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In agricultural landscapes, arable plants are negatively affected by management intensification. These species can fulfill various ecosystem functions, such as biological pest control, by supporting predators. The ecosystem functions of common plant species are widely known. By contrast, the contribution of rare arable plants (RAPs) to biocontrol in cereal fields remains poorly understood. This study investigated the effect of RAPs on biocontrol potential. We compared cropped plots with and without sowing of rare and threatened arable plant species and investigated the effects on cereal aphids and their antagonistic predators, active hunting, and web-building spiders, as well as carnivorous/omnivorous carabids. We counted the total number of aphids on cereal shoots and trapped ground-dwelling arthropods on an experimental field and on 10 agricultural farms in the vicinity of Munich, Germany, in 2018 and 2019. The effects of the presence of RAPs were analyzed using linear mixed-effect models, whereas cover of RAPs was analyzed using structural equation models. Linear models revealed that the presence of RAPs did not significantly affect the aphid density and the activity densities of spiders and carabids. Structural equation models revealed direct negative effects of RAP cover on aphid density. However, no indirect effects via the predators of aphids were detected. Direct negative effects of active hunting spiders on aphids were determined, but not of the other potential predators. Our results suggest that RAPs may impact aphid infestation, however, the activity density of spiders and carabids were unsuitable indicators for such interactions.

Background Malignant peritoneal mesothelioma (MPeM) is clinically distinct and less studied than malignant pleural mesothelioma. We report the genomic and immunophenotypic features of a prospectively collected MPeM cohort. Methods Next-generation sequencing (NGS) was performed on MPeM tumors. Genomic near-haploidization (GNH) was assessed. WT1, BAP1, mesothelin, VISTA, and PD-L1 were examined by immunohistochemistry (IHC) when tissue was available. Overall survival (OS) was stratified by selected genomic and IHC features. Results Fifty consented patients with MPeM (45 epithelioid, 5 non-epithelioid) demonstrated common alterations in BAP1 (60%; 30/50), NF2 (24%; 12/50) SETD2 (22%; 11/50), and TP53 (16%; 8/50). Seventy-six percent (38/50) of specimens were evaluable for allele-specific copy number analysis; 8% (3/38) had GNH. IHC positivity rates were 93% (37/40) for mesothelin, 96% (46/48) for WT1, 50% (19/38) for PD-L1 and 89% (34/38) for VISTA. BAP1 loss by IHC was observed in 76% (29/38), including five wildtype on NGS. Combining NGS and IHC for BAP1, OS was worse with alteration/loss compared to wildtype/retained in all patients (n=37 vs. 13, 43.8 vs. 117.3 months; p=0.04) Three of thirty patients had a pathogenic germline variant: POT1 I78T, MUTYH R109Y, BAP1 E402*. Conclusion MPeM has a distinct biology and genomic composition. CDKN2A/B alterations were rare in MPeM while BAP1, NF2, TP53, SETD2, LATS2 were common. BAP1 alteration/loss was associated with shorter survival when all patients were included. A notable minority of specimens had GNH associated with NF2, TP53, and SETDB1 mutations. Pathogenic germline mutations were found in 3 of 30 patients.

PURPOSE Tumor-intrinsic features may render large B-cell lymphoma (LBCL) insensitive to CD19-directed chimeric antigen receptor T cells (CAR-T). We hypothesized that TP53 genomic alterations are detrimental to response outcomes in LBCL treated with CD19-CAR-T. MATERIALS AND METHODS Patients with LBCL treated with CD19-CAR-T were included. Targeted next-generation sequencing was performed on pre–CAR-T tumor samples in a subset of patients. Response and survival rates by histologic, cytogenetic, and molecular features were assessed. Within a cohort of newly diagnosed LBCL with genomic and transcriptomic profiling, we studied interactions between cellular pathways and TP53 status. RESULTS We included 153 adults with relapsed or refractory LBCL treated with CD19-CAR-T (axicabtagene ciloleucel [50%], tisagenlecleucel [32%], and lisocabtagene maraleucel [18%]). Outcomes echoed pivotal trials: complete response (CR) rate 54%, median overall survival (OS) 21.1 months (95% CI, 14.8 to not reached), and progression-free survival 6 months (3.4 to 9.7). Histologic and cytogenetic LBCL features were not predictive of CR. In a subset of 82 patients with next-generation sequencing profiling, CR and OS rates were comparable with the unsequenced cohort. TP53 alterations (mutations and/or copy number alterations) were common (37%) and associated with inferior CR and OS rates in univariable and multivariable regression models; the 1-year OS in TP53-altered LBCL was 44% (95% CI, 29 to 67) versus 76% (65 to 89) in wild-type ( P = .012). Transcriptomic profiling from a separate cohort of patients with newly diagnosed lymphoma (n = 562) demonstrated that TP53 alterations are associated with dysregulation of pathways related to CAR-T-cell cytotoxicity, including interferon and death receptor signaling pathway and reduced CD8 T-cell tumor infiltration. CONCLUSION TP53 is a potent tumor-intrinsic biomarker that can inform risk stratification and clinical trial design in patients with LBCL treated with CD19-CAR-T. The role of TP53 should be further validated in independent cohorts.

We estimated HLA haplotype frequencies based on individuals homozygous for 4, 5 or 6 loci. Validation of our approach using a sample of over 3.4 million German individuals was successful. Compared to an expectation-maximization algorithm, the errors were larger. However, our approach allows the unequivocal detection of rare haplotypes.

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

The growth of ZnO clusters supported by ZnO‐bilayers on Ag(111) and the interaction of these oxide nanostructures with water have been studied by a multi‐technique approach combining temperature‐dependent infrared reflection absorption spectroscopy (IRRAS), grazing‐emission X‐ray photoelectron spectroscopy, and density functional theory calculations. Our results reveal that the ZnO bilayers exhibiting graphite‐like structure are chemically inactive for water dissociation, whereas small ZnO clusters formed on top of these well‐defined, yet chemically passive supports show extremely high reactivity ‐ water is dissociated without an apparent activation barrier. Systematic isotopic substitution experiments using H2 16O/D2 16O/D2 18O allow identification of various types of acidic hydroxyl groups. We demonstrate that a reliable characterization of these OH‐species is possible via co‐adsorption of CO, which leads to a red shift of the OD frequency due to the weak interaction via hydrogen bonding. The theoretical results provide atomic‐level insight into the surface structure and chemical activity of the supported ZnO clusters and allow identification of the presence of under‐coordinated Zn and O atoms at the edges and corners of the ZnO clusters as the active sites for H2O dissociation., Chemical nature of small ZnO clusters: The combined infrared reflection absorption spectroscopy (IRRAS) and theoretical results reveal that the Ag(111)‐supported graphitic ZnO bilayers are chemically inactive. Significant activity is observed when small ZnO clusters are supported by these bilayers. This apparently barrier‐free water dissociation takes place at under‐coordinated Zn and O atoms at the edges and corners of the ZnO nanostructures. Various acidic hydroxyl species are identified by isotopic substitution experiments and the formation of weak H bonds to coadsorbed CO molecules.

Maintaining an optimal pH that simultaneously supports plants, fish, and nitrifying microorganisms is a challenge in recirculating aquaponics systems as nitrification is optimal at a slightly alkaline pH and plant growth is optimal at a slightly acidic pH. Freshwater fish tolerate pH > 5.5. Our aim was to adapt a microbial inoculum for a recirculating aquaponics system from an operational pH of 7.6 to 5.6, compare nitrification activity and production of N2O, and describe changes in the adapted versus unadapted microbial communities. Four adaptation strategies were tested; our results indicated that a gradual reduction from pH 7.6 to 5.6, along with a gradual reduction followed by a gradual return of available ammonium, was the best strategy resulting in retention of 81% nitrification activity at pH 5.6 compared to pH 7.6. 16S rRNA gene amplicon sequencing and qPCR enumeration of nitrification-related genes showed that the composition of pH 5.6 adapted microbial communities from all four adaptation strategies was similar to one another and distinct from those operating at pH 7.6, with enrichment of comammox clade B bacteria over ammonia-oxidizing bacteria and thaumarchaeota. N2O production of the pH 5.6 adapted microbial communities was below detection in all adaptation experiments, likely due to the increased proportion of comammox bacteria. Aquaponics biofilters enriched with comammox bacteria and adapted to function at pH 5.6 can be a desirable inoculum for freshwater recirculating aquaponics systems to retain nitrification activity and improve crop yields.Key points• Microbial communities adapted from pH 7.6 to pH 5.6 retained 81% nitrification activity.• Microbial communities adapted from pH 7.6 to pH 5.6 were enriched in comammox bacteria.• Comammox-enriched microbial communities did not produce N2 O.

Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors. Significance: Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors. See related commentary by Aldea et al., p. 2674. This article is highlighted in the In This Issue feature, p. 2659

BACKGROUND Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. METHODS More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. RESULTS At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p

Physiological ageing and tumorigenesis are both associated with epigenomic alterations in human tissue cells, the most extensively investigated of which entails de novo cytosine methylation (i.e., hypermethylation) within the CpG dinucleotides of CpG islands. Genomic regions that become hypermethylated during tumorigenesis are generally believed to overlap regions that acquire methylation in normal tissues as an effect of ageing. To define the extension of this overlap, we analysed the DNA methylomes of 48 large-bowel tissue samples taken from women of different ages during screening colonoscopy: 18 paired samples of normal and lesional tissues from donors harbouring a precancerous lesion and 12 samples of normal mucosa from tumour-free donors. Each sample was subjected to targeted, genome-wide bisulphite sequencing of ~2.5% of the genome, including all CpG islands. In terms of both its magnitude and extension along the chromatin, tumour-associated DNA hypermethylation in these regions was much more conspicuous than that observed in the normal mucosal samples from older (vs. younger) tumour-free donors. 83% of the ageing-associated hypermethylated regions (n = 2501) coincided with hypermethylated regions observed in tumour samples. However, 86% of the regions displaying hypermethylation in precancerous lesions (n = 16,772) showed no methylation changes in the ageing normal mucosa. The tumour-specificity of this latter hypermethylation was validated using published sets of data on DNA methylation in normal and neoplastic colon tissues. This extensive set of genomic regions displaying tumour-specific hypermethylation represents a rich vein of putative biomarkers for the early, non-invasive detection of colorectal tumours in women of all ages.

Phytosulfokine (PSK) receptor signaling promotes root elongation, determines lateral root density, and maintains non-hair cell fate partially independent of TPST responsible for the activating sulfation of PSK., Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSK receptors (PSKRs) are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST; encoding tyrosylprotein sulfotransferase). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 169 PSK-regulated genes, several had functions in root growth and development, in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs, and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23), and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1, revealing unexpected synergistic effects of ligand and PSKR deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth, suggesting that PSKR1 may be active in the absence of sulfated ligands.

Background/aim: Kambô is a name for the secretion of the Giant Maki Frog ( Phyllomedusa bicolor), which has been used by indigenous cultures from the Amazonas basin and has recently become popular in alternative healing circles in Western countries, with a certain overlap with psychedelic self-exploration. Methods: We carried out an online-based survey in English (54.92%) and German investigating motivations for using Kambô, settings in which rituals take place, and subjective experiences during and after the application. Results: Participants ( n = 386, mean age: 38.08 years, (standard deviation = 9.95)) were well-educated individuals with an increased lifetime prevalence of the use of ayahuasca (67.88%). A plethora of motivations for using Kambô was reported, including general healing, detoxification and spiritual growth. Acute effects included severe physical reactions and mild psychoactive effects, most surprisingly, the feeling of being connected to the frog’s spirit (41.97%), whereas predominantly positive persisting psychological effects were reported. Few participants reported long-lasting physical (2.85%) or mental (1.81%) health problems which they attributed to Kambô. Of the participants, 87.31% reported an increase in personal well-being or life satisfaction, and 64.26% considered Kambô to have been at least of ‘very much’ spiritual significance for their lives. Conclusions: The majority of users claimed beneficial effects including more health-orientated behaviors, whereas only very few participants complained about new health problems which they ascribed to Kambô. In retrospect, Kambô was given a high personal and spiritual significance by many participants. Additional research is needed to determine in how far reported effects are modulated by setting and subjective expectations.

Tripartite motif containing 24 (TRIM24) is a multifunctional protein involved in p53 degradation, chromatin binding, and transcriptional modulation of nuclear receptors. Emerging research has revealed that upregulation of TRIM24 in numerous tumor types is linked to poor prognosis, attributing an important role to TRIM24 in tumor biology. In order to better understand the role of TRIM24 in prostate cancer, we analyzed its immunohistochemical expression on a tissue microarray containing >17,000 prostate cancer specimens. TRIM24 immunostaining was detectable in 61% of 15,321 interpretable cancers, including low expression in 46% and high expression in 15% of cases. TRIM24 upregulation was associated with high Gleason grade, advanced pathologic tumor stage, lymph node metastasis, higher preoperative prostate-specific antigen level, increased cell proliferation as well as increased genomic instability, and predicted prognosis independent of clinicopathologic parameters available at the time of the initial biopsy (all P

Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.

Objective: Anoctamin 7 (ANO7) is a calcium2+-dependent chloride ion channel protein. Its expression is restricted to prostate epithelial cells. The exact function is unknown. This study aimed to analyze ANO7 expression and its clinical significance in prostate cancer (PCa). Methods: ANO7 expression was assessed by immunohistochemistry in 17,747 clinical PCa specimens. Results: ANO7 was strongly expressed in normal prostate glandular cells but often less abundant in cancer cells. ANO7 staining was interpretable in 13,594 cancer tissues and considered strong in 34.4%, moderate in 48.7%, weak in 9.3%, and negative in 7.6%. Reduced staining was tightly linked to adverse tumor features [high classical and quantitative Gleason grade, lymph node metastasis, advanced tumor stage, high Ki67 labeling index, positive surgical margin, and early biochemical recurrence (P < 0.0001 each)]. The univariate Cox hazard ratio for prostate-specific antigen (PSA) recurrence after prostatectomy in patients with negative vs. strong ANO7 expression was 2.98 (95% confidence interval 2.61–3.38). The prognostic impact was independent of established pre- or postoperatively available parameters (P < 0.0001). Analysis of annotated molecular data showed that low ANO7 expression was linked to TMPRSS2:ERG fusions (P < 0.0001), elevated androgen receptor expression (P < 0.0001), as well as presence of 9 of 11 chromosomal deletions (P < 0.05 each). A particularly strong association of low ANO7 expression with phosphatase and tensin homolog (PTEN) deletion may indicate a functional relationship with the PTEN/AKT pathway. Conclusions: These data identify reduced ANO7 protein expression as a strong and independent predictor of poor prognosis in PCa. ANO7 measurement, either alone or in combination, might provide clinically useful prognostic information in PCa.

Deletion of chromosome 5q is common in prostate cancer and is linked to aggressive disease. Most previous studies focused on 5q21 where CHD1 is located, but deletion of mapping studies has identified a second deletion hotspot at 5q13. To clarify the prevalence and clinical relevance of 5q13 deletions and to determine the relative importance of 5q13 and 5q21 abnormalities, a tissue microarray containing samples from 12 427 prostate cancers was analyzed by fluorescence in situ hybridization. Deletion of 5q13 and 5q21 was found in 13.5% and 10%, respectively, of 7932 successfully analyzed cancers. Deletion was restricted to 5q13 in 49.4% and to 5q21 in 32.0% of cancers with a 5q deletion. Only 18.6% of 5q-deleted cancers had deletions of both loci. Both 5q13 and 5q21 deletions were significantly linked to advanced tumor stage, high Gleason grade, nodal metastasis and early biochemical recurrence (P < .005 each). Cancers with co-deletion of 5q13 and 5q21 had a worse prognosis than cancers with isolated 5q13 or 5q21 deletion (P = .0080). Comparison with TMPRSS2:ERG fusion status revealed that 5q21 deletions were tightly linked to ERG negativity (P < .0001) while 5q13 deletions were unrelated to the ERG status. In summary, 5q13 deletion and 5q21 deletion are common, but independent genomic alterations with different functional effects lead to aggressive prostate cancer.

Background Distant metastasis formation is the major clinical problem in prostate cancer (PCa) and the underlying mechanisms remain poorly understood. Our aim was to identify novel molecules that functionally contribute to human PCa systemic dissemination based on unbiased approaches. Methods We compared mRNA, microRNA (miR) and protein expression levels in established human PCa xenograft tumours with high (PC-3), moderate (VCaP) or weak (DU-145) spontaneous micrometastatic potential. By focussing on those mRNAs, miRs and proteins that were differentially regulated among the xenograft groups and known to interact with each other we constructed dissemination-related mRNA/miR and protein/miR networks. Next, we clinically and functionally validated our findings. Results Besides known determinants of PCa progression and/or metastasis, our interaction networks include several novel candidates. We observed a clear role of epithelial-to-mesenchymal transition (EMT) pathways for PCa dissemination, which was additionally confirmed by an independent human PCa model (ARCAP-E/-M). Two converging nodes, CD46 (decreasing with metastatic potential) and DDX21 (increasing with metastatic potential), were used to test the clinical relevance of the networks. Intriguingly, both network nodes consistently added prognostic information for patients with PCa whereas CD46 loss predicted poor outcome independent of established parameters. Accordingly, depletion of CD46 in weakly metastatic PCa cells induced EMT-like properties in vitro and spontaneous micrometastasis formation in vivo. Conclusions The clinical and functional relevance of the dissemination-related interaction networks shown here could be successfully validated by proof-of-principle experiments. Therefore, we suggest a direct pro-metastatic, clinically relevant role for the multiple novel candidates included in this study; these should be further exploited by future studies.

Background Vestibular symptoms are a frequent reason for presenting at the emergency department (ED). Underlying conditions range in severity from life-threatening to benign, but often remain undiagnosed despite extensive investigations. We aimed to identify clinical characteristics that are associated with ED consultations by patients with vestibular symptoms of unknown origin (VUO) and to quantify the ED resources consumed during the investigations. Methods This retrospective one-year, single-centre, cross-sectional study assessed ED consultations with patients whose chief complaint was ‘vestibular symptoms’. Data on risk factors, clinical characteristics, management and ED resources were extracted from the administrative database and medical records. Consultations were grouped according to the discharge diagnosis as either VUO or non-VUO. We determined clinical factors associated with VUO and compared ED resource consumption by the two patient groups using multivariable analysis. Results A total of 1599 ED consultations were eligible. Of these, 14.3% (n = 229) were consultations with patients with VUO. Clinical characteristics included in the final multivariable model to determine associations with VUO were sensory disorders, aural fullness, improvement at rest, absence of situational provocation, pre-existing neurological conditions, and age Conclusion One in seven emergency patients with vestibular symptoms is dismissed without a diagnosis. Clinical characteristics of VUO patients are distinct from patients in whom a diagnosis was made in the ED. VUO triggers higher ED resource consumption, which can be justified if appropriately indicated.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

Double unit cord blood (dCB) transplantation (dCBT) is associated with high engraftment rates but delayed myeloid recovery. We investigated adding haplo-identical CD34+ cells to dCB grafts to facilitate early haplo-identical donor-derived neutrophil recovery (optimal bridging) prior to CB engraftment. Seventy-eight adults underwent myeloablation with cyclosporine-A/mycophenolate mofetil immunoprophylaxis (no antithymocyte globulin, ATG). CB units (median CD34+ dose 1.1 × 105/kg/unit) had a median 5/8 unit-recipient human leukocyte antigen (HLA)-match. Haplo-identical grafts had a median CD34+ dose of 5.2 × 106/kg. Of 77 evaluable patients, 75 had sustained CB engraftment that was mediated by a dominant unit and heralded by dominant unit-derived T cells. Optimal haplo-identical donor-derived myeloid bridging was observed in 34/77 (44%) patients (median recovery 12 days). Other engrafting patients had transient bridging with second nadir preceding CB engraftment (20/77 (26%), median first recovery 12 and second 26.5 days) or no bridge (21/77 (27%), median recovery 25 days). The 2 (3%) remaining patients had graft failure. Higher haplo-CD34+ dose and better dominant unit-haplo-CD34+ HLA-match significantly improved the likelihood of optimal bridging. Optimally bridged patients were discharged earlier (median 28 versus 36 days). ATG-free haplo-dCBT can speed neutrophil recovery but successful bridging is not guaranteed due to rapid haplo-identical graft rejection.

The transcriptional coactivator YAP1 controls the balance between cell proliferation and apoptosis. YAP1 overexpression is linked to poor prognosis in many cancer types, yet its role in prostate cancer is unknown. Here, we applied YAP1 immunohistochemistry to a tissue microarray containing 17,747 clinical prostate cancer specimens. Cytoplasmic and nuclear YAP1 staining was seen in 81% and 63% of tumours. For both cytoplasmic and nuclear YAP1 staining, high levels were associated with advanced tumour stage, classical and quantitative Gleason grade, positive nodal stage, positive surgical margin, high KI67 labelling index, and early biochemical recurrence (p TMPRSS2:ERG fusion (p PTEN and 8p deletions (p

Monoclonal antibodies (mAbs) are increasingly exploited as vehicles for the targeted delivery of cytotoxic drugs. In antibody-drug conjugates (ADCs) antibodies specifically deliver cytotoxic compounds to cancer cells. Here, we present a technology for elevating the intracellular delivery of antibodies by the conjugation of tetrameric cell-penetrating peptides (tCPPs). The solid phase synthesis of tCPPs and their application in a chemical modification strategy for mAbs provides constructs that attain up to fourfold elevated internalization rates while retaining the mAbs target specificity. The antigen independent internalization is accompanied by beneficial pharmacokinetics limiting off-target accumulation. Applicability was proven for matuzumab, trastuzumab and the ADC Kadcyla®. Cytotoxicity studies of tCPP-conjugates of Kadcyla® resulted in a sixfold increased cytotoxicity proving the potential of chemical modification strategies to extend the applicability of biologicals. This constitutes a significant step towards next-generation antibody-based therapeutics.

In 2014, African swine fever (ASF) emerged in Latvia for the first time. The majority of cases appeared in wild boar, but the presence of ASF in these animals constitutes a permanent threat to domestic pig holdings. Recent studies have shown an increase in serologically positive and a decrease in PCR-positive ASF cases in wild boar, possibly indicating a decline of ASF incidence. We aimed to investigate the course of the ASF epidemic in wild boar in Latvia, thus attaining further insights into the ASF epidemiology in this country with the goal of assessing the stage of the epidemic. Latvian ASF surveillance data of wild boar were utilized to estimate the seroprevalence and ASF virus (ASFV) prevalence in the wild boar population. Prevalence estimates were obtained for both the eastern and western part of the country and in addition for the 2014/2015 to 2018/2019 hunting seasons. Moreover, prevalence estimates for three different age classes were calculated. An increase in serologically positive yet PCR-negative wild boar samples from active surveillance was identified over time. When comparing the age groups, wild boar younger than one year displayed the ASFV prevalence to be higher than the seroprevalence, whereas older animals shared higher seroprevalence estimates. These findings support the assumption that only a small proportion of affected animals survive an infection, leading to an accumulation of their numbers over time. As a result, ASF elimination in a country with an infected wild boar population could possibly be achieved, if effective wild boar population management and surveillance is maintained and combined with the detection and removal of wild boar carcasses to reduce the viral load in the environment. In addition, the wild boar population should be kept as small as possible to break the ASFV infection cycle.

BACKGROUND: PD-L1 expression is the only FDA-approved biomarker for immune checkpoint inhibitors (ICIs) in patients with lung adenocarcinoma, but sensitivity is modest. Understanding the impact of molecular phenotype, clinical characteristics, and tumor features on PD-L1 expression is largely unknown and may improve prediction of response to ICI. METHODS: We evaluated patients with lung adenocarcinoma in whom PD-L1 testing and targeted next-generation sequencing (MSK-IMPACT) was performed on the same tissue. Clinical and molecular features were compared across PD-L1 subgroups to examine how molecular phenotype associated with tumor PD-L1 expression. In patients treated with anti-PD-(L)1 blockade, we assessed how these interactions impacted efficacy. RESULTS: 1586 patients with lung adenocarcinoma had paired PD-L1 testing and targeted next-generation sequencing. PD-L1 negativity was more common in primary compared to metastatic samples (p

Protein phosphatase 1 nuclear-targeting subunit (PNUTS) is ubiquitously expressed and associates with PTEN and protein phosphatase 1 (PP1) to control its activity. The role of PNUTS overexpression has hardly been studied in cancer. In this study, we used immunohistochemistry to quantitate PNUTS expression on a tissue microarray containing 17,747 clinical prostate cancer specimens. As compared to normal prostate epithelium, PNUTS expression was often higher in cancer. Among 12,235 interpretable tumors, PNUTS staining was negative in 21%, weak in 34%, moderate in 35%, and strong in 10% of cases. High PNUTS expression was associated with higher tumor stage, classical and quantitative Gleason grade, nodal stage, surgical margin, Ki67 labeling index, and early biochemical recurrence (p<0.0001 each). PNUTS expression proved to be a moderate prognostic parameter with a maximal univariable Cox proportional hazard for PSA recurrence-free survival of 2.21 compared with 5.91 for Gleason grading. It was independent from established prognostic parameters in multivariable analysis. Comparison with molecular data available from earlier studies using the same TMA identified associations between high PNUTS expression and elevated androgen receptor expression (p<0.0001), presence of TMPRSS2:ERG fusion (p<0.0001), and 8 of 11 chromosomal deletions (3p13, 5q21, 8p21, 10q23, 12p13, 13q14, 16q24, and 17p13; p<0.05 each). Particularly strong associations with PTEN and 12p13 deletions (p<0.0001 each) may indicate a functional relationship, which has already been established for PNUTS and PTEN. PNUTS had no additional role on outcome in PTEN-deleted cancers. In conclusion, the results of our study identify high PNUTS protein levels as a predictor of poor prognosis possibly linked to increased levels of genomic instability. PNUTS measurement, either alone or in combination, might be of clinical utility in prostate cancers.

African swine fever (ASF) in wild boar remains a threat for the global pig industry. Therefore, surveillance is of utmost importance, not only to control the disease but also to detect new introductions as early as possible. Passive surveillance is regarded as the method of choice for an effective detection of ASF in wild boar populations. However, the relevance of wild boar killed through road traffic accidents (RTA) for passive surveillance seems to be unclear. Using comprehensive ASF wild boar surveillance data from Estonia and Latvia, the prevalence of ASF-infected wild boar was calculated and the probability of infection as measured by PCR compared for animals that were hunted, found dead, shot sick or killed in a RTA. The number of samples originating from wild boar killed in a RTA was low and so was the ASF prevalence in these animals. However, the reasons for this low number of RTA animals remain unknown. Therefore, we recommend to sample wild boar killed in a RTA to a greater extent, also to explore, if this approach can increase the detection probability, and to avoid missing disease introduction.

Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)–based analysis using the “Bethesda Panel” confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass.

Background: Immune checkpoint inhibitors (ICIs) are standard therapies for patients with advanced non-small-cell lung cancer (NSCLC) and a programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) ≥50%. Tumor mutation burden (TMB) also predicts response to ICIs but is often not available in real time for decision making in the first-line setting. Smoking exposure can be a proxy for TMB in NSCLC. The impact of smoking status on efficacy of PD-1 blockade in NSCLC patients with PD-L1 TPS ≥50% has not been well defined. Patients and methods: To investigate the relationship between smoking and activity of ICIs in NSCLC, we retrospectively studied 315 patients with NSCLC and PD-L1 TPS ≥50% at five USA academic medical centers. Objective response rates (ORRs), progression-free survival (PFS), and duration of response (DOR) were compared between never (10 pack-years) smokers. A subset of patients underwent next-generation sequencing to estimate TMB. Results: We identified 36 (11%) never, 42 (13%) light, and 237 (75%) heavy smokers with NSCLC and PD-L1 TPS ≥50% treated with ICIs. Objective responses were observed in 27%, 40%, and 40% of never, light, and heavy smokers, respectively (P = 0.180 never versus heavy; P = 1.000 light versus heavy). Median PFS and median DOR were numerically shorter in never and light smokers compared with heavy smokers (PFS 3.0 versus 4.0 versus 5.4 months; median DOR 6.9 versus 10.8 versus 17.8 months), but were not statistically different [PFS: hazard ratio (HR) 1.37, P = 0.135 and HR 1.24, P = 0.272; DOR: HR 1.92, P = 0.217 and HR 1.79, P = 0.141]. Conclusions: PD-(L)1 inhibitors are associated with antitumor activity in NSCLC with PD-L1 TPS ≥50% regardless of smoking status. Nevertheless, there is a signal of potentially decreased durability among never and light smokers that should be further evaluated. Distinct immunobiologic features may affect initial response versus durability of antitumor immunity to programmed cell death 1 (PD-1) blockade.

Drought and flooding are environmental extremes and major threats to crop production. Water uptake is achieved by plant roots which have to explore new soil spaces to alleviate water deficit during drought or to cope with water excess during flooding. Adaptation of the root system architecture helps plants cope with such extreme conditions and is crucial for plant health and survival. While for dicot plants the well studied model plant Arabidopsis thaliana has provided insight into the genetic and molecular regulation of the root system, less information is available for monocot species, which include the agronomically important cereal crops. Rice (Oryza sativa L.) is a semi-aquatic monocot plant that develops strong tolerance to flooding. Flooding tolerance of rice is closely linked to its adaptive root system. The functional root system of rice is mainly composed of crown roots and is shifted to nodal adventitious roots during flooding which allows rice to maintain oxygen supply to the roots and to survive longer periods of partial submergence as compared with other crops. Likewise, a number of drought-tolerance traits of rice are the result of an altered root system architecture. Hence, the structure of the root system adapts to, both, flooding and drought. Understanding the regulatory mechanisms that control root system adaptation to extreme environments is a key task for scientists to accelerate the breeding efforts for stress-tolerant crops. This review summarizes recently identified genes and molecular mechanisms that regulate root system architecture in rice in response to drought and flooding.

Background Buccal swab sampling constitutes an attractive noninvasive alternative to blood drawings for antibody serostatus assays. Here we describe a method to determine the cytomegalovirus immunoglobulin G (CMV IgG) serostatus from dried buccal swab samples. Methods Upon solubilization, CMV IgG is determined by an ELISA assay specifically adapted to cope with low IgG concentrations. The derived CMV titer is normalized against the total protein concentration to adjust for incorrectly or less efficiently sampled buccal swabs. Assay parameters were optimized on a set of 713 samples. Results Validation with 1784 samples revealed distinct results for > 80% of samples with 98.6% specificity and 99.1% sensitivity. Based on the analysis of 1.2 million samples we derived age- and sex-stratified CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile. To confirm accuracy of the assay in routine operation, the CMV status of 6518 donors was reassessed by independent laboratories based on conventional blood samples revealing 96.9% specificity and 97.4% sensitivity. Conclusions The assay accurately delivers the CMV IgG serostatus from dried buccal swab samples for > 80% of the participants. Thereby it provides a noninvasive alternative to plasma-based CMV monitoring for nondiagnostic purposes such as hematopoietic stem cell transplantation donor screening or population studies., We developed an assay to determine the CMV IgG status based on dried buccal swab samples. Assay validation on a set of 1784 samples revealed 99% sensitivity and specificity. We describe CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile.

Significance The cell of origin and the temporal order of oncogenic events in tumors play important roles for disease state. This is of particular interest for PCa due to its highly variable clinical outcome. However, these features are difficult to analyze in tumors. We established an in vitro murine PCa organoid model taking into account the cell of origin and the temporal order of events. We found that TIP5 primes luminal prostate cells for Pten-loss mediated oncogenic transformation whereas it is dispensable once the transformation is established. Cross-species transcriptomic analyses revealed a PTEN-loss gene signature that identified a set of aggressive tumors with PTEN-del, or low PTEN expression, and high-TIP5 expression. This paper provides a powerful tool to elucidate PCa mechanisms., Prostate cancer (PCa) is the second leading cause of cancer death in men. Its clinical and molecular heterogeneities and the lack of in vitro models outline the complexity of PCa in the clinical and research settings. We established an in vitro mouse PCa model based on organoid technology that takes into account the cell of origin and the order of events. Primary PCa with deletion of the tumor suppressor gene PTEN (PTEN-del) can be modeled through Pten-down-regulation in mouse organoids. We used this system to elucidate the contribution of TIP5 in PCa initiation, a chromatin regulator that is implicated in aggressive PCa. High TIP5 expression correlates with primary PTEN-del PCa and this combination strongly associates with reduced prostate-specific antigen (PSA) recurrence-free survival. TIP5 is critical for the initiation of PCa of luminal origin mediated by Pten-loss whereas it is dispensable once Pten-loss mediated transformation is established. Cross-species analyses revealed a PTEN gene signature that identified a group of aggressive primary PCas characterized by PTEN-del, high-TIP5 expression, and a TIP5-regulated gene expression profile. The results highlight the modeling of PCa with organoids as a powerful tool to elucidate the role of genetic alterations found in recent studies in their time orders and cells of origin, thereby providing further optimization for tumor stratification to improve the clinical management of PCa.

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies., Tumours that are deficient in mismatch-repair genes should, in theory, have higher evolvability. Here, the authors explore this theory in gastro-oesophageal tumours.

Objective Cold and warm water ear irrigation, also known as bithermal caloric testing, has been considered for over 100 years the ‘Gold Standard’ for the detection of peripheral vestibular hypofunction. Its discovery was awarded a Nobel Prize. We aimed to investigate the diagnostic accuracy of Caloric Testing when compared to the video head impulse test (vHIT) in differentiating between vestibular neuritis and vestibular strokes in acute dizziness. Design Prospective cross-sectional study (convenience sample). Setting All patients presenting with signs of an acute vestibular syndrome at the emergency department of a tertiary referral center. Participants One thousand, six hundred seventy-seven patients were screened between February 2015 and May 2020 for Acute Vestibular Syndrome (AVS), of which 152 met the inclusion criteria and were enrolled. Inclusion criteria consisted of a state of continuous dizziness, associated with nausea or vomiting, head-motion intolerance, new gait or balance disturbance and nystagmus. Patients were excluded if they were younger than 18 years, if symptoms lasted 72 h after symptom onset. Of the 152 included patients 85 completed testing. We assessed 58 vestibular neuritis and 27 stroke patients. Main outcome measures All patients underwent calorics and vHIT followed by a delayed MRI which served as a gold standard for vestibular stroke confirmation. Results The overall sensitivity and specificity for detecting stroke with a caloric asymmetry cut-off of 30.9% was 75% and 86.8%, respectively [negative likelihood ratio (NLR) 0.29] compared to 91.7% and 88.7% for vHIT (NLR 0.094). Best VOR gain cut-off was 0.685. Twenty-five percent of vestibular strokes were misclassified by calorics, 8% by vHIT. Conclusions Caloric testing proved to be less accurate than vHIT in discriminating stroke from vestibular neuritis in acute dizziness. Contrary to classic teaching, asymmetric caloric responses can also occur with vestibular strokes and might put the patient at risk for misdiagnosis. We, therefore, recommend to abandon caloric testing in current practice and to replace it with vHIT in the acute setting. Caloric testing has still its place as a diagnostic tool in an outpatient setting.

Advanced materials interfaces 2102325 (2022). doi:10.1002/admi.202102325, Published by Wiley-VCH, Weinheim

Mounting evidence indicates that immunogenic therapies engaging the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress favor proficient cancer cell-immune interactions, by stimulating the release of immunomodulatory/proinflammatory factors by stressed or dying cancer cells. UPR-driven transcription of proinflammatory cytokines/chemokines exert beneficial or detrimental effects on tumor growth and antitumor immunity, but the cell-autonomous machinery governing the cancer cell inflammatory output in response to immunogenic therapies remains poorly defined. Here, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated a NF-κB/AP-1-inflammatory stress response, which dissociated from both cell death and UPR. This stress-induced inflammation was specifically abolished by the IRE1α-kinase inhibitor KIRA6. Supernatants from immunogenic chemotherapy and KIRA6 co-treated cancer cells were deprived of proinflammatory/chemoattractant factors and failed to mobilize neutrophils and induce dendritic cell maturation. Furthermore, KIRA6 significantly reduced the in vivo vaccination potential of dying cancer cells responding to immunogenic chemotherapy. Mechanistically, we found that the anti-inflammatory effect of KIRA6 was still effective in IRE1α-deficient cells, indicating a hitherto unknown off-target effector of this IRE1α-kinase inhibitor. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry, and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the IKK-driven NF-κB pathway. In sum, our study unravels that HSP60 is a KIRA6-inhibitable upstream regulator of the NF-κB/AP-1-inflammatory stress responses evoked by immunogenic treatments. It also urges caution when interpreting the anti-inflammatory action of IRE1α chemical inhibitors. ispartof: CELL DEATH AND DIFFERENTIATION vol:29 issue:1 pages:230-245 ispartof: location:England status: published

We have conceptualized and demonstrated an approach based on the combination of hydrophobicity, a substrate-independent dip coating as porous material with double residual chemical reactivities for implementing multiplexed, miniaturized and unclonable bulk-infused patterns of different fluorophores following distinct reaction pathways. The embedded hydrophobicity (∼102°) restricted the unwanted spreading of beaded aqueous ink on the coating. The constructions of micropatterns on porous dip-coating via ink-jet printing or microchannel cantilever spotting offered orthogonal read-out and remained readable even after removal of the exterior of the coating.

Summary ParagraphProtein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzyme’s conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at Gly150 that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution., Author summary Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

Summary Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson’s disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1. publishedVersion

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggests that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe. - Introduction - Results -- Archaeological and Anthropological Findings -- The 16th Century Ellwangen Plague Victims Display Genetic Similarity with Modern Inhabitants -- Two Immunity-Related Genes Harbor Strongly Differentiated single nucleotide polymorphisms -- No Evidence for Role of CCR5-D32 in Protection from Y. pestis Infection Discussion -- Natural Selection Has Increased HLA-DRB*13 and Reduced HLA-B*51 and -C*06 Frequencies in Modern Individuals -- Higher Incidence of KIR3DL1 Interaction with HLA-B in Plague Victims Than Modern Inhabitants of Ellwangen - Discussion - Materials and Methods

Background The quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient’s survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies. However, a framework to assess the PD-L1 status with a high interobserver reproducibility on tumor cells and different types of immune cells has yet to be established. Materials and Methods To study the impact of PD-L1 expression on the tumor immune microenvironment and patient outcome, a framework for fully automated PD-L1 quantification on tumor cells and immune cells was established and validated. Automated PD-L1 quantification was facilitated by incorporating three different deep learning steps for the analysis of more than 80 different neoplasms from more than 10’000 tumor specimens using a bleach & stain 15-marker multiplex fluorescence immunohistochemistry panel (i.e., PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, CD31). Clinicopathological parameter were available for more than 30 tumor entities and overall survival data were available for 1517 breast cancer specimens. Results Comparing the automated deep-learning based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p Conclusions In conclusion, multiplex immunofluorescence PD-L1 assessment provides cutoff-free/continuous PD-L1 data which are superior to the conventional percentage of PD-L1+ tumor cells and of high prognostic relevance. The combined analysis of spatial PD-L1/PD-1 data and more than 20 different immune cell subtypes of the immune tumor microenvironment revealed distinct PD-L1 immune phenotypes. Disclosure Information N.C. Blessin: None. E. Bady: None. T. Mandelkow: None. C. Yang: None. J. Raedler: None. R. Simon: None. C. Fraune: None. M. Lennartz: None. S. Minner: None. E. Burandt: None. D. Hoflmayer: None. G. Sauter: None. S.A. Weidemann: None.

Background CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study aimed at a comparative analysis of CTLA-4+ cells between different tumor entities. Materials and Methods To quantify CTLA-4+ cells, 4,582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Results Comparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r=0.87; p Conclusions Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4. Disclosure Information D. Dum: None. T.L.C. Henke: None. T. Mandelkow: None. E. Bady: None. R. Simon: None. G. Sauter: None. S. Steuerer: None. W. Wilczak: None. E. Burandt: None. J. Raedler: None. M. Lennartz: None. N.C. Blessin: None.

African swine fever (ASF) emerged in Estonia in 2014. From February 2019 to August 2020, no pigs or wild boar tested positive for ASF virus (ASFV), only ASFV-specific antibodies could be detected in shot wild boar. However, ASF recently re-emerged in wild boar. We tested three hypotheses that might explain the current situation: (i) ASFV may have been present throughout, but at a prevalence below the detection limit, (ii) seropositive wild boar may have remained infectious (i.e., virus-carriers) and kept the epidemic going, or (iii) ASF was gone for 1.5 years, but was recently re-introduced. Using Estonian surveillance data, the sensitivity of the surveillance system and the confidence in freedom from ASF were estimated. Furthermore, the detection probability was determined and cluster analyses were performed to investigate the role of serological positive wild boar. The results suggest that the surveillance system was not able to detect virus circulation at a design prevalence below 1%. With respect to the confidence in freedom from ASF, the results indicate that circulating virus should have been detected over time, if the prevalence was ≥2%. However, the decreasing wild boar population density and ongoing surveillance activities made ASFV circulation at a low prevalence unlikely. Cluster analyses provided no evidence for a significant accumulation of serologically positive wild boar in temporal connection to the re-emergence of ASFV. Further targeted research, such as long-term experimental studies and molecular epidemiology, is necessary to improve our knowledge on the epidemiology of ASF and to control the disease more effectively.

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of ‘large’ and ‘dual-expanded’ clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.