miR-20b and miR-451a Are Involved in Gastric Carcinogenesis through the PI3K/AKT/mTOR Signaling Pathway: Data from Gastric Cancer Patients, Cell Lines and Ins-Gas Mouse Model

Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP, and miR-451a: CAV1 and TSC1. Loss-of-function experiments revealed that down-regulation of miR-20b and up-regulation of miR-451a expression exhibits an anti-tumor effect in vitro (miR-20b: reduced viability, colony formation, increased apoptosis rate, and miR-451a: reduced colony forming ability). To summarize, the present study identified that expression of miR-20b and miR-451a are deregulated in vitro and in vivo and have a tumor suppressive role in GC through regulation of the PI3K/AKT/mTOR signaling pathway

Electrotransfection efficiency dependence on electroporation medium conductivity and cell line

Šiame darbe vykdyta plazmidinės DNR elektrotransfekcija skirtingo laidumo elektroporacijos terpėse su skirtingų ląstelių kultūrų tipais. Naudotos kininio žiurkėno sukultūrintos kiaušidžių ląstelės (CHO) ir kininio žiurkėno sukultūrintos plaučių fibroblastų ląstelės (DC-3F). Buvo naudotos 3 rūšių elektroporacijos terpės: SMEM (1,3 S/m 270 mOsm.), EP (0,1 S/m 270 mOsm.), EP (1,3 S/m 690 mOsm.). Darbe bandoma rasti elektrotransfekcijos skirtumus priklausomai nuo ląstelių tipų, elektroporacijos terpės osmosinio slėgio ir elektrinio laidumo. Eksperimentiškai įrodyti elektroforetinės jėgos įtaką ląstelių elektrotransfekcijai. Dažniausiai naudoti elektriniai impulsai HV (1200 V/cm 100 µs) ir LV (100 V/cm 100 ms). Tarpas tarp impulsų 1 s. HV elektrinis impulsas kuria elektrinį lauką, kuris ląstelės membranoje sukuria elektroporas ir sukelia nedidelę elektroforetinę jėga. LV elektrinis impulsas kuria elektrinį lauką, kuris nekuria elektroporų, bet sukelia didesnę elektroforetinę jėgą. Iš darbo rezultatų matyti, kad atskiri ląstelių tipai turi skirtingas optimalias elektrotransfekcijos sąlygas. Be to sumažinus plazmidinės DNR koncentraciją elektroporacijos terpėje pasimato LV impulso (elektroforetinės jėgos) įtaka elektrotransfekcijos efektyvumui. Taip pat aptiktas plazmidinės DNR pagausėjimas tame pačiame ląstelių kiekyje, kai didinamas HV impulsų skaičius. Be to pastebėtas ląstelių gyvybingumo ir elektrotransfekcijos efektyvumo pokytis priklausomai nuo elektroporacijos terpės osmosinio slėgio.Two cell lines types were electrotransfected in different electroporation mediums. Chinese hamster ovary (CHO) and Chinese hamster lung fibroblast (DC-3F) cell culture lines were used. Electrotransfection was made with three different electroporation mediums: SMEM (1,3 S/m 270 mOsm), EP (0,1 S/m 270 mOsm), EP (1,3 S/m 690 mOsm). The aim of the study is to obtain effective electrotransfection with used cell culture line types depending on conductivity and osmolarity of electroporation medium and to prove the effect of electrophoretic force in the process of electrotransfection. HV (1200 V/cm 100 µs) and LV (100 V/cm 100 ms) electric pulses in various combinations were mainly used. There was always 1 s. lag between pulses. Electric field that makes electropores was created by HV electric pulse. Electric field that makes electrophoretic forces was created by LV electric pulse. Work results have shown major differences in electrotransfection effectiveness comparing two cell line types. In addition a higher electrotransfection and cell viability was obtained with the increase of osmolarity in electroporation medium. Also LV pulse (responsible for electrophoretic force) influence in efficiency of cell electrotransfection was found. In acknowledgement to the last statement higher number of HV pulses increase plasmid DNA in the same amount of cells.Vytauto Didžiojo universiteta

Application of STEAM teaching method to remote learning (iSTEAM) at Kaunas queen's Morta school in 5-6 th grade

Tradiciniai mokymo metodai susideda iš kelių pagrindinių punktų: 1) mokytojai sukuria pamokos planą; 2) studentai naudoja knygas / pratimus. Mokymas vyksta tada, kai mokytojas aiškina mokiniams pamoką. Tokios pamokos metu mokiniai sprendžia modelio užduotis iš pratimų. Toks studentų mokymasis tapo neefektyvus dėl dažnai naudojamų prieš dešimtmečius technologijų, kurios dabar turi kur kas pažangesnes šiuolaikines alternatyvas. Vienas tokių yra „STEAM“. Šis mokymo metodas sujungia penkis atskirus elementus (mokslą (Science), technologijas (Technology), inžineriją (Engeneering), menus (Art) ir matematiką (Math)). Sujungus šias sritis ir nukreipiant jas į vieną koncepciją, kuris skatina besimokantįjį kurti naujas žinias sprendžiant problemas ar kuriant produktą. Iš šios subjektų kombinacijos genezės slypi naujo požiūrio į inovacijas, vadinamo STEAM, stiprybė. Šių metų pradžioje Pasaulio sveikatos organizacija (PSO) paskelbė pandemiją, kurią sukėlė CoVid-2 virusas. Viena svarbiausių nemedikamentinių intervencijų siekiant sustabdyti šios ligos plitimą yra karantino taikymas ir socialinė izoliacija. Socialinė izoliacija veikia ne tik įvairias susitikimų vietas, tokias kaip koncertų salės, barai ar sporto centrai, bet ir mokyklas. Dėl šios priežasties daugelyje nukentėjusių šalių mokymas švietimo sistemoje buvo perkeltas iš internetinio į mokymą. „STEAM“ daugiausia remiasi praktine patirtimi, kurią sunku suderinti su nuotoliniu mokymusi, nes kiekvieno studento namuose reikia rasti įrankių. Todėl kilo klausimas (problema), ar galima skambinti STEAM mokymosi būdu? Todėl šio tyrimo objektas, kuris čia pateiktas, yra STEAM mokymosi taikymas nuotoliniame mokyme. Čia pateiktas tikslas (inovacija) yra: STEAM pritaikymas nuotoliniu būdu (iSTEAM) 5-6 klasių koncentre Kauno karalienės Mortos mokykloje Metodai. Šis tyrimas tęsėsi 3 pamokas. Pirmoje pamokoje buvo pateiktas vertinimo testas, temos paaiškinimas ir projekto sudedamoji dalis (kristalų auginimo paruošimas). Antroji pamoka buvo skirta konsultacijoms ir projekto skaidrių sudarymui. Trečioje pamokoje vyko vertinimo testas ir projekto pristatymai. Tyrimų vertinimai buvo 3 tipai: vertinimo testas, mokytojo interviu ir stebėjimo pastabos. Vertinimo parametrai buvo pasirinkti taip: testas, kuris mokiniams pateikiamas prieš STEAM paskaitas ir po jų; mokytojo interviu, kuris moko tiriamuosius mokinius daugiau nei metai; stebėtojo, kuris atliko šiuos tyrimus, užrašai. Rezultatai: testai rodo ne tik padidėjusias žinias apie pamokos tema, bet ir padidintą motyvaciją sukurti ką nors naujo ar konstruoti dalykus. Panašūs rezultatai gauti ir iš interviu. Šio interviu metu mokytojas nurodė, kad mokiniai, kuriems nerūpėjo mokymasis, pakeitė savo nuomonę ir yra labiau į tai įsitraukę. Stebėjimo analizėje pabrėžta ta pati nuomonė. Be to, buvo teigiama, kad šio metodo trūkumas yra daug ilgesnis pasirengimo laikas bei parengiamosios medžiagos (vadovėlių) trūkumas norint atlikti STEAM mokymą Lietuvos mokyklose. Šio tyrimo išvados yra šios: a) Šiuolaikinių technologijų pagalba galima pritaikyti ir kitokį nei standartinį mokymo, pavyzdžiui STEAM. Šis metodas skatina mokinį generuoti naujas žinias per problemų sprendimą ar produktų kūrimą. Deja, bet nėra pakankamai žinių, kaip STEAM būtų galimas plėtoti nuotolinio mokymo kontekste. b) Iš atlikto tyrimo galima teigti, kad iSTEAM tikrai veikia ir gali būti pritaikoma nuotoliniame mokyme. Per pravestas tris pamokas, mokiniai atliko projektą (augino kristalą iš prisotinto tirpalo). Antros pamokos metu buvo individualus darbas, mokiniai ruošėsi pristatymams, bei buvo individualiai konsultuojami. Trečios pamokos metu mokiniai grupelėmis pristatė savo projektus ir gavo savo įvertinimus. c) Individualus nuotolinės konsultacijos laikas, suplanuotas kaip neatskiriama STEAM mokymo dalis, padėjo individualiai skirti laiką mokiniams. Dėl to mokiniai daugiau įsisavino žinių. Dėl to, pristatymų metu mokiniai parodė savo žinias aiškiau. Tyrimo metu pasimatė mokinių kūrybingumas. Kai kurie mokiniai pristatymo metu sugalvojo šio metodo pritaikymą, bei ypač kūrybingai apipavidalino pristatymą.Traditional teaching methods consist of several key points: 1) teachers create a lesson plan; 2) students use books / exercises. Teaching then takes place when the teacher is explaining the lesson to the students. During such a lesson, students solve pattern problems from exercises. Such student learning has become ineffective due to the frequent use of technologies developed decades ago, which now has much more advanced modern alternatives. One of such is STEAM. This teaching method combines five separate elements (Science, Technology, Engineering, Arts, and Math). By combining these areas and directing them into a single concept that encourages the learner to generate new knowledge through problem solving or product development. From this genesis of combination of subjects lies the strength of a new approach of innovation termed STEAM. Earlier this year, the World Health Organization (WHO) declared a pandemic caused by the CoVid-2 virus. One of the most important non-pharmaceutical interventions to curb the spread of this disease is the application of quarantine and social isolation. Social isolation affects not only various meeting places, such as concert halls, bars or sports centres, but also schools. For this reason, teaching in the education system has been shifted from online to education in many of the countries affected. STEAM is largely based on hands-on experience, which is difficult to reconcile with distance learning because of the need for tools to be found in each student’s home. Therefore, a question (problem) rose whether calls could be held in a STEAM learning manner? Therefore, the object of this study that is presented here is STEAM learning application in distant learning. The aim (innovation) that is presented here is the examination of possibilities to apply STEAM in a distance learning (iSTEAM) in the 6th grade class at Kaunas Queen Morta School. Methods. This research continued for 3 lectures. First being the evaluation test, explanation of the topic, and the project making part (preparation of crystal growing). Second lecture was for consultations and making of the project slides. And in the third lecture the evaluation test and the project presentations were ongoing. There, were 3 types of research evaluations: evaluation test, interview of the teacher and the observation notes.Švietimo akademij

The Dependence of electrotransfection efficiency on the duration of cell growth passage

ONLINE ISSN: 2335-8718In clinical applications, such as DNA vaccination and gene therapy, gene electrotransfer is used as one of the most promising and efficient technique. The proper establishment of the gene transfer method ensures the improvement of gene therapy protocols. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. Electroporation is a relatively safe and simple technique to deliver nucleic acids to the cell that acts by rendering cells transiently permeable using short periods of high voltage. To achieve the maximal introduction of plasmid DNA into cells and, at the same time, to prevent undesirable cell deaths, electro transfection conditions should be determined for every single cell type individually. In the present study, we determined the duration of cell growth passage for in vitro transfection of CHO cells. Time of 24 h and 48 h cell passage before the experiment was chosen. Electrotransfection efficiency with all plasmid concentrations significantly differed when comparing 24 h and 48 h passage time. Transfection efficiency from 47.28±0.41% after 24 h passage time fell to 19.87±1.02% after 48 h of cell passage time using same electric field parameters. But there was very less viability change when comparing cell passage time of 24h and 48 h. The cell passage time, if optimized, may generate a reproducibly high proportion of transfected cells among the different cell typesBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

In vitro analysis of the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation

Electroporation is a process when as a result of electric fields effect induced transmembrane potential create transient pores in the plasma membrane, leading to a large increase in drug transport, delivery of macromolecules, a quick drug effect onset. Hence, anticancer drugs can be easily transported into targeted tumour cells by using this technique. Nowadays, electrochemotherapy is used in clinics for the cancer treatment. Electrochemotherapy is a process, when electroporation is combined with the injection of cytotoxic drugs (e.g. bleomycin (BLM). A single molecule of bleomycin can cause around 8–10 DNA breaks causing the cell death, which explains its high cytotoxicity when present inside the cell. However, it is only a local response. In order to initiate a systemic response rate, one has to induce immune response. One way to do it is to transfect a gene that express a compound that enhance immune response. In this study, we show the possibility to simultaneously transfer bleomycin and plasmid DNA in cells via electroporation. Chinese hamster ovary (CHO) cell line was used for experiments. The final concentration of BLM in experiments ranged from 0.1 ng/ml to 20 ng/ml. For that different concentrations of anticancer drugs were used together with pDNA transfection. pMAX FGP coding plasmid in concentraton of 200 μg/ml was used. Electroporation was performed by using combination of 1 electric pulse that induced electric fields at the amplitude of 1400 V/cm for the duration of 100 μs. Cells were electroporated in laboratory made EP medium (pH 7.1, conductivity 0.1 S/m, osmolarity 270 mOsm). Afterwards, comet assay was performed to evaluate DNA damage. In addition, clonogenic assay was done to evaluate cell viability. Transfection efficiency was measured using flow cytometry (BD accuri C6).[...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Bleomycin electrotransfer enhancement by using Plasmid Dna

Over the last decade a new cancer treatment method termed electrochemotherapy has emerged. Such anticancer therapy is based on the process of electroporation. This process initiates when applied electric field is increasing transmembrane voltage of the affected cells, hence increasing cell membrane permeability that leads to increased uptake of exogenous molecules such as anticancer drugs. Currently, the combination of anticancer drug (mainly bleomycin) and the method of electroporation is used in clinics. Once inside the cell, the anticancer drug bleomycin induces the cellular DNA cleavage, that in turn leads to cell death. Previously it has been shown that big molecules (dextrans) with high net charge increase electrotransfer of bleomycin into the cells. Here we show the enhanced bleomycin electrotransfer when plasmid DNA (as a big molecule with charge) was in the media during the electroporation We analysed bleomycin electrotransfer in Chinese Hamster Ovary (CHO) cells. For that different concentrations of anticancer drugs were used together with pDNA transfection. pMAX FGP coding plasmid in concentraton of 200 µg/ml was used. The anticancer drug bleomycin was used for electrotransfer experiments in the concentrations ranging from 0.1 to 20 ng/ml. Electroporation was performed by using combination of 1 electric pulse that induced electric fields at the amplitude of 1400 V/cm for the duration of 100 µs. Afterwards, comet assay was performed to evaluate DNA damage. In addition, clonogenic assay was done to evaluate cell viability. Obtained results showed bigger bleomycin electrotransfer after usage of combination of bleomycin, pDNA and electroporation. In conditions of 1HV electroporation intensity, 200 µg/ml pDNA and 20 ng/ml BLM concentrations, DNA cleavage reaches up to 10% [...]Aplinkos tyrimų centrasGamtos mokslų fakultetasVytauto Didžiojo universiteta

Cell sensitization induced by application of microsecond electric fields in vitro

Electroporation (EP) is a method that is used to induce a temporal increase of cell membrane permeability for hydrophilic molecules. Moreover, other cellular properties change as a result of cell membrane permeability deviations caused by EP. EP is triggered by inducing transmembrane potential that can be estimated with Schwan’s equation. According to the stated equation the transmembrane potential has a direct dependence on the cell size. There had been articles published that indicate cell swelling after application of electric fields [1]. Induced cell swelling can increase electric field triggered transmembrane potential. Hence, the cell sensitivity to electric field can change. Such phenomena is termed electrosensitization [2,3]. Such processes could influence membrane permeability for hydrophilic molecule and are important for modelling the applications of EP. The aim of this research is to examine influence of cell size changes to the subsequent electroporation induced molecule electrotransfer. CHO cells were subcultured a day before the experiment. For cell size measurements we used high voltage (HV) electric pulses with amplitude from 800-2000 V/cm for the duration of 100 µs, after electroporation and incubation of 10 min we took photos of cells by using Kern OCO-255 inverted microscope. Sizes of the cells were measured by using open source ImageJ software. For PI electrotransfer efficiency measurements, we used 2 or 8 HV, 1400 V/cm pulses and after 1, 5, 10 and 15 min time interval PI (40 µM) was put with an application of additional 1 HV pulse. Then, cells were plated for flow cytometry measurements were performed 10 min after the incubation procedure. The results indicate that cells can decrease or increase in size depending on applied electric field [...]Biologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta