Rapid hydrogel-based phage susceptibility test for pathogenic bacteria

Phage therapy is one alternative to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds to thousands of phages are required to cover the diversity of bacterial pathogens. In personalized phage therapy, fast selection of the phages for individual patients is essential for successful therapy. The aims of this study were to set up a rapid hydrogel-based liquid phage susceptibility assay (PST) for the selection of phages for therapeutic use and to establish a “ready-to-screen” plate concept, where phages are readily stored in hydrogel as small droplets in microtiter plate wells. We first tested four commercially available hydrogels (GrowDex, Askina, Purilon, and Intrasite) for their suitability as phage matrices in PSTs with four phages, two of which infecting Escherichia coli and two Staphylococcus aureus. Of these four hydrogels, GrowDex was the best matrix for PST, as it did not inhibit bacterial growth, released phages quickly when mixed with bacterial culture, and maintained phage viability well. We then optimized the assay for both optical density and microscopy readers using GrowDex as matrix with 23 bacterial strains representing 10 different species and 23 phages possessing different morphologies and genome sizes. When the bacterial growth was monitored by microscopy reader, the PST was executed in just 3 hours, and there was no need for overnight culturing bacterial cells prior to the assay, whereas using optical density reader, bacteria had to be pre-cultured overnight, and the assay time was five hours. Finally, we evaluated the effect of three different chemical stabilizers (trehalose, hyaluronic acid, and gelatin) in a six-month stability assay with six model phages. These phages assay behaved very differently in respect to the chemical stabilizers, and there was not a single stabilizer suitable for all phages. However, when gelatin (0.01%) or hyaluronic acid (0.2 mg/ml) was used as stabilizer, all tested phages were still considered as positives in PST after a six-month storage in 1 ml volume. In “ready-to-screen” plates, the differences in phage stabilities were even more profound, varying from two to six months for the most and least stable phages, respectively.Peer reviewe

Long-term measurement of sub-3 nm particles and their precursor gases in the boreal forest

The knowledge of the dynamics of sub-3 nm particles in the atmosphere is crucial for our understanding of the first steps of atmospheric new particle formation. Therefore, accurate and stable long-term measurements of the smallest atmospheric particles are needed. In this study, we analyzed over 5 years of particle concentrations in size classes 1.1-1.7 and 1.7-2.5 nm obtained with the particle size magnifier (PSM) and 3 years of precursor vapor concentrations measured with the chemical ionization atmospheric pressure interface time-of-flight mass spectrometer (CI-APi-ToF) at the SMEAR II station in Hyytiala, Finland. The results show that there are significant seasonal differences in median concentrations of sub-3 nm particles, but the two size classes behave partly differently. The 1.1-1.7 nm particle concentrations are highest in summer, while the 1.7-2.5 nm particle concentrations are highest in springtime. The 1.7-2.5 nm particles exhibit a daytime maximum in all seasons, while the 1.1-1.7 nm particles have an additional evening maximum during spring and summer. Aerosol precursor vapors have notable diurnal and seasonal differences as well. Sulfuric acid and highly oxygenated organic molecule (HOM) monomer concentrations have clear daytime maxima, while HOM dimers have their maxima during the night. HOM concentrations for both monomers and dimers are the highest during summer and the lowest during winter following the biogenic activity in the surrounding forest. Sulfuric acid concentrations are the highest during spring and summer, with autumn and winter concentrations being 2 to 3 times lower. A correlation analysis between the sub-3 nm concentrations and aerosol precursor vapor concentrations indicates that both HOMs (particularly their dimers) and sulfuric acid play a significant role in new particle formation in the boreal forest. Our analysis also suggests that there might be seasonal differences in new particle formation pathways that need to be investigated further.Peer reviewe

Mitigation Impact of Different Harvest Scenarios of Finnish Forests That Account for Albedo, Aerosols, and Trade-Offs of Carbon Sequestration and Avoided Emissions

The pressure to increase forest and land carbon stocks simultaneously with increasing forest based biomass harvest for energy and materials emphasizes the need for dedicated analyses of impacts and possible trade-offs between these different mitigation options including also forest related biophysical factors, surface albedo and the formation of biogenic Secondary Organic Aerosols (SOA). We analyzed the change in global radiative forcing (RF) due to changes in these climatic agents as affected by the change in state of Finnish forests under increased or decreased harvest scenarios from a baseline. We also included avoided emissions due to wood material and energy substitution. Increasing harvests from baseline (65% of Current Annual Increment) decreased the total carbon sink (carbon in trees, soil and harvested wood products) at least for 50 years. When we coupled this change in carbon with other biosphere responses, surface albedo and aerosols, decreasing harvests from the baseline produced the largest cooling effect during 50 years. Accounting also for the avoided emissions due to increased wood use, the RF responses of the two lowest harvest scenarios were within uncertainty range. Our results show that the effects of forest management on SOA formation should be included in the analyses trying to deduce the net climate impact of forest use. The inclusion of the rarely considered SOA effects enforces the view that the lower the harvest, the more climatic cooling boreal forests provide. These results should act as a caution mark for policy makers who are emphasizing the increased utilization of forest biomass for short-living products and bioenergy as an efficient measure to mitigate climate change.Peer reviewe

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease

Ulkoisella signaalilla aktivoituvat liposomaaliset lääkkeenkuljetusjärjestelmät

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.Lääketutkimus tuottaa runsaasti uusia potentiaalisia lääkeaineita, mutta joissain tapauksissa lääkeaine ei tuota elimistössä haluttua vaikutusta koska sen kulkeutuminen vaikutuspaikalleen on riittämätöntä. Lääkkeenkuljetus onkin yksi farmaseuttisen tutkimuksen päähaasteista. Lukuisia lääkekantajia, kuten liposomeja, on kehitetty kuljetuksen ja kohdentamisen tehostamiseksi. Kun lääkeaine kohdennetaan elimistössä vaikutuspaikalleen, hoidon teho paranee ja haitalliset sivuvaikutukset vähenevät. Liposomit ovat kooltaan nanometri-luokkaa olevia pallomaisia, rasvamolekyyleistä koostuvia rakenteita, joita on laajasti tutkittu lääkekantajina. Lääkeaine voidaan sijoittaa liposomeihin, ja lisäksi niihin voidaan liittää kohdentajia sekä aistivia materiaaleja joiden avulla liposomien aktivoitumista pystytään kontrolloimaan. Tässä tutkimuksessa kehitettiin erilaisia ulkoisella signaalilla aktivoituvia liposomeja lääkekuljetukseen. Signaaleilla voidaan säädellä lääkeaineen kulkeutumista tai vapautumista liposomi-formulaatiosta, ja ne mahdollistavat aktivaation ajan, paikan ja määrän kontrollin. Työssä tutkittiin kolmea erilaista signaalia: sähköä, lämpöä ja valoa. Sähköä käytettiin parantamaan liposomaalisen DNA:n kuljetusta ihon läpi. Geenitransfektiota tutkittiin ihoa mallintavassa solulinjassa mittaamalla erittyvän proteiinin pitoisuutta solujen kasvatusliuoksessa. Liposomi/DNA-kompleksit tuottivat geeniekspression, mutta sähkövirta ei parantanut tehoa merkittävästi. Lämpöherkät liposomit valmistettiin päällystämällä liposomit biohajoavalla, lämpöherkällä polymeerilla (HPMA-mono/dilaktaatti). Tutkittiin liposomien aggregoitumista ja sisällön vapautumista lämpötilan vaikutuksesta. Polymeerilla päällystetyt liposomit aggregoituivat ja vapauttivat sisältönsä lämpötilan nostamisen myötä, mutta päällystämättömät liposomit pysyivät muuttumattomina. Lämpötilan noustessa polymeeri sakkautuu ja vaikuttaa liposomin kuoren rakenteeseen. Todennäköisesti tämä lisää liposomin läpäisevyyttä ja aiheuttaa sisällön vapautumisen. Liposomit herkistettiin valolle lisäämällä niihin pieniä (< 5 nm) kultananopartikkeleita. Sekä rasvaliukoisia että vesiliukoisia kultananopartikkeleita sisällytettiin liposomeihin, ja tutkittiin UV valo –altistuksen aiheuttamaa sisällön vapautumista. UV valon aiheuttamia liposomin rakennemuutoksia tutkittiin pienkulmaröntgensironnalla (SAXS), ja liposomien valoaktivaation osoitettiin toimivan myös soluviljelmässä. Kultananopartikkelit absorboivat valoenergiaa ja muuttavat sen lämmöksi, mikä aikaansaa faasimuutoksia liposomien rakenteessa ja sisällön vapautumisen. Yhteenvetona voidaan todeta että ulkoisella signaalilla aktivoituvat liposomit tarjoavat lähtökohdan monille uusille sovelluksille lääkkeenkuljetuksessa ja -kohdennuksessa. Lääkeaineen vapautuminen liposomeista pystytään rajoittamaan haluttuun aikaan ja paikkaan ulkoisella signaalilla. Näin lääkehoidon tehoa voidaan parantaa ja haitallisia sivuvaikutuksia vähentää. Aktivoiva signaali ja liposomiformulaatio voidaan valita halutun sovelluksen perusteella

Efficiency of GrowDex^® nanofibrillar cellulosic hydrogel when generating homotypic and heterotypic 3D tumor spheroids

In recent times, homotypic and heterotypic 3D tumor spheroid (HTS) models have been receiving increasing attention and come to be widely employed in preclinical studies. The present study is focused on the generation of homotypic (A549 and MDA-MB-231, separately) and heterotypic (A549 + NIH/3T3; MDA-MB-231 + NIH/3T3) 3D tumor spheroids by using GrowDex® nanofibrillar cellulosic (NFC) hydrogel as the scaffold. Light microscopic observations and F-actin staining confirmed the formation of spheroids. The proliferation efficiency indicated an expansion of cell population and an increase in spheroid size over time. The distribution, interaction pattern and influence of fibroblasts on the epithelial cell types were observed in terms of the size and shape of the HTS against homo-spheroids. An interesting observation was that, with an increase in the size of HTSs, many more fibroblast cells were found to occupy the core region, which, perhaps, was due to the faster growth of tumor cells over normal cells. Thus, normal and tumor cells, especially with origins from two different species, can be cultured together in 3D format, and this can potentially enhance our knowledge of tumor microenvironments and cell-cell interaction. These spheroids could be used to improve microphysiological systems for drug discovery and to better understand the tumor microenvironment

Event‐recurring multiband SWIFT functional MRI with 200‐ms temporal resolution during deep brain stimulation and isoflurane‐induced burst suppression in rat

Purpose To develop a high temporal resolution functional MRI method for tracking repeating events in the brain. Methods We developed a novel functional MRI method using multiband sweep imaging with Fourier transformation (SWIFT), termed event-recurring SWIFT (EVER-SWIFT). The method is able to image similar repeating events with subsecond temporal resolution. Here, we demonstrate the use of EVER-SWIFT for detecting functional MRI responses during deep brain stimulation of the medial septal nucleus and during spontaneous isoflurane-induced burst suppression in the rat brain at 9.4 T with 200-ms temporal resolution. Results The EVER-SWIFT approach showed that the shapes and time-to-peak values of the response curves to deep brain stimulation significantly differed between downstream brain regions connected to the medial septal nucleus, resembling findings obtained with traditional 2-second temporal resolution. In contrast, EVER-SWIFT allowed for detailed temporal measurement of a spontaneous isoflurane-induced bursting activity pattern, which was not achieved with traditional temporal resolution. Conclusion The EVER-SWIFT technique enables subsecond 3D imaging of both stimulated and spontaneously recurring brain activities, and thus holds great potential for studying the mechanisms of neuromodulation and spontaneous brain activity.peerReviewe

Structural Elucidation of Light Activated Vesicles

In the present study, synchrotron small-angle X-ray scattering (SAXS) combined with a UV light source (in situ SAXS-UV irradiation) was used to determine the structure response of gold nanoparticle (NP)-loaded vesicles. The investigated system consisted of multilamellar vesicles of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) containing hydrophilic gold NPs with a size of 40 Å. Our results indicate drastic optothermically induced structural changes in these gold NP-loaded aqueous dispersions, which are accompanied by strong alterations in the phospholipid membrane permeability. A structural mechanism from well-ordered multilamellar vesicles in the fluid lamellar (L_α) phase to an inverted type hexagonal liquid crystalline (H₂) phase via the formation of an intermediate phase of uncorrelated fluid bilayers is proposed. In general, these investigations are also crucial for the understanding of the potential of gold NP-loaded model phospholipid systems as efficient drug nanocarriers aiming to improve drug targeting and releasing on demand