Publications

A problem of systemic pharmacotherapy is off-target activity, which causes adverse effects. Outstanding examples include neuroinhibitory medications like antiseizure drugs, which are used against epilepsy and neuropathic pain but cause systemic side effects. There is a need of drugs that inhibit nerve signals locally and on-demand without affecting other regions of the body. Photopharmacology aims to address this problem with light-activated drugs and localized illumination in the target organ. Here, we have developed photoswitchable derivatives of the widely prescribed antiseizure drug carbamazepine. For that purpose, we expanded our method of ortho azologization of tricyclic drugs to meta/para and to N-bridged diazocine. Our results validate the concept of ortho cryptoazologs (uniquely exemplified by Carbazopine-1) and bring to light Carbadiazocine (8), which can be photoswitched between 400-590 nm light (using violet LEDs and halogen lamps) and shows good drug-likeness and predicted safety. Both compounds display photoswitchable activity in vitro and in translucent zebrafish larvae. Carbadiazocine (8) also offers in vivo analgesic efficacy (mechanical and thermal stimuli) in a rat model of neuropathic pain and a simple and compelling treatment demonstration with non-invasive illumination.

JTD Keywords: Antiepileptic drugs, Anxiet, Azobenzene, Diazocine, Epileps, Ion channels, Neuromodulation, Optical control, Pain, Photopharmacology, Rat, Receptors, Release, Spatiotemporal control, Tricyclic drugs, Zebrafish

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

JTD Keywords: Animals, Barrier, Cell line, tumor, Dna, Drug delivery, Drug-delivery, Enzyme catalysis, Gene delivery, Gene transfer techniques, Humans, Lactic acid, Mice, Nanobots, Nanoparticles, Pdna, Plasmids, Polyglycolic acid, Polylactic acid-polyglycolic acid copolymer, Swarming, Transfectio, Transfection, Urease, Urinary bladder neoplasms

Several pathogenic Escherichia coli strains cause diarrhea. Enteroaggregative E. coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC cells form a "stacked-brick" arrangement over the intestinal epithelial cells. EAEC isolates express, among other virulence determinants, the AggR transcriptional activator and the aggregative adherence fimbriae (AAF). Overexpression of the aggR gene results in increased expression of virulence factors such as the aff genes, as well as several genes involved in specific metabolic pathways such as fatty acid degradation (fad) and arginine degradation (ast). To support the hypothesis that induction of the expression of some of these pathways may play a role in EAEC virulence, in this study we used a murine infection model to evaluate the impact of the expression of these pathways on infection parameters. Mice infected with a mutant derivative of the EAEC strain 042, characterized by overexpression of the aggR gene, showed increased disease symptoms compared to those exhibited by mice infected with the wild type (wt) strain 042. Several of these symptoms were not increased when the infecting mutant, which overexpressed aggR, lacked the fad and ast pathways. Therefore, our results support the hypothesis that different metabolic pathways contribute to EAEC virulence.

JTD Keywords: Adherence, Aggr, Burde, Chain fatty-acids, Children, Enteroaggregative e. coli, Escherichia-coli, Etiology, Infection, Mice, Microbiota, Persistent diarrhea, Protein, Scfa, Sex-differences

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

JTD Keywords: Ai, Consumption, Culture, Embryo development, Fluorescence, Hyperspectral imagin, Implantation, In vitro fertilization, Infertility, Label-free imaging, Microscopy, Morphokinetics, Oxygen concentrations, Selectio, Time-lapse

Electronic noses have improved in terms of reliability in the last two decades. Their design has allowed for model training and validation for predicting odour emissions and classifying odour sources. In the last few years, interest has turned to designing e-noses capable of flying in drones. Fast 3D mapping of areas where odour might become a problem, such as Wastewater Treatment Plants, Compost Plants, and Refineries, has been the main target of some recent studies. Here, a fully functional design of a drone-embedded, E-Nosebased measurement platform is presented. The design of its ENose and its fast-sampling frequency will allow for fast tracking of plumes and fast 3D odour mapping. In this contribution, a description of the system is provided in addition to preliminary measurements in the laboratory characterizing the fast response of the E-Nose to an odour stimulus.

JTD Keywords: Autonomous, Drone, E-nose, Iot, Machine learnin, Odour

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.Copyright © 2023 Liang, Nilsson, Kragh, Hedal, Alcàcer-Almansa, Kiilerich, Andersen and Tolker-Nielsen.

JTD Keywords: aggregates, antibiotic tolerance, biofilm formation, extracellular matrix, genome, growth, lungs, molecular-mechanisms, mutations, polysaccharide, pseudomonas aeruginosa, psl, system, Aggregates, Antibiotic tolerance, Biofilm, Extracellular matrix, Pseudomonas aeruginosa, Small-colony variants

Development of liver fibrosis is paralleled by contraction of hepatic stellate cells (HSCs), the main profibrotic hepatic cells. Yet, little is known about the interplay of neprilysin (NEP) and its substrate neuropeptide Y (NPY), a potent enhancer of contraction, in liver fibrosis. We demonstrate that HSCs are the source of NEP. Importantly, NPY originates majorly from the splanchnic region and is cleaved by NEP in order to terminate contraction. Interestingly, NEP deficiency (Nep-/-) showed less fibrosis but portal hypertension upon liver injury in two different fibrosis models in mice. We demonstrate the incremental benefit of Nep-/- in addition to AT1R blocker (ARB) or ACE inhibitors for fibrosis and portal hypertension. Finally, oral administration of Entresto, a combination of ARB and NEP inhibitor, decreased hepatic fibrosis and portal pressure in mice. These results provide a mechanistic rationale for translation of NEP-AT1R-blockade in human liver fibrosis and portal hypertension.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: activation, cirrhosis, cirrhotic rats, cp: cell biology, expression, hepatic stellate cell, identification, inhibition, mechanisms, modulation, neprilysin, neuropeptide y, neuropeptide y receptor 1, portal hypertension, portal-hypertension, web server, Renin-angiotensin system

Escherichia coli is one of the most common members of the intestinal microbiota. Many of its strains are associated with various inflammatory infections, including urinary or gut infections, especially when displaying antibiotic resistance or in patients with suppressed immune systems. According to recent reports, the biofilm-forming potential of E. coli is a crucial factor for its increased resistance against antibiotics. To overcome the limitations of using antibiotics against resistant E. coli strains, the world is turning once more towards bacteriophage therapy, which is becoming a promising candidate amongst the current personalized approaches to target different bacterial infections. Although matured and persistent biofilms pose a serious challenge to phage therapy, they can still become an effective alternative to antibiotic treatment. Here, we assess the efficiency of clinically isolated phages in phage therapy against representative clinical uropathogenic and invasive biofilm-forming E. coli strains. Our results demonstrate that irrespective of host specificity, bacteriophages producing clear plaques with a high burst size, and exhibiting depolymerizing activity, are good candidates against biofilm-producing E. coli pathogens as verified from our in vitro and in vivo experiments using Galleria mellonella where survival was significantly increased for phage-therapy-treated larvae.

JTD Keywords: antibiotic resistance, assay, bacteriophage, bacteriophages, biofilm-forming potential, infection, inflammatory infections, mechanisms, Galleria-mellonella, Intestinal microflora

The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.© 2022 The Author(s).

JTD Keywords: bilayer, curvature, diffusion-coefficients, identification, membrane-scission, phase-diagram, reveals, sorting complex, structural basis, Biophysics, Biotechnology, Cell biology, Giant vesicles, Membranes

Designing useful functionalities in clinically validated, old antibiotics holds promise to provide the most economical solution for the global lack of effective antibiotics, as undoubtedly a serious health threat. Here we show that using the surface chemistry of the cyclodextrin (beta CD) cycle and arginine (arg) as a linker, provides more stable ternary antibiotic complex (beta CD-arg-cpx). In contrast to classical less stable inclusion complexes, which only modify antibiotic solubility, here-presented ternary complex is more stable and controls drug release. The components of the complex intensify interactions with bacterial membranes and increase the drug's availability inside bacterial cells, thereby improving its antimicrobial efficacy and safety profile. Multifunctional antibiotics, formulated as drug delivery systems per se, that take the drug to the site of action, maximize its efficacy, and provide optical detectability are envisaged as the future in fighting against infections. Their role as a tool against multiresistant strains remains as interesting challenge open for further research.; Ternary cyclodextrin- arginine- ciprofloxacin complexes show improved stability and increased efficacy against P. aeruginosa in Galleria mellonella worms.

JTD Keywords: Antibiotic-resistance, Beta-cyclodextrin, Dissolution, Drugs, Salts

According to circular economy, wine-making by-products represent a fascinating biomass, which can be used for the sustainable exploitation of polyphenols and the development of new nanotechnological health-promoting products. In this study, polyphenols contained in the grape pomace were extracted by maceration with ethanol in an easy and low dissipative way. The obtained extract, rich in malvidin-3-glucoside, quercetin, pro-cyanidin B2 and gallic acid, was incorporated into phospholipid vesicles tailored for intestinal delivery. To improve their performances, vesicles were enriched with gelatine or a maltodextrin (Nutriose (R)), or their com-bination (gelatine-liposomes, nutriosomes and gelatine-nutriosomes). The small (-147 nm) and negatively charged (--50mV) vesicles were stable at different pH values mimicking saliva (6.75), gastric (1.20) and intestinal (7.00) environments. Vesicles effectively protected intestinal cells (Caco-2) from the oxidative stress and promoted the biofilm formation by probiotic bacteria. A preliminary evaluation of the vesicle feasibility at industrial levels was also performed, analysing the economic and energetic costs needed for their production.

JTD Keywords: Adhesion, Antioxidant activity, Caco-2 cells, Dextrin, Grape pomace extract, Lactobacillus-reuteri, Manufacturing costs, Oxidative stress, Ph, Phospholipid vesicles, Polyphenols, Probiotic bacteria, Protein

The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 mu M DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies.

JTD Keywords: antibiotics, antimalarial drugs, domiphen bromide, malaria, plasmodium falciparum, Antibiotics, Antimalarial drugs, Antimalarial-drug, Artemisinin, Combination therapies, Domiphen bromide, Intraerythrocytic stages, Isoprenoid biosynthesis, Malaria, Methyl erythritol phosphate pathway, Nonmevalonate pathway, Plasmodium falciparum, Plasmodium-falciparum apicoplast, Red-blood-cells, Targeted delivery

We investigated the diffusion of three cyclic boronates formulated as beta-lactamase inhibitors through the porin OmpF to evaluate their potential to cross OM via the porin pathway. The three nonbeta-lactam molecules diffuse through the porin eyelet region with the same mechanism observed for beta-lactam molecules and diazobicyclooctan derivatives, with the electric dipole moment aligned with the transversal electric field. In particular, the BOH group can interact with both the basic ladder and the acidic loop L3, which is characteristic of the size-constricted region of this class of porins. On one hand, we confirm that the transport of small molecules through enterobacter porins has a common general mechanism; on the other, the class of cyclic boronate molecules does not seem to have particular difficulties in diffusing through enterobacter porins, thus representing a good scaffold for new anti-infectives targeting Gram-negative bacteria research.

JTD Keywords: beta-lactamase inhibitors, cyclic boronates, diffusion current, metadynamics, molecular dynamics simulations, permeation, Antibiotics, Beta-lactamase inhibitors, Cyclic boronates, Diffusion, Diffusion current, Discovery, Electric-field, Metadynamics, Molecular dynamics simulations, Molecular-dynamics simulations, Nanopores, Permeability, Permeation, Porins, Rules, Translocation

It is not well understood why diabetic individuals are more prone to develop severe COVID-19. To this, we here established a human kidney organoid model promoting early hallmarks of diabetic kidney disease development. Upon SARS-CoV-2 infection, diabetic-like kidney organoids exhibited higher viral loads compared with their control counterparts. Genetic deletion of the angiotensin-converting enzyme 2 (ACE2) in kidney organoids under control or diabetic-like conditions prevented viral detection. Moreover, cells isolated from kidney biopsies from diabetic patients exhibited altered mitochondrial respiration and enhanced glycolysis, resulting in higher SARS-CoV-2 infections compared with non-diabetic cells. Conversely, the exposure of patient cells to dichloroacetate (DCA), an inhibitor of aerobic glycolysis, resulted in reduced SARS-CoV-2 infections. Our results provide insights into the identification of diabetic-induced metabolic programming in the kidney as a critical event increasing SARS-CoV-2 infection susceptibility, opening the door to the identification of new interventions in COVID-19 pathogenesis targeting energy metabolism.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: complications, coronavirus, cultured-cells, disease, distal tubule, mouse, protein, reveals, spike, Ace2, Angiotensin-converting enzyme 2, Angiotensin-converting enzyme-2, Covid-19, Diabetes 2, Human kidney organoids, Sars-cov-2

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

JTD Keywords: antibiotic-resistance, antimicrobial peptides, autonomous treatment, bacterial infection, delivery, ll-37, nanomotors, nanoparticles, peptide, self-propulsion, tissue, vitro, wasp venom, Antibiotic-resistance, Antimicrobial peptides, Autonomous treatment, Bacterial infection, Delivery, Ll-37, Mesoporous silica nanoparticles, Nanomotors, Nanoparticles, Peptide, Self-propulsion, Tissue, Vitro, Wasp venom

The impact of diet and digestive disorders in flatus composition remains largely unexplored. This is partially due to the lack of standardized sampling collection methods, and the easy atmospheric contamination. This paper describes a method to quantitatively determine the major gases in flatus and their application in a nutritional intervention. We describe how to direct sample flatus into Tedlar bags, and simultaneous analysis by gas chromatography–thermal conductivity detection (GC–TCD). Results are analyzed by univariate hypothesis testing and by multilevel principal component analysis. The reported methodology allows simultaneous determination of the five major gases with root mean measurement errors of 0.8% for oxygen (O2), 0.9% for nitrogen (N2), 0.14% for carbon dioxide (CO2), 0.11% for methane (CH4), and 0.26% for hydrogen (H2). The atmospheric contamination was limited to 0.86 (95% CI: [0.7–1.0])% for oxygen and 3.4 (95% CI: [1.4–5.3])% for nitrogen. As an illustration, the method has been successfully applied to measure the response to a nutritional intervention in a reduced crossover study in healthy subjects. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: breath, colonic microbiota, diet effect on flatus, disorders, evacuation, excretion, flatulence, hydrogen gas, major flatus gas components, multilevel principal component analysis, rectal gas collection, systems, volume, Atmospheric contamination, Carbon dioxide, Conductivity detection, Diet effect on flatus, Gas chromatography, Gas collections, Gas component, Gases, Major flatus gas component, Major flatus gas components, Multilevel principal component analyse, Multilevel principal component analysis, Multilevels, Nitrogen, Nutrition, Oxygen, Principal component analysis, Principal-component analysis, Rectal gas collection, Volatile organic-compounds

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence

Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.

JTD Keywords: agents, antibiotic-free technology, arginine, charged amino acids, contact-based antimicrobials, discovery, enzyme-resistant antimicrobials, functionalized gold, peptides, polymers, resistant, Antibiotic-free technology, Charged amino acids, Contact-based antimicrobials, Enzyme-resistant antimicrobials, Functionalized gold, Nanoparticles

Multifunctional hydrogels are a class of materials offering new opportunities for interfacing living organisms with machines due to their mechanical compliance, biocompatibility, and capacity to be triggered by external stimuli. Here, we report a dual magnetic- and electric-stimuli-responsive hydrogel with the capacity to be disassembled and reassembled up to three times through reversible cross-links. This allows its use as an electronic device (e.g., temperature sensor) in the cross-linked state and spatiotemporal control through narrow channels in the disassembled state via the application of magnetic fields, followed by reassembly. The hydrogel consists of an interpenetrated polymer network of alginate (Alg) and poly(3,4-ethylenedioxythiophene) (PEDOT), which imparts mechanical and electrical properties, respectively. In addition, the incorporation of magnetite nanoparticles (Fe3O4 NPs) endows the hydrogel with magnetic properties. After structural, (electro)chemical, and physical characterization, we successfully performed dynamic and continuous transport of the hydrogel through disassembly, transporting the polymer-Fe3O4 NP aggregates toward a target using magnetic fields and its final reassembly to recover the multifunctional hydrogel in the cross-linked state. We also successfully tested the PEDOT/Alg/Fe3O4 NP hydrogel for temperature sensing and magnetic hyperthermia after various disassembly/re-cross-linking cycles. The present methodology can pave the way to a new generation of soft electronic devices with the capacity to be remotely transported.

JTD Keywords: conductive hydrogel, constructs, magnetic field, magnetite nanoparticle, nanoindentation, soft electronics, spatiotemporal control, Conductive hydrogel, Conductive hydrogels, Magnetic field, Magnetite nanoparticle, Soft electronics, Spatiotemporal control

A plethora of applications using polysaccharides have been developed in recent years due to their availability as well as their frequent nontoxicity and biodegradability. These polymers are usually obtained from renewable sources or are byproducts of industrial processes, thus, their use is collaborative in waste management and shows promise for an enhanced sustainable circular economy. Regarding the development of novel delivery systems for biotherapeutics, the potential of polysaccharides is attractive for the previously mentioned properties and also for the possibility of chemical modification of their structures, their ability to form matrixes of diverse architectures and mechanical properties, as well as for their ability to maintain bioactivity following incorporation of the biomolecules into the matrix. Biotherapeutics, such as proteins, growth factors, gene vectors, enzymes, hormones, DNA/RNA, and antibodies are currently in use as major therapeutics in a wide range of pathologies. In the present review, we summarize recent progress in the development of polysaccharide-based hydrogels of diverse nature, alone or in combination with other polymers or drug delivery systems, which have been implemented in the delivery of biotherapeutics in the pharmaceutical and biomedical fields. © 2021 American Chemical Society.

JTD Keywords: biodegradable dextran hydrogels, biotherapeutics, bone morphogenetic protein-2, carrageenan-based hydrogels, chitosan-based hydrogels, controlled delivery, controlled-release, cross-linked hydrogels, growth-factor delivery, hydrogels, in-vitro characterization, polysaccharides, self-healing hydrogel, stimuli-responsiveness, tissue engineering, Antibodies, Bioactivity, Biodegradability, Biomedical fields, Biomolecules, Biotherapeutics, Chemical modification, Circular economy, Controlled delivery, Controlled drug delivery, Delivery systems, Drug delivery system, Functional polymers, Hyaluronic-acid hydrogels, Hydrogels, Industrial processs, Polysaccharides, Recent progress, Renewable sources, Stimuli-responsiveness, Targeted drug delivery, Tissue engineering, Waste management

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns

Asthma is the most common inflammatory disease affecting the lungs, which can be caused by intrauterine or postnatal insults depending on the exposure to environmental factors. During early life, the exposure to different risk factors can influence the microbiome leading to undesired changes to the immune system. The modulations of the immunity, caused by dysbiosis during development, can increase the susceptibility to allergic diseases. On the other hand, immune training approaches during pregnancy can prevent allergic inflammatory diseases of the airways. In this review, we focus on evidence of risk factors in early life that can alter the development of lung immunity associated with dysbiosis, that leads to asthma and affect childhood and adult life. Furthermore, we discuss new ideas for potential prevention strategies that can be applied during pregnancy and postnatal period.

JTD Keywords: asthma, dysbiosis, early life immunity, lung microbiome, Adulthood, Antibiotic exposure, Asthma, Childhood, Disease, Disease exacerbation, Dysbiosis, Early life immunity, Gut microbiome, Human, Immunity, Intestine flora, Lung development, Lung microbiome, Lung microbiota, Nonhuman, Perinatal period, Pregnancy, Prevention, Prevention strategies, Review, Risk, Risk factor, Sensitization, Supplementation, Vitamin-d, Wheeze

© 2020 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers. Biodistribution studies are essential in drug carrier design and translation, and radiotracing provides a sensitive quantitation for this purpose. Yet, for biodegradable formulations, small amounts of free-label signal may arise prior to or immediately after injection in animal models, causing potentially confounding biodistribution results. In this study, we refined a method to overcome this obstacle. First, we verified free signal generation in animal samples and then, mimicking it in a controllable setting, we injected mice intravenously with a radiolabeled drug carrier formulation (125I-antibody/3DNA) containing a known amount of free radiolabel (125I), or free 125I alone as a control. Corrected biodistribution data were obtained by separating the free radiolabel from blood and organs postmortem, using trichloroacetic acid precipitation, and subtracting the confounding signal from each tissue measurement. Control free 125I-radiolabel was detected at ≥85% accuracy in blood and tissues, validating the method. It biodistributed very heterogeneously among organs (0.6–39 %ID/g), indicating that any free 125I generated in the body or present in an injected formulation cannot be simply corrected to the free-label fraction in the original preparation, but the free label must be empirically measured in each organ. Application of this method to the biodistribution of 125I-antibody/3DNA, including formulations directed to endothelial target ICAM-1, showed accurate classification of free 125I species in blood and tissues. In addition, this technique rendered data on the in vivo degradation of the traced agents over time. Thus, this is a valuable technique to obtain accurate measurements of biodistribution using 125I and possibly other radiotracers.

JTD Keywords: biodistribution data correction, degradation, drug delivery carriers, free label, in vivo biodistribution, radiotracing, trichloroacetic acid precipitation, Biodistribution data correction, Degradation, Drug delivery carriers, Free label, In vivo biodistribution, Radiotracing, Trichloroacetic acid precipitation

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving endolysins.

JTD Keywords: antibiotic resistance, bacteriophages, Antibiotic resistance, Bacteriophages, Endolysin

Grape extract-loaded fibre-enriched vesicles, nutriosomes, were prepared by combining antioxidant extracts obtained from grape pomaces and a prebiotic, soluble fibre (Nutriose®FM06). The nutriosomes were small in size (from ∼140 to 260 nm), homogeneous (polydispersity index < 0.2) and highly negative (∼ −79 mV). The vesicles were highly stable during 12 months of storage at 25 °C. When diluted with warmed (37 °C) acidic medium (pH 1.2) of high ionic strength, the vesicles only displayed an increase of the mean diameter and a low release of the extract, which were dependent on Nutriose concentration. The formulations were highly biocompatible and able to protect intestinal cells (Caco-2) from oxidative stress damage. In vivo results underlined that the composition of mouse microbiota was not affected by the vesicular formulations. Overall results support the potential application of grape nutriosomes as an alternative strategy for the protection of the intestinal tract.

JTD Keywords: Antioxidant activity, Grape pomace, Gut microbiota, In vivo studies, Intestinal cells, Nutriosomes, Phospholipid vesicles, Prebiotic activity

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

JTD Keywords: COVID-19, Angiotensin converting enzyme 2, Blood vessels, Human organoids, Kidney, Severe acute respiratory syndrome coronavirus, Spike glycoproteins, Treatment

We propose a method to control chimera states in a ring-shaped network of nonlocally coupled phase oscillators. This method acts exclusively on the network’s connectivity. Using the idea of a pacemaker oscillator, we investigate which is the minimal action needed to control chimeras. We implement the pacemaker choosing one oscillator and making its links unidirectional. Our results show that a pacemaker induces chimeras for parameters and initial conditions for which they do not form spontaneously. Furthermore, the pacemaker attracts the incoherent part of the chimera state, thus controlling its position. Beyond that, we find that these control effects can be achieved with modifications of the network’s connectivity that are less invasive than a pacemaker, namely, the minimal action of just modifying the strength of one connection allows one to control chimeras.

JTD Keywords: Complex networks, Oscillators, Spatiotemporal phenomena

Nature has developed processes and robust materials, which possess superior physical, chemical, and electromagnetic properties that can withstand the most extreme conditions. We need to take inspiration from nature to obtain a more sustainable development. By combining our knowledge of processes and the knowledge of natural systems, we can create “biomimetic” solutions to the problems that we are facing as a consequence of the over-exploitation of our natural resources. Nice, France, the capital city of the French Riviera, once again welcomes the 4th edition of the International Conference on Bioinspired and Biobased Chemistry and Materials (“Nature Inspires Creativity Engineers” or N.I.C.E. 2018 Conference) from 14 to 17 of October, 2018. As in the previous editions, we are expecting hundreds of scientists and engineers to share the latest developments in the growing field of bioinspired and biobased chemistry and materials. It is a unique opportunity to understand the new challenges, to initiate new collaborations and to envisage sustainable solutions for the future.

JTD Keywords: Nanotechnology, Biotech, Smartech

One of the treatments for recurrent or complicated osteomyelitis is by local antibiotherapy mediated by suitable bone grafts. β–Tricalcium Phosphate (β–TCP) bioceramic is a resorbable bone graft. Its microporosity allows for incorporation of drugs, but a too fast release is often obtained. Complex strategies have been explored to obtain controlled drug release. In this work, plasma polymerization of a biocompatible polymer was investigated on β-TCP. Polyethyleneglycol (PEG)-like polymer coatings of different thickness were deposited on microporous β-TCP loaded with antibiotics. A highly hydrophobic surface was obtained despite the hydrophilicity of the PEG-like layer produced, which was associated to the roughness of the β-TCP substrate. The bioceramics nevertheless retained their suitable biological behavior with regard to human osteoblast cells. The microbiological activity of the antibiotics was preserved, and the coatings reduced the total amount of drug released as a function of the increasing plasma treatment time.

JTD Keywords: Plasma polymerization, β–Tricalcium phosphate, PEG-like polymer, Antibiotics, Drug release, Biocompatibility

A simple method for constructing versatile ordered biotin/avidin arrays on UV-curable perfluoropolyethers (PFPEs) is presented. The goal is the realization of a versatile platform where any biotinylated biological ligands can be further linked to the underlying biotin/avidin array. To this end, microcontact arrayer and microcontact printing technologies were developed for photobiotin direct printing on PFPEs. As attested by fluorescence images, we demonstrate that this photoactive form of biotin is capable of grafting onto PFPEs surfaces during irradiation. Bioaffinity conjugation of the biotin/avidin system was subsequently exploited for further self-assembly avidin family proteins onto photobiotin arrays. The excellent fouling release PFPEs surface properties enable performing avidin assembly step simply by arrays incubation without PFPEs surface passivation or chemical modification to avoid unspecific biomolecule adsorption. Finally, as a proof of principle biotinylated heparin was successfully grafted onto photobiotin/avidin arrays.

JTD Keywords: Antifouling, Heparin, Malaria, Microcontact arrayer, Microcontact printing, Micropatterning, Perfluoropolyether, Photobiotin, Polymers, Soft lithography

The first part of this paper reviews the current development and key issues on implantable multi-sensor devices for in vivo theranostics. Afterwards, the authors propose an innovative biomedical multisensory system for in vivo biomarker monitoring that could be suitable for customized theranostics applications. At this point, findings suggest that cross-cutting Key Enabling Technologies (KETs) could improve the overall performance of the system given that the convergence of technologies in nanotechnology, biotechnology, micro&nanoelectronics and advanced materials permit the development of new medical devices of small dimensions, using biocompatible materials, and embedding reliable and targeted biosensors, high speed data communication, and even energy autonomy. Therefore, this article deals with new research and market challenges of implantable sensor devices, from the point of view of the pervasive system, and time-to-market. The remote clinical monitoring approach introduced in this paper could be based on an array of biosensors to extract information from the patient. A key contribution of the authors is that the general architecture introduced in this paper would require minor modifications for the final customized bio-implantable medical device.

JTD Keywords: Biocompatible, Biosensor, Biotelemetry, Implantable multi-sensor, Innovation, KET, Nanomedicine, Personalized medicine, Biotelemetry, Innovation, Medical nanotechnology, Biocompatible, Implantable system, In-vivo, KET, Multi sensor, Personalized medicines, Theranostics, Biosensors

Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.

JTD Keywords: Antibiotic, Bioceramic, Biomaterial, Bone regeneration, Calcium phosphate cement, Ceramic matrix, Growth factor, Hydroxyapatite, Ions, Protein

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JTD Keywords: Biotin, Click chemistry, Dip-pen nanolithography, Oligonucleotides, Surfaces

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell–adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm2), with a threshold value of 4.0 pmol/cm2 for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis.

JTD Keywords: RGD gradient, Cell adhesion, Poly(methyl methacrylate), Hydrolysis, Biotin-streptavidin

A method for the simultaneous (bio)chemical and topographical patterning of enclosed structures in poly(dimethyl siloxane) (PDMS) is presented. The simultaneous chemical and topography transference uses a water-soluble chitosan sacrificial mold to impart a predefined pattern with micrometric accuracy to a PDMS replica. The method is compared to conventional soft-lithography techniques on planar surfaces. Its functionality is demonstrated by the transference of streptavidin directly to the surface of the three-dimensional PDMS structures as well as indirectly using streptavidin-loaded latex nanoparticles. The streptavidin immobilized on the PDMS is tested for bioactivity by coupling with fluorescently labeled biotin. This proves that the streptavidin is immobilized on the PDMS surface, not in the bulk of the polymer, and is therefore accessible for use as signaling/binding element in micro and bioengineering. The use of a biocompatible polymer and processes enables the technique to be used for the chemical patterning of tissue constructions.

JTD Keywords: Biotechnology, Chitosan, Microfabrication, MEMs, Soft lithography

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

JTD Keywords: Tuning fork, Atomic force microscopy, Shear-force microscopy, Molecular recognition, Avidin-biotin