Detection of Water Contaminants by Organic Transistors as Gas Sensors in a Bottom-Gate/Bottom-Contact Cross-Linked Structure.

Detecting volatile organic compounds is a fundamental step in water quality analysis. Methylisoborneol (MIB) provides a lousy odor to water, whereas geosmin (GEO) is responsible for its sour taste. A widely-used technique for their detection is gas-phase chromatography. On the other hand, an electronic nose from organic thin-film transistors is a cheaper and faster alternative. Poly(2,5-bis(3-tetradecyl-thiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT-C14) features semiconducting properties suitable for organic electronics. However, in order to expose the active layer in a bottom-gate transistor structure with photolithographically patterned electrodes, a cross-linked dielectric such as poly(4-vinyl phenol) (PVP) is necessary. In this work, the cross-linking was demonstrated using FTIR and Raman spectroscopies, as well as high-k capacitors with a dielectric constant of 5.3. The presence of enhanced crystallinity with terrace formation in the semiconducting film was confirmed with UV-visible spectrophotometry, atomic force microscopy, and X-ray diffraction. Finally, for the first time, a PBTTT-C14 transistor on cross-linked PVP was shown to respond to isoborneol with a sensitivity of up to 6% change in mobility per ppm. Due to its similarity to MIB, a system comprising these sensors must be investigated in the future as a tool for sanitation companies in real-time water quality monitoring.

Review of Bacterial Nanocellulose-Based Electrochemical Biosensors: Functionalization, Challenges, and Future Perspectives.

Electrochemical biosensing devices are known for their simple operational procedures, low fabrication cost, and suitable real-time detection. Despite these advantages, they have shown some limitations in the immobilization of biochemicals. The development of alternative materials to overcome these drawbacks has attracted significant attention. Nanocellulose-based materials have revealed valuable features due to their capacity for the immobilization of biomolecules, structural flexibility, and biocompatibility. Bacterial nanocellulose (BNC) has gained a promising role as an alternative to antifouling surfaces. To widen its applicability as a biosensing device, BNC may form part of the supports for the immobilization of specific materials. The possibilities of modification methods and in situ and ex situ functionalization enable new BNC properties. With the new insights into nanoscale studies, we expect that many biosensors currently based on plastic, glass, or paper platforms will rely on renewable platforms, especially BNC ones. Moreover, substrates based on BNC seem to have paved the way for the development of sensing platforms with minimally invasive approaches, such as wearable devices, due to their mechanical flexibility and biocompatibility.

Ligand-independent oligomerization of TACI is controlled by the transmembrane domain and regulates proliferation of activated B cells.

In mature B cells, TACI controls class-switch recombination and differentiation into plasma cells during T cell-independent antibody responses. TACI binds the ligands BAFF and APRIL. Approximately 10% of patients with common variable immunodeficiency (CVID) carry TACI mutations, of which A181E and C172Y are in the transmembrane domain. Residues A181 and C172 are located on distinct sides of the transmembrane helix, which is predicted by molecular modeling to spontaneously assemble into trimers and dimers. In human B cells, these mutations impair ligand-dependent (C172Y) and -independent (A181E) TACI multimerization and signaling, as well as TACI-enhanced proliferation and/or IgA production. Genetic inactivation of TACI in primary human B cells impaired survival of CpG-activated cells in the absence of ligand. These results identify the transmembrane region of TACI as an active interface for TACI multimerization in signal transduction, in particular for ligand-independent signals. These functions are perturbed by CVID-associated mutations.

Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

CD20 as a gatekeeper of the resting state of human B cells.

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.

Organic Thin-Film Transistors as Gas Sensors: A Review.

Organic thin-film transistors (OTFTs) are miniaturized devices based upon the electronic responses of organic semiconductors. In comparison to their conventional inorganic counterparts, organic semiconductors are cheaper, can undergo reversible doping processes and may have electronic properties chiefly modulated by molecular engineering approaches. More recently, OTFTs have been designed as gas sensor devices, displaying remarkable performance for the detection of important target analytes, such as ammonia, nitrogen dioxide, hydrogen sulfide and volatile organic compounds (VOCs). The present manuscript provides a comprehensive review on the working principle of OTFTs for gas sensing, with concise descriptions of devices' architectures and parameter extraction based upon a constant charge carrier mobility model. Then, it moves on with methods of device fabrication and physicochemical descriptions of the main organic semiconductors recently applied to gas sensors (i.e., since 2015 but emphasizing even more recent results). Finally, it describes the achievements of OTFTs in the detection of important gas pollutants alongside an outlook toward the future of this exciting technology.

Starch-Mediated Immobilization, Photochemical Reduction, and Gas Sensitivity of Graphene Oxide Films.

This work unveils the roles played by potato starch (ST) in the immobilization, photochemical reduction, and gas sensitivity of graphene oxide (GO) films. The ST/GO films are assembled layer by layer (LbL) onto quartz substrates by establishing mutual hydrogen bonds that drive a stepwise film growth, with equal amounts of materials being adsorbed in each deposition cycle. Afterward, the films are photochemically reduced with UV irradiation (254 nm), following a first-order kinetics that proceeds much faster when GO is assembled along with ST instead of a nonoxygenated polyelectrolyte, namely, poly(diallyl dimethylammonium) hydrochloride (PDAC). Finally, the gas-sensing performance of ST/reduced graphene oxide (RGO) and PDAC/RGO sensors fabricated via LbL atop of gold interdigitated microelectrodes is evaluated at different relative humidity levels and in different concentrations of ammonia, ethanol, and acetone. In comparison to the PDAC/RGO sensor, the ones containing ST are much more sensitive, especially when operating in a high-relative-humidity environment. An array comprising these chemical sensors provides unique electrical fingerprints for each of the investigated analytes and is capable of discriminating and quantifying them in a wide range of concentrations, from 10 to 1000 ppm.

Rapid Antigen and Antibody-Like Molecule Discovery by Staphylococcal Surface Display.

Ever since the discovery of antibodies, they have been generated by complicated multi-step procedures. Typically, these involve sequencing, cloning, and screening after expression of the antibodies in a suitable organism and format. Here, a staphylococcal nanobody display is described that omits many the abovementioned intermediate steps and allows for simultaneous screening of multiple targets without prior knowledge nor expression of the binders. This paper reports a detailed, general step-by-step protocol to achieve nanobodies of high affinity. Apart from its focus on radioactive and fluorescent targets, it gives options for various other target formats and additional applications for the staphylococcal library; including flow cytometry and immunoprecipitation. This provides a system for antibody engineers that can be easily adopted to their specific needs.

A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.

Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)- and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.

A Two-Step Approach for the Design and Generation of Nanobodies.

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

Immediate loading of three (fixed-on-3) vs four (fixed-on-4) implants supporting cross-arch fixed prostheses: 1-year results from a multicentre randomised controlled trial.

PURPOSE: To evaluate the outcome of three (fixed-on-3 = Fo3) vs four (fixed-on-4 = Fo4) implants immediately restored with metal-resin screw-retained cross-arch prostheses in fully edentulous jaws. MATERIALS AND METHODS: Forty-eight edentulous or to be rendered edentulous patients were randomised in six centres (eight patients per centre) to the Fo3 group (24 patients: 12 maxillae and 12 mandibles) and to the Fo4 group (24 patients: 12 maxillae and 12 mandibles) according to a parallel group design. To be immediately loaded, implants had to be inserted with a minimum torque of 40 Ncm. Outcome measures were prosthesis and implant failures, complications and peri-implant marginal bone level changes evaluated up to 1 year post-loading. RESULTS: One maxillary prosthesis per group was delayed loaded because implants could not be placed with a torque of at least 40 Ncm. Ten patients in the Fo3 group and four in the Fo4 group had implants placed flapless. One year after loading no drop out occurred. One patient of the Fo3 group lost three implants vs three patients of the Fo4 group who lost four implants, the difference being no statistically significant (risk difference = -0.08; 95% CI: -0.27 to 0.10; Fisher's exact test P = 0.609). One mandibular Fo3 and one maxillary Fo4 prosthesis failed. Six Fo3 patients were affected by complications vs three Fo4 patients (risk difference = 0.12; 95% CI: -0.10 to 0.34; Fisher's exact test; P = 0.461). Both groups lost marginal bone in a statistically significant way (0.22 ± 0.20 mm for Fo3 patients and 0.40 ± 0.21 mm for Fo4 patients), with Fo3 group showing less marginal peri-implant bone loss than Fo4 group (estimate of the difference = -0.18 (standard error: 0.06) mm; 95% CI: -0.30 to -0.06; P = 0.005). There were no differences in clinical outcomes between the six operators. CONCLUSIONS: These preliminary results suggest that immediately loaded cross-arch prostheses of both jaws can be supported by only three dental implants at least up to 1 year post-loading, though longer follow-ups are definitively needed.

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells.

Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igß (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igß can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igß and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.

Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries.

Unbiased and simultaneous identification of a specific antibody and its target antigen has been difficult without prior knowledge of at least one interaction partner. Immunization with complex mixtures of antigens such as whole organisms and tissue extracts including tumoral ones evokes a highly diverse immune response. During such a response, antibodies are generated against a variety of epitopes in the mixture. Here, we propose a surface display design that is suited to simultaneously identify camelid single domain antibodies and their targets. Immune libraries of single-domain antigen recognition fragments from camelid heavy chain-only antibodies (VHH) were attached to the peptidoglycan of Gram-positive Staphylococcus aureus employing its endogenous housekeeping sortase enzyme. The sortase transpeptidation reaction covalently attached the VHH to the bacterial peptidoglycan. The reversible nature of the reaction allowed the recovery of the VHH from the bacterial surface and the use of the VHH in downstream applications. These staphylococcal surface display libraries were used to rapidly identify VHH as well as their targets by immunoprecipitation (IP). Our novel bacterial surface display platform was stable under harsh screening conditions, allowed fast target identification, and readily permitted the recovery of the displayed VHH for downstream analysis.

From Immunologically Archaic to Neoteric Glycovaccines.

Polysaccharides (PS) are present in the outermost surface of bacteria and readily come in contact with immune cells. They interact with specific antibodies, which in turn confer protection from infections. Vaccines with PS from pneumococci, meningococci, Haemophilus influenzae type b, and Salmonella typhi may be protective, although with the important constraint of failing to generate permanent immunological memory. This limitation has in part been circumvented by conjugating glycovaccines to proteins that stimulate T helper cells and facilitate the establishment of immunological memory. Currently, protection evoked by conjugated PS vaccines lasts for a few years. The same approach failed with PS from staphylococci, Streptococcus agalactiae, and Klebsiella. All those germs cause severe infections in humans and often develop resistance to antibiotic therapy. Thereby, prevention is of increasing importance to better control outbreaks. As only 23 of more than 90 pneumococcal serotypes and 4 of 13 clinically relevant Neisseria meningitidis serogroups are covered by available vaccines there is still tremendous clinical need for PS vaccines. This review focuses on glycovaccines and the immunological mechanisms for their success or failure. We discuss recent advances that may facilitate generation of high affinity anti-PS antibodies and confer specific immunity and long-lasting protection.

Enhanced Sensitivity of Gas Sensor Based on Poly(3-hexylthiophene) Thin-Film Transistors for Disease Diagnosis and Environment Monitoring.

Electronic devices based on organic thin-film transistors (OTFT) have the potential to supply the demand for portable and low-cost gadgets, mainly as sensors for in situ disease diagnosis and environment monitoring. For that reason, poly(3-hexylthiophene) (P3HT) as the active layer in the widely-used bottom-gate/bottom-contact OTFT structure was deposited over highly-doped silicon substrates covered with thermally-grown oxide to detect vapor-phase compounds. A ten-fold organochloride and ammonia sensitivity compared to bare sensors corroborated the application of this semiconducting polymer in sensors. Furthermore, P3HT TFTs presented approximately three-order higher normalized sensitivity than any chemical sensor addressed herein. The results demonstrate that while TFTs respond linearly at the lowest concentration values herein, chemical sensors present such an operating regime mostly above 2000 ppm. Simultaneous alteration of charge carrier mobility and threshold voltage is responsible for pushing the detection limit down to units of ppm of ammonia, as well as tens of ppm of alcohol or ketones. Nevertheless, P3HT transistors and chemical sensors could compose an electronic nose operated at room temperature for a wide range concentration evaluation (1-10,000 ppm) of gaseous analytes. Targeted analytes include not only biomarkers for diseases, such as uremia, cirrhosis, lung cancer and diabetes, but also gases for environment monitoring in food, cosmetic and microelectronics industries.

The use of 18F-2-fluorodeoxyglucose (FDG) to label antibody fragments for immuno-PET of pancreatic cancer.

We generated 18F-labeled antibody fragments for PET imaging using a sortase-mediated reaction to install a transcyclooctene (TCO)-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields >25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon-fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional18F-FDG imaging.

Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization.

UNLABELLED: Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of influenza virus-encoded proteins. We generated a panel of camelid-derived single-domain antibody fragments (VHHs) against influenza virus nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza virus genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the viral ribonucleoprotein (vRNP) complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. IMPORTANCE: Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function and determining how to disrupt said function through pharmaceutical intervention are difficult. We present a novel method based on single-domain-antibody technology that permits specific targeting and disruption of an essential influenza virus protein in the absence of genetic manipulation of influenza virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.

A semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice.

Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen α-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, long-lived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydrate-based vaccines against other major bacterial pathogens.

Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells.

Human T cells that express a T cell antigen receptor (TCR) containing γ-chain variable region 9 and δ-chain variable region 2 (Vγ9Vδ2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I-related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vγ9Vδ2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vγ9Vδ2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vγ9Vδ2 T cells.

Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus.

The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire.