Significance The αβ T-cell receptor (TCR) on mammalian T lymphocytes recognizes intracellular pathogens to afford protective immunity. Detection of various foreign peptides bound to MHC molecules as ...TCR ligands occurs during immune surveillance where mechanical forces are generated through cell movement. Using single-molecule optical tweezer assays, we show with isolated and complete receptors on single T cells that both sensitivity and specificity of the biological T-lymphocyte response is dependent upon force-based interactions. Our work demonstrates a catch-and-release αβTCR structural conversion correlating with ligand potency wherein a strongly binding/compact state transitions to a weakly binding/extended state. An allosteric mechanism controls bond strength and lifetime, supporting a model in which quaternary αβTCR subunit associations regulate TCR recognition under load.
The αβ T-cell receptor (TCR) on each T lymphocyte mediates exquisite specificity for a particular foreign peptide bound to a major histocompatibility complex molecule (pMHC) displayed on the surface of altered cells. This recognition stimulates protection in the mammalian host against intracellular pathogens, including viruses, and involves piconewton forces that accompany pMHC ligation. Physical forces are generated by T-lymphocyte movement during immune surveillance as well as by cytoskeletal rearrangements at the immunological synapse following cessation of cell migration. The mechanistic explanation for how TCRs distinguish between foreign and self-peptides bound to a given MHC molecule is unclear: peptide residues themselves comprise few of the TCR contacts on the pMHC, and pathogen-derived peptides are scant among myriad self-peptides bound to the same MHC class arrayed on infected cells. Using optical tweezers and DNA tether spacer technology that permit piconewton force application and nanometer scale precision, we have determined how bioforces relate to self versus nonself discrimination. Single-molecule analyses involving isolated αβ-heterodimers as well as complete TCR complexes on T lymphocytes reveal that the FG loop in the β-subunit constant domain allosterically controls both the variable domain module’s catch bond lifetime and peptide discrimination via force-driven conformational transition. In contrast to integrins, the TCR interrogates its ligand via a strong force-loaded state with release through a weakened, extended state. Our work defines a key element of TCR mechanotransduction, explaining why the FG loop structure evolved for adaptive immunity in αβ but not γδTCRs or immunoglobulins.
Abstract
The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of ...multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described, providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment.
Bacterial pathogens bearing capsular polysaccharides identical to mammalian glycans benefit from an additional level of protection from host immune response.
Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, ...and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T p.Leu412Phe), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism.
The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin ...glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.
Pediatric therapy-related myeloid neoplasms (tMN) occur in children after exposure to cytotoxic therapy and have a dismal prognosis. The somatic and germline genomic alterations that drive these ...myeloid neoplasms in children and how they arise have yet to be comprehensively described. We use whole exome, whole genome, and/or RNA sequencing to characterize the genomic profile of 84 pediatric tMN cases (tMDS: n = 28, tAML: n = 56). Our data show that Ras/MAPK pathway mutations, alterations in RUNX1 or TP53, and KMT2A rearrangements are frequent somatic drivers, and we identify cases with aberrant MECOM expression secondary to enhancer hijacking. Unlike adults with tMN, we find no evidence of pre-existing minor tMN clones (including those with TP53 mutations), but rather the majority of cases are unrelated clones arising as a consequence of cytotoxic therapy. These studies also uncover rare cases of lineage switch disease rather than true secondary neoplasms.
Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all ...anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet).
Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall.
An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.
In the past decade, Houston, Texas has been virtually overrun by an unidentified ant species, the sudden appearance and enormous population sizes and densities of which have received national media ...attention. The Rasberry Crazy Ant, as it has become known due to its uncertain species status, has since spread to neighboring states and is still a major concern to pest control officials. Previous attempts at identifying this species have resulted in widely different conclusions in regards to its native range, source, and biology. We identify this highly invasive pest species as Nylanderia fulva (Mayr) using morphometric data measured from 14 characters, molecular sequence data consisting of 4,669 aligned nucleotide sites from six independent loci and comparison with type specimens. This identification will allow for the study and control of this emerging pest species to proceed unencumbered by taxonomic uncertainty. We also show that N. fulva has a much wider distribution than previously thought and has most likely invaded all of the Gulf Coast states.
Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for ...studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state,
activation with cytokines and co-culture, and
activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and
versus
treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.
•Strategies to promote civic and political engagement of urban youth in three cities are outlined.•These include youth work, deliberative forums, volunteerism, arts and technology.•Challenges ...associated with youth civic and political engagement are explored.
Over the past decade and more, there has been a significant emphasis in western societies on policy processes to engage youth in civic and political life. Concerns about the extent to which marginalized urban young people in particular have become disengaged from civic and political life has been a common, and contested, theme in youth policy. Currently, there is a dearth of literature exploring how policy provisions have been translated into practice on the ground. In this paper, we draw on semi-structured interviews with 66 policymakers, public officials, and youth work practitioners in London, Belfast, and Dublin to explore the strategies used to engage urban youth and to identify the key challenges associated with these efforts. The strategies used in youth engagement efforts include youth work, deliberative forums, volunteerism, arts, sports and media, non-formal education and technology and social media. Challenges associated with youth civic and political engagement include youth alienation from their communities and institutions, capacity and resource limitations, ritual rather than real engagement and challenges of inclusion.
Abstract Background Repetitive transcranial magnetic stimulation (rTMS) can temporarily interrupt or facilitate activity in a focal brain region. Several lines of evidence suggest that rTMS of the ...dorsolateral prefrontal cortex (DLPFC) can affect processes involved in drug addiction. We hypothesized that a single session of low-frequency rTMS of the left DLPFC would modulate cue-induced craving for methamphetamine (MA) when compared to a sham rTMS session. Methods In this single-blind, sham-controlled crossover study, 10 non-treatment seeking MA-dependent users and 8 healthy controls were randomized to receive 15 min of sham and real (1 Hz) DLPFC rTMS in two experimental sessions separated by 1 h. During each rTMS session, participants were exposed to blocks of neutral cues and MA-associated cues. Participants rated their craving after each cue block. Results In MA users, real rTMS over the left DLPFC increased self-reported craving as compared to sham stimulation (17.86 ± 1.46 vs. 24.85 ± 1.57, p = 0.001). rTMS had no effect on craving in healthy controls. One Hertz rTMS of the left DLPFC was safe and tolerable for all participants. Conclusions Low frequency rTMS of the left DLPFC transiently increased cue-induced craving in MA participants. These preliminary results suggest that 1 Hz rTMS of the left DLPFC may increase craving by inhibiting the prefrontal cortex or indirectly activating subcortical regions involved in craving.
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