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Human leukocyte antigen (HLA) class I has more than 18,000 alleles, each of which binds to a set of unique peptides from the cellular degradome. Deciphering the interaction between antigenic peptides and HLA proteins is crucial for understanding immune responses in autoimmune diseases and cancer. In this study, we aimed to characterize the peptidome that binds to HLA-A*33:03, which is one of the most prevalent HLA-A alleles in the Northeast Asian population, but poorly studied. For this purpose, we analyzed the HLA-A*33:03 monoallelic B cell line using immunoprecipitation of HLA-A and peptide complexes, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we identified 5731 unique peptides that were associated with HLA A*33:03, and experimentally validated the affinity of 40 peptides for HLA-A*33:03 and their stability in HLA A*33:03-peptides complexes. To our knowledge, this study represents the largest dataset of peptides associated with HLA-A*33:03. Also, this is the first study in which HLA A*33:03-associated peptides were experimentally validated.
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Antígenos HLA-A , Espectrometria de Massas em Tandem , Cromatografia Líquida , Epitopos , Humanos , ImunoprecipitaçãoRESUMO
BACKGROUND: Dysregulation of DNA damage response and altered DNA methylation in acute myeloid leukemia (AML) have been reported, but the impact of methylation of DNA repair genes has not yet been researched. We aimed to predict the prognosis of non-APL AML patients based on the known CpG site methylation levels of DNA repair genes through The Cancer Genome Atlas AML project (TCGA-LAML). METHODS: We utilized TCGA-LAML cohort (174 non-APL AML) for the methylation data of 22 DNA repair genes. RESULTS: In univariate analysis among 174 non-APL AML patients of the TCGA-LAML cohort, the hypermethylation of MLH1, RAD51, and ATM showed superior overall survival (OS) than non-hypermethylated groups, while hypermethylation of RAD23A, RAD23B, MLH1, MSH2, BRCA1, BRCA2, RAD50, and PARP1 was associated with poor OS. We demonstrated that CpG hypermethylation levels of DNA repair genes differed according to the AML cytogenetic risk groups. In multivariate analysis, hypermethylation of MLH1 and RAD51 showed better OS than non-hypermethylated patients, but hypermethylation of MSH2 and RAD50 showed worse OS than non-hypermethylated patients. CONCLUSION: Methylation of 4 DNA repair genes, such as MLH1, RAD51, MSH2, and RAD50, have the potential to be independent risk factors in non-APL AML patients.
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Leucemia Mieloide Aguda , Biomarcadores , Metilação de DNA , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteína 2 Homóloga a MutS/genética , PrognósticoRESUMO
BACKGROUND: (1-3)-ß-D-glucan (BDG) is a fast and simple assay to diagnose invasive fungal infection. In this study, we evaluated the performance of the Goldstream BDG assay (Beijing Gold Mountainriver Tech Development) performed on the automated analyzer, IGL-200 (Genobio Pharmaceutical). METHODS: The precision and linearity of the Goldstream BDG assay were evaluated according to Clinical and Laboratory Standards Institute procedures. BDG results performed on the IGL-200 were compared to a manual photometer, MB-80A (Genobio Pharmaceutical). The manufacturer-provided reference interval was verified. RESULTS: Within-laboratory imprecision (% Coefficient of Variation) was 9.4%. The best polynomial fit was third-order within the manufacturer's claimed linear range (32.0 - 830.0 pg/mL). The BDG assay performed on IGL-200 and MB-80A showed a total agreement of 97.6%. All healthy subjects were within range of the manufacture provided reference interval. CONCLUSIONS: The analytical performance of the Goldstream BDG assay was clinically acceptable.
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Infecções Fúngicas Invasivas , beta-Glucanas , Fungos , Humanos , Infecções Fúngicas Invasivas/diagnóstico , Sensibilidade e EspecificidadeRESUMO
The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the capping of CD20 after ofatumumab binding was also confirmed using Ramos B cells. In the functional equivalence tests, the binding affinities and complement-dependent cell cytotoxicity efficacy of plant-ofatumumab-HDEL and plant-ofatumumab without HDEL were significantly reduced compared to those of CHO-derived ofatumumab. Therefore, we suggest that although ofatumumab is not a good candidate as a template for plant-derived monoclonal antibodies because of its decreased affinity when produced in plants, it is an interesting target to study the differences between post-translational modifications in mammals and plants.
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Anticorpos Monoclonais Humanizados/genética , Fragmentos Fab das Imunoglobulinas/química , Nicotiana/metabolismo , Folhas de Planta/metabolismo , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Antígenos CD20/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose , Linfócitos B , Células CHO , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cricetulus , Citotoxicidade Imunológica/efeitos dos fármacos , Células HEK293 , Humanos , Conformação Proteica , Rituximab/metabolismoRESUMO
Background: EDTA-induced pseudothrombocytopenia (PTCP) during whole blood collection requires significant laboratory resources to obtain accurate results. We evaluated platelet-deaggregation function in EDTA-induced PTCP and platelet-clump flagging by the BC-6800Plus hematology analyzer using integrated digital image analysis. Methods: We prospectively collected 132 whole blood samples suspected of platelet clumping (102 in EDTA and 30 in sodium citrate) from 88 individuals. We compared platelet counts determined using the platelet count by impedance (PLT-I) function of the DxH 900 hematology analyzer and the PLT-I or optical platelet count (PLT-O) function of the BC-6800Plus. Platelet clumping was verified through manual inspection and the MC-80 digital image analyzer. Results: Among the 132 whole blood samples, 43 EDTA samples showed platelet clumping. The DxH 900 PLT-I and BC-6800Plus PLT-I results demonstrated a strong correlation (r=0.711) for the EDTA samples but only a moderate correlation with the BC-6800Plus PLT-O results (r=0.506 and 0.545, respectively). The BC-6800Plus PLT-O results were consistent with the sodium citrate platelet counts, with a median dissociation rate of 102.5% (range, 74.9%-123.1%). The DxH 900 and BC-6800Plus analyzers had sensitivity values of 0.79 and 0.72, respectively, for platelet-clump flagging. When integrating the MC-80 digital image analysis results, the sensitivity of BC-6800Plus improved to 0.89 (standard mode) or 1.0 (PLT-Pro mode). Conclusions: BC-6800Plus PLT-O measurement results are close to the actual values obtained by platelet deaggregation with PTCP samples. Integrating the BC-6800Plus with a digital imaging analyzer effectively improved the diagnosis of PTCP and reduced the requirement for additional laboratory procedures.
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In this study, we investigated the antimicrobial susceptibility and molecular characteristics of antimicrobial resistance of Acinetobacter colistiniresistens strains isolated from the bloodstream using whole-genome sequencing. Clinical isolates identified as Acinetobacter baumannii and showing colistin resistance at the time of detection were collected. Antimicrobial susceptibility was determined using the VITEK2 system (bioMérieux) and Sensititre system (Thermo Fisher Scientific). Species identification and antimicrobial resistance gene searches were performed through whole-genome sequencing. Through whole-genome sequencing, three colistin-resistant strains from the bloodstream were identified as A. colistiniresistens. All three A. colistiniresistens strains were resistant to two or more antimicrobial agents except for colistin, and two of them were resistant to carbapenems. Genes involved in aminoglycoside [AAC(3)-â ¡b, AAC(6')-â j, aadA2, ANT(3â³)-â ¡b, APH(3')-â ¥a], macrolide (mphD, msrE), carbapenem and cephalosporin (OXA-420, VIM-2), fluoroquinolone and tetracycline (adeF), and sulfonamide (sul1, sul2) resistance were detected. We report multidrug-resistant A. colistiniresistens strains isolated from the bloodstream through whole-genome sequencing. Two strains carried carbapenemase genes, and this is the first report of VIM-2-producing A. colistiniresistens.
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Infecções por Acinetobacter , Acinetobacter , Antibacterianos , Colistina , Farmacorresistência Bacteriana Múltipla , beta-Lactamases , Humanos , Masculino , Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Antibacterianos/farmacologia , Bacteriemia/microbiologia , Proteínas de Bactérias/genética , beta-Lactamases/genética , Carbapenêmicos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Sequenciamento Completo do GenomaRESUMO
We evaluated the impact of the FilmArray blood culture identification (BCID) panel on the time taken to administer effective antibiotics and the clinical outcomes of bloodstream infections. We retrospectively screened patients with bloodstream infections who underwent BCID testing and compared them to a historical control group that received conventional culture testing. A total of 144 and 214 patients who underwent BCID and conventional cultures, respectively, were compared. The 30-day mortality (BCID: 9.7% vs. conventional method: 10.7%, p = 0.755), time to effective antibiotic administration (3 h for both BCID and conventional method, p = 0.789), and time to appropriate antibiotic administration did not differ significantly between the groups. BCID was not significantly associated with 30-day mortality after adjusting for the Pitt bacteremia score and the Charlson comorbidity index (adjusted OR = 0.833, CI; 0.398-1.743). Compared with conventional methods, BCID reduced the time to administration of effective antibiotics in cases of carbapenem-resistant Enterobacterales (CRE) (39 h vs. 93 h, p = 0.012) and vancomycin-resistant enterococci (VRE) (50 h vs. 92 h, p < 0.001) bacteremia. BCID did not affect the clinical outcomes of overall bloodstream infections; however, it contributed to the early administration of effective antibiotics in cases of CRE and VRE bacteremia.
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The prompt implementation of optimal antibacterial therapy through the rapid identification of the causative organisms is essential for improving outcomes for critically ill patients with bloodstream infections. We evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel for rapidly identifying causative pathogens in the bloodstream using large-scale clinical samples. We analyzed the results of identification using a BCID panel performed on 2005 positive blood culture bottles from September 2019 to June 2022. Pathogen detection efficiency and interval from Gram staining to identification using the BCID panel were compared to those of conventional identification systems-VITEK MS MALDI-TOF Mass Spectrometer and Vitek2-and antibiotic susceptibility testing-Vitek2. We detected 2167 isolates from 2005 positive blood culture bottles. In these isolates, the BCID panel showed 93% full agreement-both organisms and antimicrobial resistance genes were matched, and no off-target organisms were detected. Species-level discordance was found in 0.6% of tests. Sixty-five isolates (3.0%) were only detected by BCID, whereas 22 isolates (1.0%) from the on-target panel were not detected by BCID. This large-scale study demonstrated that the BCID panel was a reliable and rapid identification method for directly identifying bloodstream pathogens in a positive blood culture.
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The rapid identification of patients infected with COVID-19 during the SARS-CoV-2 pandemic is critical to operating emergency rooms effectively. Xpert Xpress SARS-CoV-2 (Xpert) assays are increasingly being used in the rapid screening of COVID-19. We evaluated the clinical performance of Xpert by comparing findings with those of qRT-PCR evaluations and included the clinical features of patients visiting the emergency department. Positive results with Xpert testing (n = 370) were compared with qRT-PCR findings, demonstrating a 91.9% intertest agreement. We reviewed the subsequent COVID-19 test results and SARS-CoV-2 infection histories for individuals showing discrepancies in Xpert and qRT-PCR testing and determined whether the findings were true-positive or false-positive. The true-positive rate for Xpert testing was 95.4% (353/370); the remaining 17 samples (4.6%) were false-positive. All false-positive data for Xpert testing showed N2 signals amplified to Ct values of ≥40 with no E gene signals. Rapid Xpert testing is highly sensitive and shows a good performance overall in challenging situations, such as an emergency room. However, we considered the possibility of false-positive Xpert results given an N2 gene signal only, especially given high Ct values. We recommend interpreting test data with caution and considering retesting over time.
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PURPOSE: Prostate cancer is one of the most heritable cancers and prostate cancer with germline mutations is associated with aggressive features and a poor prognosis. We investigated germline variants in unselected Korean men with prostate cancer. MATERIALS AND METHODS: In this study, we prospectively collected buccal swab DNA from 120 unselected Korean men with prostate cancer, and performed massively parallel sequencing. Identified germline variants were interpreted according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology 2015 guidelines. RESULTS: Of the 120 patients, 30 had regional or metastatic disease and 10, 34, 25, and 21 patients were categorized as having low, intermediate, high, or very high-risk disease, respectively. Of the 88 germline variants, 6 pathologic or likely pathogenic variants were identified in 7 patients (5.8%) with BRCA2 (1.7%), HOXB13 (1.7%), PALB2 (0.8%), ATM (0.8%), and MSH2 (0.8%). Of 7 patients, 2 possessed intermediate risk disease that was not included in the recommendation for genetic testing. We identified the Gly132Glu variant, which was different from the Gly84Glu variant of the HOXB13 gene in Western populations. CONCLUSIONS: This study presents the first analysis of germline variants in unselected Korean men with prostate cancer. Our results showed comparable germline prevalence with previous studies and provides evidence for the necessity of genetic testing in Korean men with prostate cancer.
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Mutação em Linhagem Germinativa , Neoplasias da Próstata , Predisposição Genética para Doença , Testes Genéticos , Células Germinativas/patologia , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , República da CoreiaRESUMO
c-Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small-cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti-c-Kit antibody-drug conjugate was developed in this study to treat wild-type and mutant c-Kit-positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (to give NN2101-DM1). The antitumor activity of NN2101-DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101-DM1 exhibited potent growth-inhibitory activities against c-Kit-positive cancer cell lines. In a mouse xenograft model, NN2101-DM1 exhibited potent growth-inhibitory activities against imatinib-resistant GIST and SM cells. In addition, NN2101-DM1 exhibited a significantly higher anti-cancer effect than carboplatin/etoposide against SCLC cells where c-Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101-DM1 with imatinib in imatinib-sensitive GIST cells induced complete remission compared with treatment with NN2101-DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101-DM1 is a potential therapeutic agent for wild-type and mutant c-Kit-positive cancers.
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Tumores do Estroma Gastrointestinal , Imunoconjugados , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Liquid biopsies have increasingly shown clinical utility. Although next-generation sequencing has been widely used for the detection of somatic mutations from plasma, performance characteristics vary by platform. Therefore, thorough validation is mandatory for clinical use. This study aimed to evaluate the analytical validity of the Oncomine Pan-Cancer Cell-Free Assay. A massively parallel sequencing for the assay was performed using the Ion S5 XL System with Ion 540 kit. The analytical sensitivity and precision were evaluated using pre-characterized reference materials. The specificity was evaluated using plasma from healthy subjects. A comparison with the Cobas EGFR Mutation Test v2 was performed using reference materials and plasma from lung cancer patients. For SNVs and short indels, the analytical sensitivities at variant allele frequencies (VAFs) of 0.1%, 0.5%, and 1% were 50%, 93.4%, and 100% with 20 ng of input, respectively. The overall precision of the true positive variants was 98% at a VAF of 1% with 20 ng input. The assay showed a similar sensitivity to that of the Cobas EGFR Mutation Test v2 at a VAF of 0.5% with 20 ng of input and 100% concordance on clinical samples. The Pan-Cancer Cell-Free Assay can be applied to detect EGFR mutations in advanced lung cancer patients, although follow-up studies will be needed to evaluate the analytical validity for other types of genes and aberrations using clinical samples.
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Primary myelofibrosis (PMF) and paroxysmal nocturnal hemoglobinuria (PNH) are very rare diseases, respectively, and it is uncommon to have both diseases together. Mutational profiling using next-generation sequencing in PMF and PNH detected additional mutations associated with myeloid neoplasms, suggesting a step-wise clonal evolution. We present here a very rare case with PMF and PNH with JAK2 V617F, U2AF1 and SETBP1 mutations at the time of diagnosis. The combination of these two diseases and three genetic mutations is difficult to interpret at once. (i.e., the sequence of these two clonal diseases or the time points of acquiring these mutations). Our report suggests that when diagnosing or treating patients with PMF, it is necessary to keep in mind that PNH may be present at the same time or sometimes new. The genetic mutations simultaneously found in this patient require further research to elucidate the clinical significance and their genetic associations fully.
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The spread of delta variants (B.1.671.2) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a severe global threat. Multiplex real-time PCR is a common method for confirming SARS-CoV-2 infection, however, additional tests, such as whole genomic sequencing, are required to reveal the presence or type of viral mutation. Moreover, applying whole genomic sequencing to all SARS-CoV-2 positive samples is challenging due to time and cost constraints. Here, we report that the double or low amplification curve observed during RNA-dependent RNA polymerase (RdRp) gene/S gene amplification in the Allplex SARS-CoV-2 Assay is related to delta/alpha variants. We analyzed the RdRp/S gene amplification curve using 94 samples confirmed as SARS-CoV-2 infection by the Allplex SARS-CoV-2 Assay from January to August, 2021. These positive samples identified variant types using the Novaplex SARS-CoV-2 Variants I and IV Assays. Overall, 17 samples showing a double curve and 11 samples showing a low amplification pattern were associated with alpha-/delta-type strains with variants in the P681 region. The double or low curve shown in the RdRp gene amplification curve had 100% sensitivity and 100% specificity for diagnosing delta/alpha variants. During the SARS-CoV-2 virus diagnostic RT-PCR test using the Allplex SARS-CoV-2 Assay, we could consider the presence of delta/alpha variants in the samples with double or low amplification curve of the RdRp/S gene channel. This PCR amplification curve abnormality enables rapid and cost-effective variant type prediction during SARS-CoV-2 diagnostic testing in clinical laboratories.
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Stickler syndrome is an inherited connective tissue disorder of collagen. There are relatively few reports of East Asian patients, and no large-scale studies have been conducted in Korean patients yet. In this study, we retrospectively analyzed the genetic characteristics and clinical features of Korean Stickler syndrome patients. Among 37 genetically confirmed Stickler syndrome patients, 21 types of gene variants were identified, of which 12 were novel variants. A total of 30 people had variants in the COL2A1 gene and 7 had variants in the COL11A1 gene. Among the types of pathogenic variants, missense variants were found in 11, nonsense variants in 8, and splice site variants in 7. Splicing variants were frequently associated with retinal detachment (71%) followed by missense variants. This is the first large-scale study of Koreans with Stickler syndrome, which will expand the spectrum of genetic variations of Stickler syndrome.
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Artrite/genética , Colágeno Tipo II/genética , Colágeno Tipo XI/genética , Doenças do Tecido Conjuntivo/genética , Perda Auditiva Neurossensorial/genética , Miopia/genética , Descolamento Retiniano/genética , Adolescente , Adulto , Artrite/epidemiologia , Artrite/patologia , Povo Asiático/genética , Criança , Pré-Escolar , Doenças do Tecido Conjuntivo/epidemiologia , Doenças do Tecido Conjuntivo/patologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Perda Auditiva Neurossensorial/epidemiologia , Perda Auditiva Neurossensorial/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação/genética , Miopia/epidemiologia , Miopia/patologia , Linhagem , Fenótipo , República da Coreia/epidemiologia , Descolamento Retiniano/epidemiologia , Descolamento Retiniano/patologia , Adulto JovemRESUMO
Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.
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Medições Luminescentes/métodos , Pontos Quânticos , Animais , Diagnóstico por Imagem , Transferência de Energia , Fluorescência , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Semicondutores , Sensibilidade e EspecificidadeRESUMO
N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100⯵g/mL to 4000⯵g/mL in 25⯵L of water. The determination of coefficient (r2 >â¯0.9992), recovery (88.992% ~ 111.198%), limit of detection (LODâ¯<â¯193.274⯵g/mL), limit of quantification (LOQâ¯<â¯585.679⯵g/mL), and precision (Intra-dayâ¯<â¯2.317%RSD and Inter-dayâ¯<â¯4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.
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Anticorpos Monoclonais/análise , Imunoglobulina G/análise , Polissacarídeos/análise , Adalimumab/análise , Bevacizumab/análise , Cromatografia Líquida de Alta Pressão , Humanos , Infliximab/análise , Espectrometria de Massas , Rituximab/análise , Trastuzumab/análiseRESUMO
BACKGROUND: Although BRCA1 or BRCA2 (BRCA1/2) genetic testing plays an important role in determining treatment modalities in patients with hereditary breast and ovarian cancer, sequence variants with unknown clinical significance or variant of uncertain significance (VUS) have limited use in medical decision-making. With vast quantities of gene-related data being updated, the clinical significance of VUS may change over time. We reinterpreted the sequence variant previously reported as BRCA1/2 VUS results in patients with breast or ovarian cancer and assessed whether the clinical significance of VUS was changed. METHODS: We retrospectively reviewed medical records of 423 breast or ovarian cancer patients who underwent BRCA1/2 genetic testing from 2010 to 2017. The VUSs in BRCA1/2 were reanalyzed using the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology standards and guidelines (ACMG/AMP 2015 guidelines) and the VUS was reclassified into five categories: "pathogenic", "likely pathogenic", "VUS", "likely benign", and "benign". RESULTS: A total of 75 patients (48 sequence types of VUS) were identified as carrying either one or more VUS in BRCA1/2. Among the 75 patients, two patients (2.7%) were reclassified as "likely pathogenic", 30 patients (40.0%) were reclassified as either "benign" or "likely benign", and the remaining 43 patients (57.3%) were still classified as VUS category. CONCLUSIONS: Since the clinical significance of VUS in BRCA1/2 may vary from time to time, reinterpretation of the VUS results could contribute to clinical decision-making.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Humanos , Guias de Prática Clínica como AssuntoRESUMO
Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123-421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5-80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17-1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.
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We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.