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Platelet-rich plasma (PRP) is an orthobiologic treatment that has gained popularity as a potential alternative treatment for various musculoskeletal conditions. The physiologic role of platelets in the healing cascade provides clarity regarding its potential as it releases various growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF). However, there are various characteristics of PRP treatments including platelet count, presence or absence of leukocytes and red blood cells, as well as the use of an activating agent that introduces heterogeneity among preparations. This aim of this article is to provide clarity, where available, regarding the optimal characteristics for PRP treatments regarding tendon and ligament injuries as well as articular and muscular pathology.

Background Malignant peripheral nerve sheath tumors (MPNSTs) are rare aggressive sarcomas with poor prognosis. More than half of MPNSTs develop from benign precursor tumors associated with neurofibromatosis type 1 (NF1) which is a tumor suppressor gene disorder. Early detection of malignant transformation in NF1 patients is pivotal to improving survival. The primary aim of this study was to evaluate the role of immuno-modulators as candidate biomarkers of malignant transformation in NF1 patients with plexiform neurofibromas as well as predictors of response to immunotherapeutic approaches. Methods Sera from a total of 125 NF1 patients with quantified internal tumor load were included, and 25 of them had MPNSTs. A total of six immuno-modulatory factors (IGFBP-1, PD-L1, IFN-α, GM-CSF, PGE-2, and AXL) were measured in these sera using respective ELISA. Results NF1 patients with MPNSTs had significantly elevated PD-L1 levels in their sera compared to NF1 patients without MPNSTs. By contrast, AXL concentrations were significantly lower in sera of NF1-MPNST patients. IGFBP-1 and PGE2 serum levels did not differ between the two patient groups. IFN-α and GM-CSF were below the detectable level in most samples. Conclusion The immuno-modulator PD-L1 is upregulated in MPNST patients and therefore may provide as a potential biomarker of malignant transformation in patients with NF1 and as a response predictor for immunotherapeutic approaches.

Patients with neurofibromatosis type 1 (NF1) and type 1 NF1 deletions often exhibit more severe clinical manifestations than patients with intragenic NF1 gene mutations, including facial dysmorphic features, overgrowth, severe global developmental delay, severe autistic symptoms and considerably reduced cognitive abilities, all of which are detectable from a very young age. Type 1 NF1 deletions encompass 1.4 Mb and are associated with the loss of 14 protein-coding genes, including NF1 and SUZ12. Atypical NF1 deletions, which do not encompass all 14 protein-coding genes located within the type 1 NF1 deletion region, have the potential to contribute to the delineation of the genotype/phenotype relationship in patients with NF1 microdeletions. Here, we review all atypical NF1 deletions reported to date as well as the clinical phenotype observed in the patients concerned. We compare these findings with those of a newly identified atypical NF1 deletion of 698 kb which, in addition to the NF1 gene, includes five genes located centromeric to NF1. The atypical NF1 deletion in this patient does not include the SUZ12 gene but does encompass CRLF3. Comparative analysis of such atypical NF1 deletions suggests that SUZ12 hemizygosity is likely to contribute significantly to the reduced cognitive abilities, severe global developmental delay and facial dysmorphisms observed in patients with type 1 NF1 deletions.

Rapid antigen tests (RATs) are an integral part of SARS-CoV-2 containment strategies. As emerging variants of concern (VOCs) displace the initially circulating strains, it is crucial that RATs do not fail to detect these new variants. In this study, four RATs for nasal swab testing were investigated using cultured strains of B.1.1 (non-VOC), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). Based on dilution series in cell culture medium and pooled saliva, the limit of detection of these RATs was determined in a laboratory setting. Further investigations on cross-reactivity were conducted using recombinant N-protein from seasonal human coronaviruses (hCoVs). RATs evaluated showed an overall comparable performance with cultured strains of the non-VOC B.1.1 and the VOCs Alpha, Beta, Gamma, and Delta. No cross-reactivity was detected with recombinant N-protein of the hCoV strains HKU1, OC43, NL63, and 229E. A continuous evaluation of SARS-CoV-2 RAT performance is required, especially with regard to evolving mutations. Moreover, cross-reactivity and interference with pathogens and other substances on the test performance of RATs should be consistently investigated to ensure suitability in the context of SARS-CoV-2 containment.

Birch (Betula pendula Roth.) and beech (Fagus sylvatica L.) solid wood and plywood were overmolded with polyamide 6 (PA 6) and polypropylene (PP) to investigate their mechanical properties and interfacial adhesion. In the case of PA 6, maximum tensile shear strengths values of more than 8 to 9 MPa were obtained for birch and beech, respectively. The values are comparable to bond strengths of commercial joints bonded with formaldehyde-containing amino-plastics. Perpendicular to the wood elements, bond strength values of 3 MPa was achieved for PA 6. The penetration of the polymers into the wood structure results in a non-densified interphase and subsequent plastic deformation of the wood structure beyond the interphase. These compressed areas influenced the interfacial adhesion and mechanical interlocking. SEM and XPS analysis revealed different interpenetration behavior of the polymers into the wood structure, with chemical interaction confirmed only for wood and PA 6 but not PP.

On degraded DNA samples a multiplex PCR mixture containing polynucleotide primers can provide superior genotyping of Short Tandem Repeats (STRs) to the current commercial kits based on standard oligonucleotides.

Agricultural by-products can be upcycled into environmentally-sustainable, inexpensive chitinous materials and nanofibers derived from fungal mycelium for composites, cosmetics, pharmaceuticals and water treatment applications. This study determined the suitability of common agricultural by-products as medium for fungal growth. Growth was measured by quantifying ergosterol, a unique fungal product, in solid and liquid media. The results reveal that fungi grew less on rice hull, sugarcane bagasse and wheat straw agricultural by-products than on commercial wheat grains. However, the liquid agricultural by-product blackstrap molasses facilitated very high biomass production, outperforming the commonly used laboratory nutrient malt extract. Hyphal fusion, sheet formation and hyphal diameter metrics of fungi growing on each substrate were evaluated by SEM to assess suitability for chitin nanofiber production. Utilising these materials offers a cheap, renewable, easily-isolated, and abundant alternative to problematic crustacean chitin that when implemented on a large scale could rapidly upcycle low-value agricultural by-products into high-value chitinous materials.

Epstein-Barr virus (EBV), a member of the large herpesvirus family is a very complex humanγ-herpesvirus. Its complexity with respect to viral gene regulation, its preferred latent life style and technical difficulties are major obstacles, which hinder efficient virus generationin vitroto mass-produce or establish recombinant wild-type EBV or mutant derivatives. To explore conditions optimizing and improving virus production, we established and tested an EBV gene library with 77 expression plasmids and a set of designed shRNAs. With this tool set we investigated the contributions of individual viral genes in the context of virus synthesis and virion functions. Engineered virus stocks were systematically analyzed with respect to physical and bioparticle concentration, virus titer and virus uptake by primary human B cells, EBV’s target cellsin vivo. To quantitate virus uptake by these cells, we developed a novelß-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells. Based on our findings, EBV does not encode a dominant regulator that governs virus production, but our results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a transcriptional regulator of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality, and virus uptake by primary human B cells.ImportanceFor more than 20 years HEK293 cells have been instrumental to produce virus stocks of recombinant EBV. The identification of this cell line as a source of infectious EB virions was unexpected because HEK293 cells are very distant from B cells and in particular plasma cells which produce EBV progenyin vivo. To our knowledge, no systematic analysis has addressed the fundamental question whether virus yield with respect to virus concentration, virion composition and functionality can be improved to produce recombinant EBV stocks in HEK293 cells. We tackled this question and analyzed 77 individual EBV genes for their possible contribution to virus yield. To analyze and compare different virus stocks we used established and developed novel assays to characterize important parameters of EB virions and their functions.

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03315-6.

SARS-CoV-2 variants of concern (VOC) should not escape molecular surveillance. We investigated if SARS-CoV-2 rapid antigen tests (RATs) could detect B.1.1.7 and B.1.351 VOCs in certain laboratory conditions. Infectious cell culture supernatants containing B.1.1.7, B.1.351 or non-VOC SARS-CoV-2 were respectively diluted both in DMEM and saliva. Dilutions were analysed with Roche, Siemens, Abbott, nal von minden and RapiGEN RATs. While further studies with appropriate real-life clinical samples are warranted, all RATs detected B.1.1.7 and B.1.351, generally comparable to non-VOC strain.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications., This work was funded by Deutsche Krebshilfe grant 70112499, the NCT Heidelberg and an Illumina Medical Research Grant. Part of this work was funded by the National Institute of Health Research (to S.B. and Z.J.) and to UCLH Biomedical research centre (BRC399/NS/RB/101410). Human tissues were obtained from University College London NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK, Ref: 18/004) which is funded by the Medical Research Council and Brain Tumour Research UK. The methylation profiling at NYU is supported by a grant from the Friedberg Charitable Foundation (to M.Sn.). M.Mi. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant). Open Access funding enabled and organized by Projekt DEAL.

Background Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

Background By targeted DNA sequencing of 305 diagnostic follicular lymphoma (FL) biopsies, we identified somatic mutations of Cathepsin S (CTSS) in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). Another 13% of FL had CTSS amplifications (37/286), associated with higher CTSS expression (P=0.05). CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells.1 CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). Materials and Methods We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). We purified pro-CTSS WT and Y132D and assayed the in vitro autocatalytic cleavage over time. We then tested the impact of CTSS on CD4+ T-cell activation in co-culture assays, in a previously described in vivo model2 which we slightly modified to reflect FL-like conditions, and in primary patient samples. Results Single-cell derived Y132D mutant Karpas422 clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001) which was mediated by an accelerated conversion from pro-CTSS to active CTSS (11 minutes for CTSS Y132D vs 17 minutes for CTSS WT; N=3, P=0.04). Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2A between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which could facilitate intramolecular cleavage. The higher substrate cleavage activity of CTSS Y132D came along with a high capacity to stimulate antigen specific CD4+ T cell responses in vitro and in vivo. Additionally, CTSS overexpression could phenocopy this high CD4+ T cell activation. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012). Conclusions Here, we provide biochemical, structural, functional and clinical evidence that aberrant CTSS activity induces a supportive immune microenvironment in FL. We propose that aberrant CTSS activity can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. References Riese, R.J., et al., Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity 1996; 4(4): p. 357–66. Kim, K.J., et al., Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol 1979; 122(2): p. 549–54. Disclosure Information J.A. Hildebrand: None. D. Bararia: None. S. Stolz: None. S. Habe: None. F. Osorio-Barrios: None. M.D. Bartoschek: None. E. Gaitzsch: None. V. Jurinovic: None. K. Rautter: None. C. Ludwig: None. S. Bultmann: None. H. Leonhardt: None. S. Eustermann: None. K. Hopfner: None. W. Hiddemann: None. M. Bergwelt: None. M. Schmidt-Supprian: None. M.B. Sarosi: None. M. Rudelius: None. V. Passerini: None. J. Mautner: None. O. Weigert: None.

Post-transcriptional gene regulation is complex, dynamic and ensures proper T cell function. The targeted transcripts can simultaneously respond to various factors as evident for Icos, an mRNA regulated by several RNA binding proteins (RBPs), including Roquin. However, fundamental information about the entire RBPome involved in post-transcriptional gene regulation in T cells is lacking. Here, we applied global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS) to human and mouse primary T cells and identified the core T cell RBPome. This defined 798 mouse and 801 human proteins as RBPs, unexpectedly containing signaling proteins like Stat1, Stat4 and Vav1. Based on the vicinity to Roquin-1 in proximity labeling experiments, we selected ∼50 RBPs for testing coregulation of Roquin targets. Induced expression of these candidate RBPs in wildtype and Roquin-deficient T cells unraveled several Roquin-independent contributions, but also revealed Celf1 as a new Roquin-1-dependent and target-specific coregulator of Icos.One sentence statementWe provide an atlas of RNA-binding proteins in human and mouse T helper cells as a resource for studying higher order post-transcriptional gene regulation.

Senescence was recently linked to neurodegeneration and astrocytes are one of the major cell types to turn senescent under neurodegenerative conditions. Senescent astrocytes were detected in Parkinson's disease (PD) patients' brains besides reactive astrocytes, yet the difference between senescent and reactive astrocytes is unclear. We aimed to characterize senescent astrocytes in comparison to reactive astrocytes and investigate differences and similarities. In a cell culture model of human fetal astrocytes, we determined a unique senescent transcriptome distinct from reactive astrocytes, which comprises dysregulated pathways. Both, senescent and reactive human astrocytes activated a proinflammatory pattern. Astrocyte senescence was at least partially depending on active mechanistic-target-of-rapamycin (mTOR) and DNA-damage response signaling, both drivers of senescence. To further investigate how PD and senescence connect to each other, we asked if a PD-linked environmental factor induces senescence and if senescence impairs midbrain neurons. We could show that the PD-linked pesticide rotenone causes astrocyte senescence. We further delineate, that the senescent secretome exaggerates rotenone-induced neurodegeneration in midbrain neurons differentiated from human induced pluripotent stem cells (hiPSC) of PD patients with alpha-synuclein gene (SNCA) locus duplication.

Summary Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Cellular immunotherapy is a proven approach against Epstein-Barr virus (EBV)-driven lymphoproliferation in recipients of hematopoietic stem cells. Extending the applicability and improving the response rates of such therapy demands improving the knowledge base. We studied 23 healthy donors for specific CD4(+) T cell responses against the viral tegument protein BNRF1 and found such T cells in all seropositive donors, establishing BNRF1 as an important immune target in EBV. We identified 18 novel immune epitopes from BNRF1, all of them generated by natural processing of the full-length protein from virus-transformed lymphoblastoid cell lines (LCL). BNRF1-specific CD4(+) T cells were measured directly ex vivo by a cytokine-based method, thus providing a tool to study the interaction between immunity and infection in health and disease. T cells of the cytotoxic Th1 type inhibited the proliferation of autologous LCL as well as virus-driven transformation. We infer that they are important in limiting reactivations to subclinical levels during health and reducing virus propagation during disease. The information obtained from this work will feed into data sets that are indispensable in the design of patient-tailored immunotherapeutic approaches, thereby enabling the stride toward broader application of T cell therapy and improving clinical response rates. IMPORTANCE Epstein-Barr virus is carried by most humans and can cause life-threatening diseases. Virus-specific T cells have been used in different clinical settings with variable success rates. One way to improve immunotherapy is to better suit T cell generation protocols to viral targets available in different diseases. BNRF1 is present in viral particles and therefore likely available as a target for T cells in diseases with virus amplification. Here, we studied healthy Epstein-Barr virus (EBV) carriers for BNRF1 immunogenicity and report our results indicating BNRF1 to be a dominant target of the EBV-specific CD4(+) T cell response. BNRF1-specific CD4(+) T cells were found to be cytotoxic and capable of limiting EBV-driven B cell transformation in vitro. The findings of this work contribute to forwarding our understanding of host-virus interactions during health and disease and are expected to find direct application in the generation of specific T cells for immunotherapy.

More than 50% of the world's population is infected with the bacterium Helicobacter pylori. If left untreated, infection with H. pylori can cause chronic gastritis and peptic ulcer disease, which may progress into gastric cancer. Owing to the limited efficacy of anti-H. pylori antibiotic therapy in clinical practice, the development of a protective vaccine to combat this pathogen has been a tempting goal for several years. In this study, a chimeric gene coding for the antigenic parts of H. pylori FliD, UreB, VacA, and CagL was generated and expressed in bacteria and the potential of the resulting fusion protein (rFUVL) to induce humoral and cellular immune responses and to provide protection against H. pylori infection was evaluated in mice. Three different immunization adjuvants were tested along with rFUVL: CpG oligodeoxynucleotides (CpG ODN), Addavax, and Cholera toxin subunit B. Compared to the control group that had received PBS, vaccinated mice showed significantly higher cellular recall responses and antigen-specific IgG2a, IgG1, and gastric IgA antibody titers. Importantly, rFUVL immunized mice exhibited a reduction of about three orders of magnitude in their stomach bacterial loads. Thus, adjuvanted rFUVL might be considered as a promising vaccine candidate for the control of H. pylori infection.

The Epstein-Barr virus (EBV), a ubiquitous γ-herpesvirus, has been implicated in the etiology of several acute and chronic inflammatory, autoimmune, and malignant diseases. Although considered a genetically stable virus, recent sequence information obtained from a large number of viral isolates from around the world revealed that numerous viral variants exist. Whether these different strains differ in pathogenicity and immunogenicity and thereby contribute to the varying incidence rates of several EBV-associated diseases in different geographical regions is now studied intensively. The recent identification of amino acid sequence polymorphisms in a high percentage of all known virus-specific CD4+ and CD8+ T-cell epitopes, and of holes in the individual T-cell repertoire against epitopes derived from strain variants, may suggest that antiviral immunity is incompletely cross-protective against diverse EBV strains. These findings may have implications for immunological approaches seeking to prevent, monitor, or treat EBV-associated diseases.

Precise characterization of nonallelic homologous recombination (NAHR) breakpoints is key to identifying those features that influence NAHR frequency. Until now, analysis of NAHR-mediated rearrangements has generally been performed by comparison of the breakpoint-spanning sequences with the human genome reference sequence. We show here that the haplotype diversity of NAHR hotspots may interfere with breakpoint-mapping. We studied the transmitting parents of individuals with germline type-1 NF1 deletions mediated by NAHR within the paralogous recombination site 1 (PRS1) or paralogous recombination site 2 (PRS2) hotspots. Several parental wild-type PRS1 and PRS2 haplotypes were identified that exhibited considerable sequence differences with respect to the reference sequence, which also affected the number of predicted PRDM9-binding sites. Sequence comparisons between the parental wild-type PRS1 or PRS2 haplotypes and the deletion breakpoint-spanning sequences from the patients (method #2) turned out to be an accurate means to assign NF1 deletion breakpoints and proved superior to crude reference sequence comparisons that neglect to consider haplotype diversity (method #1). The mean length of the deletion breakpoint regions assigned by method #2 was 269-bp in contrast to 502-bp by method #1. Our findings imply that paralog-specific haplotype diversity of NAHR hotspots (such as PRS2) and population-specific haplotype diversity must be taken into account in order to accurately ascertain NAHR-mediated rearrangement breakpoints.

The most frequent recurring mutations in neurofibromatosis\ud type 1 (NF1) are large deletions encompassing\ud the NF1 gene and its flanking regions (NF1 microdeletions).\ud The majority of these deletions encompass 1.4-Mb\ud and are associated with the loss of 14 protein-coding genes\ud and four microRNA genes. Patients with germline type-1\ud NF1 microdeletions frequently exhibit dysmorphic facial\ud features, overgrowth/tall-for-age stature, significant delay\ud in cognitive development, large hands and feet, hyperflexibility\ud of joints and muscular hypotonia. Such patients\ud also display significantly more cardiovascular anomalies\ud as compared with patients without large deletions and\ud often exhibit increased numbers of subcutaneous, plexiform\ud and spinal neurofibromas as compared with the general\ud NF1 population. Further, an extremely high burden of\ud internal neurofibromas, characterised by >3000 ml tumour\ud volume, is encountered significantly, more frequently,\ud in non-mosaic NF1 microdeletion patients than in NF1\ud patients lacking such deletions. NF1 microdeletion patients\ud also have an increased risk of malignant peripheral nerve\ud sheath tumours (MPNSTs); their lifetime MPNST risk is\ud 16–26%, rather higher than that of NF1 patients with intragenic\ud NF1 mutations (8–13%). NF1 microdeletion patients,\ud therefore, represent a high-risk group for the development\ud of MPNSTs, tumours which are very aggressive and difficult to treat. Co-deletion of the SUZ12 gene in addition\ud to NF1 further increases the MPNST risk in NF1 microdeletion\ud patients. Here, we summarise current knowledge\ud about genotype–phenotype relationships in NF1 microdeletion\ud patients and discuss the potential role of the genes\ud located within the NF1 microdeletion interval whose haploinsufficiency may contribute to the more severe clinical phenotype

Infections with Epstein–Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome., Infection with Epstein–Barr virus (EBV) is associated with increased risk of cancer development. Here the authors show that EBV particles, and more specifically the viral protein BNRF1, induce centrosome amplification and chromosomal instability in host cells in the absence of chronic infection.

Exceptional variability in the shell of the pancake tortoise Malacochersus tornieri, both in the keratinous surficial scutes and the underlying bones, in addition to its remarkably fenestrated bony shell are unique among tortoises. Based on 14 individuals of different sizes and ages, the observed variation in M. tornieri was described in detail, with additional notes on the typically testudinid skull, inner ear and brain endocast using microCT-scan data, as well as the limbs. Similar degrees of variation have not yet been described in any other extant turtle species and therefore seem notable in M. tornieri, and might be related to the species' unique lifestyle. Within the carapace, the peripherals and suprapygals are most variable in number. Furthermore, different combinations of peripherals are participating in the central plastral fontanelle and in some individuals additional bones take part in the formation of the plastron. J. Morphol., 2017. © 2017 Wiley Periodicals, Inc.

Schwannomatosis is characterized by the predisposition to develop multiple schwannomas and, less commonly, meningiomas. Despite the clinical overlap with neurofibromatosis type 2 (NF2), schwannomatosis is not caused by germline NF2 gene mutations. Instead, germline mutations of either the SMARCB1 or LZTR1 tumour suppressor genes have been identified in 86% of familial and 40% of sporadic schwannomatosis patients. In contrast to patients with rhabdoid tumours, which are due to complete loss-of-function SMARCB1 mutations, individuals with schwannomatosis harbour predominantly hypomorphic SMARCB1 mutations which give rise to the synthesis of mutant proteins with residual function that do not cause rhabdoid tumours. Although biallelic mutations of SMARCB1 or LZTR1 have been detected in the tumours of patients with schwannomatosis, the classical two-hit model of tumorigenesis is insufficient to account for schwannoma growth, since NF2 is also frequently inactivated in these tumours. Consequently, tumorigenesis in schwannomatosis must involve the mutation of at least two different tumour suppressor genes, an occurrence frequently mediated by loss of heterozygosity of large parts of chromosome 22q harbouring not only SMARCB1 and LZTR1 but also NF2. Thus, schwannomatosis is paradigmatic for a tumour predisposition syndrome caused by the concomitant mutational inactivation of two or more tumour suppressor genes. This review provides an overview of current models of tumorigenesis and mutational patterns underlying schwannomatosis that will ultimately help to explain the complex clinical presentation of this rare disease. Electronic supplementary material The online version of this article (doi:10.1007/s00439-016-1753-8) contains supplementary material, which is available to authorized users.

We coincidently detected an atypical deletion of at least 1.3-Mb, encompassing the NF1 tumor suppressor gene and several adjacent genes at an apparent heterozygous level in the blood of a 65-year-old female patient. She had multiple subcutaneous tumors that appeared with a certain similarity of subcutaneous neurofibromas, which, however, was revealed as lipomas by histological examination. Comprehensive and exhaustive clinical and radiological examinations did not detect any neurofibromatosis type 1-related clinical symptoms in the patient. Multiplex ligation-dependent probe amplification detected no or only very low level of the 1.3-Mb NF1 deletion in six lipomas and two skin biopsies. Digital polymerase chain reaction estimated the proportion of cells carrying a heterozygous NF1 deletion at 87% in the blood, and 8%, 10%, 13%, 17%, and 20%, respectively, in the five lipomas investigated by this method, confirming our hypothesis of mosaicism. Our findings suggest that de novo cases of genetic disease are potentially mosaic regardless of finding the mutation at an apparently heterozygous level in the blood and that the possibility of mosaicism should be considered in genotype-phenotype studies and genetic counseling.

BackgroundNeurofibromatosis type 1 (NF1) predisposes to breast cancer (BC), but no genotype-phenotype correlations have been described.MethodsConstitutional NF1 mutations in 78 patients with NF1 with BC (NF1-BC) were compared with the NF1 Leiden Open Variation Database (n=3432).ResultsNo cases were observed with whole or partial gene deletions (HR 0.10; 95% CI 0.006 to 1.63; p=0.014, Fisher’s exact test). There were no gross relationships with mutation position. Forty-five (64.3%; HR 6.4–83) of the 70 different mutations were more frequent than expected (pBRCA1/2 testing, revealing one BRCA2 mutation.DiscussionThese data strongly support the hypothesis that certain constitutional mutation types, and indeed certain specific variants in NF1 confer different risks of BC. The lack of large deletions and excess of nonsenses and missenses is consistent with gain of function mutations conferring risk of BC, and also that neurofibromin may function as a dimer. The observation that somatic NF1 amplification can occur independently of ERBB2 amplification in sporadic BC supports this concept. A prospective clinical-molecular study of NF1-BC needs to be established to confirm and build on these findings, but regardless of NF1 mutation status patients with NF1-BC warrant testing of other BC-predisposing genes.

Different types of large NF1 deletion are distinguishable by breakpoint location and potentially also by the frequency of mosaicism with normal cells lacking the deletion. However, low-grade mosaicism with fewer than 10% normal cells has not yet been excluded for all NF1 deletion types since it is impossible to assess by the standard techniques used to identify such deletions, including MLPA and array analysis. Here, we used ultra-deep amplicon sequencing to investigate the presence of normal cells in the blood of 20 patients with type-1 NF1 deletions lacking mosaicism according to MLPA. The ultra-deep sequencing entailed the screening of 96 amplicons for heterozygous SNVs located within the NF1 deletion region. DNA samples from three previously identified patients with type-2 NF1 deletions and low-grade mosaicism with normal cells as determined by FISH or microsatellite marker analysis were used to validate our methodology. In these type-2 NF1 deletion samples, proportions of 5.3%, 6.6% and 15.0% normal cells, respectively, were detected by ultra-deep amplicon sequencing. However, using this highly sensitive method, none of the 20 patients with type-1 NF1 deletions included in our analysis exhibited low-grade mosaicism with normal cells in blood, thereby supporting the view that the vast majority of type-1 deletions are germline deletions.

The use of long ssDNA polynucleotides in PCR of degraded DNA samples can discriminate Short Tandem Repeat (STR) genotypes otherwise missed by current commercial kits. Fil: Mautner, Martin E.. Biodynamics; Argentina Fil: Caputo, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina Fil: Corach, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina

The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection., Author summary Human herpesviruses are tremendously successful pathogens that establish lifelong infection in a substantial proportion of the population. The oncogenic γ-herpesvirus EBV, like other herpesviruses, expresses a plethora of open-reading frames throughout its multifaceted life cycle. We have developed a prophylactic vaccine candidate in the form of immunogenic particles that contain several EBV antigens. This is in stark contrast to the vast majority of EBV vaccines candidates that contain only one or two EBV antigens. Our immunogenic particles were shown capable of stimulating several EBV-specific T-cell clones in vitro. The immunogenic particles were also capable of expanding cytolytic EBV-specific T cells ex vivo and provided a protective benefit in vivo when used as a prophylactic vaccine.

Neurofibromatosis type-1 (NF1) patients suffer from cutaneous and subcutaneous neurofibromas (CNF) and large plexiform neurofibromas (PNF). Whole gene deletions of the NF1 gene can cause a more severe phenotype compared to smaller intragenic changes. Two distinct groups of NF1 whole gene deletions are type-1 deletions and atypical deletions. Our aim was to assess volumes and averaged annual growth-rates of CNF and PNF in patients with NF1 whole gene deletions and to compare these with NF1 patients without large deletions of the NF1 gene. We retrospectively evaluated 140 whole-body MR examinations of 38 patients with NF1 whole gene deletions (type-1 group: n = 27/atypical group n = 11) and an age- and sex matched collective of 38 NF1-patients. Age-dependent subgroups were created (0–18 vs >18 years). Sixty-four patients received follow-up MRI examinations (NF1whole gene deletion n = 32/control group n = 32). Whole-body tumor-volumes were semi-automatically assessed (MedX, V3.42). Tumor volumes and averaged annual growth-rates were compared. Median tumor-burden was significantly higher in the type-1 group (418ml; IQR 77 – 950ml, p = 0.012) but not in the atypical group (356ml;IQR 140–1190ml, p = 0.099) when compared to the controls (49ml; IQR 11–691ml). Averaged annual growth rates were significantly higher in both the type-1 group (14%/year; IQR 45–36%/year, p = 0.004) and atypical group (11%/year; IQR 5–23%/year, p = 0.014) compared to the controls (4%/year; IQR1–8%/year). Averaged annual growth rates were significantly higher in pediatric patients with type-1 deletions (21%/year) compared with adult patients (8%/year, p = 0.014) and also compared with pediatric patients without large deletions of the NF1 gene (3.3%/year, p = 0.0015). NF1 whole gene deletions cause a more severe phenotype of NF1 with higher tumor burden and higher growth-rates compared to NF1 patients without large deletions of the NF1 gene. In particular, pediatric patients with type-1 deletions display a pronounced tumor growth., Author summary Neurofibromatosis type-1 (NF1) is an autosomal dominantly inherited tumor predisposition syndrome and is caused by a variety of mutations of the NF1 gene. Large deletions encompassing the entire NF1 gene are known to be associated with a more severe clinical phenotype. A hallmark feature of NF1 is the development of different benign tumors: cutaneous or subcutaneous neurofibromas (CNF) and the potentially large plexiform neurofibromas (PNF). Importantly, PNF can undergo transformation into malignant peripheral nerve sheath tumors, which contribute to the increased mortality of NF1 patients. Identified risk factors for malignant transformation are NF1 whole gene deletions, large whole-body tumor burden and a high number of subcutaneous neurofibromas. We performed semi-automated volumetry of these tumors to determine volumes and growth rates of CNF and PNF in patients with whole gene deletions and a control group of NF1 patients without large deletions of the NF1 gene. Our results indicate that NF1 whole gene deletions lead to higher tumor burden and higher growth rates. In particular, pediatric NF1 patients with a specific type of whole gene deletion (type-1 deletion) exhibit rapid growth of PNF. We therefore suggest that these patients should be monitored in close intervals to evaluate tumor growth and risk of malignant transformation.

EBV reduces the activation of cytotoxic CD4+ effector T cells by inducing a state of reduced immunogenicity in infected B cells. EBV-derived miRNAs suppress release of proinflammatory cytokines, interfere with peptide processing and presentation on HLA class II, repress differentiation of naive CD4+ T cells to Th1 cells, and ultimately avoid killing of infected B cells., Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.

Introduction Inflammation is part of the physiological phases of wound healing. It serves the purpose to attract different immune cells in order to clean the wound from pathogens and debris and create an ideal environment for fibroblasts and keratinocytes to differentiate, migrate and divide in order to close the wound. Pathological inflammation caused by many different factors is often the underlying cause of prolonged wound healing and unfavourable outcome in burn patients. We wanted to investigate what influence an induced hyper-inflammation on wound healing has in a controlled environment. Methods In a porcine wound model, pigs received full-thickness wounds on their backs where hyper-inflammation was induced by application of a chemotherapeutic for 6 days. Control wounds were left without induction. Wound were scored on a daily basis for 16 days in total and biopsies were taken for RNA analysis as well as histological assessment. Results The results showed a rise in inflammation of the wounds and the surrounding tissue due to the induction. The wounds healed slower, needing up to seven more days to completely heal. The results of the RNA analysis showed high values of pro-inflammatory cytokines after the induction in comparison to the baseline and control wounds. Histological assessment showed thick layers of necrosis, adipocyte as well as leucocyte infiltration in the hyper-inflamed wounds compared to completely healed control wounds with all layers of epidermis visible. Conclusions Hyper-inflammation, even just for a short period of time, can significantly prolong the time of wound healing. These findings underline the importance of preventing and treating wound infections of burn patients in order to support physiological wound healing as well as to avert complications such as infections with multi-resistant microbes that are associated with prolonged hospital stays. Applicability of Research to Practice The findings of these experiments cannot be directly applied to practice however, the influence of inflammation should kept in mind when treating burn patients.

Objective Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas characterized by high recurrence rates and early metastases. These tumors arise more frequently within neurofibromatosis type 1 (NF1) and present with resistance during standard chemotherapy leading to increased mortality and morbidity in those patients. In vitro all-trans retinoic acid (ATRA) and MEK inhibitors (MEKi) were shown to inhibit tumor proliferation, especially when applied in combination. Therefore, we established a nude mouse model to investigate if treatment of xenografts derived from NF1 associated S462 and T265 MPNST cells respond to ATRA and the MEKi PD0325901. Results We demonstrated that human NF1 associated MPNST derived from S462 but not T265 cells form solid subcutaneous tumors in Foxn1 nude mice but not in Balb/c, SHO or Shorn mice. We verified a characteristic staining pattern of human MPNST xenografts by immunohistochemistry. Therapeutic effects of ATRA and/or MEKi PD0325901 on growth of S462 MPNST xenografts in Foxn1 nude mice were not demonstrated in vitro, as we did not observe significant suppression of MPNST growth compared with placebo treatment. Electronic supplementary material The online version of this article (10.1186/s13104-018-3630-0) contains supplementary material, which is available to authorized users.

The breakpoints of type-1 NF1 deletions encompassing 1.4-Mb are located within NF1-REPa and NF1-REPc, which exhibit a complex structure comprising different segmental duplications in direct and inverted orientation. Here, we systematically assessed the proportion of type-1 NF1 deletions caused by nonallelic homologous recombination (NAHR) and those mediated by other mutational mechanisms. To this end, we analyzed 236 unselected type-1 deletions and observed that 179 of them (75.8%) had breakpoints located within the NAHR hotspot PRS2, whereas 39 deletions (16.5%) had breakpoints located within PRS1. Sixteen deletions exhibited breakpoints located outside of these NAHR hotspots but were also mediated by NAHR. Taken together, the breakpoints of 234 (99.2%) of the 236 type-1 NF1 deletions were mediated by NAHR. Thus, NF1-REPa and NF1-REPc are strongly predisposed to recurrent NAHR, the main mechanism underlying type-1 NF1 deletions. We also observed a non-random overlap between type-1 NF1-deletion breakpoints and G-quadruplex forming sequences (GQs) as well as regions flanking PRDM9A binding-sites. These findings imply that GQs and PRDM9A binding-sites contribute to the clustering of type-1 deletion breakpoints. The co-location of both types of sequence was at its highest within PRS2, indicative of their synergistic contribution to the greatly increased NAHR activity within this hotspot.

Neurofibromatosis type 1 (NF1) is caused, in 4.7–11% of cases, by large deletions encompassing the NF1 gene and its flanking regions within 17q11.2. Different types of large NF1 deletion occur which are distinguishable by their breakpoint location and underlying mutational mechanism. Most common are the type-1 NF1 deletions of 1.4 Mb which exhibit recurrent breakpoints caused by nonallelic homologous recombination (NAHR), also termed unequal crossover. Here, we analyzed 37 unrelated families of patients with de novo type-1 NF1 deletions by means of short tandem repeat (STR) profiling to determine the parental origin of the deletions. We observed that 33 of the 37 type-1 deletions were of maternal origin (89.2% of cases; p

The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all cancers in humans. Some of these EBV-associated tumors have been successfully treated by the infusion of virus-specific T-cell lines. Recent sequence analyses of a large number of viral isolates suggested that distinct EBV strains have evolved in different parts of the world. Here, we assessed the impact of such sequence variations on EBV-specific T-cell immunity. With the exceptions of EBNA2 and the EBNA3 family of proteins, an overall low protein sequence disparity of about 1% was noted between Asian viral isolates, including the newly characterized M81 strain, and the prototypic EBV type 1 and type 2 strains. However, when T-cell epitopes including their flanking regions were compared, a substantial proportion was found to be polymorphic in different EBV strains. Importantly, CD4+ and CD8+ T-cell clones specific for viral epitopes from one strain often showed diminished recognition of the corresponding epitopes in other strains. In addition, T-cell recognition of a conserved epitope was affected by amino acid exchanges within the epitope flanking region. Moreover, the CD8+ T-cell response against polymorphic epitopes varied between donors and often ignored antigen variants. These results demonstrate that viral strain heterogeneity may impair antiviral T-cell immunity and suggest that immunotherapeutic approaches against EBV should preferably target broad sets of conserved epitopes including their flanking regions.

Short tandem repeats (STRs) are the markers of choice for purposes of human forensic identification because of their considerable degree of polymorphism. This variability may occasionally become a challenge for the analyst when a new variant invades the allele distribution range of the neighboring locus. We present here a novel variant at locus D1S1656 showing a molecular length of 211.32 bp corresponding to 21.3 repeating units. This variant is superimposed on the D12S391 locus when the Global Filer™ (Thermo Fisher Scientific Inc., USA) is used, invading the shortest allele range and being assigned as Off-ladder (OL). In contrast, typing with PowerPlex® Fusion (Promega Corp., Madison, USA) overlaps the D2S441 locus, generating an allele that is recognized as 8. In both cases the invasion was observed as a tri-allele pattern. The results were confirmed by DNA re-extraction and typing with the two independent commercial megaplexes described above. The variant was detected during analysis of a reference sample throughout a paternity exclusion case. This is the first time this variant has been described and reported to the NIST STR Base. Since D1S1656 is included in widely-used multiplex kits from several vendors - Biotype (Dresden, Germany) Qiagen (Venlo, The Netherlands), Promega (Madison, USA) and Thermo Fisher (Foster City, CA, USA) - it would then be recommendable that other forensic labs be aware of this new micro-variant in dealing with similar interpretation challenges and that the kit producers take this fact into account in designing new multiplex kits. Fil: Marcucci, Valeria Cecilia. Universidad Nacional de la Patagonia Austral; Argentina Fil: Cano, H.. Laboratorio Regional de Investigación Forense; Argentina Fil: Coggiola, Liliana. Laboratorio Regional de Investigación Forense; Argentina Fil: Mautner, M. E.. Biodynamics Sa; Argentina Fil: Sala, Adriana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina Fil: Corach, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina

The cytosolic protein Sharpin is a component of the linear ubiquitin chain assembly complex, which regulates NF-κB signaling in response to specific ligands, such as TNF-α. Its inactivating mutation in chronic proliferative dermatitis mutation (Cpdm) mice causes multiorgan inflammation, yet this phenotype is not transferable into wild-type mice by hematopoietic stem cell transfer. Recent evidence demonstrated that Cpdm mice additionally display low bone mass, and that this osteopenia is corrected by Tnf deletion. Because the cellular mechanism underlying this pathology, however, was still undefined, we performed a thorough skeletal phenotyping of Cpdm mice on the basis of nondecalcified histology and cellular and dynamic histomorphometry. We show that the trabecular and cortical osteopenia in Cpdm mice is solely explained by impaired bone formation, whereas osteoclastogenesis is unaffected. Consistently, Cpdm primary calvarial cells display reduced osteogenic capacity ex vivo, and the same was observed with CD11b− bone marrow cells. Unexpectedly, short-term treatment of these cultures with TNF-α did not reveal an impaired molecular response in the absence of Sharpin. Instead, genome-wide and gene-specific expression analyses revealed that Cpdm mesenchymal cells display increased responsiveness toward TNF-α–induced expression of specific cytokines, such as CXCL5, IL-1β, and IL-6. Therefore, our data not only demonstrate that the skeletal defects of Cpdm mice are specifically caused by impaired differentiation of osteoprogenitor cells, they also suggest that increased cytokine expression in mesenchymal bone marrow cells contributes to the inflammatory phenotype of Cpdm mice.

Purpose Vitamin D is well known for its effects on bone mineralisation but has also been attributed immunomodulatory properties. It positively influences human health, but in vivo data describing vitamin D effects on the human gut microbiome are missing. We aimed to investigate the effects of oral vitamin D3 supplementation on the human mucosa-associated and stool microbiome as well as CD8+ T cells in healthy volunteers. Methods This was an interventional, open-label, pilot study. Sixteen healthy volunteers (7 females, 9 males) were endoscopically examined to access a total of 7 sites. We sampled stomach, small bowel, colon, and stools before and after 8 weeks of vitamin D3 supplementation. Bacterial composition was assessed by pyrosequencing the 16S rRNA gene (V1–2), and CD8+ T cell counts were determined by flow cytometry. Results Vitamin D3 supplementation changed the gut microbiome in the upper GI tract (gastric corpus, antrum, and duodenum). We found a decreased relative abundance of Gammaproteobacteria including Pseudomonas spp. and Escherichia/Shigella spp. and increased bacterial richness. No major changes occurred in the terminal ileum, appendiceal orifice, ascending colon, and sigmoid colon or in stools, but the CD8+ T cell fraction was significantly increased in the terminal ileum. Conclusion Vitamin D3 modulates the gut microbiome of the upper GI tract which might explain its positive influence on gastrointestinal diseases, such as inflammatory bowel disease or bacterial infections. The local effects of vitamin D demonstrate pronounced regional differences in the response of the GI microbiome to external factors, which should be considered in future studies investigating the human microbiome. Electronic supplementary material The online version of this article (doi:10.1007/s00394-015-0966-2) contains supplementary material, which is available to authorized users.

Two new mononuclear Ni(II) complexes, namely [Ni(HL)(SCN)2(H2O)] (1) and [Ni(HL)(SCN)2(4,4′-bipy)] (2) [HL = 2-[(2-piperazin-1-yl-ethylimono)-methyl]phenol] have been synthesized and structurally characterized. The uncoordinated nitrogen atom of the piperazine moiety is protonated to provide electrical neutrality to the system. The configuration of the isothiocyanato ligands is different in the two complexes, cis located in 1 and trans in complex 2. The change of configuration of the isothiocyanato groups on addition of the neutral spacer 4,4-bipyridine to complex 1 has been monitored by conductometric and FTIR spectral studies and the most probable mechanistic pathway has been proposed. DFT calculations have been performed and the outcome corroborates well with the experimental facts. The anticancer activity of the two Ni(II) complexes has been evaluated in human cervical (HeLa) cancer and breast cancer (MCF-7) cell lines. The cytotoxic effects of these complexes were determined by an MTT assay. The fluorescent intensity obtained in HeLa cells reveals the generation of ROS by the complexes using DCFH-DA (2,7-dichlorofluorescein diacetate) dye. The apoptotic cell death was determined by fluorescent staining with acridine orange and ethidium bromide, which confirmed the presence of apoptotic cells. Further, flow cytometry analysis was done, suggesting an arrest of the cell cycle in the S phase of the HeLa cells. Although a detailed molecular mechanism for the anticancer activity of the two complexes was not ascertained, the experimental results suggest that both complexes are effective anticancer agents, with complex 2 seeming to be more promising. The result also indicates that the apoptotic cell death in the cancer cell might be triggered by the process of ROS generation with the complexes. Thermo gravimetric analysis of two complexes hints that NiO particles are the final product after decomposition of the complexes at 600 °C.

Neurofibromatosis Type 2 (NF2) is an autosomal disorder caused by mutations of the NF2 gene. More than half of all NF2 patients have unaffected parents and carry de novo mutations, which may be of prezygotic or postzygotic origin. The latter can result in mosaicism, which is relatively common in NF2 patients. Previous studies indicated that, in 50% of patients with mosaic NF2 mutations, the mutant allele is only detectable by Sanger sequencing of PCR products amplified from tumor tissue but not from blood samples. In order to establish a highly sensitive method that has the power to detect low levels of NF2 mutant alleles from blood samples of mosaic NF2 patients, we performed ultra deep sequencing and calculated the percentage of mutant and wildtype NF2 alleles. The mutant allele frequencies detected ranged from 2.6% to 19.7%. In three patients, however, the NF2 mutation previously identified in tumor tissue was not identified in blood samples by means of deep sequencing, suggesting absence of mutant cells in the blood. Remarkably, we observed a correlation between the age at onset of the disease and the mutant allele frequency. Our study indicates that ultra deep sequencing is an effective and highly sensitive method to determine the mutant allele frequency in patients with mosaic NF2 gene mutations, which enables extended phenotype/correlations in these patients. © 2015 Wiley Periodicals, Inc.

Regulatory T cells (Tregs) use different mechanisms to exert their suppressive function, among them the conversion of ATP to adenosine initiated by the ectonucleotidase CD39. In humans, the expression of CD39 on Tregs shows a high interindividual variation, and is especially high at sites of inflammation, like the synovia of patients with arthritis. How CD39 expression is regulated, and the functional consequences of different levels of CD39 expression is not known. We show here that stimulation of CD39 − Tregs results in a modest upregulation of CD39, which cannot explain the high levels observed in many donors. Moreover, CD39 + Tregs are present in naive compartments such as cord blood and thymus, and the individual frequency of CD39 + Tregs remains stable over time, suggesting inherent regulation of CD39 expression. Indeed, we show that a single nucleotide polymorphism in the CD39 gene determines expression levels in Tregs. CD39 + Tregs suppress T cell proliferation and inflammatory cytokine production more efficiently than CD39 − Tregs. Accordingly, Tregs from donors with the GG (high CD39) genotype have a higher capacity to suppress IFN-γ and IL-17 production by effector cells than Tregs from AA (low CD39) donors. Our study demonstrates that the expression of CD39 in Tregs is primarily genetically driven, and this may determine interindividual differences in the control of inflammatory responses.

Peptides presented on major histocompatibility complex (MHC) class I molecules are generated via cytosolic proteolysis. However, the nature of the endogenous peptide precursors and the intracellular processing steps preceding protein degradation remain poorly defined. Here, we assessed whether ubiquitination is an essential signal for proteasomal cleavage of antigen substrates in human cells. Conversion into antigenic peptides occurred in the absence of any detectable N-terminal ubiquitination of the model antigens, and did not require the presence of any of the four types, nor a minimum number of ubiquitinatable amino acids within the antigen substrate. However, the knockdown of ubiquitin, expression of a lysine 48 (K48) ubiquitin mutant, or inhibition of proteasome-associated deubiquitinases significantly impaired antigen presentation. The results presented here are consistent with a model in which the binding of the antigen substrate by an adaptor protein leads to its K48-polyubiquitination and the subsequent delivery of the antigen cargo for degradation by the 26S proteasome. Altogether, these findings show an important but indirect role of K48-polyubiquitination in preproteasomal antigen sampling.

Astroecology concerns the relations between life and space resources, and cosmo-ecology extrapolates these relations to cosmological scales. Experimental astroecology can quantify the amounts of life that can be derived from space resources. For this purpose, soluble carbon and electrolyte nutrients were measured in asteroid/meteorite materials. Microorganisms and plant cultures were observed to grow on these materials, whose fertilities are similar to productive agricultural soils. Based on measured nutrient contents, the 1022 kg carbonaceous asteroids can yield 1018 kg biomass with N and P as limiting nutrients (compared with the estimated 1015 kg biomass on Earth). These data quantify the amounts of life that can be derived from asteroids in terms of time-integrated biomass [BIOTAint = biomass (kg) × lifetime (years)], as 1027 kg-years during the next billion years of the Solar System (a thousand times the 1024 kg-years to date). The 1026 kg cometary materials can yield biota 10 000 times still larger. In the galaxy, potential future life can be estimated based on stellar luminosities. For example, the Sun will develop into a white dwarf star whose 1015 W luminosity can sustain a BIOTAint of 1034 kg-years over 1020 years. The 1012 main sequence and white and red dwarf stars can sustain 1046 kg-years of BIOTAint in the galaxy and 1057 kg-years in the universe. Life has great potentials in space, but the probability of present extraterrestrial life may be incomputable because of biological and ecological complexities. However, we can establish and expand life in space with present technology, by seeding new young solar systems. Microbial representatives of our life-form can be launched by solar sails to new planetary systems, including extremophiles suited to diverse new environments, autotrophs and heterotrophs to continually form and recycle biomolecules, and simple multicellulars to jump-start higher evolution. These programs can be motivated by life-centered biotic ethics that seek to secure and propagate life. In space, life can develop immense populations and diverse new branches. Some may develop into intelligent species that can expand life further in the galaxy, giving our human endeavors a cosmic purpose.

Endocarditis presenting with invasion of the myocardium by gas-forming organisms is rare, previously limited to Escherichia coli and Clostridia species. We describe a case of native valve emphysematous endocarditis presenting with stroke and ultimately leading to left ventricular rupture and

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). Fil: Mautner, Martín E.. Biodynamics SRL; Argentina Fil: Perez Santangelo, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Torcuato Di Tella; Argentina Fil: Corti Bielsa, Rodrigo M.. Biodynamics SRL; Argentina Fil: Sala, Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Ginart, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Fil: Corach, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina

The Cu(II) complexes [Cu(bpdmpz)Cl]ClO4 (1), [Cu(bdmpzp)Cl]ClO4 (2-ClO4), [Cu(bdmpzp)Cl]PF6 (2-PF6) and [Cu(tdmpza)Cl]ClO4 (3), bpdmpzp=[bis[((2-pyridylmethyl)-di(3,5-dimethyl-1H-pyrazolyl)methyl)]amine, bdmpzp=[bis((di(3,5-dimethyl-1H-pyrazolyl)methyl)-(2-pyridylmethyl)]amine and tdmpza=tris[di(3,5-dimethyl-1H-pyrazolyl)-methyl)]amine were synthesized and characterized by elemental analysis, magnetic and conductivity measurements, electrospray-ionization mass spectrometry, infrared and electronic spectroscopy, and X-ray crystallography. The magnetic properties of the complexes, measured at variable temperature, revealed weak antiferromagnetic intermolecular interactions. The cytotoxicity of the complexes 1, 2-ClO4, 3, and 4 ([Cu(bedmpzp)Cl]PF6, where bedmpzp=[bis(3,5-dimethyl-1H-pyrazol-1-yl-1-ethyl)-(2-pyridylmethyl)]amine), was investigated against four human cancer cell lines: A2780 (ovarian), A2780R (cisplatin-resistant variant), HOS (aggressive bone tumors), CaCo2 (epithelial colorectal adenocarcinoma) and on healthy human hepatocytes. The complex 4 was the most cytotoxic one, with IC50=1.4μM (A2780), 8.3μM (A2780R), 4.7μM (HOS) and 10.8μM (CaCo2). The mass spectrometry-based interaction studies, involving selected sulfur-containing biomolecules and small model proteins, revealed pro-oxidant effects of complexes 1 and 4 and differences in stability of both complexes in the mixtures containing the model protein cytochrome c after 24h incubation, complex 1 formed 1:1 adduct, the formation of which was accompanied by the loss of one dimethylpyrazole pendant arm from the bpdmpz ligand, while the complex 4 composition remained intact and the complex formed both 1:1 and 1:2 adducts (cytochrome c vs. Cu(II)-complex).

Nearly half of the world's population is infected with Helicobacter pylori. Clinical manifestations of this infection range from gastritis and peptic ulcers to gastric adenocarcinoma and lymphoma. Due to the emerging of antibiotic resistant strains and poor patient compliance of the antibiotic therapy, there is increasing interest in the development of a protective vaccine against H. pylori infection. The bacterial protein FliD forms a capping structure on the end of each flagellum which is critical to prevent depolymerization and structural degradation. In this study, the potential of FliD as a prospective H. pylori subunit vaccine was assessed. For this purpose, immunogenicity and protective efficacy of recombinant FliD (rFliD) from H. pylori was evaluated in C57BL/6 mice. Purified rFliD was formulated with different adjuvants and administered via subcutaneous or oral route. Subcutaneous immunization with rFliD elicited predominantly mixed Th1 and Th17 immune responses, with high titers of specific IgG(1) and IgG(2a). Splenocytes of immunized mice exhibited strong antigen-specific memory responses, resulting in the secretion of high amounts of IFN-gamma and IL-17, and low levels of IL-4. Immunization with rFliD caused a significant reduction in H. pylori bacterial load relative to naive control mice (p < 0.001), demonstrating a robust protective effect. Taken together, these results suggest that subcutaneous vaccination with rFliD formulated with CpG or Addavax could be considered as a potential candidate for the development of a subunit vaccine against H. pylori infection.