Usp14 is required for spermatogenesis and ubiquitin stress responses in Drosophila melanogaster.

Deubiquitylating (DUB) enzymes free covalently linked ubiquitin moieties from ubiquitin-ubiquitin and ubiquitin-protein conjugates, and thereby maintain the equilibrium between free and conjugated ubiquitin moieties and regulate ubiquitin-mediated cellular processes. Here, we performed genetic analyses of mutant phenotypes in Drosophila melanogaster and demonstrate that loss of Usp14 function results in male sterility, with defects in spermatid individualization and reduced testicular free monoubiquitin levels. These phenotypes were rescued by germline-specific overexpression of wild-type Usp14. Synergistic genetic interactions with Ubi-p63E and cycloheximide sensitivity suggest that ubiquitin shortage is a primary cause of male sterility. In addition, Usp14 is predominantly expressed in testes in Drosophila, indicating a higher demand for this DUB in testes that is also reflected by testis-specific loss-of-function Usp14 phenotypes. Collectively, these results suggest a major role of Usp14 in maintaining normal steady state free monoubiquitin levels during the later stages of Drosophila spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Designer covalent heterobivalent inhibitors prevent IgE-dependent responses to peanut allergen.

Allergies are a result of allergen proteins cross-linking allergen-specific IgE (sIgE) on the surface of mast cells and basophils. The diversity and complexity of allergen epitopes, and high-affinity of the sIgE-allergen interaction have impaired the development of allergen-specific inhibitors of allergic responses. This study presents a design of food allergen-specific sIgE inhibitors named covalent heterobivalent inhibitors (cHBIs) that selectively form covalent bonds to only sIgEs, thereby permanently inhibiting them. Using screening reagents termed nanoallergens, we identified two immunodominant epitopes in peanuts that were common in a population of 16 allergic patients. Two cHBIs designed to inhibit only these two epitopes completely abrogated the allergic response in 14 of the 16 patients in an in vitro assay and inhibited basophil activation in an allergic patient ex vivo analysis. The efficacy of the cHBI design has valuable clinical implications for many allergen-specific responses and more broadly for any antibody-based disease.

Self-Consistent Potential Correction for Charged Periodic Systems.

Supercell models are often used to calculate the electronic structure of local deviations from the ideal periodicity in the bulk or on the surface of a crystal or in wires. When the defect or adsorbent is charged, a jellium counter charge is applied to maintain overall neutrality, but the interaction of the artificially repeated charges has to be corrected, both in the total energy and in the one-electron eigenvalues and eigenstates. This becomes paramount in slab or wire calculations, where the jellium counter charge may induce spurious states in the vacuum. We present here a self-consistent potential correction scheme and provide successful tests of it for bulk and slab calculations.

Covalent Heterobivalent Inhibitor Design for Inhibition of IgE-Dependent Penicillin Allergy in a Murine Model.

Drug allergies occur when hapten-like drug metabolites conjugated to serum proteins, through their interactions with specific IgE, trigger allergic reactions that can be life threatening. A molecule termed covalent heterobivalent inhibitor (cHBI) was designed to specifically target drug hapten-specific IgE to prevent it from binding drug-haptenated serum proteins. cHBI binds the two independent sites on a drug hapten-specific Ab and covalently conjugates only to the specific IgE, permanently inhibiting it. The cHBI design was evaluated via ELISA to measure cHBI-IgE binding, degranulation assays of rat basophil leukemia cells for in vitro efficacy, and mouse models of ear swelling and systemic anaphylaxis responses for in vivo efficacy. The cHBI design was evaluated using two separate models: one specific to inhibit penicillin G-reactive IgE and another to inhibit IgE specific to a model compound, dansyl. We show that cHBI conjugated specifically to its target Ab and inhibited degranulation in cellular degranulation assays using rat basophil leukemia cells. Furthermore, cHBIs demonstrated in vivo inhibition of allergic responses in both murine models. We establish the cHBI design to be a versatile platform for inhibiting hapten/IgE interactions, which can potentially be applied to inhibit IgE-mediated allergic reactions to any drug/small-molecule allergy.

Proper surface termination for luminescent near-surface NV centers in diamond.

By accurate quantum mechanical simulations, we show that typical diamond surfaces possess image states with sub-bandgap energies, and compromise the photostability of NV centers placed within a few nm of the surface. This occurs due to the mixture of the NV-related gap states and the surface image states, which is a novel and distinct process from the well-established band bending effect. We also find that certain types of coverages on the diamond surface may lead to blinking or bleaching due to the presence of acceptor surface states. We identify a combination of surface terminators that is perfect for NV-center based nanoscale sensing.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

A novel interplay between the ubiquitin­proteasome system and serine proteases during Drosophila development.

The concentrations of the Drosophila proteasomal and extraproteasomal polyubiquitin receptors fluctuate in a developmentally regulated fashion. This fluctuation is generated by a previously unidentified proteolytic activity. In the present paper, we describe the purification, identification and characterization of this protease (endoproteinase I). Its expression increases sharply at the L1-L2 larval stages, remains high until the second half of the L3 stage, then declines dramatically. This sharp decrease coincides precisely with the increase of polyubiquitin receptor concentrations in late L3 larvae, which suggests a tight developmental co-regulation. RNAi-induced down-regulation of endoproteinase I results in pupal lethality. Interestingly, we found a cross-talk between the 26S proteasome and this larval protease: transgenic overexpression of the in vivo target of endoproteinase I, the C-terminal half of the proteasomal polyubiquitin receptor subunit p54/Rpn10 results in transcriptional down-regulation of endoproteinase I and consequently a lower level of proteolytic elimination of the polyubiquitin receptors. Another larval protease, Jonah65A-IV, which degrades only unfolded proteins and exhibits similar cross-talk with the proteasome has also been purified and characterized. It may prevent the accumulation of polyubiquitylated proteins in larvae contrary to the low polyubiquitin receptor concentration.

Possibility of a field effect transistor based on Dirac particles in semiconducting anatase-TiO2 nanowires.

In Dirac materials, like graphene or topological insulators, massless pseudorelativistic electrons promise new, very fast electronic devices by utilizing the partial suppression of backscattering. However, the semimetal nature of graphene makes the realization of practical field effect transistors difficult, due to small on-off current ratios. Here, we propose a new concept, based on Dirac states inside the conduction (or valence) band of a lightly doped wide band gap semiconductor. With the application of a gate voltage, the Dirac states become populated; that is, the Fermi level is switched between the "classical" high-resistivity semiconducting and the relativistic high-mobility metallic range. We demonstrate by theoretical calculations that such a transition can be realized, for example, in thin anatase nanowires, which have been synthesized before. Ta-doped anatase nanowires offer an excellent possibility to build field effect transistors with high speed and good on-off ratio. Guidelines for finding similar "Dirac semiconductors" are provided.

The kinetics of carbon pair formation in silicon prohibits reaching thermal equilibrium.

Thermal equilibrium is reached when the system assumes its lowest energy. This can be hindered by kinetic reasons; however, it is a general assumption that the ground state can be eventually reached. Here, we show that this is not always necessarily the case. Carbon pairs in silicon have at least three different configurations, one of them (B-configuration) is the G photoluminescence centre. Experiments revealed a bistable nature with the A-configuration. Electronic structure calculations predicted that the C-configuration is the real ground state; however, no experimental evidence was found for its existence. Our calculations show that the formation of the A- and B-configurations is strongly favoured over the most stable C-configuration which cannot be realized in a detectable amount before the pair dissociates. Our results demonstrate that automatized search for complex defects consisting of only the thermodynamically most stable configurations may overlook key candidates for quantum technology applications.

The ubiquitin thioesterase YOD1 ameliorates mutant Huntingtin induced pathology in Drosophila.

Huntington's disease (HD) is a neurodegenerative disorder caused by a dominant gain-of-function mutation in the huntingtin gene, resulting in an elongated polyglutamine repeat in the mutant Huntingtin (mHtt) that mediates aberrant protein interactions. Previous studies implicated the ubiquitin-proteasome system in HD, suggesting that restoring cellular proteostasis might be a key element in suppressing pathology. We applied genetic interaction tests in a Drosophila model to ask whether modulating the levels of deubiquitinase enzymes affect HD pathology. By testing 32 deubiquitinase genes we found that overexpression of Yod1 ameliorated all analyzed phenotypes, including neurodegeneration, motor activity, viability, and longevity. Yod1 did not have a similar effect in amyloid beta overexpressing flies, suggesting that the observed effects might be specific to mHtt. Yod1 overexpression did not alter the number of mHtt aggregates but moderately increased the ratio of larger aggregates. Transcriptome analysis showed that Yod1 suppressed the transcriptional effects of mHtt and restored the expression of genes involved in neuronal plasticity, vesicular transport, antimicrobial defense, and protein synthesis, modifications, and clearance. Furthermore, Yod1 overexpression in HD flies leads to the upregulation of genes involved in transcriptional regulation and synaptic transmission, which might be part of a response mechanism to mHtt-induced stress.

High-throughput screening identification of novel immunomodulatory combinations for the generation of tolerogenic dendritic cells.

Introduction: Tolerogenic Dendritic Cells (tolDCs) have an exceptional promise as a potential therapy for autoimmune disease and transplantation rejection. TolDCs are a unique phenotype of antigen presenting cells (APCs) that can influence naïve T cells into antigen specific T regulatory cells (Tregs), which can re-establish tolerance against auto/allo-antigens in the long term. Despite their promise, tolDCs have not found clinical success. Most strategies seek to generate tolDCs ex vivo by differentiating naïve dendritic cells (DCs) with immunosuppressive agents. Recently, we developed a tolDC generation strategy, which we call Push/Pull Immunomodulation (PPI). In PPI, DCs are treated with combinations of toll-like-receptor (TLR) agonists and immunomodulatory agents, which generate more robust, Treg-inducing tolDCs than previous strategies. Here, we seek to identify more potent and clinically viable PPI formulations using data from a high-throughput screening project. Methods: Over 40,000 combinations of pathogen-associated molecular patterns (PAMPs) and immunomodulatory small molecules were screened using a modified murine macrophage line, RAW dual cells, to observe the effect of these combinations on two major immune regulatory transcription factors, NF-κB and IRF. Combinations were further screened for inflammatory cytokine activity using a human monocyte cell line, THP-1, then on murine DCs. Leading candidates were co-cultured with T cells to assess antigen specific T cell responses. Results: From this data, we identified 355 combinations that showed low or moderate IRF activity, low NF-κB activity, low inflammatory cytokine generation and good viability: all hallmarks of tolerogenic potential. We further screened these 355 combinations using bone marrow derived DCs (BMDCs) and identified 10 combinations that demonstrated high IL-10 (tolerogenic) and low TNF-α (inflammatory) secretion. After further optimizing these combinations, we identified two combinations that generate robust tolDCs from BMDCs ex vivo. We further show that these PPI-tolDCs can also generate antigen specific Tregs but do not increase overall Treg populations. Discussion: These second-generation PPI formulations have significant potential to generate robust tolDCs and strong antigen specific Tregs.

Cell-targeted vaccines: implications for adaptive immunity.

Recent advancements in immunology and chemistry have facilitated advancements in targeted vaccine technology. Targeting specific cell types, tissue locations, or receptors can allow for modulation of the adaptive immune response to vaccines. This review provides an overview of cellular targets of vaccines, suggests methods of targeting and downstream effects on immune responses, and summarizes general trends in the literature. Understanding the relationships between vaccine targets and subsequent adaptive immune responses is critical for effective vaccine design. This knowledge could facilitate design of more effective, disease-specialized vaccines.

Ubiquitylation of Drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors.

Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Drosophila 26S proteasome revealed that the site of ubiquitylation is the C-terminal cluster of lysines, which is conserved in higher eukaryotes. Extraproteasomal p54 was extensively multiubiquitylated, but only very modest modification was detected in the proteasome-assembled subunit. Ubiquitylation of p54 seriously jeopardizes one of its most important functions, i.e., the interaction of its ubiquitin-interacting motifs with the ubiquitin-like domain of Dsk2 and Rad23 extraproteasomal polyubiquitin receptors. This modification of p54 supports the previous notion that p54 is a shuttling subunit of the 26S proteasome with a specific extraproteasomal function. This assumption is supported by the observation that, while transgenic p54 can fully rescue the lethal phenotype of the Δp54 null mutation, its derivative from which the cluster of conserved lysines is deleted shifts the lethality from the early pupa to pharate adult stage but cannot rescue the Δp54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa-adult transition.

CalpB modulates border cell migration in Drosophila egg chambers.

BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

Robust tolerogenic dendritic cells via push/pull pairing of toll-like-receptor agonists and immunomodulators reduces EAE.

A failure of central immune tolerance driven by autoantigen specific T regulatory (Treg) cells is a major cause of many autoimmune diseases. Restoration of proper autoantigen Treg specific response holds promise as a highly effective, long-term therapy for a wide variety of autoimmune diseases. Generating autoantigen specific Tregs remains a challenge due to the non-specific nature of most tolerizing agents and the complexities of generating Tregs in vivo. Here we show a new push/pull method for inducing antigen-specific Treg tolerance via induction of tolerogenic dendritic cells (tolDCs). We identified a combination of three tolerogenic drugs, dexamethasone, simvastatin and SC-514, which when used in combination with toll-like-receptor (TLR) agonists induces an active tolDC phenotype. When the tolerogenic combination was packaged into a liposome with a model antigen such as ovalbumin (OVA), these tolDCs induce differentiation of OVA specific Tregs both ex vivo and in vivo. We examined the tolerizing potential of the combination in an experimental autoimmune encephalomyelitis (EAE) disease model. Given the antigen specificity of this technique, this paper presents an attractive preclinical autoimmune therapy.

Fundamental Limit of Selectivity in Photocatalytic Denitrification over Titania.

Although photocatalytic decomposition of NO (deNO) into N2 and O2 is low-cost and non-polluting, it has a low NO conversion efficiency. Establishing the activity and selectivity trend among active sites is an important base to explore and improve the deNO processes. Because the experimental performances are determined by the reaction rate, it is worthwhile to investigate the kinetic limiting steps calculated by comparative microkinetic modeling. We found that, without illumination, N2 production is inactive over various TiO2 surfaces/sites, but photogenerated holes can break the scaling relation of the dark condition by weakening O2* adsorption, leading to a significant increase in deNO activity on defective titania surfaces. However, the low N2 selectivity can be attributed to the small strength of N2O adsorption. In contrast, the N2 selectivity is enhanced in Ti-modified zeolite because of a stronger N2O* adsorption. We demonstrate here that the reaction phase diagram analysis can clearly establish a global picture of reaction activity and selectivity over various catalytic sites. In combination with microkinetic modeling, it can effectively determine the kinetic limits, providing insights to improve the design of photocatalysts.

Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses.

SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Here, using a genetically non-redundant Drosophila model, we show that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. We find that accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response.Abbreviations: Atg8a: Autophagy-related 8a; cnc: cap-n-collar; IFM: indirect flight muscle; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1: Phox and Bem1; ref(2)P: refractory to sigma P; SAR: selective autophagy receptor; UBA: ubiquitin-associated.

Usp5, Usp34, and Otu1 deubiquitylases mediate DNA repair in Drosophila melanogaster.

Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, we applied the direct repeat (DR)-white reporter system with which we induced site-specific DSBs and affirmed the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways using Drosophila. Our results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process.

Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses.

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of "first responders" (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and T cell responses in vivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.