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The diagnostic workout of ulnar sided wrist pain may be challenging, since there can be many different causes for it, varying from ulnar nerve problems to fractures. Congenital lunotriquetral synostosis may present as a source of pain in some cases, but it is a rare diagnosis. The author presents a case of post-traumatic ulnar sided wrist pain in a patient with Minnaar’s type 1 congenital lunotriquetral synostosis.

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses., (© 2024. The Author(s).)

Competing Interests: The authors declare no conflict of interest.

Syphilis has reemerged as a serious public health problem in Brazil and worldwide, disproportionately affecting men who have sex with men (MSM) and travestis and transgender women (TrTGW). Studies on sexually transmitted infections (STI) in adolescents from these key populations are relatively scarce. This is a Brazilian multi-center, cross study with prevalence analysis, using as baseline the PrEP1519 cohort of sexually active MSM and TrTGW adolescents, recruited from April 2019 to December 2020. Analyses were made using the dimensions of vulnerability to STI/HIV and logistic regression models were conducted to estimate the odds ratios of the association between the predictor variables and positive treponemal test for syphilis at the moment of entry in the study. In total, 677 participants were analyzed; participants' median age was 18.9 years (IQR: 18.1-19.5); 70.5% (477) self-declared as black; 70.5% (474), as homosexuals/gays; and 48 (7.1%), as trans women or travestis. The baseline prevalence of syphilis was 21.3%. In the final logistic regression model, higher chance of syphilis was associated with: self-reported episode of STI in the last 12 months (OR = 5.92; 95%CI: 3.74-9.37), sex worker (OR = 3.39; 95%CI: 1.32-8.78), and < 11 years of schooling (OR = 1.76; 95%CI: 1.13-2.74). The prevalence of syphilis among MSM/TrTGW adolescents aged from 15 to 19 years was alarming, much higher than the described for the general population within this age range and associated with vulnerability factors. This reinforces the urgent need to strengthen public health programs to debate about race, gender, sexuality, and prevention.

Background: Despite being first identified in 1947, Zika virus-related outbreaks were first described starting from 2007 culminating with the 2015 Latin American outbreak. Hypotheses indicate that the virus has been circulating in Asia for decades, but reports are scarce., Methods: We performed serological analysis and screened placental samples isolated in 2008 for the presence of Zika virus from pregnant women in Ho Chi Minh City (Vietnam)., Results: None of the placental samples was positive for Zika virus. Four serum samples out of 176 (2.3%) specifically inhibited Zika virus, with variable degrees of cross-reactivity with other flaviviruses. While one of the four samples inhibited only Zika virus, cross-reactivity with other flaviviruses not included in the study could not be ruled out., Conclusion: Our results support the conclusion that the virus was not present among pregnant women in the Vietnamese largest city during the initial phases of the epidemic wave., (© 2023. The Author(s).)

Higher education students have faced several changes in their lives due to the COVID-19 pandemic. This study aims to explore the effect of dispositional optimism in students' fear of COVID-19 and to test the mediating role of general anxiety in the relationship between optimism and fear. Using an online survey, data were collected during the second wave of the pandemic in Portugal. The sample included 312 higher education students (76% females) aged 18-25 years old, who completed measures of dispositional optimism, general anxiety and fear of COVID-19. The results showed that higher optimism and lower general anxiety reduce fear of COVID-19. Moreover, the link between optimism and fear is fully mediated by general anxiety, showing that optimism reduces fear of COVID-19 indirectly through the reduction of students' anxiety. The role of optimism, anxiety and fear in higher education students is discussed and topics for further research are presented.

Child routines have been recognized as positive contributors to children's development. However, in Portugal there is still a lack of instruments available to assess school-age child routines. The purpose of this study was to present the translation, adaptation, and validation studies of the Portuguese version of the Child Routines Questionnaire (CRQ), a parent self-report measure developed to assess school-age child routines. A total of 460 parents of children aged between 6 and 12 years-old participated in the study. Two studies were conducted to define the CRQ-PT factor structure. In Study 1 ( n  = 204 children from 6 to 12 years-old), findings from the exploratory factor analysis provided evidence for a four-factor structure (for 32 items), which explained 43.53% of the total variance. In Study 2 ( n  = 256 children from 6 to 9 years-old), results from confirmatory factor analysis showed good model fit indices (CFI = 0.84, RMSEA = 0.06). The total scale of the CRQ-PT ( α  = 0.89) and its subscales showed good internal consistency. Further evidence of construct validity was shown by weak to moderate correlations with measures of parental sense of competence and family mealtime routines. Relevant contributions of the study are underscored, namely the availability and usefulness of a reliable and valid assessment tool to evaluate the routines of Portuguese school-age children for clinical practice and research purposes., Competing Interests: The authors declare that there is no conflict of interests., (© The Author(s) 2023.)

Here, we present a protocol to analyze the T cell profiles of the neonatal ovine lung during respiratory syncytial virus (RSV) infection. The protocol delivers standardized multiparameter flow cytometry (FCM) analysis of CD4 + , CD8 + , regulatory, and γδ T cells isolated from lung, lymph nodes, and bronchoalveolar lavages (BALs). We detail the preparation of RSV and transtracheal inoculation of newborn lambs. We then describe tissue isolation and preparation of cell suspensions, followed by FCM acquisition to identify different T cell subsets. For complete details on the use and execution of this protocol, please refer to Démoulins et al. (2021)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Peanuts (PN) and tree nuts (TN) are major causes of anaphylaxis worldwide. We aimed to determine the clinical and demographic characteristics associated with anaphylaxis in patients sensitized to PN and/or TN in a Mediterranean population. Methods: We conducted a retrospective study, which included 198 patients allergic to PN and/or TN (allergy symptoms plus specific immunoglobulin E [sIgE] sensitization), evaluated in consultations from January 2015 to December 2020. Univariate analysis and multivariate logistic regression models were developed, including demographic, clinical, and laboratory data as independent variables, and anaphylaxis to each PN and/or TN as a dependent variables. Results: Anaphylaxis was associated with an earlier age of onset of allergy to PN, cashew and/or pistachio, and pine nut allergy but not to other TN allergies. Gender, atopic comorbidities, and cofactors were not associated with PN and/or TN anaphylaxis. Anaphylaxis to PN, cashew and/or pistachio, and pine nut were associated with reactivity to a fewer number of PN and/or TN foods. Although sIgE sensitization to lipid transfer proteins (LTP) was highly prevalent in our population, only seed storage protein (SSP) positivity was associated with anaphylaxis in PN allergy. The absence of pathogenesis-related protein family 10 sensitization correlated with PN and hazelnut anaphylaxis. A higher level of sIgE to almond extract predicted anaphylaxis but the level of sIgE to other PN and/or TN extracts did not predict it. Conclusion: The high prevalence of sensitization to the pan-allergen LTP did not seem to have a significant impact in PN and/or TN allergy severity in our study. Instead, other factors, such as early age of onset and positivity for SSPs, seem to strongly associate with anaphylaxis to specific PN and/or TN. These findings may contribute to individual risk assessment in these populations.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance., (© 2022. The Author(s).)

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery., Competing Interests: Declaration of interests Part of the results presented herein have been used in the patent application EP-A-20213494.6., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

We present a protocol to generate an advanced ex vivo model of human placenta. We use a vibrating tissue slicer to obtain precision-cut slices representative of the entire thickness of human placenta. This approach delivers standardized cultures with a preserved microstructure and cellular composition comparable to the native tissue. We applied this system to study SARS-CoV-2 infection at the maternal-fetal interface. Moreover, this system can be used to investigate the basic functions of the human placenta in health and disease. For complete details on the use and execution of this protocol, please refer to Fahmi et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

The ongoing SARS-CoV-2 pandemic continues to lead to high morbidity and mortality. During pregnancy, severe maternal and neonatal outcomes and placental pathological changes have been described. We evaluate SARS-CoV-2 infection at the maternal-fetal interface using precision-cut slices (PCSs) of human placenta. Remarkably, exposure of placenta PCSs to SARS-CoV-2 leads to a full replication cycle with infectious virus release. Moreover, the susceptibility of placental tissue to SARS-CoV-2 replication relates to the expression levels of ACE2. Viral proteins and/or viral RNA are detected in syncytiotrophoblasts, cytotrophoblasts, villous stroma, and possibly Hofbauer cells. While SARS-CoV-2 infection of placenta PCSs does not cause a detectable cytotoxicity or a pro-inflammatory cytokine response, an upregulation of one order of magnitude of interferon type III transcripts is measured. In conclusion, our data demonstrate the capacity of SARS-CoV-2 to infect and propagate in human placenta and constitute a basis for further investigation of SARS-CoV-2 biology at the maternal-fetal interface., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The current challenging organizational context demands that organizations adapt quickly and continuously in order to survive and maintain their competitive advantage. Considering this need, one of the responses given by companies has been the valorization of work teams and their capacity for innovation, as well as fostering positive skills and emergent states in employees, such as emotional carrying capacity and affective commitment, respectively. The aim of this research is thus to study the relationship between emotional carrying capacity and group innovation, considering affective commitment as the mediating variable. To test these relationships, an empirical cross-sectional study was conducted including 138 Portuguese work teams belonging to different sectors of activity, composed of 625 members and their respective leaders. The results were analyzed through structural equation modeling (SEM) and showed positive relationships between emotional carrying capacity and affective commitment, as well as between affective commitment and group innovation. In addition, the mediating role of affective commitment in the relationship between emotional carrying capacity and group innovation was also supported. Therefore, the results suggest that a work context in which members openly express their emotions contributes to reinforcing their affective attachment to the group, making them feel more involved and available to test and implement new ideas and procedures. The findings reinforce the benefits of promoting the expression of emotions and the development of healthy bonds between team members., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Damasceno, Dimas, Lourenço, Rebelo and Alves.)

Nasal epithelial cells (NECs) are among the first cells to be exposed to air pollutants and respiratory viruses. Although it is known that air pollution exposure and rhinovirus infections increase the risk for asthma development independently, it is unclear how these risk factors interact on a cellular level. Therefore, we aimed to investigate how exposure to diesel particulate matter (DPM) modifies the response of primary NECs to rhinovirus (RV) infection in vitro. Exposure of re-differentiated, primary NECs (49 healthy children [0-7 years], 12 adults) to DPM modified the mRNA expression of viral cell-surface receptors, pattern recognition receptors, and pro-inflammatory response (also protein levels). After exposure to DPM, we additionally infected the NECs with RV-1b and RV-16. Viral loads (assessed by titration assays) were significantly higher in DPM-exposed compared with non-exposed NECs. Exposure to DPM prior to RV infection resulted in a significant upregulation of pro-inflammatory cytokines (mRNA and protein level) and β-defensins mRNA, and significant downregulation of pattern recognition receptors mRNA and CXCL10 (mRNA and protein levels). There was no difference between all outcomes of NECs from children and adults. We can conclude that exposure to DPM prior to RV infection increases viral loads by downregulation of viral defense receptors and upregulation of pro-inflammatory cytokines. Our findings indicate a strong interaction between air pollution and the antiviral response to RV infection in NECs. We provide mechanistic evidence that exposure to air pollution increases susceptibility to RV infection., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications., Competing Interests: The authors have declared that no competing interests exist.

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity., Competing Interests: The authors have declared that no competing interests exist.

The study aims to test how the association between leader's centrality (outdegree and betweenness) in the group network, considering both workflow and friendship ties between leader and members, and the perception of team performance is mediated by the leader's satisfaction with the team. The research included a total of 74 formal leaders of organizational teams from several organizations. Total, direct and indirect effects were calculated through the estimation of an OLS regression-based mediation model, controlling for team size. Results revealed that only leader's outdegree and betweenness centrality in the team friendship network positively predicted the leader's perception of team performance. In contrast to the predictions, a significant negative indirect effect of outdegree centrality of the leader within the team workflow network on the evaluation of group performance through leader's satisfaction was observed. Also, both leader´s outdegree and betweenness centrality levels in the friendship network were shown to have a positive effect on leader's assessment of team performance through leader's satisfaction with the team. Overall, findings point to the negative effects of leader's centrality in the workflow team network and the positive effects of leader's centrality in the friendship team network on his/her attitudes toward the team. The effects of the more or less central position of the leader within each of the group networks are discussed.

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues., (Copyright © 2020. Published by Elsevier B.V.)

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM + ) fraction, which diminished with age. The mesenchymal fraction composed of CD90 + and CD90 + CD73 + cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM + cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM + cells supported by CD90 + subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90 + and CD90 + PDGFRα + cells. In postnatal lung, a subset of CD90 + cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90 + CD146 + cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

With substantial progress of nanotechnology, there is rising concern about possible adverse health effects related to inhalation of nanomaterials, such as multi-walled carbon nanotubes (MWCNT). In particular, individuals with chronic respiratory disorders, such as chronic obstructive pulmonary disease (COPD), may potentially be more susceptible to adverse health effects related to inhaled MWCNT. Hazard assessment of such inhaled nanomaterials therefore requires timely clarification. This was assessed in this study using a mouse model of COPD by exposing animals to 0.08 µg/cm 2 of MWCNT administered by intratracheal instillation. Treatment with MWCNT induced an accumulation of alveolar macrophages (AMφ) in bronchoalveolar lavage fluid (BALF) in COPD mice that increased from 24 h to 7 d. In COPD mice, MWCNT induced a dynamic shift in macrophage polarization as measured by expression of CD38 and CD206, and increased AMφ and lung parenchyma macrophage (LPMΦ) activation with upregulation of co-stimulatory markers CD40 and CD80. Moreover, MWCNT treatment increased the frequencies of pulmonary dendritic cells (DC), leading to an expansion of the CD11b + CD103 - DC subset. Although MWCNT did not trigger lung functional or structural changes, they induced an increased expression of the muc5AC transcript in mice with COPD. Our data provide initial evidence that inhaled MWCNT affect the pulmonary mucosal immune system by altering the numbers, phenotype, and activation status of antigen-presenting cell populations. Extrapolating these in vivo mouse findings to human pulmonary MWCNT exposure, caution is warranted in limiting exposure when handling inhalable nanofibers.

The molecular dynamics of the Pseudomonas fluorescens 07A metalloprotease in the presence of structural Ca 2+ and Mn 2+ ions was evaluated. Seven Ca 2+ ions are primarily bound to the C-terminus, while a divalent cation is located at the catalytic site, acting as a cofactor. The observed enzyme's experimental activity suggests that Mn 2+ could compete for the active site of the enzyme with Ca 2+ , Zn 2+ or other divalent cations, thus providing greater catalytic power to the enzyme. Our molecular dynamics simulations suggest that these ions partially protect the enzyme's structure from thermal denaturation. Moreover, our simulations have shown a collective movement of opening-closing of the active-site in simulations with structural Ca 2+ and Mn 2+ ions bound, leading to a proposal of a dynamical model of P. fluorescens 07A metalloprotease active and inactive conformations. These findings can support the development of measures to control the activity of P. fluorescens and other spoilage microorganism proteases., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Flaviviruses replicate in a wide variety of species and have a broad cellular tropism. They are isolated from various body fluids, and Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) RNAs have been detected in nasopharyngeal swabs. Consequently, we evaluated the cellular tropism and host responses upon ZIKV, JEV, WNV, and Usutu virus (USUV) infection using a relevant model of the human upper respiratory tract epithelium based on primary human nasal epithelial cells (NECs) cultured at the air-liquid interface. NECs were susceptible to all the viruses tested, and confocal analysis showed evidence of infection of ciliated and non-ciliated cells. Each flavivirus productively infected NECs, leading to apical and basolateral live virus shedding with particularly high basal release for JEV and WNV. As demonstrated by a paracellular permeability assay, the integrity of the epithelium was not affected by flavivirus infection, suggesting an active release of live virus through the basolateral surface. Also, we detected a significant secretion of interferon type III and the pro-inflammatory cytokine IP-10/CXCL10 upon infection with JEV. Taken together, our data suggest that the human upper respiratory tract epithelium is a target for flaviviruses and could potentially play a role in the spread of infection to other body compartments through basolateral virus release. Undoubtedly, further work is required to evaluate the risks and define the adapted measures to protect individuals exposed to flavivirus-contaminated body fluids.

Introduction: Rhinovirus (RV) infection is a major cause of cystic fibrosis (CF) lung morbidity with limited therapeutic options. Various diseases involving chronic inflammatory response and infection are associated with endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), an adaptive response to maintain cellular homeostasis. Recent evidence suggests impaired ER stress response in CF airway epithelial cells, this might be a reason for recurrent viral infection in CF. Therefore, assuming that ER stress inducing drugs have antiviral properties, we evaluated the activation of the UPR by selected ER stress inducers as an approach to control virus replication in the CF bronchial epithelium., Methods: We assessed the levels of UPR markers, namely the glucose-regulated protein 78 (Grp78) and the C/EBP homologous protein (CHOP), in primary CF and control bronchial epithelial cells and in a CF and control bronchial epithelial cell line before and after infection with RV. The cells were also pretreated with ER stress-inducing drugs and RV replication and shedding was measured by quantitative RT-PCR and by a TCID 50 assay, respectively. Cell death was assessed by a lactate dehydrogenate (LDH) activity test in supernatants., Results: We observed a significantly impaired induction of Grp78 and CHOP in CF compare to control cells following RV infection. The ER stress response could be significantly induced in CF cells by pharmacological ER stress inducers Brefeldin A, Tunicamycin, and Thapsigargin. The chemical induction of the UPR pathway prior to RV infection of CF and control cells reduced viral replication and shedding by up to two orders of magnitude and protected cells from RV-induced cell death., Conclusion: RV infection causes an impaired activation of the UPR in CF cells. Rescue of the ER stress response by chemical ER stress inducers reduced significantly RV replication in CF cells. Thus, pharmacological modulation of the UPR might represent a strategy to control respiratory virus replication in the CF bronchial epithelium., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Differentiation of primary alveolar type II epithelial cells (AEC II) to AEC type I in culture is a major barrier in the study of the alveolar epithelium in vitro. The establishment of an AEC II cell line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity to study alveolar epithelial cell biology, for instance, in the context of lung injury, fibrosis, and repair. In the present study, we generated long-lasting AEC II from iPSC (LL-iPSC-AEC II). LL-iPSC-AEC II displayed morphological characteristics of AEC II, including growth in a cobblestone monolayer, the presence of lamellar bodies, and microvilli, as shown by electron microscopy. Also, LL-iPSC-AEC II expressed AEC type II proteins, such as cytokeratin, surfactant protein C, and LysoTracker DND 26 (a marker for lamellar bodies). Furthermore, the LL-iPSC-AEC II exhibited functional properties of AEC II by an increase of transepithelial electrical resistance over time, secretion of inflammatory mediators in biologically relevant quantities (IL-6 and IL-8), and efficient in vitro alveolar epithelial wound repair. Consistent with the AEC II phenotype, the cell line showed the ability to uptake and release surfactant protein B, to secrete phospholipids, and to differentiate into AEC type I. In summary, we established a long-lasting, but finite AEC type II cell line derived from iPSC as a novel cellular model to study alveolar epithelial cell biology in lung health and disease.

The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs. IMPORTANCE JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV., (Copyright © 2018 García-Nicolás et al.)

Zika virus recently re-emerged and caused global outbreaks mainly in Central Africa, Southeast Asia, the Pacific Islands and in Central and South America. Even though there is a declining trend, the virus continues to spread throughout different geographical regions of the world. Since its re-emergence in 2015, massive advances have been made regarding our understanding of clinical manifestations, epidemiology, genetic diversity, genomic structure and potential therapeutic intervention strategies. Nevertheless, treatment remains a challenge as there is no licensed effective therapy available. This review focuses on the recent advances regarding research models, as well as available experimental tools that can be used for the identification and characterization of potential antiviral targets and therapeutic intervention strategies.

The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments. This evolutionarily conserved strategy facilitates efficient viral genome replication and contributes to evasion from host cell cytosolic defense mechanisms. We have previously described the identification of a small-compound inhibitor, K22, which exerts a potent antiviral activity against a broad range of coronaviruses by targeting membrane-bound viral RNA replication. To analyze the antiviral spectrum of this inhibitor, we assessed the inhibitory potential of K22 against several members of the Flaviviridae family, including the reemerging Zika virus (ZIKV). We show that ZIKV is strongly affected by K22. Time-of-addition experiments revealed that K22 acts during a postentry phase of the ZIKV life cycle, and combination regimens of K22 together with ribavirin (RBV) or interferon alpha (IFN-α) further increased the extent of viral inhibition. Ultrastructural electron microscopy studies revealed severe alterations of ZIKV-induced intracellular replication compartments upon infection of K22-treated cells. Importantly, the antiviral activity of K22 was demonstrated against several other members of the Flaviviridae family. It is tempting to speculate that K22 exerts its broad antiviral activity against several positive-strand RNA viruses via a similar mechanism and thereby represents an attractive candidate for development as a panviral inhibitor., (Copyright © 2018 García-Nicolás et al.)

While Zika virus (ZIKV) circulated for decades (African lineage strains) without report of outbreaks and severe complications, its emergence in French Polynesia and subsequently in the Americas (Asian lineage strains) was associated with description of severe neurological defects in newborns/neonates and adults. With the aim to identify virus lineage-dependent factors, we compared cell susceptibility, virus replication, cell death and innate immune responses following infection with two African and three contemporary Asian lineage strains of ZIKV. To this end, we used green monkey Vero and Aedes albopictus C6/36 cells and human monocyte-derived dendritic cells (DCs). The latter are involved in the pathogenesis of several mosquito-borne Flavivirus infections. In Vero and C6/36 cells, we observed strain- but not lineage-dependent differences in infection profiles. Nevertheless, in human DCs, no significant differences in susceptibility and virus replication were found between lineages and strains. ZIKV induced antiviral interferon type I/III in a limited fashion, with the exception of one African strain. None of the strains induced cell death or DC maturation in terms of MHC II, CD40, CD80/86 or CCR7 expression. Taken together, our data suggest that a large collection of virus isolates needs to be investigated before conclusions on lineage differences can be made.

This work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the strain was observed after incubation for 12 h at 25°C while the strain was in the logarithmic growth phase. After 24 h, protease production significantly increased and remained constant for up to 48 h, a time range during which the strain remained in the stationary phase. Conversely, at refrigeration temperatures, at 12 h the strain was still in the lag phase and expressed the protease at higher levels than when the logarithmic phase was reached. Casein fractions were highly degraded by P. fluorescens 07A, the purified protease, and the bacterial pellet on d 7 of incubation at 25°C and to a lesser extent at 10°C for the sample incubated with the bacterium. Heat treatment at 90°C for 5 min completely inactivated the proteolytic activity of the purified protease and the bacterial pellet. This work contributes to the knowledge about the conditions of milk storage that influence the production and activity of this extracellular metalloprotease. The results demonstrate the need to find alternative strategies to control the synthesis and activity of proteolytic enzymes in the dairy industry to ensure the quality of processed products., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients., Methods: Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay., Results: Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight., Conclusion: In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection. We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice. ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC 50 after a single intraperitoneal injection. In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV., (Copyright © 2017. Published by Elsevier B.V.)

The World Health Organization (WHO) published 2 alcohol-based formulations to be used in healthcare settings and for outbreak-associated infections, but inactivation efficacies of these products have not been determined against (re-)emerging viruses. In this study, we evaluated the virucidal activity of these WHO products in a comparative analysis. Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) as (re-)emerging viral pathogens and other enveloped viruses could be efficiently inactivated by both WHO formulations, implicating their use in healthcare systems and viral outbreak situations., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Zika virus (ZIKV) was first isolated in 1947 in a rhesus monkey from the Zika forest of Uganda. Until 2007, only 14 human cases were reported. The first large human outbreak occurred in 2007 (Yap Island, Federated States of Micronesia, Pacific) followed by French Polynesia in 2013 and Brazil in 2015. The virus is mainly transmitted through Aedes mosquito bites, but sexual and post-transfusion transmissions have been reported. Symptoms include low-grade fever, maculopapular rash, conjunctivitis, myalgia, arthralgia, and asthenia. During the recent outbreaks in French Polynesia and Brazil, ZIKV infection has been associated with two major complications: microcephaly and Guillain-Barré syndrome. Since fetal infection includes other birth defects, congenital Zika syndrome has been used to define in utero infection. The majority of sexual transmission occurred from a symptomatic male to a female, but female-to-male and male-to-male transmission have been reported. Asymptomatic male-to-female transmission has also been described. Importantly, ZIKV RNA can persist at least 6 months in semen. The male urogenital tract may therefore act as a reservoir for the virus. ZIKV RNA was detected in a cervical swab of a patient 3 days after presenting the classic symptoms suggesting a potential tropism for the female genital tract. Long-lasting presence of ZIKV RNA might not indicate that the individual is infectious but makes recommendation for couples potentially exposed to the virus and willing to conceive difficult. It will also be important to determine whether genital ZIKV infection might have a deleterious effect on male and female fertility., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Chronic obstructive pulmonary disease (COPD) has been associated with periodontal disease (PD), and periodontal treatment (PT) has been connected to reduction of lung disease exacerbations. Bronchiectasis has many clinical similarities with COPD but, although it is also a chronic lung disease, to date it has not been studied with relation to PD. The aim of this study is to evaluate whether PT associated with photodynamic therapy (PDT) reduces the number of exacerbations, improves pulmonary function, periodontal clinical parameters and quality of life after 1 year of periodontal treatment follow-up., Methods: Bronchiectasis patients will undergo medical anamnesis and periodontal examination. Participants with periodontitis will be divided into two groups and PT will be performed as G1 control group (n = 32) - OHO (oral hygiene orientation) + supragingival treatment + simulation of using photodynamic therapy (PDT); G2 experimental (n = 32) - scaling and root planing + PDT + OHO. Lung function will be assessed both at baseline and after 1 year by spirometry, exacerbation history will be analyzed through clinical records monitoring. Three instruments for quality of life assessment will also be applied - Saint George's Respiratory Questionnaire and Impact Profile Analysis Oral health (OHIP-14). It is expected that periodontal treatment can improve the analyzed parameters after 1 year., Discussion: Although only one study evaluates exacerbation in COPD after 1 year of PT, bronchiectasis has not been studied in the dentistry field to date., Trial Registration: NCT02514226. Version #1. This study protocol receives grant from FAPESP (São Paulo Research Foundation) #2015/20535-1. First received: July 22, 2015, 1 st version. This protocol has been approved by the Research Ethics Committee of Nove de Julho University.

Chronic periodontitis (CP) is an infectious inflammatory disease that affects tooth-supporting structures and in which dental plaque bacteria, immune mechanisms and genetic predisposition play important roles. Interleukin 4 (IL-4) is a key anti-inflammatory cytokine with relevant action in imbalances in inflamed periodontal tissue. Individuals carrying the TCI/CCI genotype (S-haplotype) of the IL-4 gene are 5 times more susceptible to CP, whereas the CTI/TTD genotype (P-haplotype) confers protection against CP. Compared with the S-haplotype, subjects with the P-haplotype produce higher levels of the IL-4 protein after non-surgical periodontal therapy. The present in vitro study aimed to investigate the functionality of IL-4 haplotypes in immune cells to obtain insight into the influence of these genetic variations in regulating immune responses to CP-associated bacteria. Peripheral blood was collected from 6 subjects carrying each haplotype, and their immune cells were challenged with periodontopathogens to compare responses of the different haplotypes with regard to gene expression, protein secretion and the immunophenotype of T helper responses. We found higher IL-4 mRNA and protein levels in the P-haplotype, which also presented higher levels of anti-inflammatory cytokines. In contrast, cells from S-haplotype subjects responded with higher levels of pro-inflammatory cytokines. S-haplotype individuals exhibited significantly greater polarization toward the Th1 phenotype, whereas the P-haplotype was associated with an attenuated response to periodontopathogens, with suggestive skewing toward Th2/M2 phenotypes. In conclusion, IL-4 genetic variations associated with susceptibility to or protection against chronic periodontitis are directly associated with influencing the response of immune cells to periodontopathogens., Competing Interests: The authors have declared that no competing interests exist.

Zika virus infection during pregnancy poses a serious risk for pregnant women as it can cause severe birth defects. Even though the virus is mainly transmitted via mosquitos, human-to-human transmission has been described. Infectious viral particles have been detected in breast milk of infected women which raised concerns regarding the safety of breastfeeding in areas of Zika virus transmission or in case of a suspected or confirmed Zika virus infection. In this study, we show that Zika virus is effectively inactivated in human breast milk after prolonged storage or upon pasteurization of milk., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective Comparing the working environment of nurse managers and nursing assistants in the hospital context. Method A mixed methods research with concomitant triangulation of data developed in a university hospital in the South of Brazil. Participants in the quantitative study were 94 nursing assistants and 12 nurse managers. The data were collected using the Brazilian Nursing Work Index - Revised (B-NWI-R) and analyzed through descriptive and inferential statistics. Eight (8) nurse managers and 18 nursing assistants were interviewed for the qualitative study. The data were analyzed through thematic analysis. Results The total B-NWI-R mean score for nurse managers was 2.15±0.39, and for nursing assistants it was 2.22±0.39. No statistical significance was identified in the comparison between the groups (p=0.508). The qualitative results show the existence of collaborative relationships between nurse managers and nursing assistants. Conclusion The working environment was similarly evaluated by nurse managers and nursing assistants in the hospital context.

This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Introduction: The frequent recolonization of treated sites by periodontopathogens and the emergence of antibiotic resistance have led to a call for new therapeutic approaches for managing periodontal diseases. As probiotics are considered a new tool for combating infectious diseases, we systematically reviewed the evidences for their effectiveness in the management of periodontitis., Areas Covered: An electronic search was performed in the MEDLINE, SCOPUS and Cochrane Library databases up to March 2016 using the terms 'periodontitis', 'chronic periodontitis', 'probiotic(s)', 'prebiotic(s)', 'symbiotic(s)', 'Bifidobacterium and 'Lactobacillus'. Only randomized controlled trials (RCTs) were included in the present study. Analysis of 12 RCTs revealed that in general, oral administration of probiotics improved the recognized clinical signs of chronic and aggressive periodontitis such as probing pocket depth, bleeding on probing, and attachment loss, with a concomitant reduction in the levels of major periodontal pathogens. Continuous probiotic administration, laced mainly with Lactobacillus species, was necessary to maintain these benefits. Expert commentary: Oral administration of probiotics is a safe and effective adjunct to conventional mechanical treatment (scaling) in the management of periodontitis, specially the chronic disease entity. Their adjunctive use is likely to improve disease indices and reduce the need for antibiotics.

The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins.