Search

Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these “kiss-and-run” interactions directlyin vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell–cell interactions across multiple biological systems.

As a response to the Russian invasion of Crimea, NATO returned to the core military concepts of deterrence and collective defence. This NATO adaption has recently come to include attempts to integrate offensive cyber effects into NATO force structure and response doctrine. The article argues—counter to what is publicly declared—that such an integration does little to strengthen NATO's deterrence posture and is unlikely to deter non-military, hybrid cyber activity below the threshold of collective defence. The article identifies several practical challenges to the current integration effort, which include the temporal dimension of developing exploits, battle damage assessment and deconfliction. With these challenges in mind, the article suggests that deploying minor and less resourceful cyber effects that cause persistent ‘cyber annoyances’ holds an unappreciated potential as they can drain opponent resources, disturb vital IT-systems and complicate decision-making. The article ends by arguing that NATO should not adapt its collective defence clause to cyberspace. A more active NATO in cyberspace risks undermining the cyber-intelligence norm that so far has prevented escalation and thereby increasing the likelihood that Russia misinterprets intelligence and active cyber defence activities as military preparation, armament or an attack in the making.

Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys(6) solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.

Cucurbit[7]uril (CB[7]) is a supramolecular binding host for peptides and proteins with N-terminal Phe. However, the low occurrence of such peptides and proteins limits broader applications of this unique host-guest interaction. Here, we report a strategy to expand the scope of CB[7]-peptide interaction by site-specifically introducing N-terminal substitutions (e.g. benzyl groups) using reductive alkylation. N-terminal benzylated peptides have similar affinity to CB[7] as native peptides with N-terminal Phe and even stronger interactions can be achieved using better ligands. We further expanded this host-guest interaction to be stimuli responsive. By introducing benzyl carboxylate substituents, the CB[7]-peptide interaction shows pH-dependent binding. Furthermore, benzyl boronate substituents led to saccharide-dependent CB[7]-peptide interactions. We demonstrated that using this strategy to introduce stronger CB[7] binders to the N-terminus of human calcitonin (hCT) results in increased aggregation stability in the presence of CB[7]. This strategy to expand CB[7]-peptide interaction scope opens opportunities for future applications in peptides and proteins.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.

Regulating germinal center contractionGerminal centers (GCs) in secondary lymphoid organs are where mature B cells expand and differentiate. Although GC formation is well studied, the control of GC duration and contraction is less well understood. Using intravital imaging of mouse GCs and single-cell RNA sequencing, Jacobsenet al.report that T follicular helper (TFH) cells are a critical player in this process. They found that some late-GC TFHcells upregulate the transcription factor FOXP3 and acquire a regulatory T cell–like phenotype. These cells are distinct from T follicular regulatory (TFR) cells and, unlike TFRcells, are needed to shut down the GC reaction. Tweaking this process may be key to extending GC lifetimes and enhancing antibody responses in the context of vaccination.Science, abe5146, this issue p.eabe5146

We employ time- and angle-resolved photoemission spectroscopy to study the spin- and valley-selective photoexcitation and dynamics of free carriers at the $\overline{\mathit{K}}$ and ${\overline{\mathit{K}}}^{\ensuremath{'}}$ points in singly oriented single-layer ${\mathrm{WS}}_{2}/\mathrm{Au}(111)$. Our results reveal that in the valence band maximum an ultimate valley polarization of free holes of 84% can be achieved upon excitation with circularly polarized light at room temperature. Notably, we observe a significantly smaller valley polarization for the photoexcited free electrons in the conduction band minimum. Clear differences in the carrier dynamics between electrons and holes imply intervalley scattering processes into dark states being responsible for the efficient depolarization of the excited electron population.

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency gamma-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex(+) T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.

Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, TFH, and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans. The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines., Vaccine targeting: Enhancement of responses by MHC class II targeting The germinal center (GC) reaction underpins the robust antibody responses that are the goal of most vaccine approaches. Bjarne Bogen and colleagues at the University of Oslo develop an adjuvant-free engineered vaccine that can enhance the GC reaction. The vaccine is composed of an antibody fragment targeting a mouse major histocompatability complex (MHC) class II (I-Ed) linked to an experimental myeloma antigen. When tested in B-cell and T-cell receptor transgenic mice specific for the myeloma antigen, GC responses, antibody titers and affinity as well as B and T cell proliferation are all enhanced compared to non-MHC class II targeted vaccine. MHC class II targeting can also enhance antibody responses to influenza antigen using a DNA vaccination approach in wildtype mice. Targeting antigens to MHC class II enhances the GC reaction and has potential implications for the design of more potent and durable vaccines.

Significance B and T lymphocytes collaborate during immune responses to antigens. B cells use membrane-bound antibody as part of their antigen receptor while T cells use a different receptor that recognizes antigen fragments bound to MHC molecules. We show here that T cells can recognize the variable parts of the B cell receptor when these are presented on MHC molecules. A prerequisite for such receptor cross-talk is that the B cell receptor binds antigen. The cross-talk results in collaboration between B and T cells and production of antibodies directed against the antigen. The findings have implications for basic immune regulation. The results may also help us understand the mechanism behind the development of SLE-like autoimmune diseases and B cell lymphomas., The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4+ T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id+ B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4+ T cells, extrafollicular T–B cell collaboration and some germinal center formation, and production of Id+ IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

Antibodies are currently the fastest-growing class of therapeutics. Although naked antibodies have proven valuable as pharmaceutical agents, they have some limitations, such as low tissue penetration and a long circulatory half-life. They have been conjugated to toxic payloads, PEGs, or radioisotopes to increase and optimize their therapeutic efficacy. Although nonspecific conjugation is suitable for most in vitro applications, it has become evident that site specifically modified antibodies may have advantages for in vivo applications. Herein we describe a novel approach in which the antibody fragment is tagged with two handles: one for the introduction of a fluorophore or F isotope, and the second for further modification of the fragment with a PEG moiety or a second antibody fragment to tune its circulatory half-life or its avidity. Such constructs, which recognize Class II MHC products and CD11b, showed high avidity and specificity. They were used to image cancers and could detect small tumors., Cancer Research Institute (New York, N.Y.), United States. National Institutes of Health (R01-AI087879-06), United States. National Institutes of Health (DP1-GM106409-03), United States. National Institutes of Health (R01-GM100518-04)

Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation., National Institute of Allergy and Infectious Diseases (Grant R01AI119006), National Institute of Allergy and Infectious Diseases (Grant R21AI138020)

We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-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 conventional 18F-FDG imaging., 18F-2-Deoxyfluoroglucose combined with sortase is used to site-specifically label antibody fragments. A new mouse Class II MHC specific 18F-labeled VHH not only decorated lymphoid organs but also detected a heterotopic pancreatic tumor (∼1.5 mm) due to the presence of immune cells. 1, 2, 3: lymph nodes, 4: thymus, 5: tumor.

Estimates of the thickness variation in lateritic weathering profiles (LWPs) are important in tropical areas underlain by young basalt lavas like those found in Hawaii. Seismic shear-wave velocity data were obtained by a new application of multichannel analysis of surface waves (MASW) to map variations in the LWP and to derive the downward rate of advance of the weathering front in basaltic lavas. The MASW technique proved highly capable of imaging the internal structure and base of the critical zone, as confirmed by borehole data and direct field measurements. Profile thickness thus obtained, rapidly and without drilling, has applications to engineering and geochemical studies. The rate of advance of the weathering front derived from MASW in Oahu ranged from 0.010 m/ka to 0.026 m/ka in mesic zones (∼1500 mm/a rainfall), whereas an area with ∼800 mm/a revealed rates from 0.005 m/ka to 0.011 m/ka. These rates are comparable to those derived from recent solute-based mass balance studies of ground and surface water. Conventional P-wave seismic reflection did not perform as well for detecting boundaries due to a gradational seismic velocity structure within the weathering profile. Shear-wave velocity models showed internal variations that may be caused by textural differences in parental lava flows. Limitations in imaging depth were overcome by innovative experiment designs. Increasing source-receiver offsets and merging surface-wave dispersion curves allowed for a more objective derivation of velocity-frequency relations. Further improvements were made from a recently developed form of the combined active and passive source technique. These advances allowed for more detailed and deeper imaging of the subsurface with greater confidence. Velocity models derived from MASW can thus describe the LWP in terms of depth and variability in stiffness.

We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing pre-rearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR/Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR and affinity maturation.

The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst.

International audience; Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (“helping hands”) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.

Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of L-TNFα along with the mirror-image D-protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory D-peptide with the potential to improve current anti-TNFα therapeutic strategies.

Diversity reigns in antibody responses During the course of an immune response, B cells specific for an invading pathogen divide. The antibodies they produce increase in affinity via somatic mutation in specialized lymph node structures called germinal centers. Tas et al. used multiphoton microscopy and sequencing to determine how different B cell clones compete with one another within mouse germinal centers. Multiple B cell clones can seed individual germinal centers, and germinal centers lose diversity at disparate rates. Such heterogeneity suggests that manipulating minor clonal populations to gain an advantage during vaccination may one day be possible. Science , this issue p. 1048

Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

The decontamination of a rotating cutting tool used for slicing in the meat industry by means of atmospheric pressure plasmas is investigated. The target is Listeria monocytogenes, a bacterium which causes listeriosis and can be found in plants and food. The non-pathogenic species, Listeria innocua, is used for the experiments. A rotating knife was inoculated with L. innocua. The surface of the rotating knife was partly exposed to an atmospheric pressure dielectric barrier discharge operated in air, where the knife itself served as a ground electrode. The rotation of the knife ensures a treatment of the whole cutting tool. A log 5 reduction of L. innocua is obtained after 340 s of plasma operation. The temperature of the knife after treatment was found to be below 30 °C. The design of the setup allows a decontamination during slicing operation.

Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG)2K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and micro-rheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, time-course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability. Keywords: biodegradable, click chemistry, hydrogels, peptide crosslinks, poly(ethylene glycol) Introduction Hydrogels were the first biomaterials designed for clinical use.[1] They have found applications in numerous areas, including tissue engineering, drug delivery, sensing, diagnostics, and microfluidics.[2,3] Several factors need to be considered when designing biocompatible hydrogel biomaterials including controlled chemistry for the formation of reproducible, reversible 3D networks with precisely defined structures, and biodegradability. Living radical polymerization has been used for the control of the molecular weight distribution of the primary chains of the network;[4,5] copper catalyzed[6,7] or copper-free [8] Huisgen cycloaddition of azides and alkynes (click chemistry) and Michael addition[9] have been used to control crosslinking and/or attachment of biologically active molecules. The combination of synthetic polymers with sequences/chains of natural polymers is a promising route for the design and synthesis of enzymatically degradable hybrid polymers/hydrogels.[2,10] To render a hybrid polymer/hydrogel degradable, the structure of the degradable sequences should match the active site of respective enzyme(s); oligopeptide sequences have been frequently used as degradable crosslinks in hydrogels. Hydrogels, containing oligopeptide crosslinks susceptible to chymotrypsin-catalyzed hydrolysis were synthesized by crosslinking N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing reactive side-chains (terminated in p-nitrophenoxy groups) with oligopeptide-containing diamines.[10] The degradability of hydrogels was dependent on the length and detailed structure of the oligopeptide sequence and on the equilibrium degree of swelling (network density); the higher the degree of swelling, the faster the rate of degradation. The degree of swelling also has an impact on surface versus bulk degradation of the hydrogel. If the enzyme cannot diffuse into the hydrogel interior, only surface degradation takes place.[11] Similar HPMA-based hydrogels degradable by cathepsin B, a lysosomal thiol proteinase, have also been evaluated.[12] In further experiments, HPMA-based hydrogels with degradable crosslinks were shown to release FITC-dextran and daunomycin (covalently bound via oligopeptide spacer) during incubation with a mixture of lysosomal enzymes (Tritosomes) or chymotrypsin.[13] Recently, Plunkett et al. studied acrylamide-based hydrogels containing oligopeptides degradable by chymotrypsin.[14] Poly(ethylene glycol) (PEG) is a hydrophilic polymer that has been used in several clinical applications. PEG-based hydrogels have been extensively studied. Hubbell’s laboratory used multiarm-PEG and Michael-type addition to synthesize extracellular matrix mimicking hydrogels degradable by cell-excreted matrix metalloproteinases.[9,15] PEG-based hydrogels containing Schiff base linkages were designed for low molecular weight (doxorubicin) drug delivery.[16] Azide-alkyne click chemistry was also used for the synthesis of PEG-based hydrogels.[6,17] Hawker and coworkers[6] synthesized hydrogels by reacting a tetraazide-modified tetraethylene glycol with telechelic alkyne terminated PEGs of varying molecular weight. Yang and coworkers employed 4-arm alkyne terminated PEG and crosslinked it with a series of RGD containing dialkyne-peptides. However, both alkyne groups were attached to the N-terminal lysine. Consequently, the side-chains were degradable, but the hydrogel crosslinked backbone remained stable.[17] In this study we synthesized two 4-arm azide-terminated PEGs differing in molecular weight (2 100 and 8 800) and two alkyne-terminated peptides, [alkyne]-GFLGK-[alkyne] (GFLG1) and ([alkyne]-GFLG)2K (GFLG2). The hydrogels were synthesized by Huisgen cycloaddition (click chemistry) of PEG azides with peptide alkynes and contained enzymatically degradable crosslinks. The kinetics of the crosslinking reaction was monitored by dynamic rheology. The hydrogels were characterized by the equilibrium degree of swelling, their morphology, and bulk and micro-rheology. The relationship between the structure of the hydrogels and their degradability by papain, a thiol proteinase, was also evaluated.

If the potential for a boom in the global hydrogen economy is realized, there will be an increase in the need for accurate hydrogen thermodynamic property standards. Based on current and anticipated needs, new fundamental equations of state for parahydrogen, normal hydrogen, and orthohydrogen were developed to replace the existing property models. To accurately predict thermophysical properties near the critical region and in liquid states, the quantum law of corresponding states was applied to improve the normal hydrogen and orthohydrogen formulations in the absence of available experimental data. All three equations of state have the same maximum pressure of 2000MPa and upper temperature limit of 1000K. Uncertainty estimates in this paper can be considered to be estimates of a combined expanded uncertainty with a coverage factor of 2 for primary data sets. The uncertainty in density is 0.04% in the region between 250 and 450K and at pressures up to 300MPa. The uncertainties of vapor pressures and saturated liquid densities vary from 0.1% to 0.2%. Heat capacities are generally estimated to be accurate to within 1%, while speed-of-sound values are accurate to within 0.5% below 100MPa.

The Alzheimer's amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activationin vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.

As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) over-expressing OTK18 by recombinant adenoviral gene transfer. Viral infection of MDM strongly increased the processing of OTK18 into its N-terminal fragment. Treatment of OTK18-expressing MDM with calpain- and proteasome- inhibitors significantly accumulated either full-length or processed OTK18 fragments in time and dose-dependent manners. A series of OTK18 truncation mutants and synthetic peptides were tested to locate the calpain cleavage sites after arginine 359. Finally, we developed an enhanced cyan and yellow fluorescent protein (ECFP and EYFP)-based intramolecular fluorescent resonance energy transfer (intramolecular FRET) system to monitor the OTK18 endoproteolysis in human microglia cell line. Inhibition of proteasome activity significantly increased the intramolecular FRET signal in the nucleus. These data suggest that calpain and proteasome are involved in OTK18 endoproteolysis and degradation. Additionally, intramolecular FRET has proven to be a useful tool for monitoring the processing in live cells.

The EU Water Framework Directive (WFD) requires an integrated approach to river basin management in order to meet environmental and ecological objectives. This paper presents concepts and full-scale application of an integrated modelling framework. The Ringkoebing Fjord basin is characterized by intensive agricultural production and leakage of nitrate constitute a major pollution problem with respect groundwater aquifers (drinking water), fresh surface water systems (water quality of lakes) and coastal receiving waters (eutrophication). The case study presented illustrates an advanced modelling approach applied in river basin management. Point sources (e.g. sewage treatment plant discharges) and distributed diffuse sources (nitrate leakage) are included to provide a modelling tool capable of simulating pollution transport from source to recipient to analyse the effects of specific, localized basin water management plans. The paper also includes a land rent modelling approach which can be used to choose the most cost-effective measures and the location of these measures. As a forerunner to the use of basin-scale models in WFD basin water management plans this project demonstrates the potential and limitations of comprehensive, integrated modelling tools.

At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.

A widely used form of an equation of state explicit in Helmholtz energy has been modified with new terms to eliminate certain undesirable characteristics in the two phase region. Modern multiparameter equations of state exhibit behavior in the two phase that is inconsistent with the physical behavior of fluids. The new functional form overcomes this dilemma and results in equations of state for pure fluids that are more fundamentally consistent. With the addition of certain nonlinear fitting constraints, the new equation now achieves proper phase stability, i.e., only one solution exists for phase equilibrium at a given state. New fitting techniques have been implemented to ensure proper extrapolation of the equation at low temperatures, in the vapor phase at low densities, and at very high temperatures and pressures. A formulation is presented for the thermodynamic properties of refrigerant 125 (pentafluoroethane, CHF2–CF3) using the new terms and fitting techniques. The equation of state is valid for te...

Mixture models explicit in Helmholtz energy have been developed to calculate the thermodynamic properties of refrigerant mixtures containing R-32, R-125, R-134a, R143a, and R-152a. The Helmholtz energy of the mixture is the sum of the ideal gas contribution, the compressibility (or real fluid) contribution, and the contribution from mixing. The independent variables are the density, temperature, and composition. The model may be used to calculate the thermodynamic properties of mixtures, including dew and bubble point properties, within the experimental uncertainties of the available measured properties. It incorporates the most accurate equations of state available for each pure fluid. The estimated uncertainties of calculated properties are 0.1% in density and 0.5% in heat capacities and in the speed of sound. Calculated bubble point pressures have typical uncertainties of 0.5%.

A new formulation for the thermodynamic properties of trifluoromethane (R-23) is presented. The formulation is valid for single-phase and saturation states for temperatures from the triple point (118.02 K) to 475 K, pressures up to 120 MPa, and densities up to 24.31 mol/dm3. The formulation includes a fundamental equation and ancillary functions for the estimation of saturation properties. The experimental data used to determine the fundamental equation included pressure–density–temperature (p–ρ–T), isobaric heat capacity (cp–p–T), isochoric heat capacity (cν–ρ–T), saturation heat capacity (cσ), speed of sound (w–p–T), and vapor pressure. A nonlinear regression algorithm was used to determine the constants and exponents of various functions within the formulation. Experimental data and values computed using the formulation are compared to verify the uncertainties in the calculated properties. The formulation presented may be used to compute densities to within ±0.1%, heat capacities to within ±0.5%, speed...

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.

Significance This paper addresses a fundamental question: Can natural chaperones fold mirror-image proteins? Mirror-image proteins (composed of d -amino acids) are only accessible by chemical synthesis, but are protease resistant and therefore have tremendous potential as long-lived drugs. Many large/complex proteins depend on chaperones for efficient folding. Here we describe the total chemical synthesis of a 312-residue chaperone-dependent protein (DapA) in natural ( l -) and mirror-image ( d -) forms, the longest fully synthetic proteins yet reported. Using these proteins we show that the natural bacterial GroEL/ES chaperone is “ambidextrous”—i.e., it can fold both natural and mirror-image proteins via nonspecific hydrophobic interactions. Our study also provides proof-of-concept for the use of natural GroEL/ES to fold d -proteins for mirror-image drug discovery and synthetic biology applications.

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id+ Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 107 M−1) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4+ T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell–B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id+ Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell–B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II–restricted presentation of Id+ Ig and suggest that Id-specific T cell–B cell collaboration is of physiological relevance.

A plot a la Dalitz for angles between transverse momenta gives information about the reaction mechanism for AB → AXB at high energy.

The purpose of this article is to update the common knowledge on characteristic features of empirical multiparameter equations of state, to increase the confidence of potential users, and possibly to attract other scientists to theoretical and experimental work that is relevant for the future development of these kinds of thermodynamic property models. To do so, the most important features of current multiparameter equations of state and of the algorithms which are used to develop such formulations are briefly explained. Future challenges are outlined with regard both to the development of multiparameter equations of state and to the underlying experimental basis. Relevant references are given for further studies.

The reaction pp -> pf (pi0pi0pi0) ps has been studied at 450 GeV/c. The pi0pi0pi0 effective mass spectrum shows clear eta(547) and pi2(1670) signals. Branching ratios for the eta(547) and pi_2(1670) are given as well as upper limits for the decays of the omega(782), a1(1260) and a2(1320) into 3pi0., 10 pages, Latex, 4 Figures

A new formulation for the thermodynamic properties of nitrogen has been developed. Many new data sets have become available, including high accuracy data from single and dual-sinker apparatuses which improve the accuracy of the representation of the pρT surface of gaseous, liquid, and supercritical nitrogen, including the saturation states. New measurements of the speed of sound from spherical resonators yield accurate information on caloric properties in gaseous and supercritical nitrogen. Isochoric heat capacity and enthalpy data have also been published. Sophisticated procedures for the optimization of the mathematical structure of equations of state and special functional forms for an improved representation of data in the critical region were used. Constraints regarding the structure of the equation ensure reasonable results up to extreme conditions of temperature and pressure. For calibration applications, the new reference equation is supplemented by a simple but also accurate formulation, valid only for supercritical nitrogen between 250 and 350 K at pressures up to 30 MPa. The uncertainty in density of the new reference equation of state ranges from 0.02% at pressures less than 30 MPa up to 0.6% at very high pressures, except in the range from 270 to 350 K at pressures less than 12 MPa where the uncertainty in density is 0.01%. The equation is valid from the triple point temperature to temperatures of 1000 K and up to pressures of 2200 MPa. From 1000 to 1800 K, the equation was validated with data of limited accuracy. The extrapolation behavior beyond 1800 K is reasonable up to the limits of chemical stability of nitrogen, as indicated by comparison to experimental shock tube data.