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Background Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Biofilms are considered one of the most challenging areas of modern medicine. Existing antibiotics have been developed against free-floating bacterial cells, and thus, many treatments of biofilm-related infection fail. In this study, we compared the effects of different media on biofilm growth of clinical reference strains of Staphylococci and Enterococci, including multi-drug resistant representatives. Further, we optimized the resazurin-based assay for determining the minimal biofilm inhibitory concentration (MBIC) of standard antibiotics, and evaluated its use for the determination of minimal biofilm eradication concentration (MBEC). Results We showed that tryptic soy broth supplemented with 1% glucose was an optimal media for maximum biofilm growth of all strains tested, with an extended incubation time for Enterococci. A range of parameters were tested for the resazurin assay, including concentration, temperature and time of incubation. Using quality parameters to analyze the assay’s performance, the conditions for the resazurin assay were set as follows: 4 μg/mL and 8 μg/mL, with incubation at 25 °C for 20 min and 40 min for Staphylococci and Enterococci, respectively. Conclusions In summary, we defined conditions for optimal biofilm growth and for standardized resazurin assay for MBIC determination against six Gram-positive clinical reference strains. We also observed that MBEC determination by the resazurin-based assay is limited due to the poor detection limit of the assay. Complementary cell counting data is needed for precise determination of MBEC. Electronic supplementary material The online version of this article (10.1186/s12866-018-1321-6) contains supplementary material, which is available to authorized users.

Funding Information: V. Känkänen and H.A. Santos acknowledge Bayer Oy (Finland) for financial support. H.A. Santos acknowledges also the Sigrid Jusélius Foundation and the UMCG Research Fund for financial support. Publisher Copyright: © 2022 The Authors The use of amphiphilic block copolymers to generate colloidal delivery systems for hydrophobic drugs has been the subject of extensive research, with several formulations reaching the clinical development stages. However, to generate particles of uniform size and morphology, with high encapsulation efficiency, yield and batch-to-batch reproducibility remains a challenge, and various microfluidic technologies have been explored to tackle these issues. Herein, we report the development and optimization of poly(ethylene glycol)-block-(ε-caprolactone) (PEG-b-PCL) nanoparticles for intravenous delivery of a model drug, sorafenib. We developed and optimized a glass capillary microfluidic nanoprecipitation process and studied systematically the effects of formulation and process parameters, including different purification techniques, on product quality and batch-to-batch variation. The optimized formulation delivered particles with a spherical morphology, small particle size (dH < 80 nm), uniform size distribution (PDI < 0.2), and high drug loading degree (16 %) at 54 % encapsulation efficiency. Furthermore, the stability and in vitro drug release were evaluated, showing that sorafenib was released from the NPs in a sustained manner over several days. Overall, the study demonstrates a microfluidic approach to produce sorafenib-loaded PEG-b-PCL NPs and provides important insight into the effects of nanoprecipitation parameters and downstream processing on product quality.

Inspired by natural resources, such as peptides and carbohydrates, glycopolypeptide biopolymer has recently emerged as a new form of biopolymer being recruited in various biomedical applications. Glycopolypeptides with well-defined secondary structures and pendant glycosides on the polypeptide backbone have sparked lots of research interest and they have an innate ability to self-assemble in diverse structures. The nanostructures of glycopolypeptides have also opened up new perspectives in biomedical applications due to their stable three-dimensional structures, high drug loading efficiency, excellent biocompatibility, and biodegradability. Although the development of glycopolypeptide-based nanocarriers is well-studied, their clinical translation is still limited. The present review highlights the preparation and characterization strategies related to glycopolypeptides-based copolymers, followed by a comprehensive discussion on their biomedical applications with a specific focus on drug delivery by various stimuli-responsive (e.g., pH, redox, conduction, and sugar) nanostructures, as well as their beneficial usage in diagnosis and regenerative medicine.

In this article, formulation studies for terbinafine hydrochloride nanoemulsions, prepared by high-energy ultrasonication technique, are described. Pseudo-ternary phase diagram was constructed in order to find out the optimal ratios of oil and surfactant/co-solvent mixture for nanoemulsion production. Clove and olive oils were selected as oil phase. Based on the droplet size evaluation, maximum nanoemulsion region were determined for formulation development. Further characterization included polydispersity index (PDI), zeta potential, Fourier transform infrared (FT-IR) spectroscopy, morphology, pH, viscosity, refractive index, ex vivo skin permeation, skin irritation, and histopathological examination. Droplet sizes of optimized formulations were in colloidal range. PDI values below 0.35 indicated considerably homogeneous nanoemulsions. Zeta potential values were from 13.2 to 18.1 mV indicating good stability, which was also confirmed by dispersion stability studies. Ex vivo permeation studies revealed almost total skin permeation of terbinafine hydrochloride from the nanoemulsions (96-98%) in 6 hours whereas commercial product reached only 57% permeation at the same time. Maximum drug amounts were seen in epidermis and dermis layers. Skin irritation and histopathological examination demonstrated dermatologically safe formulations. In conclusion, olive oil and clove oil-based nanoemulsion systems have potential to serve as promising carriers for topical terbinafine hydrochloride delivery.

Lymphatic vessels are an integral part of the immune system that defends us against bacteria and viruses. These vessels are a drainage system for returning interstitial tissue fluid and immune cells into the blood circulation. A dysfunctional lymphatic system causes swelling of tissues due to excess fluid, known as lymphedema. The primary factor that stimulates the growth of lymphatic vessels (lymphangiogenesis) is vascular endothelial growth factor-C (VEGF-C). Although this has been known since 1997, many mechanistic details of VEGF-C’s action have only been recently uncovered. VEGF-C needs to be activated by the removal of its C- and N-terminal propeptides before it can generate new vessels, and this activation requires the CCBE1 helper protein and one of several proteases. VEGF-C can be a drug target for diseases involving the lymphatics, such as lymphedema, cancer, and heart diseases. However, the main limitation is our incomplete understanding of the actual mechanisms of VEGF-C activation for developing a functional lymphatic vasculature. The primary aim of my studies has been to identify additional puzzle pieces in VEGF-C activation, which will prove instrumental in future attempts to therapeutically target VEGF-C. In study I, we have characterized the role of the individual domains of VEGF-C and CCBE1 in the activation of VEGF-C. Our study determined the crucial role of the C-terminal domain of VEGF-C for efficient VEGF-C activation. In addition, we established differential roles of C- and N-terminal domains of CCBE1 for VEGF-C activation. The N-terminal domain is essential for mobilizing VEGF-C to the endothelial cell surface to form the VEGF-C/ADAMTS3/CCBE1 cleavage complex, whereas the C-terminal domain aids ADAMTS3-mediated VEGF-C processing. We have also proposed a model of VEGF-C activation where VEGF-C can be activated when bound to VEGFR-3/HSPGs on the cell surface or to ECM as well as in the soluble phase. In study II, we discovered additional proteases activating VEGF-C. We identified both VEGF-C and VEGF-D as substrates for KLK3 and cathepsin D. The presence of KLK3, VEGF-C, and CCBE1 in sperm plasma suggested a role of KLK3-activated VEGF-C in reproduction. KLK3 and cathepsin D cleavage of VEGF-C and VEGF-D generated N-terminally different forms that varied in their affinities towards VEGFR-2 and VEGFR-3. Cathepsin D-cleaved VEGF-C was VEGFR-3 specific and lost its angiogenic properties, whereas cathepsin-D-activated VEGF-D was VEGFR-2 specific and lost its lymphangiogenic properties. Identification of novel VEGF-C activating proteases has opened ways to therapeutically target VEGF-C in reproduction and metastatic cancer besides lymphedema treatment. In study III, we described for the first time a method to produce biologically active VEGF-C from E. coli using a combination of redox-modified Origami strain and maltose binding protein (MBP) tag. There are no reports of successful attempts to produce active VEGF-C in E. coli without the need for refolding from inclusion bodies. Such bacterial VEGF-C could be a readily available cost- and time-effective source of VEGF-C for in vitro studies. In study IV, we analyzed the occurrence of PDGF/VEGF homologs in different animal clades. We identified Cnidaria as the simplest animals containing PDGF/VEGF-like proteins. Almost all cnidarian PDGF/VEGFs contain the BR3P repeat characteristic for the C-terminal domain of VEGF-C. This finding suggests a VEGF-C-like protein as the earliest PDGF/VEGF to emerge during evolution. Interestingly, we showed a complete absence of PlGF in Amphibia and VEGF-B in crocodiles and birds. In addition, the presence of VEGF-F was not limited to venomous reptiles but could also be found in non-venomous lizards and gekkos. Due to recent advancements in our understanding of the development of fish vasculature, we also analyzed the heterogeneity of PDGF/VEGF molecules in fishes, including verification of gene prediction with mRNA sequencing data. This study provides a basis for choosing appropriate animal models for vascular biology research. With these studies, we primarily advance the understanding of VEGF-C biology and thus lay the groundwork for developing VEGF-C into a therapeutic target for diseases involving the lymphatic system. Imusuonet ovat olennainen osa immuunijärjestelmää, joka suojaa meitä bakteereilta ja viruksilta. Nämä suonet keräävät interstitiaalisen kudosnesteen ja immuunisolut palauttaakseen ne takaisin verenkiertoon. Viallinen imusuonisto aiheuttaa kudosten turvotusta ylimääräisen nesteen vuoksi, mikä tunnetaan nimellä lymfedeema. Ensisijainen imusuonten kasvua (lymfangiogeneesiä) stimuloiva tekijä on verisuonten endoteelin kasvutekijä-C (VEGF-C). Ennen kuin VEGF-C voi luoda uusia suonia, se on aktivoitava poistamalla sen C- ja N-terminaaliset propeptidit, ja tämä aktivaatio vaatii CCBE1-auttajaproteiinin sekä yhden useista proteaaseista. VEGF-C voi olla lääkinnällinen kohde sairauksissa, jotka liittyvät imusuonistoon, kuten lymfedeema, syöpä ja sydänsairaudet. Suurin rajoitus liittyy kuitenkin epätäydelliseen ymmärrykseemme VEGF-C:n aktivaation todellisista mekanismeista toimivan imusuoniston kehittämiseksi. Tämän tutkimuksen ensisijaisena tavoitteena oli tunnistaa lisää sellaisia palapelin palasia VEGF-C:n aktivaatiossa, jotka osoittautuisivat tärkeiksi tulevissa yrityksissä VEGF-C:n terapeuttisessa kohdistamisessa. Tutkimuksessa I on karakterisoitu VEGF-C:n ja CCBE1:n yksittäisten domeenien rooli VEGF-C:n aktivoinnissa. Tutkimus osoitti VEGF-C:n C-terminaalisen domeenin ratkaisevan roolin tehokkaassa VEGF-C-aktivaatiossa. Lisäksi selvitettiin CCBE1:n C- ja N-terminaalisten domeenien erilaiset roolit VEGF-C-aktivaatiossa. N-terminaalinen domeeni on välttämätön VEGF-C:n mobilisoimiseksi endoteelisolujen pintaan VEGF-C/ADAMTS3/CCBE1-kompleksin muodostamiseksi, kun taas C-terminaalinen domeeni auttaa ADAMTS3-välitteistä VEGF-C:n prosessointia. Tutkimuksessa on myös esitetty malli VEGF-C-aktivaatiosta, jossa VEGF-C voidaan aktivoida sekä solun pinnalla VEGFR-3/HSPG:eihin sitoutuneena, että liukoisena solunulkoisessa väliaineessa. Tutkimuksessa II havaittiin lisää proteaaseja, jotka aktivoivat VEGF-C:tä. Sekä VEGF-C että VEGF-D ovat substraatteja KLK3:lle ja katepsiini D:lle. KLK3:n, VEGF-C:n ja CCBE1:n läsnäolo siemenplasmassa viittasi KLK3-aktivoidun VEGF-C:n rooliin lisääntymisessä. KLK3 ja katepsiini D pilkkovat VEGF-C:n ja VEGF-D:n N-terminaaliset päät niin, että muodostuu N-terminaalisesti erilaisia muotoja, joiden affiniteetit vaihtelevat VEGFR-2:ta ja VEGFR-3:a kohtaan. Katepsiini D:llä pilkottu VEGF-C oli VEGFR-3-spesifinen ja menetti angiogeeniset ominaisuutensa, kun taas katepsiini-D-aktivoitu VEGF-D oli VEGFR-2-spesifinen ja menetti lymfangiogeeniset ominaisuutensa. Uusien VEGF-C:tä aktivoivien proteaasien tunnistaminen on avannut tapoja kohdistaa VEGF-C:tä terapeuttisesti hedelmöityshoidoissa ja metastaattisissa syövissä lymfedeemahoidon lisäksi. Tutkimuksessa III kuvattiin ensimmäistä kertaa menetelmä biologisesti aktiivisen VEGF-C:n tuottamiseksi E. colissa. Tässä hyödynnettiin redox-modifioidun Origami-kannan ja maltoosia sitovan proteiinin (MBP) yhdistelmää. Tämä menetelmä tuotti biologisesti aktiivista proteiinia, joka on aiemmissa raportoiduissa yrityksissä vaatinut uudelleenlaskostamisen inkluusiokappaleista. Tällainen bakteeriperäinen VEGF-C voisi olla helposti saatavilla oleva kustannus- ja aikatehokas VEGF-C:n lähde in vitro -tutkimuksiin. Tutkimuksessa IV analysoitiin PDGF/VEGF-homologien esiintymistä eri eläinkladeissa. Cnidaria-pääjaksoon kuuluvat lajit tunnistettiin yksinkertaisimmiksi PDGF/VEGF:n kaltaisia proteiineja sisältäviksi eläimiksi. Havaittiin, että VEGF-C:n kaltainen proteiini on varhaisin muoto PDGF/VEGF-tyyppisistä proteiineista, joka on ilmaantunut evoluution aikana. Mielenkiintoista on, että PlGF puuttui täysin sammakkoeläimistä ja VEGF-B puuttui krokotiileista ja linnuista. Lisäksi huomattiin, että VEGF-F:n esiintyminen ei rajoittunut vain myrkyllisiin matelijoihin, vaan sitä voitiin löytää myös ei-myrkyllisistä liskoista ja gekkoista. Tämä tutkimus tarjoaa pohjan sopivien eläinmallien valinnalle verisuonibiologian tutkimuksiin. Näillä tutkimuksilla edistetään ensisijaisesti VEGF-C:n biologian ymmärtämistä ja luodaan pohja imusuoniston sairauksien hoidolle.

Azulene is a rare ring structure in drugs, and we investigated whether it could be used as a biphenyl mimetic in known orexin receptor agonist Nag 26, which is binding to both orexin receptors OX1 and OX2 with preference towards OX2. The most potent azulene-based compound was identified as an OX1 orexin receptor agonist (pEC50 = 5.79 +/- 0.07, maximum response = 81 +/- 8% (s.e.m. of five independent experiments) of the maximum response to orexin-A in Ca2+ elevation assay). However, the azulene ring and the biphenyl scaffold are not identical in their spatial shape and electron distribution, and their derivatives may adopt different binding modes in the binding site.

This study investigates pharmacy students’ reasoning while solving a case task concerning an acute patient counselling situation in a pharmacy. Participants’ (N = 34) reasoning processes were investigated with written tasks utilizing eye-tracking in combination with verbal protocols. The case was presented in three pages, each page being followed by written questions. Eye movements were recorded during case processing. Success in the task required differentiating the relevant information from the task redundant information, and initial activation of several scripts and verification of the most likely one, when additional information became available. 2nd (n = 16) and 3rd (n = 18) -year students’ and better and worse succeeding students’ processes were compared. The results showed that only a few 2nd-year students solved the case correctly, whereas almost all of the 3rd-year students were successful. Generally, the average total processing times of the case material did not differ between the groups. However, better-succeeding and 3rd-year students processed the very first task-relevant sentences longer, indicating that they were able to focus on relevant information. Differences in the written answers to the 2nd and 3rd question were significant, whereas differences regarding the first question were not. Thus, eye-tracking seems to be able to capture illness script activation during case processing, but other methods are needed to depict the script verification process. Based on the results, pedagogical suggestions for advancing pharmacy education are discussed. This study investigates pharmacy students’ reasoning while solving a case task concerning an acute patient counselling situation in a pharmacy. Participants’ (N=34) reasoning processes were investigated with written tasks utilizing eye-tracking in combination with verbal protocols. The case was presented in three pages, each page being followed by written questions. Eye movements were recorded during case processing. Success in the task required differentiating the relevant information from the task redundant information, and initial activation of several scripts and verification of the most likely one, when additional information became available. 2nd (n=16) and 3rd (n=18)-year students’ and better and worse succeeding students’ processes were compared. The results showed that only a few 2nd-year students solved the case correctly, whereas almost all of the 3rd-year students were successful. Generally, the average total processing times of the case material did not differ between the groups. However, better-succeeding and 3rd-year students processed the very first task-relevant sentences longer, indicating that they were able to focus on relevant information. Differences in the written answers to the 2nd and 3rd question were significant, whereas differences regarding the first question were not. Thus, eye-tracking seems to be able to capture illness script activation during case processing, but other methods are needed to depict the script verification process. Based on the results, pedagogical suggestions for advancing pharmacy education are discussed.

Organoboron compounds are well known for their use as synthetic building blocks in several significant reactions, e.g., palladium-catalyzed Suzuki-Miyaura cross-coupling. As an element, boron is fascinating; as part of a molecule it structurally resembles a three-valent atom, but if there is a lone pair of electrons nearby, the boron atom’s empty p-orbital may capture the lone pair and form a covalent bond. This is the main aspect that is challenging chemistry during the synthesis of boron containing molecules and may lead into unexpected reactions and products. To study this, we synthesized and studied novel aromatic azaborines for better understanding of their structures and reactions. Here, we report a one-pot method for the synthesis of substituted aromatic azaborines and computational studies of their structure to explain their observed chemical properties.

Background Biological drugs are generally expensive and produce a continuously growing share of drug costs. Yet they are essential in the treatment of many chronic diseases. Biosimilars, clinically equivalent to biological originator products, are expected to restrain drug costs in the biological market. Objective This study aimed to examine the impact of the biosimilar market entry on the prices of the reference products in outpatient care in Finland, investigate the impact of biosimilar market entries on price competition among biological medicinal products, and examine how the prices and market shares of outpatient biosimilars have developed in Finland during 2009-2020. Methods This retrospective register study applied data from IQVIA covering national community pharmacy wholesale data between 1 January, 2009, and 31 August, 2020, for somatropin, epoetin, filgrastim, follitropin, insulin glargine, insulin lispro, etanercept, pegfilgrastim, adalimumab, teriparatide, and enoxaparin biosimilars and their reference products, in addition to two relevant insulin products. We determined the monthly wholesale amounts in defined daily doses and wholesale weighted average prices (excluding value-added tax) per defined daily dose for each product. We analyzed the evolution of the price and market shares. We performed a linear segmented regression analysis to examine the impact of the market entry of biosimilars on the prices of reference products. Results The prices of the reference products mainly decreased after the biosimilar entered the market. If the reference product price was not reduced, it was no longer reimbursable after evaluation under the Health Insurance Act, leading to marginal market shares. The changes in the prices of biosimilars were not as remarkable as the changes in the prices of reference products after the biosimilar market entry. For most active substances, biosimilar prices were stable or decreased. The utilization of biosimilars varied widely between different active substances at the end of the observation period. Conclusions Changes in pricing policy and the public reimbursement scheme related to the market entry of biosimilars were the main reasons for the decrease in the prices of reference products. Therefore, biosimilars did not generate genuine price competition between biological products. In many of the drug groups examined, the market shares of biosimilars have growth potential in the future.

Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. The intratumoral administration of these repurposed vaccines can induce immune cell infiltra-tion into the treated tumor. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV cancer vaccine platform. The intratumoral administration of this novel MMR-containing PeptiENV cancer vaccine significantly increased both intratumoral as well as systemic tumor-specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, improved tumor growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy. Importantly, mice pre-vaccinated with the MMR vaccine responded equally well, if not better, to the PeptiENV therapy, indicating that pre-existing immunity against the MMR vaccine viruses does not compromise the use of this novel cancer vaccine platform.

Background There is a need for effective and cost-effective interprofessional care models that support older people to maintain their quality of life (QoL) and physical performance to live longer independently in their own homes. Objectives The objectives were to evaluate effectiveness, QoL and physical performance, and cost-utility of a people-centred care model (PCCM), including the contribution of clinically trained pharmacists, compared with that of usual care in primary care. Methods A randomised controlled trial (RCT) with a two-year follow-up was conducted. The participants were multimorbid community-living older people, aged ≥75 years. The intervention comprised an at-home patient interview, health review, pharmacist-led clinical medication review, an interprofessional team meeting, and nurse-led care coordination and health support. At the baseline and at the 1-year and 2-year follow-ups, QoL (SF-36, 36-Item Short-Form Health Survey) and physical performance (SPPB, Short Performance Physical Battery) were measured. Additionally, a physical dimension component summary in the SF-36 was calculated. The SF-36 data were transformed into SF-6D scores to calculate quality-adjusted life-years (QALYs). Healthcare resource use were collected and transformed into costs. A healthcare payer perspective was adopted. Incremental cost-effectiveness ratio (ICER) was calculated, and one-way sensitivity analysis was performed. Results No statistically or clinically significant differences were observed between the usual care (n = 126) and intervention group (n = 151) patients in their QoL; at the 2-year follow-up the mean difference was −0.02, (95 % CI -0.07; 0.04,p = 0.56). While the mean difference between the groups in physical performance at the 2-year follow-up was −1.02, (−1.94;-0.10,p = 0.03), between the physical component summary scores it was −7.3, (−15.2; 0.6,p = 0.07). The ICER was −73 638€/QALY, hence, the developed PCCM dominated usual care, since it was more effective and less costly. Conclusions The cost-utility analysis showed that the PCCM including pharmacist-led medication review dominated usual care. However, it had no effect on QoL and the effect towards physical performance remained unclear.

Publisher Copyright: © 2023 The Authors A series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 μM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.

The repertoire of naturally presented peptides within the MHC (major histocompatibility complex) or HLA (human leukocyte antigens) system on the cellular surface of every mammalian cell is referred to as ligandome or immunopeptidome. This later gained momentum upon the discovery of CD8 + T cells able to recognize and kill cancer cells in an MHC-I antigen-restricted manner. Indeed, cancer immune surveillance relies on T cell recognition of MHC-I-restricted peptides, making the identification of those peptides the core for designing T cell-based cancer vaccines. Moreover, the breakthrough of antibodies targeting immune checkpoint molecules has led to a new and strong interest in discovering suitable targets for CD8 +T cells. Therapeutic cancer vaccines are designed for the artificial generation and/or stimulation of CD8 +T cells; thus, their combination with ICIs to unleash the breaks of the immune system comes as a natural consequence to enhance anti-tumor efficacy. In this context, the identification and knowledge of peptide candidates take advantage of the fast technology updates in immunopeptidome and mass spectrometric methodologies, paying the way to the rational design of vaccines for immunotherapeutic approaches. In this review, we discuss mainly the role of immunopeptidome analysis and its application for the generation of therapeutic cancer vaccines with main focus on HLA-I peptides. Here, we review cancer vaccine platforms based on two different preparation methods: pathogens (viruses and bacteria) and not (VLPs, nanoparticles, subunits vaccines) that exploit discoveries in the ligandome field to generate and/or enhance anti-tumor specific response. Finally, we discuss possible drawbacks and future challenges in the field that remain still to be addressed.

Remarkable progress in phototherapy has been made in recent decades, due to its non-invasiveness and instant therapeutic efficacy. In addition, with the rapid development of nanoscience and nanotechnology, phototherapy systems based on nanoparticles or nanocomposites also evolved as an emerging hotspot in nanomedicine research, especially in cancer. In this review, first we briefly introduce the history of phototherapy, and the mechanisms of phototherapy in cancer treatment. Then, we summarize the representative development over the past three to five years in nanoparticle-based phototherapy and highlight the design of the innovative nanoparticles thereof. Finally, we discuss the feasibility and the potential of the nanoparticle-based phototherapy systems in clinical anticancer therapeutic applications, aiming to predict future research directions in this field. Our review is a tutorial work, aiming at providing useful insights to researchers in the field of nanotechnology, nanoscience and cancer.

Background: Poor medication management may negatively impact the health and functional capacity of older adults. This cross-sectional study aimed to identify medication-related risk factors in home-dwelling residents using a validated self-assessment as part of comprehensive health screening. Methods: The data were derived from comprehensive health screening (PORI75) for older adults of 75 years living in Western Finland in 2020 and 2021. One of 30 validated measures in health screening focused on identifying medication-related risk factors (LOTTA Checklist). The Checklist items were divided into (1) systemic risk factors (10 items) and (2) potentially drug-induced symptoms (10 items). Polypharmacy was categorized according to the number of used drugs: (1) no polypharmacy (5 drugs, that is, having polypharmacy. Of the systemic risk factors most common was that the resident had more than one physician responsible for the treatment (48% of the residents), followed by missing drug list (43%), missing regular monitoring (35%), and unclear durations of the medication (35%). The most experienced potentially drug-induced symptoms were self-reported constipation (21%), urinating problems (20%), and unusual tiredness (17%). An increasing number of drugs in use, particularly excessive polypharmacy, was associated with various medication-related risk factors. Conclusion: As a part of comprehensive health screening the LOTTA Checklist provides useful information to prevent medication-related risk factors in home-dwelling older adults. The Checklist could be used to guide planning and implementing health services in the future. publishedVersion

Dissolving microneedles (dMNs) are promising versatile drug delivery systems for the transdermal delivery of numerous drugs, enabling their use in a wide range of biomedical and pharmaceutical applications. Being made of water-soluble polymers, dMNs own several advantages, including fast dissolution and short application time which enhance patients’ compliance and minimize the damage to skin tissue. Moreover, they possess no biohazard risk as they leave no sharp waste behind. For these reasons, the research on dMNs has increased dramatically in recent years. The formulation of successful dMNs requires a well-defined pre-set design, considering the goal and the payloads that will be used. Every aspect of formulation as patch design, needles geometry, polymer composition, method of formation and payloads, has a direct effect on the mechanical properties of the MNs, affecting their administration and efficacy. Thus, there is the need to understand how each factor affects the final formulation and how to optimize each MN. Taking this into consideration, this review serves as a guide for dMN formulation, discussing the different setbacks of each step and possible strategies to overcome them, improving their administration, and enhancing the loading of various molecules and their controlled release

The oral route is highly desirable for colorectal cancer (CRC) treatment because it allows concentrating the drug in the colon and achieving a localized effect. However, orally administered drugs are often metabolized in the liver, resulting in reduced efficacy and the need for higher doses. Nanoparticle-based drug delivery systems can be engineered to prevent the release of the drug in the stomach, addressing the release at the target site and enhancing the efficacy of the delivered drug. Here, we develop an orally administrable galunisertib delivery system made of gelatin-covered diatomite nanoparticles modified with an antibody targeting the L1-CAM on metastatic cells, and further encapsulated in an enteric matrix by microfluidics. The gastro-resistant polymer protects the nanoparticles from the action of the digestive enzymes and allows for a sustained release of galunisertib at the intestinal pH. The efficacy of antibody-antigen interactions to drive the internalization of nanoparticles in the targeted cells is investigated in CRC cells expressing abnormal (SW620) or basal levels (Caco-2, HT29-MTX) of L1-CAM. The combination of local drug release and active targeting enhances the effect of the delivered galunisertib, which inhibits the migration of the SW620 cells with greater efficiency compared to the free drug. This article is protected by copyright. All rights reserved.

The blood-brain barrier significantly limits effective drug delivery to central nervous system (CNS) targets. The recently characterized glymphatic system offers a perivascular highway for intrathecally (i.t.) administered drugs to reach deep brain structures. Although periarterial cerebrospinal fluid (CSF) influx and concomitant brain drug delivery can be enhanced by pharmacological or hyperosmotic interventions, their effects on drug delivery to the spinal cord, an important target for many drugs, have not been addressed. Hence, we studied in rats whether enhancement of periarterial flow by systemic hypertonic solution might be utilized to enhance spinal delivery and efficacy of i.t. morphine. We also studied whether the hyperosmolar intervention affects brain or cerebrospinal fluid drug concentrations after systemic administration. Periarterial CSF influx was enhanced by intraperitoneal injection of hypertonic saline (HTS, 5.8%, 20 ml/kg, 40 mOsm/kg). The antinociceptive effects of morphine were characterized, using tail flick, hot plate and paw pressure tests. Drug concentrations in serum, tissue and microdialysis samples were determined by liquid chromatography-tandem mass spectrometry. Compared with isotonic solution, HTS increased concentrations of spinal i.t. administered morphine by 240% at the administration level (T13-L1) at 60 min and increased the antinociceptive effect of morphine in tail flick, hot plate, and paw pressure tests. HTS also independently increased hot plate and paw pressure latencies but had no effect in the tail flick test. HTS transiently increased the penetration of intravenous morphine into the lateral ventricle, but not into the hippocampus. In conclusion, acute systemic hyperosmolality is a promising intervention for enhanced spinal delivery of i.t. administered morphine. The relevance of this intervention should be expanded to other i.t. drugs and brought to clinical trials.

Rapid precorneal loss of topically applied eye drops limits ocular drug absorption. Controlling release and precorneal residence properties of topical formulations may improve ocular drug bioavailability and duration of action. In this study, we evaluated in vivo ocular pharmacokinetics of dexamethasone in rabbits after application of a drug solution (0.01%), suspension (Maxidex (R) 0.1%), and hydrogels of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AAc) copolymers. The rabbits received a single eyedrop (solution or suspension) or dexamethasone-loaded hydrogel topically. Dexamethasone in tear fluid was sampled with glass capillaries and quantitated by LC-MS/MS. Higher dexamethasone exposure (AUC) in the tear fluid was observed with the suspension (approximate to 3.6-fold) and hydrogel (12.8-fold) as compared to the solution. During initial 15 min postapplication, the highest AUC of dissolved dexamethasone was seen after hydrogel application (368 min*mu g/ mL) followed by suspension (109.9 min*mu g/mL) and solution (28.7 min*mu g/mL. Based on kinetic simulations, dexamethasone release from hydrogels in vivo and in vitro is comparable. Our data indicate that prolonged exposure of absorbable dexamethasone in tear fluid is reached with hydrogels and suspensions. Pharmacokinetic understanding of formulation behavior in the lacrimal fluid helps in the design of dexamethasone delivery systems with improved ocular absorption and prolonged duration of action.

The development of cell membrane-modified biomimetic nanoparticles has extensively increased during the past years due to their exceptional biocompatibility, evasion from the immune system, and targeting ability. Known as a cutting-edge area of research in nanomedicine, such novel nanoplatforms can mimic different functions of the primary cells, while successfully delivering their cargos to the defect site with the aim of enhancing the therapeutic responses and reducing the side effects. Platelet is a key factor for haemostasis and a major player in wound healing, inflammation, and many other biological functions and pathological conditions. As a highly responsive cell, platelets can adapt to environment modifications and release several soluble biomolecules, such as growth factors, coagulant factors, and extracellular vesicles. Additionally, platelets are capable of immune system evasion, sub-endothelial adhesion, and pathogen interaction. These characteristics have inspired the design of several platelet membrane-coated nanoparticles as drug delivery systems. This review describes the current developments in platelet membrane-coated nanoparticles for targeted therapy, specifically, their advantages compared to other biomimetic cell-derived nanoparticles and their applicability in the medical field are elucidated. Finally, the challenges and future perspectives associated with this nanoplatform are summarised.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located pro-tein with cytoprotective effects in neurons and pancreatic b cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1a, and we identify the binding interface between MANF and IRE1a. The expression of wild-type MANF, but not its IRE1a binding-deficient mutant, attenuates UPR signaling by decreasing IRE1a oligomerization; phosphor-ylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1a interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1a as an intracellular receptor for MANF and regulator of neuronal survival.

IntroductionImmune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer, but preclinical testing of hypotheses such as combination therapies has been complicated, in part due to species incompatibility issues. For example, one of few known permissive animal models for oncolytic adenoviruses is the Syrian hamster, for which an ICI, mainly an anti-PD-L1 monoclonal antibody (mAb) was not previously available. In this study, we developed an anti-Syrian hamster PD-L1 mAb to enable the evaluation of safety and efficacy, when combining anti-PD-L1 with an oncolytic adenovirus encoding tumour necrosis factor alpha (TNFα) and interleukin-2 (IL-2) (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2 or TILT-123).MethodsRecombinant Syrian hamster PD-L1 was expressed and mice immunized for mAb formation using hybridoma technology. Clonal selection through binding and functional studies in vitro, in silico and in vivo identified anti-PD-L1 clone 11B12-1 as the primary mAb candidate for immunotherapy modelling. The oncolytic virus (OV) and ICI combination approach was then evaluated using 11B12-1 and TILT-123 in a Syrian hamster model of pancreatic ductal adenocarcinoma (PDAC).ResultsSupernatants from hybridoma parent subclone 11B12B4 provided the highest positive PD-L1 signal, on Syrian hamster PBMCs and three cancer cell lines (HT100, HapT1 and HCPC1). In vitro co-cultures revealed superior immune modulated profiles of cell line matched HT100 tumour infiltrating lymphocytes when using subclones of 7G2, 11B12 and 12F1. Epitope binning and epitope prediction using AlphaFold2 and ColabFold revealed two distinct functional epitopes for clone 11B12-1 and 12F1-1. Treatment of Syrian hamsters bearing HapT1 tumours, with 11B12-1 induced significantly better (pConclusionsNovel Syrian hamster anti-PD-L1 clone 11B12-1 induces tumour growth control in a hamster model of PDAC. Combining 11B12-1 with oncolytic adenovirus TILT-123 improves tumour growth control further and demonstrates good safety and toxicity profiles.

Drug-drug interactions (DDIs) are a major concern for the safe use of medications. Breast cancer resistance protein (BCRP) is a clinically relevant ATP-binding cassette (ABC) transporter for drug disposition. Inhibition of BCRP increases the plasma concentrations of BCRP substrate drugs, which potentially could lead to adverse drug reactions. The aim of the present study was to identify BCRP inhibitors amongst a library of 232 commonly used drugs and anticancer drugs approved by the United States Food and Drug Administration (FDA). BCRP inhibition studies were carried out using the vesicular transport assay. We found 75 drugs that reduced the relative transport activity of BCRP to less than 25% of the vehicle control and were categorized as strong inhibitors. The concentration required for 50% inhibition (IC50) was determined for 13 strong inhibitors that were previously poorly characterized for BCRP inhibition. The IC50 ranged from 1.1 to 11 mu M, with vemurafenib, dabigatran etexilate and everolimus being the strongest inhibitors. According to the drug interaction guidance documents from the FDA and the European Medicines Agency (EMA), in vivo DDI studies are warranted if the theoretical intestinal luminal concentration of a drug exceeds its IC50 by tenfold. Here, the IC50 values for eight of the drugs were 100-fold lower than their theoretical intestinal luminal concentration. Moreover, a mechanistic static model suggested that vemurafenib, bexarotene, dabigatran etexilate, rifapentine, aprepitant, and ivacaftor could almost fully inhibit intestinal BCRP, increasing the exposure of concomitantly administered rosuvastatin over 90%. Therefore, clinical studies are warranted to investigate whether these drugs cause BCRP-mediated DDIs in humans.

Parkinson’s disease (PD) is a progressive neurodegenerative disease that comprises a range of motor and nonmotor symptoms. Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopamine neuronsin vitroandin vivo, and intracranial delivery of GDNF has been tested in six clinical trials for treating PD. However, clinical trials with ectopic GDNF have yielded variable results, which could in part result from abnormal expression site and levels caused by ectopic overexpression. Therefore, an important open question is whether an increase in endogenous GDNF expression could be potent in reversing PD progression. Here, we tested the therapeutic potential of endogenous GDNF using mice in which endogenous GDNF can be conditionally upregulated specifically in cells that express GDNF naturally (conditional GDNF hypermorphic mice;GdnfcHyper). We analyzed the impact of endogenous GDNF upregulation in both neuroprotection and neurorestoration procedures, and for both motor and nonmotor symptoms in the proteasome inhibitor lactacystin (LC) model of PD. Our results showed that upregulation of endogenous GDNF in the adult striatum is not protective in LC-induced PD model in mice. Since age is the largest risk factor for PD, we also analyzed the effect of deletion of endogenous GDNF in agedGdnfconditional knock-out mice. We found that GDNF deletion does not increase susceptibility to LC-induced damage. We conclude that endogenous GDNF does not impact the outcome in the LC-induced proteasome inhibition mouse model of Parkinson’s disease.

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.

In the last years there has been a booming in the development of biological pharmaceuticals, and various nanoparticle delivery systems have been developed to overcome biological barriers to maximize the therapeutic potentials. Especially, when these drug-loaded nanoparticle systems arrive at the target cells, they must be successfully internalized into the cells, followed by the endosome escape, to release the payload in the cytoplasm. This is crucial so that the payloads are not degraded in the endosome (around pH 6.8–6.0) or lysosome (around pH 4.5). Considering the acid environment of the endosome, several studies have shown the design of different acid-labile nanoparticle delivery systems attempting to achieve endosome escape by, for example, endosomal rupture, membrane destabilization, and membrane fusion. In this mini-review, we summarize current designs in acid-labile chemical bonds for endosome escape of nanosystems. First, we briefly introduce the structural design of acid-labile nanoparticles and their endosomal escape mechanisms. Then, we review recent research work on the topic, highlighting how the nanoparticle designs can be used in endosome escape for different biomedical applications. Finally, we discuss the challenges and future perspectives in this field.

Background Craving for alcohol, in other words powerful desire to drink after withdrawal, is an important contributor to the development and maintenance of alcoholism. Here, we studied the role of GDNF (glial cell line-derived neurotrophic factor) and BDNF (brain-derived neurotrophic factor) on alcohol-seeking behavior in group-housed female mice. Methods We modeled alcohol-seeking behavior in C57Bl/6J female mice. The behavioral experiments in group-housed female mice were performed in an automated IntelliCage system. We conducted RT-qPCR analysis of Gdnf, Bdnf, Manf and Cdnf expression in different areas of the female mouse brain after alcohol drinking conditioning. We injected an adeno-associated virus (AAV) vector expressing human GDNF or BDNF in mouse nucleus accumbens (NAc) after ten days of alcohol drinking conditioning and assessed alcohol-seeking behavior. Behavioral data were analyzed by two-way repeated-measures ANOVA, and statistically significant effects were followed by Bonferroni’s post hoc test. The student’s t-test was used to analyze qPCR data. Results The RT-qPCR data showed that Gdnf mRNA level in NAc was more than four times higher (p Manf mRNA (p = 0.04) and Cdnf mRNA (p = 0.02) levels in the hippocampus and Manf mRNA in the VTA (p = 0.04) after alcohol consumption. Two-fold endogenous overexpression of Gdnf mRNA and lack of CDNF did not affect alcohol-seeking behavior. The AVV-GDNF overexpression in nucleus accumbens suppressed alcohol-seeking behavior while overexpression of BDNF did not. Conclusions The effect of increased endogenous Gdnf mRNA level in female mice upon alcohol drinking has remained unknown. Our data suggest that an increase in endogenous GDNF expression upon alcohol drinking occurs in response to the activation of another mesolimbic reward pathway participant.

Hydrophilic derivatives of an earlier described series of carbonic anhydrase inhibitors have been designed, prepared and profiled against a panel of carbonic anhydrase isoforms, including the glaucoma-related hCA II. For all hydrophilic derivatives, computational prediction of intraocular permeability routes showed the predominance of conjunctival rather than corneal absorption. The potentially reactive primary or secondary amine periphery of these compounds makes them suitable candidates for bioconjugation to polymeric drug carriers. As was shown previously, the most active hCA II inhibitor is efficacious in alleviating intraocular pressure in normotensive rabbits with efficacy matching that of dorzolamide.

The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports. publishedVersion

Increased digitalization and technological development raises the demand for rare and precious metals (RPM). Due to their rarity, mining RPMs from the earth is becoming increasingly difficult. Traditional urban mining methods to recover RPMs from printed circuit boards (PCB) need to separate the RPMs from non-metallic substances, e.g. plastic. This separation requires toxic substances and causes unwanted and toxic by-products and emissions. The ability to identify regions-of-interest on PCBs, i.e. the gold pads, and to extract RPMs from only the desired areas would reduce the need for toxic substances. In this study, a single 12 MHz high-intensity focused-ultrasound transducer was used to 1) image a PCB to locate the gold pads, and 2) to subsequently induce inertial cavitation to remove gold from three extraction areas on the selected gold pad. The sonication was performed in water without additional chemicals. Gold removal was verified by imaging the pad with a coded-excitation scanning acoustic microscope (fc = 375 MHz). Average areas and volumes of the three extraction regions were A = (12.2 ± 0.5)·103 μm2 and V = (18 ± 2)·103 μm3, respectively. The total amount of removed gold and nickel (from beneath the gold plating) from all three extraction areas was estimated to mAu,tot = (570 ± 20) ng and mNi,tot = (440 ± 30) ng. This study constitutes a first step towards more environmentally friendly, non-toxic urban mining of RPMs.

Cavitation-induced surface erosion has been studied for decades. High-intensity focused ultrasound (HIFU) enables localized erosion, with applications in many fields. However, no research has been published on machining solely with HIFU. Compared to existing micro-machining technologies, HIFU exhibits a unique set of benefits: inexpensive, minimal maintenance due to non-contact machining without slurry, mitigated chemical load, and monitoring capability. We demonstrate controlled surface machining of mirror-polished aluminium (AW-5754) using high-frequency (12 MHz) HIFU-induced cavitation erosion. Optimal sonication parameters (transducer-sample distance, amplitude, cycles per burst, number of bursts, and pulse repetition frequency) for stationary surface erosion were first identified experimentally. These parameters served as a basis for studying the effect of sonication parameters during on-the-fly erosion, i.e., engraving lines. The effect of stage translation velocity and the number of repeated passes across the engraved line were also studied. Subsequently, the acronym of our laboratory, “ETLA”, was engraved, with a 500 µm letter height and an average line width of 53 µm.

Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, β-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

Background New competence requirements have emerged for pharmacists as a result of changing societal needs towards more patient-centred practices. Today, medication review competence can be considered as basic pharmaceutical competence. Medication review specific competence criteria and tools for self-assessing the competence are essential in building competences and a shared understanding of medication reviews as a collaborative practice. The aim of this study was to develop and pilot a self-assessment tool for medication review competence among practicing pharmacists in Finland. Methods The development of the self-assessment tool was based on the national medication review competence criteria for pharmacists established in Finland in 2017 and piloting the tool among practicing pharmacists in a national online survey in October 2018. The pharmacists self-assessed their medication review competence with a five-point Likert scale ranging from 1 for "very poor/not at all" to 5 for "very good". Results The internal consistency of the self-assessment tool was high as the range of the competence areas' Cronbach's alpha was 0.953-0.973. The competence areas consisted of prescription review competence (20 items, Cronbach's alpha 0.953), additional statements for medication review competence (11 additional items, Cronbach's alpha 0.963) and medication review as a whole, including both the statements of prescription review and medication review competence (31 items, Cronbach's alpha 0.973). Competence items closely related to routine dispensing were most commonly self-estimated to be mastered by the practicing pharmacists who responded (n = 344), while the more clinical and patient-centred competence items had the lowest self-estimates. This indicates that the self-assessment tool works logically and differentiates pharmacists according to competence. The self-assessed medication review competence was at a very good or good level among more than half (55%) of the respondents (n = 344). Conclusion A self-assessment tool for medication review competence was developed and validated. The piloted self-assessment tool can be used for regular evaluation of practicing pharmacists' medication review competence which is becoming an increasingly important basis for their contribution to patient care and society.

Colorectal cancer is the third most commonly occurring cancer in men and the second in women. The global burden of colorectal cancer is projected to increase to over 2 million new cases with over 1 million deaths within the next 10 years, and there is a great need for new compounds with novel mechanisms of action. Our group has developed protein kinase C (PKC)-modulating isophthalic acid derivatives that induce cytotoxicity toward human cervical and prostate cancer cell lines. In this study, we investigated the effects of 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) on colorectal cancer cell lines (Caco-2, Colo205, and HT29). HMI-1a3 inhibited cell proliferation, decreased cell viability, and induced an apoptotic response in all studied cell lines. These effects, however, were independent of PKC. Using serine/threonine kinome profiling and pharmacological kinase inhibitors, we identified activation of the cAMP/PKA pathway as a new mechanism of action for HMI-1a3-induced anticancer activity in colorectal cancer cell lines. Our current results strengthen the hypothesis for HMI-1a3 as a potential anticancer agent against various malignancies. SIGNIFICANCE STATEMENT: Colorectal cancer (CRC) is a common solid organ malignancy. This study demonstrates that the protein kinase C (PKC)-C1 domain-targeted isophthalatic acid derivative 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) has anticancer activity on CRC cell lines independently of PKC. We identified PKA activation as a mechanism of HMI-1a3-induced anticancer effects. The results reveal a new anticancer mechanism of action for the partial PKC agonist HMI-1a3 and thus provide new insights for the development of PKC and PKA modulators for cancer therapy.

Inhibition of membrane‐bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure‐activity relationships around a new scaffold having a pyrazolo[1,5‐a]pyrimidine core. The most promising pyrazolo[1,5‐a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay., Chemical space exploration: Novel low micromolar membrane‐bound pyrophosphatase (mPPase) inhibitors based on an exploratory screening against Thermotoga maritima mPPase followed by early SARs around a pyrazolo[1,5‐a]pyrimidine scaffold. Compound 17 a retained activity against Plasmodium falciparum mPPase in membranes, and inhibited parasite growth ex vivo.

Immune cells play a crucial regulatory role in inflammatory phase and proliferative phase during skin healing. How to programmatically activate sequential immune responses is the key for scarless skin regeneration. In this study, an “Inner-Outer” IL-10-loaded electrospun fiber with cascade release behavior was constructed. During the inflammatory phase, the electrospun fiber released a lower concentration of IL-10 within the wound, inhibiting excessive recruitment of inflammatory cells and polarizing macrophages into anti-inflammatory phenotype “M2c” to suppress excessive inflammation response. During the proliferative phase, a higher concentration of IL-10 released by the fiber and the anti-fibrotic cytokines secreted by polarized “M2c” directly acted on dermal fibroblasts to simultaneously inhibit extracellular matrix overdeposition and promote fibroblast migration. The “Inner-Outer” IL-10-loaded electrospun fiber programmatically activated the sequential immune responses during wound healing and led to scarless skin regeneration, which is a promising immunomodulatory biomaterial with great potential for promoting complete tissue regeneration., Graphical abstract Image 1, Highlights • Macrophages play a crucial regulatory role during sequential skin regeneration process. • “Inner-Outer” IL-10-loaded electrospun fiber released the loaded cytokine in cascade. • IL-10 of low concentration within the wound inhibited the excessive inflammatory response. • Cytokines secreted by “M2c” inhibited the extracellular matrix overproduction. • The “Inner-Outer” IL-10-loaded electrospun fiber programmatically activated the sequential immune responses.

Gossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow–derived DCs (BM-DC) expressing dysfunctional β2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional β2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo–cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, β2-integrin–mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting β2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

Objectives This study investigated severe medication errors (MEs) reported to the National Supervisory Authority for Welfare and Health (Valvira) in Finland and evaluated how the incident documentation applies to learning from errors. Methods This study was a retrospective document analysis consisting of medication-related complaints and authoritative statements investigated by Valvira in 2013 to 2017 (n = 58). Results Medication errors caused death or severe harm in 52% (n = 30) of the cases (n = 58). The majority (83%; n = 48) of the incidents concerned patients older than 60 years. Most likely, the errors occurred in prescribing (n = 38; 47%), followed by administration (n = 15; 19%) and monitoring (n = 14; 17%). The error process often included many failures (n = 24; 41%) or more than one health professional (n = 16; 28%). Antithrombotic agents (n = 17; 13%), opioids (n = 10; 8%), and antipsychotics (n = 10; 8%) were the therapeutic groups most commonly involved in the errors. Almost all error cases (91%; n = 53) were assessed as likely or potentially preventable. In 60% (n = 35) of the cases, the organization reported actions taken to improve medication safety after the occurrence of the investigated incident. Conclusions Medication errors reported to the national health care supervisory authority provide a valuable source of risk information and should be used for learning from severe errors at the level of health care systems. High age remains a key risk factor to severe MEs, which may be associated with a wide range of medications including those not typically perceived as high-alert medications or high-risk administration routes. Despite being complex processes, the severe MEs have a great potential to lead to developing systems, processes, resources, and competencies of health care organizations.

Quantitative understanding of pharmacokinetics of topically applied ocular drugs requires more research to further understanding and to eventually allow predictive in silico models to be developed. To this end, a topical cocktail of betaxolol, timolol and atenolol was instilled on albino rabbit eyes. Tear fluid, corneal epithelium, corneal stroma with endothelium, bulbar conjunctiva, anterior sclera, iris-ciliary body, lens and vitreous samples were collected and analysed using LC-MS/MS. Iris-ciliary body was also analysed after intracameral cocktail injection. Non-compartmental analysis was utilized to estimate the pharmacokinetics parameters. The most lipophilic drug, betaxolol, presented the highest exposure in all tissues except for tear fluid after topical administration, followed by timolol and atenolol. For all drugs, iris-ciliary body concentrations were higher than that of the aqueous humor. After topical instillation the most hydrophilic drug, atenolol, had 3.7 times higher AUCiris-ciliary body than AUCaqueous humor, whereas the difference was 1.4 and 1.6 times for timolol and betaxolol, respectively. This suggests that the non-corneal route (conjunctival-scleral) was dominating the absorption of atenolol, while the corneal route was more important for timolol and betaxolol. The presented data increase understanding of ocular pharmacokinetics of a cocktail of drugs and provide data that can be used for quantitative modeling and simulation.

Pathological angiogenesis related to neovascularization in the eye is mediated through vascular endothelial growth factors (VEGFs) and their receptors. Ocular neovascular-related diseases are mainly treated with anti-VEGF agents. In this study we evaluated the efficacy and safety of novel gene therapy using adeno associated virus 2 vector expressing a truncated form of soluble VEGF receptor-2 fused to the Fc-part of human IgG1 (AAV2-sVEGFR-2-Fc) to inhibit ocular neovascularization in laser induced choroidal neovascularization (CNV) in mice. The biological activity of sVEGFR-2-Fc was determined in vitro. It was shown that sVEGFR-2-Fc secreted from ARPE-19 cells was able to bind to VEGF-A165 and reduce VEGF-A165 induced cell growth and survival. A single intravitreal injection (IVT) of AAV2-sVEGFR-2-Fc (1 mu l, 4.7 x 1012 vg/ml) one-month prior laser photocoagu-lation did not cause any changes in the retinal morphology and significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post-lasering compared to controls. Macrophage infiltration was observed after the injection of both AAV2-sVEGFR-2-Fc and PBS. Our findings indicate that AAV2 mediated gene delivery of the sVEGFR-2-Fc efficiently reduces formation of CNV and could be developed to a therapeutic tool for the treatment of retinal diseases associated with neovascularization.

Photothermal therapy (PTT) in combination with other treatment modalities has shown great potential to activate immunotherapy against tumor metastasis. However, the nanoparticles (NPs) that generate PTT have served as the photothermal agent only. Moreover, researchers have widely utilized highly immunogenic tumor models to evaluate the immune response of these NPs thus giving over-optimistic results. In the present study black porous silicon (BPSi) NPs were developed to serve as both the photothermal agent and the adjuvant for PTT-based antitumor immunotherapy. We found that the poorly immunogenic tumor models such as B16 are more valid to evaluate NP-based immunotherapy than the widely used immunogenic models such as CT26. Based on the B16 cancer model, a cocktail regimen was developed that combined BPSi-based PTT with doxorubicin (DOX) and cytosine-phosphate-guanosine (CpG). BPSi-based PTT was an important trigger to activate the specific immunotherapy to inhibit tumor growth by featuring the selective upregulation of TNF-α. Either by adding a low dose DOX or by prolonging the laser heating time, a similar efficacy of immunotherapy was evoked to inhibit tumor growth. Moreover, BPSi acted as a co-adjuvant for CpG to significantly boost the immunotherapy. The present study demonstrates that the BPSi-based regimen is a potent and safe antitumor immunotherapy modality. Moreover, our study highlighted that tuning the laser heating parameters of PTT is an alternative to the toxic cytostatic to evoke immunotherapy, paving the way to optimize the PTT-based combination therapy for enhanced efficacy and decreased side effects. STATEMENT OF SIGNIFICANCE: Tumor metastasis causes directly or indirectly more than 90% of cancer deaths. Combination of photothermal therapy (PTT), chemotherapy and immunotherapy based on nanoparticles (NPs) has shown great potential to inhibit distant and metastatic tumors. However, these NPs typically act only as photothermal agents and many of them have been evaluated with immunogenic tumor models. The present study developed black porous silicon working as both the photothermal conversion agent and the immunoadjuvant to inhibit distant tumor. It was recognized that the poorly immunogenic tumor model B16 is more appropriate to evaluate immunotherapy than the widely used immunogenic model CT26. The coordination mechanism of the PTT-based combination therapy regimen was discovered in detail, paving the way to optimize cancer immunotherapy for enhanced efficacy and decreased side effects.

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune check-point programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 +/- 25 mu g (PNA) mg(DNPs)(-1). These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200 mu g mL(-1) and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.

Low-molecular synthetic fluorophores are convenient tools in bioimaging applications. Several derivatives of Safirinium dyes as well as their reactive N-hydroxysuccinimide (NHS) esters bearing diverse substituents were synthesized and evaluated experimentally in terms of their lipophilicity by means of reverse-phase and immobilized artificial membrane high-performance liquid chromatography. Subsequently, the selected compounds were employed as novel cellular imaging agents for staining Gram-positive and Gram-negative bacteria, human kidney cell line, as well as human skin tissue. The analyzed dyes allowed for visualization of cellular structures such as mitochondria, endoplasmic reticulum, and cellular nuclei. They proved to be useful in fluorescent staining of stratum corneum, especially in the aspect of xenobiotic exposure and its penetration into the skin. The best results were obtained with the use of moderately lipophilic NHS esters of Safirinium Q. The development of Safirinium dyes is a promising alternative for commercially available dyes since the reported molecules have low molecular masses and exhibit efficient staining and remarkable water solubility. Moreover, they are relatively simple and low-cost in synthesis.

The purpose of this study was to evaluate the contribution of the anterior elimination route for four anti-vascular endothelial growth factor (anti-VEGF) macromolecules (aflibercept, bevacizumab, pegaptanib and ranibizumab) after intravitreal injection using published human and rabbit data and three previously described pharmacokinetic (PK) modeling methods. A PubMed search was used to identify published studies with concentration-time data. The data were utilized only if the intravitreally injected drugs were used as plain solutions and several criteria for a well-performed PK study were fulfilled. The three methods to analyze rabbit data were (1) the equation for vitreal elimination halflife based molecular size assuming anterior elimination, (2) Maurice equation and plot for the ratio of aqueous humor (AH) to vitreal concentration assuming anterior elimination, and (3) the equation for amount of macromolecule eliminated anteriorly based on the area under the curve in AH. The first and third methods were used for human data. In the second and third methods, AH flow rate is a key model parameter, and it was varied between 2 and 3 ill/min. The methods were applied to data from 9 rabbit studies (1 for aflibercept, 5 for bevacizumab, and 3 for ranibizumab) and 5 human studies (1 for aflibercept, 3 for bevacizumab, and 1 for ranibizumab). Experimental half-lives of anti-VEGF macromolecules in both vitreous and aqueous humor were close to those calculated with the equations for vitreal elimination half-life in humans and rabbits. Rabbit data analyzed with Maurice plot indicated that the contribution of anterior elimination was usually at least 75%. In most human and rabbit studies, the calculated percentage of anterior elimination was at least 51%. Variability between studies was extensive for bevacizumab and ranibizumab. The results suggest that the anterior elimination route dominates after intravitreal injection of anti-VEGF macromolecules. However, the clinical data are sparse and variability is extensive, the latter emphasizing the need of proper experimental design.

Cardiovascular and cerebrovascular diseases (CCVDs) describe abnormal vascular system conditions affecting the brain and heart. Among these, ischemic heart disease and ischemic stroke are the leading causes of death worldwide, resulting in 16% and 11% of deaths globally. Although several therapeutic approaches are presented over the years, the continuously increasing mortality rates suggest the need for more advanced strategies for their treatment. One of these strategies lies in the use of stimuli-responsive biomaterials. These "smart" biomaterials can specifically target the diseased tissue, and after "reading" the altered environmental cues, they can respond by altering their physicochemical properties and/or their morphology. In this review, the progress in the field of stimuli-responsive biomaterials for CCVDs in the last five years, aiming at highlighting their potential as early-stage therapeutics in the preclinical scenery, is described.

Antibacterial screenings are most commonly targeted at planktonic bacteria but less effort is dedicated to the exploration of agents acting on biofilms. Here, a natural compounds library was screened against Staphylococcus aureus using a 384-well plate platform to identify compounds preventing biofilm formation. Five structurally diverse hits were selected for follow-up studies: honokiol, tschimganidin, ferutinin, oridonin and deoxyshikonin. The compounds were evaluated against different bacterial species for their capacity to prevent and disrupt biofilms. The development of resistance and cytotoxicity were also investigated. Ferutinin displayed the best antibacterial activity, with a minimum inhibitory, bactericidal and biofilm preventive concentration of 25 mu M against S. aureus. It efficiently disrupted pre-formed biofilms (over 5-log reduction of viable cells) and reduced biofilm formation on a catheter in the presence of neutrophils. This work provides new information on the antibacterial activity of five natural compounds and identified ferutinin as a promising candidate against S. aureus biofilms.

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP+-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and seemed to protect dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying, therapy for PD.

Porous silicon (PSi) nanoparticles have been applied in various fields, such as catalysis, imaging, and biomedical applications, because of their large specific surface area, easily modifiable surface chemistry, biocompatibility, and biodegradability. For biomedical applications, it is important to precisely control the surface modification of PSi-based materials and quantify the functionalization density, which determines the nanoparticle’s behavior in the biological system. Therefore, we propose here an optimized solution to quantify the functionalization groups on PSi, based on the nuclear magnetic resonance (NMR) method by combining the hydrolysis with standard 1H NMR experiments. We optimized the hydrolysis conditions to degrade the PSi, providing mobility to the molecules for NMR detection. The NMR parameters were also optimized by relaxation delay and the number of scans to provide reliable NMR spectra. With an internal standard, we quantitatively analyzed the surficial amine groups and their sequential modification of polyethylene glycol. Our investigation provides a reliable, fast, and straightforward method in quantitative analysis of the surficial modification characterization of PSi requiring a small amount of sample.