Comparative analysis of asian carps parvalbumin reveals the divergence pattern of major fish allergen.

BACKGROUND: Asian carps, a popular freshwater fish globally, are valued for their flavor and serve as a crucial protein source, especially for infants. However, grass carp parvalbumin is highly allergenic, surpassing the allergenicity of fish like salmon and cod. The allergenic potential of parvalbumin in other Asian carps remains unknown, underscoring the need for allergen identification to improve the precision of fish allergy diagnosis and treatment. OBJECTIVE: To identify all parvalbumin homologs in Asian carps and investigate the role of gene divergence in allergenic homolog formation. METHODS: Three annotated genomes of Asian carp, including grass carp, black carp and bighead carp, were constructed using a hybrid assembly approach. Through sequence homology at the genomic level, all the homologs of major fish allergens were identified. Bioinformatics tools were then employed to reveal the gene structures, expression levels, and protein conformations of parvalbumin. RESULTS: Grass carp genome analysis showed nine parvalbumin homologs, with Cid_PV2 most similar to Cten i 1. Bighead and black carp genomes had ten homologs, including potentially allergenic Mpi_PV7 and Hno_PV7. Tissue-specific expression patterns revealed alternative usage of parvalbumin homologs. Gene duplication events expanded parvalbumin copies in bony fish, with two gene clusters identified in Asian carp genomes. CONCLUSION: All the homologs of Asian carps' parvalbumin were accurately identified and gene divergence contributed to the formation of allergenic homologs. Together with a comprehensive gene sequence profile of carps' parvalbumin, those could be applied to achieve a more precise clinical diagnostic test.

Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations.

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing ∼1-2% of all allergic diseases globally; however, their evolutionary origin and diverse lifestyles including reversible parasitism have not been illustrated at the genomic level, which hampers allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the diversification of astigmatic mites. In monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, and then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases. Gene diversification after tandem duplications provides many genetic resources for adaptation to sensing environmental signals, digestion, and detoxification in rapidly changing household environments. Many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UDP glucuronosyltransferases and several important fungal cell wall lytic enzymes, which enable detoxification and digestive functions and provide perfect drug targets for pest control. This comparative study sheds light on the divergent evolution and quick adaptation to human household environments of astigmatic mites and provides insights into the genetic adaptations and even control of human household pests.

Performance evaluation of the TheraTyper-GJB2 assay for detection of GJB2 gene mutations.

Mutations in the GJB2 gene are the most common cause of congenital hearing loss in many populations. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based minisequencing assay, TheraTyper-GJB2, for the detection of c.35delG, c.167delT, and c.235delC mutations in the GJB2 gene. This assay was evaluated for analytic performance, including detection limit, interference, cross-reactivity, and precision, using GJB2 reference standards prepared by site-directed mutagenesis of a molecular clone. The detection limit was as low as 0.040 ng of human genomic DNA per PCR. No cross-reactivity with bacteria and viruses and no negative effects of increased levels of various potential interfering substances was observed. A precision test involving repetitive analysis of 2400 replicates showed 99.9% agreement (2397 of 2,400) with 99.8% (95% CI, 99.7%-99.8%) sensitivity and 100.0% (95% CI, 99.3%-100.0%) specificity. TheraTyper-GJB2 and direct sequencing assays showed 100% concordance for detecting mutations in 1,113 clinical specimens. Overall, TheraTyper-GJB2 showed comparable performance for detecting GJB2 mutations in reference and clinical samples with that of direct sequencing, and easier interpretation of results for analysis of a large quantity of samples. Therefore, the TheraTyper-GJB2 assay will be practically useful for the diagnosis of GJB2 mutations associated with congenital hearing loss with faster, cheaper, more reliable, and high-throughput capability.

Comparison of digene hybrid capture 2, GeneMatrix PapilloScreen, and a PCR sequencing assay in detecting high-risk and probable high-risk oncogenic HPV genotypes in specimens from Korean women.

Most cervical cancers are caused by 15 high-risk (HR) and three probable high-risk (pHR) oncogenic types of human papillomavirus (HPV). However, current commercial HR HPV screening test products do not include pHR HPV genotypes. Recently, PapilloScreen has been developed to detect the 15 HR and three pHR HPV types. In this study, we evaluated the concordance levels and clinical performance of Hybrid Capture 2 (HC2), PapilloScreen, and a PCR sequencing assay in detecting HR and pHR HPV. The PapilloScreen (96.8 %) and PCR sequencing assay (96.8 %) demonstrated higher sensitivity than HC2 (80.7 %) for detecting HR and pHR HPV. The three assays showed similar specificities and positive or negative predictive values. The concordance levels were 86.5 % (κ = 0.68) and 86.5 % (κ = 0.67) between HC2 and PapilloScreen and between HC2 and PCR sequencing, respectively. A near-perfect concordance was observed between PapilloScreen and PCR sequencing (97.8 %, κ = 0.95). Overall, the agreement between the three assays suggests that the results obtained by the HC2 assay are more often discordant (12.6 %) than the PCR-based tests. In conclusion, PapilloScreen is highly sensitive for detecting high-grade CIN or cervical cancer. The PapilloScreen assay should be considered an accurate and sensitive method for detecting HR and pHR HPV infections and an epidemiological tool for prevalence studies as well as early diagnosis and intervention in HR and pHR HPV infections.

Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.

BACKGROUND: The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. OBJECTIVES: We evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. STUDY DESIGN: The RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. RESULTS: Overall, 216 specimens (93.1%, κ = 0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9-3173.3) was higher than the LA assay (67.0, 95% CI: 8.2-2887.0). CONCLUSIONS: The RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer.

Performance evaluation of the HepB Typer-Entecavir kit for detection of entecavir resistance mutations in chronic hepatitis B.

BACKGROUND/AIMS: Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. METHODS: The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. RESULTS: Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. CONCLUSIONS: The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B.

Mutational complex genotype of the hepatitis B virus X /precore regions as a novel predictive marker for hepatocellular carcinoma.

This study explored the combined effect of number and pattern of mutations in the X/precore regions of the hepatitis B virus (HBV) genome, mutational complex genotype (MCG), on hepatocellular carcinoma (HCC) development. Sequence variations were determined by direct sequencing and multiplex restriction fragment mass polymorphism analysis in 150 age-, sex- and hepatitis B e antigen (HBeAg) status-matched patients with and without HCC. In addition, a longitudinal study and an external validation of MCG were conducted. All were HBV subgenotype C2. Eight high-frequency mutations (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) were significantly associated with HCC. Whereas C1653T, T1753V, G1764A and A1846T were independent mutational factors for HCC, the significance of these individual mutations was negligible when analyzed with all clinico-virological variables. The total number of mutations was the only independent viral factor for HCC, irrespective of HBeAg status. There was a significant dose-risk relationship between the number of mutations and HCC, in which high risks for HCC were associated with mutation numbers ≥ 6. Pattern analysis of the mutations revealed disparity in distribution among the top seven high-risk mutation combination patterns, which accounted for 40 and 2.7% of HCC and non-HCC cases, respectively. The predictive accuracy of the high-risk mutations for HCC was similar to that of α-fetoprotein. Longitudinal and external validation studies also supported the association of mutation number with HCC development. MCG in the HBV X/precore regions is a risk indicator for HCC, and might serve as a new guide to the HCC screening scheme for chronic HBV carriers.

Comparison of multiplex restriction fragment mass polymorphism and sequencing analyses for detecting entecavir resistance in chronic hepatitis B.

BACKGROUND: Drug resistance is a major limitation to the long-term efficacy of controlling chronic hepatitis B (CHB). There is a growing need to analyse multiple mutations associated with drug resistance because sequential or combinational use of antivirals is increasingly being used in treatment. In this study, we introduced a multiplex restriction fragment mass polymorphism (RFMP) assay for detecting mutations conferring entecavir and lamivudine resistance, and compared its performance with direct or clonal sequencing assays. METHODS: Multiplex PCR was performed with mixed primers designed to interrogate rt184, rt202, rt204 and rt250. The PCR products were digested with restriction enzymes and the resulting fragments were analysed by mass spectrometry. A total of 251 serum samples, taken serially from 45 patients who received entecavir treatment after confirmed diagnosis of lamivudine resistance and inadequate adefovir dipivoxil response, were analysed by the multiplex RFMP assay. RESULTS: The multiplex RFMP assay correctly identified known viral sequences with sufficient analytical sensitivity to detect as few as 100 IU/ml of HBV and with superior ability to determine haplotypes composed of neighbouring variations. Complex mutational patterns and relative abundances determined by multiplex RFMP assay were in good concordance with results obtained by direct or clonal sequencing analyses. Defined mixtures were successfully and consistently identified at 2% relative concentration of mutant versus wild-type virus by the assay. CONCLUSIONS: The multiplex RFMP assay is an accurate and sensitive means to detect entecavir and lamivudine resistance mutations, simultaneously. The method is expected to enable early and efficient diagnosis of multiple drug resistance mutations for optimal management of CHB.

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay.

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI-TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.

Analytical and clinical performances of a restriction fragment mass polymorphism assay for detection and genotyping of a wide spectrum of human papillomaviruses.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay was adapted to human papillomavirus (HPV) genotyping. The analytical sensitivity and the clinical utility were evaluated by testing defined HPV genome equivalents and a total of 426 specimens composed of normal cytology, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and invasive squamous cell carcinoma. The RFMP assay was able to detect 38.4-114.6 genomic equivalents of a wide variety of HPV types. The RFMP assay detected 34 different HPV genotypes in cervical samples of which 8% were found to be multiple-type infections. The high-risk HPV positivity rate according to the histological diagnosis was 7.9% (8/101), 31.7% (38/120), 50% (55/110), 86% (37/43), 96.2% (50/52) in normal, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion, high grade squamous intraepithelial lesion and squamous cell carcinoma subgroups, respectively. Diagnostic sensitivities/specificities for the cervical lesions of squamous cell carcinoma and high grade squamous intraepithelial lesion or worse histology were found to be 96.2%/92.1% and 91.6%/92.1%, respectively. The sensitivity, accuracy, wide range of genotype identification and high-throughput capacity with cost-effectiveness of the test consumables make the RFMP assay suitable for mass screening and monitoring of HPV-associated cervical cancer.

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry.

We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.

Inhibition of APP intracellular domain (AICD) transcriptional activity via covalent conjugation with Nedd8.

The processing of amyloid precursor protein (APP) by gamma-secretase generates the APP intracellular domain (AICD), which functions as a transcriptional factor for target gene activation following localization into the nucleus. In this study, we demonstrate that AICD could be modified via covalent conjugation with Nedd8, a ubiquitin-like protein. Domain analysis and site-directed substitution of neddylation sites showed that multiple lysine residues of the APP C-terminal C99 fragment including AICD were acceptor sequences for Nedd8 conjugation. AICD-mediated transcriptional activation was inhibited by Nedd8 conjugation. Furthermore, the transcriptional activity of the neddylation-defective AICD mutant was not altered by Nedd8 expression. Nedd8 conjugation of AICD inhibited its interaction with Fe65, and consequently resulted in the impairment of AICD-Fe65-Tip60 complex formation for the transcriptional activation of the target gene. These results illustrate the regulatory mechanisms by which AICD transcriptional activity might be regulated via covalent conjugation with Nedd8.

Polymorphisms of XRCC1 gene, alcohol consumption and colorectal cancer.

To evaluate contribution of polymorphisms of the XRCC1 gene to the risk of colorectal cancer, we conducted a case-control study of 209 colorectal cancer cases and 209 age- and gender-matched controls in the Korean population. We tested the hypothesis by constructing allele combinations with known SNP. Allelic variants of the XRCC1 gene at codons 194, 280 and 399 were analyzed in lymphocyte DNA by PCR-RFLP. We observed an increased risk of colorectal cancer associated with the 399Gln allele. The odds ratio (OR) was 1.61 (95% confidence interval [CI] 1.09-2.39) for the 399Gln allele. When combined allele-specific OR were calculated after estimating frequencies, 3 common allele combinations were found to be associated with an increased risk of colorectal cancer. The OR for the 194Trp-280Arg-399Arg was 1.48 (95% CI = 1.06-2.07) using 194Arg-280Arg-399Arg as the reference. The OR for the 194Arg-280His-399Arg and the 194Arg-280Arg-399Gln were 1.78 (95% CI = 1.09-2.89) and 1.78 (95% CI = 1.23-2.59), respectively. Analysis after controlling for smoking, exercise and dietary habits indicated that alcohol consumption (> or =80 g/week) is a significant risk factor of colorectal cancer (OR = 2.60, 95% CI = 1.46-4.62). An increased risk for colorectal cancer was identified in alcohol drinkers with the risky allele combinations. Our results suggest that polymorphisms in the XRCC1 genes may contribute to colorectal cancer susceptibility, and some evidence was obtained of a genetic modification for the relationship between alcohol intake and colorectal cancer.

Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis.

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK(-) cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK(-) osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK(-) cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.