7 research outputs found
Separation of fiber bundles from willow bark using sodium bicarbonate and their novel use in yarns for superior UV protection and antibacterial performance
The development of a mild and green method for separating natural fiber bundles from willow bark is an essential step in exploring and preserving their natural functions. The isolation of well-oriented fiber bundles from the bark of a fast-growing willow hybrid solely using sodium bicarbonate under mild conditions was successfully demonstrated. Additionally, Lyocell fibers were mixed with an equal amount of the willow bark fiber bundles and proved their ability to convert into spun yarns, which provided excellent protection for ultraviolet radiation (UPF > 140). Moreover, these yarns demonstrated strong antibacterial activity (A > 8) against the Gram-positive pathogen Staphylococcus aureus, resulting in complete eradication of viable bacteria after 24 -h incubation with the material. A laundering treatment had no effect on the UV protection or the antibacterial performance. Utilizing these inherent properties from natural fibers for technical textile applications is very promising.Peer reviewe
Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs
Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC50 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure–activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed
Installation of an aryl boronic acid function into the external section of N-aryl-oxazolidinones : Synthesis and antimicrobial evaluation
N-aryl-oxazolidinones is a prominent family of antimicrobials used for treating infections caused by clinically prevalent Gram-positive bacteria. Recently, boron-containing compounds have displayed intriguing potential in the antibiotic discovery setting. Herein, we report the unprecedented introduction of a boron-containing moiety such as an aryl boronic acid in the external region of the oxazolidinone structure via a chemoselective acyl coupling reaction. As a result, we accessed a series of analogues with a distal aryl boronic pharmacophore on the oxazolidinone scaffold. We identified that a peripheric linear conformation coupled with freedom of rotation and no further substitution on the external aryl boronic ring, an amido linkage with hydrogen bonding character, in addition to a para-relative disposition between boronic group and linker, are the optimal combination of structural features in this series for antimicrobial activity. In comparison to linezolid, the analogue comprising all those features, compound 20b, displayed levels of antimicrobial activity augmented by an eight-fold to a thirty-two-fold against a panel of Gram-positive strains, and a near one hundred-fold against Escherichia coli JW5503, a Gram-negative mutant strain with a defective efflux capability.Peer reviewe
Effect of Hybrid Type and Harvesting Season on Phytochemistry and Antibacterial Activity of Extracted Metabolites from Salix Bark
Hundreds of different fast-growing Salix hybrids have been developed mainly for energy crops. In this paper, we studied water extracts from the bark of 15 willow hybrids and species as potential antimicrobial additives. Treatment of ground bark in water under mild conditions extracted 12-25% of the dry material. Preparative high-performance liquid chromatography is proven here as a fast and highly efficient tool in the small-scale recovery of raffinose from Salix bark crude extracts for structural elucidation. Less than half of the dissolved material was assigned by chromatographic (gas chromatography and liquid chromatography) and spectroscopic (mass spectrometry and nuclear magnetic resonance spectroscopy) techniques for low-molecular-weight compounds, including mono- and oligosaccharides (sucrose, raffinose, and stachyose) and aromatic phytochemicals (triandrin, catechin, salicin, and picein). The composition of the extracts varied greatly depending on the hybrid or species and the harvesting season. This information generated new scientific knowledge on the variation in the content and composition of the extracts between Salix hybrids and harvesting season depending on the desired molecule. The extracts showed high antibacterial activity on Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.6-0.8 mg/mL; however, no inhibition was observed against Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. MIC of triandrin (i.e., 1.25 mg/mL) is reported for the first time. Although antibacterial triandrin and (+)-catechin were present in extracts, clear correlation between the antibacterial effect and the chemical composition was not established, which indicates that antibacterial activity of the extracts mainly originates from some not yet elucidated substances. Aquatic toxicity and mutagenicity assessments showed the safe usage of Salix water extracts as possible antibacterial additives.Peer reviewe
Gramnegatiivisten bakteerien viestintä ja AI-2-välitteisen signaloinnin häirintä
Bakteerit kommunikoivat keskenään quorum sensing (QS) -ilmiön avulla. QS-ilmiössä bakteerit tuottavat ja vapauttavat ympäristöönsä pieniä molekyylejä, jotka toimivat signaalimolekyyleinä viestinnässä. Bakteerit käyttävät viestintää tilanteissa, joissa on hyödyllistä toimia koko bakteeripopulaation laajuisesti. QS-viestintä on tärkeässä osassa esim. virulenssitekijöiden ja biofilmien muodostuksessa.
QS-viestintäjärjestelmiä on useita erilaisia. Gramnegatiiviset bakteerit käyttävät viestinnässään mm. AI-1-, AI-2-, AI-3- ja CAI-1-viestintäjärjestelmiä. Jokainen viestintäjärjestelmä perustuu kyseisen järjestelmän signalointimolekyylin konsentraation nousuun bakteerien ympäristössä sitä tuottavien bakteerien määrän kasvaessa. Signalointimolekyylin konsentraation noustessa tarpeeksi korkeaksi, aktivoituu signalointijärjestelmä, mikä johtaa sen alaisuudessa olevien geenien aktivoitumiseen.
AI-2-järjestelmän oletetaan olevan universaali eli se toimii eri bakteerilajien välisessä kommunikoinnissa. AI-2-signalointijärjestelmässä bakteerit tuottavat 4,5-dihydroksi-2,3-pentaanidionia (DPD), joka toimii AI-2-järjestelmän signalointimolekyylinä. DPD:n kuljetus Escherichia coli ja Salmonella typhimurium -bakteerisolujen sisään tapahtuu ATP:tä sitovan ABC-tyyppisen kuljettajaproteiinin avulla. Bakteerisolun sisällä LsrK-kinaasientsyymi fosforyloi DPD-molekyylit, jotka tämän jälkeen sitoutuvat LsrR-säätelijäproteiiniin. LsrR toimii lsr-operonin vaimentimena operonin promoottorialueella ja fosforyloitujen DPD-molekyylien sitoutuminen irrottaa LsrR:n promoottorialueelta aktivoiden lsr-operonin geenit. Vibrio harveyin LuxP- ja LuxQ-proteiinit muodostavat bakteerin pinnalla proteiinikompleksin, joka tunnistaa DPD-molekyylit. DPD-konsentraation noustessa bakteerien ympäristössä, LuxPQ-kompleksi muuttuu kinaasista fosfataasiksi, mikä aikaansaa reaktioketjun jossa LuxU-fosfaatinsiirtäjä siirtää fosfaattiryhmän LuxO-säätelijäproteiinilta, jonka defosforylaatio vapauttaa LuxR-transkriptiotekijän toiminnan ja aktivoi AI-2-säädellyt geenit.
Tämän työn tarkoituksena oli optimoida kaksi koetta, joita käytetään seulomaan yhdisteitä, jotka estävät AI-2-viestintää. Ensimmäinen koe oli bioreportteripohjainen koe, jossa käytettiin V. harveyi BB120 -bioreportterikantaa. Toinen koe oli entsyymipohjainen koe, jossa käytettiin työssä tuotettua LsrK-entsyymiä, jonka aktiivisuutta seurattiin ATP:n tai ADP:n määrää mittaavien testimenetelmien avulla.
Optimoinnin kohteina olivat kokeissa käytettävät bakteeri-, LsrK- ja DPD-konsentraatiot. Lisäksi tutkittiin kokeiden dimetyylisulfoksiditoleranssia (DMSO) ja LsrK-kokeessa käytettyjen testimenetelmien stabiiliutta sekä LsrK-kokeessa käytettävää puskuriliuosta. Optimaatiokokeiden perusteella V. harveyi BB120 -kokeeseen valikoitui bakteerikonsentraatioksi 100000 pmy/ml ja DPD-konsentraatioksi 1 µM. LsrK-kokeen entsyymikonsentraatioksi valikoitui 300 nM ja DPD-konsentraatioksi 300 µM. DMSO:lla ei LsrK-kokeessa huomattu olevan vaikutusta ATP-konsentraatiota mittaavaan testipakkaukseen, mutta korkeimmat testatut DMSO-konsentraatiot vaikuttivat hieman ADP-konsentraatiota mittaavaan testipakkaukseen. LsrK-kokeeseen valittiin reaktiopuskuriksi trietanoliamiinia, magnesiumkloridia ja naudan seerumin albumiinia sisältävä puskuriliuos.
Optimoidulla LsrK-kokeella suoritettiin seulonta synteettisille yhdisteille, joista ei kuitenkaan löytynyt osumia. Lisäksi optimoitua LsrK-koetta käytettiin erillisessä seulonnassa löytyneen AI-2-viestintää estävän yhdisteen, jota kutsutaan koodilla FIMM000642, annosvastekokeen suoritukseen. FIMM000642-yhdisteelle suoritettiin myös annosvastekoe glyserolikinaasientsyymiä vastaan, koska haluttiin tutkia vaikuttaako FIMM000642 toisen samaan proteiiniperheeseen kuuluvan kinaasin aktiivisuuteen vai onko se spesifinen estäjä LsrK:lle. FIMM000642 esti tulosten perusteella myös glyserolikinaasin toimintaa.Bacteria can communicate with each other using phenomenon called quorum sensing (QS). In QS the bacteria produce and release small signaling molecules which they use to communicate. Bacteria use QS in situations where it is beneficial to act on population level. QS has an important role e.g. in the formation of virulence factors and biofilms.
There are several different QS systems. Gram-negative bacteria use i.a AI-1, AI-2, AI-3, and CAI-1 systems to communicate. All QS systems are based on the accumulation of signaling molecules when the bacterial concentration increases. When the concentration of signal molecules reaches the threshold level, the system activates. The activation of the signaling system then activates the expression of the genes controlled by the QS system.
AI-2 signaling is assumed to be universal. That means that bacteria can use AI-2 signaling system in interspecies communication. In AI-2 signaling bacteria produce and release 4,5-dihydroxy-2,3-pentanedione (DPD) which works as a signaling molecule in the AI-2 system. Escherichia coli and Salmonella typhimurium use an ATP binding cassette ABC-type transporter to transport DPD molecules into the cell where LsrK kinase phosphorylates the DPD molecules. The phosphorylated DPD molecules bind to the LsrR regulator protein which acts as a suppressor of the lsr operon. The binding of the phosphorylated DPD molecules releases the LsrR from the lsr promoter region enabling the expression of the lsr genes. In Vibrio harveyi the surface proteins LuxP and LuxQ form a protein complex that recognizes DPD molecules. When the DPD concentration increases, the LuxPQ complex transform from kinase to phosphatase and the reaction chain, where LuxU phosphate transfer protein transfers a phosphate group from LuxO regulator protein, activates. The dephosphorylation of of LuxO releases the LuxR transcription factor and activates the expression of QS controlled genes.
The aim of this thesis was to optimize two assays which can be used to screen for compounds that disrupt AI-2 signaling. The first assay was a bioreporter based assay where V. harveyi BB120 bioreporter strain was used. The second assay was protein based LsrK assay where the LsrK activity was monitored using assay kit which measures the concentration of ATP or ADP.
The concentrations of bacteria, LsrK, and DPD used in the assays were optimized. The dimethyl sulfoxide (DMSO) tolerance of both assays were tested, the stability of the kits used in the LsrK assays was tested and the reaction buffer for the LsrK assay was selected from the two tested buffer options. The selected bacterial concentration for the V. harveyi BB120 assay was 100000 CFU/ml and DPD concentration 1 µM. The selected enzyme concentration for the LsrK assay was 300 nM and DPD concentration 300 µM. The tested DMSO concentrations had no effect on the kit measuring ATP but the highest concentrations tested had a small effect on the kit measuring ADP. A buffer containing triethanolamine, magnesium chloride, and bovine serum albumin was selected as the reaction buffer for the LsrK assay.
Using the optimized LsrK assay, a screening was performed for a synthesized compound library. None of the compounds showed any LsrK inhibiting activity. The optimized assay was also used to make dose-response experiment to one LsrK inhibiting compound, named FIMM000642, which was found in a separate screening. The FIMM000642 dose-response as-say was also done against glycerol kinase to see if the compound would inhibit another enzyme from the same protein family or if the compound was a specific inhibitor to LsrK. FIMM000642 inhibited also the activity of glycerol kinase
Soluproliferaatiomenetelmien validoiminen virtaussytometrian avulla
Insinöörityön tarkoituksena oli testata solujen proliferaation ja solusyklin tutkimiseen tarkoitettuja värjäystapoja ja verrata niiden toimivuutta käyttäen näytteiden analysoimiseen virtaussytometriä.
Virtaussytometria on yksittäisiä soluja ja niiden ominaisuuksia tutkiva tieteenala. Näytteen solupopulaatioiden tunnistaminen perustuu nestevirrassa liikkuvien solujen ja virtaussytometrin valonlähteen vuorovaikutuksen mittaamiseen. Useiden erilaisten kaupallisten fluoresoivien väriaineiden avulla voidaan virtaussytomerillä analysoida mm. solun nukleiinihappojen määrää, jolloin saadaan tietoa missä solusyklin vaiheessa solu on.
Solusykli on tapahtuma, jonka aikana solun DNA kahdentuu ja jonka päätteeksi solu jakaantuu kahdeksi tytärsoluksi. Solusykli jakaantuu neljään eri vaiheeseen eli G1-, S-, G2- ja M-vaiheisiin. Solusyklin tutkiminen antaa tietoa solun tilasta ja hyvinvoinnista.
Värjäyksissä mitattiin solujen DNA-pitoisuutta, jonka avulla saatiin tietää, missä vaiheessa solusykliä solut olivat. Tieto siitä, kuinka iso osa tutkittavista soluista oli kussakin solusyklin vaiheessa, antoi tietoa solujen jakaantumisen tilasta. Työssä käytettiin PI-värjäystä ja kaupallisia EdU- ja BrdU-värjäystestejä. Tutkittavina soluina työssä käytettiin HeLa- ja HEK 293 -soluja.
Tulosten perusteella PI-värjäys on toimiva tapa solusyklin tutkimiseen. Testatuista kaupallisista värjäystesteistä BrdU-testi osoittautui luotettavammaksi ja paremmin toimivaksi kuin EdU-testi, kun haluttiin tutkia solusykliä ja solujen proliferaatiota yhtä aikaa.The aim of this thesis was to test cell proliferation and cell cycle dyes and compare their performance using a flow cytometer.
In flow cytometry the attributes of single cells are being researched. The detection of the cell populations of the sample is based on measuring the interactions between the cells moving in a liquid flow and the light source of the flow cytometer. Several commercial fluorescent dyes are available which can be used to analyze i.a. the amount of nucleic acids in the cells. The nucleic acid concentration relates to the phase of cell cycle the cell is currently in.
Cell cycle is the series of events during which the DNA of the cell doubles and finally the cell divides into two daughter cells. The cell cycle can be divided into four phases called G1, S, G2 and M. Research of the cell cycle gives information of the state and health of the cell.
The DNA concentration was measured by dyeing the nucleic acids and the results gave information of the phase of the cell cycle the cell was in during the process. The knowledge of how many of the cells were in each phase of the cell cycle provided information of the state of the cell proliferation. PI dyeing and commercial EdU and BrdU kits were used to measure the DNA concentration. HeLa and HEK 293 cells were used as the cell samples.
According to the results PI dyeing is a functional method to analyze cell cycle. The commercial BrdU kit proved to be more reliable than the EdU kit when both cell cycle and cell proliferation were studied at the same time
Antibacterial and antibiofilm activity of permanently ionized quaternary ammonium fluoroquinolones
Publisher Copyright: © 2023 The AuthorsA series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 μM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.Peer reviewe