Search

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

We recently identified fingolimod as a potent antibiofilm compound by screening FDA-approved drugs. To study if the antibacterial activity of fingolimod could be further improved and to explore in-depth structure-activity relationships, we synthesized 28 novel fingolimod derivatives and evaluated their efficacy against Staphylococcus aureus grown in planktonic/single cell and biofilms. The most effective derivatives were tested on preformed S. aureus biofilms and against Gram-negative bacteria Acinetobacter baumannii and Pseudomonas aeruginosa, using fingolimod as the reference compound. Seven derivatives were more effective against S. aureus, while five other derivatives showed improved activity against P. aeruginosa and/or A. baumannii, with no apparent change in cytotoxicity on human cells. The most interesting derivatives, compounds 43 and 55, displayed a broader spectrum of antibacterial activity, possibly exerted by the change of the para-hydrocarbon chain to a meta position for 43 and by an additional hydroxyl group for 55.

Oxidative stress (OS) is a common toxic feature in various neurodegenerative diseases. Therefore, reducing OS could provide a potential approach to achieve neuroprotection. Prolyl oligopeptidase (PREP) is a serine protease that is linked to neurodegeneration, as endogenous PREP inhibits autophagy and induces the accumulation of detrimental protein aggregates. As such, inhibition of PREP by a small-molecular inhibitor has provided neuroprotection in preclinical models of neurodegenerative diseases. In addition, PREP inhibition has been shown to reduce production of reactive oxygen species (ROS) and the absence of PREP blocks stress-induced ROS production. However, the mechanism behind PREP-related ROS regulation is not known. As we recently discovered PREP's physiological role as a protein phosphatase 2A (PP2A) regulator, we wanted to characterize PREP inhibition as an approach to reduce OS. We studied the impact of a PREP inhibitor, KYP-2047, on hydrogen peroxide and ferrous chloride induced ROS production and on cellular antioxidant response in HEK-293 and SHSY5Y cells. In addition, we used HEK-293 and SH-SY5Y PREP knock-out cells to validate the role of PREP on stress-induced ROS production. We were able to show that absence of PREP almost entirely blocks the stressinduced ROS production in both cell lines. Reduced ROS production and smaller antioxidant response was also seen in both cell lines after PREP inhibition by 10 mu M KYP-2047. Our results also revealed that the OS reducing mechanism of PREP inhibition is related to reduced activation of ROS producing NADPH oxidase through enhanced PP2A activation. In conclusion, our results suggest that PREP inhibition could also provide neuroprotection by reducing OS, thus broadening the scope of its beneficial effects on neurodegeneration.

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE–CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.

Based on social cognitive theory, this study focused on how self-efficacy beliefs were related to assessment practices among experienced university teachers. The data consisted of 16 thematic interviews of university teachers from various faculties who had received awards of excellence in teaching. The thematic interviews were content analyzed both inductively and deductively. Academics' self-efficacy experiences were related in four assessment modes: assessment in general, diagnostic assessment, formative assessment and summative assessment. Sources of self-efficacy experience were identified by mastery experience, vicarious experience, social persuasions and physiological and affective states. The mastery experience was the most common source of self-efficacy in assessment practice, and for experienced teachers, fairness was the prominent feature in assessment.

Oncolytic viruses (OVs) have been shown to induce anti-cancer immunity and enhance cancer immunotherapies, such as immune checkpoint inhibitor therapies. OV therapies can be further improved by arming OVs with immunostimulatory molecules, including various cytokines or chemokines. Here, we have developed a novel adenovirus encoding two immunostimulatory molecules: cluster of differentiation 40 ligand (CD40L) and tumor necrosis factor receptor superfamily member 4 ligand (OX40L). This novel virus, designated VALO-D102, is designed to activate both innate and adaptive immune responses against tumors. CD40L affects the innate side by licensing antigen-presenting cells to drive CD8+ T cell responses, and OX40L increases clonal expansion and survival of CD8+ T cells and formation of a larger pool of memory T cells. VALO-D102 and its murine surrogate VALO-mD901, expressing murine OX40L and CD40L, were used in our previously developed PeptiCRAd cancer vaccine platform. Intratumoral administration of PeptiCRAd significantly increased tumor-specific T cell responses, reduced tumor growth, and induced systemic anti-cancer immunity in two mouse models of melanoma. In addition, PeptiCRAd therapy, in combination with anti-PD-1 immune checkpoint inhibitor therapy, significantly improved tumor growth control as compared to either monotherapy alone., Graphical Abstract, Ylösmäki et al. develop an oncolytic adenovirus expressing immunostimulatory cytokines OX40L and CD40L. This virus was tested in a peptide-based cancer vaccine platform called PeptiCRAd. Intratumoral administration of PeptiCRAd induced systemic and intratumoral tumor-specific T cell responses, reduced tumor growth, and sensitized tumors to immune checkpoint inhibitor therapy.

The solubility and dissolution rates of chemical compounds are crucial properties in several fields of industry and research. However, accurate, rapid and green methods for their measurement, which only consume micrograms of compound, are lacking. Here, the unique approach of non-specific, image-based single particle analysis (SPA) for solubility testing is directly compared to and thus validated on the mid-solubility range with the current gold standard shake-flask method with UV-Vis spectroscopy employed for determining sample concentrations. Five biologically active compounds representing a range of physicochemical properties including pK(a) and logP were analyzed with both methods. The comparison of SPA and the shake-flask (SF) analysis shows excellent linear correlation (R-2 = 0.99). Higher variability of the SPA method is attributed to variability between the properties of individual particles, which cannot be detected with traditional methods. Due to the similar average solubility values compared to those produced with SF, it is concluded that the SPA method has great potential as an analytical tool for small-scale solubility studies. It also has several practical advantages over the current gold standard shake-flask method, such as speed, low consumables consumption, and no requirement for prior knowledge of compound chemistry.

Micrometer-sized hydrogels are cross-linked three-dimensional network matrices with high-water contents and dimensions ranging from several to hundreds of micrometers. Due to their excellent biocompatibility and capability to mimic physiological microenvironments in vivo, micrometer-sized hydrogels have attracted much attention in the biomedical engineering field. Their biological properties and applications are primarily influenced by their chemical compositions and geometries. However, inhomogeneous morphologies and uncontrollable geometries limit traditional micrometer-sized hydrogels obtained by bulk mixing. In contrast, microfluidic technology holds great potential for the fabrication of micrometer-sized hydrogels since their geometries, sizes, structures, compositions, and physicochemical properties can be precisely manipulated on demand based on the excellent control over fluids. Therefore, micrometer-sized hydrogels fabricated by microfluidic technology have been applied in the biomedical field, including drug encapsulation, cell encapsulation, and tissue engineering. This review introduces micrometer-sized hydrogels with various geometries synthesized by different microfluidic devices, highlighting their advantages in various biomedical applications over those from traditional approaches. Overall, emerging microfluidic technologies enrich the geometries and morphologies of hydrogels and accelerate translation for industrial production and clinical applications.

Cancer Immunotherapy relies on harnessing a patient's immune system to fine-tune specific anti-tumor responses and ultimately eradicate cancer. Among diverse therapeutic approaches, oncolytic viruses (OVs) have emerged as a novel form of cancer immunotherapy. OVs are a naturally occurring or genetically modified class of viruses able to selectively kill cancer cells, leaving healthy cells unharmed; in the last two decades, the role of OVs has been redefined to act beyond their oncolytic activity. Indeed, the immunogenic cancer cell death mediated by OVs induces the release of tumor antigens that in turn induces anti-tumor immunity, allowing OVs to act as in situ therapeutic cancer vaccines. Additionally, OVs can be engineered for intratumoral delivery of immunostimulatory molecules such as tumor antigens or cytokines to further enhance anti-tumor response. Moreover, OVs can be used in combination with other cancer immunotherapeutic approaches such as Immune Checkpoint Inhibitors and CAR-T cells. The current review first defines the three main mechanisms of action (MOA) of OVs currently used in cancer therapy that are: i) Oncolysis, ii) OV-induced cancer-specific immune activation, and iii) Exploiting preexisting anti-viral immunity to enhance cancer therapy. Secondly, we focus on how OVs can induce and/or improve anti-cancer immunity in a specific or unspecific fashion, highlighting the importance of these approaches. Finally, the last part of the review analyses OVs combined with other cancer immunotherapies, revising present and future clinical applications. (c) 2021 Published by Elsevier Inc.

Funding Information: The authors are thankful to the Institute of Chemical Technology, Mumbai, India for providing facilities to carry out this work. V.J.is supported by a personal research grant from the Finnish Cultural Foundation (Ingrid, Toini and Olavi Martelius foundation), and thanks Arto Urtti for suggestions that improved the manuscript. Publisher Copyright: © 2022 The Authors Age-related macular degeneration (AMD) is a macular degenerative eye disease, the major cause of irreversible loss of central vision. In this review, we highlight current progress and future perspectives of novel and investigational therapeutic strategies in the drug pipeline, including anti-vascular endothelial growth factor (VEGF) agents, bispecific antibodies, biosimilars, small molecules, gene therapy, and long-acting drug delivery strategies for both dry and wet AMD. We anticipate that biologics with dual functionalities and combined therapies with long-acting capabilities will lead the wet AMD pipeline. Sustained-release platforms also show potential. However, significant breakthroughs are yet to be made for dry AMD. The personalized approach might be well suited in the scenario of diverse genetic variations in both conditions.

BackgroundThe segmentation of 3D cell nuclei is essential in many tasks, such as targeted molecular radiotherapies (MRT) for metastatic tumours, toxicity screening, and the observation of proliferating cells. In recent years, one popular method for automatic segmentation of nuclei has been deep learning enhanced marker-controlled watershed transform. In this method, convolutional neural networks (CNNs) have been used to create nuclei masks and markers, and the watershed algorithm for the instance segmentation. We studied whether this method could be improved for the segmentation of densely cultivated 3D nuclei via developing multiple system configurations in which we studied the effect of edge emphasizing CNNs, and optimized H-minima transform for mask and marker generation, respectively.ResultsThe dataset used for training and evaluation consisted of twelve in vitro cultivated densely packed 3D human carcinoma cell spheroids imaged using a confocal microscope. With this dataset, the evaluation was performed using a cross-validation scheme. In addition, four independent datasets were used for evaluation. The datasets were resampled near isotropic for our experiments. The baseline deep learning enhanced marker-controlled watershed obtained an average of 0.69 Panoptic Quality (PQ) and 0.66 Aggregated Jaccard Index (AJI) over the twelve spheroids. Using a system configuration, which was otherwise the same but used 3D-based edge emphasizing CNNs and optimized H-minima transform, the scores increased to 0.76 and 0.77, respectively. When using the independent datasets for evaluation, the best performing system configuration was shown to outperform or equal the baseline and a set of well-known cell segmentation approaches.ConclusionsThe use of edge emphasizing U-Nets and optimized H-minima transform can improve the marker-controlled watershed transform for segmentation of densely cultivated 3D cell nuclei. A novel dataset of twelve spheroids was introduced to the public.

Vascular endothelial growth factor-C (VEGF-C) stimulates lymphatic vessel growth in transgenic models, via viral gene delivery, and as a recombinant protein. Expressing eukaryotic proteins like VEGF-C in bacterial cells has limitations, as these cells lack specific posttranslational modifications and provisions for disulfide bond formation. However, given the cost and time savings associated with bacterial expression systems, there is considerable value in expressing VEGF-C using bacterial cells. We identified two approaches that result in biologically active Escherichia coli-derived VEGF-C. Expectedly, VEGF-C expressed from a truncated cDNA became bioactive after in vitro folding from inclusion bodies. Given that VEGF-C is one of the cysteine-richest growth factors in humans, it was unclear whether known methods to facilitate correct cysteine bond formation allow for the direct expression of bioactive VEGF-C in the cytoplasm. By fusing VEGF-C to maltose-binding protein and expressing these fusions in the redox-modified cytoplasm of the Origami (DE3) strain, we could recover biological activity for deletion mutants lacking the propeptides of VEGF-C. This is the first report of a bioactive VEGF growth factor obtained from E. coli cells circumventing in-vitro folding.

Funding Information: We wish to thank MSc Jarkko Etula for kindly providing us the ta-C containing samples, and PhD Toni Pasanen for kindly providing us the antireflective black silicon material that was used in upright fluorescence imaging. We wish to also thank PhD Christoffer Kauppinen for sharing his knowledge in lift-off processing, and PhD Ali Shah for great discussion and sharing his expertise in cryogenic etching. We acknowledge the provision of facilities by Aalto University at OtaNano - Micronova Nanofabrication Centre and we would also like to thank the Biomedicum Imaging Unit (BIU), Helsinki, for microscopy services. This work was supported by European Union's Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01 (No. 824070), Academy of Finland (No. 321996 and No. 328854), and Jane and Aatos Erkko Foundation. Publisher Copyright: © 2022 The Author(s) | openaire: EC/H2020/824070/EU//CONNECT Vertically aligned carbon nanofibers (VACNFs) are promising material candidates for neural biosensors due to their ability to detect neurotransmitters in physiological concentrations. However, the expected high rigidity of CNFs could induce mechanical mismatch with the brain tissue, eliciting formation of a glial scar around the electrode and thus loss of functionality. We have evaluated mechanical biocompatibility of VACNFs by growing nickel-catalyzed carbon nanofibers of different lengths and inter-fiber distances. Long nanofibers with large inter-fiber distance prevented maturation of focal adhesions, thus constraining cells from obtaining a highly spread morphology that is observed when astrocytes are being contacted with stiff materials commonly used in neural implants. A silicon nanopillar array with 500 nm inter-pillar distance was used to reveal that this inhibition of focal adhesion maturation occurs due to the surface nanoscale geometry, more precisely the inter-fiber distance. Live cell atomic force microscopy was used to confirm astrocytes being significantly softer on the long Ni-CNFs compared to other surfaces, including a soft gelatin hydrogel. We also observed hippocampal neurons to mature and form synaptic contacts when being cultured on both long and short carbon nanofibers, without having to use any adhesive proteins or a glial monoculture, indicating high cytocompatibility of the material also with neuronal population. In contrast, neurons cultured on a planar tetrahedral amorphous carbon sample showed immature neurites and indications of early-stage apoptosis. Our results demonstrate that mechanical biocompatibility of biomaterials is greatly affected by their nanoscale surface geometry, which provides means for controlling how the materials and their mechanical properties are perceived by the cells. Statement of significance: Our research article shows, how nanoscale surface geometry determines mechanical biocompatibility of apparently stiff materials. Specifically, astrocytes were preventedfrom obtaining highly spread morphology when their adhesion site maturation was inhibited, showing similar morphology on nominally stiff vertically aligned carbon fiber (VACNF) substrates as when being cultured on ultrasoft surfaces. Furthermore, hippocampal neurons matured well and formed synapses on these carbon nanofibers, indicating high biocompatibility of the materials. Interestingly, the same VACNF materials that were used in this study have earlier also been proven to be capable for electrophysiological recordings and sensing neurotransmitters at physiological concentrations with ultra-high sensitivity and selectivity, thus providing a platform for future neural probes or smart culturing surfaces with superior sensing performance and biocompatibility.

Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.

Biopharmaceutical products are composed of complex or ordinate combinations of proteins, lipids, sugars, and nucleic acids or living cells or tissues. Due to the intrinsic variability of biological systems and the complexity of the bioprocesses involved in the production of these products, new technologies are required to monitor and characterize, the production processes and the final products. Biophotonic techniques, particularly Raman and Infrared (IR) spectroscopy are rapid, robust, operator independent, non-destructive and label free, thus particularly suitable for these purposes. This dissertation first investigates the use of biophotonic techniques in research of Extracellular Vesicles (EVs). EVs act as intercellular messengers and therefore have considerable potential in drug delivery system, diagnostic biomarker, or therapeutic agent. Subsequently highlighting the need and potential of a new kind of time gated Raman spectrometer to be created. Raman and IR spectroscopy are methods able to characterize and assess the quality of EV suspensions with different degree of purity. These vibrational spectroscopic techniques show different intrinsic advantages, as they are label free and operator independent methods. Particularly, Raman might be the most suitable technology since it is less sensitive to water compared to IR. Raman spectroscopy reveals the information on the chemical composition and physical status of the analyte. However, it does not provide information of the analyte environment which the spectrofluorometer can gather. EVs studied by spectrofluorometer are required to be labelled with a fluorescent dye. Results obtained by fluorescence lifetime imaging spectroscopy underline that the cell uptake of the fluorescently labelled EVs is feasible. Attention must be paid to the efficacy of the labelling and further to the elimination of the unbound dye since the labelling may severely compromise the results or lead to wrong conclusions on EV functionality. The combined advantage of Raman spectroscopy and fluorescence decay are obtained by a previously in-house developed time resolved Raman spectrometer. Thanks to the peculiar sensor of the spectrometer, the width of the time gate can be modified which is used to separate the Raman signal from the fluorescence tail. The modifications can be done even in the data post-processing phase, to obtain the best possible Raman signal-to-noise ratios. The simultaneously detected Raman spectra and time-resolved fluorescence decay curves are used to study the diffusion of small molecular drugs in a hydrogel. The data reveal the chemical composition, physical status, and the interaction with the environment of the samples. Taken together the obtained results suggest that the quality of EV suspensions can be assessed by Raman spectroscopy and their cell uptake detected by fluorescence lifetime spectroscopy. Both Raman spectra and fluorescence decay can be measured simultaneously by a second generation of time-resolved Raman spectrometer. Biofarmaseuttiset tuotteet koostuvat proteiinien, lipidien, sokereiden ja nukleiinihappojen tai elävien solujen tai kudosten järjestäytyneistä tai monimutkaisista yhdistelmistä. Biologisten järjestelmien luontaisen vaihtelevuuden ja näiden tuotteiden valmistukseen liittyvien bioprosessien monimutkaisuuden vuoksi tarvitaan uusia teknologioita tuotantoprosessien ja lopputuotteiden seurantaa ja karakterisointia varten. Biofotoniset tekniikat, erityisesti Raman- ja Infrapuna (IR) -spektroskopia ovat nopeita, luotettavia, hellävaraisia, leimattomia ja käyttäjästä riippumattomia tekniikoita, jotka soveltuvat erityisen hyvin biofarmaseuttisten tuotteiden määritykseen. Tämä väitöskirjan alussa tutkitaan biofotonisten tekniikoiden käyttöä solunulkoisten vesikkeleiden (EV:iden) tutkimuksessa. EV:t toimivat solujenvälisessä viestinnässä ja siksi niillä on huomattava potentiaali lääkekehityksessä, diagnostisina biomarkkerina ja terapeuttisina tekijöinä. Myöhemmin tutkimuksessa korostetaan uudenlaisen aikaerotteisen Raman-spektrometrin tarvetta ja mahdollisuuksia biofarmaseuttisten tuotteiden määrityksessä. Raman- ja IR-spektroskopia ovat menetelmiä, joilla voidaan karakterisoida ja arvioida eri puhtausasteen omaavia EV-näytteiden laatua. Nämä värähtelyspektroskooppiset tekniikat ovat leimattomia ja käyttäjästä riippumattomia menetelmiä. Verrattuna IR-spektroskopiaan, Raman on luonteeltaan mahdollisesti käyttökelpoisin tekniikka EV:iden karakterisointiin, sillä veden läsnäolo näytteessä ei häiritse mittausta. Raman-spektroskopian avulla saadaan tietoa analyytin kemiallisesta koostumuksesta sekä fysikaalisesta tilasta ja spektrofluorometrin avulla saadaan kerättyä informaatiota analyytin ympäristöstä, mihin Raman ei puolestaan pysty. Spektrofluorometrillä tutkitut EV:t on leimattava fluoresoivalla väriaineella ja fluoresenssin eliniän kuvantamisspektroskopialla saadut tulokset korostavat, että fluoresenssileimattujen EV:iden soluunotto on mahdollista. Leimauksen tehokkuuteen ja sitoutumattoman väriaineen eliminointiin on kiinnitettävä huomiota, sillä virheellinen leimaus voi väärentää tuloksia tai johtaa virheellisiin johtopäätöksiin EV:iden funktionaalisuudesta. Talon sisällä rakennetun laitteiston avulla on saatu yhdistettyä Raman-spektroskopia ja fluoresenssin eliniän vaimeneminen. Spektrometrin omalaatuisen anturin ansiosta kuvantamisen aikaikkunaa pystytään muokkaamaan, minkä avulla Raman-signaali pystytään erottamaan fluoresenssisignaalista. Muutokset voidaan tehdä myös datan jälkikäsittelyvaiheessa parhaan mahdollisen Raman-signaali-kohinasuhteen saamiseksi. Pienmolekyylisten lääkkeiden diffuusion tutkimiseen hydrogeelissä voidaan käyttää samanaikaisesti havaittuja Raman-spektrejä ja aikaerotteisia fluoresenssin vaimenemiskäyriä. Kerätty aineisto paljastaa näytteiden kemiallisen koostumuksen, fysikaalisen tilan ja vuorovaikutuksen ympäristön kanssa. Yhdessä saadut tulokset viittaavat siihen, että EV-suspensioiden laatua voidaan arvioida Raman-spektroskopialla ja niiden soluunotto havaita fluoresenssielinaikaspektroskopialla, kun Raman-spektri ja fluoresenssin heikkeneminen voidaan mitata samanaikaisesti toisen sukupolven aikaerotteisella Raman-spektrometrillä.

New classes of antibiotics are urgently needed in the fight against multidrug-resistant bacteria. Drug repurposing has emerged as an alternative approach to accelerate antimicrobial research and development. In this study, we screened a library of sphingosine-1-phosphate receptor (S1PR) modulators against Staphylococcus aureus and identified five active compounds. Among them, etrasimod (APD334), an investigational drug for the treatment of ulcerative colitis, displayed the best inhibitory activity against S. aureus when growing as free-floating planktonic cells and within biofilms. In follow-up studies, etrasimod showed bactericidal activity and drastic reduction of viable bacteria within 1 h of exposure. It also displayed a potent activity against other Gram-positive bacteria, including penicillin- and methicillin-resistant S. aureus strains, S. epidermidis, and Enterococcus faecalis, with a minimum inhibitory concentration (MIC) ranging from 5 to 10 μM (2.3–4.6 μg/mL). However, no inhibition of viability was observed against Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, showing that etrasimod preferably acts against Gram-positive bacteria. On the other hand, etrasimod was shown to inhibit quorum sensing (QS) signaling in Chromobacterium violaceum, suggesting that it may block the biofilm formation by targeting QS in certain Gram-negative bacteria. Furthermore, etrasimod displayed a synergistic effect with gentamicin against S. aureus, thus showing potential to be used in antibiotic combination therapy. Finally, no in vitro toxicity toward mammalian cells was observed. In conclusion, our study reports for the first time the potential of etrasimod as a repurposed antibacterial compound against Gram-positive bacteria.

The current situation, heavily influenced by the ongoing pandemic, puts vaccines back into the spotlight. However, the conventional and traditional vaccines present disadvantages, particularly related to immunogenicity, stability, and storage of the final product. Often, such products require the maintenance of a “cold chain,” impacting the costs, the availability, and the distribution of vaccines. Here, after a recall of the mode of action of vaccines and the types of vaccines currently available, we analyze the past, present, and future of vaccine formulation. The past focuses on conventional formulations, the present discusses the use of nanoparticles for vaccine delivery and as adjuvants, while the future presents microneedle patches as alternative formulation and administration route. Finally, we compare the advantages and disadvantages of injectable solutions, nanovaccines, and microneedles in terms of efficacy, stability, and patient-friendly design. Graphical abstract Different approaches to vaccine formulation development, the conventional vaccine formulations from the past, the current development of lipid nanoparticles as vaccines, and the near future microneedles formulations are discussed in this review.

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (D-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Among different cereals, oat is becoming more popular due to its unique composition and health benefits. The increase in oat production is associated with an increase in related side streams, comprising unutilized biomass that is rich in valuable components, such as polysaccharides, proteins, and antioxidants. To valorize such biomass, it is fundamental that side streams enter back into the food production chain, in respect of the circular economy model. Here, we propose the use of soluble and insoluble oat-production side-stream in suspensions and emulsions, avoiding any further extraction, fractionation, and/or chemical derivatization. Our approach further increases the value of these side streams. To this aim, we first studied the effect of thermal and mechanical processes on the behavior and properties of both soluble and insoluble oat side-stream fractions in water and at air/water interface. Then, we characterized the emulsifying and stabilizing abilities of these materials in oil-in-water emulsions. Interestingly, we found that the insoluble fraction was able to form stable suspensions and emulsions after mechanical treatment. The oil droplets in the emulsions were stabilized by anchoring at the surface of the insoluble particles. On the other hand, the soluble fraction formed only stable viscous solutions. Finally, we demonstrated that the two fractions can be combined to increase the storage stability of the resulting emulsion.Our results highlight that oat production side streams can be used as novel bio-based emulsifiers, showing the great potential behind the underutilized cereal-side-stream biomass. Graphical Abstract

Minimal inhibitory concentration of antimicrobials, determined by the broth microdilution method, requires visual assessment or absorbance measurement using a spectrophotometer. Both procedures are usually performed manually, requiring the presence of an operator to assess the plates at specific time point. To increase the throughput of antimicrobial susceptibility testing, and concurrently convert into an automatic assay, the Biolog OmniLog(R) system was validated for a new, label-free application using standard 96-well microplates. OmniLog was evaluated for its signal strength to ensure that the signal intensity, detected and measured by the system's camera, was satisfactory. Variability due to the plate location inside the OmniLog incubator, as well as variation between wells, was investigated. Then the system was validated by determining the minimal inhibitory concentration of ciprofloxacin, piperacillin and linezolid against a selected Gram-negative and Gram-positive strains. No significant difference was observed in relation to position of the plates within the system. Plate edge effects were noticeable, thus the edge wells were not included in further experiments. Minimal inhibitory concentration results were comparable to those obtained by conventional protocol as well as to values defined by the Clinical Laboratory Standards Institute or published in the literature.

In recent years, several studies have shown that the use of solid lipid nanoparticles (SLN) as a colloidal drug delivery system was more advantageous than lipid emulsions, liposomes and polymeric nanoparticles. SLNs have numerous advantages of different nanosystems and rule out many of their drawbacks. Despite the numerous advantages of SLNs, translation from the preclinical formulation to the industrial scale-up is limited. In order to provide a reproducible and reliable method of producing nanoparticles, and thus, obtain an industrial scale-up, several methods of synthesis of nanoparticles by microfluidic have been developed. Microfluidic technique allows a good control and a continuous online synthesis of nanosystems compared to synthesis in bulk, leading to a narrow size distribution, high batch-to-batch reproducibility, as well as to the industrial scale-up feasibility. This work described the optimization process to produce SLNs by microfluidics. The SLNs produced by microfluidics were characterized by complementary optical and morphological techniques and compared with those produced by bulk method. SLNs were loaded with paclitaxel and sorafenib, used as model drugs. The anti-cancer efficiency of the SLNs formulation was estimated with 2D and 3D tumour models of two different cell lines, and the cellular uptake was also studied with fluorescence-assisted measurements. Statement of significance In this work, we describe the production of a single step continuous production for solid lipid nanoparticles (SLNs) via glass capillary-based microfluidic-chip. Comparing to conventional bulk methods, the current synthesis method showed several advantages, including a continuous production with high yield, good reproducibility and precise control over the properties of SLNs, which are critical pre-conditions for its successful industrialization. The superiority of this microfluidic-based method was confirmed by an overall physicochemical characterization of the produced SLNs. The size of the SLNs was controlled by altering the microfluidic parameters, and SLNs with dimensions ca. 100 nm were feasibly fabricated through parameters optimization. The microfluidics production of SLNs offered a good encapsulation efficiency and drug loading degree for a sustained release manner . (c) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Tissue oxygen levels are known to be critical to regulation of many cellular processes, including the hepatic metabolism of therapeutic drugs, but its impact is often ignored in in vitro assays. In this study, the material-induced oxygen scavenging property of off-stoichiometric thiol-enes (OSTE) was exploited to create physiologically relevant oxygen concentrations in microfluidic immobilized enzyme reactors (IMERs) incorporating human liver microsomes. This could facilitate rapid screening of, for instance, toxic drug metabolites possibly produced in hypoxic conditions typical for many liver injuries. The mechanism of OSTE-induced oxygen scavenging was examined in depth to enable precise adjustment of the on-chip oxygen concentration with the help of microfluidic flow. The oxygen scavenging rate of OSTE was shown to depend on the type and the amount of the thiol monomer used in the bulk composition, and the surface-to-volume ratio of the chip design, but not on the physical or mechanical properties of the bulk. Our data suggest that oxygen scavenging takes place at the polymer-liquid interface, likely via oxidative reactions of the excess thiol monomers released from the bulk with molecular oxygen. Based on the kinetic constants governing the oxygen scavenging rate in OSTE microchannels, a microfluidic device comprising monolithically integrated oxygen depletion and IMER units was designed and its performance validated with the help of oxygen-dependent metabolism of an antiretroviral drug, zidovudine, which yields a cytotoxic metabolite under hypoxic conditions.

Porous silicon (PSi) is a biocompatible and biodegradable material, which can be utilized in biomedical applications. It has several favorable properties, which makes it an excellent material for building engineered nanosystems for drug delivery and diagnostic purposes. One significant hurdle for commercial applications of PSi is the lack of industrial scale production of nanosized PSi particles. Here, we report a novel two-step production method for PSi nanoparticles. The method is based on centrifuge chemical vapor deposition (cCVD) of elemental silicon in an industrial scale reactor followed by electrochemical post-processing to porous particles. Physical properties, biocompatibility and in vivo biodistribution of the cCVD produced nanoparticles were investigated and compared to PSi nanoparticles conventionally produced from silicon wafers by pulse electrochemical etching. Our results demonstrate that the cCVD production provides PSi nanoparticles with comparable physical and biological quality to the conventional method. This method may circumvent several limitations of the conventional method such as the requirements for high purity monocrystalline silicon substrates as starting material and the material losses during the top-down milling process of the pulse-etched films to porous nanoparticles. However, the electroless etching required for the porosification of cCVD-produced nanoparticles limited control over the pore size, but is amenable for scaling of the production to industrial requirements.

Drug delivery to the posterior segment of the eye is challenging due to several anatomical and physiological barriers. Thus, there is a need for prolonged action and targeted drug delivery to treat retinal diseases. Intravitreal injections avoid anterior eye barriers, but the vitreoretinal interface and inner limiting membrane (ILM) may prevent access of drug delivery systems to the retina. Existing data on retinal permeation of intravitreal nanoparticles are sparse and probably misleading due to the inter-species differences of retinal structures in rodents and humans. To bridge this gap, retinal permeation of light-activated liposomes was studied in an ex vivo bovine explant system that simulates the structure of vitreoretinal interface and intact ILM. Our findings indicate that the particle size plays a significant role in determining the retinal penetration as the liposomes of >100 nm sized failed to overcome the ILM and could not permeate into the retina. In addition, our results demonstrate the impact of surface charge and PEG-coating on retinal penetration. Small (≈ 50 nm) anionic liposomes with PEG coating showed the most extensive distribution and cellular localization in the retina. In summary, this study extends understanding of ocular barriers, and provides valuable information to augment design of retinal drug delivery systems.

We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV, based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed: varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To understand the mechanism of action and binding mode, we have obtained a crystal structure of compound 16a, bearing a primary amino group, in complex with the N-terminal domain of E. coli gyrase B (24 kDa) (PDB: 6YD9). Compound 15a, with a low molecular weight of 383 Da, potent inhibitory activity on E. coli gyrase (IC50 = 9.5 nM), potent antibacterial activity on E. faecalis (MIC = 3.13 [micro]M), and efflux impaired E. coli strain (MIC = 0.78 [micro]M), is an important contribution for the development of novel gyrase and topoisomerase IV inhibitors in Gram-negative bacteria. Abstract. Bibliografija: str. 2439-2440. EU H2020 ITN-ETN Project INTEGRATE EU FP7 Integrated Project MAREX Academy of Finland ARRS Biotechnology and Biosciences Research Council (UK) Institute Strategic Programme Grant B Wellcome Trust - (Investigator Award)

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-IgP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membrane-associated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Recently, there has been an increasing interest for utilizing the host immune system to fight against cancer. Moreover, cancer vaccines, which can stimulate the host immune system to respond to cancer in the long term, are being investigated as a promising approach to induce tumor-specific immunity. In this work, we prepared an effective cancer vaccine (denoted as vacosome) by reconstructing the cancer cell membrane, monophosphoryl lipid A as a toll-like receptor 4 agonist, and egg phosphatidylcholine. The vacosome triggered and enhanced bone marrow dendritic cell maturation as well as stimulated the antitumor response against breast cancer 4T1 cells in vitro. Furthermore, an immune memory was established in BALB/c mice after three-time preimmunization with the vacosome. After that, the immunized mice showed inhibited tumor growth and prolonged survival period (longer than 50 days). Overall, our results demonstrate that the vacosome can be a potential candidate for clinical translation as a cancer vaccine.

Aims To understand the genetics involved in surface attachment and biofilm formation of Listeria monocytogenes. Methods and Results An in vitro screen of a Himar1 transposon library of L. monocytogenes strain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01 mprF::Himar1, which had a transposon insertion in the mprF gene, was selected 29 for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as 30 stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01 31 mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic 32 antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, 33 suggesting virulence properties are compromised by the inactivation of mprF. 34 Conclusions 35 Biofilm formation and gallidermin resistance of L. monocytogenes is influenced by mprF, but this trait is 36 associated with a compromise in invasiveness. 37 Significance 38 The presence of pathogenic microorganisms in the food processing environment can cause a significant 39 problem, especially when these microorganisms are established as biofilms. This study shows that the 40 inactivation of the mprF gene results in enhanced biofilm formation and abiotic surface attachment of 41 Listeria monocytogenes.

Colorectal cancer (CRC) is the third most common and the second deadliest type of cancer worldwide, urging the development of more comprehensive models and of more efficient treatments. Although the combination of nanotechnology with chemo- and immuno-therapy has represented a promising treatment approach, its translation to the clinic has been hampered by the absence of cellular models that can provide reliable and predictive knowledge about the in vivo efficiency of the formulation. Herein, a 3D model based on CRC multicellular tumor spheroids (MCTS) model was developed by combining epithelial colon cancer cells (HCT116), human intestinal fibroblasts and monocytes. The developed MCTS 3D model mimicked several tumor features with cells undergoing spatial organization and producing extracellular matrix, forming a mass of tissue with a necrotic core. Furthermore, monocytes were differentiated into macrophages with an anti-inflammatory, pro-tumor M2-like phenotype. For a combined chemoimmunotherapy effect, spermine-modified acetalated dextran nanoparticles (NPs) loaded with the chemotherapeutic Nutlin-3a (Nut3a) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were produced and tested in 2D cultures and in the MCTS 3D model. NPs were successfully taken-up by the cells in 2D, but in a significant less extent in the 3D model. However, these NPs were able to induce an anti-proliferative effect both in the 2D and in the 3D models. Moreover, Nut3a was able to partially shift the polarization of the macrophages present in the MCTS 3D model towards an anti-tumor M1-like phenotype. Overall, the developed MCTS 3D model showed to recapitulate key features of tumors, while representing a valuable model to assess the effect of combinatorial nano-therapeutic strategies in CRC. In addition, the developed NPs could represent a promising approach for CRC treatment.

Nanoparticle morphology (size, shape, and composition) and surface chemistry are the determining factors underpinning the efficacy of such materials in therapeutic applications. The size, shape, and surface chemistry of a nanoparticle can strongly influence key properties such as interactions with diverse biological fluids and interfaces and, in turn, impact the delivery of bioactive cargo, modulating therapeutic performance. This is exemplified in ocular drug delivery, where potential therapeutics must navigate complex biological media such as the gel-like vitreal fluid and the retina. Biodegradable block copolymer amphiphiles are a robust tool for the engineering of various types of self-assembled nanoparticles with diverse morphologies ranging from spherical and tubular polymersomes to spherical and worm-like micelles. Here, we explore the effect of morphological features such as shape and surface chemistry upon the interactions of a series of copolymer nanoparticles with retinal (ARPE-19) cells and the release of a low solubility drug (dexamethasone) that is currently used in ocular therapy and study their diffusion in vitreous using ex vivo eyes. We demonstrate that both aspect ratio and surface chemistry of nanoparticles will influence their performance in terms of cell uptake, drug release, and diffusion with high aspect ratio shapes demonstrating enhanced properties in relation to their spherical counterparts.

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme., Graphical abstract Image 1, Highlights • An easy-to-use assay for sensing UGT1A1 activities has been developed, using LC-FD. • The newly developed assay is fully validated. • The LC-FD based assay is currently the most sensitive UGT1A1 activity assay.

Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson’s disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic., Graphical Abstract, Ectopic GDNF delivery raises concerns about efficacy in clinical trials of Parkinson’s disease and safety issues in preclinical studies. Andressoo and colleagues showed that a long-term, 2-fold increase in endogenous GDNF levels in mice is safe and improves motor and dopaminergic system function, supporting further development of endogenous GDNF-based therapies.

The varying levels and quality of students’ prior knowledge pose a challenge for instruction at university. Due to the scarcity of studies in pharmacy, in this study pharmacy students (N=126) prior knowledge of biosciences was measured at the beginning of their first study year using a questionnaire comprising ten multiple choice questions and a case task. The results of multiple-choice and open-ended questions revealed serious gaps and different-level misconceptions in students’ answers partly related to the absence of elementary knowledge. The level of prior knowledge correlated with study progress measured at the end of the first study year. The study’s findings suggest that it is possible to identify students at risk of delayed studies using this kind of relatively light pre-test. Problem-solving tasks such as case descriptions can be used to detect potential misconceptions. The pedagogical implications of these results are discussed.

Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n = 45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcussonʼs type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01 – 0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (−)-carvone, as most active against both bacteria, had the lowest MIC90 values. (+)-Menthyl acetate, (−)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus. Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.

This paper presents a novel microflow-based concept for studying the permeability of in vitro cell models or ex vivo tissues. Using the proposed concept, we demonstrate how to maintain physiologically relevant test conditions and produce highly reproducible permeability values for a range (31) of drug compounds. The apparent permeability coefficients (Papp) showed excellent correlation (0.89) with the values from experiments performed with a conventional Ussing chamber. Additionally, the microflow-based concept produces notably more concentrated samples than the conventional Ussing chamber-based approach, despite the fact that more than 10 times smaller quantities of test compounds and biological membranes are needed in the microflow-based concept. publishedVersion

Objective: Skin graft donor site management is a concern particularly for elderly patients and patients with poor wound healing competence, and also because donor sites are a source of pain and discomfort. Although different types of dressings exist, there is no consensus regarding optimal dressing type on donor site care to promote healing, reduce pain, and improve patients' comfort. Approach: This prospective, single-center clinical trial evaluated the performance of nanofibrillar cellulose (NFC) wound dressing (FibDex (R) by UPM-Kymmene Corporation) for treatment of donor sites compared with a polylactide-based copolymer dressing. The study enrolled 24 patients requiring skin grafting with mean age of 49 +/- 18. The primary outcome measure was wound healing time. Secondary outcomes, the epithelialization, subjective pain, the scar appearance assessed using the Patient and Observer Scar Assessment Scale (POSAS), and skin elasticity and transepidermal water loss (TEWL), were evaluated at 1 and 6 months postoperatively. Results: No statistically significant differences were observed between NFC and copolymer dressings regarding wound healing time, epithelialization, experience of pain, or TEWL. Significant differences were observed in the POSAS results for thickness and vascularity in the Observer score, in the favor of NFC over copolymer dressing. Moreover, skin elasticity was significantly improved with NFC dressing in terms of viscoelasticity and elastic modulus at 1 month postoperatively. Innovation: NFC dressing is a new, green sustainable product for wound treatment without animal or human-origin components. Conclusion: NFC dressing provides efficient wound healing at skin graft donor sites and is comparable or even preferable compared with the copolymer dressing.

Doxorubicin is a widely used anticancer drug that causes dose-related cardiotoxicity. The exact mechanisms of doxorubicin toxicity are still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose doxorubicin treatments. Here, we developed an in vitro model of long-term low-dose administration of doxorubicin utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Moreover, given that current strategies for prevention and management of doxorubicin-induced cardiotoxicity fail to prevent cancer patients developing heart failure, we also investigated whether the GATA4-targeted compound 3i-1000 has cardioprotective potential against doxorubicin toxicity both in vitro and in vivo. The final doxorubicin concentration used in the chronic toxicity model in vitro was chosen based on cell viability data evaluation. Exposure to doxorubicin at the concentrations of 1–3 µM markedly reduced (60%) hiPSC-CM viability already within 48 h, while a 14-day treatment with 100 nM doxorubicin concentration induced only a modest 26% reduction in hiPCS-CM viability. Doxorubicin treatment also decreased DNA content in hiPSC-CMs. Interestingly, the compound 3i-1000 attenuated doxorubicin-induced increase in pro-B-type natriuretic peptide (proBNP) expression and caspase-3/7 activation in hiPSC-CMs. Moreover, treatment with 3i-1000 for 2 weeks (30 mg/kg/day, i.p.) inhibited doxorubicin cardiotoxicity by restoring left ventricular ejection fraction and fractional shortening in chronic in vivo rat model. In conclusion, the results demonstrate that long-term exposure of hiPSC-CMs can be utilized as an in vitro model of delayed doxorubicin-induced toxicity and provide in vitro and in vivo evidence that targeting GATA4 may be an effective strategy to counteract doxorubicin-induced cardiotoxicity. Electronic supplementary material The online version of this article (10.1007/s00204-020-02711-8) contains supplementary material, which is available to authorized users.

This commentary article conveys the views of the board of the Nanomedicine and Nanoscale Delivery Focus Group of the Controlled Release Society regarding the decision of the United States National Cancer Institute (NCI) in halting funding for the Centers of Cancer Nanotechnology Excellence (CCNEs), and the subsequent editorial articles that broadened this discussion.

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine-105 (S105) for the actions of GATA4 in an angiotensin II (AngII)-induced hypertension rat model. Adenoviral constructs overexpressing wild type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg x kg-1 x h-1) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT-qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII?induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult-to-foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild type or mutated GATA4. Our results indicate that GATA4 reduces AngII-induced responses by interfering with pro-fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.

Oncolytic virotherapy is an emerging therapeutic approach based on replication-competent viruses able to selectively infect and destroy cancer cells, inducing the release of tumor-associated antigens and thereby recruiting immune cells with a subsequent increase in antitumoral immune response. To increase the anticancer activity, we engineered a specific oncolytic adenovirus expressing a single-chain variable fragment of an antibody against PD-L1 to combine blockage of PD-1/PD-L1 interaction with the antitumoral activity of Onc.Ad5. To assess its efficacy, we infected B16.OVA cells, a murine model of melanoma, with Ad5Δ24 -anti-PD-L1-scFv and then co-cultured them with C57BL/6J naïve splenocytes. We observed that the combinatorial treatments were significantly more effective in inducing cancer cell death. Furthermore, we assessed the efficacy of intratumoral administrations of Ad5Δ24-anti-PD-L1-scFv in C57BL/6J mice engrafted with B16.OVA and compared this treatment to that of the parental Ad5Δ24 or placebo. Treatment with the scFv-expressing Onc.Ad induced a marked reduction of tumor growth concerning the parental Onc.Ad. Additionally, the evaluation of the lymphocytic population infiltrating the treated tumor reveals a favorable immune profile with an enhancement of the CD8+ population. These data suggest that Onc.Ad-mediated expression of immune checkpoint inhibitors increases oncolytic virotherapy efficacy and could be an effective and promising tool for cancer treatments, opening a new way into cancer therapy.

Biopolymeric patches show enormous potential for the regeneration of infarcted myocardium tissues. However, most of them usually lack appropriate mechanical performance, stability in water, and important functionalities; for instance, antioxidant activity. Protein nanofibrils, such as lysozyme nanofibrils (LNFs), are biocompatible nanostructures with excellent mechanical performance, water insolubility, and antioxidant activity exploited to fabricate materials for different biomedical applications. In this study, LNFs are used to produce gelatin electrospun nanocomposite cardiac patches with improved properties. The addition of the LNFs to the gelatin electrospun patches enhance their mechanical properties, increasing the patches Young's modulus from 3 to 6 MPa, in their wet state, which agrees with the requirements of myocardial contractility. Additionally, it is observed an increment of the antioxidant activity to 80%, by adding only 5% (w/w) of LNFs, and the bioresorbability rate is shortened to 30-35 d, compared to 45 d for the gelatin-only patches, while maintaining their morphology, and biocompatibility toward cardiomyoblasts and fibroblasts. Furthermore, 15% of a model drug is burst released from the patches and preserved for 21 d. Overall, these results demonstrate that LNFs have a great potential as functional reinforcements to fabricate biopolymeric electrospun patches for myocardial infarcted tissue regeneration.

Slurry studies are useful for exhaustive polymorph and solid-state stability screening of drug compounds. Raman spectroscopy is convenient for monitoring crystallization in such slurries, as the measurements can be performed in situ even in aqueous environments. While the mid-frequency region (400-4000 cm(-1)) is dominated by intramolecular vibrations and has traditionally been used for such studies, the low-frequency spectral region (

Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.

The purpose of our study was to improve the solubility, bioavailability, and efficacy of zotepine (ZTP) by brain-targeted intranasal delivery of microemulsion (ME) and its physicochemical properties, the pharmacokinetic and pharmacodynamic parameters were evaluated. The optimized ME formulations contain 10% w/w of oil (Capmul MCM C8, monoglycerides, and diglycerides of caprylic acid), 50% w/w of S-mix (Labrasol and Transcutol HP, and 40% w/w of water resulting in a globule size of 124.6 +/- 3.52 nm with low polydispersity index (PDI) (0.212 +/- 0.013) and 2.8-fold higher permeation coefficient through porcine nasal mucosa compared to pure drug). In vitro cell line studies on RPMI 2650, Beas-2B, and Neuro-2A revealed ZTP-ME as safe. ZTP-ME administered intranasally showed higher AUC(0-t24) (18.63 +/- 1.33 h x mu g/g) in the brain by approximately 4.3-fold than oral ME (4.30 +/- 0.92 h x mu g/g) and 7.7-fold than intravenous drug solutions (2.40 +/- 0.36 h x mu g/g). In vivo anti-schizophrenic activity was conducted using catalepsy test scores, the formulation showed better efficacy via the intranasal route; furthermore, there was no inflammation or hemorrhage in the nasal cavity. The results concluded that the ZTP microemulsion as a safe and effective strategy could greatly enhance brain distribution by intranasal administration.

Fibrotic diseases remain a substantial health burden with few therapeutic approaches. A hallmark of fibrosis is the aberrant activation and accumulation of myofibroblasts, which is caused by excessive profibrotic cytokines. Conventional anticytokine therapies fail to undergo clinical trials, as simply blocking a single or several antifibrotic cytokines cannot abrogate the profibrotic microenvironment. Here, biomimetic nanoparticles based on autologous skin fibroblasts are customized as decoys to neutralize multiple fibroblast-targeted cytokines. By fusing the skin fibroblast membrane onto poly(lactic-co-glycolic) acid cores, these nanoparticles, termed fibroblast membrane-camouflaged nanoparticles (FNPs), are shown to effectively scavenge various profibrotic cytokines, including transforming growth factor-beta, interleukin (IL)-11, IL-13, and IL-17, thereby modulating the profibrotic microenvironment. FNPs are sequentially prepared into multiple formulations for different administration routines. As a proof-of-concept, in three independent animal models with various organ fibrosis (lung fibrosis, liver fibrosis, and heart fibrosis), FNPs effectively reduce the accumulation of myofibroblasts, and the formation of fibrotic tissue, concomitantly restoring organ function and indicating that FNPs are a potential broad-spectrum therapy for fibrosis management.

Thrombosis and infections are the two major complications associated with extracorporeal circuits and indwelling medical devices, leading to significant mortality in clinic. To address this issue, here, we report a biomimetic surface engineering strategy by the integration of mussel-inspired adhesive peptide, with bio-orthogonal click chemistry, to tailor the surface functionalities of tubing and catheters. Inspired by mussel adhesive foot protein, a bioclickable peptide mimic (DOPA) 4 -azide-based structure is designed and grafted on an aminated tubing robustly based on catechol-amine chemistry. Then, the dibenzylcyclooctyne (DBCO) modified nitric oxide generating species of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelated copper ions and the DBCO-modified antimicrobial peptide (DBCO-AMP) are clicked onto the grafted surfaces via bio-orthogonal reaction. The combination of the robustly grafted AMP and Cu-DOTA endows the modified tubing with durable antimicrobial properties and ability in long-term catalytically generating NO from endogenous s-nitrosothiols to resist adhesion/activation of platelets, thus preventing the formation of thrombosis. Overall, this biomimetic surface engineering technology provides a promising solution for multicomponent surface functionalization and the surface bioengineering of biomedical devices with enhanced clinical performance.

Biomaterial aerogel fabrication by freeze-drying must be further improved to reduce the costs of lengthy freeze-drying cycles and to avoid the formation of spongy cryogels and collapse of the aerogel structures. Residual water content is a critical quality attribute of the freeze-dried product, which can be monitored in-line with near-infrared (NIR) spectroscopy. Predictive models of NIR have not been previously applied for biomaterials and the models were mostly focused on the prediction of only one formulation at a time. We recorded NIR spectra of different nanofibrillated cellulose (NFC) hydrogel formulations during the secondary drying and set up a partial least square regression model to predict their residual water contents. The model can be generalized to measure residual water of formulations with different NFC concentrations and the excipients, and the NFC fiber concentrations and excipients can be separated with the principal component analysis. Our results provide valuable information about the freeze-drying of biomaterials and aerogel fabrication, and how NIR spectroscopy can be utilized in the optimization of residual water content.

Background Drug shortages are a growing global problem, posing clinical and economic challenges. To understand them better, we conducted an inventory of national public drug shortage registers and their comparability in Europe and the USA. Methods The study was based on openly accessible drug shortage notifications published by national drug authorities. These data were obtained from all national data sources mentioned on the European Medicines Agency’s (EMA’s) web page and FDA in the USA. After selection of the countries with comparable data, descriptive statistics were used to present characteristics of the shortages both across countries and within countries for 9 months (January–September) in 2020. We studied whether the shortages that occurred in these countries were the same, and how shortages were distributed by therapeutic uses and formulations. We also investigated price variation between the United States and Finland among drugs in shortage in one formulation category (creams and gels). Results Finland, Sweden, Norway, Spain, and the United States had suitable registers and were included. Altogether 5132 shortage reports from Finland (n = 1522), Sweden (n = 890), Norway (n = 800), Spain (n = 814), and the United States (n = 1106) were published during the study period. Of active ingredient level shortages 54% occurred in only one country, and 1% occurred in all five. However, at the country level, where there was one or more shortage notifications in an ATC active ingredient category, 19–41% were in a single country. The distributions by ATC therapeutic class and drug formulation differed substantially between countries, particularly between the USA and European countries. Injectables had a high shortage risk in the USA (57% of all shortages versus 17–31% of all shortages in the European countries). By contrast, shortages in gels and creams occurred only in European data (4–6% of all shortages). In the price comparison, creams and gels in shortage in Finland were 160% more expensive in the USA where these shortages were not detected. Conclusions Public drug shortage registers are vital data sources for proactively maintaining and managing a reliable drug supply. However, our study demonstrates that much work remains to standardize the contents and quality of public register data. Shortages may not be solely a consequence of manufacturing disruptions but may reflect other contributing factors in the international drug distribution and supply mechanisms, including price differences and profit margins between national pharmaceutical markets. Data to perform practical and useful international comparisons to understand these shortages are required.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5′ ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5′ ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.