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BackgroundTumor-associated macrophages (TAMs) are alternatively activated cells induced by interleukin-4 (IL-4)-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs) circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells.ResultsWe utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO) that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway.ConclusionsWe conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC(50)) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies.

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds doublestranded DNA, dependent on the presence of a 5 singlestranded overhang, indicating a 5 to 3 polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT 24 binding over 10 times more strongly than dA 24 as observed by the apparent K m .

Dear Editor, The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global public health, and is imposing severe burdens on human society. Several candidate vaccines against SARS-CoV-2 are now undergoing clinical trials. The Spike (S) protein of SARS-CoV-2 is widely considered as a promising antigen. However, limited information about the protective immune response against SARS-CoV-2 has been reported. 1 In vivo or in natura data of the immune response in patients, including major immune responses to S protein, are currently lacking. The development of effective and safe vaccines against SARS-CoV-2 is urgently needed because of some potential adverse events including antibodydependent enhancement (ADE), 2 which might be difficult to avoid in current vaccine designs. Therefore, it is important to mine serological information from COVID-19 patients. In this study, we analysed the correlation between S-or Nucleocapsid (N) protein-specific antibody

The incompleteness of the fossil record obscures the origin of many of the more derived clades of vertebrates. One such group is the Ichthyopterygia, a clade of obligatory marine reptiles that appeared in the Early Triassic epoch, without any known intermediates. Here we describe a basal ichthyosauriform from the upper Lower Triassic (about 248 million years ago) of China, whose primitive skeleton indicates possible amphibious habits. It is smaller than ichthyopterygians and had unusually large flippers that probably allowed limited terrestrial locomotion. It also retained characteristics of terrestrial diapsid reptiles, including a short snout and body trunk. Unlike more-derived ichthyosauriforms, it was probably a suction feeder. The new species supports the sister-group relationships between ichthyosauriforms and Hupehsuchia, the two forming the Ichthyosauromorpha. Basal ichthyosauromorphs are known exclusively from south China, suggesting that the clade originated in the region, which formed a warm and humid tropical archipelago in the Early Triassic. The oldest unequivocal record of a sauropterygian is also from the same stratigraphic unit of the region.

Background: Wildtype mice are not susceptible to SARS-CoV-2 infection. Emerging SARS-CoV-2 variants, including B.1.1.7, B.1.351, P.1, and P.3, contain mutations in spike that has been suggested to associate with an increased recognition of mouse ACE2, raising the postulation that these SARS-CoV-2 variants may have evolved to expand species tropism to wildtype mouse and potentially other murines. Our study evaluated this possibility with substantial public health importance. Methods: We investigated the capacity of wildtype (WT) SARS-CoV-2 and SARS-CoV-2 variants in infecting mice (Mus musculus) and rats (Rattus norvegicus) under in vitro and in vivo settings. Susceptibility to infection was evaluated with RT-qPCR, plaque assays, immunohistological stainings, and neutralization assays. Findings: Our results reveal that B.1.1.7 and other N501Y-carrying variants but not WT SARS-CoV-2 can infect wildtype mice. High viral genome copies and high infectious virus particle titres are recovered from the nasal turbinate and lung of B.1.1.7-inocluated mice for 4-to-7 days post infection. In agreement with these observations, robust expression of viral nucleocapsid protein and histopathological changes are detected from the nasal turbinate and lung of B.1.1.7-inocluated mice but not that of the WT SARS-CoV-2-inoculated mice. Similarly, B.1.1.7 readily infects wildtype rats with production of infectious virus particles. Interpretation: Our study provides direct evidence that the SARS-CoV-2 variant, B.1.1.7, as well as other N501Y-carrying variants including B.1.351 and P.3, has gained the capability to expand species tropism to murines and public health measures including stringent murine control should be implemented to facilitate the control of the ongoing pandemic. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Modulation of transcription factor activity leading to changes in cell behavior (e.g., differentiation versus proliferation) is one of the critical outcomes of receptor tyrosine kinase (RTK) stimulation. In the early Drosophila embryo, activation of the torso (tor) RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. Our analysis of the tor response element (tot-RE} in the tll promoter indicates that the key activity modulated by the tor RTK pathway is a repressor present throughout the embryo. We have mapped the tor-RE to an ll-bp sequence; using this sequence as the basis for protein purification, we have determined that the proteins GAGA and NTF-1 (also known as Elf-l, product of the grainyhead gene) bind to the tot-RE. We demonstrate that NTF-1 can be phosphorylated by MAPK (mitogen-activated protein kinase), and that tll expression is expanded in embryos lacking maternal NTF-1 activity; these results make NTF-1 a likely target for modulation by the tor RTK pathway in vivo. The data presented here support a model in which activation of the tor RTK at the poles of the embryos leads to inactivation of the repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo.[Key Words: Torso tyrosine kinase receptor; transcription factor; GAGA; NTF-1; Elf-l; tailless; Trithorax-like; grainyhead] Received June 20, 1995; revised version accepted October 24, 1995.A multitude of extracellular signals can lead to a change in the program of cellular gene activity. An important mechanism by which extracellular signals {growth factors) are transduced is by receptor tyrosine kinases (RTKs). Ligand stimulation of an RTK leads to activation of a series of proteins: Transient generation of Ras-GTP leads to activation of a cascade of serine-threonine kinases in the order Raf-1, mitogen-associated protein (MAP) kinase kinase (MAPKK), and MAP kinase {MAPK) (for review, see Hill and Treisman 1995). This "cassette" of sequentially acting proteins functions in mammalian cultured cells in the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGFI activated pathways, in Drosophila in the sevenless and torso receptor-activated pathways, and in nematodes in the Let-23 (an EGF receptor homolog)-activated pathway (for review, see Perrimon 1993).Modification of the transcriptional program resulting from RTK activation can be ascribed, at least in part, to the function of activated MAPK in phosphorylating vat4Corresponding author.ious ets family transcription factors (for review, see Hill and Treisman 1995). Although our understanding of the cassette of proteins involved in the phosphorylation cascade is fairly robust, the subsequent steps that ultimately lead to altered cell behavior are less well defined. Neither all of the transcription factors modulated, nor all of the genes targeted by any one activated RTK are known.T...