Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses.

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.

Electrochemical and chemical cycle for high-efficiency decoupled water splitting in a near-neutral electrolyte.

Green hydrogen produced by water splitting using renewable electricity is essential to achieve net-zero carbon emissions. Present water electrolysis technologies are uncompetitive with low-cost grey hydrogen produced from fossil fuels, limiting their scale-up potential. Disruptive processes that decouple the hydrogen and oxygen evolution reactions and produce them in separate cells or different stages emerge as a prospective route to reduce system cost by enabling operation without expensive membranes and sealing components. Some of these processes divide the hydrogen or oxygen evolution reactions into electrochemical and chemical sub-reactions, enabling them to achieve high efficiency. However, high efficiency has been demonstrated only in a batch process with thermal swings that present operational challenges. This work introduces a breakthrough process that produces hydrogen and oxygen in separate cells and supports continuous operation in a membraneless system. We demonstrate high faradaic and electrolytic efficiency and high rate operation in a near-neutral electrolyte of NaBr in water, whereby bromide is electro-oxidized to bromate concurrent with hydrogen evolution in one cell, and bromate is chemically reduced to bromide in a catalytic reaction that evolves oxygen in another cell. This process may lead the way to high-efficiency membraneless water electrolysis that overcomes the limitations of century-old membrane electrolysis.

Protein Engineering: Advances in Phage Display for Basic Science and Medical Research.

Functional Protein Engineering became the hallmark in biomolecule manipulation in the new millennium, building on and surpassing the underlying structural DNA manipulation and recombination techniques developed and employed in the last decades of 20th century. Because of their prominence in almost all biological processes, proteins represent extremely important targets for engineering enhanced or altered properties that can lead to improvements exploitable in healthcare, medicine, research, biotechnology, and industry. Synthetic protein structures and functions can now be designed on a computer and/or evolved using molecular display or directed evolution methods in the laboratory. This review will focus on the recent trends in protein engineering and the impact of this technology on recent progress in science, cancer- and immunotherapies, with the emphasis on the current achievements in basic protein research using synthetic antibody (sABs) produced by phage display pipeline in the Kossiakoff laboratory at the University of Chicago (KossLab). Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.

Structural basis for the recognition of transiently structured AU-rich elements by Roquin.

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.

The Association between the Alcohol Biomarker Phosphatidylethanol (PEth) and Self-Reported Alcohol Consumption among Russian and Norwegian Medical Patients.

AIMS: Valid measures to identify harmful alcohol use are important. Alcohol Use Disorders Identification Test (AUDIT) is a validated questionnaire used to self-report harmful drinking in several cultures and settings. Phosphatidylethanol 16:0/18:1 (PEth) is a direct alcohol biomarker measuring alcohol consumption levels. The aim of this study was to investigate how PEth levels correlate with AUDIT-QF and weekly grams of alcohol consumed among patients in two urban hospitals. In addition, we wanted to investigate the predictive value of PEth in identifying harmful alcohol use as defined by AUDIT-QF and weekly grams of alcohol cutoffs. METHODS: A cross-sectional study comprising acute medically ill patients with measurable PEth levels (≥0.030 µM) admitted to two urban hospitals in Oslo, Norway (N = 931) and Moscow, Russia (N = 953) was conducted using PEth concentrations in whole blood, sociodemographic data and AUDIT-QF questionnaires. RESULTS: PEth levels from patients with measurable PEth were found to be positively correlated with AUDIT-QF scores, with PEth cutpoints of 0.128 µM (Oslo) and 0.270 µM (Moscow) providing optimal discrimination for harmful alcohol use defined by AUDIT-QF (the difference between cities probably reflecting different national drinking patterns in QF). When converting AUDIT-QF into weekly grams of alcohol consumed, the predictive value of PEth improved, with optimal PEth cutpoints of 0.327 (Oslo) and 0.396 (Moscow) µM discriminating between harmful and non-harmful alcohol use as defined in grams (≥350 grams/week). CONCLUSIONS: By using PEth levels and converting AUDIT-QF into weekly grams of alcohol it was possible to get an improved rapid and sensitive determination of harmful alcohol use among hospitalized patients.

Structures and Chemical Bonding in Antimony(III) Bromide Complexes with Pyridine.

Weakly or "partially" bonded molecules are an important link between the chemical and van der Waals interactions. Molecular structures of six new SbBr3 -Py complexes in the solid state have been determined by single-crystal X-ray diffraction analysis. In all complexes all Sb atoms adopt a pseudo-octahedral coordination geometry which is completed by additional Sb⋅⋅⋅Br contacts shorter than the sum of the van der Waals radii, with Br-Sb⋅⋅⋅Br angles close to 180°. To reveal the nature of Sb-Br and Sb-N interactions, the DFT calculations were performed followed by the analysis of the electrostatic potentials, the orbital interactions and the topological analysis. Based on Natural Bond Orbital (NBO) analysis, the Sb-Br interactions range from the covalent bonds to the pnictogen bonds. A simple structural parameter, non-covalence criterion (NCC) is defined as a ratio of the atom-atom distance to the linear combination of sums of covalent and van der Waals radii. NCC correlates with E(2) values for Sb-N, Sb-Cl and Sb-Br bonds, and appears to be useful criterion for a preliminary evaluation of the bonding situation.

Complex beryllium amidoboranes: Structures, stability, and evaluation of their potential as hydrogen storage materials.

Complex beryllium amidoboranes Mx [Be(NH2 BH3 )x+2 ] (M = Li-Cs, x = 1,2) have been computationally studied at M06-2X/def2-TZVPPD//B3LYP/def2-TZVPPD level of theory. Compounds are predicted to be stable at room temperature but release H2 on heating. Agostic Be…HB bonds play an important role in stabilization of oligomeric beryllium imidoboranes. Polymeric imidoborane, hydrogen, and ammonia are expected as major thermal decomposition products of complex beryllium amidoboranes. Ammonia evolution is predicted to proceed at slightly higher temperatures than hydrogen evolution. Based on thermodynamic analysis, Li[Be(NH2 BH3 )3 ] and Li2 [Be(NH2 BH3 )4 ] are the most perspective synthetic targets. Synthetic approaches to these potentially efficient hydrogen storage materials have been proposed. © 2016 Wiley Periodicals, Inc.

Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase.

Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

Competitive reaction pathways for the gas-phase reactivity of [Me2 AlNH2 ]3.

Reaction energy profiles for [Me2 AlNH2 ]3 have been computationally explored by using density functional theory. Both intra- and intermolecular methane elimination reactions, as well as Al-N bond-breaking pathways, were considered. The results show that the energy required for Al-N bond breaking in cyclic [Me2 AlNH2 ]3 is of the same order of magnitude as the activation energies for the first (limiting) step of methane elimination (for both mono- and bimolecular mechanisms). Thus, dissociative and associative reaction pathways are competitive. Low-temperature/high-pressure conditions will favor the bimolecular pathway, whereas at high temperatures, either intramolecular methane elimination or Al-N bond-breaking dissociative pathways will be operational.

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides.

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O-helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

Do solid-state structures reflect Lewis acidity trends of heavier group 13 trihalides? Experimental and theoretical case study.

Lewis acidity trends of aluminum and gallium halides have been considered on the basis of joint X-ray and density functional theory studies. Structures of complexes of heavier group 13 element trihalides MX(3) (M = Al, Ga; X = Cl, Br, I) with monodentate nitrogen-containing donors Py, pip, and NEt(3) as well as the structure of the AlCl(3)·PPh(3) adduct have been established for the first time by X-ray diffraction studies. Extensive theoretical studies (B3LYP/TZVP level of theory) of structurally characterized complexes between MX(3) and nitrogen-, phosphorus-, arsenic-, and oxygen-containing donor ligands have allowed us to establish the Lewis acidity trends Al > Ga, Cl ≈ Br > I. Analysis of the experimental and theoretical results points out that the solid state masks the Lewis acidity trend of aluminum halides. The difference in the Al-N bond distances between AlCl(3)·D and AlBr(3)·D complexes in the gas phase is small, while in the condensed phase, shorter Al-N distances for AlBr(3)·D complexes are observed with 9-fluorenone, mdta, and NEt(3) donors. The model based on intermolecular (H···X) interactions in solid adducts is proposed to explain this phenomenon. Thus, the donor-acceptor bond distance in the solid complexes cannot always be used as a criterion of Lewis acidity.

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death.

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load.

In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of compartments. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2 × 10(2) to 4.0 × 10(6) molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8 × 10(3) to 1.2 × 10(7) molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at a dynamic range of 1.7 × 10(2) to 2.0 × 10(7) molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in other work by Kreutz et al.

Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.

X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase.

Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a 5-bp stem-3 nt loop hairpin structure. Here, we describe the 2.0-A resolution x-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the full-length vRNAP. The structure resembles a "fisted right hand" with Fingers, Palm and Thumb subdomains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with W129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription.

Effect of the Synthetic Method on the Properties of Ni-Based Hydrogen Oxidation Catalysts.

The latest progress in alkaline anion-exchange membranes has led to the expectation that less costly catalysts than those of the platinum-group metals may be used in anion-exchange membrane fuel cell devices. In this work, we compare structural properties and the catalytic activity for the hydrogen-oxidation reaction (HOR) for carbon-supported nanoparticles of Ni, Ni3Co, Ni3Cu, and Ni3Fe, synthesized by chemical and solvothermal reduction of metal precursors. The catalysts are well dispersed on the carbon support, with particle diameter in the order of 10 nm, and covered by a layer of oxides and hydroxides. The activity for the HOR was assessed by voltammetry in hydrogen-saturated aqueous solutions of 0.1 mol dm-1 KOH. A substantial activation by potential cycling of the pristine catalysts synthesized by solvothermal reduction is necessary before these become active for the HOR; in situ Raman spectroscopy shows that after activation the surface of the Ni/C, Ni3Fe, and Ni3Co catalysts is fully reduced at 0 V, whereas the surface of the Ni3Cu catalyst is not. The activation procedure had a smaller but negative impact on the catalysts synthesized by chemical reduction. After activation, the exchange-current densities normalized with respect to the ECSA (electrochemically active surface area) were approximately independent of composition but relatively high compared to catalysts of larger particle diameter.

Unusual molecular complexes of antimony fluoride dimers with acetonitrile and pyridine: structures and bonding.

The structures of two new molecular complexes of antimony pentafluoride with pyridine (Py) and acetonitrile (AN), SbF5·Py and Sb2F10·AN, and a molecular complex of antimony trifluoride Sb2F6·Py and its ionic derivative [HPy]+[Sb2F7]- in the solid state have been determined by single crystal X-ray structural analysis. The complexes Sb2F10·AN and Sb2F6·Py are the first structurally characterized compounds of dimeric antimony fluorides. To reveal the nature of bonding in the complexes and their stability, DFT computations of the electronic structure and thermodynamic characteristics were performed, in particular the analysis of the electrostatic potentials, the orbital interactions and the topology. The results indicate that the intermolecular Sb⋯F interactions can be described as a network of pnictogen bonds.

Nanoliter multiplex PCR arrays on a SlipChip.

The SlipChip platform was tested to perform high-throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high-throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays.

Structure and stability of M6N8 clusters (M = Si, Ge, Sn, Ti).

The structures and stabilities of the M(6)N(8) clusters (M = Si, Ge, Sn, Ti) have been theoretically studied at DFT and ab initio levels of theory. Two new isomers have been considered: cage-like molecules and propeller-like molecules. It is shown that only for M = Si are both isomers true minima on the potential energy surface. The thermodynamics of the dissociation process (1/6)M(6)N(8) --> (1/3)M(3)N(4) is discussed. For each M(3)N(4) molecule, four structures with different multiplicity are considered. The thermodynamic analysis shows that independently of the multiplicity of M(3)N(4) nitrides all M(6)N(8) clusters are stable in the gas phase in a wide temperature range and could be potential intermediates in chemical vapor deposition of the nitride materials.

Necessity of the target discrimination in the P300-based complex trial protocol test for concealed information.

The two most common types of ERP-based protocols to detect concealed information are the 3-stimulus protocol (3SP) and Complex Trial Protocol (CTP). Both protocols traditionally include presentation of a target (a stimulus with assigned significance requiring a unique behavioral response). The intention of the target presentation is forcing subjects to pay attention to all stimuli, especially to guilty knowledge stimuli, called probes. It was unclear though, how the presence of a targets influences probe recognition, and thus, the concealed information test (CIT) effect-the difference in P300 response to the probe and Irrelevant (crime-unrelated) stimuli. The question of target necessity was first raised in relation to the 3SP, and it was found that although omitting target stimuli reduced P300 amplitudes for all probe and irrelevant stimuli, the CIT effect was not reduced. The current study investigated how the presence or absence of the target/nontarget discrimination in the CTP affects the CIT effect, by comparing two CTP groups both with (T) and without (NT) the target/nontarget discrimination. The results demonstrated that this discrimination significantly increases the P300 effect. We found a greater P300 CIT effect in the T group than in the NT group, suggesting that for field use, it is better to retain the target discrimination in the CTP. CIT effects were also seen with P300 latency, but not reaction time.