Identification of TRDV-TRAJ V domains in human and mouse T-cell receptor repertoires

Here, we describe the identification of two T-cell receptors (TRs) containing TRDV genes in their TRA chains, the first one in human and the second one in mouse. First, using 5’RACE on a mixed lymphocyte-tumor cell culture (MLTC), we identified TRDV1 5’-untranslated region (UTR) and complete coding sequence rearranged productively to TRAJ24. Single-cell TR RNA sequencing (RNA-seq) of the MLTC, conducted to identify additional clonotypes, revealed that the analysis software detected the hybrid TRDV-TRAJ TRA (TRA) chain but excluded it from the final results. In a separate project, we performed TR sequencing of tumor-infiltrating lymphocytes (TILs) in a murine tumor model. Here, the predominant clonotype contained a TRA chain with a TRDV2-2-TRAJ49 rearrangement. Again, the hybrid TRA chain was not reported in the final results. Transfection of both TR cDNAs resulted in cell surface localization of TR together with CD3, suggesting a productive protein in both cases. Tumor recognition of the Homo sapiens (Homsap) TRDV1-containing TR could be demonstrated by IFN Gamma ELISA ELISpot kit, whereas the Mus musculus (Musmus) TR did not recognize a tumor-derived cell line. To determine whether the TRDV-containing TRA chains we detected were rare events or whether TRDV genes are commonly incorporated into TRA chains, we queried the NCBI Sequence Read Archive for TR single-cell RNA-seq data and analyzed 21 human and 23 murine datasets. We found that especially Homsap TRDV1, Musmus TRDV1, and to some extent Musmus TRDV2-2 are more commonly incorporated into TRA chains than several TRAV genes, making those TRDV genes a relevant contribution to TRA diversity. TRDV-containing TRA chains are currently excluded from the final results of V-(D)-J dataset analyses with the CellRanger software. We provide a work-around to avoid exclusion of those hybrid TRA chains from the final analysis results

Coincident Pre- and Postsynaptic Activation Induces Dendritic Filopodia via Neurotrypsin-Dependent Agrin Cleavage

SummaryThe synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS

Localization and Characterization of STRO-1+ Cells in the Deer Pedicle and Regenerating Antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1+ cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process

Tracheostomy care and decannulation during the COVID-19 pandemic. A multidisciplinary clinical practice guideline.

PURPOSE: Traditional critical care dogma regarding the benefits of early tracheostomy during invasive ventilation has had to be revisited due to the risk of COVID-19 to patients and healthcare staff. Standard practises that have evolved to minimise the risks associated with tracheostomy must be comprehensively reviewed in light of the numerous potential episodes for aerosol generating procedures. We meet the urgent need for safe practise standards by presenting the experience of two major London teaching hospitals, and synthesise our findings into an evidence-based guideline for multidisciplinary care of the tracheostomy patient. METHODS: This is a narrative review presenting the extensive experience of over 120 patients with tracheostomy, with a pragmatic analysis of currently available evidence for safe tracheostomy care in COVID-19 patients. RESULTS: Tracheostomy care involves many potentially aerosol generating procedures which may pose a risk of viral transmission to staff and patients. We make a series of recommendations to ameliorate this risk through infection control strategies, equipment modification, and individualised decannulation protocols. In addition, we discuss the multidisciplinary collaboration that is absolutely fundamental to safe and effective practise. CONCLUSION: COVID-19 requires a radical rethink of many tenets of tracheostomy care, and controversy continues to exist regarding the optimal techniques to minimise risk to patients and healthcare workers. Safe practise requires a coordinated multidisciplinary team approach to infection control, weaning and decannulation, with integrated processes for continuous prospective data collection and audit

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Observation of a coherence length effect in exclusive ρ(0) electroproduction

Exclusive incoherent electroproduction of the ρ0(770) meson from 1H, 2H, 3He, and 14N targets has been studied by the HERMES experiment at squared four-momentum transfer Q^2>0.4 GeV^2 and positron energy loss ν from 9 to 20 GeV. The ratio of the 14N to 1H cross sections per nucleon, known as the nuclear transparency, was found to decrease with increasing coherence length of quark-antiquark fluctuations of the virtual photon. The data provide clear evidence of the interaction of the quark-antiquark fluctuations with the nuclear medium

HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient’s HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy