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1.
Front Cell Infect Microbiol ; 11: 717068, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34804989

RESUMO

This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen's column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component.


Assuntos
COVID-19 , RNA Viral , Endopeptidase K , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2
2.
J Synchrotron Radiat ; 28(Pt 5): 1386-1392, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34475287

RESUMO

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.


Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Manejo de Espécimes/métodos , Álcool Desidrogenase/química , Cristalização , Endo-1,4-beta-Xilanases/química , Endopeptidase K/química , Proteínas de Plantas/química , Conformação Proteica
3.
PLoS One ; 16(9): e0257615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34547058

RESUMO

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 µg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.


Assuntos
Biomarcadores/urina , Endopeptidase K/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Tuberculose/diagnóstico , Endopeptidase K/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Lipopolissacarídeos/urina , Papel , Temperatura
4.
PLoS One ; 16(6): e0245708, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34133441

RESUMO

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.


Assuntos
Bacillus cereus/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Bacillus cereus/genética , Bacillus cereus/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/farmacologia , Desoxirribonuclease I/farmacologia , Endopeptidase K/farmacologia , Endopeptidases/farmacologia , Enzimas/metabolismo , Enzimas/farmacologia , Matriz Extracelular/microbiologia , Glucana 1,4-alfa-Glucosidase/farmacologia , Esporos Bacterianos/efeitos dos fármacos , alfa-Amilases/farmacologia
5.
Acta Vet Hung ; 69(1): 88-93, 2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33844641

RESUMO

Prion disease is a fatal neurodegenerative disease with a broad host range in humans and animals. It is caused by proteinase K-resistant prion protein (PrPres). In previous studies, a heterogeneous infection in Cervidae and Caprinae was reported. Chronic wasting disease (CWD) has been frequently reported as the only prion disease in Korea that occurs in livestock. Thus, there is a possibility of transmission of CWD to Korean native black goats. However, PrPres has not been investigated thus far in Korean native black goats. We found strong linkage disequilibrium between c.126G>A and c.414T>C (r2 = 1) and between c.718C>T and c.126G>A (r2 = 0.638). In addition, the haplotype GTGTAAAC (representing codons 42, 102, 127, 138, 143, 146, 218 and 240) showed the highest frequency with 45.1%. Among 41 Korean native black goats, 20 animals (48.78%) were homozygous for the susceptible haplotypes (histidine at codon 143, asparagine at codon 146 and arginine at codon 154). Interestingly, we did not detect PrPres bands in any of the tested animals, including the 20 animals carrying potential scrapie susceptible haplotypes.


Assuntos
Doenças das Cabras , Doenças Neurodegenerativas , Proteínas Priônicas/genética , Príons , Scrapie , Animais , Endopeptidase K , Doenças das Cabras/epidemiologia , Doenças das Cabras/genética , Cabras , Haplótipos , Doenças Neurodegenerativas/veterinária , Príons/genética , República da Coreia/epidemiologia , Ovinos , Doenças dos Ovinos
6.
ACS Appl Mater Interfaces ; 13(16): 18545-18553, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33853319

RESUMO

Two major issues in cell-mediated drug delivery systems (c-DDS) are the availability of free cell surfaces for the binding of the cells to the target or to their microenvironment and internalization of the cytotoxic drug. In this study, the Janus structure, MOF nanoparticles, and tannic acid (TA) are utilized to address these issues. Janus carrier cells coated with metal-organic frameworks (MOFs) are produced by asymmetrically immobilizing the nanoparticles of a MOF based on zinc with cytotoxic enzymes that are internally encapsulated on the surface of carrier cells. By maintaining the biological and structural features of regular living cells, the MOF-coated Janus cells developed in the present study preserve the intrinsic binding capacity of the cells to their microenvironment. Interconnected MOFs loaded onto the other face of the Janus cells cannot penetrate the cell. Therefore, the carrier cells are protected from the cytotoxic drug contained in MOFs. These MOF-Janus carrier cells are demonstrated to successfully eliminate three-dimensional (3D) tumor spheroids when a chemotherapeutic protein of proteinase K is released from the MOF nanoparticles in an acid environment. The ease with which the MOF-Janus carrier cells are prepared (in 15 min), and the ability to carry a variety of enzymes and even multiple ones should make the developed system attractive as a general platform for drug delivery in various applications, including combination therapy.


Assuntos
Portadores de Fármacos/química , Endopeptidase K/química , Endopeptidase K/toxicidade , Estruturas Metalorgânicas/química , Linhagem Celular Tumoral , Humanos , Nanopartículas/química , Microambiente Tumoral/efeitos dos fármacos , Zinco/química
7.
J Virol Methods ; 293: 114131, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33798606

RESUMO

The World Health Organization (WHO) has declared a pandemic of COVID-19, the disease caused by the recently described SARS-CoV-2. The relevance and importance of mass diagnosis in order to find the asymptomatic individuals is widely recognized as a mandatory tool to reinforce the control measures for monitoring virus circulation and reduce the spreading of SARS-CoV-2. Here, we described quickness and cheaper strategies of direct RT-qPCR (in the absence of RNA isolation) and compared the results to those obtained using standard RNA isolation procedure. The tests varied using pure, diluted samples, combined with Proteinase K (PK) or Lysis Buffer. Our findings showed consistently that PK pre-treated samples in the absence of RNA extraction procedures presents similar results to those obtained by standard RNA isolation procedures. On average, 16 samples extracted with the MagMAX™ CORE Kit, take around 2 h, costing an average of USD 5, the pre-treatment of samples using PK, on the other hand, would cut the value to less than USD 0.30 and reduce the time of procedure in more than 1 ½ hours. The present study suggests the use of PK treatment instead of RNA isolation in order to reduce costs and time in processing samples for molecular diagnosis of SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Endopeptidase K/farmacologia , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Teste de Ácido Nucleico para COVID-19/economia , Humanos , SARS-CoV-2/genética
8.
Biochemistry ; 60(10): 765-779, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33656846

RESUMO

Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.


Assuntos
Distrofina/química , Endopeptidase K/metabolismo , Éxons , Distrofina/genética , Distrofina/metabolismo , Humanos , Conformação Proteica , Estabilidade Proteica , Proteólise
9.
PLoS One ; 16(3): e0248885, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33760876

RESUMO

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol-samples submitted to proteinase K treatment (3 µg/µL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Endopeptidase K/metabolismo , Humanos , Pandemias , Peru/epidemiologia , RNA/genética , RNA/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética
10.
PLoS One ; 16(2): e0247248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33600459

RESUMO

The conversion of cellular prion protein (PrPC) to disease-provoking conformer (PrPSc) is crucial in the pathogenesis of prion diseases. Heparin has been shown to enhance mammalian prion protein misfolding. As spontaneous prion disease has not been reported in non-mammalian species, such as chicken, it is interesting to explore the influence of heparin on the conversion of chicken prion protein (ChPrP). Herein, we investigated the influences of heparin on biochemical properties of full-length recombinant ChPrP, with murine prion protein (MoPrP) as control. The results showed that at low heparin concentration (10 µg/mL), a great loss of solubility was observed for both MoPrP and ChPrP using solubility assays. In contrast, when the concentration of heparin was high (30 µg/mL), the solubility of MoPrP and ChPrP both decreased slightly. Using circular dichroism, PK digestion and transmission electron microscopy, significantly increased ß-sheet content, PK resistance and size of aggregates were observed for MoPrP interacted with 30 µg/mL heparin, whereas 30 µg/mL heparin-treated ChPrP showed less PK resistance and slight increase of ß-sheet structure. Therefore, heparin can induce conformational changes in both MoPrP and ChPrP and the biochemical properties of the aggregates induced by heparin could be modified by heparin concentration. These results highlight the importance of concentration of cofactors affecting PrP misfolding.


Assuntos
Endopeptidase K/metabolismo , Heparina/farmacologia , Proteínas Priônicas/química , Proteínas Priônicas/genética , Animais , Galinhas , Dicroísmo Circular , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas Priônicas/metabolismo , Conformação Proteica , Conformação Proteica em Folha beta , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade
11.
PLoS One ; 16(2): e0247792, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33635936

RESUMO

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Endopeptidase K/química , SARS-CoV-2/isolamento & purificação , Animais , Chlorocebus aethiops , Temperatura Alta , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Células Vero
12.
Clin Chem ; 67(2): 415-424, 2021 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-33098427

RESUMO

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Saliva/virologia , COVID-19/epidemiologia , Colorimetria/métodos , Endopeptidase K/química , Humanos , Limite de Detecção , Pandemias , Testes Imediatos , SARS-CoV-2/química
13.
Mol Neurobiol ; 58(1): 375-390, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32959170

RESUMO

Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrPC) to the scrapie prion (PrPSc). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrPC/PrPSc molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrPC and PrPSc particle sizes and we also measured seeding activity and resistance to proteases. For cellular analysis in brain tissue, we measured inflammatory markers and synaptic damage, and used the isotropic fractionator to measure neuronal loss; these techniques were applied at different timepoints in a cross-sectional study of disease progression. Our analyses align with previous reports defining significant decreases in PrPC levels at pre-clinical stages of the disease and demonstrate that these decreases become significant before neuronal loss. We also identified the earliest PrPSc assemblies at a timepoint equivalent to 40% elapsed time for the disease incubation period; we propose that these assemblies, mostly composed of proteinase K (PK)-sensitive species, play an important role in triggering disease pathogenesis. Lastly, we show that the PK-resistant assemblies of PrPSc that appear at timepoints close to the terminal stage have similar biophysical characteristics, and hence that preparative use of PK-digestion selects for this specific subpopulation. In sum, our data argue that qualitative, as well as quantitative, changes in PrP conformers occur at the midpoint of subclinical phase; these changes affect quaternary structure and may occur at the threshold where adaptive responses become inadequate to deal with pathogenic processes.


Assuntos
Progressão da Doença , Regulação para Baixo , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Scrapie/patologia , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Morte Celular , Endopeptidase K/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/patologia , Camundongos , Peso Molecular , Proteínas PrPSc/metabolismo , Estrutura Quaternária de Proteína , Solubilidade , Sinapses/patologia , Fatores de Tempo
14.
Sci Rep ; 10(1): 21090, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273563

RESUMO

Bacteria can form biofilms, complex microbial communities protected from environmental stress, on food contact surfaces. Brassicaceae plant has been shown to contain bioactive compounds with antimicrobial activities. The objective of this study was to evaluate the synergistic effects of Brassicaceae species and proteinase K against E. coli O157:H7 biofilm. We determined the minimum biofilm inhibitory concentration, the fractional inhibitory concentration indexes, and the synergistic inhibitory effect of Raphanus sativus var. longipinnatus, R. sativus, and Brassica oleracea var. acephala extracts with proteinase K on E. coli O157:H7. The biofilm showed a 49% reduction with 2 mg/mL R. sativus. The combination of proteinase K 25 µg/mL significantly increased the effect of 2 mg/mL R. sativus var. longipinnatus and the combined treatment yielded up to 2.68 log reduction on stainless steel coupons. The results showed that the combination of R. sativus var. longipinnatus extract and proteinase K could serve as an anti-biofilm agent with synergistic effects for inhibiting E. coli O157:H7 biofilm on stainless steel surfaces.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Brassicaceae/química , Endopeptidase K/farmacologia , Escherichia coli/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sinergismo Farmacológico , Escherichia coli/fisiologia , Aço Inoxidável
15.
Sci Rep ; 10(1): 21169, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273617

RESUMO

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T. marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells' transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T. marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.


Assuntos
Antígenos de Fungos/imunologia , Transição de Fase , Saccharomyces cerevisiae/imunologia , Talaromyces/metabolismo , Temperatura , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Carboidratos/química , Citocinas/metabolismo , Endopeptidase K/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Proteínas Fúngicas/imunologia , Glicosilação , Humanos , Mediadores da Inflamação/metabolismo , Lectinas de Ligação a Manose/imunologia , Microscopia de Fluorescência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Fagocitose , Lectinas de Plantas/imunologia , Esporos Fúngicos/fisiologia , Células THP-1 , Talaromyces/citologia
17.
Part Fibre Toxicol ; 17(1): 43, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917232

RESUMO

BACKGROUND: The quantification of nanomaterials accumulated in various organs is crucial in studying their toxicity and toxicokinetics. However, some types of nanomaterials, including carbon nanomaterials (CNMs), are difficult to quantify in a biological matrix. Therefore, developing improved methodologies for quantification of CNMs in vital organs is instrumental in their continued modification and application. RESULTS: In this study, carbon black, nanodiamond, multi-walled carbon nanotube, carbon nanofiber, and graphene nanoplatelet were assembled and used as a panel of CNMs. All CNMs showed significant absorbance at 750 nm, while their bio-components showed minimal absorbance at this wavelength. Quantification of CNMs using their absorbance at 750 nm was shown to have more than 94% accuracy in all of the studied materials. Incubating proteinase K (PK) for 2 days with a mixture of lung tissue homogenates and CNMs showed an average recovery rate over 90%. The utility of this method was confirmed in a murine pharyngeal aspiration model using CNMs at 30 µg/mouse. CONCLUSIONS: We developed an improved lung burden assay for CNMs with an accuracy > 94% and a recovery rate > 90% using PK digestion and UV-Vis spectrophotometry. This method can be applied to any nanomaterial with sufficient absorbance in the near-infrared band and can differentiate nanomaterials from elements in the body, as well as the soluble fraction of the nanomaterial. Furthermore, a combination of PK digestion and other instrumental analysis specific to the nanomaterial can be applied to organ burden analysis.


Assuntos
Endopeptidase K/metabolismo , Pulmão/fisiopatologia , Nanotubos de Carbono/toxicidade , Espectrofotometria , Animais , Digestão , Grafite , Pulmão/diagnóstico por imagem , Camundongos , Nanoestruturas , Raios Ultravioleta
18.
J Virol Methods ; 286: 113965, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32891677

RESUMO

Pandemic SARS-CoV-2 infection has rapidly developed into a socioeconomic and humanitarian catastrophe. Basic principles to prevent SARS-CoV-2 transmission are social distancing, face masks, contact tracing and early detection of SARS-CoV-2. To meet these requirements, virtually unlimited test capacities delivering results in a rapid and reliable manner are a prerequisite. Here, we provide and validate such a rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA, termed COVID-quick-DET. This straightforward method operates with simple proteinase K treatment and repetitive heating steps with a sensitivity of 94.6% in head-to-head comparisons with kit-based isolation methods. This result is supported by data obtained from serially diluted SARS-CoV-2 virus stocks. Given its cost- and time-effective operation, COVID-quick-DET might be best suited for countries with general shortage or temporary acute scarcity of resources and equipment.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico , COVID-19 , Teste para COVID-19 , Testes Diagnósticos de Rotina , Endopeptidase K/química , Calefação , Humanos , Pandemias , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
19.
J Biol Chem ; 295(46): 15677-15691, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32900851

RESUMO

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.


Assuntos
Núcleo Celular/genética , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Ensaios de Triagem em Larga Escala/métodos , Mitocôndrias/genética , Automação , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/metabolismo , Endopeptidase K/metabolismo , Humanos , Magnetismo , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , Temperatura
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