Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling

Minotaur is critical for primary piRNA biogenesis

Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur

Automated virtual reality cognitive therapy for people with psychosis: Protocol for a qualitative investigation using peer research methods

BACKGROUND: Many people with psychosis experience difficulties in everyday social situations. Anxiety can make life challenging, leading to withdrawal. Cognitive therapy, using active in vivo learning, enables people to overcome fears. These treatments are not readily available to people with psychosis. Automated virtual reality (VR) therapy is a potential route to increase accessibility. The gameChange automated VR cognitive therapy is designed to help people overcome anxious avoidance and build confidence in everyday social situations. A virtual coach guides the person through the treatment. Understanding user experience is key to facilitating future implementation. Peer research methods, in which people with lived experience of the issues being studied are involved in collecting and analyzing data, may be useful in developing this understanding. This encourages researchers to draw on their lived experience to explore participant perspectives and co-create knowledge. OBJECTIVE: The primary objective is to use a peer research approach to explore the participant experience of a novel automated VR therapy for anxious social avoidance. This includes understanding (1) the experience of anxious social avoidance in people with psychosis, (2) the experience of the gameChange automated VR cognitive therapy, and (3) any potential impact of the therapy in people’s lives. This will inform future implementation strategies. The secondary objective is to explore how peer research can be used to co-create knowledge. METHODS: Semistructured interviews will be conducted with approximately 25 people with psychosis participating in the gameChange trial (ISRCTN17308399). Participants will be recruited from the five trial centers based in National Health Service mental health trusts across England. Interviews will be conducted by two researchers. One is a peer researcher with similar lived experience to the trial participants. The other has lived experiences of mental health issues that do not directly overlap with those of the trial participants. Interview questions will focus on an individual’s experience of anxious social avoidance, experiences of participating in the gameChange VR therapy, and any changes or impact following therapy. The interview schedule was developed in collaboration with the gameChange Lived Experience Advisory Panel (LEAP), comprising 10 project advisors with lived experience of psychosis. Interpretative phenomenological analysis and template analysis will be used to explore individual accounts. The LEAP will contribute to the analysis. RESULTS: Data collection will be conducted from April to September 2021, and analysis will be conducted from June to October 2021. As of September 28, 2021, 20 participants had been interviewed, and coding is underway. CONCLUSIONS: The study, employing a peer research approach, may provide a unique insight into the experiences of anxious social avoidance in people with psychosis and its treatment using automated VR therapy. This will inform potential future implementation of VR automated therapies in mental health services. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/3174

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity

Automated virtual reality therapy to treat agoraphobic avoidance and distress in patients with psychosis (gameChange): a multicentre, parallel-group, single-blind, randomised, controlled trial in England with mediation and moderation analyses

Background: Automated delivery of psychological therapy using immersive technologies such as virtual reality (VR) might greatly increase the availability of effective help for patients. We aimed to evaluate the efficacy of an automated VR cognitive therapy (gameChange) to treat avoidance and distress in patients with psychosis, and to analyse how and in whom it might work. // Methods: We did a parallel-group, single-blind, randomised, controlled trial across nine National Health Service trusts in England. Eligible patients were aged 16 years or older, with a clinical diagnosis of a schizophrenia spectrum disorder or an affective diagnosis with psychotic symptoms, and had self-reported difficulties going outside due to anxiety. Patients were randomly assigned (1:1) to either gameChange VR therapy plus usual care or usual care alone, using a permuted blocks algorithm with randomly varying block size, stratified by study site and service type. gameChange VR therapy was provided in approximately six sessions over 6 weeks. Trial assessors were masked to group allocation. Outcomes were assessed at 0, 6 (primary endpoint), and 26 weeks after randomisation. The primary outcome was avoidance of, and distress in, everyday situations, assessed using the self-reported Oxford Agoraphobic Avoidance Scale (O-AS). Outcome analyses were done in the intention-to-treat population (ie, all participants who were assigned to a study group for whom data were available). We performed planned mediation and moderation analyses to test the effects of gameChange VR therapy when added to usual care. This trial is registered with the ISRCTN registry, 17308399. // Findings: Between July 25, 2019, and May 7, 2021 (with a pause in recruitment from March 16, 2020, to Sept 14, 2020, due to COVID-19 pandemic restrictions), 551 patients were assessed for eligibility and 346 were enrolled. 231 (67%) patients were men and 111 (32%) were women, 294 (85%) were White, and the mean age was 37·2 years (SD 12·5). 174 patients were randomly assigned to the gameChange VR therapy group and 172 to the usual care alone group. Compared with the usual care alone group, the gameChange VR therapy group had significant reductions in agoraphobic avoidance (O-AS adjusted mean difference –0·47, 95% CI –0·88 to –0·06; n=320; Cohen's d –0·18; p=0·026) and distress (–4·33, –7·78 to –0·87; n=322; –0·26; p=0·014) at 6 weeks. Reductions in threat cognitions and within-situation defence behaviours mediated treatment outcomes. The greater the severity of anxious fears and avoidance, the greater the treatment benefits. There was no significant difference in the occurrence of serious adverse events between the gameChange VR therapy group (12 events in nine patients) and the usual care alone group (eight events in seven patients; p=0·37). // Interpretation: Automated VR therapy led to significant reductions in anxious avoidance of, and distress in, everyday situations compared with usual care alone. The mediation analysis indicated that the VR therapy worked in accordance with the cognitive model by reducing anxious thoughts and associated protective behaviours. The moderation analysis indicated that the VR therapy particularly benefited patients with severe agoraphobic avoidance, such as not being able to leave the home unaccompanied. gameChange VR therapy has the potential to increase the provision of effective psychological therapy for psychosis, particularly for patients who find it difficult to leave their home, visit local amenities, or use public transport. // Funding: National Institute of Health Research Invention for Innovation programme, National Institute of Health Research Oxford Health Biomedical Research Centre

The evolutionary history of 2,658 cancers

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

CSIRO Environmental Modelling Suite (EMS): scientific description of the optical and biogeochemical models (vB3p0)

Abstract. Since the mid-1990s, Australia's Commonwealth Science Industry and Research Organisation (CSIRO) has been developing a biogeochemical (BGC) model for coupling with a hydrodynamic and sediment model for application in estuaries, coastal waters and shelf seas. The suite of coupled models is referred to as the CSIRO Environmental Modelling Suite (EMS) and has been applied at tens of locations around the Australian continent. At a mature point in the BGC model's development, this paper presents a full mathematical description, as well as links to the freely available code and user guide. The mathematical description is structured into processes so that the details of new parameterisations can be easily identified, along with their derivation. In EMS, the underwater light field is simulated by a spectrally resolved optical model that calculates vertical light attenuation from the scattering and absorption of 20+ optically active constituents. The BGC model itself cycles carbon, nitrogen, phosphorous and oxygen through multiple phytoplankton, zooplankton, detritus and dissolved organic and inorganic forms in multiple water column and sediment layers. The water column is dynamically coupled to the sediment to resolve deposition, resuspension and benthic–pelagic biogeochemical fluxes. With a focus on shallow waters, the model also includes detailed representations of benthic plants such as seagrass, macroalgae and coral polyps. A second focus has been on, where possible, the use of geometric derivations of physical limits to constrain ecological rates. This geometric approach generally requires population-based rates to be derived from initially considering the size and shape of individuals. For example, zooplankton grazing considers encounter rates of one predator on a prey field based on summing relative motion of the predator with the prey individuals and the search area; chlorophyll synthesis includes a geometrically derived self-shading term; and the bottom coverage of benthic plants is calculated from their biomass using an exponential form derived from geometric arguments. This geometric approach has led to a more algebraically complicated set of equations when compared to empirical biogeochemical model formulations based on populations. But while being algebraically complicated, the model has fewer unconstrained parameters and is therefore simpler to move between applications than it would otherwise be. The version of EMS described here is implemented in the eReefs project that delivers a near-real-time coupled hydrodynamic, sediment and biogeochemical simulation of the Great Barrier Reef, northeast Australia, and its formulation provides an example of the application of geometric reasoning in the formulation of aquatic ecological processes. </jats:p

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression