imagine (Institute of molecular genetics and genetic engineering)
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Physiological and gene expression changes during imbibition in maize seeds under low temperature conditions
Get PDFMaize is one of the most important crop species worldwide, but also extremely susceptible to the effects of increasingly higher temperatures and drought during the summer and its flowering and grain filling stage. Different strategies are being utilized to ensure a satisfying yield potential and quality even in the extremely unfavourable environmental conditions, which are the result of climate change. Some of them are cropping pattern changes and sowing alterations, including earlier sowing. Since this implies the exposure to suboptimal temperatures during early developmental stages, it leads to a demand for developing maize lines tolerant to low temperatures during these stages. This research focuses on the first phase of germination, imbibition. Maize tolerance to low temperatures is a complex trait that includes different mechanisms and strategies that all work together to ensure adaptation and survival, such as cell membrane changes, antioxidative system activation, etc. Additionally, the low temperature response of different maize inbreds varies substantially, so recognizing the different ways in which they respond to low temperatures during imbibition and other earlier stages of development is crucial. To accomplish this, seeds of two maize inbred lines of contrasting susceptibility to low temperatures were selected and exposed to control (20 degrees C) and low temperature (8 degrees C) conditions during the first 24h of imbition and then further analyzed to assess their response. This included germination and tetrazolium assays; ascertaining membrane integrity by evaluating cell leakage and lipid peroxidation; determining the antioxidative capacity by assessing superoxide dismutase (SOD) and catalase (CAT) activity; and expression analysis of four genes included in the low temperature response (gibberellin insensitive dwarf 1 gibberellin receptor, gid1; fatty acid desaturases 2 and 6, fad2 and fad6; plastid-lipid-associated 2 protein, pap2). The results showed that, while there is not a significant difference in their germination rate, they differ in their survival rate, with more seeds of the tolerant genotype surviving the low temperature period. Significant differences between them were found in cell leakage (p lt 0,01), as well as gid1(p lt 0,05) and fad6 (p lt 0,05) gene expression assays. The present research brings light to our understanding of the effect of low temperatures on the first germination stage, -imbibition. It highlights the importance of choosing the right inbreds for earlier sowing and points to certain routes that could be taken for improving and accelerating the breeding process for low temperature tolerance
Inter-donor variability in dendritic cells capacity to respond to stimulation in vitro associates with donors gut microbiota composition
No full textFunctionalization of polyamide microfiltration membranes by supercritical solvent impregnation
No full textThe study reports the application of supercritical solvent impregnation to deliver antibacterial substance thymol to the defined polymeric structure of commercial polyamide microfiltration membranes. The process performed at a temperature of 40 degrees C and pressures of 10, 15, and 20 MPa was characterized by the fast impregnation in the first 30 min with thymol loadings up to 29 wt%. The maximal achieved thymol loading in the process was around 42 wt%, regardless the pressure. The scanning electron and ion microscopy analyses revealed that the membranes' microstructure was preserved with a thymol content of 20 wt%, while a more prominent swelling effect was observed in samples with 35 wt% of thymol. The cross-flow filtration tests showed that the impregnation process did not harm the membranes' functionality in samples with 20 wt% of thymol. These membranes exhibited strong anti-biofilm activity, and were less prone to the blockage with bacteria in comparison to neat membranes
SOX transcription factors and glioma stem cells: Choosing between stemness and differentiation
Get PDFGlioblastoma (GBM) is the most common, most aggressive and deadliest brain tumor. Recently, remarkable progress has been made towards understanding the cellular and molecular biology of gliomas. GBM tumor initiation, progression and relapse as well as resistance to treatments are associated with glioma stem cells (GSCs). GSCs exhibit a high proliferation rate and self-renewal capacity and the ability to differentiate into diverse cell types, generating a range of distinct cell types within the tumor, leading to cellular heterogeneity. GBM tumors may contain different subsets of GSCs, and some of them may adopt a quiescent state that protects them against chemotherapy and radiotherapy. GSCs enriched in recurrent gliomas acquire more aggressive and therapy-resistant properties, making them more malignant, able to rapidly spread. The impact of SOX transcription factors (TFs) on brain tumors has been extensively studied in the last decade. Almost all SOX genes are expressed in GBM, and their expression levels are associated with patient prognosis and survival. Numerous SOX TFs are involved in the maintenance of the stemness of GSCs or play a role in the initiation of GSC differentiation. The fine-tuning of SOX gene expression levels controls the balance between cell stemness and differentiation. Therefore, innovative therapies targeting SOX TFs are emerging as promising tools for combatting GBM. Combatting GBM has been a demanding and challenging goal for decades. The current therapeutic strategies have not yet provided a cure for GBM and have only resulted in a slight improvement in patient survival. Novel approaches will require the fine adjustment of multimodal therapeutic strategies that simultaneously target numerous hallmarks of cancer cells to win the battle against GBM
Unravelling Anti-Melanogenic Potency of Edible Mushrooms Laetiporus sulphureus and Agaricus silvaticus In Vivo Using the Zebrafish Model
Get PDFSevere drawbacks associated with the topical use of depigmenting agents in treatments of skin hyperigmentations impose a great demand for novel, effective, and safe melanogenesis inhibitors. Edible and medicinal mushrooms, known for numerous health-promoting properties, represent a rich reservoir of anti-melanogenic compounds, with the potential to be applied in preventing excessive skin pigmentation. Herein, using zebrafish (Danio rerio) as a preclinical animal model, we have demonstrated that ethanol extract of Laetiporus sulphureus (LSE) and Agaricus silvaticus (ASE) are not toxic at high doses up to 400-500 mu g/mL while effectively inhibit melanogenesis in a dose-dependent manner. At depigmenting doses, the explored extracts showed no adverse effects on zebrafish embryos melanocytes. Even more, they did not provoke inflammation or neutropenia when applied at the highest dose ensuring almost complete the cells depigmentation. Since LSE and ASE have demonstrated significantly higher the therapeutic potential than kojic acid and hydroquinone, two well-known depigmenting agents, overall results of this study strongly suggest that the explored mushrooms extracts could be used as efficient and safe topical agents in treatments of skin hyperpigmentation disorders
Identification and characterization of cells factors involved in regeneration of the Ustilago maydis population after oxidative stress
No full textUstilago maydis je patogena gljiva kukuruza, otporna na ekstremne doze UV i jonizujućeg zračenja.
U prirodi U. maydis prolazi kroz dva životna stadijuma, tokom kojih je izložen različitim vrstama
stresa. Studije su pokazale da DNK reparacija ima veliki doprinos u oporavku populacije nakon
snažnog stresa. Međutim, razumevanje molekularne osnove ekološkog uspeha U. maydis je ostalo
nepotpuno. Cilj ovog istraživanja je bilo pružanje jasnijeg uvida u mehanizme koji omogućavaju
opstanak populacije ustilaga u nepovoljnim uslovima. Kao metodološki pristup za istraživanje
oporavka od snažnog oksidativnog stresa, primenjen je LH test (Liquid Holding)-praćenje
vijabilnosti tretiranih ćelija tokom inkubacije u vodi. Rezultati su pokazali da U. maydis ima visok
stepen opravka populacije i da se ova obnova ostvaruje multiplikacijom preživelih ćelija
zahvaljujući njihovoj sposobnosti da preuzmu i recikliraju produkte degradacije umrlih ćelija.
Identifikovano je i okarakterisano 10 ćelijskih faktora (Adr1, Did4, Kel1, Tbp1, Snf8, Slm1, Chk1,
Gmt, Snf5, Hsf1) uključenih u proces, putem izolovanja mutanata koji su izgubili sposobnost
obnove vijabilnosti populacije (RUS). Poznate su uloge identifikovanih ćelijskih faktora u
različitim ćelijskim procesima, a ovo je istraživanje pokazalo da su neki od RUS-faktora uključeni
u održavanje integriteta genoma. Takođe je pokazano i da je ustilago metabolički aktivan u vodi i
da nakon pada brojnosti populacije usled autogenerisane hipoksije, koristeći RUS, uspeva da
obnovi populaciju i u normoksičnim i u hipoksičnim uslovima. Sumirano, RUS bi mogao da
predstavlja adaptaciju koja povećava fitnes U. maydis u nepovoljnim uslovima, putem repopulacije
nakon delovanja različitih stresogenih faktora.Ustilago maydis is a pathogenic corn fungus, resistant to extreme doses of UV and ionizing
radiation. In nature, U. maydis passes through two life stages, during which it is exposed to
different types of stresses. Studies have shown that DNA reparation makes a major contribution to
the recovery of the population after severe stress. However, understanding the molecular basis of
environmental success of U. maydis remained incomplete. The aim of this research was to provide a
clear insight into the mechanisms that enable survival of U. maydis population in unfavorable
conditions. As methodoligical approach for investigation of recovery from strong oxidative stress,
LH assay (Liquid Holding)- monitoring of viability of treated cells during incubation in water, was
applied. The results showed that U. maydis had a high degree of population recovery, and that this
renewal was accomplished by multiplication of the surviving cells at the expense of the released
biomolecules from dead cells. Ten cellular factors (Adr1, Did4, Kel1, Tbp1, Snf8, Slm1, Chk1,
Gmt, Snf5, Hsf1) involved in the process were identified and characterized, by isolating mutants
that lost the ability to restore population viability (RUS). The roles in various cellular processes of
the identified factors heave been already known, and this research showed that some of the RUS
factors were involved in maintainance of genome integrity. It was also shown that U. maydis was
metabolically active in water and that after population decline due to autogenerated hypoxia, it
managed to reconstitute the population in both normoxic and hypoxic conditions, employing RUS.
In summary, RUS could be an adaptation that increases fitness of U. maydis in adverse conditions,
by repopulation after expsoure to various stressors
loop-mediated isothermal amplification (lamP) as a point-of-care SarS-covdetection method
Get PDFPravovremeno testiranje većeg broja ljudi na SARS-CoV-2 virus je povezano sa nižim mortalitetom od
COVID-19 oboljenja. Međutim, većina zemalja nema mogućnosti za takvo masivno testiranje putem metode
“PCR u realnom vremenu”, zbog visoke cene neophodne opreme i potrebe za stručnim osobljem.
Zbog toga se razvijaju brze i ekonomičnije metode, koje se najčešće zasnivaju na izotermalnim metodama
amplifikacije DNK. Za ove metode nije potreban ciklični termostat, zbog čega su primenljivije za
terensku upotrebu. Fokus je na izotermalnoj amplifikaciji posredovanoj petljom (lamP), zbog njene
visoke specifičnosti, mogućnosti za upotrebu neprečišćenih uzoraka i jednostavnosti merenja signala. Autori
predstavljaju pregled najvažnijih radova o LAMP metodi za detekciju SARS-CoV-2 virusa objavljenih
tokom 2020. godine, kao i opise nekoliko komercijalnih kompleta na bazi LAMP metode za COVID-19 testiranje.Massive testing for SARS-CoV-2 virus is related to lower mortality rates of COVID-19. Most countries face
challenges to perform massive testing with the currently available methods (real-time PCR), due to expensive
equipment and requirement of highly skilled personnel. To overcome these challenges, faster
and cost-effective alternative detection methods are being developed, primarily based on isothermal
methods of nucleic acid amplification (isoNAAs). PCR depends on precision instruments, high cleanliness
of operating conditions and cannot be easily used on-site, while isoNAAs, which do not require
thermal cyclers, are more applicable for point-of-care (PoC) use. Loop-mediated isothermal amplification
(LAMP) is one of the isoNAA most in focus for COVID-19 tests due to its high specificity and possibility to
use unpurified specimens in combination with simplified detection setup. The article gives a review of
the most significant publications on use of LAMP for SARS-CoV-2 detection and of the several commercial
LAMP-based COVID-19 testing kits.Ministarstva prosvete, nauke i tehnološkog razvoja
Republike Srbije – broj ugovora 451-03-68/2020-14/ 200358 za autore M. Dj, T.K i Lj.J i broj ugovora 451-03-9/2021-
14/200125 za Ž.D.P.
Autori zahvaljuju Jeleni Ognjenov na pomoći u pripremi ilustracija za rad
Molecular basis of primary ciliary dyskinesia
Get PDFPrimarna cilijarna diskinezija (PCD) predstavlja redak genetički poremećaj motornih cilija koji se najčešće
nasleđuje na autozomno recesivan način, a uočeno je i nasleđivanje vezano za hromozom X i autozomno
dominantno nasleđivanje ovog oboljenja. Osnovne karakteristike PCD-a su abnormalnosti pokretnih cilija,
koje su posledica patogenih varijanti u genima koji kodiraju za proteine koji su neophodni za pravilnu strukturu
i funkciju ovih organela. PCD predominantno obuhvata respiratorni trakt i reproduktivne organe, a
utiče i na lateralnost unutrašnjih organa. Klinička prezentacija nije specifična, već obuhvata simptome različitih
respiratornih oboljenja, što često dovodi do odloženog uspostavljanja precizne dijagnoze, zbog čega
je upotreba dijagnostičkih analiza i genetičkog profilisanja neophodna za utvrđivanje PCD-a. Metode sekvenciranja
nove generacije omogućile su poslednjih godina detekciju velikog broja novih gena uzročnika i gena
kandidata za PCD, pa je do sada opisano 45 gena uzročnika koje su odgovorni za nastanak PCD-a i 200 gena
kandidata, a smatra se da je taj broj mnogo veći, s obzirom da 2500 proteina učestvuje u izgradnji i pravilnom
funkcionisanju cilija. Nakon detekcije genetičkih varijanti u uzorcima pacijenata suspektnih na PCD,
molekularna karakterizacija varijanti, upotrebom in silico i/ili eksperimentalnih metoda, omogućava determinaciju
njihove patogenosti i uspostavljanje korelacije između genotipa i fenotipa pacijenata. Terapijski
protokoli kojima se leče PCD pacijenti nisu specifični za ovo oboljenje već se ekstrapoliraju iz terapijskih protokola
drugih plućnih oboljenja sa sličnom kliničkom prezentacijom bolesti. Stoga su istraživanja terapeutika
na animalnim model sistemima od ključnog značaja za razvoj adekvatne terapije za PCD pacijente.
ključne reči: Primarna cilijarna diskinezija (PCD), dijagnostički testovi, genomsko profilisanje PCD pacijenata,
funkcionalna karakterizacija varijanti, model sistemi za PCD, terapija za PCD.Primary ciliary dyskinesia (PCD) is a rare genetic disorder that is most often inherited in an autosomal recessive
manner, nevertheless X-linked and autosomal dominant inheritance of this disease have been reported. The
main characteristics of PCD are abnormalities of motile cilia, which are a consequence of pathogenic variants
in genes encoding proteins necessary for the proper structure and function of these organelles. PCD predominantly
involves the respiratory tract and reproductive organs, and also affects the laterality of internal organs.
The clinical presentation is not specific, and most often includes the symptoms of various respiratory
diseases, which leads to the delayed establishment of an accurate diagnosis. Consequently, the use of diagnostic
and genetic analyses is necessary for the determination of PCD. Next-generation sequencing methods
have enabled the detection of a large number of novel PCD disease-causing and candidate genes in recent
years, and so far, 45 PCD disease-causing and 200 candidate genes have been described, and this number is
thought to be much higher since 2500 proteins participate in cilia formation. After detection of genetic variants
in samples of patients suspected of PCD, molecular characterization of variants, using in silico and/or experimental
methods, allows determination of their pathogenicity and establishment of genotype-phenotype
correlation in patients. Therapeutic protocols for treating PCD patients are not specific for this disease rather,
the therapy is extrapolated from other similar lung diseases. Therefore, research on therapeutics on animal
model systems is crucial for the development of adequate therapy for PCD patients
Predictive and prognostic role of p1 Ink a, p1 arf and kraS genes in humnan rectal carcinoma
Get PDFKarcinom rektuma (KR) čini oko 30% svih slučajeva kolorektalnih karcinoma (KRK), koji se u zadnje vreme
proučava kao zaseban klinički entitet. Aktuelan terapijski pristup u lečenju lokalno uznapredovalih stadijuma
KR podrazumeva primenu preoperativne hemioradioterapije (HRT), nakon koje sledi hirurško uklanjanje
tumora. Iako se ovim tretmanom postiže bolja lokalna kontrola bolesti, individualni odgovor na
primenjenu terapiju je varijabilan i ukazuje na značajnu biološku heterogenost tumora u okviru istog kliničkog
stadijuma. Stoga je u cilju kreiranja i primene personalizovane terapije neophodno definisati prediktivne
i prognostičke molekularne parametre. Primenom PCR metode specifične za metilaciju (MSP), ispitivan
je metilacioni status gena p16INK4a i p14ARF, dok je automatskim sekvenciranjem analiziran mutacioni status
gena KRAS u pre-tretmanskim biopsijama tumorskog tkiva obolelih od KR u lokalno uznapredovalom stadijumu,
kao bi se utvrdio njihov potencijalni prediktivni i prognostički značaj. Rezultati ove studije su pokazali
da ispitivane promene u genima p16INK4a, p14ARF i KRAS pojedinačno gledano, nisu značajni prediktivni i
prognostički faktori kod obolelih od KR, mada je istovremeno prisustvo metilacije gena p14ARF i mutacije
gena KRAS povezano sa agresivnijim ponašanjem tumora, dok je istovremeno prisustvo izmena u sva tri ispitivana
gena povezano sa kraćim ukupnim preživljavanjem. Međutim, kombinovane analize genetičkih, epigenetičkih
i imunohistohemijskih parametara (EGFR, VEGF, Bcl-2 i Ki-67), mogu imati klinički značaj u
identifikaciji podgrupa obolelih, koje se međusobno razlikuju kako prema odgovoru na HRT, tako i prema
toku i ishodu bolesti.Rectal carcinoma (RC) represents approximately 30% cases of colorectal carcinomas (CRC), which is
nowadays studing as a distinct clinical entity. The current management of locally advanced rectal carcinoma
involves neoadjuvant chemoradiotherapy (CRT) prior to surgery. Despite improved local control rate,
individual patient response to CRT is variable and may reflect heterogeneous biological properties among
tumors of the same clinical stage. So, there is utility to define predictive and prognostic molecular parameters
of response and to personalize therapeutic approach. Methylation-specific polymerase chain reaction (MSP)
was used to examine p16INK4a and p14ARFmethylation status, while KRASmutation status was analyzed by
direct sequencing in pretreatment tumor biopsies of patients with locally advanced RC, in order to evaluate
its potential predictive and/or prognostic role. According to the results of this study, examined molecular
changes of p16INK4a, p14ARF and KRAS genes, considering separately, were not proven to be significant
predictive and prognostic factors in RC patients, although simultaneous presence of p14ARFmethylation and
KRAS mutation was associated with more aggressive tumor behavior, while concurrent presence of
genetic/epigenetic alteration in all three examined genes was correlated with shorter overall survival.
However, combined analyses of genetic, epigenetic and immunohistochemical parameters (EGFR, VEGF, Bcl-
2 and Ki-67), could have important clinical relevance in identification of RC patients subgroups, with distinct
pattern of response to CRT and disease outcome
Bioinformatics analysis of eukaryotic positively oriented single stranded RNA viruses
Get PDFPositively oriented single stranded RNA viruses [ssRNA(+)] persistently affect health and well-being of all
eukaryotes, including plants, animals and humans (i.e. SARS-CoV-2, yellow fever, hepatitis C, zika, West Nile,
pepper mild mottle virus, etc.). How come these viruses are so wide spread and hard to eradicate? Besides
their high changeability, another major reason is their ability to mimic host processes upon entering the host.
Only recently it was revealed that ssRNA(+) viruses undergo methylation inside the host in the process that
is similar to the methylation of the hosts’ own mRNAs. Such process may enable or disable virus to avoid
some of the host’s defense mechanisms, but it inevitably impacts viral stability and fitness.
Studies on this topic have only started, opening even more questions, with major ones being: how ssRNA(+)
methylation, that occurs in the host, impacts viral pathogenicity and are these methylation patterns different
in different hosts and for different ssRNA(+) viruses or do these viral methylomes share more universal
pattern in concordance with their similar genome organization? Among numerous different methylation
patterns of RNA, this research focused on N6-methyladenosine (m6A), as the most common and abundant
methylation in eukaryotes, which was confirmed to be present in ssRNA(+) viruses as well.
This study searched for patterns in the primary sequences and secondary structures of ssRNA(+) that are
associated to m6A methylation sites relying on the experimentally obtained m6A datasets for eukaryotes and
eukaryotic ssRNA(+) viruses. The results are discussed in view of datasets characteristics and study approach.Book of Abstracts: Belgrade BioInformatics Conference 202