Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting rings in Caenorhabditis elegans embryos. Laser cutting causes rings to snap open. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms and is repaired by recruitment of new material in an actin polymerization-dependent manner. An open ring is able to constrict, and rings repair from successive cuts. After gap repair, an increase in constriction velocity allows cytokinesis to complete at the same time as controls. Our analysis demonstrates that tension in the ring increases while net cortical tension at the site of ingression decreases throughout constriction and suggests that cytokinesis is accomplished by contractile modules that assemble and contract autonomously, enabling local repair of the actomyosin network. Consequently, cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure.

Upper respiratory obstruction is a common sequela in children with Zika-related microcephaly (ZRM). As a cross-sectional analysis nested in a cohort study, this study aims to investigate the prevalence of adenoid hypertrophy (AH) in children with ZRM and symptoms of respiratory obstruction. The data were collected in the first three years of life from children with ZRM who were followed in two reference centers for otorhinolaryngological care of patients with congenital Zika syndrome. Out of 92 children with confirmed ZRM, 57 were evaluated by nasopharyngoscopy after presenting with upper respiratory obstruction symptoms. In this study, 31 of the 57 (54%) children with ZRM who were evaluated had obstructive AH. Thirteen children with obstructive AH were submitted to surgery, which resulted in the complete resolution of symptoms for 11, partial resolution in 1, and no improvement in 1. No evidence of direct involvement by Zika virus (ZIKV) infection in the adenoid tissues was demonstrated by histology or immunohistochemistry. Our results suggest that there is a high prevalence and early presentation of AH in children with ZRM, with consequent upper airway obstruction causing upper airway obstructive disorder, secretory otitis media, and dysphagia.

Cytokinesis is the last step of cell division that physically partitions the mother cell into two daughter cells. Cytokinesis requires the assembly and constriction of a contractile ring, a circumferential array of filamentous actin (F-actin), non-muscle myosin II motors (myosin), and actin-binding proteins that forms at the cell equator. Cytokinesis is accompanied by long-range cortical flows from regions of relaxation toward regions of compression. In the C. elegans one-cell embryo, it has been suggested that anterior-directed cortical flows are the main driver of contractile ring assembly. Here, we use embryos co-expressing motor-dead and wild-type myosin to show that cortical flows can be severely reduced without major effects on contractile ring assembly and timely completion of cytokinesis. Fluorescence recovery after photobleaching in the ingressing furrow reveals that myosin recruitment kinetics are also unaffected by the absence of cortical flows. We find that myosin cooperates with the F-actin crosslinker plastin to align and compact F-actin bundles at the cell equator, and that this cross-talk is essential for cytokinesis. Our results thus argue against the idea that cortical flows are a major determinant of contractile ring assembly. Instead, we propose that contractile ring assembly requires localized concerted action of motor-competent myosin and plastin at the cell equator.

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube-polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.

Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas.

Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.

Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

Background The recent epidemics of Zika virus (ZIKV) implicated it as the cause of serious and potentially lethal congenital conditions such microcephaly and other central nervous system defects, as well as the development of the Guillain-Barré syndrome in otherwise healthy patients. Recent findings showed that anti-Dengue antibodies are capable of amplifying ZIKV infection by a mechanism similar to antibody-dependent enhancement, increasing the severity of the disease. This scenario becomes potentially catastrophic when the global burden of Dengue and the advent of the newly approved anti-Dengue vaccines in the near future are taken into account. Thus, antiviral chemotherapy should be pursued as a priority strategy to control the spread of the virus and prevent the complications associated with Zika. Methods Here we describe a fast and reliable cell-based, high-content screening assay for discovery of anti-ZIKV compounds. This methodology has been used to screen the National Institute of Health Clinical Collection compound library, a small collection of FDA-approved drugs. Results and conclusion From 725 FDA-approved compounds triaged, 29 (4%) were found to have anti-Zika virus activity, of which 22 had confirmed (76% of confirmation) by dose-response curves. Five candidates presented selective activity against ZIKV infection and replication in a human cell line. These hits have abroad spectrum of chemotypes and therapeutic uses, offering valuable opportunities for selection of leads for antiviral drug discovery.

Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two non-muscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles.This article has an associated 'The people behind the papers' interview.

The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions.

High 17β-Estradiol (E2) levels are known to cause alterations of spermatogenesis and environments throughout the male reproductive tract. Sertoli cells (SCs) ensure an adequate environment inside the seminiferous tubule. Glycerol stands as essential for the maintenance of blood⁻testis barrier created by SCs, however, the role of E2 in this process is not known. Herein, we hypothesized that the effect of E2 on glycerol permeability in mouse SCs (mSCs) could be mediated by aquaglyceroporins. The expression of aquaglyceroporins was assessed by RT-PCR and qRT-PCR. Glycerol permeability was evaluated by stopped-flow light scattering. We were able to identify the expression of AQP3 and AQP9 in mSCs where AQP9 is more abundant than AQP3. Our results show that high E2 levels decrease AQP9 mRNA abundance with no influence on AQP3 in mSCs. Interestingly, high E2 levels decreased mSCs' permeability to glycerol, while downregulating AQP9 expression, thus suggesting a novel mechanism by which E2 modulates fluid secretion in the testis. In conclusion, E2 is an important regulator of mSCs physiology and secretion through changes in AQP9 expression and function. Thus, alterations in glycerol permeability induced by E2 may be the cause for male infertility in cases associated with the presence of high E2 levels.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

Glioblastoma (GBM) is a primary malignant tumor of the central nervous system responsible for the most deaths among patients with primary brain tumors. Current therapies for GBM are not effective, with the average survival of GBM patients after diagnosis being limited to a few months. Chemotherapy is difficult in this case due to the heterogeneity of GBM and the high efficacy of the blood-brain barrier, which makes drug absorption into the brain extremely difficult. In a previous study, 3',4',3,4,5-trimethoxychalcone (MB) showed antiproliferative and anti-invasion activities toward GBM cells. Polymersomes (PMs) are an attractive, new type of nanoparticle for drug administration, due to their high stability, enhanced circulation time, biodegradability, and sustained drug release. In the present study, different MB formulations, PEG2000-PCL and PEG5000-PCL, were synthesized, characterized, and compared in terms of 14-day stability and in vitro cytotoxicity (hCMEC/D3 and U-373 MG).

We analyse new genomic data (0.05-2.95x) from 14 ancient individuals from Portugal distributed from the Middle Neolithic (4200-3500 BC) to the Middle Bronze Age (1740-1430 BC) and impute genomewide diploid genotypes in these together with published ancient Eurasians. While discontinuity is evident in the transition to agriculture across the region, sensitive haplotype-based analyses suggest a significant degree of local hunter-gatherer contribution to later Iberian Neolithic populations. A more subtle genetic influx is also apparent in the Bronze Age, detectable from analyses including haplotype sharing with both ancient and modern genomes, D-statistics and Y-chromosome lineages. However, the limited nature of this introgression contrasts with the major Steppe migration turnovers within third Millennium northern Europe and echoes the survival of non-Indo-European language in Iberia. Changes in genomic estimates of individual height across Europe are also associated with these major cultural transitions, and ancestral components continue to correlate with modern differences in stature.

Several arboviruses, including dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV), transmitted by Aedes mosquitoes, circulate in northeast Brazil. Diseases caused by these viruses are of great public health relevance, however, their epidemiological features in areas where the three viruses co-circulate are scarce. Here, we present analyses of molecular and serological diagnostics in a prospective study of acute febrile patients recruited from May 2015 to May 2016 in Recife, Brazil.

We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples.

The complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity.

Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching.

The diagnostic of arbovirus-related obstetric complications in high-risk pregnancy and childbirth care is challenging, especially in endemic areas. We conducted a prospective study to track active or recent Zika (ZIKV), dengue (DENV), or chikungunya (CHIKV) virus infection among hospitalized pregnant women (PW) with obstetric complications in a hospital at the epicenter of Zika outbreak and ZIKV-related microcephaly in Brazil. Clinical data and blood samples were collected at enrollment and 10 days after the admission of study participants, between October 2018 and May 2019. Further clinical data were extracted from medical records. Samples were screened by molecular and serological tests. Out of 780 participants, 93.1% (95% CI: 91.1-94.7%) presented previous DENV exposure (IgG). ZIKV, CHIKV, and/or DENV laboratory markers of recent or active infection were detected in 130 PW, yielding a prevalence of 16.6% (95% CI: 14.2-19.5%); 9.4% (95% CI: 7.4-11.7%), 7.4% (95% CI: 5.7-9.7%), and 0.38% (95% CI: 0.1-1.2%) of CHIKV, ZIKV, and DENV infections, respectively. Most ZIKV infections were detected by molecular assays (89.6%), while CHIKV infections were detected by serology (95.9%). Our findings highlight the need for arbovirus infections screening in PW with obstetrical complications, potentially associated to these infections in endemic areas regardless of the signs or symptoms suggestive of arboviral disease.