The telomerase enzyme enables unlimited proliferation of most human cancer cells by elongating telomeres and preventing replicative senescence. Despite the critical importance of telomerase in cancer ...biology, challenges detecting telomerase activity and expression in individual cells have hindered the ability to study patterns of telomerase expression and function across heterogeneous cell populations. While sensitive assays to ascertain telomerase expression and function exist, these approaches have proven difficult to implement at the single cell level. Here, we validate in situ RNAscope detection of the telomerase TERT mRNA and couple this assay with our recently described TSQ1 method for in situ detection of telomere elongation. This approach enables detection of TERT expression, telomere length, and telomere elongation within individual cells of the population. Using this assay, we show that the heterogeneous telomere elongation observed across a HeLa cell population is in part driven by variable expression of the TERT gene. Furthermore, we show that the absence of detectable telomere elongation in some TERT-positive cells is the result of inhibition by the telomeric shelterin complex. This combined assay provides a new approach for understanding the integrated expression, function, and regulation of telomerase at the single cell level.
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a ...possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.
Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in ...the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.
Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.
Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of ...genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.
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•A method for genome-wide CRISPR screens in primary human T cells•Screens identify regulators of T cell stimulation and immunosuppression•Candidate hits can boost T cell activation and in vitro cancer cell killing•Pooled perturbations with single-cell RNA-seq revealed affected gene programs
CRISPR screening in primary human T cells combined with RNA sequencing identifies regulators of T cell stimulation and suppression with implications for immunotherapy.
Aims. We present the characteristics and some early scientific results of the first instrument at the Large Binocular Telescope (LBT), the Large Binocular Camera (LBC). Each LBT telescope unit will ...be equipped with similar prime focus cameras. The blue channel is optimized for imaging in the $UV-B$ bands and the red channel for imaging in the VRIz bands. The corrected field-of-view of each camera is approximately 30 arcmin in diameter, and the chip area is equivalent to a 23$\times$23 arcmin2 field. In this paper we also present the commissioning results of the blue channel. Methods. The scientific and technical performance of the blue channel was assessed by measurement of the astrometric distortion, flat fielding, ghosts, and photometric calibrations. These measurements were then used as input to a data reduction pipeline applied to science commissioning data. Results. The measurements completed during commissioning show that the technical performance of the blue channel is in agreement with original expectations. Since the red camera is very similar to the blue one we expect similar performance from the commissioning that will be performed in the following months in binocular configuration. Using deep UV image, acquired during the commissioning of the blue camera, we derived faint UV galaxy-counts in a ~ 500 sq. arcmin sky area to U(Vega) = 26.5. These galaxy counts imply that the blue camera is the most powerful UV imager presently available and in the near future in terms of depth and extent of the field-of-view. We emphasize the potential of the blue camera to increase the robustness of the UGR multicolour selection of Lyman break galaxies at redshift z ~ 3.
The telomerase enzyme plays a critical role in human aging and cancer biology by maintaining telomere length and extending the proliferative lifespan of most stem cells and cancer cells. Despite the ...importance of this enzyme, our understanding of the mechanisms that regulate its activity and establish telomere length homeostasis in mammalian cells is incomplete, in part because the perfect repetitive nature of telomeric sequence hampers in situ detection of telomere elongation patterns. Here, we describe a novel assay using a mutant telomerase that adds a well-tolerated variant telomeric repeat sequence to telomere ends. By specifically detecting the addition of these variant repeats, we can directly visualize telomere elongation events in human cells. We validate this approach by in situ mapping of telomere elongation patterns within individual nuclei and across a population of cells.
•The performance of the SOLARIS concentrator are presented and discussed as function of the direct normal irradiance.•The performance have been estimated as function of the number of residential ...utilities, considering an entire year.•The optimal number of utilities connected to the SOLARIS was found considering both the energy and economic point of view.•The optimal size of electric and thermal storage have been defined.•The maximum capital cost of the system has been estimated in order to achieve a roi in ten or twelve years.
The research on photovoltaic conversion is continuously overtaking technological challenges and modern PV cells can nowadays be efficiently combined with solar concentrators. In this paper a new photovoltaic solar concentrator model based on non-imaging optics and embedding high efficiency multi-junctions cells is presented. The concentrator has been optimized to maximize the electricity production but it is thought to work in cogeneration to allow also for thermal energy by recovering the residual heat, since an active cooling system for the cells is necessary. The efficiency enhancement is obtained thanks to the high matching between the collected solar irradiance and the receiver electrical features.
The aim of the paper is the definition of the number of residential utilities connected to the system in order to minimize the exchange of energy with the network and also the use of auxiliary boiler. Also the optimal size of electric and thermal storage to be integrated to the concentrator, has been determined as function of the number of utilities.
To achieve these goals a parametric analysis has been carried out by the use of an in-house-developed calculation code considering a whole year of operation in order to estimate the energy fluxes and the economic performance. Furthermore, the maximum capital cost of the system has been estimated in order to achieve a return on the investment in ten or twenty years.
According to the Authors’ knowledge, the novelty of this study regards the implementation of this kind of photovoltaic solar concentrator – for combined heat and power production – in a residential application.
ABSTRACT
We present a novel analysis of a young star cluster in the Large Magellanic cloud, R136-like, as seen by the Extremely Large Telescope (ELT). The main aim of this study is to quantify ...precision and accuracy of stellar proper motion measurements in crowded field when using an ELT working at its diffraction limit. This can serve as a reference study for future development of ELT scientific cases. In particular, we investigate our future ability to detect the dynamical signature of intermediate-mass black holes (IMBHs) with mass ∼104 M⊙ through detailed measurements of stellar proper motions. We have simulated two N-body dynamical cluster models with and without an IMBH. For each model, we have chosen two snapshots temporally spaced by 5 yr. Stellar fluxes in IJHK filters and star positions have been used to create ELT mock images for both single- and multiconjugate adaptive optics observing modes following the requierements given by ESO technical specifications for the first light imager. These images have been analysed using a classical software for seeing-limited data reduction, daophot/allstar. We make accurate photometry till the very faint pre-main-sequence stars, i.e. depending on the adaptive optics (AO) mode, magnitudes down to K ∼ 24 mag (single-conjugate AO) or K ∼ 22 mag (multiconjugate AO) in a total integration time of 20 min on target. Although daophot suite of programs is not devoted to precise astrometry, the astrometric accuracy is impressive, reaching few μas yr−1 or km s−1. In these assumptions, we are able to detect the IMBH signature at the centre of the cluster.
Abstract
The new class of large telescopes, like the future Extremely Large Telescope (ELT), are designed to work with a laser guide star (LGS) tuned to a resonance of atmospheric sodium atoms. This ...wavefront sensing technique presents complex issues when applied to big telescopes for many reasons, mainly linked to the finite distance of the LGS, the launching angle, tip–tilt indetermination and focus anisoplanatism. The implementation of a laboratory prototype for the LGS wavefront sensor (WFS) at the beginning of the phase study of MAORY (Multi-conjugate Adaptive Optics Relay) for ELT first light has been indispensable in investigating specific mitigation strategies for the LGS WFS issues. This paper presents the test results of the LGS WFS prototype under different working conditions. The accuracy within which the LGS images are generated on the Shack–Hartmann WFS has been cross-checked with the MAORY simulation code. The experiments show the effect of noise on centroiding precision, the impact of LGS image truncation on wavefront sensing accuracy as well as the temporal evolution of the sodium density profile and LGS image under-sampling.
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