RESUMO
Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.
Assuntos
COVID-19/complicações , Cardiotônicos/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Cardiopatias/tratamento farmacológico , Quinazolinonas/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citocinas/metabolismo , Feminino , Cardiopatias/etiologia , Células-Tronco Embrionárias Humanas , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.
Assuntos
Deficiência Intelectual , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Humanos , Spliceossomos/genética , Transtornos do Neurodesenvolvimento/genética , Deficiência Intelectual/genética , Deficiência Intelectual/complicações , Síndrome , Malformações do Sistema Nervoso/genética , Perda de Heterozigosidade , FenótipoRESUMO
Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 µg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.
Assuntos
Aedes , Alphavirus , Vírus Chikungunya , Doenças Reumáticas , Vacinas de Partículas Semelhantes a Vírus , Animais , Camundongos , Vacinas de Partículas Semelhantes a Vírus/genética , Camundongos Endogâmicos C57BL , Alphavirus/genética , Brasil , Anticorpos Neutralizantes , MamíferosRESUMO
How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.
Assuntos
COVID-19 , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/genética , Modelos Animais de Doenças , Expressão Gênica , Humanos , Pulmão , Camundongos , Camundongos Transgênicos , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2/genéticaRESUMO
The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal pathogen priority list. This systematic review aimed to evaluate the epidemiology and impact of infections caused by Talaromyces marneffei, Coccidioides species, and Paracoccidioides species. PubMed and Web of Sciences databases were searched to identify studies published between 1 January 2011 and 23 February 2021 reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 25, 17, and 6 articles were included for T. marneffei, Coccidioides spp. and Paracoccidioides spp., respectively. Mortality rates were high in those with invasive talaromycosis and paracoccidioidomycosis (up to 21% and 22.7%, respectively). Hospitalization was frequent in those with coccidioidomycosis (up to 84%), and while the duration was short (mean/median 3-7 days), readmission was common (38%). Reduced susceptibility to fluconazole and echinocandins was observed for T. marneffei and Coccidioides spp., whereas >88% of T. marneffei isolates had minimum inhibitory concentration values ≤0.015 µg/ml for itraconazole, posaconazole, and voriconazole. Risk factors for mortality in those with talaromycosis included low CD4 counts (odds ratio 2.90 when CD4 count <200 cells/µl compared with 24.26 when CD4 count <50 cells/µl). Outbreaks of coccidioidomycosis and paracoccidioidomycosis were associated with construction work (relative risk 4.4-210.6 and 5.7-times increase, respectively). In the United States of America, cases of coccidioidomycosis increased between 2014 and 2017 (from 8232 to 14 364/year). National and global surveillance as well as more detailed studies to better define sequelae, risk factors, outcomes, global distribution, and trends are required.
Assuntos
Antifúngicos , Coccidioides , Paracoccidioides , Talaromyces , Organização Mundial da Saúde , Talaromyces/isolamento & purificação , Talaromyces/classificação , Talaromyces/efeitos dos fármacos , Humanos , Paracoccidioides/isolamento & purificação , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/classificação , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Coccidioides/isolamento & purificação , Coccidioides/classificação , Coccidioides/efeitos dos fármacos , Micoses/epidemiologia , Micoses/microbiologia , Micoses/mortalidade , Paracoccidioidomicose/epidemiologia , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/tratamento farmacológico , Coccidioidomicose/epidemiologia , Coccidioidomicose/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Diphtheria is a re-emerging infectious disease and public health concern worldwide and in Vietnam with increasing cases in recent years. This study aimed to assess the anti-diphtheria toxoid antibodies status in Khanh Hoa Province and identify factors contributing to the vaccination policy in the south-central coast of Vietnam. METHODS: This was a cross-sectional study to evaluate the seroprevalence of anti-diphtheria toxoid antibodies among 1,195 participants, aged 5 - 40 years in Khanh Hoa Province, Vietnam. Immunoglobulin G antibody levels against diphtheria were detected using a commercial anti-diphtheria toxoid enzyme-linked immunosorbent assay (SERION ELISA classic Diphtheria Immunoglobulin G) and were categorized following the World Health Organization guidelines. RESULTS: The mean anti-diphtheria toxoid antibody levels were 0.07 IU/ml (95% Confidence Interval: 0.07-0.08). Anti-diphtheria toxoid antibody levels were found to be associated with age and history of diphtheria vaccination. The 5-15 years age group had the highest levels (0.09 IU/ml), while the older age group had the lowest antibody level (p < 0.001). Individuals who received three doses (adjusted Odds ratio: 2.34, 95%CI: 1.35 - 4.07) or 4+ doses (adjusted Odds ratio: 2.45, 95%CI: 1.29 - 4.64) had a higher antibody level compared to those who received only one dose regardless of age. CONCLUSION: It is crucial to promote routine vaccination coverage to over 95% for children under one year of age with three primary doses of the diphtheria-containing vaccine, including additional doses at 18 months and 7 years of age. Booster doses should be promoted and administered to adolescents and adults every 10 years.
Assuntos
Anticorpos Antibacterianos , Toxoide Diftérico , Difteria , Vacinação , Humanos , Estudos Transversais , Vietnã/epidemiologia , Adolescente , Adulto , Estudos Soroepidemiológicos , Masculino , Criança , Feminino , Adulto Jovem , Difteria/prevenção & controle , Difteria/imunologia , Difteria/epidemiologia , Anticorpos Antibacterianos/sangue , Pré-Escolar , Toxoide Diftérico/imunologia , Toxoide Diftérico/administração & dosagem , Vacinação/estatística & dados numéricos , Imunoglobulina G/sangue , Ensaio de Imunoadsorção EnzimáticaRESUMO
Type 1 diabetes (T1D) is characterized by the loss of immune self-tolerance, resulting in an aberrant immune responses against self-tissue. A few therapeutics have been partially successful in reverting or slowing down T1D progression in patients, and the infusion of autologous hematopoietic stem cells (HSCs) is emerging as an option to be explored. In this study, we proposed to pharmacologically enhance by ex vivo modulation with small molecules the immunoregulatory and trafficking properties of HSCs to provide a safer and more efficacious treatment option for patients with T1D and other autoimmune disorders. A high-throughput targeted RNA sequencing screening strategy was used to identify a combination of small molecules (16,16-dimethyl PGE2 and dexamethasone), which significantly upregulate key genes involved in trafficking (e.g., CXCR4) and immunoregulation (e.g., programmed death ligand 1). The pharmacologically enhanced, ex vivo-modulated HSCs (regulatory HSCs [HSC.Regs]) have strong trafficking properties to sites of inflammation in a mouse model of T1D, reverted autoimmune diabetes in NOD mice, and delayed experimental multiple sclerosis and rheumatoid arthritis in preclinical models. Mechanistically, HSC.Regs reduced lymphocytic infiltration of pancreatic ß cells and inhibited the activity of autoreactive T cells. Moreover, when tested in clinically relevant in vitro autoimmune assays, HSC.Regs abrogated the autoimmune response. Ex vivo pharmacological modulation enhances the immunoregulatory and trafficking properties of HSCs, thus generating HSC.Regs, which mitigated autoimmune diabetes and other autoimmune disorders.
Assuntos
Doenças Autoimunes , Diabetes Mellitus Tipo 1 , Transplante de Células-Tronco Hematopoéticas , Animais , Doenças Autoimunes/terapia , Diabetes Mellitus Tipo 1/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NODRESUMO
Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses-often de novo-contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.
Assuntos
Variações do Número de Cópias de DNA , Sistema Nervoso Entérico/crescimento & desenvolvimento , Redes Reguladoras de Genes , Doença de Hirschsprung/genética , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Sistema Nervoso Entérico/química , Epistasia Genética , Predisposição Genética para Doença , Haplótipos , Humanos , Camundongos , Peixe-ZebraRESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Infecção Hospitalar , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Tetraciclinas , beta-Lactamases , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/enzimologia , Humanos , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/epidemiologia , Tetraciclinas/farmacologia , Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/epidemiologia , beta-Lactamases/genética , Países Baixos/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Surtos de Doenças , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Unidades de Terapia IntensivaRESUMO
In this study, essential oils and waste hydrosols of leaves of Ocimum tenuiflorum in four different geographical locations were extracted by hydrodistillation method and using gas chromatography / mass spectrometry (GC/MS) for chemical composition analysis. All four essential oil samples contained the main components (E)-ß-caryophyllene (27.8-49.0%), trans-ß-elemene (20.3-37.1%) and eugenol (9.0-44.0%). Three of the four hydrosol samples had eugenol in absolute content (94.5-98.6%), while the remaining hydrosol sample had two main components, elemicin (77.8%) and eugenol (14.2%). Essential oils and hydrosols demonstrated larvicidal activities against four important disease-transmitting mosquito species including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex fuscocephala with 24-h LC50 values in the range 15.42-56.01 µg/mL and 53.88-97.80 µg/mL for the essential oils and the hydrosols, respectively. Essential oils and hydrosols strongly inhibited the acetylcholinesterase (AChE) enzyme of electric eels with IC50 values in the range of 25.35-107.19 µg/mL. Microemulsion (ME) can be considered as a sustainable pesticide formulation over 300 days and has improved larvicidal activity compared to free essential oil. The O. tenuiflorum in Vietnam can be considered a low-cost source of eugenol, botanical pesticides that control disease-transmitting mosquitoes, as well as having therapeutic potential to be further investigated.
RESUMO
The seeds of Annona glabra L., an invasive plant in Vietnam, were first employed as a new biosorbent for the adsorption of methylene blue (MB) from aqueous media. The characterizations of the material using FT-IR, SEM, nitrogen adsorption-desorption analysis, and point of zero charge reveals that it possesses a rough and irregular surface, various polar functional groups, and pHpzc of 5.5. Certain adsorption conditions including adsorbent dose, solution pH, contact time, and initial concentration of MB were found to affect adsorption efficiency. The kinetic data are well fitted with pseudo-second-order model with the adsorption rate of 0.002 g mg-1 min-1 and initial rate of 4.46 mg g-1 min-1. For the adsorption isotherm, three nonlinear models were used to analyze the experiment data, including Langmuir, Freundlich, and Temkin. The results indicate that the Langmuir model best describes the adsorption of Annona glabra L. seeds powder (AGSP) with a maximum adsorption capacity of 98.0 mg g-1. The investigation underpins the adsorption mechanism, whereby the electrostatic attraction between positively charged MB and negatively charged surface of AGSP is expected to be the predominant mechanism, together with hydrogen bonding and pi-pi interaction. These results make AGSP an interesting biosorbent concerning its environmental friendliness, cost-effectiveness, and relatively high dye adsorption capacity.
Assuntos
Annona , Poluentes Químicos da Água , Azul de Metileno/análise , Azul de Metileno/química , Espectroscopia de Infravermelho com Transformada de Fourier , Poluentes Químicos da Água/análise , Concentração de Íons de Hidrogênio , Sementes/química , Adsorção , CinéticaRESUMO
Mpox was diagnosed in 2 women returning to Vietnam from the United Arab Emirates. The monkeypox viruses belonged to an emerging sublineage, A.2.1, distinct from B.1, which is responsible for the ongoing multicountry outbreak. Women could contribute to mpox transmission, and enhanced genomic surveillance is needed to clarify pathogen evolution.
Assuntos
Monkeypox virus , Mpox , Humanos , Feminino , Mpox/diagnóstico , Mpox/epidemiologia , Emirados Árabes Unidos/epidemiologia , Vietnã/epidemiologiaRESUMO
The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.
Assuntos
Alelos , Deficiências do Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/genética , Sequência de Bases , Criança , Pré-Escolar , Cílios/fisiologia , Feminino , Humanos , Lactente , Masculino , Modelos Moleculares , Neoplasias/genética , Proteínas do Tecido Nervoso , Proteínas Nucleares , Linhagem , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Perfilação da Expressão Gênica , Lentivirus , SARS-CoV-2 , Transdução Genética , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células VeroRESUMO
Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/ß receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.
Assuntos
Proteínas do Envelope Viral/genética , Virulência/genética , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/patogenicidade , Animais , Barreira Hematoencefálica , Permeabilidade Capilar , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Zika virus/metabolismoRESUMO
Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1ß, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design.
Assuntos
Vetores Genéticos/administração & dosagem , Imunidade Inata/imunologia , Reação no Local da Injeção/imunologia , Vacinação/métodos , Vacinas Sintéticas/administração & dosagem , Vacínia/imunologia , Infecção por Zika virus/imunologia , Animais , Feminino , Vetores Genéticos/genética , Genoma Viral , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Vacinas Sintéticas/imunologia , Vacínia/genética , Vacínia/metabolismo , Vacínia/virologia , Vaccinia virus/isolamento & purificação , Vacinologia , Zika virus/isolamento & purificação , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 µg/ml for itraconazole, 0.004-0.13 µg/ml for voriconazole, 0.03-0.13 µg/ml for posaconazole, 0.06-0.5 µg/ml for flucytosine, 0.5-1 µg/ml for amphotericin B, 0.5-4 µg/ml for caspofungin, and 0.5-16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.
We developed a colorimetric alamarBlue method to determine the susceptibility of antifungal drugs against Talaromyces marneffei. We observed excellent agreement between the alamarBlue method and the Clinical Laboratory Standard Institute broth microdilution method, and the alamarBlue method had substantially higher inter-rater agreement.
Assuntos
Antifúngicos , Talaromyces , Animais , Antifúngicos/farmacologia , Colorimetria/veterinária , Reprodutibilidade dos Testes , Voriconazol/farmacologia , Testes de Sensibilidade Microbiana/veterináriaRESUMO
INTRODUCTION: Cigarette smoking continues to pose a threat to public health. Identifying individual risk factors for smoking initiation is essential to further mitigate this epidemic. To the best of our knowledge, no study today has used machine learning (ML) techniques to automatically uncover informative predictors of smoking onset among adults using the Population Assessment of Tobacco and Health (PATH) study. AIMS AND METHODS: In this work, we employed random forest paired with Recursive Feature Elimination to identify relevant PATH variables that predict smoking initiation among adults who have never smoked at baseline between two consecutive PATH waves. We included all potentially informative baseline variables in wave 1 (wave 4) to predict past 30-day smoking status in wave 2 (wave 5). Using the first and most recent pairs of PATH waves was found sufficient to identify the key risk factors of smoking initiation and test their robustness over time. The eXtreme Gradient Boosting method was employed to test the quality of these selected variables. RESULTS: As a result, classification models suggested about 60 informative PATH variables among many candidate variables in each baseline wave. With these selected predictors, the resulting models have a high discriminatory power with the area under the specificity-sensitivity curves of around 80%. We examined the chosen variables and discovered important features. Across the considered waves, two factors, (1) BMI, and (2) dental and oral health status, robustly appeared as important predictors of smoking initiation, besides other well-established predictors. CONCLUSIONS: Our work demonstrates that ML methods are useful to predict smoking initiation with high accuracy, identifying novel smoking initiation predictors, and to enhance our understanding of tobacco use behaviors. IMPLICATIONS: Understanding individual risk factors for smoking initiation is essential to prevent smoking initiation. With this methodology, a set of the most informative predictors of smoking onset in the PATH data were identified. Besides reconfirming well-known risk factors, the findings suggested additional predictors of smoking initiation that have been overlooked in previous work. More studies that focus on the newly discovered factors (BMI and dental and oral health status,) are needed to confirm their predictive power against the onset of smoking as well as determine the underlying mechanisms.
Assuntos
Fumar Cigarros , Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Adulto , Humanos , Estudos Longitudinais , Uso de Tabaco/epidemiologia , Fumar Cigarros/epidemiologia , Fatores de RiscoRESUMO
Distribution patterns of 10 phthalic acid diesters (PAEs) and four cyclic volatile methylsiloxanes (cVMSs) were investigated in fine particulate matter (PM0.1 and PM0.5) collected from Bac Ninh, an industrial province in Vietnam during September-October in 2021. Total concentrations of PAEs found in PM0.1 and PM0.5 were in the ranges of 1.76-372 (median: 34.0 ng/m3) and 2.23-895 ng/m3 (median: 15.4 ng/m3), respectively. Among PAEs, di-n-butyl phthalate (DBP) was the most abundant compound found in PM0.1, whereas, di-2-(ethyl)hexyl phthalate (DEHP) was measured at the highest concentration in PM0.5. Total concentrations of cVMSs measured in PM0.1 and PM0.5 were in the ranges of method quantification limit (MQL)-203 (median: 2.10 ng/m3) and MQL-537 ng/m3 (median: 0.389 ng/m3), respectively. Among cVMSs, decamethylcyclopentasiloxane (D5) was found at the highest concentration in both PM0.1 and PM0.5 fractions of particulate matter. The concentration ratios between PAEs and cVMSs in PM0.1/PM0.5 were greater than 1 (except di-n-octyl phthalate: DnOP), suggesting that these chemicals tend to sorb to PM0.1 more preferentially than PM0.5. Among sampling locations, high concentrations of PAEs and cVMSs were found at traffic intersections (Que Vo district) and a craft village (Tu Son city). Relatively stronger correlations existed between cVMSs pairs in PM0.1 and PM0.5 (correlation coefficient: 0.73-1) than those of PAEs (-0.83-0.90). The human exposure doses to PAEs and cVMSs through inhalation of particulate matter were estimated based on the measured concentrations in PM0.1 and PM0.5 fractions. The estimated exposure doses of PAEs and cVMSs for infants (7.1 ng/kg-bw/d and 2.5 ng/kg-bw/d) were higher than those for adults (2.6 ng/kg-bw/d and 0.9 ng/kg-bw/d).
RESUMO
BACKGROUND: Tracking the US smoking cessation rate over time is of great interest to tobacco control researchers and policymakers since smoking cessation behaviors have a major effect on the public's health. Recent studies have employed dynamic models to estimate the US cessation rate through observed smoking prevalence. However, none of those studies has provided annual estimates of the cessation rate by age group. Hence, the primary objective of this study is to estimate annual smoking cessation rates specific to different age groups in the US from 2009 to 2017. METHODS: We employed a Kalman filter approach to investigate the annual evolution of age-group-specific cessation rates, unknown parameters of a mathematical model of smoking prevalence, during the 2009-2017 period using data from the 2009-2018 National Health Interview Surveys. We focused on cessation rates in the 25-44, 45-64 and 65 + age groups. RESULTS: The findings show that cessation rates followed a consistent u-shaped curve over time with respect to age (i.e., higher among the 25-44 and 65 + age groups, and lower among 45-64-year-olds). Over the course of the study, the cessation rates in the 25-44 and 65 + age groups remained nearly unchanged around 4.5% and 5.6%, respectively. However, the rate in the 45-64 age group exhibited a substantial increase of 70%, from 2.5% to 2009 to 4.2% in 2017. The estimated cessation rates in all three age groups tended to converge to the weighted average cessation rate over time. CONCLUSIONS: The Kalman filter approach offers a real-time estimation of cessation rates that can be helpful for monitoring smoking cessation behavior.