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The brine shrimp, Artemia franciscana, has a body plan composed of 11 thoracic segments, followed by 2 genital segments, and then 6 additional abdominal segments. Previous studies of Artemia reported that expression of the posterior-most Hox gene, Abdominal-B (Abd-B), is restricted to the genital segments and is not observed posteriorly in the abdomen at any developmental stage. This report was remarkable because it suggested that the Artemia abdomen posterior to the genital segments was a novel body region of 6 segments that bore no homology to any region in other crustaceans and was unique amongst arthropods in being a Hox-free segmented domain outside of the head. In this study, we used RT-PCR, antibody staining, and in situ hybridization on various stages of Artemia nauplii to show that Abd-B mRNA and protein are in fact expressed throughout the abdominal segments during Artemia development, but this expression later retracts to the two genital segments (G1, G2) and the T11 appendages. This suggests that Abd-B does play a role in specifying abdominal segment identity in all crustaceans that have been examined and suggests a common evolutionary origin for the crustacean abdomen.

Hypothalamic interleukin-6 (IL6) exerts a broad metabolic control. Here, we demonstrated that IL6 activates the ERK1/2 pathway in the ventromedial hypothalamus (VMH), stimulating AMPK/ACC signaling and fatty acid oxidation in mouse skeletal muscle. Bioinformatics analysis revealed that the hypothalamic IL6/ERK1/2 axis is closely associated with fatty acid oxidation– and mitochondrial-related genes in the skeletal muscle of isogenic BXD mouse strains and humans. We showed that the hypothalamic IL6/ERK1/2 pathway requires the α2-adrenergic pathway to modify fatty acid skeletal muscle metabolism. To address the physiological relevance of these findings, we demonstrated that this neuromuscular circuit is required to underpin AMPK/ACC signaling activation and fatty acid oxidation after exercise. Last, the selective down-regulation of IL6 receptor in VMH abolished the effects of exercise to sustain AMPK and ACC phosphorylation and fatty acid oxidation in the muscle after exercise. Together, these data demonstrated that the IL6/ERK axis in VMH controls fatty acid metabolism in the skeletal muscle.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase of PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

The COVID-19 disease caued by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has two characteristics that distinguish it from other viral infections. It affects more severely people with pre-existing comorbidities and viral load peaks prior to the onset of the symptoms. Investigating factors that could contribute to these characteristics, we found increased mTOR signaling and suppressed genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2. Transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients revealed that COVID-19 severity is associated with increased expression of genes related to mTOR signaling and decreased expression of genes related to autophagy, lysosome function, and vesicle fusion. SARS-CoV-2 infection in Vero E6 cells also resulted in virus retention inside the cells and trafficking of virus-bearing vesicles between neighboring cells. Our findings support a scenario where SARS-CoV-2 benefits from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling, which might relate to undetectable proliferation and evasion of the immune system.

Regeneration is classically demonstrated in mammals using mice digit tip. In this study, we compared different amputation plans and show that distally amputated digits regrow with morphology close to normal but fail to regrow the fat pad. Proximally amputated digits do not regrow the phalangeal bone, but the remaining structures (nail, skin and connective tissue), all with intrinsic regenerative capacity, re-establishing integrity indistinguishably in distally and proximally amputated digits. Thus, we suggest that the bone growth promoted by signals and progenitor cells not removed by distal amputations is responsible for the re-establishment of a drastically different final morphology after distal or proximal digit tip amputations. Despite challenging the use of mouse digit tip as a model system for limb regeneration in mammals, these findings evidence a main role of bone growth in digit tip regeneration and suggest that mechanisms that promote joint structures formation should be the main goal of regenerative medicine for limb and digit regrowth.

Background Mutations accrued by SARS-CoV-2 lineage P.1—first detected in Brazil in early January, 2021—include amino acid changes in the receptor-binding domain of the viral spike protein that also are reported in other variants of concern, including B.1.1.7 and B.1.351. We aimed to investigate whether isolates of wild-type P.1 lineage SARS-CoV-2 can escape from neutralising antibodies generated by a polyclonal immune response. Methods We did an immunological study to assess the neutralising effects of antibodies on lineage P.1 and lineage B isolates of SARS-CoV-2, using plasma samples from patients previously infected with or vaccinated against SARS-CoV-2. Two specimens (P.1/28 and P.1/30) containing SARS-CoV-2 lineage P.1 (as confirmed by viral genome sequencing) were obtained from nasopharyngeal and bronchoalveolar lavage samples collected from patients in Manaus, Brazil, and compared against an isolate of SARS-CoV-2 lineage B (SARS.CoV2/SP02.2020) recovered from a patient in Brazil in February, 2020. Isolates were incubated with plasma samples from 21 blood donors who had previously had COVID-19 and from a total of 53 recipients of the chemically inactivated SARS-CoV-2 vaccine CoronaVac: 18 individuals after receipt of a single dose and an additional 20 individuals (38 in total) after receipt of two doses (collected 17–38 days after the most recent dose); and 15 individuals who received two doses during the phase 3 trial of the vaccine (collected 134–230 days after the second dose). Antibody neutralisation of P.1/28, P.1/30, and B isolates by plasma samples were compared in terms of median virus neutralisation titre (VNT50, defined as the reciprocal value of the sample dilution that showed 50% protection against cytopathic effects). Findings In terms of VNT50, plasma from individuals previously infected with SARS-CoV-2 had an 8·6 times lower neutralising capacity against the P.1 isolates (median VNT50 30 [IQR Interpretation SARS-CoV-2 lineage P.1 might escape neutralisation by antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Continuous genomic surveillance of SARS-CoV-2 combined with antibody neutralisation assays could help to guide national immunisation programmes. Funding São Paulo Research Foundation, Brazilian Ministry of Science, Technology and Innovation and Funding Authority for Studies, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health. Translation For the Portuguese translation of the abstract see Supplementary Materials section.

Microbiota-derived molecules called short-chain fatty acids (SCFAs) play a key role in the maintenance of the intestinal barrier and regulation of immune response during infectious conditions. Recent reports indicate that SARS-CoV-2 infection changes microbiota and SCFAs production. However, the relevance of this effect is unknown. In this study, we used human intestinal biopsies and intestinal epithelial cells to investigate the impact of SCFAs in the infection by SARS-CoV-2. SCFAs did not change the entry or replication of SARS-CoV-2 in intestinal cells. These metabolites had no effect on intestinal cells’ permeability and presented only minor effects on the production of anti-viral and inflammatory mediators. Together our findings indicate that the changes in microbiota composition of patients with COVID-19 and, particularly, of SCFAs do not interfere with the SARS-CoV-2 infection in the intestine.

Background: A new SARS-CoV-2 lineage, named P.1 (20J/501Y.V3), has recently been detected in Brazil. Mutations accrued by the P.1 lineage include amino acid changes in the receptor-binding domain of the spike protein that also are reported in variants of concern in the United Kingdom (B.1.1.7) and South Africa (B.1.325). Methods: We isolated two P.1-containing specimens from nasopharyngeal and bronchoalveolar lavage samples of patients of Manaus, Brazil. We measured neutralization of the P.1 virus after incubation with the plasma of 19 COVID-19 convalescent blood donors and recipients of the chemically-inactivated CoronaVac vaccine and compared these results to neutralization of a SARS-CoV-2 B-lineage previously circulating in Brazil. Findings: The immune plasma of COVID-19 convalescent blood donors had 6-fold less neutralizing capacity against the P.1 than against the B-lineage. Moreover, five months after booster immunization with CoronaVac, plasma from vaccinated individuals failed to efficiently neutralize P.1 lineage isolates. Interpretation: These data indicate that the P.1 lineage may escape from neutralizing antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Funding: Sao Paulo Research Foundation, MCTI/FINEP, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health. Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGMBiopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. Ethical Approval: All procedures followed the ethical standards of the responsible committee on humanexperimentation and approved by the ethics committees from the University of Campinas, Brazil (Approval number CONEP 4.021.484 for plasma collection of blood donors, CAEE32078620.4.0000.5404 and 30227920.9.0000.5404 for the sampling of vaccinated and viral genome sequencing, respectively). All patient data were anonymized before study inclusion.Informed consent was obtained from all subjects for being included in the study.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality1,2. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here we show that SARS-CoV-2 infects human CD4+T helper cells, but not CD8+T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Summary Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while pro-amniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings., Graphical abstract, Highlights • Increased-size embryos present a delay in embryonic morphogenesis • Embryo size determines the mechanism of pro-amniotic cavity formation • Apoptosis is required for the formation of a single cavity in enlarged embryos • Cavity formation and size regulation have distinct molecular underpinnings, In this article Shahbazi, Zernicka-Goetz, and colleagues explore how increasing embryo size affects the mechanism and timing of embryonic morphogenesis. Enlarged embryos present a delay in pro-amniotic cavity formation, an essential morphogenetic event that happens concomitantly with the regulation of embryo size. However, whereas pro-amniotic cavity formation requires apoptosis of embryonic cells lacking basement membrane contact, size regulation occurs independently of cell death.

Made available in DSpace on 2020-12-10T17:31:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Analysis of the transcriptome of organisms exposed to toxicants offers new insights for ecotoxicology, but further research is needed to enhance interpretation of results and effectively incorporate them into useful environmental risk assessments. Factors that must be clarified to improve use of transcriptomics include assessment of the effect of organism sex within the context of toxicant exposure. Amphipods are well recognized as model organisms for toxicity evaluation because of their sensitivity and amenability to laboratory conditions. To investigate whether response to metals in crustaceans differs according to sex we analyzed the amphipod Parhyale hawaiensis after exposure to AgCl and Ag nanoparticles (AgNP) via contaminated food. Gene specific analysis and whole genome transcriptional profile of male and female organisms were performed by both RT-qPCR and RNA-seq. We observed that expression of transcripts of genes glutathione transferase (GST) did not differ among AgCl and AgNP treatments. Significant differences between males and females were observed after exposure to AgCl and AgNP. Males presented twice the number of differentially expressed genes in comparison to females, and more differentially expressed were observed after exposure to AgNP than AgCl treatments in both sexes. The genes that had the greatest change in expression relative to control were those genes related to peptidase and catalytic activity and chitin and carbohydrate metabolic processes. Our study is the first to demonstrate sex specific differences in the transcriptomes of amphipods upon exposure to toxicants and emphasizes the importance of considering gender in ecotoxicology. (C) 2020 Elsevier Ltd. All rights reserved. Univ Sao Paulo, Sch Pharmaceut Sci, BR-05508000 Sao Paulo, Brazil Univ Estadual Campinas, Sch Technol, BR-13484332 Limeira, SP, Brazil Univ Estadual Campinas, Sch Appl Sci, Lab Biotechnol, BR-13484350 Limeira, SP, Brazil Sao Paulo State Univ, Inst Biosci, BR-13506900 Rio Claro, SP, Brazil Univ Estadual Campinas, Life Sci Core Facil LaCTAD, BR-13083886 Campinas, SP, Brazil Heriot Watt Univ, Sch Energy Geosci Infrastruct & Soc, Inst Life & Earth Sci, Edinburgh EH14 4AS, Midlothian, Scotland Univ Tennessee, Ctr Environm Biotechnol, 676 Dabney Hall,1416 Circle Dr, Knoxville, TN 37996 USA Univ Estadual Campinas, Dept Biochem & Tissue Biol, BR-13083970 Campinas, SP, Brazil Sao Paulo State Univ, Inst Biosci, BR-13506900 Rio Claro, SP, Brazil FAPESP: 2014/08829-7 CNPq: 400362/2014-7 CAPES: 001

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-AandArginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet forT. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliverin planta-transcribed dsRNA to target insect genes.Tuta absolutalarvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage byT. absolutain these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

Background Providing double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The tomato leafminer (Tuta absoluta Meyrick) is a major Solanum lycopersicum (tomato) pest that causes significant yield losses and has recently invaded Europe, from where it is spreading at an alarming rate. To explore RNA interference (RNAi) against T. absoluta, sequence information on potential target genes is necessary, but only a few sequences are available in public databases. Results We sequenced six libraries from RNA samples from eggs, adults, and larvae at four stages, obtaining an overall total of around 245 million reads. The assembled T. absoluta transcriptome contained 93,477 contigs with an average size of 1,574 bp, 59.8 % of which presented positive Blast hits, with 19,995 (21.4 %) annotated by gene ontology. From the transcriptome, most of the core genes of the RNAi mechanism of Lepidoptera were identified indicating the potential suitability of T. absoluta for gene silencing. No contigs displayed significant similarity with a RNA-dependent RNA Polymerase. Genes from the juvenile hormone and ecdysteroid biosynthetic pathways were identified, representing potential target genes for systemic silencing. Comparisons of transcript profiles among stages revealed 1,577 genes differentially expressed at earlier larval stages, from which potential gene targets were identified. Five of these genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, which were fed to 1st instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes. Conclusions The transcriptome we generated represents a valuable genomic resource for screening potential gene targets that affect the development or survival of T. absoluta larvae. Five novel genes that showed greater expression at the 1st larval stage were demonstrated to be effective potential RNAi targets by reducing larval weight and can be considered good candidates for use in RNAi-mediated crop protection. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1841-5) contains supplementary material, which is available to authorized users.

The identification of subpharyngeal cardiac precursors has had a strong influence on the way we think about early cardiac development. From this discovery was born the concept of multiple heart fields. Early support for the concept came from gene expression, genetic retrospective fate mapping, and gene targeting studies, which collectively suggested the existence of a second heart field (SHF) on the basis of specific Islet-1 (Isl-1) expression, presence of two cardiac ancestral lineages, and compatible cardiac knockout phenotypes, respectively. A decade after the original studies, support for the SHF concept is dwindling. This is because in all bilaterian models studied, Isl expression in heart progenitors is not SHF-specific, because lineage data are best explained by alternative models including an older, truly ancestral, lineage of cardiac pioneers with unrestricted contribution to all cardiac segments and, finally, because the inflow-to-outflow segmental nature of the early vertebrate peristaltic heart has been reaffirmed with novel, less invasive, methodologies. Altogether, the paradigms derived from the discovery of subpharyngeal cardiac progenitors helped us shift from relatively simple models, which rely predominantly either on patterning, gene expression patterns or lineages, to a much more sophisticated body of knowledge in which all these parameters must be accounted. Thus, it is well possible that due consideration of the key elements contained in the inflow/outflow, pioneer/scaffold, ballooning, and SHF hypotheses may provide us with a unified framework of the early stages of cardiac development. Here, we advance into this direction by suggesting an intuitive model of early heart development based on the concept of an inflow/outflow scaffold erected by cardiac pioneers, one that is required to assemble all the subsequent cell contribution that emigrates from cardiac progenitor areas.

The gap gene hunchback in Drosophila acts during syncytial blastoderm stage via a short-range gradient and concentration-dependent activation or repression of target genes. Orthologues of hunchback can be easily found in other insects, but it has been unclear how well its functions are conserved. The segmentation process in most insect embryos occurs under cellular conditions, which should not allow the formation of diffusion-controlled transcription factor gradients. We have studied here in detail the function of hunchback in the short germ embryo of Tribolium using parental RNAi and interaction with possible target genes. We find that hunchback is a major regulator of the trunk gap genes and Hox genes in Tribolium, but may only indirectly be required to regulate other segmentation genes. The core function of hunchback appears to be the setting of the Ultrabithoraxexpression border via a repression effect, and the activation of the Krüppel expression domain. These regulatory effects are likely to be direct and are conserved between Drosophila and Tribolium. We find no evidence for a classical gap phenotype in the form of loss of segments in the region of expression of hunchback. However, the phenotypic effects in Tribolium are highly comparable with those found for other short germ embryos, i.e. the core functions of hunchback in Tribolium appear to be the same in these other insects, although they are evolutionarily more distant to Tribolium,than Tribolium is to Drosophila. These results allow the disentanglement of the conserved role of hunchback in insects from the derived features that have been acquired in the lineage towards Drosophila. Given that the gap phenotype appears to occur only in long germ embryos and that the main role of hunchback appears to be the regionalization of the embryo, it may be appropriate to revive an alternative name for the class of gap genes, namely `cardinal genes'.

Summary Segmentation genes in insects are required for generating the subdivisions of the early embryo. We describe here a new member of the gap family of segmentation genes in the flour beetle Tribolium , mille-pattes ( mlpt ). mlpt knockdown leads to transformation of the abdominal segments into thoracic segments, providing embryos with up to ten pairs of legs. We show that there are crossregulatory interactions between mlpt and the known gap genes in Tribolium , suggesting that mlpt is itself a gap gene. The mlpt gene reveals an unusual structure, as it encodes a polycistronic mRNA that codes for four peptides. mlpt appears to be the prototype of this previously unknown gene structure in eukaryotes, as we find homologous genes with the same polycistronic arrangement in other insect genomes as well.

Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila. Electronic supplementary material The online version of this article (doi:10.1007/s00427-008-0240-1) contains supplementary material, which is available to authorized users.