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Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

In various organisms, sequencing of selectively bred lines at apparent selection limits has demonstrated that genetic variation can remain at many loci, implying that evolution at the genetic level may continue even if the population mean phenotype remains constant. We compared selection signatures at generations 22 and 61 of the High Runner mouse experiment, which includes 4 replicate lines bred for voluntary wheel-running behavior (HR) and 4 non-selected control (C) lines. Previously, we reported multiple regions of differentiation between the HR and C lines, based on whole-genome sequence data for 10 mice from each line at generation 61, which was >31 generations after selection limits had been reached in all HR lines. Here, we analyzed pooled sequencing data from ~20 mice for each of the 8 lines at generation 22, around when HR lines were reaching limits. Differentiation analyses of allele frequencies at ~4.4 million SNP loci used the regularized T-test and detected 258 differentiated regions with FDR = 0.01. Comparable analyses involving pooling generation 61 individual mouse genotypes into allele frequencies by line produced only 11 such regions, with almost no overlap among the largest and most statistically significant peaks between the two generations. These results implicate a sort of genetic churn that continues at loci relevant for running. Simulations indicate that loss of statistical power due to random genetic drift and sampling error are insufficient to explain the differences in selection signatures. The 13 differentiated regions at generation 22 with strict culling measures include 79 genes related to a wide variety of functions. Gene ontology identified pathways related to olfaction and vomeronasal pathways as being overrepresented, consistent with generation 61 analyses, despite those specific regions differing between generations. Genes Dspp and Rbm24 are also identified as potentially explaining known bone and skeletal muscle differences, respectively, between the linetypes.

Abstract Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

BackgroundDiabetic retinopathy (DR) affects about 25% of people with diabetes in Canada. Early detection of DR is essential for preventing vision loss. ObjectiveWe evaluated the real-world performance of an artificial intelligence (AI) system that analyzes fundus images for DR screening in a Quebec tertiary care center. MethodsWe prospectively recruited adult patients with diabetes at the Centre hospitalier de l’Université de Montréal (CHUM) in Montreal, Quebec, Canada. Patients underwent dual-pathway screening: first by the Computer Assisted Retinal Analysis (CARA) AI system (index test), then by standard ophthalmological examination (reference standard). We measured the AI system's sensitivity and specificity for detecting referable disease at the patient level, along with its performance for detecting any retinopathy and diabetic macular edema (DME) at the eye level, and potential cost savings. ResultsThis study included 115 patients. CARA demonstrated a sensitivity of 87.5% (95% CI 71.9-95.0) and specificity of 66.2% (95% CI 54.3-76.3) for detecting referable disease at the patient level. For any retinopathy detection at the eye level, CARA showed 88.2% sensitivity (95% CI 76.6-94.5) and 71.4% specificity (95% CI 63.7-78.1). For DME detection, CARA had 100% sensitivity (95% CI 64.6-100) and 81.9% specificity (95% CI 75.6-86.8). Potential yearly savings from implementing CARA at the CHUM were estimated at CAD $245,635 (US $177,643.23, as of July 26, 2024) considering 5000 patients with diabetes. ConclusionsOur study indicates that integrating a semiautomated AI system for DR screening demonstrates high sensitivity for detecting referable disease in a real-world setting. This system has the potential to improve screening efficiency and reduce costs at the CHUM, but more work is needed to validate it.

Even though actions we observe in everyday life seem to unfold in a continuous manner, they are automatically divided into meaningful chunks, that are single actions or segments, which provide information for the formation and updating of internal predictive models. Specifically, boundaries between actions constitute a hub for predictive processing since the prediction of the current action comes to an end and calls for updating of predictions for the next action. In the current study, we investigated neural processes which characterize such boundaries using a repertoire of complex action sequences with a predefined probabilistic structure. Action sequences consisted of actions that started with the hand touching an object (T) and ended with the hand releasing the object (U). These action boundaries were determined using an automatic computer vision algorithm. Participants trained all action sequences by imitating demo videos. Subsequently, they returned for an fMRI session during which the original action sequences were presented in addition to slightly modified versions thereof. Participants completed a post-fMRI memory test to assess the retention of original action sequences. The exchange of individual actions, and thus a violation of action prediction, resulted in increased activation of the action observation network and the anterior insula. At U events, marking the end of an action, increased brain activation in supplementary motor area, striatum, and lingual gyrus was indicative of the retrieval of the previously encoded action repertoire. As expected, brain activation at U events also reflected the predefined probabilistic branching structure of the action repertoire. At T events, marking the beginning of the next action, midline and hippocampal regions were recruited, reflecting the selected prediction of the unfolding action segment. In conclusion, our findings contribute to a better understanding of the various cerebral processes characterizing prediction during the observation of complex action repertoires.

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic [SRSF2.sup.P95H/+] mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. [SRSF2.sup.P95H]-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of [SRSF2.sup.P95H/+] cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in [SRSF2.sup.P95H/+] cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in [SRSF2.sup.P95H] mutant MDS and AML., Introduction Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by hyperproliferative bone marrow, dysplastic hematopoietic cells, peripheral cytopenias, and high risk of progression to acute myeloid leukemia (AML). Recurrent [...]

Background: MRI-guidance may aid better discrimination between Organs at Risk (OARs) and target volumes in proximity of the mediastinum. We report the first clinical experiences with Stereotactic Body Radiotherapy (SBRT) of (ultra)central lung tumours on a 1.5 T MR-linac. Materials and Methods: Patients with an (ultra)central lung tumour were selected for MR-linac based SBRT treatment. A T2-weighted 3D sequence MRI acquired during free breathing was used for daily plan adaption. Prior to each fraction, contours of Internal Target Volume (ITV) and OARs were deformably propagated and amended by a radiation oncologist. Inter-fractional changes in volumes and coverage of target volumes as well as doses in OARs were evaluated in offline and online treatment plans. Results: Ten patients were treated and completed 60 Gy in 8 or 12 fractions. In total 104 fractions were delivered. The median time in the treatment room was 41 min with a median beam-on time of 8.9 min. No grade ≥3 acute toxicity was observed. In two patients, the ITV significantly decreased during treatment (58 % and 37 %, respectively) due to tumour shrinkage. In the other patients, 81 % of online ITVs were within ±15 % of the volume of fraction 1. Comparison with the pre-treatment plan showed that ITV coverage of the online plan was similar in 52 % and improved in 34 % of cases. Adaptation to meet OAR constraints, led to decreased ITV coverage in 14 %. Conclusions: We describe the workflow for MR-guided Radiotherapy and the feasibility of using 1.5 T MR-linac for SBRT of (ultra) central lung tumours.

In various organisms, sequencing of selectively bred lines at apparent selection limits has demonstrated that genetic variation can remain at many loci, implying that evolution at the genetic level may continue even if the population mean phenotype remains constant. We compared selection signatures at generations 22 and 61 of the "High Runner" mouse experiment, which includes 4 replicate lines bred for voluntary wheel-running behavior (HR) and 4 non-selected control (C) lines. Previously, we reported multiple regions of differentiation between the HR and C lines, based on whole-genome sequence data for 10 mice from each line at generation 61, which was >31 generations after selection limits had been reached in all HR lines. Here, we analyzed pooled sequencing data from ~20 mice for each of the 8 lines at generation 22, around when HR lines were reaching limits. Differentiation analyses of allele frequencies at ~4.4 million SNP loci used the regularized T-test and detected 258 differentiated regions with FDR = 0.01. Comparable analyses involving pooling generation 61 individual mouse genotypes into allele frequencies by line produced only 11 such regions, with almost no overlap among the largest and most statistically significant peaks between the two generations. These results implicate a sort of "genetic churn" that continues at loci relevant for running. Simulations indicate that loss of statistical power due to random genetic drift and sampling error are insufficient to explain the differences in selection signatures. The 13 differentiated regions at generation 22 with strict culling measures include 79 genes related to a wide variety of functions. Gene ontology identified pathways related to olfaction and vomeronasal pathways as being overrepresented, consistent with generation 61 analyses, despite those specific regions differing between generations. Genes Dspp and Rbm24 are also identified as potentially explaining known bone and skeletal muscle differences, respectively, between the linetypes.

Genetic approaches in model organisms have consistently demonstrated that molecular traits such as gene expression are under genetic regulation, similar to clinical traits. The resulting expression quantitative trait loci (eQTL) have revolutionized our understanding of genetic regulation and identified numerous candidate genes for clinically relevant traits. More recently, these analyses have been extended to other molecular traits such as protein abundance, metabolite levels, and miRNA expression. Here, we performed global hepatic eQTL and microRNA expression quantitative trait loci (mirQTL) analysis in a population of Diversity Outbred mice fed two different diets. We identified several key features of eQTL and mirQTL, namely differences in the mode of genetic regulation (cis or trans) between mRNA and miRNA. Approximately 50% of mirQTL are regulated by a trans-acting factor, compared to ∼25% of eQTL. We note differences in the heritability of mRNA and miRNA expression and variance explained by each eQTL or mirQTL. In general, cis-acting variants affecting mRNA or miRNA expression explain more phenotypic variance than trans-acting variants. Finally, we investigated the effect of diet on the genetic architecture of eQTL and mirQTL, highlighting the critical effects of environment on both eQTL and mirQTL. Overall, these data underscore the complex genetic regulation of two well-characterized RNA classes (mRNA and miRNA) that have critical roles in the regulation of clinical traits and disease susceptibility.

Abstract During preimplantation development, contractile forces generated at the apical cortex segregate cells into inner and outer positions of the embryo, establishing the inner cell mass (ICM) and trophectoderm. To which extent these forces influence ICM-trophectoderm fate remains unresolved. Here, we found that the nuclear lamina is coupled to the cortex via an F-actin meshwork in mouse and human embryos. Actomyosin contractility increases during development, upregulating Lamin-A levels, but upon internalization cells lose their apical cortex and downregulate Lamin-A. Low Lamin-A shifts the localization of actin nucleators from nucleus to cytoplasm increasing cytoplasmic F-actin abundance. This results in stabilization of Amot, Yap phosphorylation and acquisition of ICM over trophectoderm fate. By contrast, in outer cells, Lamin-A levels increase with contractility. This prevents Yap phosphorylation enabling Cdx2 to specify the trophectoderm. Thus, forces transmitted to the nuclear lamina control actin organization to differentially regulate the factors specifying lineage identity.

Abstract During the second long shutdown period of the CERN accelerator complex (LS2, 2019-2021), several upgrade activities took place at the n_TOF facility. The most important have been the replacement of the spallation target with a next generation nitrogen-cooled lead target. Additionally, a new experimental area, at a very short distance from the target assembly (the NEAR Station) was established. In this paper, the core commissioning actions of the new installations are described. The improvement in the n_TOF infrastructure was accompanied by several detector development projects. All these upgrade actions are discussed, focusing mostly on the future perspectives of the n_TOF facility. Furthermore, some indicative current and future measurements are briefly reported.

The biological basis of exercise behavior is increasingly relevant for maintaining healthy lifestyles. Various quantitative genetic studies and selection experiments have conclusively demonstrated substantial heritability for exercise behavior in both humans and laboratory rodents. In the High Runner selection experiment, four replicate lines of Mus domesticus were bred for high voluntary wheel running (HR), along with four nonselected control (C) lines. After 61 generations, the genomes of 79 mice (9-10 from each line) were fully sequenced and single nucleotide polymorphisms (SNPs) were identified. We used nested ANOVA with MIVQUE estimation and other approaches to compare allele frequencies between the HR and C lines for both SNPs and haplotypes. Approximately 61 genomic regions, across all somatic chromosomes, showed evidence of differentiation; 12 of these regions were differentiated by all methods of analysis. Gene function was inferred largely using Panther gene ontology terms and KO phenotypes associated with genes of interest. Some of the differentiated genes are known to be associated with behavior/motivational systems and/or athletic ability, including Sorl1, Dach1, and Cdh10 Sorl1 is a sorting protein associated with cholinergic neuron morphology, vascular wound healing, and metabolism. Dach1 is associated with limb bud development and neural differentiation. Cdh10 is a calcium ion binding protein associated with phrenic neurons. Overall, these results indicate that selective breeding for high voluntary exercise has resulted in changes in allele frequencies for multiple genes associated with both motivation and ability for endurance exercise, providing candidate genes that may explain phenotypic changes observed in previous studies.

This paper provides an in-depth review of deep learning techniques to address the challenges of odometry and global ego-localization using frequency modulated continuous wave (FMCW) radar sensors. In particular, we focus on the prediction of odometry, which involves the determination of the ego-motion of a system by external sensors, and loop closure detection, which concentrates on the determination of the ego-position typically on an existing map. We initially emphasize the significance of these tasks in the context of radar sensors and underscore the motivations behind them. The subsequent sections delve into the practical implementation of deep learning approaches, strategically designed to effectively address the aforementioned challenges. We primarily focus on spinning and automotive radar configurations within the domain of autonomous driving. Additionally, we introduce publicly available datasets that have been instrumental in addressing these challenges and analyze the importance and struggles of current methods used for radar based odometry and localization. In conclusion, this paper highlights the distinctions between the addressed tasks and other radar perception applications, while also discussing their differences from challenges posed by alternative sensor modalities. The findings contribute to the ongoing discourse on advancing radar sensor capabilities through the application of deep learning methodologies, particularly in the context of enhancing odometry and ego-localization for autonomous driving applications.

The neutron Time-of-Flight (TOF) research facility at CERN, n_TOF, has been a pioneering platform for neutron cross-section measurements since its inception in 2001. It boasts three distinct experimental areas, each tailored to address a specific range of neutron energies. This paper delves into the intricacies of the n_TOF facility, including its recent upgrade during the Long Shutdown 2 (LS2) at CERN. Additionally, it highlights the key characteristics of the detectors employed for capture and fission cross-section measurements, paving the way for future research endeavors.

Chromium is a very relevant element regarding criticality safety in nuclear reactors because of its presence in stainless steel, an important structural material. Currently, there are serious discrepancies between the different evaluations regarding the neutron capture cross sections of 50Cr and 53Cr, most probably related to the difficulty of reducing and then estimating the very large neutron scattering effects on the shape of the resonances. In this context, there is a recent entry in the Nuclear Energy Agency (NEA) High Priority Request List (HPRL) to measure these reactions between 1 and 100 keV with an accuracy of 8-10%. In response to this request, we have performed a time-of-flight experiment at CERN n_TOF (Switzerland) and a complementary activation experimenton 50Crat30and90keVatCNAHiSPANoS(Spain).Theexperiments are presented herein, together with a discussion on the quality of the preliminary data and the results to be expected.

The experimental setup of the new measurement of 239Pu fission and capture cross-section in the n_TOF time-of-flight facility at CERN is presented. The measurement aims to address the needs and demands of nuclear data users. The experiment incorporates an innovative fast Fission Fragment Detector and the n_TOF Total Absorption Calorimeter, enabling the implementation of the fission tagging technique. Preliminary results exhibit the robust performance of the detector systems, along with the high quality of the new 239Pu samples. These samples were exclusively produced for this measurement by the European Commission’s Joint Research Centre in Geel.