Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Using advanced racial and ethnic identity demographics to improve surveillance of work-related conditions in an occupational clinic setting.

BACKGROUND: Although racial and ethnic identities are associated with a multitude of disparate medical outcomes, surveillance of these subpopulations in the occupational clinic setting could benefit enormously from a more detailed and nuanced recognition of racial and ethnic identity. METHODS: The research group designed a brief questionnaire to capture several dimensions of this identity and collected data from patients seen for work-related conditions in four occupational medicine clinics from May 2019 through March 2020. Responses were used to calculate the sensitivity and specificity of extant racial/ethnic identity data within our electronic health records system, and were compared to participants' self-reported industry and occupation, coded according to North American Industry Classification System and Standard Occupational Classification System listings. RESULTS: Our questionnaire permitted collection of data that defined our patients' specific racial/ethnic identity with far greater detail, identified patients with multiple ethnic identities, and elicited their preferred language. Response rate was excellent (94.2%, n = 773). Non-White participants frequently selected a racial/ethnic subcategory (78.1%-92.2%). Using our race/ethnicity data as a referent, the electronic health record (EHR) had a high specificity (>87.1%), widely variable sensitivity (11.8%-82.2%), and poorer response rates (75.1% for race, 82.5% for ethnicity, as compared to 93.8% with our questionnaire). Additional analyses revealed some industries and occupations disproportionately populated by patients of particular racial/ethnic identities. CONCLUSIONS: Our project demonstrates the usefulness of a questionnaire which more effectively identifies racial/ethnic subpopulations in an occupational medicine clinic, permitting far more detailed characterization of their occupations, industries, and diagnoses.

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as ß-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.

Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine.

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5-7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations.

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme ß-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with ß-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.

Experimental Simulation of Clinical Borderline Situations in Temporal Bone Specimens After Ossiculoplasty.

OBJECTIVES: One reason for insufficient hearing improvement with a distinct air-bone gap after ossiculoplasty with implantation of partial or total ossicular replacement prostheses can be the dislocation or minimal shifting of the prosthesis. The aim of this study was the simulation of common clinical borderline situations with minimal shifting of the prosthesis in temporal bone specimens after ossiculoplasty. It was furthermore the goal to identify these specific situations through imaging by cone beam computed tomography (cbCT) and direct visual inspection using the operation microscope. Additionally, the functional status was evaluated using laser-Doppler vibrometry (LDV). DESIGN: We used a total of four temporal bone specimens for this study. A reconstruction with a partial ossicular replacement prostheses was performed in three specimens and with a total ossicular replacement prostheses in one specimen, with good initial acoustic properties. Subsequently, one specific type of prosthesis failure was simulated in each specimen, respectively, by minimally shifting, tilting, or bending the prostheses from their initial positions. These changes were introduced step-by-step until a borderline situation just short of complete acoustic decoupling was reached. Each step was examined using both LDV and cbCT and observed through the operation microscope. RESULTS: LDV was able to quantify the mechanic function of the ossicular chain after most of the manipulation steps by demonstrating the effect of any shifting of the prosthesis on the middle ear transfer function. However, in some situations, the middle ear transfer function was better with a visually more advanced failure of the prosthesis. In addition, cbCT showed most of the steps with excellent resolution and was able to delineate changes in soft tissue (e.g., cartilage covering). CONCLUSION: cbCT seems to be a promising imaging technique for middle ear problems. As cbCT and LDV exhibited slightly different advantages and disadvantages regarding the demonstration of borderline situations, the combination of both techniques allowed for a more precise evaluation of middle ear reconstructions. Knowledge of the specific characteristics of these methods and their possible combination might help otologists and otosurgeons to refine indications for revision surgery and improve their personal patient counseling.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

Right ventricular function assessed by tissue Doppler echocardiography in older subjects without evidence for structural cardiac disease.

The aim of our study was to obtain right ventricular (RV) tissue Doppler imaging (TDI) data in older subjects (n = 95, mean age: 74.5 ± 4.6 years) without evidence of hemodynamically significant structural heart disease recruited from a large population-based cohort (ActiFE-Ulm study). Our data indicate that aging may be accompanied by decreasing RV diastolic function and at most little alterations of RV systolic function. Mean values of all parameters were still within the guideline-suggested reference range with most of them closer to the abnormality thresholds. On an individual basis, respective thresholds were also exceeded in some subjects (almost all parameters <20 %) despite the absence of evidence for structural cardiac disease. RV-TDI is a feasible method for evaluation of RV systolic and diastolic function also in a geriatric population as sufficient TDI data was obtainable in the majority of our participants. Published reference values also seem to be mostly suitable although among older subjects, presumed pathological measures might still be compatible with physiological age-related alterations. Therefore, they always have to be interpreted across the clinical context and in relation to other parameters of morphology and function obtained by other ultrasound imaging techniques (M-mode, B-mode, etc.) in the context of echocardiographic evaluation of the right heart.

The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals.

SIRT6 is a NAD(+)-dependent deacetylase that modulates chromatin structure and safeguards genomic stability. Until now, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules, interact with various stress granule components and induce their assembly. Loss of SIRT6 or inhibition of its catalytic activity in mouse embryonic fibroblasts impairs stress granule formation and delays disassembly during recovery, whereas deficiency of SIR-2.4 diminishes maintenance of P granules and decreases survival of C. elegans under stress conditions. Our findings uncover a novel, evolutionary conserved function of SIRT6 in the maintenance of stress granules in response to stress.

Real-time and three-dimensional MRI for diagnosis of pharyngoceles.

In the evaluation of patients with local pathologic dilatation inside the upper airway a pressure-related testing seems important for understanding its pathophysiology and for developing a concept of intra-individually adjusted therapy. Commonly used diagnostic techniques like endoscopy or medical imaging including ultrasound, barium swallow or computer-assisted tomography (CT) have shown limitations either in evaluating a dynamic process or assessing the entirety of cervical structures. This article presents a case report of a professional trumpet player with bilateral pharyngoceles, introducing real-time and three-dimensional (3D) MRI as a helpful tool in the diagnosis of pressure dependent pathologies in the upper airway. With the use of MRI the complete sub- and supraglottic airway can be viewed simultaneously, avoiding the distortion which can occur with endoscopy. Thus, it was possible to evaluate the pharyngoceles pressure-related pathophysiology, from which a successful therapy could be conceived which included modifying the musician's blowing technique.

Analysis of selected and designed chimeric D- and L-α-helix assemblies.

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V.

BACKGROUND: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. RESULTS: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. CONCLUSIONS: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed.

Temporally constrained respiratory gating improves continuously moving table MRI during free breathing.

PURPOSE: To evaluate a novel breathing motion correction algorithm for continuously moving table magnetic resonance imaging (CMT-MRI) that optimizes motion consistency in a fixed time span. MATERIALS AND METHODS: In 22 patients CMT-MRI was performed during free breathing. During a preparatory phase (constant) or continuously during the scan (adaptive) gating thresholds were computed from breathing states that should allow for motion consistent k-space sampling. After data from a first k-space traversal was acquired irrespective of breathing motion, subsequently k-space lines with discordant breathing states were reacquired below the gating threshold. Time constraints of CMT-MRI were respected, because a fixed time was allocated for reacquisition. Image quality and lesion depiction were evaluated on images reconstructed from the first traversal and motion-corrected images. RESULTS: Compared to constant thresholds, gating with adaptive thresholds led to a higher number of reacquired k-space lines (60.1%/41.7%) and a larger fraction of motion consistent final k-space lines (96.6%/78.8%). Adaptive gating induced a significant increase in image quality for all regions affected by breathing motion. Only one of 22 lesions was not depicted on the adaptively corrected images, whereas 15 were readily appreciable. CONCLUSION: Temporally constrained respiratory gating with adaptive thresholds allows for fully sampled, motion-corrected CMT-MRI acquisitions during free breathing.

Orthogonal translation with 5-cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding.

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile-containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi-rational approach to engineer a tyrosyl-tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5-cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well-established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW-specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin-binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non-invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

Revisionary bariatric surgery: indications and outcome of 100 consecutive operations at a single center.

BACKGROUND: A growing number of revisionary and secondary bariatric operations have been performed in recent years, with the number of operations doubling each year at the authors' center. Diagnostics, indications, and most revisionary operations should be performed by an experienced bariatric surgeon. This study was undertaken to evaluate indications and outcomes of revisionary bariatric operations at a specialized center. METHODS: At the Centre of Obesity and Metabolic Surgery (University of Freiburg, Germany), 100 consecutive revisionary bariatric operations performed between March 2007 and September 2009 were analyzed concerning indications and outcomes. RESULTS: Only 9 of the 100 revisions were due to early complications (<30 days after the primary operation). The indication for most revisions was poor weight loss (n = 55). A mean body mass index reduction of 10 points could be achieved in 1 year, which equals a 56% excess weight loss (EWL). No significant difference in weight reduction between restrictive and malabsorptive revisions was observed. Revisions due to implant-related problems also were frequent (n = 25). Laparoscopic revision was possible in 95% of the cases. CONCLUSION: Insufficient weight loss is the most frequent indication for revisionary bariatric surgery. The surgery can be performed laparoscopically in most cases, and a significant EWL (> 50%) can be achieved in 1 year if the right revisionary procedure is chosen.

Peripheral vessel scout imaging based on continuously moving table acquisition of projection data.

A fast and spatially seamless peripheral vessel scout is desirable for subsequent planning of magnetic resonance (MR) angiography. We implemented a continuously moving table sequence providing projection data with time-of-flight contrast of the entire lower extremities in less than 2 minutes. Variation of arterial signal during the cardiac cycle and autocorrelation were exploited to enhance vessel-to-background contrast. Subjective image analysis revealed excellent vessel depiction, indicating that the proposed scout allows for seamless expedited visualization of major arterial structures.

Engineered bacterial host for genetic encoding of physiologically stable protein nitration.

Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology.

Halogenation of tyrosine perturbs large-scale protein self-organization.

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools.