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SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex
No full text(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums
Patogeneza varijanti tafazina u Barthovom sindromu: bioinformatička analiza
No full textBarth syndrome (BTHS) is an X-linked genetic disease characterized by severe cardiovascular defects, skeletal muscle weakness and neutropenia. Defects in the protein tafazzin, which is encoded by the TAZ gene, have been identified to give rise to BTHS through impaired capability of remodeling the mitochondrial phospholipid cardiolipin (CL). Even though the genetic defects responsible for the disease are known, the links between specific TAZ mutation variants and mechanisms of BTHS pathogenicity haven't been thoroughly examined. The aim of this work was to present bioinformatics methods which would facilitate this type of research and, using these methods, examine tafazzin protein features, map all known benign and pathogenic tafazzin protein changes to its corresponding domains, identify the mutations that are found in evolutionarily conserved regions, and highlight potentially important domains and mutations for future in silico and in vivo research. The variants were compiled from the Barth Syndrome Foundation's Tafazzin Human Variants Database. Databases UniProt, ClinVar, OMIM and GnomAD were used in addition. Seven pathogenicity prediction softwares were used to analyze potential pathogenicity of missense variants based on protein structure and evolutionary conservation. A species alignment analysis was done using ClustalOmega. Our results show that tafazzin is not subject to binding CL via induced-fit, but that the interaction is rather based on conformational selection, indicating a potential conformational flexibility that might enable interactions with diverse phospholipids without significant structural changes caused by ligand binding. We hypothesize that tafazzin's conformational flexibility may be influenced by the surrounding microenvironment, including membrane composition and neighboring molecules. We demonstrate pathogenic mutational enrichment in five of the protein's domains and show that 34 out of 62 pathogenic missense variants are predicted to be detrimental to the protein's function. Through further refinement, we show that two domains of the protein, HX4D and MT1, are highlighted as significantly enriched in pathogenic variants. D74 and D75 mutations in the HX4D domain and R94 mutations in the MT1 domain, identified as pathogenic through our methods, suggest the likelihood of interactions between these domains due to the complementary charges of aspartate and arginine, hinting at a potential active site. Finally, we discuss the potential of studying these two domains and TAZ mutation variants in general by applying molecular dynamics simulations and using the model organism Saccharomyces cerevisiae.Barthov sindrom (BTHS) je X-vezana genetska bolest karakterizirana teškim kardiovaskularnim defektima, slabošću skeletnih mišića i neutropenijom. Defekti u proteinu tafazinu kodiranom sa strane TAZ gena identificirani su kao uzrok BTHS-a, djelujući pogubno na sposobnost remodeliranja mitohondrijskog fosfolipida kardiolipina (CL). Iako su genetski poremećaji odgovorni za bolest poznati, poveznice između specifičnih TAZ varijanti i mehanizama BTHS patogenosti nisu detaljno istražene. Cilj ovog rada bio je predstaviti bioinformatičke metode koje bi omogućile takvo istraživanje, te pomoću njih proučiti proteinske značajke tafazina, mapirati sve do sada poznate benigne i patogene promjene proteina na njegove domene, identificirati mutacije koje se nalaze na evolucijski konzerviranim mjestima proteina, te istaknuti potencijalno važne domene i mutacije za buduća in silico i in vivo istraživanja. Varijante u ovom radu su primarno kompilirane iz Barth syndrome foundation baze podataka Tafazzin Human Variants, a potom su korištene i baze podataka UniProt, ClinVar, OMIM i GnomAD. Sedam programa za predviđanje patogenosti korišteno je u svrhu analize patogenosti missense varijanti tafazina na temelju proteinske strukture i evolucijske konzervacije. Analiza poravnanja proteinskih sekvenci između vrsti napravljena je pomoću programa ClustalOmega. Predstavljamo rezultate koji ukazuju na to da tafazin ne podliježe vezanju CL-a putem inducirane prilagodbe, već konformacijske selekcije, te da stoga postoji potencijalna konformacijska fleksibilnost proteina koja omogućuje interakcije s raznim fosfolipidima bez značajnih strukturalnih promjena uzrokovanih vezanjem liganada. Prilažemo hipotezu da konformacijska fleksibilnost tafazina podliježe utjecaju mikrookoliša, uključujući kompoziciju membrane i susjedne molekule. Pokazujemo patogeno obogaćenje u pet domena tafazina, te da su 34 od 62 patogene missense varijante predviđene kao pogubne za funkciju proteina. Izdvajamo dvije domene tafazina, HX4D i MT1, kao značajno obogaćene patogenim varijantama. Identificirane kao patogene pomoću naših metoda, D74 i D75 mutacije u HX4D domeni, te R94 mutacije u MT1 domeni sugeriraju na mogućnost interakcija između ove dvije domene zbog komplementarnih naboja aspartata i arginina, što potencijalno ukazuje i na aktivno mjesto proteina. Naposlijetku, raspravljamo o potencijalu izučavanja ove dvije domene i TAZ mutacijskih varijanti pomoću metoda kao što su simulacije molekularne dinamike i korištenje modelnog organizma Saccharomyces cerevisiae
Analyza ADAR proteina tijekom produktivne infekcije HSV-1
No full textADAR proteins are enzymes involved in the editing of dsRNA transcripts by
deamination in which adenosine is changed to Inosine. This alteration
ultimately affects protein translation. ADAR1 has two isoforms; ADAR1p110
and ADAR1p150, ADAR2 and ADAR3 make up the mammalian ADAR family.
Both isoforms of ADAR1 and ADAR2 are catalytically active while ADAR3 is
not. ADARs edit both self and non-self dsRNA. Several studies have shown
the role of ADAR proteins mostly in RNA viruses, however, the role of these
proteins in dsDNA viruses is largely unknown. During viral infection, they can
play an antiviral role by forming part of the cell’s innate immune response, a
proviral role by blocking viral dsRNA from being recognized by the immune
sensors, thereby facilitating viral replication in the host cell, or both roles as
observed in Influenza A virus.
Based on in vitro experiments, we have established cell models to analyse
the role of ADAR proteins during productive HSV-1 infection. ADAR protein
and mRNA levels during productive HSV-1 infection were determined,
showing upregulation of ADAR during the infection. We observed an
upregulation of ADAR proteins in U2OS, HeLa, HEK293T, and HFF cells
infected with wild-type HSV-1 using MOIs 5 and 10, but not with MOI of 1.
Interestingly, we observed ADAR cells-specific response to infection with UV
inactivated HSV-1, which we cannot explain at this point. To investigate the
functional relevance of ADAR1 and ADAR2 proteins for virus replication, we
transfected cells with ADAR1 and ADAR2 expressing plasmids to overexpress
ADAR in cells. Surprisingly, we did not observe any effect on virus DNA
replication, indicating that ADAR1 and ADAR2 might not play a role in
productive HSV-1 infection. However further investigation is needed to
answer this question.ADAR protein su enzimi uklučeni u uređivanje dsRNA transkripata
deaminacijom u kojoj se adenozin mijenja u inozin. Ova promjena u
konačnici može utjecati na stabilnost RNA te na translaciju proteina. Obitelj
ADAR proteina čine ADAR1, koji ima dvije izoforme: ADAR1p110 i
ADAR1p150, ADAR2 i ADAR3. ADAR1 i ADAR2 su katalitički aktivni dok
ADAR3 nije. ADAR uređuju vlastite i brojne druge dsRNA. Nekoliko je studija
pokazalo ulogu proteina ADAR većinom u RNA virusima, , međutim njihova
uloga kod infekcije s dsDNA virusima slabo je istražena. Tijekom virusne
infekcije, oni mogu imati antivirusnu ulogu čineći dio urođenog imunološkog
odgovora stanica, ali i provirusnu ulogu, blokirajući virusnu dsRNA da je
prepozna imunološki senzor, čime se olakšava replikacija virusa u stanici
domaćinu, ili obje uloge kao što je primijećeno kod Influenza A virus.
Na temelju in vitro eksperimenata, uspostavili smo stanične modele za
analizu uloge ADAR proteina tijekom productivene HSV-1 infekcije. Određene
su razine ADAR proteina i mRNA tijekom produktivne HSV-1 infekcije. Uočili
smo pojačanu regulaciju ADAR u U2OS, HeLa, HEK293T i HFF stanicama
zaraženim divljim tipom HSV-1 te je takva pojačana ekspresija ovisila o
količini virusa kojom se inficira stanica. Zanimljivo je da smo uočili različiti
ADAR odgovor na infekciju s UV inaktiviranim virusom kod različitih vrsta
stanica, što u ovom trenutku ne možemo objasniti. Nadalje, kako bismo
istražili funkcionalnu relevantnost ADAR1 i ADAR2 proteina za replikaciju
virusa, transfecirali smo stanice s ADAR1 i ADAR2 ekspresijskim plazmidima
za prekomjernu ekspresiju ADAR-a. Naime, nismo primijetili nikakav učinak
na replikaciju DNA virusa, što ukazuje da ADAR1 i ADAR2 možda nemaju
ulogu u produktivnoj HSV-1 infekciji. Međutim, potrebna su daljnja
istraživanja kako bi se odgovorilo na ova pitanja
Replikacija respiratornog sincicijalnog virusa: Inhibicija interferirajućim česticama defektnog virusa gripe A
No full textRespiratory syncytial virus (RSV) is a major cause of acute lower respiratory
infections in infants and susceptible adults. Although the epidemiology and
immunobiology of the virus have been widely investigated, no safe vaccine has
yet been approved, and treatment with the antiviral ribavirin is limited to severe
cases. Nonetheless, new antiviral agents are undergoing development1
.
A new approach encompasses influenza A virus (IAV) defective interfering
particles (DIPs). IAV DIPs have been proposed as an antiviral treatment against
interferon (IFN)-sensitive respiratory viruses such as influenza A and B, severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and yellow fever virus
(YFV). In this study, we established a method for RSV production and assessed
the virus’ replication dynamics. Moreover, the inhibitory potential of IAV DIPs
against RSV propagation was studied in in vitro coinfection experiments.
Specifically, we investigated the antiviral activity of DI244, a prototypic, wellcharacterized DIP that harbors a large internal deletion in Segment 1 of the viral
genome, and of OP7, a newly discovered DIP that presents 37 point mutations
on Segment 7 of the viral RNA (vRNA)2,3
. We report that DI244 and especially
OP7 are able to partially suppress RSV replication in IFN-competent cells.
Furthermore, we show that the inhibitory potential of IAV DIPs is dependent on
the innate immune response stimulation. It appears that DI vRNAs were
recognized by the retinoic acid inducible gene I (RIG-I), leading to upregulation
of type I and type III interferons (IFNs). IFNs activated the janus kinase-signal
transducers and activators of transcription (JAK/STAT) signaling cascade, which
culminated in the expression of interferon stimulated genes (ISGs).
Our results suggest that the IAV DIPs DI244 and OP7 may represent a promising
antiviral agent for the treatment and prophylaxis of RSV infection.Respiratorni sincicijski virus (RSV) glavni je uzročnik akutnih infekcija donjeg
dišnog sustava u dojenčadi i osjetljivih odraslih osoba. Iako su epidemiologija i
imunobiologija virusa opsežno istražene, sigurno cjepivo još nije odobreno, a
liječenje antivirotikom ribavirinom ograničeno je na teške slučajeve. Usprkos
tome, razvijaju se novi antivirusni lijekovi1
.
Novi pristup obuhvaća defektne interferirajuće čestice (DIP) virusa influence A
(IAV). DIP-ovi IAV predloženi su kao antivirusni tretman protiv respiratornih
virusa osjetljivih na interferon (IFN) kao što su gripa A i B, teški akutni
respiratorni sindrom coronavirus 2 (SARS-CoV-2) i virus žute groznice (YFV). U
ovoj studiji smo uspostavili metodu za proizvodnju RSV-a i procijenili dinamiku
replikacije virusa. Nadalje, inhibicijski potencijal DIP-a IAV protiv širenja RSV-a
proučavan je u in vitro eksperimentima koinfekcije. Konkretno, istražili smo
antivirusnu aktivnost DI244, prototipskog, dobro karakteriziranog DIP-a koji
sadrži veliku unutarnju deleciju u segmentu 1 virusnog genoma, i OP7,
novootkrivenog DIP-a koji sadrži 37 točkastih mutacija na segmentu 7 virusne
RNA (vRNA)2,3. Naši rezultati pokazuju da DI244, a posebno OP7, mogu
djelomično suzbiti RSV replikaciju u stanicama kompetentnim za IFN. Nadalje,
pokazujemo da inhibitorni potencijal DIP IAV ovisi o stimulaciji urođenog
imunološkog odgovora. DI vRNA prepoznaje gen I inducibilan retinoičnom
kiselinom (RIG-I), što dovodi do pojačane ekspresije interferona tipa I i tipa III
(IFN). IFN-i dovode do aktivacije Janus kinaze i STAT proteina putem JAK/STAT
signalnog puta, što kulminira ekspresijom gena stimuliranih interferonom (ISG).
Naši rezultati sugeriraju da su DIP-ovi IAV, DI244 i OP7, obećavajuća antivirusna
sredstva za liječenje i profilaksu RSV infekcije
Influence of mica functionalization with nickel chloride and poly-L-lysine on binding and visualization of extracellular vesicles from human cerebrospinal fluid by atomic force microscopyInfluence of mica functionalization with nickel chloride and poly-L-
No full textIzvanstanične vezikule (IV) su membranske nanočestice koje sve stanice izlučuju i igraju važnu ulogu u komunikaciji između stanica, ali njihova nanometarska veličina zahtijeva napredne tehnike vizualizacije u cilju istraživanja njihove morfologije. Mikroskopija atomskih sila (engl. Atomic Force Microscopy) predložena je kao jedna od obećavajućih metoda za vizualizaciju, a upravo korištenjem načina tapkanja vršnom silom postignut je napredak u samoj tehnologiji snimanjem slika visoke razlučivosti. Tinjac se često koristi kao nosač uzorka zbog ravnine površine na razini atoma. Iako se uslijed elektrostatskih interakcija negativno nabijene IV vežu za tinjac, u cilju jačeg vezivanja potrebno je provesti funkcionalizaciju površine pozitivnim nabojem, primjerice niklovim(II) kloridom (NiCl2) ili poli-L-lizinom (PLL-om). Cilj ovog istraživanja bio je uspostaviti i optimizirati protokole vezivanja IV-a na tinjac prethodno funkcionaliziran s NiCl2 ili PLL-om te istražiti njihov učinak na vizualizaciju IV-a mikroskopom atomskih sila (AFM-om). Vizualizacija IV-a je izvedena AFM-om u načinu tapkanja, a tretman IV-a prije snimanja uključivao je; fiksaciju paraformaldehidom (PFA-om, 3%) i glutaraldehidom (GA-om, 1,5%), dehidraciju s etanolom ili 2,2-dimetoksipropanom i sušenje s heksametildisilazanom ili pri kritičnoj točki ugljikovog dioksida (CPD). Najbolji protokol za vizualizaciju IV-a i za primjenu u daljnjim istraživanjima uključuje funkcionalizaciju s NiCl2, fiksaciju s PFA-om/GA-om, dehidraciju s etanolom i CPD. Ova metoda se pokazala optimalnom jer jedina pokazuje ujednačen broj detektiranih čestica prema promjeru i visini te veću srednju vrijednost promjera i visine s većom standardnom devijacijom. Svaka korištena metoda identificirala je određene oblike IV-a; konkavne, konveksne, multirežnjaste i nepravilne. Budući da se ove metode primjenjuju kako bi se omogućila identifikacija po sferičnosti, promjeru i visini, još nije moguće razdvojiti IV od čestica iste veličine. S tim razlogom, sljedeći korak istraživanja trebala bi biti imuno-interakcija IV-a s antitijelima.Extracellular vesicles (EV) are membrane nanoparticles that all cells secrete and play an important role in communication between cells, but their nanometer size requires advanced visualization techniques in order to investigate their morphology. Atomic force microscopy has been proposed as one of the promising methods for EV visualization, and due to the use of peak force tapping methods, advances have been made in the technology itself by capturing high-resolution images. Mica is often used as a sample carrier due to the plane surface at the atomic level. Although due to electrostatic interactions negatively charged EV binds to mica, in order to strengthen binding it is necessary to carry out surface functionalization with positive charges such as nickel(II) chloride (NiCl2) and poly-L-lysine (PLL). The aim of this study was to establish and optimize protocols of EV binding to mica previously functionalized with NiCl2 or PLL and to investigate their effect on EV visualization by atomic force microscope (AFM). EV visualization was performed on AFM in tapping mode, and pre-recording EV treatment included; fixation with paraformaldehyde (PFA, 3%) and glutaraldehyde (GA, 1,5%), dehydration with ethanol or 2,2-dimethoxypropane and drying with hexamethyldisilazane or at the critical point of carbon dioxide (CPD). The best protocol for EV visualization and for use in further research includes functionalization with NiCl2, fixation with PFA/GA, dehydration with ethanol, and CPD. This method proved to be optimal because it is the only one that shows a uniform number of detected particles according to diameter and height and a higher mean value of diameter and height with a higher standard deviation. Each method used identified certain forms of EV; concave, convex, multi-lobed and irregular. Because these methods are applied to allow identification by sphericity, diameter, and height, it is not yet possible to separate EV from particles of the same size. For this reason, the next step in research should be immuno-interaction of EV with antibodies
Epigenetic factors of human infertility
No full textNeplodnost pogađa oko 10 % svjetske populacije. Definira se kao
nemogućnost postizanja kliničke trudnoće nakon više od 12 mjeseci
redovitih nezaštićenih spolnih odnosa. Na nastanak neplodnosti mogu
utjecati različiti mehanizmi epigenetičkih modifikacija. Epigenetičke
modifikacije podrazumijevaju promjene u funkciji gena koje ne uključuju
promjenu slijeda nukleotida u DNA molekuli, a obuhvaćaju DNA metilaciju,
modifikacije histona i djelovanje nekodirajućih RNA molekula. Metilacija
DNA podrazumijeva dodavanje metilnih skupina na specifična mjesta unutar
DNA molekule, što utječe na aktivnost gena. Modifikacije histona utječu na
to koliko čvrsto je DNA omotana oko histonskih proteina djelujući time na
njenu dostupnost za ekspresiju gena. Konačno, nekodirajuće RNA
podrazumijevaju RNA molekule koje ne kodiraju proteine, već također
reguliraju ekspresiju gena specifičnim mehanizmima djelovanja.
Istraživanja su pokazala da kod muškaraca promjene u metilaciji DNA
tijekom spermatogeneze mogu djelovati na kvalitetu nastalih muških
spolnih stanica, dok histonske modifikacije, kao što su metilacija i acetilacija
histona, mogu narušiti sposobnost spermija da oplodi jajnu stanicu. Slično
tome, kod žena, promjene u DNA metilaciji i modifikacijama histona utječu
na oogenezu, potencijalno dovodeći do otežane oplodnje ili poremećaja u
razvoju nastalih zametaka. Razumijevanje epigenetičkih mehanizama i
njihovih uloga u ljudskoj plodnosti može pružiti uvid u uzroke i potencijalne
tretmane neplodnosti.Infertility affects about 10% of the world's population. It is defined as the inability to achieve a clinical pregnancy after more than 12 months of regular unprotected sexual intercourse. The occurrence of infertility can be influenced by various mechanisms of epigenetic modifications. Epigenetic modifications are changes in gene function that are not associated with a change in the nucleotide sequence in the DNA molecule. These include DNA methylation, histone modifications and the effect of non-coding RNA molecules. In DNA methylation, methyl groups are added to specific sites on the DNA molecule, which influences gene activity. Histone modifications affect how tightly the DNA is wrapped around histone proteins and thus influence the availability of DNA for gene expression. Finally, non-coding RNAs include RNA molecules that do not code for proteins but also regulate gene expression through specific mechanisms of action. Research has shown that in men, changes in DNA methylation during spermatogenesis can affect the quality of the male gametes formed, while histone modifications such as histone methylation and acetylation can affect the ability of sperm to fertilize an egg. Similarly, in women, changes in DNA methylation and histone modifications affect oogenesis, which can lead to impaired fertilization or development of the resulting embryos. Understanding the epigenetic mechanisms and their roles in human fertility may help to identify the causes and potential treatments of infertility
Uloga TRPA1 kanala u patologiji pluća
No full textThe goal of this work is to better understand how lung disorders
including asthma and chronic obstructive pulmonary disease (COPD)
are affected by the TRPA1 channel, a crucial molecular sensor. The
TRPA1 channel is involved in controlling inflammation, pain
perception, and the respiratory system's reaction to damaging stimuli.
It is activated by a variety of chemical irritants. The desire to find
novel treatment targets and get a deeper understanding of the
molecular mechanisms behind the course of lung illness is what
motivates this research's scientific setting.
The study entails a careful review of the body of literature already in
existence, with an emphasis on the findings of knockout (KO) studies,
which employed mice deficient in the TRPA1 gene to investigate its
function. The precise functions of the TRPA1 channel in the
pathophysiology of lung diseases have been made clearer by these
models. Furthermore, the possibility of pharmacologically blocking
the TRPA1 channel is taken into consideration as a potential
treatment approach.
According to the research, the TRPA1 channel plays a significant role
in several processes that lead to inflammation and lung tissue
destruction. As a result, blocking this channel may lessen symptoms
and delay the course of conditions like asthma and COPD. The study's
conclusion highlights the need for ongoing research to maximize
therapeutic agent selectivity and gain a deeper understanding of the
roles played by the TRPA1 channel in various tissues, as well as the
potential for new avenues in the development of targeted therapies
for lung diseases.Ovo istraživanje usmjereno je na razumijevanje uloge TRPA1 kanala,
ključnog molekularnog senzora, u plućnim bolestima poput astme i
kronične opstruktivne plućne bolesti (KOPB). TRPA1 kanal, poznat po
svojoj aktivaciji različitim kemijskim iritantima, sudjeluje u regulaciji
upale, osjetu boli i reakcijama na štetne podražaje u dišnom sustavu.
Znanstveni kontekst ovog rada temelji se na potrebi za dubljim
razumijevanjem molekularnih mehanizama koji stoje iza progresije
plućnih bolesti i na identificiranju novih terapijskih ciljeva.
Istraživanje koristi pristup koji uključuje kritičku analizu postojeće
literature, s posebnim naglaskom na rezultate knockout pokusa u
kojima su miševi bez TRPA1 gena korišteni za proučavanje njegove
funkcije. Ovi modeli omogućili su otkrivanje specifičnih uloga TRPA1
kanala u patofiziologiji plućnih bolesti. Uz to, razmatra se
farmakološka inhibicija TRPA1 kanala kao potencijalna terapijska
strategija.
Rezultati pokazuju da je TRPA1 kanal uključen u brojne ključne
procese koji pridonose upali i oštećenju plućnog tkiva, te da inhibicija
ovog kanala može smanjiti simptome i progresiju bolesti kao što su
astma i KOPB. Zaključci rada sugeriraju da daljnje istraživanje TRPA1
kanala može otvoriti nove puteve u razvoju ciljanih terapija za plućne
bolesti, te ističu potrebu za daljnjim istraživanjima kako bi se
optimizirala selektivnost terapijskih sredstava i bolje razumjele
funkcije ovog kanala u različitim tkivima
Chemical characterization and biological effect of olive leaf extract
No full textMaslina (Olea europaea L.) je glavna biljka mediteranskog područja, poznata po svojim plodovima i ulju. Osim plodova, važan je i list masline, koji sadrži brojne bioaktivne spojeve s dokazanim antioksidativnim, protuupalnim i antikancerogenim svojstvima. Cilj istraživanja bio je provesti karakterizaciju ekstrakta lista masline, Magdis praha, pomoću LC-MS analize, i ispitati njegovo citotoksično djelovanje na određenim vrstama humanih karcinoma. Ispitivanje se provelo MTT testom citotoksičnosti na trima tumorskim staničnim linijama (MDA-MB-231, MeWo i HeLa) i humanoj staničnoj liniji fibroblasta (HFF). Za tretman na stancama te su pripremljena dva ekstrakta; prvi tretiran etanolom pa otopljen u dimetilsulfoksidu, dok je drugi ekstrakt samo otopljen u dimetilsulfksidu.
Upotrebom LC-MS analize, kao glavni fenolni spoj, detektiran je oleuropein čiji je učinak najviše zaslužan za citotoksičnost na stanicama. Od ostalih, u najvišim koncentracijama detektirani su luteolin-7-glukozid, verbaskozid, kvercentin. Ispitivajući citotoksičnost ekstrakta međusobno, nije se pokazala značajna razlika u citotoksičnom učinku na tumorske stanične linije. Oba testirana ekstrakta masline smanjuju proliferaciju stanica svih ispitivanih tumorskih staničnih linija gdje je najveći citotoksični učinak zabilježen na HeLa stanicama (IC50 = 0,36 ± 0,03 odnosno 0,55 ± 0,05 mg/ml), potom na MeWo (IC50 = 0,37 ± 0,05 odnosno 0,36 ± 0,03 mg/ml), a najmanji je učinak uočen kod MDA-MB-231 stanica (IC50 = 0,43 ± 0,10 odnosno 0,41 ± 0,11 mg/ml). Usporedbom dobivenih IC50 vrijednosti pokazana je relativna selektivnost ekstrakta masline prema tumorskim stanicama u usporedbi s netumorskim HFF (IC50 = 0,68 ± 0,04 odnosno 0,75 ± 0,03 mg/ml) stanicama.
Zaključno, oba ekstrakta su pokazali visoki citotoksičnost, najveću na HeLa stanicama za što su zaslužne bioaktivne komponente lista masline, s naglaskom na polifenole. Ovime se pokazao potencijal ekstrakta lista masline kao selektivnog antikancerogenog agensa
Istraživanje agregacije izoformi CRMP1 povezanih sa shizofrenijom
No full textThe majority of chronic mental illnesses (CMI) involve risk factors that
contribute to the illness’s onset and do not have a single underlying cause.
No specific singular cause or mechanism for the development of mental
illnesses have been established yet.
Prior studies have revealed the role of protein aggregation as a potential
cause of chronic mental illness. CRMP1 is one of the most recently found
and least studied proteins whose aggregation is associated with mental
illness, particularly schizophrenia. Different versions of the CRMP1 Lv
isoform, with deletions of different parts of N-terminal region were explored
in this research to determine the influence of N-terminal region on
aggregation.
CRMP1 isoforms were expressed in vitro using HEK293 and SH-SY5Y cells
and examined using fluorescent microscopy and a quantitative blinded test
assay.
While previous studies have suggested CRMP1 Lv to be more prone to
aggregation than CRMP1 Sv, in this more relevant and through experiment
both CRMP1 Lv and CRMP1 Sv appear to have similar levels of aggregation.
Compared to other studies, a relatively small, FLAG
tag is used in this research, which is less likely to obstruct expression and
function of protein than GFP fusion proteins used previously, and using
human neuroblastoma cells SH-SY5Y provides a more realistic setting than
in prior research.
Although the results were not statistically significant, they do suggest that
the unique N-terminus region of CRMP1 Lv may be required for its
aggregation.
Thus, further research is needed, and it should include examining the effects
of proteasome inhibitor and/or introduction reactive oxygen species
triggering the cell stress. Co-aggregation with DISC1 should also be
investigated further with different isoforms of CRMP1 LvVećina mentalnih bolesti ima nekoliko rizičnih čimbenika koji pridonose
nastanku bolesti i nemaju jedan temeljni uzrok. Specifični pojedinačni
uzroci ili mehanizmi za nastanak mentalnih bolesti još uvijek nisu utvrđeni.
U prethodnim studijama prepoznata je uloga agregacije proteina kao
potencijalnog uzroka kroničnih mentalnih bolesti. CRMP1 je jedan od
nedavno otkrivenih i najmanje proučavanih proteina čija je agregacija
povezana s mentalnim bolestima, posebice shizofrenijom.
Istraženi su različiti izoformi CRMP1 Lv s delecijama različitih dijelova Nterminalne regije kako bi se odredio utjecaj N-terminalne regije na agregaciju. Izoforme CRMP1 eksprimirane su in vitro korištenjem HEK293 i
SH-SY5Y stanica te su proučavane korištenjem fluorescentne mikroskopije
i zaslijepljenog testa.
Dok su prethodne studije sugerirale da je CRMP1 Lv skloniji agregaciji u
usporedbi s CRMP1 Sv, u ovom, relevantnijem istraživanju čini se da i
CRMP1 Lv i CRMP1 Sv imaju slične razine agregacije. U usporedbi s drugim
studijama, relativno mala FLAG oznaka korištena u ovom istraživanju
smanjuje vjerojatnost ometanja ekspresije i funkcije proteina, a korištenje
stanica ljudskog neuroblastoma SH-SY5Y predstavlja prirodnije i pogodnije
uvjete za eksperiment, stoga su i dobiveni rezultati realističniji I relevantniji
u usporedbi s dosadašnjim istraživanjima.
Iako rezultati nisu bili statistički značajni, oni sugeriraju da je jedinstvena
N-terminalna regija CRMP1 Lv možda potrebna za njegovu agregaciju.
Stoga su potrebna daljnja istraživanja koja bi trebala uključivati ispitivanje
učinaka inhibitora proteasoma i/ili uvođenja reaktivnih vrsta kisika radi
izazivanja oksidativnog stresa. Daljnji koraci trebali bi uključivati i
ispitivanje koagregacije izoforma CRMP1 Lv s DISC1