Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK-cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK-recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNADNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.Until recently, genetic and molecular experiments on homologous recombination in cultured mammalian cells have been limited to viral systems such as simian virus 40 and adenovirus (1)(2)(3)(4)(5). In these studies the presence of viral recombination function is not precluded, although the design of certain experiments (3-5) makes this problem seem unlikely. The development of DNA-mediated gene transfer (6, 7) and recombinant DNA techniques provides a means for the study of homologous recombination between defined selectable DNA sequences in mammalian cells free of potential viral-encoded recombination functions. While our studies were in progress, several reports appeared presenting findings that are consistent with homologous recombination after introduction of different plasmid DNA molecules into mouse cells (8)(9)(10). One of these studies used microinjection of physically marked sequences (8). Two other investigations applied selective systems (9, 10), similar to the one described here, in which mutant genes introduced into mammalian cells were shown to recombine to yield wild-type genes. In addition, when truncated H2 mouse genes were introduced into mouse cells, complete H2 gene products were observed at low frequency (11). The authors state that these findings are consistent, at least, with homologous recombination between incoming and resident H2 sequences. However, in this latter study, evidence for recombination at the DNA level is at present lacking.In this communication, we present evidence for homologous recombination between various mutant genes of herpes simplex virus type 1 thymidine kinase (HTK). These mutants result from insertion of 8-base-pair (bp) synthetic oligonucleotide linkers that specify a unique recognition site for the restriction endonuclease Xho I. These mutations represent subtle alterations of the HTK coding region and yet provide an easy means for distinguishing between mutant and wild-type genes. As expected, when defective HTK genes bearing frameshift mutations are introduced into thymidine kinase (TK)-deficient mouse L cells, transformation frequencies are considera...

This article was fast-tracked under a recently instituted interim policy in which editors may, at their discretion, accept coronavirus-related manuscripts submitted for the Review, Perspectives, and Hypotheses categories without additional review.

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Highlights d R53-induced gliomas present higher recruitment of peripheral immune cells d Neutrophils levels are higher from initiation through progression in R53 tumors d Depletion of neutrophils at initiation stage facilitates tumor growth d Bone marrow-derived neutrophils switch from anti-to protumorigenic phenotype

Eosinophils are multifunctional, evolutionary conserved leukocytes that are involved in a plethora of responses ranging from regulation of tissue homeostasis to host defense and cancer. Eosinophils have been studied mostly in the context of Type 2 inflammatory responses such as those found in allergy. Nonetheless, it is now evident that they participate in Type 1 inflammatory responses and can respond to Type 1 cytokines such as IFN-γ. Recent data suggest that the pleotropic roles of eosinophils are due to heterogeneous responses to environmental cues. Despite this, the activation profile of eosinophils, in response to various stimuli is yet to be defined. To better understand the transcriptional spectrum of eosinophil activation, we exposed eosinophils to Type 1 (e.g. IFN-γ, E. coli) vs. Type 2 (e.g. IL-4) conditions and subjected them to global RNA sequencing. Our analyses show that IL-4, IFN-γ, E. coli and IFN-γ in the presence of E. coli (IFN-γ/E. coli)-stimulated eosinophils acquire distinct transcriptional profiles, which polarize them towards what we termed Type 1 and Type 2 eosinophils. Bioinformatics analyses using Gene Ontology based on biological processes revealed that different stimuli induced distinct pathways in eosinophils. These pathways were confirmed using functional assays by assessing cytokine/chemokine release (i.e. CXCL9, CCL24, TNF-α and IL-6) from eosinophils following activation. In addition, analysis of cell surface markers highlighted CD101 and CD274 as potential cell surface markers that distinguish between Type 1 and Type 2 eosinophils, respectively. Finally, the transcriptome signature of Type 1 eosinophils resembled that of eosinophils that were obtained from mice with experimental colitis whereas the transcriptome signature of Type 2 eosinophils resembled that of eosinophils from experimental asthma. Our data demonstrate that eosinophils are polarized to distinct “Type 1” and “Type 2” phenotypes following distinct stimulations. These findings provide fundamental knowledge regarding the heterogeneity of eosinophils and support the presence of transcriptional differences between Type 1 and Type 2 cells that are likely reflected by their pleotropic activities in diverse disease settings.

COVID-19 exerts deleterious cardiopulmonary effects, leading to a worse prognosis in the most affected. This retrospective multi-center observational cohort study aimed to analyze the trajectories of key vitals amongst hospitalized COVID-19 patients using a chest-patch wearable providing continuous remote patient monitoring of numerous vital signs. The study was conducted in five COVID-19 isolation units. A total of 492 COVID-19 patients were included in the final analysis. Physiological parameters were measured every 15 min. More than 3 million measurements were collected including heart rate, systolic and diastolic blood pressure, cardiac output, cardiac index, systemic vascular resistance, respiratory rate, blood oxygen saturation, and body temperature. Cardiovascular deterioration appeared early after admission and in parallel with changes in the respiratory parameters, showing a significant difference in trajectories within sub-populations at high risk. Early detection of cardiovascular deterioration of COVID-19 patients is achievable when using frequent remote patient monitoring.

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