Nicotinic acetylcholine receptors: Diversity and physiological importance for neurodegenerative disorders and development of organophosphate antidotes

The communication between the nervous and other systems in the organism is carried out by the transmission of nerve impulses. Diverse neurotransmitters are released into the synaptic cleft and bind to the specific receptors at the neighbouring cell to transmit the signal further. One of such receptors are nicotinic acetylcholine receptors (nAChR), integrated membrane proteins (ligand-gated ion channels) activated by the binding of a neurotransmitter acetylcholine. nAChR\u27s main characteristic is their diversity, as they consist of five of the same or mutually different subunits, which contribute to the specific receptors properties and biological activity. During the assembly of a pentameric protein structure, various combinations of subunits are linked together. After the discovery of nAChR’s involvement in various diseases, they became an important therapeutic target, for example in the treatment of neurodegenerative diseases (Alzheimer\u27s and Parkinson\u27s) and in the treatment of organophosphorus compound poisoning. This paper presents an overview of current knowledge on nicotinic receptors and an accompanying discussion on diseases, poisonings, potential drugs and treatments is given.</p

Overexpression of selected genes and resistance of yeast Saccharomyces cerevisiae on growth inhibitors

Proizvodnja bioetanola druge generacije temelji se na korištenju lignoceluloznih sirovina kao supstrata, no osnovni problem je nesposobnost proizvodnog mikroorganizma, kvasca Saccharomyces cerevisiae, da koristi ovakav supstrat za alkoholnu fermentaciju. Sirovinu je potrebno hidrolizirati, pri čemu nastaju spojevi koje kvasac može koristiti, ali i različiti spojevi koji djeluju kao inhibitori rasta i fermentacije (octena i levulinska kiselina, 2-furaldehid), zbog čega se nastoji konstruirati proizvodne sojeve koji bi bili rezistentni na inhibitore. Cilj istraživanja bio je utvrditi da li pojačana ekspresija kvaščevih gena ATR1, FLR1, YAP1, GSH1, CTA1 i ZWF1 povećava rezistenciju odabranih sojeva na inhibitore rasta. U ovome radu korišteni su kontrolni homozigotni i hibridni heterozigotni diploidni sojevi kvasca transformirani plazmidima koji sadrže pojedinačne navedene gene. Utvrđeno je da pojačana ekspresija gena ATR1 i YAP1 rezultira povećanom rezistencijom sojeva na inhibitore rasta (octenu i levulinsku kiselinu te 2-furaldehid) u odnosu na ishodne sojeve.Second generation bioethanol production is based on the use of lignocellulosic composites as substrates, but the main problem is that the producer microorganism, the yeast S. cerevisiae, cannot use this substrate for fermentation. Therefore composites are hydrolysed to produce compounds that can be used by the yeast, but during hydrolysis different compounds that inhibit yeast growth are produced. Therefore, the aim is to construct producer strains that will be resistant to different growth inhibitors (acetic and levulinic acid and 2-furaldehyde). The aim of this research was to determine if the overexpression of yeast genes ATR1, FLR1, YAP1, GSH1, CTA1 or ZWF1 can increase the resistance of producer strains to inhibitors. In this research, homozygous control and heterozygous diploid strains were transformed with plasmids that allow overexpression of selected genes. It was determined that the overexpression of ATR1 and YAP1 genes in different strains results in the increased resistance to growth inhibitors (acetic and levulinic acid and 2-furaldehyde) compared to starting ones

Učinci imidazolijevih i kloriranih bispiridinijevih oksima povezani s njihovom toksičnosti na stanicama SH-SY5Y

Current research has shown that several imidazolium and chlorinated bispyridinium oximes are cytotoxic and activate different mechanisms or types of cell death. To investigate this further, we analysed interactions between these oximes and acetylcholine receptors (AChRs) and how they affect several signalling pathways to find a relation between the observed toxicities and their effects on these specific targets. Chlorinated bispyridinium oximes caused time-dependent cytotoxicity by inhibiting the phosphorylation of STAT3 and AMPK without decreasing ATP and activated ERK1/2 and p38 MAPK signal cascades. Imidazolium oximes induced a time-independent and significant decrease in ATP and inhibition of the ERK1/2 signalling pathway along with phosphorylation of p38 MAPK, AMPK, and ACC. These pathways are usually triggered by a change in cellular energy status or by external signals, which suggests that oximes interact with some membrane receptors. Interestingly, in silico analysis also indicated that the highest probability of interaction for all of our oximes is with the family of G-coupled membrane receptors (GPCR). Furthermore, our experimental results showed that the tested oximes acted as acetylcholine antagonists for membrane AChRs. Even though oxime interactions with membrane receptors need further research and clarification, our findings suggest that these oximes make promising candidates for the development of specific therapies not only in the field of cholinesterase research but in other fields too, such as anticancer therapy via altering the Ca2+ flux involved in cancer progression.Praćenjem učinka odabranih imidazolijevih i kloriranih bispiridinijevih oksima utvrđeno je da uzrokuju citotoksičnost i aktiviraju različite mehanizme ili tipove stanične smrti. Kako bismo to detaljnije istražili, analizirali smo aktivaciju nekoliko signalnih putova, kao i interakcije acetilkolinskih receptora (AChR) s navedenim oksimima te procijenili može li se opaženi toksični učinak objasniti njihovim utjecajem na ove specifične mete. Rezultati su pokazali da su klorirani bispiridinijevi oksimi prouzročili vremenski-ovisnu citotoksičnost, bez smanjenja razine ATP-a uz aktivaciju ERK1/2 i p38 MAPK-vezanih signalnih kaskada i inhibiciju fosforilacije STAT3 i AMPK proteina. Imidazolijevi oksimi djelovali su vremenski neovisno, uz značajno smanjenje razine ATP-a i inhibiciju ERK1/2 signalnog puta te fosforilaciju p38 MAPK, AMPK i ACC proteina. Navedeni signalni putovi obično se aktiviraju ili promjenom unutarnjega staničnog statusa, osobito energetskoga, ili vanjskim signalima, što upućuje na moguće interakcije oksima s nekim membranskim receptorima. Zanimljivo, in silico analizom procijenjeno je da je najvjerojatnija interakcija testiranih oksima s porodicom G-protein-spregnutih membranskih receptora (GPCR). K tomu, eksperimentalno je potvrđeno da testirani oksimi djeluju kao mogući antagonisti acetilkolina za vezanje na membranske AChR, potvrđujući tako i računalnu in silico procjenu. Iako interakcije ispitanih oksima s membranskim receptorima treba dodatno potvrditi, takve bi ih interakcije učinile kandidatima za razvoj specifičnih terapija u drugim područjima istraživanja, osim u istraživanjima povezanima s kolinesterazama, npr. kao moguće protutumorske lijekove, putem utjecaja na fluks iona Ca2+ uključenoga u progresiju tumora

Research on teachers' perceptions of a more inclusive teaching process in the school context

O presente estudo justifica-se pelas inquietações dos pesquisadores perante ao ensino de alunos surdos, pois tem-se resultados de estudos de outros autores, que muitas vezes os professores não têm qualificação adequada para atender a demanda de alunos com algum tipo de deficiência. Assim as perguntas que envolvem essa pesquisa estão centradas em: como está a qualificação docente perante questões inclusivas? A prática pedagógica está centrada em metodologias inclusivas? Nesse sentido, o objetivo principal é investigar de que forma o professor está atuando no contexto inclusivo a fim de contribuir com o processo de ensino aprendizagem de alunos surdos, que convivem num contexto escolar regular com diversas realidades. A metodologia está baseada na análise das informações supracitadas, que foram organizadas por meio da realização de um questionário e os resultados demonstraram que a maioria dos professores já tiveram ou tem alunos com alguma deficiência e que na sua maioria, não usam metodologias diferenciadas para ensino, mas se mostraram preocupados em querer se qualificar e melhorar o processo de ensino aprendizagem dos alunos com deficiência auditiva. Conclui-se que existe diversas metodologias que podem ser utilizadas pelos professores em sala de aula para trabalhar com alunos surdos e que possibilita a construção do conhecimento e melhoria no processo de ensino, além de disponibilizar aos professores novas formas de organizar sua prática pedagógica.The present study is justified by the concerns of researchers regarding the teaching of deaf students, as there are results from studies by other authors, that teachers often do not have adequate qualifications to meet the demand of students with some type of disability. So the questions that involve this research are centered on: how is the teacher qualification facing inclusive issues? Is the pedagogical practice centered on inclusive methodologies? In this sense, the main objective is to investigate how the teacher is acting in the inclusive context in order to contribute to the teaching-learning process of deaf students, who live in a regular school context with different realities. The methodology is based on the analysis of the aforementioned information, which was organized by means of a questionnaire and the results showed that most teachers have had or have students with a disability and that most of them do not use different methodologies for teaching, but they were concerned about wanting to qualify and improve the teaching-learning process of students with hearing impairment. It is concluded that there are several methodologies that can be used by teachers in the classroom to work with deaf students and that enable the construction of knowledge and improvement in the teaching process, in addition to providing teachers with new ways of organizing their pedagogical practice

Učinak ketamina na vijabilnost, primarna oštećenja DNA i parametre oksidacijskog stresa u stanicama HepG2 i SH-SY5Y

Ketamine is a dissociative anaesthetic used to induce general anaesthesia in humans and laboratory animals. Due to its hallucinogenic and dissociative effects, it is also used as a recreational drug. Anaesthetic agents can cause toxic effects at the cellular level and affect cell survival, induce DNA damage, and cause oxidant/antioxidant imbalance. The aim of this study was to explore these possible adverse effects of ketamine on hepatocellular HepG2 and neuroblastoma SH-SY5Y cells after 24-hour exposure to a concentration range covering concentrations used in analgesia, drug abuse, and anaesthesia (0.39, 1.56, and 6.25 μmol/L, respectively). At these concentrations ketamine had relatively low toxic outcomes, as it lowered HepG2 and SH-SY5Y cell viability up to 30 %, and low, potentially repairable DNA damage. Interestingly, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) remained unchanged in both cell lines. On the other hand, oxidative stress markers [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] pointed to ketamine-induced oxidant/antioxidant imbalance.Ketamin je disocijativni anestetik koji se koristi za izazivanje opće anestezije u određenim medicinskim postupcima kod ljudi, kao i u anesteziji laboratorijskih životinja. Zbog svojih halucinogenih i disocijativnih učinaka koristi se i kao rekreacijska droga. Anestetici također mogu prouzročiti toksične učinke na staničnoj razini i, utječući na preživljavanje stanica, izazvati oštećenje DNA te neravnotežu oksidansa i antioksidansa. Cilj ove studije bio je istražiti moguće štetne učinke ketamina na hepatocelularne HepG2 i neuroblastoma SH-SY5Y stanice nakon 24-satne izloženosti širokom rasponu koncentracija, uključujući koncentracije relevantne u slučajevima korištenja u analgeziji, zlouporabi droga i anesteziji (0,39, 1,56 odnosno 6,25 μmol/L). Naši rezultati pokazali su da je ketamin u ovim ispitivanim koncentracijama izazvao relativno nisku citotoksičnost, budući da je do 30 % smanjio preživljenje stanica HepG2 i SH-SY5Y, ali je uočen neznatan porast razine primarnih oštećenja DNA. Zanimljivo je da su razine reaktivnih kisikovih vrsta (ROS), malondialdehida (MDA) i glutationa (GSH) ostale nepromijenjene u objema staničnim linijama. S druge strane, markeri oksidacijskog stresa [suporeksid dismutaza (SOD), glutation peroksidaza (GPx), katalaza (CAT)] upućivali su na oksidacijsko-redukcijsku neravnotežu izazvanu ketaminom

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids

Za proizvodnju bioetanola iz lignoceluloznih hidrolizata potreban je proizvodni soj koji dobro podnosi prisutnost inhibitora rasta i fermentacije te veliku koncentraciju etanola. Stoga smo konstruirali hibridne diploide kvasca Saccharomyces cerevisiae međusobnim križanjem dvaju prirodnih izolata, Yllcl 7_E5 i UWOPS87-2421. Soj Yllcl 7_E5 izoliran je iz vina kao dobar proizvođač etanola, a soj UWOPS87-2421 izoliran je iz cvijeta kaktusa Opuntia megacantha i otporan je na inhibitore koji se mogu naći u lignoceluloznim hidrolizatima. Hibridni sojevi rasli su brže od ishodnih sojeva u odsutnosti i prisutnosti octene i levulinske kiseline te 2-furaldehida, koji se često nalaze kao inhibitori rasta u lignoceluloznim hidrolizatima, a pojačana ekspresija gena YAPI povećala je preživljenje testiranih sojeva. Također, pojedini su hibridni sojevi, iako potječu od dvaju istih ishodnih sojeva, pokazali različit fermentativni potencijal u testu proizvodnje C02, što upućuje na njihovu genetičku varijabilnost koja omogućava daljnju selekciju poželjnih svojstava. Iz naših se rezultata može zaključiti da se kombiniranjem konstrukcije hibridnih sojeva i metoda genetičkog inženjerstva mogu oplemeniti i razviti novi biotehnološki relevantni sojevi kvasca S. cerevisiae. Osim toga, utvrđeno je da je uspješnost ciljanja gena u prirodnim izolatima S. cerevisiae (Yllcl 7_E5a and UWOPS87-2421 a) daleko manja nego u laboratorijskim sojevima, a najčešći aberantni događaj bio je duplikacija ciljanog kromosoma.Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication

Structure – cytotoxicity relationship of oxime reactivators of phosphylated acetylcholinesterase

Primjenom novog pristupa analizi baziranog na staničnim testovima in vitro proveden je probir pet serija strukturno povezanih oksima. Odabrani citotoksični bispiridinijevi i 3-hidroksi-2-piridinski oksimi inducirali su aktivaciju apoptoze, putom ovisnom o mitohondriju, aktivacijom enzima kaspaze 9 i/ili 3 uz oštećenje DNA i aktivaciju MAPK kinaze ili fosforilaciju acetil-CoA karboksilaze. Nasuprot tome, imidazolijevi i kinuklidinijevi oksimi uzrokovali su nereguliranu smrt stanice, nekrozu, pucanjem stanične membrane popraćenu oksidacijskim stresom, oštećenjem mitohondrija te smanjenom fosforilacijom izvanstanično reguliranih kinaza ERK1/2, uključenih u regulaciju diobe stanice. Kao potencijalne nove meta djelovanja ovih oksima, predviđene molekulskim modeliranjem farmakofora, istaknute su: beta-sekretaza 1, važna u nastajanju amiloid-β peptida kod Alzheimerove bolesti, aldehid dehidrogenaza A1, koja ima ulogu u Parkinsonovoj bolesti te citokrom P450cam iz Pseudomonas putida, što sugerira mogućnost inhibicije i ljudskih citokroma P450 sustava. Ovakav probir dao je smjernice za poboljšanje i razvoj strukture novih oksima, koje se mogu koristiti u bilo kojem istraživanju oblikovanja novih lijekova.Using a new approach to the analysis of in vitro cell-based assays, five series of structurally related oximes were tested. The selected cytotoxic bispyridinium and 3-hydroxy-2-pyridine oximes induced apoptosis via the mitochondrial-dependent pathway by caspase 9 and/or 3 activation accompanied with DNA damage, MAPK kinase activation or acetyl-CoA carboxylase phosphorylation. In contrast, imidazolium and quinuclidine oximes caused unregulated cell death, necrosis, and cell membrane burst accompanied by oxidative stress, mitochondrial damage, and decreased phosphorylation of extracellularly regulated ERK1/2 kinases involved in the regulation of cell division. The potential new targets of these oximes, predicted by computational modeling of pharmacophores, are: beta-secretase 1, important in the formation of amyloid-β peptide in Alzheimer's disease, aldehyde dehydrogenase A1, which plays a role in Parkinson's disease and cytochrome P450cam from Pseudomonas putida. This suggests the possibility of inhibition of human cytochrome P450 systems. Such screening provided guidelines for improving the development of the structure of new oximes, which can be used in any research for designing new drugs

Application of ionic liquids and deep eutectic solvents to lipase-catalyzed butyl acetate synthesis

Uspostavljanje novih visokoučnikovitih i održivih procesa proizvodnje industrijski važnih kemikalija predmet je mnogih znanstvenih istraživanja u području kemijske tehnologije i biotehnologije. Stoga se ionske kapljevine i eutektična otapala, nova generacija nehlapljivih i stabilnih otapala, posljednjih godina intenzivno proučavaju kao zamjena za tradicionalna i škodljiva otapala u procesima organske sinteze i biokatalize. U okviru ovog rada ispitana je mogućnost primjene ionskih kapljevina i eutektičnog otapala u lipazom kataliziranoj sintezi butil-acetata, estera od indutrijskog značaja.Establishment of new highly productive and sustainable processes for the production of industrially important compounds is becoming a growing area of research in the field of chemical technology and biotechnology. Therefore, the use of ionic liquids and eutectic solvents as possible replacement for conventional molecular solvents for biocatalytic and organic reactions has recently been the subject of considerable attention. Within this work cholinium-based ionic liquids and eutectic solvents were prepared and tested as solvents for lipase-catalyzed butyl-acetate synthesis

Overexpression of selected genes and resistance of yeast Saccharomyces cerevisiae on growth inhibitors

Proizvodnja bioetanola druge generacije temelji se na korištenju lignoceluloznih sirovina kao supstrata, no osnovni problem je nesposobnost proizvodnog mikroorganizma, kvasca Saccharomyces cerevisiae, da koristi ovakav supstrat za alkoholnu fermentaciju. Sirovinu je potrebno hidrolizirati, pri čemu nastaju spojevi koje kvasac može koristiti, ali i različiti spojevi koji djeluju kao inhibitori rasta i fermentacije (octena i levulinska kiselina, 2-furaldehid), zbog čega se nastoji konstruirati proizvodne sojeve koji bi bili rezistentni na inhibitore. Cilj istraživanja bio je utvrditi da li pojačana ekspresija kvaščevih gena ATR1, FLR1, YAP1, GSH1, CTA1 i ZWF1 povećava rezistenciju odabranih sojeva na inhibitore rasta. U ovome radu korišteni su kontrolni homozigotni i hibridni heterozigotni diploidni sojevi kvasca transformirani plazmidima koji sadrže pojedinačne navedene gene. Utvrđeno je da pojačana ekspresija gena ATR1 i YAP1 rezultira povećanom rezistencijom sojeva na inhibitore rasta (octenu i levulinsku kiselinu te 2-furaldehid) u odnosu na ishodne sojeve.Second generation bioethanol production is based on the use of lignocellulosic composites as substrates, but the main problem is that the producer microorganism, the yeast S. cerevisiae, cannot use this substrate for fermentation. Therefore composites are hydrolysed to produce compounds that can be used by the yeast, but during hydrolysis different compounds that inhibit yeast growth are produced. Therefore, the aim is to construct producer strains that will be resistant to different growth inhibitors (acetic and levulinic acid and 2-furaldehyde). The aim of this research was to determine if the overexpression of yeast genes ATR1, FLR1, YAP1, GSH1, CTA1 or ZWF1 can increase the resistance of producer strains to inhibitors. In this research, homozygous control and heterozygous diploid strains were transformed with plasmids that allow overexpression of selected genes. It was determined that the overexpression of ATR1 and YAP1 genes in different strains results in the increased resistance to growth inhibitors (acetic and levulinic acid and 2-furaldehyde) compared to starting ones

Application of ionic liquids and deep eutectic solvents to lipase-catalyzed butyl acetate synthesis

Uspostavljanje novih visokoučnikovitih i održivih procesa proizvodnje industrijski važnih kemikalija predmet je mnogih znanstvenih istraživanja u području kemijske tehnologije i biotehnologije. Stoga se ionske kapljevine i eutektična otapala, nova generacija nehlapljivih i stabilnih otapala, posljednjih godina intenzivno proučavaju kao zamjena za tradicionalna i škodljiva otapala u procesima organske sinteze i biokatalize. U okviru ovog rada ispitana je mogućnost primjene ionskih kapljevina i eutektičnog otapala u lipazom kataliziranoj sintezi butil-acetata, estera od indutrijskog značaja.Establishment of new highly productive and sustainable processes for the production of industrially important compounds is becoming a growing area of research in the field of chemical technology and biotechnology. Therefore, the use of ionic liquids and eutectic solvents as possible replacement for conventional molecular solvents for biocatalytic and organic reactions has recently been the subject of considerable attention. Within this work cholinium-based ionic liquids and eutectic solvents were prepared and tested as solvents for lipase-catalyzed butyl-acetate synthesis