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Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.
Assuntos
Neoplasias , Humanos , Imunofenotipagem , Anticorpos , República da Coreia , Citometria de Fluxo/métodosRESUMO
Due to environmental and ecological changes and suitable habitats, the occurrence of vector-borne diseases is increasing. We investigated the seroprevalence of four major vector-borne pathogens in human patients with febrile illness who were clinically suspected of having Scrub Typhus (ST) caused by Orientia tsutsugamushi. A total of 187 samples (182 patient whole blood and sera samples, including 5 follow-up) were collected. Antibodies to Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, and Bartonella henselae were tested by using indirect immunofluorescence assays. Molecular diagnoses were performed using real-time PCR. Of the 182 cases, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases as other febrile diseases (OFDs). The seroprevalence of A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae was 51.4% (19/37), 10.8% (4/37), 86.5% (32/37), and 10.8% (4/37) among the ST group, and 42.8% (62/145), 10.4% (19/145), 57.7% (105/145), and 15.9% (29/145) among the OFD group, respectively. There were no significant differences in the seroprevalence between the ST and the OFD groups. Considering the co-occurrence, 89.0% (162/182) had at least one antibody to tick-borne pathogens, 37.0% (60/162) were positive for two pathogens, 17.3% (28/162) for three pathogens, and 6.2% (10/162) for four pathogens. In real-time PCR, O. tsutsugamushi was positive in 16 cases [15 (40.5%) in ST group and 1 (2.2%) in OFD group], and the four other pathogens were negative in all cases except one confirmed as anaplasmosis. In evaluating the five follow-up samples, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Our data highlighted that acute febrile illness and manifestations suggestive of a vector-borne infection must be recognized and further considered for coinfections in clinical practice and the laboratory.
Assuntos
Anaplasma phagocytophilum , Anaplasmose , Ehrlichiose , Rickettsia , Tifo por Ácaros , Doenças Transmitidas por Carrapatos , Animais , Humanos , Tifo por Ácaros/diagnóstico , Tifo por Ácaros/epidemiologia , Estudos Soroepidemiológicos , Anaplasmose/diagnóstico , Anaplasmose/epidemiologia , Anaplasma phagocytophilum/genética , Ehrlichiose/diagnóstico , Ehrlichiose/epidemiologia , Doenças Transmitidas por Carrapatos/diagnóstico , Doenças Transmitidas por Carrapatos/epidemiologiaRESUMO
Background: Cancer recurrence remains a significant problem, and most postoperative recurrences of non-small cell lung cancer (NSCLC) develop within 5 years after resection. We present a rare case of ultra-late recurrence of NSCLC accompanying choroidal metastasis with KIF13A-RET fusion 14 years after the definitive surgery. Case description: A 48-year-old female patient who had never-smoked presented with decreased visual acuity. She had been treated with right upper lobe lobectomy followed by adjuvant chemotherapy 14 years prior. Fundus photographs revealed bilateral choroidal metastatic lesions. Positron emission tomography-computed tomography (PET-CT) scans showed extensive bone metastases and focal hypermetabolism in the left uterine cervix. An excision biopsy of the uterus showed primary lung adenocarcinoma with immunohistochemistry of TTF-1+. Plasma next-generation sequencing (NGS) identified the presence of KIF13A-RET fusion. After 6 months of selpercatinib therapy, PET-CT revealed a partial response for bone and uterine metastasis and stable disease for choroidal lesions. Conclusion: In this case report, we are reporting a rare case of ultra-late recurrence of NSCLC in a patient with choroidal metastasis. Furthermore, the diagnosis of NSCLC with RET fusion was based on liquid-based NGS rather than tissue-based biopsy. The patient showed a good response to selpercatinib, which supports the efficacy of selpercatinib as a treatment for RET-fusion-positive NSCLC with choroidal metastasis.
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Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2-3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4-8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25-2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.
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Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Análise de Sequência de RNARESUMO
BACKGROUND: In this study, we presented the national cancer statistics on the incidence of hematologic malignancies in the Republic of Korea (ROK) over a period of 20 years, from 1999 to 2018. METHODS: We obtained data on the incidence of hematologic malignancies using the Korean Statistical Information Service (KOSIS). For each hematologic malignancy, the number of cases, crude incidence rate, and age-standardized incidence rate were calculated, and the statistical trends were confirmed by Poisson regression and Joinpoint regression analysis. RESULTS: All the investigated hematologic malignancies showed a statistically significant increase in incidence over 20 years. The 20-year trend of the age-standardized incidence rate was as follows: non-Hodgkin lymphoma [average annual percent change (AAPC)=2.26%, P-trend <0.05], leukemia (AAPC=0.94%, P-trend <0.05), myeloid leukemia (AAPC=1.44%, P-trend <0.05), multiple myeloma (AAPC=3.05%, P-trend <0.05), myeloproliferative disorders (AAPC=9.87%, P-trend <0.05), myelodysplastic syndrome (AAPC=7.59%, P-trend <0.05), malignant immunoproliferative diseases (AAPC=11.82%, P-trend <0.05), lymphoid leukemia (AAPC=2.21%, P-trend <0.05), and Hodgkin lymphoma (AAPC=4.04%, P<0.05). CONCLUSION: It was confirmed that the incidence of hematologic malignancies has increased significantly in the ROK over the past 20 years. This study can be used as foundational data source for future studies. In addition, it can aid in the necessary actions of predicting future incidences and establishing future healthcare policies.
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Intelligent video surveillance systems detect pre-configured surveillance events through background modeling, foreground and object extraction, object tracking, and event detection. Shadow regions inside video frames sometimes appear as foreground objects, interfere with ensuing processes, and finally degrade the event detection performance of the systems. Conventional studies have mostly used intensity, color, texture, and geometric information to perform shadow detection in daytime video, but these methods lack the capability of removing shadows in nighttime video. In this paper, a novel shadow detection algorithm for nighttime video is proposed; this algorithm partitions each foreground object based on the object's vertical histogram and screens out shadow objects by validating their orientations heading toward regions of light sources. From the experimental results, it can be seen that the proposed algorithm shows more than 93.8% shadow removal and 89.9% object extraction rates for nighttime video sequences, and the algorithm outperforms conventional shadow removal algorithms designed for daytime videos.
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Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.
Assuntos
Bacillus subtilis/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases/química , Proteínas Repressoras/química , Transativadores/química , Alquil e Aril Transferases/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Hidrolases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Pirimidinas/metabolismo , Proteínas Repressoras/metabolismo , Sulfatos/metabolismo , Tiamina Trifosfato/química , Tiamina Trifosfato/metabolismo , Transativadores/metabolismoRESUMO
The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.
Assuntos
Aldose-Cetose Isomerases/genética , Lactococcus lactis/enzimologia , Proteínas Recombinantes/isolamento & purificação , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/química , Mutação , Proteínas Recombinantes/biossíntese , SolubilidadeRESUMO
The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.
Assuntos
Genes Bacterianos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Tiamina Pirofosfoquinase/genética , Tiamina/metabolismo , Bacillus subtilis/genética , Escherichia coli/genética , NAD/metabolismoRESUMO
Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.
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Bacillus subtilis/enzimologia , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Tiamina Pirofosfato/biossíntese , Pirimidinas/metabolismo , Especificidade por SubstratoRESUMO
While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved. Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5-phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein. This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring. The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme.
Assuntos
Tiamina/biossíntese , Bacillus subtilis/genética , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Cinética , Espectrometria de Massas por Ionização por Electrospray , Tiamina/química , Tiamina/genéticaRESUMO
The thiO gene of Bacillus subtilis encodes an FAD-dependent glycine oxidase. This enzyme is a homotetramer with a monomer molecular mass of 42 kDa. In this paper, we demonstrate that ThiO is required for the biosynthesis of the thiazole moiety of thiamin pyrophosphate and describe the structure of the enzyme with N-acetylglycine bound at the active site. The closest structural relatives of ThiO are sarcosine oxidase and d-amino acid oxidase. The ThiO structure, as well as the observation that N-cyclopropylglycine is a good substrate, supports a hydride transfer mechanism for the enzyme. A mechanistic proposal for the role of ThiO in thiazole biosynthesis is also described.
Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/fisiologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Tiamina/biossíntese , Aminoácido Oxirredutases/deficiência , Aminoácido Oxirredutases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Deutério/química , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Cinética , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Estereoisomerismo , Especificidade por Substrato , Tiazóis/química , Tiazóis/metabolismoRESUMO
The structural characterization of proteins expressed from the genome is a major problem in proteomics. The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF). MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins.