Expression of Abdominal-B in the brine shrimp, Artemia franciscana, expands our evolutionary understanding of the crustacean abdomen.

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.

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection.

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling.

Differential expression of regulators of the canonical Wnt pathway during the compensatory beta-cell hyperplasia in prediabetic mice.

We previously reported that the canonical Wnt signaling pathway is activated during compensatory islet hyperplasia in prediabetic mice. Here, we aimed to expand our knowledge concerning the Wnt signaling partners and modulators involved in this process. We report here that Axin1, Axin2, and DACT1, inhibitors of the canonical Wnt signaling pathway, displayed no change in their expression, while GSK-3ß, a multi-functional kinase that acts as a negative regulator of this pathway as well as affects insulin secretion/action, was up-regulated in hyperplastic islets of prediabetic mice. We also observed that COUP-TFII, a protein that acts positively on Wnt-target genes related to cell proliferation, displays a significant increase in gene expression and protein content and is highly immunolabeled in islet cell nuclei of prediabetic mice compared to control islets. These findings suggest that GSK-3ß and COUP-TFII may play a role in beta-cell dysfunction and hyperplasia during type 2 prediabetes.

Antenna regeneration as an ecotoxicological endpoint in a marine amphipod: a proof of concept using dimethyl sulfoxide and diflubenzuron.

Regeneration is a widely spread process across the animal kingdom, including many species of marine crustaceans. It is strongly linked to hormonal cycles and, therefore, a great endpoint candidate for toxicology studies. We selected the amphipod Parhyale hawaiensis as test organism, already used in ecotoxicological studies and able to regenerate its body appendages. We are proposing a protocol to use the antenna regeneration as a toxicity endpoint. First, we evaluated differences in time of completion of regeneration in males and females after the amputation of one antenna of 6 months old animals. Then we compared the influence of different testing volumes in the regeneration process (100 and 5 mL). We used as testing substances, dimethyl sulfoxide (DMSO) and diflubenzuron, a chitin synthesis inhibitor. The most suitable protocol consisted of volumes of 5 mL in 12-well microplates, with 1 organism per well, 12 organisms per concentration (1:1 females/males) and test time duration of around 5 weeks. DMSO accelerated regeneration time with a NOEC of 0.06%. Diflubenzuron inhibited the time necessary to its completion with a NOEC of 0.32 µg L-1. We conclude that the Parhyale hawaiensis antenna regeneration protocol proposed here is a potential tool in ecotoxicology, but more studies are required for its validation not only to verify its utility for testing chemicals but also environmental samples.

De novo transcriptome assembly and analysis to identify potential gene targets for RNAi-mediated control of the tomato leafminer (Tuta absoluta).

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 1(st) 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 1(st) 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.

Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

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 PLA (Proximity Ligation 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 in 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.

Evidence for a neuromuscular circuit involving hypothalamic interleukin-6 in the control of skeletal muscle metabolism.

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 infects adipose tissue in a fat depot- and viral lineage-dependent manner.

Visceral adiposity is a risk factor for severe COVID-19, and a link between adipose tissue infection and disease progression has been proposed. Here we demonstrate that SARS-CoV-2 infects human adipose tissue and undergoes productive infection in fat cells. However, susceptibility to infection and the cellular response depends on the anatomical origin of the cells and the viral lineage. Visceral fat cells express more ACE2 and are more susceptible to SARS-CoV-2 infection than their subcutaneous counterparts. SARS-CoV-2 infection leads to inhibition of lipolysis in subcutaneous fat cells, while in visceral fat cells, it results in higher expression of pro-inflammatory cytokines. Viral load and cellular response are attenuated when visceral fat cells are infected with the SARS-CoV-2 gamma variant. A similar degree of cell death occurs 4-days after SARS-CoV-2 infection, regardless of the cell origin or viral lineage. Hence, SARS-CoV-2 infects human fat cells, replicating and altering cell function and viability in a depot- and viral lineage-dependent fashion.

Embryo size regulates the timing and mechanism of pluripotent tissue morphogenesis.

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.

Microbiota-derived short-chain fatty acids do not interfere with SARS-CoV-2 infection of human colonic samples.

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.

Neutralisation of SARS-CoV-2 lineage P.1 by antibodies elicited through natural SARS-CoV-2 infection or vaccination with an inactivated SARS-CoV-2 vaccine: an immunological study.

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 <20-45] for P.1/28 and 30 [<20-40] for P.1/30) than against the lineage B isolate (260 [160-400]), with a binominal model showing significant reductions in lineage P.1 isolates compared with the lineage B isolate (p≤0·0001). Efficient neutralisation of P.1 isolates was not seen with plasma samples collected from individuals vaccinated with a first dose of CoronaVac 20-23 days earlier (VNT50s below the limit of detection [<20] for most plasma samples), a second dose 17-38 days earlier (median VNT50 24 [IQR <20-25] for P.1/28 and 28 [<20-25] for P.1/30), or a second dose 134-260 days earlier (all VNT50s below limit of detection). Median VNT50s against the lineage B isolate were 20 (IQR 20-30) after a first dose of CoronaVac 20-23 days earlier, 75 (<20-263) after a second dose 17-38 days earlier, and 20 (<20-30) after a second dose 134-260 days earlier. In plasma collected 17-38 days after a second dose of CoronaVac, neutralising capacity against both P.1 isolates was significantly decreased (p=0·0051 for P.1/28 and p=0·0336 for P.1/30) compared with that against the lineage B isolate. All data were corroborated by results obtained through plaque reduction neutralisation tests. 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.

Transcriptome analysis in Parhyale hawaiensis reveal sex-specific responses to AgNP and AgCl exposure.

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.

RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum).

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-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. 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 deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae 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 by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

Delimiting the conserved features of hunchback function for the trunk organization of insects.

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 Ultrabithorax expression 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'.

The role of the segmentation gene hairy in Tribolium.

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.

A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides.

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.