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The discovery of the highly efficient site-specific nuclease system CRISPR–Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy 1 , 2 . The immunogenicity of Cas9 nuclease has been demonstrated in mice 3 , 4 . Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues 3 – 6 . S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease 7 , 8 . Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell–mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity. T cells that react to SpCas9 are observed in the peripheral blood mononuclear cells isolated from healthy humans. These findings may have implications for CRISPR–Cas9 therapeutics.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences. Aging: immune protein’s role in delayed bone fracture healing Neutralizing a key cytokine, a signaling protein affecting the immune system could rejuvenate the healing process following prolonged inflammatory responses to bone fractures in elderly patients. Healing patterns vary widely in the elderly following injuries such as bone fractures, and scientists now believe that a patient’s individual innate and adaptive immune profile directly affects the healing process. A short-lived pro-inflammatory response is needed to kickstart healthy healing, but a longer-lasting response can be damaging. In experiments on aged mouse models, the team led by Katharina Schmidt-Bleek at the Julius Wolff Institute in Berlin, Germany, demonstrated that high levels of the cytokine interleukin-22 impaired bone regeneration. Elevated interleukin-22 levels resulted from chronically elevated inflammation and inflammaging, prevalent in elderly patients. The team treated the mice to neutralize interleukin-22, which accelerated the healing process.

Patients suffering from musculoskeletal diseases must cope with a diminished quality of life and an increased burden on medical expenses. The interaction of immune cells and mesenchymal stromal cells during bone regeneration is one of the key requirements for the restoration of skeletal integrity. While stromal cells of the osteo-chondral lineage support bone regeneration, an excessive accumulation of cells of the adipogenic lineage is thought to promote low-grade inflammation and impair bone regeneration. Increasing evidence indicates that pro-inflammatory signaling from adipocytes is responsible for various chronic musculoskeletal diseases. This review aims to summarize the features of bone marrow adipocytes by phenotype, function, secretory features, metabolic properties and their impact on bone formation. In detail, the master regulator of adipogenesis and prominent diabetes drug target, peroxisome proliferator-activated receptor γ (PPARG), will be debated as a potential therapeutic approach to enhance bone regeneration. We will explore the possibilities of using clinically established PPARG agonists, the thiazolidinediones (TZDs), as a treatment strategy to guide the induction of a pro-regenerative, metabolically active bone marrow adipose tissue. The impact of this PPARG induced bone marrow adipose tissue type on providing the necessary metabolites to sustain osteogenic-as well as beneficial immune cells during bone fracture healing will be highlighted.

Background Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC . Results The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5′-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC -overexpression mutant. According to LC–MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. Conclusion Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.

In a subset of children and adolescents, SARS-CoV-2 infection induces a severe acute hyperinflammatory shock 1 termed multisystem inflammatory syndrome in children (MIS-C) at four to eight weeks after infection. MIS-C is characterized by a specific T cell expansion 2 and systemic hyperinflammation 3 . The pathogenesis of MIS-C remains largely unknown. Here we show that acute MIS-C is characterized by impaired reactivation of virus-reactive memory T cells, which depends on increased serum levels of the cytokine TGFβ resembling those that occur during severe COVID-19 (refs. 4 , 5 ). This functional impairment in T cell reactivity is accompanied by the presence of TGFβ-response signatures in T cells, B cells and monocytes along with reduced antigen-presentation capabilities of monocytes, and can be reversed by blocking TGFβ. Furthermore, T cell receptor repertoires of patients with MIS-C exhibit expansion of T cells expressing TCRVβ21.3, resembling Epstein–Barr virus (EBV)-reactive T cell clones capable of eliminating EBV-infected B cells. Additionally, serum TGFβ in patients with MIS-C can trigger EBV reactivation, which is reversible with TGFβ blockade. Clinically, the TGFβ-induced defect in T cell reactivity correlates with a higher EBV seroprevalence in patients with MIS-C compared with age-matched controls, along with the occurrence of EBV reactivation. Our findings establish a connection between SARS-CoV-2 infection and COVID-19 sequelae in children, in which impaired T cell cytotoxicity triggered by TGFβ overproduction leads to EBV reactivation and subsequent hyperinflammation. Multisystem inflammatory syndrome following SARS-CoV-2 infection results from increased serum levels of TGFβ, which impairs the reactivation of virus-specific T cells.

The climate impact of air traffic is to a large degree caused by changes in cirrus cloudiness resulting from the formation of contrails. Contrail cirrus radiative forcing is expected to increase significantly over time due to the large projected increases in air traffic. We use ECHAM5-CCMod, an atmospheric climate model with an online contrail cirrus parameterization including a microphysical two-moment scheme, to investigate the climate impact of contrail cirrus for the year 2050. We take into account the predicted increase in air traffic volume, changes in propulsion efficiency and emissions, in particular soot emissions, and the modification of the contrail cirrus climate impact due to anthropogenic climate change. Global contrail cirrus radiative forcing increases by a factor of 3 from 2006 to 2050, reaching 160 or even 180 mW m−2, which is the result of the increase in air traffic volume and a slight shift in air traffic towards higher altitudes. Large increases in contrail cirrus radiative forcing are expected over all of the main air traffic areas, but relative increases are largest over main air traffic areas over eastern Asia. The projected upward shift in air traffic attenuates contrail cirrus radiative forcing increases in the midlatitudes but reinforces it in the tropical areas. Climate change has an insignificant impact on global contrail cirrus radiative forcing, while regional changes are significant. Of the emission reductions it is the soot number emission reductions by 50 % that lead to a significant decrease in contrail cirrus optical depth and coverage, leading to a decrease in radiative forcing by approximately 15 %. The strong increase in contrail cirrus radiative forcing due to the projected increase in air traffic volume cannot be compensated for by the decrease in initial ice crystal numbers due to reduced soot emissions and improvements in propulsion efficiency.

Contrail cirrus are a major component of the climate forcing due to air traffic. For a given contrail cirrus cover, ice water content and ice crystal shape, their impact on radiation is dependent on the number and size of ice crystals. Here we use a global climate model to study the impact of a reduction in initially formed ice crystal numbers, as may be caused by reduced soot number emissions. We find that for reduced initial ice crystal numbers the ice water content is decreased and ice crystal sizes increased, leading to a reduction in contrail cirrus optical depth and doubling the fraction of contrail cirrus that cannot be detected by satellite remote sensing. Contrail cirrus lifetimes and coverage are strongly reduced leading to significant reductions in contrail cirrus radiative forcing. The global climate impact of contrail cirrus is nonlinearly dependent on the reduction in initial ice crystal numbers. A reduction in the initial ice crystal number of 80% leads to a decrease in contrail cirrus radiative forcing by 50%, whereas a twofold reduction leads to a decrease in radiative forcing by approximately 20%. Only a few contrail cirrus outbreaks explain a large percentage of the climate impact. The contrail cirrus climate impact can be effectively mitigated by reducing initial ice crystal concentrations in such outbreak situations. Our results are important for assessments dealing with mitigating the climate impact of aviation and discussions about the use of alternative fuels or lean combustion in aviation. Atmospheric science: alternative aviation fuel helps mitigate the contrail climate impact Reducing soot emissions from aircraft by changing the composition of aviation fuels can effectively mitigate the climate impact of contrail cirrus. Ulrike Burkhardt and colleagues from the German Aerospace Centre use a global climate model to estimate the impacts of reduced aircraft soot emissions on properties and the climate forcing of contrail cirrus. Under the low emission condition in which the number of initial ice crystals formed from soot particles is reduced by 80%—a level of reduction that could be obtained using a blend of biofuel and conventional jet fuels—the climate impact of contrail cirrus is estimated to be reduced by 50%. The mitigation effect occurs predominantly in weather conditions that are favourable for contrail cirrus outbreaks: with the same level of reduction in the initial ice crystal number, capturing 25% of those events can reduce 30% of the climate forcing caused by contrail cirrus.

Evidence from previous climate model simulations has suggested a potentially low efficacy of contrails to force global mean surface temperature changes. In this paper, a climate model with a state-of-the-art contrail cirrus representation is used for fixed sea surface temperature simulations in order to determine the effective radiative forcing (ERF) from contrail cirrus. ERF is expected to be a good metric for intercomparing the quantitative importance of different contributions to surface temperature and climate impact. Substantial upscaling of aviation density is necessary to ensure statistically significant results from our simulations. The contrail cirrus ERF is found to be less than 50% of the respective instantaneous or stratosphere adjusted radiative forcings, with a best estimate of roughly 35%. The reduction of ERF is much more substantial for contrail cirrus than it is for a CO₂ increase when both stratosphere adjusted forcings are of similar magnitude. Analysis of all rapid radiative adjustments contributing to the ERF indicates that the reduction is mainly induced by a compensating effect of natural clouds that provide a negative feedback. Compared to the CO₂ reference case, a less positive combined water vapor and lapse rate adjustment also contributes to a more distinct reduction of contrail cirrus ERF, but not as much as the natural cloud adjustment. Based on the experience gained in this paper, respective contrail cirrus simulations with interactive ocean will be performed as the next step toward establishing contrail cirrus efficacy. ERF results of contrail cirrus from other climate models equipped with suitable parameterizations are regarded as highly desirable.

Temporal aspects of ecosystem services have gained surprisingly little attention given that ecosystem service flows are not static but change over time. We present the first systematic review to describe and establish how studies have assessed temporal patterns in supply and demand of ecosystem services. 295 studies, 2% of all studies engaging with the ecosystem service concept, considered changes in ecosystem services over time. Changes were mainly characterised as monotonic and linear (81%), rather than non-linear or through system shocks. Further, a lack of focus of changing ecosystem service demand (rather than supply) hampers our understanding of the temporal patterns of ecosystem services provision and use. Future studies on changes in ecosystem services over time should (1) more explicitly study temporal patterns, (2) analyse trade-offs and synergies between services over time, and (3) integrate changes in supply and demand and involve and empower stakeholders in temporal ecosystem services research.

Functionally diverse CD8+ T cells develop in response to antigenic stimulation with differing capacities to couple TCR engagement to downstream signals and functions. However, mechanisms of diversifying TCR signaling are largely uncharacterized. Here we identified two alternative splice variants of scaffold protein Dlg1, Dlg1AB and Dlg1B, that diversify signaling to regulate p38-dependent and -independent effector functions in CD8+ T cells. Dlg1AB, but not Dlg1B associated with Lck, coupling TCR stimulation to p38 activation and proinflammatory cytokine production. Conversely, both Dlg1AB and Dlg1B mediated p38-independent degranulation. Degranulation depended on a Dlg1 fragment containing an intact Dlg1SH3-domain and required the SH3-ligand WASp. Further, Dlg1 controlled WASp activation by promoting TCR-triggered conformational opening of WASp. Collectively, our data support a model where Dlg1 regulates p38-dependent proinflammatory cytokine production and p38-independent cytotoxic granule release through the utilization of alternative splice variants, providing a mechanism whereby TCR engagement couples downstream signals to unique effector functions in CD8+ T cells.