Linear interaction between replication and transcription shapes DNA break dynamics at recurrent DNA break Clusters.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.

Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

A type 1 immunity-restricted promoter of the IL-33 receptor gene directs antiviral T-cell responses.

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33-ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33-ST2 axis in infections and inflammatory diseases.

Linear Interaction Between Replication and Transcription Shapes DNA Break Dynamics at Recurrent DNA Break Clusters.

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

Formation and Reactivity of heavy Group 14-15 bonds.

A series of low-valent Group 14-15 compounds were obtained starting from [(Dipp2 NacNac)MCl] (M=Ge-Pb) (I-III) (Dipp2 NacNac=HC{C(Me)N(Dipp)}2 ) and M'E(SiMe3 )2 (M'=Li, E=As; M'=K, E=Sb, Bi) (IV-VI). In the course of this investigations we were able to fully characterize all permutations except Pb-Bi for compounds of the composition [(Dipp2 NacNac)ME(SiMe3 )2 ] (1E : M=Ge, 2E : M=Sn, 3E : M=Pb). Thus, we report the first low valent tetrelene with Sn-Bi bond. All isolated compounds, were examined by NMR spectroscopy, IR spectroscopy and except compound 1As by X-ray structure analysis. Moreover, were examined UV-Vis spectroscopy and investigated the reactivity of these compounds towards different substrates in more detail. Starting with the compound [(Dipp2 NacNac)SnAs(SiMe3 )2 ] (2As ), the reaction with red selenium yields [(Dipp2 NacNac)Sn-Se-As(SiMe3 )2 ] (4) which exhibits a Sn-Se-As chain.

Laser-Induced Au Catalyst Generation for Tailored ZnO Nanostructure Growth.

ZnO nanostructures, semiconductors with attractive optical properties, are typically grown by thermal chemical vapor deposition for optimal growth control. Their growth is well investigated, but commonly results in the entire substrate being covered with identical ZnO nanostructures. At best a limited, binary growth control is achieved with masks or lithographic processes. We demonstrate nanosecond laser-induced Au catalyst generation on Si(100) wafers, resulting in controlled ZnO nanostructure growth. Scanning electron and atomic force microscopy measurements reveal the laser pulse's influence on the substrate's and catalyst's properties, e.g., nanoparticle size and distribution. The laser-induced formation of a thin SiO2-layer on the catalysts plays a key role in the subsequent ZnO growth mechanism. By tuning the irradiation parameters, the width, density, and morphology of ZnO nanostructures, i.e., nanorods, nanowires, and nanobelts, were controlled. Our method allows for maskless ZnO nanostructure designs locally controlled on Si-wafers.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Vectored antibody gene delivery restores host B and T cell control of persistent viral infection.

Passive antibody therapy and vectored antibody gene delivery (VAGD) in particular offer an innovative approach to combat persistent viral diseases. Here, we exploit a small animal model to investigate synergies of VAGD with the host's endogenous immune defense for treating chronic viral infection. An adeno-associated virus (AAV) vector delivering the lymphocytic choriomeningitis virus (LCMV)-neutralizing antibody KL25 (AAV-KL25) establishes protective antibody titers for >200 days. When therapeutically administered to chronically infected immunocompetent wild-type mice, AAV-KL25 affords sustained viral load control. In contrast, viral mutational escape thwarts therapeutic AAV-KL25 effects when mice are unable to mount LCMV-specific antibody responses or lack CD8+ T cells. VAGD augments antiviral germinal center B cell and antibody-secreting cell responses and reduces inhibitory receptor expression on antiviral CD8+ T cells. These results indicate that VAGD fortifies host immune defense and synergizes with B cell and CD8 T cell responses to restore immune control of chronic viral infection.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

Heterologous arenavirus vector prime-boost overrules self-tolerance for efficient tumor-specific CD8 T cell attack.

Therapeutic vaccination regimens inducing clinically effective tumor-specific CD8+ T lymphocyte (CTL) responses are an unmet medical need. We engineer two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus, for therapeutic cancer vaccination. In mice, life-replicating vector formats of these two viruses delivering a self-antigen in a heterologous prime-boost regimen induce tumor-specific CTL responses up to 50% of the circulating CD8 T cell pool. This CTL attack eliminates established solid tumors in a significant proportion of animals, accompanied by protection against tumor rechallenge. The magnitude of CTL responses is alarmin driven and requires combining two genealogically distantly related arenaviruses. Vector-neutralizing antibodies do not inhibit booster immunizations by the same vector or by closely related vectors. Rather, CTL immunodominance hierarchies favor vector backbone-targeted responses at the expense of self-reactive CTLs. These findings establish an arenavirus-based immunotherapy regimen that allows reshuffling of immunodominance hierarchies and breaking self-directed tolerance for efficient tumor control.

Correction to: Lymphocytic choriomeningitis virus meningitis after needlestick injury: a case report.

[This corrects the article DOI: 10.1186/s13756-019-0524-4.].

Lymphocytic choriomeningitis virus meningitis after needlestick injury: a case report.

Background: Needlestick accidents while handling of infectious material in research laboratories can lead to life-threatening infections in laboratory personnel. In laboratories working with the lymphocytic choriomeningitis virus (LCMV), the virus can be transmitted to humans through needlestick injury and lead to serious acute illness up to meningitis. Case presentation: We report of a case of LCMV meningitis in a laboratory worker who sustained a penetrating needlestick injury with a LCMV-contaminated hollow needle whilst disposing of a used syringe into the sharps waste bin. Four days after needlestick injury the laboratory worker developed a systemic disease: 11 days after exposure, she was diagnosed with meningitis with clinical signs and symptoms of meningismus, photophobia, nausea and vomiting, requiring hospitalisation. The PCR was positive for LCMV from the blood sample. 18 days after exposure, seroconversion confirmed the diagnosis of LCMV-induced meningitis with an increase in specific LCMV-IgM antibodies to 1:10'240 (day 42: 1:20'480). Ten weeks after exposure, a follow-up titre for IgM returned negative, whereas IgG titre increased to 1:20'480. Conclusions: This is the first case report of a PCR-documented LCMV meningitis, coupled with seroconversion, following needlestick injury. It highlights the importance of infection prevention practices that comprise particularly well established safety precaution protocols in research laboratories handling this pathogenic virus, because exposure to even a small amount of LCMV can lead to a severe, life-threatening infection.

Residual LCMV antigen in transiently CD4+ T cell-depleted mice induces high levels of virus-specific antibodies but only limited B-cell memory.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8+ T cells independently of CD4+ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4+ T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4+ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4+ T cell depletion was considerably reduced. Moreover, mice depleted of CD4+ T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.

Drug reaction with eosinophilia and systemic symptoms after daclizumab therapy.

OBJECTIVE: To report on 2 women with multiple sclerosis (MS) who developed severe neurologic deterioration and a drug reaction with eosinophilia and systemic symptoms (DRESS) after treatment with 2 and 4 subcutaneous injections of daclizumab, respectively. METHODS: This report includes clinical, MRI, and histopathologic data. RESULTS: Daclizumab is a humanized monoclonal antibody that binds the interleukin-2 receptor. It was approved for the treatment of relapsing MS. DRESS is an immunologic reaction to various medications that is characterized by eosinophilia as well as cutaneous and visceral manifestations. Following daclizumab treatment, both patients showed fulminant neurologic deterioration along with blood eosinophilia and skin changes, and both fulfilled the clinical criteria for the diagnosis of DRESS. They presented with multiple gadolinium-enhancing supra- and infratentorial lesions, with lesions in the basal ganglia, mesencephalon, and cerebellum. Brain biopsies revealed a pronounced inflammatory infiltrate including numerous eosinophils infiltrating demyelinating lesions, a feature that is atypical for MS but compatible with DRESS. In addition, numerous plasma cells and changes reminiscent of vasculitis were evident. CONCLUSIONS: Neurologic deterioration and DRESS occurred as severe adverse drug effects of daclizumab treatment. Early diagnosis and treatment of DRESS are essential because it is associated with complications such as new autoimmune diseases and liver failure, and may even be lethal. Because of its potential serious side effects, daclizumab was recently suspended for use in the European Union.

Neurofeedback of Slow Cortical Potentials in Children with Attention-Deficit/Hyperactivity Disorder: A Multicenter Randomized Trial Controlling for Unspecific Effects.

Background: Neurofeedback (NF) in children with attention-deficit/hyperactivity disorder (ADHD) has been investigated in a series of studies over the last years. Previous studies did not unanimously support NF as a treatment in ADHD. Most studies did not control for unspecific treatment effects and did not demonstrate that self-regulation took place. The present study examined the efficacy of NF in comparison to electromyographic (EMG) feedback to control for unspecific effects of the treatment, and assessed self-regulation of slow cortical potentials (SCPs). Methods: A total of 150 children aged 7-9 years diagnosed with ADHD (82% male; 43% medicated) were randomized to 25 sessions of feedback of SCPs (NF) or feedback of coordination of the supraspinatus muscles (EMG). The primary endpoint was the change in parents' ratings of ADHD core symptoms 4 weeks after the end of treatment compared to pre-tests. Results: Children in both groups showed reduced ADHD-core symptoms (NF 0.3, 95% CI -0.42 to -0.18; EMG 0.13, 95% CI -0.26 to -0.01). NF showed a significant superiority over EMG (treatment difference 0.17, 95% CI 0.02-0.3, p = 0.02). This yielded an effect size (ES) of d = 0.57 without and 0.40 with baseline observation carried forward (BOCF). The sensitivity analysis confirmed the primary result. Successful self-regulation of brain activity was observed only in NF. As a secondary result teachers reported no superior improvement from NF compared to EMG, but within-group analysis revealed effects of NF on the global ADHD score, inattention, and impulsivity. In contrast, EMG feedback did not result in changes despite more pronounced self-regulation learning. Conclusions: Based on the primary parent-rated outcome NF proved to be superior to a semi-active EMG feedback treatment. The study supports the feasibility and efficacy of NF in a large sample of children with ADHD, based on both specific and unspecific effects. Trial Register: Current controlled trials ISRCTN76187185, registered 5 February 2009.

Role of HYAL1 expression in primary breast cancer in the formation of brain metastases.

BACKGROUND: The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. METHODS: Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. RESULTS: Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p = 0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p = 0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. CONCLUSIONS: In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Near-infrared spectroscopy (NIRS) neurofeedback as a treatment for children with attention deficit hyperactivity disorder (ADHD)-a pilot study.

In this pilot study near-infrared spectroscopy (NIRS) neurofeedback was investigated as a new method for the treatment of Attention Deficit-/Hyperactivity Disorder (ADHD). Oxygenated hemoglobin in the prefrontal cortex of children with ADHD was measured and fed back. 12 sessions of NIRS-neurofeedback were compared to the intermediate outcome after 12 sessions of EEG-neurofeedback (slow cortical potentials, SCP) and 12 sessions of EMG-feedback (muscular activity of left and right musculus supraspinatus). The task was either to increase or decrease hemodynamic activity in the prefrontal cortex (NIRS), to produce positive or negative shifts of SCP (EEG) or to increase or decrease muscular activity (EMG). In each group nine children with ADHD, aged 7-10 years, took part. Changes in parents' ratings of ADHD symptoms were assessed before and after the 12 sessions and compared within and between groups. For the NIRS-group additional teachers' ratings of ADHD symptoms, parents' and teachers' ratings of associated behavioral symptoms, childrens' self reports on quality of life and a computer based attention task were conducted before, 4 weeks and 6 months after training. As primary outcome, ADHD symptoms decreased significantly 4 weeks and 6 months after the NIRS training, according to parents' ratings. In teachers' ratings of ADHD symptoms there was a significant reduction 4 weeks after the training. The performance in the computer based attention test improved significantly. Within-group comparisons after 12 sessions of NIRS-, EEG- and EMG-training revealed a significant reduction in ADHD symptoms in the NIRS-group and a trend for EEG- and EMG-groups. No significant differences for symptom reduction were found between the groups. Despite the limitations of small groups and the comparison of a completed with two uncompleted interventions, the results of this pilot study are promising. NIRS-neurofeedback could be a time-effective treatment for ADHD and an interesting new option to consider in the treatment of ADHD.