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Owing to its outstanding physical properties, graphene has attracted attention as a promising biosensor material. Field-effect-transistor (FET)-based biosensors are particularly promising because of their high sensitivity that is achieved through the high carrier mobility of graphene. However, graphene-FET biosensors have not yet reached widespread practical applications owing to several problems. In this review, the authors focus on graphene-FET biosensors and discuss their advantages, the challenges to their development, and the solutions to the challenges. The problem of Debye screening, in which the surface charges of the detection target are shielded and undetectable, can be solved by using small-molecule receptors and their deformations and by using enzyme reaction products. To address the complexity of sample components and the detection mechanisms of graphene-FET biosensors, the authors outline measures against nonspecific adsorption and the remaining problems related to the detection mechanism itself. The authors also introduce a solution with which the molecular species that can reach the sensor surfaces are limited. Finally, the authors present multifaceted approaches to the sensor surfaces that provide much information to corroborate the results of electrical measurements. The measures and solutions introduced bring us closer to the practical realization of stable biosensors utilizing the superior characteristics of graphene.
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Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.
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Epigênese Genética , Heterocromatina , Animais , Heterocromatina/genética , Histonas/metabolismo , Lisina/metabolismo , Mamíferos/genética , Metilação , Histona Metiltransferases/metabolismoRESUMO
Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5' ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5' end svRNAs are RIG-I agonists and induce the IFN-ß response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5'-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5' end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs.
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In field-effect transistor (FET) biosensors, charge screening in electrolyte solutions limits the sensitivity, thereby restricting the applicability of FET sensors. This is particularly pronounced in graphene FET (GFET) biosensors, where the bare graphene surface possesses a strongly negative charge, which impedes the high sensitivity of GFETs owing to nonlinear electrolytic screening at the interfaces between graphene and liquid. In this study, we counteracted the negative surface charge of graphene by decorating positively charged compounds and demonstrated the sensing of C-reactive protein (CRP) with surface-charge-modulated GFETs (SCM-GFETs). We integrated multiple SCM-GFETs with anti-CRP antibodies and nonfunctionalized GFETs into a chip and measured differentials to eliminate background changes to improve measurement reliability. The FET response corresponded to the fluorescence images, which visualized the specific adsorption of CRP. The estimated dissociation constant was consistent with previously reported values; this supports the conclusion that the results are attributed to specific adsorption. Conversely, the signal in GFETs without decoration was obscured by noise because of nonlinear electrolytic screening, further emphasizing the significance of surface-charge modulation. The limit of detection of the system was determined to be 2.9 nM. This value has the potential to be improved through further optimization of the surface charges to align with specific applications. Our devices effectively circumvent nonlinear electrolytic screening, opening the door for further advancements in GFET biosensor technology.
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SARS-CoV-2 has evolved continuously and accumulated spike mutations with each variant having a different binding for the cellular ACE2 receptor. It is not known whether the interactions between such mutated spikes and ACE2 glycans are conserved among different variant lineages. Here, we focused on three ACE2 glycosylation sites (53, 90 and 322) that are geometrically close to spike binding sites and investigated the effect of their glycosylation pattern on spike affinity. These glycosylation deletions caused distinct site-specific changes in interactions with the spike and acted cooperatively. Of note, the particular interaction profiles were conserved between the SARS-CoV-2 parental virus and the variants of concern (VOCs) Delta and Omicron. Our study provides insights for a better understanding of the importance of ACE2 glycosylation on ACE2/SARS-CoV-2 spike interaction and guidance for further optimization of soluble ACE2 for therapeutic use.
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COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , SARS-CoV-2/genética , Glicosilação , Peptidil Dipeptidase A , Ligação ProteicaRESUMO
INTRODUCTION AND AIMS: This study examined whether zinc supplementation with zinc acetate hydrate improved renal anemia with hypozincemia in patients undergoing hemodialysis. METHODS: The study participants included 21 patients undergoing hemodialysis who presented with a serum zinc level < 60 mg/dL and who were administered zinc acetate hydrate at 50 mg (reduced to 25 mg, as appropriate) for 6 months. Patients with a hemorrhagic lesion, acute-phase disease (pneumonia or cardiac failure), or hematologic disease and those whose treatment was switched from peritoneal dialysis to hemodialysis were excluded. The changes in the erythropoietin resistance index (ERI) before and after zinc acetate hydrate administration were examined. ERI was defined as the dose (IU) of erythropoiesis-stimulating agent (ESA)/week/body weight (kg)/hemoglobin content (g/dL). The differences between the two groups were analyzed using the Wilcoxon signed rank sum test, and p < 0.05 was considered statistically significant. RESULTS: The study participants included 19 men and 2 women aged 41-95 years (mean ± standard deviation (SD): 67.1 ± 13.6). The changes in the values of parameters measured before and after zinc acetate hydrate administration were as follows: Blood Hb did not change significantly, from 10.0-13.6 g/dL (11.5 ± 1.0 g/dL) to 10.2-12.4 g/dL (11.4 ± 0.7 g/dL); serum zinc concentration significantly increased, from 33.0-59.0 mg/dL µg/dL (52.4 ± 7.6 mg/dL µg/dL) to 57.0-124.0 mg/dL µg/dL (84.1 ± 16.3 mg/dL µg/dL; p < 0.01); the ESA dose significantly decreased, from 0-12,000 IU/week (5630 ± 3351 IU/week) to 0-9000 IU/week (4428 ± 2779; p = 0.04); and ERI significantly decreased, from 0.0-18.2 (8.1 ± 5.1) to 0.0-16.0 (6.3 ± 4.3; p = 0.04). CONCLUSIONS: Zinc supplementation increased the serum zinc concentration and significantly reduced the ESA dose and ERI, suggesting that a correction of hypozincemia contributes to lessening renal anemia in these patients.
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Anemia , Hematínicos , Nefropatias , Falência Renal Crônica , Masculino , Humanos , Feminino , Acetato de Zinco/efeitos adversos , Anemia/tratamento farmacológico , Anemia/etiologia , Diálise Renal/efeitos adversos , Hematínicos/farmacologia , Hematínicos/uso terapêutico , Hemoglobinas , Falência Renal Crônica/terapia , Zinco/uso terapêutico , Doença Crônica , Suplementos NutricionaisRESUMO
C-reactive protein (CRP) is an important biomarker of infection and inflammation, as CRP is one of the most prominent acute-phase proteins. CRP is usually detected using anti-CRP antibodies (Abs), where the intermolecular interactions between CRP and the anti-CRP Ab are largely affected by the pH and ionic strength of environmental solutions. Therefore, it is important to understand the environmental effects of CRP-anti-CRP Ab interactions when designing highly sensitive biosensors. Here, we investigated the efficiency of fluorescently labeled CRP-anti-CRP monoclonal antibody (mAb) interactions at different pHs and ionic strengths. Our results indicate that the affinity was insensitive to pH changes in the range of 5.9 to 8.1, while it was significantly sensitive to ionic strength changes. The binding affinity decreased by 55% at an ionic strength of 1.6 mM, when compared to that under a physiological condition (~150 mM). Based on the isoelectric focusing results, both the labeled CRP and anti-CRP mAb were negatively charged in the studied pH range, which rendered the system insensitive to pH changes, but sensitive to ionic strength changes. The decreased ionic strength led to a significant enhancement of the repulsive force between CRP and the anti-CRP mAb. Although the versality of the findings is not fully studied yet, the results provide insights into designing highly sensitive CRP sensors, especially field-effect transistor-based sensors.
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Solution-gated graphene field-effect transistors (SG-GFETs) provide an ideal platform for sensing biomolecules owing to their high electron/hole mobilities and 2D nature. However, the transfer curve often drifts in an electrolyte solution during measurements, making it difficult to accurately estimate the analyte concentration. One possible reason for this drift is that p-doping of GFETs is gradually countered by cations in the solution, because the cations can permeate into the polymer residue and/or between graphene and SiO2 substrates. Therefore, we propose doping sufficient cations to counter p-doping of GFETs prior to the measurements. For the pre-treatment, GFETs were immersed in a 15 mM sodium chloride aqueous solution for 25 h. The pretreated GFETs showed that the charge neutrality point (CNP) drifted by less than 3 mV during 1 h of measurement in a phosphate buffer, while the non-treated GFETs showed that the CNP was severely drifted by approximately 50 mV, demonstrating a 96% reduction of the drift by the pre-treatment. X-ray photoelectron spectroscopy analysis revealed the accumulation of sodium ions in the GFETs through pre-treatment. Our method is useful for suppressing drift, thus allowing accurate estimation of the target analyte concentration.
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Grafite , Cátions , Polímeros , Dióxido de Silício , Transistores EletrônicosRESUMO
Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.
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Adaptação ao Hospedeiro , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Humana/virologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Animais , Aves , Linhagem Celular , Genes Virais , Genótipo , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H9N2/classificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/virologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Infecções por Orthomyxoviridae/virologia , Filogenia , Aves Domésticas , RNA Polimerase Dependente de RNA/química , Vírus Reordenados/genética , Proteínas Virais/química , Zoonoses Virais , Virulência/genéticaRESUMO
Adaptive mutations and/or reassortments in avian influenza virus polymerase subunits PA, PB1, and PB2 are one of the major factors enabling the virus to overcome the species barrier to infect humans. The majority of human adaptation polymerase mutations have been identified in PB2; fewer adaptation mutations have been characterized in PA and PB1. Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and generally carry the human adaptation PB2-E627K substitution during their dissemination in nature. In this study, we identified other human adaptation polymerase mutations by analyzing phylogeny-associated PA mutations that H5N1 clade 2.2.1 viruses have accumulated during their evolution in the field. This analysis identified several PA mutations that produced increased replication by contemporary clade 2.2.1.2 viruses in vitro in human cells and in vivo in mice compared to ancestral clade 2.2.1 viruses. The PA mutations acted cooperatively to increase viral polymerase activity and replication in both avian and human cells, with the effect being more prominent in human cells at 33°C than at 37°C. These results indicated that PA mutations have a role in establishing contemporary clade 2.2.1.2 virus infections in poultry and in adaptation to infect mammals. Our study provided data on the mechanism for PA mutations to accumulate during avian influenza virus evolution and extend the viral host range.IMPORTANCE Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication in vitro in human cells and in vivo in mice. These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans.
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Evolução Molecular , Virus da Influenza A Subtipo H5N1/fisiologia , Infecções por Orthomyxoviridae/virologia , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/genética , Adaptação Fisiológica , Animais , Linhagem Celular , Galinhas , Egito/epidemiologia , Especificidade de Hospedeiro , Humanos , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/genética , Camundongos , Modelos Moleculares , Mutação , Filogenia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.
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Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Sequência de Aminoácidos , Animais , Galinhas , Genótipo , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H9N2/metabolismo , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Aviária/patologia , Camundongos , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Fenótipo , Filogenia , Receptores Virais , Deleção de Sequência , Relação Estrutura-Atividade , Virulência , Internalização do Vírus , Replicação ViralRESUMO
Due to its high carrier mobility, graphene is considered a suitable material for use in field-effect transistors. However, its application to immunosensing of small molecules is still elusive. To investigate the potential of graphene field effect transistors (G-FET) as a sensor for small molecules with small or no charge, we applied the open-sandwich immunoassay (OS-IA), which detects low-molecular-weight antigens noncompetitively, to G-FET. Using an antibody variable fragment VL immobilized on graphene and a hyperacidic region of amyloid precursor protein fused to the other variable fragment VH, we successfully detected a small antigen peptide consisting of 7 amino acids (BGP-C7), with a more than 100-fold increase in sensitivity compared with that measured by enzyme-linked OS-IA. Furthermore, we succeeded in detecting BGP-C7 in the presence of human serum with similar sensitivity, suggesting its potential application in clinical diagnostics.
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Antígenos/metabolismo , Grafite/química , Imunoensaio/métodos , Transistores Eletrônicos/normas , HumanosRESUMO
Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks.
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Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Humana/virologia , Mutação , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Animais , Evolução Molecular , Feminino , Células HEK293 , Especificidade de Hospedeiro/genética , Humanos , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Humana/epidemiologia , Mamíferos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Oriente Médio/epidemiologia , Modelos Moleculares , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Vírus Reordenados/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Virulência/genética , Replicação Viral/genética , Zoonoses/virologiaRESUMO
Low dark current, high-responsivity middle-wavelength infrared (IR) graphene photodetectors using photo-gating amplification of injected photo-carriers are demonstrated. A graphene/p-indium antimonide (InSb) heterojunction and graphene/insulator region were formed. The injected photo-carriers from InSb to graphene were amplified by photo-gating induced in the graphene/tetraethyl orthosilicate (TEOS) region, resulting in the high responsivity and low dark current performance. A responsivity of 14.9 A/W and an ON/OFF ratio of 2.66×104 were achieved. The photoresponse is shown to be determined by the cross-sectional area between the graphene and the TEOS-SiO2, in which the injected photo-carriers into graphene were modulated and amplified by the photo-gating effect. Our results indicate that high-performance IR photodetectors based on the developed graphene photodetectors can be realized.
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Graphene has strong potential for electrical biosensing owing to its two-dimensional nature and high carrier mobility which transduce the direct contact of a detection target with a graphene channel to a large conductivity change in a graphene field-effect transistor (G-FET). However, the measurable range from the graphene surface is highly restricted by Debye screening, whose characteristic length is less than 1 nm at physiological ionic strength. Here, we demonstrated electrical biosensing utilizing the enzymatic products of the target. We achieved quantitative measurements of a target based on the site-binding model and real-time measurement of the enzyme kinetics in femtoliter microdroplets. The combination of a G-FET and microfluidics, named a "lab-on-a-graphene-FET", detected the enzyme urease with high sensitivity in the zeptomole range in 100 mM sodium phosphate buffer. Also, the lab-on-a-graphene-FET detected the gastric cancer pathogen Helicobacter pylori captured at a distance greater than the Debye screening length from the G-FET.
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Técnicas Biossensoriais/instrumentação , Grafite/química , Transistores Eletrônicos , Canavalia/enzimologia , Desenho de Equipamento , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Humanos , Dispositivos Lab-On-A-Chip , Concentração Osmolar , Urease/análiseRESUMO
The cocirculation of H5N1 and H9N2 avian influenza viruses in birds in Egypt provides reassortment opportunities between these two viruses. However, little is known about the emergence potential of reassortants derived from Egyptian H5N1 and H9N2 viruses and about the biological properties of such reassortants. To evaluate the potential public health risk of reassortants of these viruses, we used reverse genetics to generate the 63 possible reassortants derived from contemporary Egyptian H5N1 and H9N2 viruses, containing the H5N1 surface gene segments and combinations of the H5N1 and H9N2 internal gene segments, and analyzed their genetic compatibility, replication ability, and virulence in mice. Genes in the reassortants showed remarkably high compatibility. The replication of most reassortants was higher than the parental H5N1 virus in human cells. Six reassortants were thought to emerge in birds under neutral or positive selective pressure, and four of them had higher pathogenicity in vivo than the parental H5N1 and H9N2 viruses. Our results indicated that H5N1-H9N2 reassortants could be transmitted efficiently to mammals with significant public health risk if they emerge in Egypt, although the viruses might not emerge frequently in birds.IMPORTANCE Close interaction between avian influenza (AI) viruses and humans in Egypt appears to have resulted in many of the worldwide cases of human infections by both H5N1 and H9N2 AI viruses. Egypt is regarded as a hot spot of AI virus evolution. Although no natural reassortant of H5N1 and H9N2 AI viruses has been reported so far, their cocirculation in Egypt may allow emergence of reassortants that may present a significant public health risk. Using reverse genetics, we report here the first comprehensive data showing that H5N1-N9N2 reassortants have fairly high genetic compatibility and possibly higher pathogenicity in mammals, including humans, than the parental viruses. Our results provide insight into the emergence potential of avian H5N1-H9N2 reassortants that may pose a high public health risk.
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Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus Reordenados/genética , Animais , Aves/genética , Cães , Genes Virais , Células HEK293 , Humanos , Influenza Aviária/virologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mamíferos/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Filogenia , Genética Reversa/métodos , Virulência , Replicação ViralRESUMO
Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed 'nanocell'. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using ß-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicability of digital bioassay for enzymes under non-optimum conditions or enzymes of low turnover rate. The sealing of the reactor with air would also expand the applicability, allowing the use of fluorescent dyes that leak into oil.
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Fosfatase Alcalina/análise , Ensaios Enzimáticos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , beta-Galactosidase/análise , Ensaios Enzimáticos/instrumentação , Ensaio de Imunoadsorção Enzimática/instrumentação , Escherichia coli/enzimologia , Proteínas de Escherichia coli/análise , Fluoresceínas/química , Corantes Fluorescentes/química , Concentração de Íons de HidrogênioRESUMO
Graphene, a single atom layer of carbon atoms, provides a two-dimensional platform with an extremely high sensitivity to charges due to its unique band structure and high surface-to-volume ratio. Graphene field-effect transistor (G-FET) biosensors have, indeed, demonstrated a detection limit of subnanomolar or even subpicomolar. However, in G-FET, signal is averaged throughout the whole channel, so there remains a need to visualize the spatial distribution of target molecules on a single G-FET, to provide further insight into target molecules and/or biological functions. Here, we made use of graphene as an imaging platform of charged molecules via Raman microscopy. Positively (or negatively) charged microbeads with a diameter of 1 µm were dispersed in a buffer solution and were attached on graphene. We found out that Raman peaks of graphene, where positively (or negatively) charged beads contacted, were up-shifted (or down-shifted) significantly, indicating that the carrier density in the graphene was locally modulated by the charged beads and the charge state of the beads was represented by the peak-shift direction. From the peak shift, the change in the carrier density was calculated to be +1.4 × 1012 cm-2 (or -1.0 × 1012 cm-2). By taking Raman peak-shift images, we visualized distribution of charged molecules on graphene with a spatial resolution below 1 µm. The technique described here overcomes the limitation of spatial resolution of G-FET and provides a new route to graphene-based chemical and biosensors.
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Although condensins play essential roles in mitotic chromosome assembly, Ki-67 (also known as MKI67), a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Proteínas de Ligação a DNA/genética , Humanos , Ácidos Indolacéticos/metabolismo , Antígeno Ki-67/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte ProteicoRESUMO
The rapidly evolvable influenza A virus has caused pandemics linked to millions of deaths in the past century. Influenza A viruses are categorized by H (hemagglutinin; HA) and N (neuraminidase; NA) proteins expressed on the viral envelope surface. Analyses of past pandemics suggest that the HA gene segment comes from a nonhuman virus, which is then introduced into an immunologically naïve human population with potentially devastating consequences. As a prerequisite for infection, the nonhuman HA molecules of H1-H16 viruses must be able to bind to specific sialyl receptors on the host cell surface along the human respiratory tract. Thus, additional insight into the structures of host cell glycans and how different HAs interact with different glycans might provide new insight into the mechanisms underlying sustained infection and transmission in humans. In this work, we identified the sialyl N-glycans found in normal human alveoli and characterized the influenza viruses that preferentially bound to these different structures. We also determined the amino acid changes in HA that were linked to a switch of receptor-binding preference from nonhuman to pandemic, as well as pandemic to seasonal. Our data provide insight into why seasonal viruses are associated with reduced alveolar infection and damage and suggest new considerations for designing anti-HA vaccines and drugs. The results provide a better understanding of viral tropism and pathogenesis in humans that will be important for prediction and surveillance of zoonotic, pandemic, and epidemic influenza outbreaks. DATABASE: The novel hemagglutinin nucleotide sequences reported here were deposited in GISAID under the accession numbers of EPI685738 for A/Yamaguchi/20/2006(H1N1) and EPI685740 for A/Kitakyushu/10/2006(H1N1).