antibody in both intracellular and surface staining at 48 hPI in comparison with 24 hPI which is consistent with previously published data showing accumulation of m04 during the infection.
In conclusion, the antibody m04 can be used as a marker of infected cells by fixing the cells and performing intracellular staining
DESIGN, VALIDATION AND IMPLEMENTATION OF FAST MOLECULAR DIAGNOSTIC TESTS IN EMERGENCY MEDICINE
No full textCilj istraživanja: glavni cilj istraživanja je uspostaviti brzu molekularnu dijagnostiku na
mjestu skrbi za pacijenta, tzv. POCT (od eng. „point-of-care tests“) molekularnu dijagnostiku
u odjelu hitne medicine (OHBP od Objedinjeni hitni bolnički prijam) Kliničkog bolničkog
centra Rijeka.
Ispitanici i metode: u prvi i drugi dio istraživanja u kojemu smo usporedili direktni qPCR
(dqPCR) sa standardnim qPCR (stndqPCR), optimizirali i validirali dqPCR u POCT, uključili
smo 126 ispitanika podijeljenih u 4 grupe: pacijenti, zaposlenici KBC-a Rijeka, pacijenti
hospitalizirani u COVID-19 respiracijskom centru i pacijenti s potvrđenom COVID-19
infekcijom dva ili više tjedana prije testiranja. Treći dio istraživanja u kojem smo tijekom 4
mjeseca analizirali implementiranu POCT – PCR metodu proveli smo na preko 10.000
pacijenata i zaposlenika KBC-a Rijeka čiji su se uzorci slali u naš Laboratorij na dijagnostičku
molekularnu analizu. Za istraživanje izabrali smo Seegene-ov Allplex SARS-CoV-2 paket
kemikalija koji dolazi u kompletu s računalnim programom „SARS-CoV-2 Viewer“ za
automatiziranu interpretaciju rezultata, a rezultate smo analizirali primjenom Seegene i strožih
naših kriterija.
Rezultati: analizirajući i uspoređujući osjetljivost i specifičnost dqPCR i POCT varijante
dqPCR sa stndqPCR-a broj pozitivnih, negativnih ili neodređenih rezultata bilo je u snažnom
slaganju s rezultatima dobivenih stndqPCR-om. Nadalje, korelacija sa stndqPCR-a bila je još
izraženija kada su za stratifikaciju dobivenih rezultata korišteni naši kriteriji te smo na temelju
učinjenih analiza i dobivenih rezultata zaključili da je POC-dqPCR, modificirani postupak
dqPCR-a u kojem medicinsko osoblje bez prethodnog iskustva u molekularnoj dijagnostici
može pouzdano koristiti prethodno pripremljenu reakcijsku smjesu za detekciju SARS-CoV-2
u odjelima hitne medicine i/ili sličnim odjelima koji neposredno skrbe za oboljele. Dodatno
smo validirali metodu korištenjem standardiziranih uzoraka Svjetske zdravstvene organizacije
te na zbirnim uzorcima djelatnika OHBP-a KBC Rijeka.
Zaključak: Seegene-ov Allplex SARS-CoV-2 test smo prilagodili u direktni qPCR test sličan
POCT-u koji može učinkovito i pouzdano provoditi medicinsko osoblje u Laboratoriju za brzu
molekularnu dijagnostiku u hitnoj medicini, OHBP-a KBC Rijeka.Objectives: main objective of this research is to establish fast, point – of – care molecular
diagnostics, at the Emergency department of the Clinical hospital center Rijeka.
Patients and methods: in the first and second part of research we compared direct qPCR with
standard qPCR, optimized and validated dqPCR in the POC setting. We included 126 subjects
in 4 groups: patients from ED, hospital employees, patients hospitalized in COVID-19
respiratory center and patients in whom COVID -19 infection has been confirmed two or more
weeks before testing. For the third par we included more than 10.000 patients and employee
samples which were analyzed in out Laboratory for the diagnostic molecular analysis. Seegene
Allplex SARS-CoV-2 reagents were used coupled with SEEGENE SARS-CoV-2 Viewer
software for the automated data interpretation. For data interpretation we applied both Seegene
and our – more stringent criteria.
Results: comparing sensitivity and specificity of dqPCR and POCT dqPCR with stndqPCR we
obtained strong correlation of results in terms of positive, negative, and undetermined results.
Furthermore, correlation was even more pronounced applying our criteria. Based on the results
obtained we conclude that POC-dqPCR, modified dqPCR in which medical staff without
previous molecular diagnostics can reliably use SARS-COV-2 detection reagents prepared in
advance in emergency departments and/or other departments which provide direct patient care.
We, additionally, validated the method using standardized World health organization samples
and pooled samples from the Clinical hospital center Rijeka Emergency department staff.
Conclusion: We adapted a Seegene Allplex SARS-CoV-2 test into a direct, point-of-care like,
test which can be efficiently and reliably used in Laboratory for fast molecular diagnostics in
emergency medicine of the Clinical hospital Center Rijeka
Karakterizacija prirođenog imunosnog odgovora na citomegalovirus u jajniku (CIRCO) : Plan upravljanja istraživačkim podacima
No full textKarakterizacija prirođenog imunosnog odgovora na citomegalovirus u jajniku (CIRCO) : Plan upravljanja istraživačkim podacima
No full textAnaliza transkriptoma mišijeg citomegalovirusa
No full textHuman cytomegalovirus (HCMV) is a ubiquitous human pathogen responsible for devastating
congenital disease and life-threatening complications in immune-suppressed patients.
Available treatments have many shortcomings and effective vaccine is still lacking. Major
obstacles to progress in vaccine and antiviral drug development are (1) species specificity of
HCMV, and (2) gaps in our understanding of viral genes and their interaction with host genes.
First limitation is circumvented by the use of model animal viruses, especially murine
cytomegalovirus (MCMV). We sought to alleviate the second problem by studying MCMV
transcriptome using two approaches: classical cDNA library analysis and next generation
sequencing (RNASeq). This dual analysis revealed incredible complexity of MCMV
transcriptome, detected numerous novel viral spliced and unspliced transcripts as well as
transcription from intergenic regions, and showed that expression levels of viral transcripts
vary by several orders of magnitude. Unexpectedly, most top expressed genes were of
unknown functions and were improperly annotated. Therefore, this analysis provides the first
comprehensive overview of MCMV transcriptome, underscores the necessity of
transcriptomic analyses in providing evidence-based genome annotation and could serve as
the first step towards re-annotation of MCMV genome. The most abundant viral transcript,
recently identified as a noncoding RNA regulating cellular microRNAs [18, 84], was shown
to also code for a novel protein(s). This is the first viral transcript that functions both as a
noncoding RNA and an mRNA. In this work it is also shown that this transcript’s 5’ UTR
plays a role in NK cell recognition of infected cells via activating Ly49 receptors.
Analysis of host transcriptome showed that lytic infection revealed that many unexpected
gene groups are disregulated in response to the infection. Such systematic analysis may shed
new light on cytomegalovirus pathogenesis and suggests new areas of research.Svrha istraživanja
Humani citomegalovirus široko je rasprostranjen patogen, a posebno je opasan za trudnice,
novorođenčad i imunosuprimirane pacijente. Nažalost, efikasnog cjepivo nema, a postoji
potreba i za efikasnijim i manje toksičnim antiviralnim lijekovima. Glavne prepreke razvoju
novih antiviralnih ljekova i cjepiva jesu: (1) specifičnost za vrstu i (2) praznine u našem
znanju i razumjevanju virusnih gena, interakcijama virusnih gena i domaćina te kako te
interakcije izazivaju bolest. Prva prepreka uspješno se nadvladava korištenjem animalnih
virusa, posebice mišjeg citomegalovirusa (MCMV). S ciljem nadvladavanja druge prepreke u
ovom je radu provedena detaljna analiza transkriptoma mišjeg citomegalovirusa (MCMV) te
analiza transkriptoma stanica domaćina tijekom litičke infekcije citomegalovirusom.
Materijali i metode
Transkriptom MCMV analiziran je na dva načina: klasičnom analizom cDNK knjižnice i
sekvencioniranjem dobivenih klonova te analizom transkriptoma uz pomoć sekvencioniranja
nove generacije (odnosno RNK-sekvencioniranjem, eng. RNASeq) koja omogućava paralelno
praćenje i transkriptoma domaćina uz transkriptom virusa. Analiza transkriptoma domaćina
rezultira vrlo dugačkim listama diferencijalno reguliranih gena iz kojih je teško izvući neko
biološko značenje. Stoga su liste diferencijalno reguliranih gena domaćina podvrgnute analizi
termina genske ontologije (eng. gene ontology analysis odnosno GO analiza) i analizom dereguliranih
bioloških puteva. Transkripcijski kompleksne regije genoma MCMV dodatno su
analizirane Northern hibridizacijom i metodom RT-PCR dok je korelacija između količine
transkripata i proteina odabranih gena analizirana metodom Western blot. Na kraju, funkcija
novog, prekrojenog transkripta MAT (most abundant transcript; najzastupljeniji transkript)
analizirana je uz pomoć reporterskih stanica koje nose aktivacijske Ly49 receptore.
Rezultati
Ovaj rad predstavlja prvu detaljnu analizu transkriptoma MCMV-a korištenjem
komplementarnih metoda analize cDNK knjižnice i RNASeq analizom, a rezultirala je
identifikacijom brojnih novih transkripata MCMV, uključujući i nove prekrojene transkripte
kao i transkripte koji se prepisuju sa intergenskih regija. Ustanovljeno je da najjače izraženi
virusni transkripti imaju nepoznatu funkciju i često su pogrešno anotirani. Najjače izražen
transkript (tzv. MAT transkript) prvi je virusni transkript koji ima i kodirajuću i nekodirajuću
funkciju. Naime, nedavno je pokazano da se na njegovom 3' netranslatiranom kraju (3' UTR,
od eng. 3' untranslated region) nalazi vezno mjesto za staničnu mikro-RNK [18, 84], dok je u
ovom radu pokazano da on također kodira i barem još dva proteina. Uz ove dvije navedene
funkcije, u ovom je radu otkriveno da je 5' netranslatirani kraj (5'UTR) MAT transkripta bitan
virusni faktor kojeg na inificranim stanicama prepoznaju stanice prirodne ubojice pomoću
aktivacijskih receptora Ly49.
Analiza transkriptoma stanica domaćina pokazala je da litička infekcija virusom MCMV
izaziva izrazite promjene u ekspresijskom profilu gena domaćina: ekspresija gotovo trećine
gena miša promijenila se uslijed infekcije virusom MCMV. Geni čija se ekspresija pojačava
tijekom infekcije uglavnom su geni uključeni u upalne i imunološke procese, međutim neki
pripadaju i skupini transkripcijskih faktora te genima povezanim s razvojem i
diferencijacijom. Ovi rezultati u skladu su s dosadašnjim saznanjima o CMV-u kao virusu
koji izaziva upalu te uzrokuje razvojne poremećaje tijekom kongenitalnih infekcija. Brojni
geni čija se ekspresija smanjila tijekom infekcije povezani su sa funkcijama čija je uloga u
infekciji za sada nepoznata poput dugačkih intergenskih nekodirajućih RNK, antisense RNK
ili malih nukleolarnih RNK. GO analiza rezultirala je detekcijom disreguliranih bioloških
puteva koji još do sada nisu bili povezani sa citomegalovirusnom infekcijom, a koji imaju
potencijal rasvjetljavanja nekih nepoznanica u patogenezi citomegalovirusne infekcije.
Zaključci
Jedno od najznačajnijih otkrića u ovome radu jest dokaz izuzetne kompleksnosti
transkriptoma MCMV-a koji dosad nije bio ovako sustavno istraživan niti su postojale
transkriptomske mape. Ova analiza transkriptoma MCMV-a predstavlja važan prvi korak ka
razvoju boljih genomskih mapa i reanotaciji genoma MCMV-a. Analiza odgovora stanica
domaćina na infekciju dala je novi pogled na molekularne interakcije između virusa i
domaćina i otvorila brojna nova područja istraživanja koja imaju potencijal da p pronađu nove
mogućnosti liječenja bolesti izazvanih CMV-om.
Ključne riječi
mišji citomegalovirus, MCMV, transkriptom, ekspresija gena, izbjegavanje imunološko
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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