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Optimization of isolation of coagulation factor IX
Get PDFZgrušavanje krvi odvija se nizom reakcija, a ključni je događaj aktivacija faktora zgrušavanja. Ukoliko je neki od faktora deficijentan ili ne može izvršavati svoju funkciju, neće doći do te važne fiziološke reakcije. Najčešće genetske bolesti povezane sa defektom zgrušavanja krvi su hemofilija A i B. Hemofilija A je češća i uzrokovana je nedostatkom ili defektom faktora VIII. Deficijencija faktora IX (FIX) uzrok je hemofilije B. Ova bolest je znatno rjeđa od hemofilije A, ali su simptomi ovih genetskih poremećaja jako slični. Kod nedostatka jednog od ova dva faktora dolazi do krvarenja u mišiće, zglobove i u mekom tkivu, što predstavlja problem prilikom ozljeda ili tijekom operacija. Kao terapija koristi se intravenozna infuzija deficijentnog faktora, koji se dobiva iz humane plazme ili metodom rekombinantne DNA.
Proces kojim se FIX izolira iz humane plazme uključuje različite tipove kromatografije kojima se postepeno uklanjaju ostale komponente plazme, dok se kao krajnji produkt ne dobije pročišćeni koncentrat FIX. Prilikom purifikacije FIX iz humane plazme, prinos i koagulacijska aktivnost FIX opada sa svakim korakom. Stoga je potrebno optimizirati postupak tako da se koncentrat FIX dobije u što manje koraka i sa maksimalnim prinosom.
U ovom radu je u prvom koraku sistematski ispitano nekoliko omjera mase matriksa i volumena plazme, kako bi se odredila koncentracija plazme pri kojoj dolazi do potiskivanja komponenata uzorka (engl. sample displacement), odnosno vezanja proteina prisutnih u niskim koncentracijama u plazmi za nosač. Uvjet je da ti proteini imaju znatno viši afinitet prema kromatografskom nosaču od onih koji dolaze u višoj koncentraciji, ali se vežu sa znatno nižim afinitetom. U ovom koraku je korišten poprečno vezani dekstran (Sephadex), koji je dodan plazmi u različitim koncentracijama. Ispitane su dvije vrste poprečno vezanog dekstrana različitih ionskih jakosti – DEAE (dietilaminoetil) Sephadex, i QAE (kvartarni aminoetil) Sephadex. U svrhu određivanja optimalnog
omjera između količine plazme i dodanog poprečno vezanog dekstrana (Sephadex), ionski izmjenjivač je odvojen centrifugiranjem i vezani proteini su eluirani otopinom NaCl. U sljedećem koraku je eluirani materijal dijaliziran i ponovo nanesen na različite kolone punjene anionskim izmjenjivačima. Te su kolone ili punjene zrnastim materijalom ili su korištene monolitne kolone sa anionskim izmjenjivačima različite jakosti – QAE i DEAE koji vežu FIX i prateće proteine različitim intenzitetom. Nakon svakog kromatografskog postupka provedena je elektroforeza na poliakrilamidnom gelu uz natrij dodecil-sulfat (SDS-PAGE) kako bi se odredio proteinski sastav frakcija. Razvijene su Western blot metode za potvrdu prisutnosti FIX, te glavnog kontaminanta, faktora X (FX) u pojedinim frakcijama.
Naši rezultati sugeriraju da pri optimalnom omjeru između količine plazme i količine poprečno vezanog dekstrana (Sephadex) dolazi do potiskivanja komponenata uzorka, što znači da komponente koje se sa višim afinitetom vežu na anionski izmjenjivač (u ovom slučaju poprečno vezani dekstran - Sephadex) potiskuju one vezane nižim afinitetom. Ovaj postupak i kromatografske kolone korištene u ovom istraživanju mogu se koristiti u izolaciji FIX iz humane plazme, te pomoći u daljnjoj optimizaciji procesa purifikacije ovog važnog terapeutskog proteina.Blood clotting takes place through a series of reactions, and the key one is activation of coagulation factors. If any of the factors in the clotting cascade is deficient or if it cannot perform its function, this important physiological reaction will be disturbed. Two most common genetic disorders linked to the blood clotting defect are hemophilia A and B. Hemophilia A, caused by factor VIII deficiency or defect, is more prevalent. Deficiency of coagulation factor IX (FIX) is the cause of hemophilia B. Its prevalence is significantly lower than hemophilia A, but the symptoms are similar. In case of deficiency of one of these factors, bleeding in the muscles, joints and soft tissues occurs, presenting a problem in injuries and during operations. As a therapy, intravenous infusion of FIX is given to the patient. Therapeutic FIX can be derived from human plasma or by recombinant DNA method.
The process of FIX isolation from human plasma involves various types of chromatography, by which other plasma components are removed gradually until the final product is obtained in form of purified FIX concentrate. During the purification process, the yield and enzymatic activity of FIX tend to decrease after each step. It is therefore necessary to optimize the process, so that FIX concentrate is obtained in as few steps as possible, and the maximal yield is achieved.
In this study, several matrices added to human plasma in different rations were tested, to determine the plasma concentration at which the sample displacement occurs. Under these conditions, low abundance plasma proteins bind to the matrix. This will happen only if these proteins bind to the matrix with significantly greater affinity than high abundance proteins. In this step, cross linked dextran (Sephadex) is added to plasma at different concentrations. Two types of Sephadex with different ionic strength were tested – diethylamino ethyl (DEAE) and quaternary (QAE) Sephadex. To determine the optimal Sephadex to plasma ratio, the anion exchanger was separated by centrifugation, and bound proteins were
eluted with a NaCl solution. In the next step, eluted material is dialyzed and reapplied to different columns packed with anion exchange materials. These columns are either packed with granular material or with a monolithic support with anion exchangers of different strength - QAE or DEAE, binding FIX and the accompanying proteins with different intensity. After each chromatographic step, electrophoresis on polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) was performed to determine protein composition. Western blot methods were developed to confirm the presence of FIX and its main contaminant, factor X (FX), in certain fractions.
Our results document that an optimal plasma to Sephadex ratio leads to sample displacement, meaning that the compounds which bind to the anionic exchanger (in this case Sephadex) with higher affinity, replace the compounds that bind with lower affinity. This procedure, as well as the columns used in this study can be used in FIX isolation from human plasma, which can help in further optimization of the purification process of this therapeutically important protein
IMMUNOTHERAPEUTIC APPROACHES FOR ALZHEIMERS DISEASE
No full textAlzheimerova bolest (AB) je progresivna neurodegenerativna bolest koja kulminira teškom demencijom. Pod pojmom demencija se smatra gubitak kognitivnih sposobnosti koji je kroničan i ireverzibilan. Može se javiti u bilo kojoj dobi, ali uglavnom zahvaća starije. AB zahvaća oko 4% bolesnika u dobi od 65 do 74 godina te 30% onih iznad 85 godina. Pacijenti inicijalno imaju probleme s gubitkom kratkotrajne memorije, a zatim slijedi zbunjenost, agitacija, poremećaji u ponašanju te potpuni gubitak kognitivnih sposobnosti. AB je multifaktorijalna bolest, što znači da ima genetsku i okolišnu pozadinu. Bolest se javlja u dva oblika, nasljednom i sporadičnom, a sporadični oblik može također imati genetsku podlogu u svom razvoju. Mutacije u tri različita gena povezane su s obiteljskom poviješću bolesti: u genu koji kodira za amiloid-β prekursorski protein (APP), zatim u genu za PSEN1 (Presenilin 1) i genu za PSEN2 (Presenilin 2). Sporadični oblik, koji se još naziva i kasni oblik bolesti se javlja nakon 65. godine života te je najčešći oblik Alzheimerove bolesti. Polimorfizam gena za apolipoprotein E, potvrđen je kao rizični čimbenik. Patohistološko obilježje AB su pojava plakova amiloida-β (Aβ) i neurofibrilarnih čvorova u kojima se nakuplja fibrilarni protein Tau. Postoji nekoliko hipoteza o patologiji Alzheimerove bolesti. Sve više prihvaćen model za razvoj novih terapija je amiloidna hipoteza koja kaže da je odlaganje Aβ glavni uzrok Alzheimerove bolesti, nakon čega slijedi nakupljanje Tau kao izravna posljedica ove akumulacije. Ova hipoteza sugerira da terapije otklanjanja Aβ mogu biti korisne u liječenju Alzheimerove bolesti. Ovdje ću opisati četiri različita antitijela koja se ispituju i koja ciljaju Aβ. Također, ovaj rad opisuje protutijela koja ciljaju Tau, kao jedan od glavnih ciljeva terapije Alzheimerove bolesti. Nakon desetljeća istraživanja ove bolesti, lijek koji bi je izliječio i dalje ne postoji. Korištenje imunoterapije u Alzheimerovoj bolesti je izazvalo veliko zanimanje. Iako dosadašnji pokušaji još nisu bili toliko uspješni u
Comment [JB1]: Zar se ne podrazumijeva da nasljedni oblik bolesti ima genetsku podlogu? Možda bi logičnije bilo da se to kaže samo za sporadični oblik.
usporavanju napredovanja bolesti, uspjeli su značajno smanjiti nakupljanje Aβ. Sve u svemu čini se da je imunoterapija jedno od najvećih dostignuća u borbi protiv ove teške bolesti i niz pokušaja poboljšanja te terapije su u tijeku.Alzheimer's disease (AD) is a progressive neurodegenerative disease that culminates with severe dementia. Dementia is a loss of cognition and it is chronic and irreversible. It can occur at any age, but mostly affects the elderly. It affects approximately 4% of patients aged 65 to 74 and 30% of those older than 85 years. Patients initially have problems with the loss of short-term memory, followed by confusion, agitation, behavioral disorders, and severe loss of cognitive abilities. AD is a multifactorial disease, meaning that it has a genetic and environmental background. The disease occurs in two forms; familiar and sporadic, and sporadic form can also have a genetic basis in development. Mutations in three different genes are linked to the family history of the disease; mutations in the gene encoding the amyloid-β APP protein precursor, then mutations in PSEN1 gene (Presenilin 1) and gene for PSEN2 (Presenilin 2). The sporadic form, also known as the late form of the disease, occurs after age of 65 and is the most common form of Alzheimer's disease. Polymorphism of the Apolipoprotein E gene, was confirmed as a risk factor. Characteristic features of AD are the appearance of amyloid-β (Aβ) plaques and neurofibrillary tangles, made of fibrillar protein Tau. There are several hypotheses about the pathology of AD. More accepted model for development new therapies is the amyloid hypothesis that says that deposition of amyloid β protein (Aβ) is the main cause of
Comment [JB2]: Ako je niz pokušaja, onda je jednina.
Comment [JB3]: Ja bi sve stavila malim slovom
Alzheimer disease which is followed by the accrual of tangles as the direct consequence of this accumulation. This hypothesis strongly suggests that targeting Aβ could be beneficial for treatment of AD. Here I report four different antibodies that are being tested and which target Aβ. Also, this thesis describes antibodies that target Tau, as one of the main goals in immunotherapy of AD. After decades of investigating AD, no cure is currently available for this disease. The use of immunotherapy in AD has caught some serious attention. Although attempts that have been made so far have not yet been successful in changing the disease course, they were able to substantially diminish Aβ load. Overall, it seems that immunotherapy is one of the most promising achievements in the fight against this devastating disease and efforts to improve it are undergoing
Localization and expression of heterogenic nuclear ribonuclearproteins after virus infection and stress
No full textHeterogeni nuklearni ribonukleoproteini (hnRNPs) su obitelj RNA-vezujućih proteina s bitnim ulogama u različitim aspektima metabolizma nukleinskih kiselina, kao i u regulaciji mnogih signalnih puteva u stanici. Herpes simplex virus 1 (HSV-1) važan je i čest patogen ljudi, koji najčešće dovodi do asimptomatske ili blage bolesti kao što su oralne ili facijalne lezije, ali može uzrokovati i ozbiljnija oboljenja poput encefalitisa i meningitisa. Dosadašnja istraživanja pokazala su interakciju hnRNPs s komponentama različitih virusa, uključujući HSV-1, kao i promjenu lokalizacije hnRNPs tijekom infekcije, no precizni molekularni mehanizmi ovih interakcija još uvijek nisu u potpunosti razjašnjeni. U ovom istraživanju metodama imunofluorescencije i Western blota ispitali smo dolazi li do translokacije i promjene ekspresije hnRNP proteina tijekom infekcije HSV-1, s posebnim naglaskom na multifunkcionalni protein hnRNP K. Lokalizaciju i promjenu ekspresije hnRNP također smo istražili nakon tretmana stanica farmakološkim aktivatorima interferonskog odgovora tipa I: IFN-β, LPS-om i poly I:C, kao i aktivatorom autofagije rapamicinom, te aktivatorom apoptoze staurosporinom. HSV-1 infekcija uzrokovala je translokaciju hnRNP K proteina iz jezgre u citoplazmu, što nije primijećeno kod nijednog od primijenjenih tretmana. Također, ekspresija hnRNP ostala je nepromijenjena tijekom infekcije, dok su svi farmakološki tretmani uzrokovali smanjenje ili povećanje razine ekspresije ispitivanih proteina hnRNP K, hnRNP E2 i hnRNP U. Usporedbom ekspresije i lokalizacije hnRNP proteina u HSV1 infekciji i nakon farmakoloških tretmana, ovaj rad pridonosi boljem razumijevanju načina na koji ovi proteini sudjeluju u kompleksnoj interakciji virusa i domaćina.The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins with important roles in different aspects of nucleic acid metabolism, as well as in the regulation of various cell signalling pathways. Herpes simplex virus 1 (HSV-1) is an important and widespread human pathogen that in most cases causes asimptomatic or mild disease such as oral or facial lesions, but can sometimes cause more serious conditions, like encephalitis and meningitis. Previous experiments have shown that hnRNPs interact with components of various viruses, including HSV-1, and that virus infection causes changes in subcellular localization, but the precise molecular mechanisms of these interactions are yet not understood. In this study, using immunofluorescence and Western blot, we aimed to test whether the localization and expression levels of hnRNPs change during HSV-1 infection, with special attention to the multifunctional hnRNP K protein. We also investigated changes in subcellular localization and expression levels in cells treated with pharmacological activators of type I interferon response: IFN-β, LPS and poly I:C, as well as with autophagy inducer, rapamycin and activator of apoptosis, staurosporine. HSV-1 infection caused hnRNP K translocation from the nucleus to the cytoplasm, which was not the case when cells were treated pharmacologically. Expression levels of hnRNPs stayed unchanged during the course of HSV-1 infection, but pharmacological treatments caused either a decrease or increase in expression of hnRNP K, hnRNP E2 and hnRNP U. By comparing the localization and expression of hnRNPs during HSV1 infection and farmacological treatments, this study contributes to better understanding of how these proteins participate in the complex interaction between virus and host organisms
Sinteza tieno[2,3-d]oksepinskog derivata kao prekursora za daljnji razvoj molekula s potencijalnim protuupalnim uĉinkom
No full textOpisana je sinteza etil-9-metoksi-4,5-dihidrobenzo[b]tieno[2,3-d]oksepin-2-karboksilata (5).
Ključni intermedijar za sintezu toga spoja je biciklički keton (3) dobiven intramolekulskom
ciklizacijom 4-(4-fenoksimetoksi)butanske kiseline (2). Reakcijom ketona s Vilsmeierovim
reagensom nastao je B -klorvinilni aldehid (4) kojim je u reakciji ciklizacije s etil-2-
merkaptoacetatom pripravljen tieno[2,3-d]oksepinski derivat (5).Synthesis of ethyl 9-methoxy-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxylate (5) is
described. The key intermediate was bicyclic ketone (3) formed by intramolecular cyclization
of 4-(4-methoxyphenoxy)butanoic acid (2). Reaction of ketone with Vilsmeier reagent
provided B -chlorovinyl aldehyde (4) that readily cyclized with ethyl 2-mercaptoacetate to form
thieno[2,3-d]-fused benzo-oxepine (5)
Usage and sugar levels in meat products
No full textRazličiti šećeri poput glukoze, fruktoze, saharoze, laktoze koriste se u proizvodnji fermentiranih mesnih proizvoda. Njihova glavna uloga je da djeluju kao supstrati za mliječne bakterije za proizvodnju mliječne kiseline, no odgovorni su i za specifičnu aromu proizvoda. Mliječna kiselina je neophodna u razradi mesnog nadjeva tijekom fermentacije. Fermentacija je metoda konzerviranja mesa za koju je karakterističan porast broja bakterija mliječne kiseline, glikolitička razgradnja šećera te sniženje pH vrijednosti. Niski pH djeluje kao antagonist patogenim bakterijama. Na sniženje te brzinu pada pH vrijednosti utječu maseni udio te vrsta šećera. Unatoč važnosti šećera kao dodatka u mesnim proizvodima, njihova dopuštena razina nije propisana nego varira ovisno o proizvodu.
U ovom istraživanju, analizirano je ukupno 140 uzoraka triju različitih skupina mesnih proizvoda. Unutar svake od skupina određeni su udjeli šećera: saharoze, D-glukoze i D-fruktoze. Skupine su sistematizirane sukladno Pravilniku o mesnim proizvodima (NN 131/2012) te je u istraživanju korištena akreditirana enzimatska metoda za određivanje šećera u mesnim proizvodima. Unutar skupine trajnih kobasica analiziran je 41 uzorak, toplinski obrađenih kobasica 82 uzorka te trajnih suhomesnatih proizvoda 17 uzoraka.
U ovom radu utvrđen je najveći udio saharoze unutar svake od ispitivanih skupina mesnih proizvoda u rasponu od 0,21-0,87%, nakon toga fruktoze u rasponu od 0,10-0,39%, a, najmanji udio dobiven je za glukozu, u rasponu od 0,05-0,06%. Najveću vrijednost udjela šećera je imala saharoza unutar skupine toplinski obrađenih kobasica u iznosu od 0,87%, a najmanji udio glukoza unutar skupine trajnih kobasica u iznosu od 0,05%. Unutar skupine toplinski obrađenih kobasica udio ukupnih šećera iznosio je 1,32%, trajnih kobasica 0,36%, a trajnih suhomesnatih proizvoda 0,47%.Different sugars such as glucose, fructose, sucrose, lactose are used in the production of fermented meat products. Their main role is to act as a substrate for the lactic acid bacteria, also they are responsible for the specific flavour of the product. Lactic acid is essential for the development of meat batter during fermentation. Fermentation is a method of meat preservation characterized by an increase in the number of lactic acid bacteria, glycolic sugar degradation and lower pH. Low pH acts as antagonist for pathogenic bacteria. Reduction and the rate of pH value decrease are affected by the weight percent and type of sugar. Despite the importance of sugar as an additive in meat products, the permitted level is not prescribed but varies depending on the product.
In this study a total of 140 samples of three different groups of meat products were analyzed. Within each group, sucrose, D-glucose and D-fructose were determined. The groups were systematised in accordance with the Ordinance on Meat Products (NN 131/2012) and an accredited enzymatic method for determination of sugar in meat products was used in the research. Within the gruoup of durable sausages, 41 samples, heat-treated sausages 82 samples and dry-cured meat products 17 samples were analyzed.
In this research the highest value within each of the tested groups was obtained for sucrose (0,21 to 0,87%), after that for fructose (0,10 to 0,39%) and the smallest value for glucose (0,05 to 0,06%). The highest sugar content was obtained for sucrose fraction within the group of heat-treated sausages in the amount of 0,87%, and the lowest glucose content within the group of durable sausages in the amount of 0,05%. Within the group of heat treated sausages the percent of total sugars was 1,32%, durable sausages 0,36%, and permanent cured meat products 0,47%
Synthesis and characterization supramolecular hydrogels for the drug delivery and controlled relase
No full textNovi sustavi prijenosa i kontrolirane isporuke lijekova se razvijaju s ciljem
poboljšavanja učinkovitosti lijekova i stavljanja pod nadzor postojećih
nuspojava. Sintetizirana su dva tripeptidna gelatora 1. organogelator Z-LPhe-
L-Phe-L-AlaNH2 i 2. hidrogelator Ac-L-Phe-L-Phe-L-AlaNH2 s ciljem
studiranja njihovih potencijala za skladištenje i kontrolirano otpuštanje
nesteroidnih antireumatika (NSAR). Nakon dugotrajne oralne uporabe NSAR,
poput ibuprofena i acetilsalicilne kiseline mogu se pojaviti ozbiljne nuspojave
u gastrointestinalnom traktu. Transdermalna kontrolirana isporuka lijekova
pomoću gelova predstavlja alternativnu metodu primjene lijeka pomoću koje
se zaobilaze nepoželjni učinci.
Za tekuću komponentu organogela odabrano je sojino ulje, a za tekuću
komponentu hidrogela vodeni fosfatni pufer. U tripeptidne gelove su
ugrađeni ibuprofen i acetilsalicilna kiselina, te se pratilo otpuštanje lijekova
ovisno o vremenu u fosfatni pufer pomoću UV-Vis spektroskopije.
Nadalje, proučavao se utjecaj pH prihvatnog medija na otpuštanje
ibuprofena iz organogela. Otpuštanje je mjereno pomoću dva različita
sustava. Kumulativno, uzimanjem uzorka kroz određen vremenski period i
određivanjem koncentracije lijeka pomoću UV Vis spektroskopije (u kiveti) ili
direktno optičkom sondom. Uspoređivani su rezultati dobiveni različitim
metodama mjerenja. Mehanokemijskom reakcijom hidrogelatora i ibuprofena
u kugličnom mlinu ispitivao se potencijal hidrogelatora za poboljšanje
topljivosti hidrofobnog lijeka u vodenoj otopini.
Rezultati pokazuju da su studirani gelatori veoma dobri mediji za skladištenje
navedenih lijekova. pH prihvatnog medija ima veliki utjecaj na otpuštanje
molekula ibuprofena. Alkalni medij je pogodniji za otpuštanje od kiselog. UVVis
spektrofotometrijska sonda je pogodna za praćenje otpuštanja u
stvarnom vremenu, no zbog zasićenja prihvatnog pufera ne postižu se visoke
stope otpuštanja. Kumulativnom metodom, premještanjem pola volumena
prihvatnog medija (0,1M fosfatni pufer, pH 7,4) u kivetu i dodatkom jednake
količine nove otopine prihvatnog medija, ne postiže se zasićenje prihvatne
otopine te su dobivene više stope otpuštanja. Mehanokemijskim miješanjem
hidrogelatora i lipofilnog lijeka, omogućeno je uvođenje lipofilnog lijeka u
vodenu fazu i olakšano samoudruživanje u hidrogelu.New controlled drug delivery systems are being developed to improve the
therapeutic efficacy of drugs and to keep under control existing side effects.
Two tripeptide gelators were synthesized 1. organogelator Z-L-Phe-L-Phe-LAlaNH2
and 2. hydrogelator Ac-L-Phe-L-Phe-L-AlaNH2 with the aim of
studying their potential to develop drug depot systems and controlled release
systems of nonsteroidal antireumatics (NSAR). After long-term oral
administration of NSAR, such as ibuprofen and acetylsalicylic acid, serious
side effects may occur in the gastrointestinal tract. Transdermal controlled
drug delivery by gels represents an alternative method of drug
administration by which unwanted effects are avoided.
For the liquid component of organogel a soybean oil was selected, and
phosphate buffer was selected for the hydrogel liquid component. Ibuprofen
and acetylsalicylic acid were loaded in the tripeptide gels and the release
behavior of drugs in phosphate buffer was investigated by using UV-Vis
spectroscopy. Further, the influence of the pH values of the accepting media
on the release behavior of ibuprofen from organogel was investigated.
Release behavior was measured by two different systems. Cumulative,
taking the sample through a certain period of time and determining drug
concentration using UV Vis spectroscopy (in cuvette cell) or directly using
optic probe. The results obtained by different measurement methods were
compared. The hydrogelator potential during the mechanochemical reaction
of hydrogelator and ibuprofen in the mill was studied in order to improve the
solubility of the hydrophobic drug in aqueous solution.
The results indicated that studied gelators are very good media for drug
depot systems. pH of the accepting media has a major impact on the release
behavior of ibuprofen molecules. Alkaline media is more suitable for release.
The UV-Vis spectrophotometric probe is suitable for real-time drug delivery
monitoring but due to saturation of the accepting buffer, high release rates
are not achieved. Cumulative method, by transferring the half volume of the
accepting media (0.1M phosphate buffer, pH 7.4) to a cuvette and adding
equal amounts of a new solution of the accepting media, the saturation of
the accepting solution is not achieved and higher release rates are obtained.
By mechanochemical mixing of hydrogelator and lipophilic drug, it is possible
to introduce a lipophilic drug into the aqueous phase and facilitate self
assembly in the hydrogel
Voltametrijske i spektroskopske studije interakcije DNA s riboflavinom, daunorubicinom i doksorubicinom
No full textRiboflavin (RF), vitamin B2 and anthracyclines, daunorubicin (DAU) and doxorubicin (DOX) showed the influence on electrochemical and spectral properties of the DNA molecule. The interactions were analysed using voltammetry, UV/VIS absorbance spectrophotometry and circular dichroism spectroscopy. These interactions were investigated by monitoring changes: in electrochemical properties of ctDNA and oligonucleotides interacting with RF on the surface of screen-printed carbon electrode (SPCE) surface using square-wave voltammetry (SWV), in UV/VIS absorption spectra of ctDNA, oligonucleotides and RF and in circular dichroism spectra of DNA in increasing concentration of RF and anthracyclines. The obtained results indicate that interactions between investigated compounds and DNA depend on the DNA sequence and the concentration of ligand. As the result of interactions, oxidation signals of guanine and adenine, absorbance spectra of DNA and RF, and CD spectra of oligonucleotides changes. RF binding to DNA causes decrease in number of exposed oxidative functional groups proved by decrease in oxidation signal of bases and lowered absorbance signal of DNA and RF mixture. CD investigations showed the influence of RF and anthracyclines on the DNA conformation. RF showed preference on AT regions while anthracyclines bind on different way in different DNA regions depending on concentration. At lower concentrations, they intercalate to GC and bind to the groove of AT regions while higher concentration induces conformational changes to Z-DNA.Riboflavin, vitamin B2 i antraciklini, daunorubicin (DAU) i doksorubicin (DOX) su pokazali utjecaj na elektrokemijska i spektralna svojstva DNA molekule. Interakcije su analizirane pomoću voltametrije, UV/VIS apsorpcijske spektrofotometrije i cirkularni dikroizam spektroskopije. Interakcije su analizirane praćenjem promjena u: elektrokemijskim svojstvima ctDNA i oligonukleotida u interakciji s RF na površini tiskane ugljikove electrode (SPCE) upotrebom kvadratno-valne voltametrije (SWV), UV/VIS apsorpcijskim spektrima ctDNA, oligonukleotida i RF te DNA spektru cirkularnog dikroizma u povećanju koncentracije RF i antraciklina. Dobiveni rezultati pokazuju da interakcije između DNA i ispitivanih spojeva ovise o slijedu DNA i koncentraciji liganda. Kao rezultat interakcija, mijenjaju se oksidacijski signali gvanina i adenine, apsorpcijski spektri DNA i RF te CD spektri oligonukleotida. Vezanje RF na DNA uzrokuje smanjenje broja izloženih oksidacijskih funkcionalnih skupina što je dokazano smanjenjem oksidacijskih signala baza te sniženim apsorcijskim signalom smjese DNA i RF. CD ispitivanja su pokazala utjecaj RF i antraciklina na DNA konformaciju. RF je pokazao veći afinitet prema AT regijama dok se antraciklini vežu na različite načine u različitim područjima DNA, ovisno o koncentraciji. Pri nižim koncentracijama, interkaliraju se na GC i vežu na utor AT regije, dok veća koncentracija izaziva konformacijske promjene u Z-DNA
Influence of different concentrations and combinations of antibiotics on survival of Escherichia coli in different media
No full textRezistencija na antibiotike koji se koriste za liječenje urinarne infekcije s
Escherichia coli (E. coli) postaje rastući problem diljem svijeta. Svrha rada
je utvrditi postoji li kombinacija antibiotika koja djeluje bolje od
pojedinačnih antibiotika u tretmanu E. coli. Preživljavanje bakterije mora
biti kratko da ne bi došlo do stvaranja mehanizma rezistencije. Veliki je
izbor lijekova koji se koriste u tretmanu urinarnih infekcija, a u radu su
izabrani lijekovi prema mehanizu djelovanja. Razlika između ampicilina,
ciprofloksacina, rifampicina i strepromicina je u metama djelovanja u
bakterijskoj stanici. U radu se ispitivao utjecaj različitih koncentracija i
kombinacija antibiotika: ampicillin-ciprofloksacin, ampicillin-rifampicin i
ampicillin-streptomicin, na preživljavanje E. coli. Kako bi se istražio
utjecaj količine nutrijenata u mediju na djelovanje antibiotika koristili su
se nutritivno bogat Luria-Bertani medij i nutritivno siromašan medij M9
Minimalne soli. Bakteriostatičko djelovanje antibiotika ispitalo se
određivanjem minimalne inhibicijske koncentracije (MIK) uz pomoć
krivulje rasta i antibiogramom, a krivuljom smrtnosti baktericidno
djelovanje antibiotika. Krivulja rasta bakterijske kulture E. coli utvrdila je
normalan rast bakterije prema dosadašnjim istraživanjima. Dodatkom
antibiotika u bakterijsku kulturu odredila se MIK ampicilina i
ciprofloksacina na 12,5 μg/ml, streptomicina na 25 μg/ml i rifampicina na
100 μg/ml što je u skladu s literaturnim vrijednostima. Kombinacije
antibiotika sastavljene od MIK svakog pojedinačnog antibiotika pokazuju
bolje i brže djelovanje od pojedinačnih antibiotika. Usporedbom krivulja
smrtnosti pojedinačnih antibiotika dokazalo se da količina nutrijenata u
mediju ne utječe na djelovanje antibiotika. Razlika u djelovanju
antibiotika primjećuje se ukoliko se usporede krivulje smrtnosti
kombinacija antibiotika na Luria-Bertani pločama i pločama M9 Minimalne
soli. Kombinacije antibiotika za razliku od pojedinačnih antibiotika ovise o
količini nutrijenata u mediju. Antibiogramom smo potvrdili iste rezultate
dobivene krivuljama smrtnosti. Rezultati dobiveni eksperimentalnim
radom postavljaju temelje za mnogobrojna istraživanja metabolizama
kombinacija antibiotika u budućosti.Resistance to antibiotics used to treat urinary tract infection with
Escherichia coli (E. coli) is becoming a growing problem worldwide. The
purpose of the paper is to determine whether there is a combination of
antibiotics that works better than the individual antibiotics in E. coli
treatment. Survival of the bacterium must be short to prevent the
formation of a resistance mechanism. There is a wide choice of medicines
used in the treatment of urinary infections, and in this work they are
selected according to the mechanism of action. The difference between
ampicillin, ciprofloxacin, rifampicin and strepromycin is the target of
action in the bacterial cell. The work we investigated the effect of various
concentrations and combinations of antibiotics: ampicillin-ciprofloxacin,
ampicillin-rifampicin and ampicillin-streptomycin, to survival of E. coli. In
order to investigate the effect of the amount of nutrients in the medium
on antibiotic activity, we used the nutrient-rich Luria-Bertani medium and
nutritionally poor medium M9 Minimal salts. By determining the minimum
inhibitory concentration (MIC) with the help of growth curves and
antibiograms, the bacteriostatic action of antibiotics was investigated, and
the mortality curve was bactericidal antibiotic activity. The E. coli bacterial
growth curve has established a normal growth of the bacterium according
to previous research. The minimum inhibitory concentration was
determined by the addition of antibiotics to bacterial culture. The
ampicillin and ciprofloxacin MICs are 12.5 μg / ml, streptomycin 25 μg /
ml and rifampicin 100 μg / ml, which is consistent with literature values.
Combinations of antibiotics made up of MICs of each individual antibiotic
showed better and faster action than individual antibiotics. By comparing
the kill curves of individual antibiotics, we proved that the amount of
nutrients in the medium does not affect the action of antibiotics.
Comparison of the kill curves of a combination of antibiotics on Luria-
Bertani plates and plates M9 Minimal salts suggests that there is a
difference in the action of antibiotics. Combinations of antibiotics, unlike
individual, depend on the amount of nutrients in the medium. We have
confirmed the same result of the kill curves obtained by the antibiogram.
The results obtained by experimental work lay the foundations for
numerous research into the metabolisms of antibiotic combinations in the
future
Molekularne osnove šizofrenije
No full textŠizofrenija je složeni neuropsihijatrijski poremećaj koji pogađa oko 1% populacije. Karakterizira ju prisutnost raznovrsnih i ozbiljnih simptoma koji se najčešće pojavljuju u ranoj odrasloj dobi, te narušavaju kvalitetu života pacijenata i njihovih obitelji. Unatoč dugotrajnom istraživanju neurobiološke osnove šizofrenije, jedinstvena patofiziologija bolesti i dalje nije definirana. S obzirom na slabo poznavanje temeljnih molekularnih mehanizama poremećaja, liječenje trenutno dostupnim antipsihoticima nije u potpunosti uspješno. Pronalazak novih pristupa i terapeutskih meta predstavlja krajnji cilj, no ujedno i značajan izazov znanstvenicima.
Dosadašnja istraživanja i prikupljeni rezultati ukazuju na činjenicu da je šizofrenija nasljedna bolest, te da se javlja kao rezultat djelovanja više gena kandidata, od kojih svaki ima mali pojedinačni učinak koji pridonosi podložnosti bolesti. Postoji opća suglasnost da različiti biološki, okolišni i psihosocijalni utjecaji, koji se nakupljaju tijekom života, mogu štetno djelovati na genetski podložnije pojedince te dovesti do razvoja psihičkog poremećaja. Istraživanja na životinjskim modelima ukazala su da značajnu ulogu u određivanju ishoda međudjelovanja gena i okoliša ima spol te precizan odabir pravog trenutka za izlaganje različitim utjecajima. Posljednjih godina pridaje se poseban značaj istraživanju epigenetike koja posreduje u složenom međudjelovanju okoliša i gena koje dovodi do pojave šizofrenije. Također, razvoj visokoprotočnih metoda analize poput genomike i proteomike, koje se koriste za globalnu karakterizaciju i kvantifikaciju bioloških molekula, predstavlja određenu revoluciju u molekularnoj neuropatologiji šizofrenije. Kako ove metode pružaju mogućnost razumijevanja uloge molekularnih puteva i međudjelovanja gena, a ne samo pojedinačnih gena kandidata, smatraju se obećavajućim alatom koji bi mogao omogućiti razumijevanje biokemijskih sustava uključenih u patofiziologiju šizofrenije.
U ovom radu prikazana su dosadašnja saznanja o šizofreniji, te opisane buduće smjernice u istraživanjima molekularne genetike šizofrenije. Detaljno su proučena brojna istraživanja i prikupljeni podaci kako bi se pružio uvid u najznačajnija otkrića koja bi mogla konačno razjasniti patofiziologiju bolesti, te pridonijeti pronalasku novih terapija za liječenje šizofrenije. Kao vrlo uspješna metoda pokazalo se korištenje integriranih pristupa u kojima se povezuje ono najbolje iz različitih studija, a vjeruje se da tehnologija matičnih stanica predstavlja jedan od perspektivnijih budućih terapeutskih pristupa ovoj teškoj bolesti.SUMMARY
Schizophrenia is a complex neuropsychiatric disorder that affects about 1% of the world population. It is characterized by the presence of various and serious symptoms, that mostly appear in early adulthood and lead to deterioration of the life quality of patients and their families. Despite many efforts to understand the neurobiological basis of schizophrenia, the unique pathophysiology of the disorder is still not defined. Given the poor insight into the underlying molecular mechanisms of the disorder, the treatment, using currently available antipsychotics, is not completely successful. Finding new approaches and therapeutic targets is the ultimate aim, which is a significant challenge for scientists.
Clinical studies and the collected data indicate that schizophrenia is a largely hereditary disease which depends on multiple candidate genes, each of which contributes with a small individual effect to the susceptibility of the disease. There is a general agreement that different biological, environmental and psychosocial impacts, which accumulate throughout life, may adversely affect genetically susceptible individuals and lead to the development of a mental disorder. Animal model research has shown that gender and choice of the right moment for exposure to different influences have a significant role in determining the outcome of gene and environment interaction. In recent years, special importance has been given to the epigenetic research that has the linking role and mediates the complex interaction between the environmental factors and genes leading to schizophrenia. Also, the development of high-throughput methods such as genomics and proteomics, used for global characterization and quantification of biological molecules, represent a revolution in the research of the molecular neuropathology of schizophrenia. Taking into account the fact that these methods provide an opportunity to understand not only the individual candidate genes but also the role of molecular pathways and the interaction of genes, they are considered to be a promising tool in enabling us to understand the biochemical systems involved in pathophysiology of schizophrenia.
In this work the present data related to molecular genetics of schizophrenia have been collected, as well as future guidance in this field of research. Numerous studies and data have been explored in order to provide insight into the most important findings that could serve to finally understand the pathophysiology of the disease, hopefully leading to the development of the new therapies for schizophrenia. The integrated approach, which combine the best of numerous different studies, is considered to be very promising, as well as stem cell technology, that is considered to be one of the most prospective research approaches for the development of the future new therapeutic approaches
An improved method for growing neurons: Comparison with standard protocols
Get PDFBackground: Since different culturing parameters – such as media composition or cell density – lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons – both in vivo and in vitro – is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes.
New method: Our culturing protocol is based on a novel serum-free preparation of astrocyte – conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7,14 and 60 days in vitro (DIV).
Results: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone’s size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7.
Comparison with existing method(s): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization.
Conclusions: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures