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Обнова ћелијске популације Ustilago maydis након стреса и веза са заштитом генома – нови ћелијски фактори
Get PDFPromenljivost životne sredine je konstantna, te su živa bića morala razviti
različite strategije odgovora na nepovoljne sredinske uslove. Ove strategije se
mogu svrstati u dve glavne grupe: a) mehanizmi rezistencije/tolerancije i b)
mehanizmi obnove nakon jakog stresa. Efikasan mehanizam obnove populacije
nakon snažnog oksidativnog stresa je okarakterisan u jednoćelijskoj
bazidiomiceti, Ustilago maydis i zasniva se na proliferaciji preživelih ćelija
na račun biomolekula oslobođenih iz oštećenih i umrlih ćelija.1 Fenomen je
nazvan RUS (od engl. Repopulation under starvation, RUS) i do sada je
identifikovano 10 ćelijskih faktora uključenih u ovaj proces. Ova gljiva se
karakteriše i visokim stepenom otpornosti na UV i jonizujuće zračenje u čijoj
osnovi leži efikasna popravka DNK oštećenja. Interesantno je da su neki od
otkrivenih RUS faktora uključeni i u proces zaštite integriteta genoma.2 U
cilju otkrivanja novih faktora čija je funkcija vezana i za obnovu populacije i za
stabilnost genoma, primenjena su dva metodološka pristupa: pretraživanje
mutanata i analiza transkriptoma. Ovim pristupima je identifikovano 15
kandidata, a preliminarna analiza je potvrdila da su ovi ćelijski faktori
neophodni za oba procesa. Za dalji rad su selektovani kandidati sa
najizraženijim fenotipom i kroz molekularno-genetičke i biohemijske analize
će biti detaljnije okarakterisane njihove ćelijske funkcije.Променљивост животне средине је константна, те су жива бића морала развити
различите стратегије одговора на неповољне срединске услове. Ове стратегије се
могу сврстати у две главне групе: а) механизми резистенције/толеранције и б)
механизми обнове након јаког стреса. Ефикасан механизам обнове популације
након снажног оксидативног стреса је окарактерисан у једноћелијској
базидиомицети, Ustilago maydis и заснива се на пролиферацији преживелих ћелија
на рачун биомолекула ослобођених из оштећених и умрлих ћелија.1 Феномен је
назван RUS (од енгл. Repopulation under starvation, RUS) и до сада је
идентификовано 10 ћелијских фактора укључених у овај процес. Ова гљива се
карактерише и високим степеном отпорности на УВ и јонизујуће зрачење у чијој
основи лежи ефикасна поправка ДНК оштећења. Интересантно је да су неки од
откривених РУС фактора укључени и у процес заштите интегритета генома.2 У
циљу откривања нових фактора чија је функција везана и за обнову популације и за
стабилност генома, примењена су два методолошка приступа: претраживање
мутаната и анализа транскриптома. Овим приступима је идентификовано 15
кандидата, а прелиминарна анализа је потврдила да су ови ћелијски фактори
неопходни за оба процеса. За даљи рад су селектовани кандидати са
најизраженијим фенотипом и кроз молекуларно-генетичке и биохемијске анализе
ће бити детаљније окарактерисане њихове ћелијске функције.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022
Приказ мишјег моноклонског антитела у функцији алел специфичног антитела за FcγRIIA 166R
Get PDFMonoklonsko antitelo proizvedeno u mišu, označeno kao Y7, napravljeno je
protiv prirodnog IgM antitela, izolovanog iz osobe sa Valdenstromovom
makroglobulinemijom i specifičnog za jednolančanu DNK. Y7 antitelo je tom
prilikom detaljno okarakterisano i utvrđeno je da pripada podklasi IgG1 i da je
specifično za Fab region prirodnog IgM molekula, čime je antitelo Y7
okarakterisano kao anti-idiotipsko antitelo.1 Nedavno smo metodom protočne
citometrije otkrili da se Y7 antitelo vezuje za humane leukocite, konkretno za
granulocite i monocite. Vezivanje za granulocite i monocite različitih ljudi je
pokazalo individualne varijacije, te je pretpostavljeno da se radi o alel
specifičnoj interakciji, preko Fcγ receptora. Da bi se potvrdila ova
pretpostavka urađena je genotipizacija FcγR2A H166R polimorfizma, paralelno
sa analizom protočnom citometrijom (n = 20) u dve vremenske tačke. Vezivanje Y7
bilo je istovetno u dve vremenske tačke i u potpunosti se podudaralo sa
genetskom analizom, naime, potvrđeno je da je alelni polimorfizam u pitanju
H166R, sa vezivanjem Y7 za R varijantu. Vezivanje je detektovano i kod
heterozigotnih i homozigotnih osoba, dok kod H homozigotnih osoba nije bilo
vezivanja. Specifičnost ka alelskoj varijanti je potvrđena Vestern blotom.
Inhibicija vezivanja testirana je pomoću C-reaktivnog proteina, ali nije bilo
moguće postići više od 20% inhibicije. Neke alelske varijante genetskih
polimorfizama, gde spada i rs1801274 (H166R) polimorfizam FCGR2A gena,
povezane su sa bržom progresijom zaraznih bolesti, a neke varijante se učestalije
javljaju u autoimunskim obolenjima. Dodatna istraživanja pokazaće da li se
interakcija Y7 za R varijantu može upotrebiti pri izvođenju funkcionalnih
testova.Моноклонско антитело произведено у мишу, означено као Y7, направљено је
против природног IgM антитела, изолованог из особе са Валденстромовом
макроглобулинемијом и специфичног за једноланчану ДНК. Y7 антитело је том
приликом детаљно окарактерисано и утврђено је да припада подкласи IgG1 и да је
специфично за Fab регион природног IgM молекула, чиме је антитело Y7
окарактерисано као анти-идиотипско антитело.1 Недавно смо методом проточне
цитометрије открили да се Y7 антитело везује за хумане леукоците, конкретно за
гранулоците и моноците. Везивање за гранулоците и моноците различитих људи је
показало индивидуалне варијације, те је претпостављено да се ради о алел
специфичној интеракцији, преко Fcγ рецептора. Да би се потврдила ова
претпоставка урађена је генотипизација FcγR2A H166R полиморфизма, паралелно
са анализом проточном цитометријом (n = 20) у две временске тачке. Везивање Y7
било је истоветно у две временске тачке и у потпуности се подударало са
генетском анализом, наиме, потврђено је да је алелни полиморфизам у питању
H166R, са везивањем Y7 за R варијанту. Везивање је детектовано и код
хетерозиготних и хомозиготних особа, док код H хомозиготних особа није било
везивања. Специфичност ка алелској варијанти је потврђена Вестерн блотoм.
Инхибиција везивања тестирана је помоћу Ц-реактивног протеина, али није било
могуће постићи више од 20% инхибиције. Неке алелске варијанте генетских
полиморфизама, где спада и rs1801274 (H166R) полиморфизам FCGR2A гена,
повезане су са бржом прогресијом заразних болести, а неке варијанте се учесталије
јављају у аутоимунским оболењима. Додатна истраживања показаће да ли се
интеракција Y7 за R варијанту може употребити при извођењу функционалних
тестова.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022
Thrombosis risk assessment in patients with congenital thrombophilia during COVID-19 infection
No full textBackground: Coagulation dysfunction represents a serious complication in patients during the COVID-19 infec-tion, while fulminant thrombotic complications emerge as critical issues in individuals with severe COVID-19. In addition to a severe clinical presentation, comorbidities and age significantly contribute to the development of thrombotic complications in this disease. However, there is very little data on association of congenital thrombophilia and thrombotic events in the setting of COVID-19. Our study aimed to evaluate the risk of COVID-19 associated thrombosis in patients with congenital thrombophilia. Methods: This prospective, case-control study included patients with confirmed COVID-19 infection, followed 6 months post-confirmation. The final outcome was a symptomatic thrombotic event. In total, 90 COVID-19 pa-tients, 30 with known congenital thrombophilia and 60 patients without thrombophilia within the period July 2020-November 2021, were included in the study. Evaluation of hemostatic parameters including FVIII activity and D-dimer was performed for all patients at 1 month, 3 months and 6 months post-COVID-19 diagnosis. Results: Symptomatic thrombotic events were observed in 7 out of 30 (23 %) COVID-19 patients with throm-bophilia, and 12 out of 60 (20 %) without thrombophilia, P = 0.715. In addition, the two patient groups had comparable localization of thrombotic events, time to thrombotic event, effect of antithrombotic treatment and changes in FVIII activity, while D-dimer level were significantly increased in patients without thrombophilia. Conclusion: Our findings suggest that patients with congenital thrombophilia, irrespective of their age, a mild clinical picture and absence of comorbidities, should receive anticoagulant prophylaxis, adjusted based on the specific genetic defect
Tenascin-C fibronectin D domain is involved in the fine-tuning of glial response to CNS injury in vitro
Get PDFUnderstanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an in vitro injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-alpha and IL-1 beta upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of beta 1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area
Antimicrobial Nanocomposites Based on Oxidized Cotton Fabric and in situ Biosynthesized Copper Oxides Nanostructures Using Bearberry Leaves Extract
No full textThe aim of this study was to develop antimicrobial nanocomposite textile material comprising of Cu-based nanostructures synthesized on oxidized cotton fabric using Arctostaphylos uva-ursi (L.) Spreng., Ericaceae (bearberry leaves) as a green reducing agent for adsorbed Cu2+-ions. In order to provide sufficient number of carboxyl groups for complexation with Cu2+-ions a two-step oxidation process with NaIO4 and NaClO2 was carried out. The influence of NaIO4 concentration on content of carboxyl groups and Cu-based nanoparticles was studied by FTIR and AAS. HPLC analysis identified the gallic acid known as a reducing agent in bearberry leaves extract. FESEM and XRD analyses revealed that using bearberry leaves extract and gallic acid solution as reducing agents led to a formation of spherical Cu2O/CuO nanoparticles and CuO nanosheets, respectively. These nanoparticles and nanosheets provided excellent antibacterial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. Cytotoxicity on human keratinocyte cells was shown to depend on their copper content
High pollen immigration but no gene flow via-seed into a Genetic Conservation Unit of the endangered Picea omorika after disturbance
No full textStudying how genetic diversity is transmitted between generations is critical for assessing the survival prospects of fragmented forest tree populations. This is especially urgent in the case of rare, endangered species that are at high risk of extinction and/or exceptionally susceptible to climate change, and are thus to be prioritised for conservation initiatives. We performed the first assessment of gene flow patterns by parentage analysis in the IUCN red-listed Serbian spruce [Picea omorika (Pan.c.) Purk.], within a Genetic Conservation Unit (GCU) which was exposed to fire disturbance 30 years ago. Parentage analysis was based on assigning the parentage of 104 juveniles to all the adult trees (48) present in the 2.8 ha population. All individuals were genotyped at 11 highly informative nuclear microsatellite markers. We found that fire disturbance triggered natural regeneration, usually poor to absent in Serbian spruce, and boosted pollen immigration (66%), which is reported as rather modest among undisturbed populations of this species. Seed immigration was absent, coherently with what was previously hypothesised for natural, undisturbed populations. Thus, the immigration of new gametes into the studied Serbian spruce population was accomplished exclusively through pollen gene flow, at a rate sufficient to counteract, at least in the short term, genetic drift. Furthermore, the abundant post-disturbance pollen immigration most likely accounts for the lack of fine-scale spatial genetic structure (SGS) in the juvenile cohort, contrary to the SGS observed in the adult cohort which is amongst the strongest ever recorded in anemophilous forest tree species. Finally, as generally found in forest trees, individual reproductive success was uneven among individuals, with 25% of local adults siring 70% of the sampled juveniles. Our results on post-disturbance gene flow patterns in the only Serbian spruce population with reportedly abundant regeneration complement the outcomes of previous genetic studies while providing essential information for the dynamic management and conservation of this rare and endangered species severely threatened by climate warming. Tracking the temporal development of gene flow parameters and the consequences of gene flow on genetic diversity are two cornerstones of future forest genetic monitoring programs, which are particularly important for GCUs. In particular, monitoring the genetic diversity of forest regeneration is deemed as the key for understanding changes that otherwise would remain mostly untraceable in long-lived organisms such as forest trees
A polyesterase from the Antarctic bacterium Moraxella sp. degrades highly crystalline synthetic polymers
No full textThe uncontrolled release of plastics in the environment has rendered them ubiquitous around the planet, threatening the wildlife and human health. Biodegradation and valorization of plastics has emerged as an ecofriendly alternative to conventional management techniques. Discovery of novel polymer-degrading enzymes with diversified properties is hence an important task in order to explore different operational conditions for plastic-waste upcycling. In the present study, a barely studied psychrophilic enzyme (MoPE) from the Antractic bacterium Moraxella sp. was heterologously expressed, characterized and its potential in polymer degradation was further investigated. Based on its amino acid composition and structure, MoPE resembled PET-degrading enzymes, sharing features from both mesophilic and thermophilic homologues. MoPE hydrolyzes nonbiodegradable plastics, such as polyethylene terephthalate and polyurethane, as well as biodegradablePeer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1630
Hydrogel Scaffolds Based on Alginate, Gelatin, and 2-Hydroxyethyl Methacrylate for Tissue Regeneration
No full textThe design of bioactive scaffolding materials with favorable properties is paramount for successful application in biomedical engineering. Polymeric hydrogels attract significant attention as leading candidates for scaffold engineering due to their specific compositional and structural similarities to the natural extracellular matrix. The ability to control porosity, surface morphology, and size of hydrogel scaffolds has created new approaches to overcome various issues in tissue engineering such as vascularization, tissue architecture, and simultaneous multiple cells seeding. This review imparts an overview of hydrogel scaffolds based on synthetic and natural polymeric components (alginate, gelatin, and 2-hydroxyethyl methacrylate). We made hydrogel scaffolds with unique properties. Their in vitro and in vivo biological response, morphology, mechanical properties, porosity, hydrophilicity, and degradability were tested to find optimal patterns of tissue regeneration
Insulin expression and action in the central nervous system
Get PDFNakon njegovog otkrića početkom dvadesetog veka, dugo je smatrano da se insulin isključivo sintetiše
u pankreasu i da u centralni nervni sistem (CNS) dospeva prolaskom kroz krvno-moždanu barijeru.
Međutim, razvojem molekularno-bioloških metoda potvrđeno je prisustvo iRNK za preporoinsulin, samog
(prepro)insulina, kao i C-peptida u nekim regionima mozga. Time je višestruko potvrđeno da se insulin,
osim u pankreasu, sintetiše i u CNS. Nakon otkrića da se i receptor za insulin eksprimira u CNS, ispitivanja
su bila usmerena ka upoznavanju dejstva ovog hormona u mozgu. Pokazano je da je, između ostalog,
uključen u kontrolu apetita, regulaciji reprodukcije, ali i procesa vezanih za učenje i konsolidaciju memorije.
Takođe je utvrđeno da su poremećaji dejstva insulina u CNS povezani sa razvojem nekih neurodegenerativnih
bolesti.
Iako je većina pomenutih efekata prvobitno pripisana insulinu koji iz sistemske cirkulacije dospeva u
CNS, sve više se dizajniraju studije koje su orijentisane isključivo ka upoznavaju funkcije insulina koji nastaje
u mozgu. Do sada je pokazano da se produkcija insulina u hipotalamusu smanjuje tokom stresnih situacija,
što se negativno odražava na sintezu hormona rasta u adenohipofizi i njegovu sekreciju u sistemsku
cirkulaciju. Nasuprot tome, pokazano je da kratkotrajno gladovanje povećava ekspresiju insulina u neuronima
istog moždanog regiona ali i da ovaj fenomen nije povezan sa povećanjem stope ugradnje glukoznih
transportera u ćelijsku membranu, niti sinteze glikogena u astrocitima. Biće potrebno sprovesti
dodatne studije kako bi se u većoj meri upoznala uloga insulina koji nastaje unutar CNS.After being discovered in the early twentieth century, insulin was long considered to be exclusively
produced in the β-pancreatic cells. Therefore the presence of this hormone in the central nervous system
(CNS) was accounted for solely by its transport across the blood-brain barrier. However, owing to the advances
in molecular biological techniques, it was established that the preproinsulin mRNA, the (pre)prounsulin
peptide and C-peptide were also present in the CNS parenchyma. These findings unequivocally proved
that, in addition to the pancreas, insulin was also produced in the CNS. After discovery that the insulin receptor
was also expressed in the brain, numerous studies aimed to discover the insulin actions within the
CNS. It was found that insulin was involved in the control of appetite, reproduction, learning and memory
consolidation. Moreover, it was confirmed that the impairment of insulin signaling within the certain brain
regions was associated with various neurodegenerative disorders. Although these effects were initially assigned
to the action of the insulin transported from the circulation into the CNS, more studies were designed
to uncover the roles of insulin produced in the CNS. It has been shown so far that stress decreases
hypothalamic insulin production which subsequently results in the decreased growth hormone production
and its secretion form adenohypophysis. Furthermore, short-term fasting increased hypothalamic insulin
expression in rats, albeit this phenomenon did not result in either increased glucose uptake nor
glycogen production in this brain region. More studies are needed to fully elucidate the role of the centrally
produced insulin
The usage of silicon fertilisation in order to mitigate the oxidative stress and to improve the resilience of barley subjected to drought
Get PDFDrought causes huge agricultural and economic losses worldwide. Silicon (Si) is considered abeneficial element for plants. It mitigates stress caused by salinity, drought, and high and low temperaturesby promoting antioxidant production. However, the underlying mechanisms are still notelucidated. We investigated Si fertilisation effect on photosynthetic parameters, trichome number,leaf optical properties, as well as profiles of amino acids and polyphenols in barley exposed towater shortage. Silicon was applied in three growth stages: (i) before the flag leaf emergence;(ii) prior to the grain filling phase; (iii) at the grain filling phase start. Drought negatively impactsphotochemical efficiency, stomatal conductance, photosynthetic pigment content, and leaf reflectiveand transmissive properties. Si application between flag leaf emergence and grain fillinghad the strongest effect on light reflectance. Among all analyzed phenolics, saponarin was themost abundant in all samples, irrespective of water regime and Si supply. Caffeoyl ester was theonly hydroxycinnamic acid showing significant accumulation with the latest applied Si comparedto no added Si upon drought. The major amino acids in barley leaves were glutamate, glutamine,aspartate, asparagine, and serine. Aspartate content was the highest in leaves exposed to droughtwithout Si addition, while lysine was the most accumulated in the leaves supplemented by Si atthe grain filling phase start. Proline was 2.5 times more abundant in the leaves exposed to droughtregardless of Si treatment. Taken together, although Si did not mitigate drought stress effects, itseffect was dependent on the barley growth phase prior to supplementation.Book of Abstracts / 4th International Conference on Plant Biology [and] 23rd SPPS Meeting, 6-8 October 2022, Belgrad