CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase.

BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Complex Portal 2018: extended content and enhanced visualization tools for macromolecular complexes.

The Complex Portal (www.ebi.ac.uk/complexportal) is a manually curated, encyclopaedic database that collates and summarizes information on stable, macromolecular complexes of known function. It captures complex composition, topology and function and links out to a large range of domain-specific resources that hold more detailed data, such as PDB or Reactome. We have made several significant improvements since our last update, including improving compliance to the FAIR data principles by providing complex-specific, stable identifiers that include versioning. Protein complexes are now available from 20 species for download in standards-compliant formats such as PSI-XML, MI-JSON and ComplexTAB or can be accessed via an improved REST API. A component-based JS front-end framework has been implemented to drive a new website and this has allowed the use of APIs from linked services to import and visualize information such as the 3D structure of protein complexes, its role in reactions and pathways and the co-expression of complex components in the tissues of multi-cellular organisms. A first draft of the complete complexome of Saccharomyces cerevisiae is now available to browse and download.

NeuBtracker-imaging neurobehavioral dynamics in freely behaving fish.

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

ITIH5 and ECRG4 DNA Methylation Biomarker Test (EI-BLA) for Urine-Based Non-Invasive Detection of Bladder Cancer.

Bladder cancer is one of the more common malignancies in humans and the most expensive tumor for treating in the Unites States (US) and Europe due to the need for lifelong surveillance. Non-invasive tests approved by the FDA have not been widely adopted in routine diagnosis so far. Therefore, we aimed to characterize the two putative tumor suppressor genes ECRG4 and ITIH5 as novel urinary DNA methylation biomarkers that are suitable for non-invasive detection of bladder cancer. While assessing the analytical performance, a spiking experiment was performed by determining the limit of RT112 tumor cell detection (range: 100-10,000 cells) in the urine of healthy donors in dependency of the processing protocols of the RWTH cBMB. Clinically, urine sediments of 474 patients were analyzed by using quantitative methylation-specific PCR (qMSP) and Methylation Sensitive Restriction Enzyme (MSRE) qPCR techniques. Overall, ECRG4-ITIH5 showed a sensitivity of 64% to 70% with a specificity ranging between 80% and 92%, i.e., discriminating healthy, benign lesions, and/or inflammatory diseases from bladder tumors. When comparing single biomarkers, ECRG4 achieved a sensitivity of 73%, which was increased by combination with the known biomarker candidate NID2 up to 76% at a specificity of 97%. Hence, ITIH5 and, in particular, ECRG4 might be promising candidates for further optimizing current bladder cancer biomarker panels and platforms.

Quantitative imaging of 33P in plant materials using 14C polymer references.

Phosphorus (P) research still lacks techniques for rapid imaging of P use and allocation in different soil, sediment, and biological systems in a quantitative manner. In this study, we describe a time-saving and cost-efficient digital autoradiographic method for in situ quantitative imaging of 33P radioisotopes in plant materials. Our method combines autoradiography of the radiotracer applications with additions of commercially available 14C polymer references to obtain 33P activities in a quantitative manner up to 2000 Bq cm-2. Our data show that linear standard regressions for both radioisotopes are obtained, allowing the establishment of photostimulated luminescence equivalence between both radioisotopes with a factor of 9.73. Validating experiments revealed a good agreement between the calculated and applied 33P activity (R2 = 0.96). This finding was also valid for the co-exposure of 14C polymer references and 33P radioisotope specific activities in excised plant leaves for both maize (R2 = 0.99) and wheat (R2 = 0.99). The outlined autoradiographic quantification procedure retrieved 100% ± 12% of the 33P activity in the plant leaves, irrespective of plant tissue density. The simplicity of this methodology opens up new perspectives for fast quantitative imaging of 33P in biological systems and likely, thus, also for other environmental compartments.

Advancing Surgical Vision with Fluorescence Imaging.

Surgical success depends on the accuracy with which disease and vital tissue can be intraoperatively detected. However, the dominant visualization approach, i.e., human vision, does not see under the tissue surface and operates on low contrast between sites of disease, such as cancer, and the surrounding tissue. Intraoperative fluorescence imaging is emerging as a highly effective method to improve surgical vision and offers the potential to be intergrated seamlessly into the normal workflow of the operating room without causing disruption or undue delay. We review and compare two critical fluorescence imaging directions: one that uses nonspecific fluorescence dyes, addressing tissue perfusion and viability, and one that uses targeted agents, interrogating pathophysiological features of disease. These two approaches present detection sensitivity challenges that may differ by orders of magnitude and require different detection strategies. Nevertheless, fluorescence imaging provides the surgeon with previously unavailable real-time feedback that improves surgical precision and can become essential for interventional decision-making.

Tools and data services registry: a community effort to document bioinformatics resources.

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand.Here we present a community-driven curation effort, supported by ELIXIR-the European infrastructure for biological information-that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners.As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools.

OLS Client and OLS Dialog: Open Source Tools to Annotate Public Omics Datasets.

The availability of user-friendly software to annotate biological datasets and experimental details is becoming essential in data management practices, both in local storage systems and in public databases. The Ontology Lookup Service (OLS, http://www.ebi.ac.uk/ols) is a popular centralized service to query, browse and navigate biomedical ontologies and controlled vocabularies. Recently, the OLS framework has been completely redeveloped (version 3.0), including enhancements in the data model, like the added support for Web Ontology Language based ontologies, among many other improvements. However, the new OLS is not backwards compatible and new software tools are needed to enable access to this widely used framework now that the previous version is no longer available. We here present the OLS Client as a free, open-source Java library to retrieve information from the new version of the OLS. It enables rapid tool creation by providing a robust, pluggable programming interface and common data model to programmatically access the OLS. The library has already been integrated and is routinely used by several bioinformatics resources and related data annotation tools. Secondly, we also introduce an updated version of the OLS Dialog (version 2.0), a Java graphical user interface that can be easily plugged into Java desktop applications to access the OLS. The software and related documentation are freely available at https://github.com/PRIDE-Utilities/ols-client and https://github.com/PRIDE-Toolsuite/ols-dialog.

Mechanistic Insights into Cyclic Peptide Generation by DnaE Split-Inteins through Quantitative and Structural Investigation.

Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction.

Helicobacter pylori γ-glutamyltranspeptidase impairs T-lymphocyte function by compromising metabolic adaption through inhibition of cMyc and IRF4 expression.

Helicobacter pylori (H. pylori) is a human-specific pathogen that has evolved to cope with the immune response elicited against the infection. We previously reported that H. pylori γ-glutamyltranspeptidase (gGT) impairs T-lymphocyte proliferation and thus might act as immune regulatory factor. In this study, we analysed the underlying mechanism and its implications for H. pylori persistence. We found that H. pylori gGT compromised T-cell proliferation, activation and effector cytokine expression by specifically depriving the extracellular space of glutamine. When assessing signalling cascades and transcription factors affected by H. pylori gGT, we found that expression of cMyc and IRF4, both required for metabolic adaptation of T-lymphocytes, was highly sensitive to extracellular glutamine levels and downregulated upon gGT treatment. Moreover, we could confirm decreased IRF4 expression in T-lymphocytes infiltrating the stomach of infected individuals. Thus, our results suggest that H. pylori gGT-mediated glutamine deprivation in the gastric mucosa may suppress T-cell function thereby contributing to bacterial persistence.

Subclass-specific labeling of protein-reactive natural products with customized nucleophilic probes.

Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, ß-lactams, and epoxides that irreversibly inhibit essential enzymes based on their catalytic mechanism. In the search for novel bioactive molecules, current methods are challenged by the frequent rediscovery of known chemical entities. Herein small nucleophilic probes that attack electrophilic natural products and enhance their detection by HPLC-UV and HPLC-MS are introduced. A screen of diverse probe designs revealed one compound with a desired selectivity for epoxide- and maleimide-based antibiotics. Correspondingly, the natural products showdomycin and phosphomycin could be selectively targeted in extracts of their natural producing organism, in which the probe-modified molecules exhibited superior retention and MS detection relative to their unmodified counterparts. This method may thus help to discover small, electrophilic molecules that might otherwise easily elude detection in complex samples.

Structural, Biochemical, and Computational Studies Reveal the Mechanism of Selective Aldehyde Dehydrogenase 1A1 Inhibition by Cytotoxic Duocarmycin Analogues.

Analogues of the natural product duocarmycin bearing an indole moiety were shown to bind aldehyde dehydrogenase 1A1 (ALDH1A1) in addition to DNA, while derivatives without the indole solely addressed the ALDH1A1 protein. The molecular mechanism of selective ALDH1A1 inhibition by duocarmycin analogues was unraveled through cocrystallization, mutational studies, and molecular dynamics simulations. The structure of the complex shows the compound embedded in a hydrophobic pocket, where it is stabilized by several crucial π-stacking and van der Waals interactions. This binding mode positions the cyclopropyl electrophile for nucleophilic attack by the noncatalytic residue Cys302, thereby resulting in covalent attachment, steric occlusion of the active site, and inhibition of catalysis. The selectivity of duocarmycin analogues for ALDH1A1 is unique, since only minor alterations in the sequence of closely related protein isoforms restrict compound accessibility.

A subfamily of bacterial ribokinases utilizes a hemithioacetal for pyridoxal phosphate salvage.

Pyridoxal 5'-phosphate (PLP) is the active vitamer of vitamin B6 and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4'-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5'-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs.

Fluorescence-aided tomographic imaging of synovitis in the human finger.

PURPOSE: To propose and evaluate indocyanine green (ICG)-enhanced tomographic optical imaging for detection and characterization of synovitis in affected finger joints of patients with rheumatoid arthritis and differentiation from healthy joints in comparison to 3-T magnetic resonance (MR) imaging. MATERIALS AND METHODS: This prospective pilot study was approved by the institutional ethics committee. Six arthritic proximal interphalangeal (PIP) joints in six patients (five women and one man; mean age ± standard deviation, 62.6 years ± 13.3) with clinically determined rheumatoid arthritis and six healthy PIP joints from six volunteers (four women and two men; mean age, 41.5 years ± 20.2) were examined with an ICG-enhanced fluorescence molecular tomography (FMT) system and 3-T MR imaging as the standard of reference. The degree of inflammation was graded semiquantitatively on a four-point ordinate scale according to the Outcome Measures in Rheumatology Clinical Trials Rheumatoid Arthritis MR Imaging Score, or OMERACT RAMRIS. FMT reconstructions were coregistered with the MR images. Groups were compared by using a two-sided t test, and a weighted κ coefficient was used for comparing FMT and MR imaging semiquantitative scores, as well as assessing intrareader agreement. RESULTS: FMT was used to detect synovitis in all arthritic joints. The reconstructed FMT signal correlated with MR imaging findings in intensity and spatial, transverse profile. Semiquantitative scoring of FMT correlated well with MR imaging findings (weighted κ coefficient = 0.90). The reconstructed quantitative FMT signal, denoting synovial hyperperfusion, was used to differentiate between synovitis and healthy joints (healthy joints, 1.25 ± 0.59; arthritic joints, 3.13 ± 1.03; P < .001). CONCLUSION: FMT enhanced with ICG provided depth-resolved imaging of synovitis in PIP joints. FMT may help detect synovitis in patients with rheumatoid arthritis.

Near-infrared fluorescence cholangiopancreatoscopy: initial clinical feasibility results.

BACKGROUND: The recent clinical propagation of targeted fluorescence agents brings a promising alternative in endoscopy by complementing visual disease detection with molecular biomarkers. OBJECTIVE: Development of near-infrared (NIR) fluorescence cholangiopancreatoscopy in real-time and validation of its clinical use. DESIGN: Feasibility study. SETTING: Tertiary referral center at a large university hospital. PATIENTS: Patients with pancreatic and biliary diseases. INTERVENTIONS: Routine cholangiopancreatoscopy with additional wide-field NIR fluorescence imaging. MAIN OUTCOME MEASUREMENTS: We adapted a miniature cholangioscope for real-time concurrent wide-field color and NIR fluorescence imaging. Illumination is provided through a custom-designed fiber bundle, and the acquired images are relayed via a dichroic beam splitter to 2 charge-coupled devices for simultaneous measurement. We characterize the sensitivity and resolution and demonstrate the clinical feasibility by detecting indocyanine green localization in 2 patients. RESULTS: A spatial optical resolution of approximately 50 µm was achieved, and fluorescent dye concentrations of 17.3 nM could be detected. Elevated fluorescence signals were detected during clinical measurements, and biopsy specimens confirmed the presence of malignancy in both patients. LIMITATIONS: Feasibility study, limited number of patients. CONCLUSIONS: The results demonstrate that real-time wide-field fluorescence detection in the NIR range is possible in humans by using adapted endoscopes. The feasibility of detecting indocyanine green in the pancreatobiliary ducts is verified, suggesting that cancer screening at a molecular level might play an increasingly important role in the future.

Structure Elucidation of Aroma-Active Bicyclic Benzofuran Derivatives Formed by Cystostereum murrayi.

Among the monoterpenoid aroma compounds formed by the basidiomycete Cystostereum murrayi are highly potent bicyclic benzofuran derivatives. In addition to the dill ethers previously described in a few fungi, two stereoisomers of the rare 3,6-dimethyl-3a,4,5,6,7,7a-hexahydro-3H-1-benzofuran-2-one (1a and 2c), also known as dihydromenthofurolactones, and a C3-unsaturated analogue (3a) are formed by C. murrayi. The analysis of synthesized reference standards of the lactones allowed an unambiguous assignment of the stereoisomers formed by the fungus. Despite a similar structure, two key differences in the stereochemistry of the lactones and dill ethers emerged. The analysis of submerged cultures further revealed the formation of additional, so far unknown, fungal terpenoids, including limonen-10-ol (7) and the corresponding aldehyde limonen-10-al (8). Analysis of chiral terpenoids as well as supplementation studies, including stable isotope-labeled compounds, indicated independent biogenesis pathways for dill ethers and lactones.

Short-wave infrared fluorescence imaging of near-infrared dyes with robust end-tail emission using a small-animal imaging device.

Commercially available near-infrared (NIR) dyes, including indocyanine green (ICG), display an end-tail of the fluorescence emission spectrum detectable in the short-wave infrared (SWIR) window. Imaging methods based on the second NIR spectral region (1,000-1,700 nm) are gaining interest within the biomedical imaging community due to minimal autofluorescence and scattering, allowing higher spatial resolution and depth sensitivity. Using a SWIR fluorescence imaging device, the properties of ICG vs. heptamethine cyanine dyes with emission >800 nm were evaluated using tissue-simulating phantoms and animal experiments. In this study, we tested the hypothesis that an increased rigidity of the heptamethine chain may increase the SWIR imaging performance due to the bathochromic shift of the emission spectrum. Fluorescence SWIR imaging of capillary plastic tubes filled with dyes was followed by experiments on healthy animals in which a time series of fluorescence hindlimb images were analyzed. Our findings suggest that higher spatial resolution can be achieved even at greater depths (>5 mm) or longer wavelengths (>1,100 nm), in both tissue phantoms and animals, opening the possibility to translate the SWIR prototype toward clinical application.