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The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans. We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen’s design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90. RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40). Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development.

Objective To explore the definition and operationalization of resilience in health system performance assessments in European Union countries. Methods We conducted multiple empirical case study analyses. We identified relevant cases through a literature review from 2014 to 2023 using Google Scholar and through a snowball technique to retrieve additional information. We included only documents that explicitly mentioned resilience in health system performance assessments. We performed a content analysis to identify common patterns in defining resilience. Findings The final sample consisted of six countries: Belgium, Croatia, Czechia, Estonia, Ireland and Italy. Each country adopted a distinct approach to conceptualizing resilience, with countries prioritizing specific aspects based on lessons learnt from the coronavirus disease 2019 (COVID-19) pandemic. Some countries focused on maintaining essential health-care services and protecting vulnerable groups. Other countries prioritized management capacity, staff preparedness, digital health utilization and strengthening of primary health care. Content analysis revealed six resilience definitions derived from the key performance indicators: addressing unmet needs and maintaining outcomes; protecting vulnerable groups; acquiring and using resources; having trained and prepared staff in place; using digital health; and strengthening primary health care. Conclusion Integration of resilience into the health profiles of European Union countries preceded its inclusion in national health system performance assessments, the latter of which became more prominent after the COVID-19 pandemic. Variations in interpretations within health system performance assessments reflect differences in indicators and policy responses. [phrase omitted] [phrase omitted] Objectif Explorer la definition et la mise en reuvre de la resilience dans l'evaluation des performances des systemes de sante au sein des pays de l'Union europeenne. Methodes Nous avons effectue plusieurs analyses d'etudes de cas empiriques. Nous avons identifie les cas pertinents en procedant a une revue de la litterature publiee entre 2014 et 2023 sur Google Scholar et en appliquant la technique de type <> pour obtenir des informations supplementaires. Nous n'avons retenu que les documents qui mentionnaient explicitement la resilience dans les evaluations des performances des systemes de sante. Enfin, nous avons examine les contenus afin de reperer les modeles les plus courants dans la definition de la resilience. Resultats Lechantillon final etait compose de six pays : la Belgique, la Croatie, l'Estonie, l'Irlande, l'Italie et la Republique tcheque. Chacun de ces pays a adopte une approche differente dans sa conceptualisation de la resilience, certains ayant donne la priorite a des aspects specifiques en fonction des lecons qu'ils ont tirees de la pandemie de maladie a coronavirus 2019 (COVID-19). Quelques-uns se sont concentres sur le maintien des services de sante essentiels et la protection des groupes vulnerables. D'autres ont privilegie les capacites de gestion, la preparation du personnel, l'utilisation des technologies numeriques et le renforcement des soins de sante primaires. Lanalyse des contenus a revele six definitions de la resilience derivees des indicateurs cles de performance : repondre aux besoins non satisfaits et assurer le maintien des resultats ; proteger les groupes vulnerables ; acquerir et exploiter les ressources ; disposer de personnel forme et prepare ; deployer des technologies numeriques ; et enfin, consolider les soins de sante primaires. Conclusion La resilience figurait dans les profils sanitaires de certains pays de l'Union europeenne avant d'etre integree dans les mecanismes d'evaluation des performances des systemes de sante nationaux, ayant gagne en importance apres la pandemie de COVID-19. Les divergences d'interpretation observees dans le cadre de ces evaluations refletent les variations au niveau des indicateurs et des solutions politiques. [phrase omitted] Objetivo Explorar la definicion y la puesta en practica de la resiliencia en las evaluaciones del rendimiento de los sistemas sanitarios en los paises de la Union Europea. Metodos Se realizaron multiples analisis de estudios de casos empiricos. Se identificaron casos relevantes mediante una revision bibliografica de 2014 a 2023 utilizando Google Scholar y mediante una tecnica de bola de nieve para recuperar informacion adicional. Se incluyeron solo los documentos que mencionaban explicitamente la resiliencia en las evaluaciones del rendimiento del sistema sanitario. Se realizo un analisis de contenido para identificar patrones comunes en la definicion de resiliencia. Resultados La muestra final estaba formada por seis paises: Belgica, Croacia, Chequia, Estonia, Irlanda e Italia. Cada pais adopto un enfoque distinto para conceptualizar la resiliencia, dando prioridad a aspectos especificos basados en las lecciones aprendidas de la pandemia de la enfermedad por coronavirus de 2019 (COVID-19). Algunos paises se centraron en mantener los servicios sanitarios esenciales y proteger a los grupos vulnerables. Otros paises priorizaron la capacidad de gestion, la preparacion del personal, la utilizacion de la salud digital y el fortalecimiento de la atencion primaria. El analisis de contenido revelo seis definiciones de resiliencia derivadas de los indicadores clave de rendimiento: abordar las necesidades insatisfechas y mantener los resultados; proteger a los grupos vulnerables; adquirir y utilizar recursos; contar con personal capacitado y preparado; utilizar la salud digital; y fortalecer la atencion primaria de salud. Conclusion La integracion de la resiliencia en los perfiles sanitarios de los paises de la Union Europea precedio a su inclusion en las evaluaciones del rendimiento de los sistemas sanitarios nacionales, estas ultimas mas destacadas tras la pandemia de la COVID-19. Las variaciones en las interpretaciones dentro de las evaluaciones del rendimiento de los sistemas sanitarios reflejan diferencias en los indicadores y las respuestas politicas., Introduction The coronavirus disease 2019 (COVID-19) pandemic posed a considerable challenge for governments, affecting health, the economy and citizens' well-being. (1) The pandemic exposed weaknesses in health systems, such as [...]

Abstract Optimal immune function is crucial in preventing cancer development and growth and for the success of anti-cancer therapies. Here, we characterized the peripheral immunological status of 83 steroids-naïve pediatric patients with central nervous system neoplasia at the disease onset. Tumors were classified into low-grade gliomas (LGG), high-grade gliomas (HGG), medulloblastoma, and other tumors. We revealed that glioma patients showed an altered lymphocyte pool. T-cells of HGG patients shifted from naïve to effector memory phenotype. LGG patients exhibited T-cell central memory expansion and higher T-cell activation. Interestingly, HGG patients displayed reduced thymic function. Furthermore, LGG and HGG patients showed reduced activated B-cells and suboptimal B-cell formation. Our data demonstrate that glioma patients have reduced lymphopoiesis at the disease onset, which could contribute to the systemic lymphopenia characterizing these patients. This study offers novel insights into the immunological status of brain tumor patients which may help in designing more effective treatments.

Abstract Molten silicates at depth are crucial for planetary evolution, yet their local structure and physical properties under extreme conditions remain elusive due to experimental challenges. In this study, we utilize in situ X-ray diffraction (XRD) at the Matter in Extreme Conditions (MEC) end-station of the Linear Coherent Linac Source (LCLS) at SLAC National Accelerator Laboratory to investigate liquid silicates. Using an ultrabright X-ray source and a high-power optical laser, we probed the local atomic arrangement of shock-compressed liquid (Mg,Fe)SiO3 with varying Fe content, at pressures from 81(9) to 385(40) GPa. We compared these findings to ab initio molecular dynamics simulations under similar conditions. Results indicate continuous densification of the O-O and Mg-Si networks beyond Earth’s interior pressure range, potentially altering melt properties at extreme conditions. This could have significant implications for early planetary evolution, leading to notable differences in differentiation processes between smaller rocky planets, such as Earth and Venus, and super-Earths, which are exoplanets with masses nearly three times that of Earth.

Objective: The aim of this study was to evaluate the clinical outcomes of breast cancer (BC) patients treated with neoadjuvant chemotherapy (NAC) followed by mastectomy, focusing on cases achieving pathologic complete response (pCR). The implications of residual ductal carcinoma in situ (DCIS) on prognosis and survival were examined. Materials and Methods: A retrospective cohort study included BC patients treated with NAC followed by mastectomy at the breast unit of IRCCS Humanitas Research Hospital between March 2010 and October 2021. Patients were sub-grouped into two: Those with residual DCIS (ypTis) and those with complete response without residual tumor (ypT0). Key variables such as demographics, tumor characteristics, treatment regimens, and survival outcomes were analyzed. Results: Of 681 patients treated with NAC, 175 achieved pCR, with 60 undergoing mastectomy. Among these 60 patients, 24 had residual DCIS (ypTis) while 36 had no residual invasive or in situ disease (ypT0). Patients with ypTis had higher rates of multifocal disease (62.5% vs. 27.8%, p = 0.006) and stage III disease (37.5% vs. 11.1%, p = 0.046). Triple-negative breast cancer was more prevalent in the ypT0 group (55.6% vs. 20.8%, p = 0.005). During a mean follow-up of 47 months, 11 patients experienced recurrence, with no significant differences in disease-free survival (DFS) and overall survival (OS) between the groups (p = 0.781, p = 0.963, respectively). Conclusion: Residual DCIS after NAC did not significantly impact DFS or OS compared to complete pathologic response without residual DCIS. This study underscores the need for further research to refine pCR definitions and improve NAC’s prognostic and therapeutic roles in BC management.