Ultrashort single-pulse laser ablation of stainless steel, aluminium, copper and its dependence on the pulse duration.

In this work, we investigate single-pulse laser ablation of bulk stainless steel (AISI304), aluminium (Al) and copper (Cu) and its dependence on the pulse duration. We measured the reflectivity, ablation thresholds and volumes under the variation of pulse duration and fluence. The known drop of efficiency with increasing pulse duration is confirmed for single-pulse ablation in all three metals. We attribute the efficiency drop to a weakened photomechanically driven ablation process and a stronger contribution of photothermal phase explosion. The highest energetic efficiency and precision is achieved for pulse durations below the mechanical expansion time of 3-5 ps, where the stress confinement condition is fulfilled.

Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library.

BACKGROUND: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. RESULTS: We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. CONCLUSION: We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

GenomeCRISPR - a database for high-throughput CRISPR/Cas9 screens.

Over the past years, CRISPR/Cas9 mediated genome editing has developed into a powerful tool for modifying genomes in various organisms. In high-throughput screens, CRISPR/Cas9 mediated gene perturbations can be used for the systematic functional analysis of whole genomes. Discoveries from such screens provide a wealth of knowledge about gene to phenotype relationships in various biological model systems. However, a database resource to query results efficiently has been lacking. To this end, we developed GenomeCRISPR (http://genomecrispr.org), a database for genome-scale CRISPR/Cas9 screens. Currently, GenomeCRISPR contains data on more than 550 000 single guide RNAs (sgRNA) derived from 84 different experiments performed in 48 different human cell lines, comprising all screens in human cells using CRISPR/Cas published to date. GenomeCRISPR provides data mining options and tools, such as gene or genomic region search. Phenotypic and genome track views allow users to investigate and compare the results of different screens, or the impact of different sgRNAs on the gene of interest. An Application Programming Interface (API) allows for automated data access and batch download. As more screening data will become available, we also aim at extending the database to include functional genomic data from other organisms and enable cross-species comparisons.

Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of FZD1, FZD2, and FZD7 and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand-receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of FZD genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.-Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens.

MOTIVATION: Genetic screens by CRISPR/Cas9-mediated genome engineering have become a powerful tool for functional genomics. However, there is currently a lack of end-to-end software pipelines to analyze CRISPR/Cas9 screens based on next generation sequencing. RESULTS: The CRISPR-AnalyzeR for pooled screens (caRpools) is an R package for exploratory data analysis that provides a complete workflow to analyze CRISPR/Cas9 screens. To further support the analysis of large-scale screens, caRpools integrates screening documentation and generation of standardized analysis reports. AVAILABILITY AND IMPLEMENTATION: caRpools, manuals and an open virtual appliance are available at http://github.com/boutroslab/caRpools.

Autophagy regulates IL-23 secretion and innate T cell responses through effects on IL-1 secretion.

Autophagy controls IL-1ß secretion by regulating inflammasome activation and by targeting pro-IL-1ß for degradation. In this article, we show that inhibition of autophagy, either with the PI3K inhibitors 3-methyladenine, wortmannin, and LY294002 or with small interfering RNA against autophagy proteins augmented the secretion of IL-23 by human and mouse macrophages and dendritic cells in response to specific TLR agonists. This process occurred at the transcriptional level and was dependent on reactive oxygen species and IL-1R signaling; it was abrogated with an IL-1R antagonist or with IL-1-neutralizing Abs, whereas treatment with either rIL-1α or IL-1ß induced IL-23 secretion. Dendritic cells treated with LPS and 3-methyladenine secreted enhanced levels of both IL-1ß and IL-23, and supernatants from these cells stimulated the innate secretion of IL-17, IFN-γ, and IL-22 by γδ T cells. These data demonstrate that autophagy has a potentially pivotal role to play in the induction and regulation of inflammatory responses by innate immune cells, largely driven by IL-1 and its consequential effects on IL-23 secretion.

The epistemic integrity of scientific research.

We live in a world in which scientific expertise and its epistemic authority become more important. On the other hand, the financial interests in research, which could potentially corrupt science, are increasing. Due to these two tendencies, a concern for the integrity of scientific research becomes increasingly vital. This concern is, however, hollow if we do not have a clear account of research integrity. Therefore, it is important that we explicate this concept. Following Rudolf Carnap's characterization of the task of explication, this means that we should develop a concept that is (1) similar to our common sense notion of research integrity, (2) exact, (3) fruitful, and (4) as simple as possible. Since existing concepts do not meet these four requirements, we develop a new concept in this article. We describe a concept of epistemic integrity that is based on the property of deceptiveness, and argue that this concept does meet Carnap's four requirements of explication. To illustrate and support our claims we use several examples from scientific practice, mainly from biomedical research.

Intelligent diagnosing of intellectual disabilities in offenders: food for thought.

Research on offenders with intellectual disabilities (IDs) in the criminal justice arena is on the rise, reflected by a growing number of relevant publications each year. However, there is a long recognized methodological problem that hampers the comparability of empirical studies and that raises doubts about the accuracy of prevalence rates, comorbidities, and various correlates and characteristics. In this paper we will argue that the crux of the problem can, on the one hand, be found in the plurality of assessment methods for intelligence and adaptive functioning, which are not all sufficiently reliable and valid. On the other hand, assessment of IQ in criminal justice and mental health-related areas appears to be informed more by practical aspects and needs rather than grounded in a solid theoretical model. Hence, we suggest that the Cattell-Horn-Carroll (CHC) model of intelligence has potential value in this regard, and deserves a closer look. Finally, we will discuss its incorporation into, and possible implications for, criminal justice practice and future study designs.

Image analysis approach for development of a decision support system for detection of malaria parasites in thin blood smear images.

This paper describes development of a decision support system for diagnosis of malaria using color image analysis. A hematologist has to study around 100 to 300 microscopic views of Giemsa-stained thin blood smear images to detect malaria parasites, evaluate the extent of infection and to identify the species of the parasite. The proposed algorithm picks up the suspicious regions and detects the parasites in images of all the views. The subimages representing all these parasites are put together to form a composite image which can be sent over a communication channel to obtain the opinion of a remote expert for accurate diagnosis and treatment. We demonstrate the use of the proposed technique for use as a decision support system by developing an android application which facilitates the communication with a remote expert for the final confirmation on the decision for treatment of malaria. Our algorithm detects around 96% of the parasites with a false positive rate of 20%. The Spearman correlation r was 0.88 with a confidence interval of 0.838 to 0.923, p<0.0001.

Genome scale CRISPR Cas9a knockout screen reveals genes that control glioblastoma susceptibility to the alkylating agent temozolomide.

Glioblastoma is the most fatal of all primary human brain tumors with 14 months median survival. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy and tumor treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impact their overall survival and outcome. However, the lack of progress in this area could be attributed to lack of tools to probe unbiasedly at the genome wide level the coding and non-coding elements contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients. CRISPR Cas9a has emerged as a functional genomics tool to study at genome level the factors that control cancer resistance to drugs. Recently, we used genome wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understand genes that control glioblastoma response to temozolomide by using genome scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

PURPOSE: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. RESULTS: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. CONCLUSIONS: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to temozolomide (TMZ) via NELFB and RPS6KB2 interaction.

Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5-methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC-mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6-mediated 5mC deposition regulates transcriptional pause by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, and TFIIE) on the preinitiation complex at the TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.

Manipulative therapy in addition to usual medical care accelerates recovery of shoulder complaints at higher costs: economic outcomes of a randomized trial.

BACKGROUND: Shoulder complaints are common in primary care and have unfavourable long term prognosis. Our objective was to evaluate the clinical effectiveness of manipulative therapy of the cervicothoracic spine and the adjacent ribs in addition to usual medical care (UMC) by the general practitioner in the treatment of shoulder complaints. METHODS: This economic evaluation was conducted alongside a randomized trial in primary care. Included were 150 patients with shoulder complaints and a dysfunction of the cervicothoracic spine and adjacent ribs. Patients were treated with UMC (NSAID's, corticosteroid injection or referral to physical therapy) and were allocated at random (yes/no) to manipulative therapy (manipulation and mobilization). Patient perceived recovery, severity of main complaint, shoulder pain, disability and general health were outcome measures. Data about direct and indirect costs were collected by means of a cost diary. RESULTS: Manipulative therapy as add-on to UMC accelerated recovery on all outcome measures included. At 26 weeks after randomization, both groups reported similar recovery rates (41% vs. 38%), but the difference between groups in improvement of severity of the main complaint, shoulder pain and disability sustained. Compared to the UMC group the total costs were higher in the manipulative group (€1167 vs. €555). This is explained mainly by the costs of the manipulative therapy itself and the higher costs due sick leave from work. The cost effectiveness ratio showed that additional manipulative treatment is more costly but also more effective than UMC alone. The cost-effectiveness acceptability curve shows that a 50%-probability of recovery with AMT within 6 months after initiation of treatment is achieved at €2876. CONCLUSION: Manipulative therapy in addition to UMC accelerates recovery and is more effective than UMC alone on the long term, but is associated with higher costs. INTERNATIONAL STANDARD RANDOMIZED CONTROLLED TRIAL NUMBER REGISTER: ISRCTN11216.

Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids.

Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor ß (TGF-ß) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

Ribosomal protein S11 influences glioma response to TOP2 poisons.

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.

Performance Evaluation of MHC Class-I Binding Prediction Tools Based on an Experimentally Validated MHC-Peptide Binding Data Set.

Knowing whether a protein can be processed and the resulting peptides presented by major histocompatibility complex (MHC) is highly important for immunotherapy design. MHC ligands can be predicted by in silico peptide-MHC class-I binding prediction algorithms. However, prediction performance differs considerably, depending on the selected algorithm, MHC class-I type, and peptide length. We evaluated the prediction performance of 13 algorithms based on binding affinity data of 8- to 11-mer peptides derived from the HPV16 E6 and E7 proteins to the most prevalent human leukocyte antigen (HLA) types. Peptides from high to low predicted binding likelihood were synthesized, and their HLA binding was experimentally verified by in vitro competitive binding assays. Based on the actual binding capacity of the peptides, the performance of prediction algorithms was analyzed by calculating receiver operating characteristics (ROC) and the area under the curve (AROC). No algorithm outperformed others, but different algorithms predicted best for particular HLA types and peptide lengths. The sensitivity, specificity, and accuracy of decision thresholds were calculated. Commonly used decision thresholds yielded only 40% sensitivity. To increase sensitivity, optimal thresholds were calculated, validated, and compared. In order to make maximal use of prediction algorithms available online, we developed MHCcombine, a web application that allows simultaneous querying and output combination of up to 13 prediction algorithms. Taken together, we provide here an evaluation of peptide-MHC class-I binding prediction tools and recommendations to increase prediction sensitivity to extend the number of potential epitopes applicable as targets for immunotherapy.

ERAP1 overexpression in HPV-induced malignancies: A possible novel immune evasion mechanism.

Immune evasion of tumors poses a major challenge for immunotherapy. For human papillomavirus (HPV)-induced malignancies, multiple immune evasion mechanisms have been described, including altered expression of antigen processing machinery (APM) components. These changes can directly influence epitope presentation and thus T-cell responses against tumor cells. To date, the APM had not been studied systematically in a large array of HPV+ tumor samples. Therefore in this study, systematic expression analysis of the APM was performed on the mRNA and protein level in a comprehensive collection of HPV16+ cell lines. Subsequently, HPV+ cervical tissue samples were examined by immunohistochemistry. ERAP1 (endoplasmic reticulum aminopeptidase 1) was the only APM component consistently altered - namely overexpressed - in HPV16+ tumor cell lines. ERAP1 was also found to be overexpressed in cervical intraepithelial neoplasia and cervical cancer samples; expression levels were increasing with disease stage. On the functional level, the influence of ERAP1 expression levels on HPV16 E7-derived epitope presentation was investigated by mass spectrometry and in cytotoxicity assays with HPV16-specific T-cell lines. ERAP1 overexpression did not cause a complete destruction of any of the HPV epitopes analyzed, however, an influence of ERAP1 overexpression on the presentation levels of certain HPV epitopes could be demonstrated by HPV16-specific CD8+ T-cells. These showed enhanced killing toward HPV16+ CaSki cells whose ERAP1 expression had been attenuated to normal levels. ERAP1 overexpression may thus represent a novel immune evasion mechanism in HPV-induced malignancies, in cases when presentation of clinically relevant epitopes is reduced by overactivity of this peptidase.

CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries.

BACKGROUND: Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. RESULTS: Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. CONCLUSION: CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld.

The epistemic integrity of NASA practices in the Space Shuttle Program.

This article presents an account of epistemic integrity and uses it to demonstrate that the epistemic integrity of different kinds of practices in NASA's Space Shuttle Program was limited. We focus on the following kinds of practices: (1) research by working engineers, (2) review by middle-level managers, and (3) communication with the public. We argue that the epistemic integrity of these practices was undermined by production pressure at NASA, i.e., the pressure to launch an unreasonable amount of flights per year. Finally, our findings are used to develop some potential strategies to protect epistemic integrity in aerospace science.