Mutational rescue of the activity of high-fidelity Cas9 enzymes.

Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.

Preassembly of specific Gßγ subunits at GABAB receptors through auxiliary KCTD proteins accelerates channel gating.

GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gßγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gßγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gßγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gßγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gß subunits. The binding site is shared between Gß1 and Gß2, limiting the interaction of KCTD proteins to these particular Gß isoforms. Substituting residues in the KCTD binding site of Gß1 with those from Gß3 hinders the preassembly of Gßγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gß interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.

Transcriptional immunogenomic analysis reveals distinct immunological clusters in paediatric nervous system tumours.

BACKGROUND: Cancer immunotherapies including immune checkpoint inhibitors and Chimeric Antigen Receptor (CAR) T-cell therapy have shown variable response rates in paediatric patients highlighting the need to establish robust biomarkers for patient selection. While the tumour microenvironment in adults has been widely studied to delineate determinants of immune response, the immune composition of paediatric solid tumours remains relatively uncharacterized calling for investigations to identify potential immune biomarkers. METHODS: To inform immunotherapy approaches in paediatric cancers with embryonal origin, we performed an immunogenomic analysis of RNA-seq data from 925 treatment-naïve paediatric nervous system tumours (pedNST) spanning 12 cancer types from three publicly available data sets. RESULTS: Within pedNST, we uncovered four broad immune clusters: Paediatric Inflamed (10%), Myeloid Predominant (30%), Immune Neutral (43%) and Immune Desert (17%). We validated these clusters using immunohistochemistry, methylation immune inference and segmentation analysis of tissue images. We report shared biology of these immune clusters within and across cancer types, and characterization of specific immune cell frequencies as well as T- and B-cell repertoires. We found no associations between immune infiltration levels and tumour mutational burden, although molecular cancer entities were enriched within specific immune clusters. CONCLUSIONS: Given the heterogeneity of immune infiltration within pedNST, our findings suggest personalized immunogenomic profiling is needed to guide selection of immunotherapeutic strategies.

Archaeal type IV pili stabilize Haloferax volcanii biofilms in flow.

Multicellular communities of contiguous cells attached to solid surfaces called biofilms represent a common microbial strategy to improve resilience in adverse environments.1,2,3 While bacterial biofilms have been under intense investigation, whether archaeal biofilms follow similar assembly rules remains unknown.4,5Haloferax volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili (T4Ps). These play a role in surface attachment and biofilm formation in both archaea and bacteria. In this study, we employed biophysical experiments to identify the function of T4Ps in H. volcanii biofilm morphogenesis. H. volcanii expresses not one but six types of major pilin subunits that are predicted to compose T4Ps. Non-invasive imaging of T4Ps in live cells using interferometric scattering (iSCAT) microscopy reveals that piliation varies across mutants expressing single major pilin isoforms. T4Ps are necessary to secure attachment of single cells to surfaces, and the adhesive strength of pilin mutants correlates with their level of piliation. In flow, H. volcanii forms clonal biofilms that extend in three dimensions. Notably, the expression of PilA2, a single pilin isoform, is sufficient to maintain levels of piliation, surface attachment, and biofilm formation that are indistinguishable from the wild type. Furthermore, we discovered that fluid flow stabilizes biofilm integrity; as in the absence of flow, biofilms tend to lose cohesion and disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity and that flow is a key factor regulating archaeal biofilm formation.

Recurrent atypical teratoid/rhabdoid tumors (AT/RT) reveal discrete features of progression on histology, epigenetics, copy number profiling, and transcriptomics.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

Porcine spermadhesin AQN-3 binds to negatively charged phospholipids.

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization.

Renal Medullary Carcinomas Harbor a Distinct Methylation Phenotype and Display Aberrant Methylation of Genes Related to Early Nephrogenesis.

Renal medullary carcinomas (RMC) are rare aggressive tumors of the kidneys, characterized by a loss of SMARCB1. Characteristically, these tumors arise in patients with sickle cell trait or other hemoglobinopathies. Recent characterization efforts have unraveled oncogenic pathways that drive tumorigenesis. Among these, gene sets that characterize replicative stress and the innate immune response are upregulated in RMCs. Despite comprehensive genetic and transcriptomic characterizations, commonalities or differences to other SMARCB1 deficient entities so far have not been investigated. We analyzed the methylome of seven primary RMC and compared it to other SMARCB1 deficient entities such as rhabdoid tumors (RT) and epithelioid sarcomas using 850 K methylation arrays. Moreover, we evaluated the differential gene expression of RMC using RNA-sequencing in comparison to other rhabdoid tumors. In accordance with previous gene expression data, we found that RMCs separate from other SMARCB1 deficient entities, pointing to a potentially different cell of origin and a role of additional genetic aberrations that may drive tumorigenesis and thus alter the methylome when compared to rhabdoid tumors. In a focused analysis of genes that are important for nephrogenesis, we particularly detected genes that govern early nephrogenesis such as FOXI1 to be hypomethylated and expressed at high levels in RMC. Overall, our analyses underscore the fact that RMCs represent a separate entity with limited similarities to rhabdoid tumors, warranting specific treatment tailored to the aggressiveness of the disease.

Frankenstein Cas9: engineering improved gene editing systems.

The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

Infants and Newborns with Atypical Teratoid Rhabdoid Tumors (ATRT) and Extracranial Malignant Rhabdoid Tumors (eMRT) in the EU-RHAB Registry: A Unique and Challenging Population.

Introduction: Malignant rhabdoid tumors (MRT) predominantly affect infants and young children. Patients below six months of age represent a particularly therapeutically challenging group. Toxicity to developing organ sites limits intensity of treatment. Information on prognostic factors, genetics, toxicity of treatment and long-term outcomes is sparse. Methods: Clinical, genetic, and treatment data of 100 patients (aged below 6 months at diagnosis) from 13 European countries were analyzed (2005-2020). Tumors and matching blood samples were examined for SMARCB1 mutations using FISH, MLPA and Sanger sequencing. DNA methylation subgroups (ATRT-TYR, ATRT-SHH, and ATRT-MYC) were determined using 450 k / 850 k-profiling. Results: A total of 45 patients presented with ATRT, 29 with extracranial, extrarenal (eMRT) and 9 with renal rhabdoid tumors (RTK). Seventeen patients demonstrated synchronous tumors (SYN). Metastases (M+) were present in 27% (26/97) at diagnosis. A germline mutation (GLM) was detected in 55% (47/86). DNA methylation subgrouping was available in 50% (31 / 62) with ATRT or SYN; for eMRT, methylation-based subgrouping was not performed. The 5-year overall (OS) and event free survival (EFS) rates were 23.5 ± 4.6% and 19 ± 4.1%, respectively. Male sex (11 ± 5% vs. 35.8 ± 7.4%), M+ stage (6.1 ± 5.4% vs. 36.2 ± 7.4%), presence of SYN (7.1 ± 6.9% vs. 26.6 ± 5.3%) and GLM (7.7 ± 4.2% vs. 45.7 ± 8.6%) were significant prognostic factors for 5-year OS. Molecular subgrouping and survival analyses confirm a previously described survival advantage for ATRT-TYR. In an adjusted multivariate model, clinical factors that favorably influence the prognosis were female sex, localized stage, absence of a GLM and maintenance therapy. Conclusions: In this cohort of homogenously treated infants with MRT, significant predictors of outcome were sex, M-stage, GLM and maintenance therapy. We confirm the need to stratify which patient groups benefit from multimodal treatment, and which need novel therapeutic strategies. Biomarker-driven tailored trials may be a key option.

Computationally designed hyperactive Cas9 enzymes.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

SMARCB1-deficient and SMARCA4-deficient Malignant Brain Tumors With Complex Copy Number Alterations and TP53 Mutations May Represent the First Clinical Manifestation of Li-Fraumeni Syndrome.

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant central nervous system tumor predominantly affecting infants. Mutations of SMARCB1 or (rarely) SMARCA4 causing loss of nuclear SMARCB1 or SMARCA4 protein expression are characteristic features, but further recurrent genetic alterations are lacking. Most AT/RTs occur de novo, but secondary AT/RTs arising from other central nervous system tumors have been reported. Malignant gliomas, IDH wild-type, arising in patients with Li-Fraumeni syndrome typically show somatic mutations of TP53 as well as complex copy number alterations, but little is known about the loss of SMARCB1 or SMARCA4 protein expression in this context. Here, we report 2 children in whom malignant supratentorial brain tumors with SMARCB1 deficiency, complex copy number alterations, and somatic TP53 mutations lead to the discovery of pathogenic/likely pathogenic TP53 variants in the germline. Screening of the molecularneuropathology.org dataset for cases with similar genetic and epigenetic alterations yielded another case with SMARCA4 deficiency in a young adult with Li-Fraumeni syndrome. In conclusion, SMARCB1-deficient or SMARCA4-deficient malignant brain tumors with complex copy number alterations and somatic TP53 mutations in children and young adults may represent the first clinical manifestation of Li-Fraumeni syndrome and should prompt genetic counseling and investigation for TP53 germline status.

Nonequilibrium band occupation and optical response of gold after ultrafast XUV excitation.

Free electron lasers offer unique properties to study matter in states far from equilibrium as they combine short pulses with a large range of photon energies. In particular, the possibility to excite core states drives new relaxation pathways that, in turn, also change the properties of the optically and chemically active electrons. Here, we present a theoretical model for the dynamics of the nonequilibrium occupation of the different energy bands in solid gold driven by exciting deep core states. The resulting optical response is in excellent agreement with recent measurements and, combined with our model, provides a quantitative benchmark for the description of electron-phonon coupling in strongly driven gold. Focusing on sub-picosecond time scales, we find essential differences between the dynamics induced by XUV and visible light.

Current and Emerging Therapeutic Approaches for Extracranial Malignant Rhabdoid Tumors.

Extracranial malignant rhabdoid tumors (extracranial MRT) are rare, highly aggressive malignancies affecting mainly infants and children younger than 3 years. Common anatomic sites comprise the kidneys (RTK - rhabdoid tumor of kidney) and other soft tissues (eMRT - extracranial, extrarenal malignant rhabdoid tumor). The genetic origin of these diseases is linked to biallelic pathogenic variants in the genes SMARCB1, or rarely SMARCA4, encoding subunits of the SWI/SNF chromatin-remodeling complex. Even if extracranial MRT seem to be quite homogeneous, recent epigenome analyses reveal a certain degree of epigenetic heterogeneity. Use of intensified therapies has modestly improved survival for extracranial MRT. Patients at standard risk profit from conventional therapies; most high-risk patients still experience a dismal course and often therapy resistance. Discoveries of clinical and molecular hallmarks and the exploration of experimental therapeutic approaches open exciting perspectives for clinical and molecularly stratified experimental treatment approaches. To ultimately improve the outcome of patients with extracranial MRTs, they need to be characterized and stratified clinically and molecularly. High-risk patients need novel therapeutic approaches including selective experimental agents in phase I/II clinical trials.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant: A clinical and histopathological distinct entity showing epigenetic similarity with ATRT-MYC.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant, is a histopathological distinct low-grade lesion encountered in older children and young adults that shows epigenetic similarity with ATRT-MYC and has the potential for malignant progression.

Boar spermadhesin AWN: novel insights in its binding behavior and localization on sperm.

As a major spermadhesin first found in the seminal plasma (SP) of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN (natAWN) in/on epididymal, ejaculated, capacitated, and acrosome-reacted boar sperm using epifluorescence and electron microscopy as well as an analysis of potential lipid-binding partners of natAWN and recombinant AWN (recAWN). By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment (EQS) of ejaculated, capacitated, and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the EQS during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine (PE) and in line with our previous study describing an interaction with phosphatidic acid (PA), the current results show a rather electrostatically driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the EQS, which suggest a possible role in sperm-oocyte fusion.

Cui Bono? Identifying Patient Groups That May Benefit From Granulocyte Transfusions in Pediatric Hematology and Oncology.

INTRODUCTION: Granulocyte transfusions have long been used to bridge the time to neutrophil recovery in patients with neutropenia and severe infection. Recent randomized controlled trials did not prove a beneficial effect of granulocyte transfusions, but were likely underpowered and suffered from very heterogeneous study populations. METHODS: We retrospectively reviewed data of all patients treated with granulocyte transfusions at our pediatric center from 2004 to 2019. To identify parameters that predict the success of granulocyte transfusions, we stratified patients in 3 groups. Patients in group 1 cleared their infection, whereas patients in group 2 succumbed to an infection in neutropenia despite granulocyte transfusions. A third group included all patients who died of causes that were not related to infection. RESULTS: We demonstrate that patients without respiratory or cardiocirculatory insufficiency are enriched in group 1 and more likely to benefit from granulocyte transfusions than patients who already require these intensive care measures. The effect of granulocyte transfusions correlates with the cell dose per body weight applied per time. With our standard twice weekly dosing, patients with a body weight below 40 kg are more likely to achieve a sufficient leukocyte increment and clear their infection in comparison to patients with a higher body weight. DISCUSSION/CONCLUSIONS: We suggest that future studies on the benefits of granulocyte transfusions stratify patients according to clinical risk factors that include the need for respiratory or cardiocirculatory support and strive for a sufficient dose density of granulocyte transfusions.