50 GHz Four-Port Coupling-Reduced Probe Card Utilizing Pogo Pins Housed in Custom Metallic Socket.

A design for a pogo-pin probe card featuring a metallic socket is proposed to eliminate signal leakage and coupling loss in a multi-port environment. The proposed metallic pogo-pin socket includes a metal wall structure between adjacent pogo pins, ensuring complete isolation. This metal wall offers an advantage in removing coupling issues between pogo pins that can occur with typical dielectric pogo-pin sockets. The designed probe card is fabricated as a prototype and verified for its performance. Measurement results using a test through line show that coupled power is minimized, providing a low-loss transmission performance of -2.14 dB to an RF chip at 50 GHz, all within a compact size. Although the dielectric spacer used to secure the pogo pins allows for some leakage, it can maintain a low coupling performance of under -15 dB in the millimeter-wave band. The prototype probe card can deliver an RF signal to a 5G circuit with a low loss of -0.7 dB at 28 GHz and -1.9 dB at 39 GHz frequency. The designed probe card is capable of transmitting multiple RF signals to the RF system without signal distortion in a multi-port environment.

A Machine Learning Algorithm Facilitates Prognosis Prediction and Treatment Selection for Barcelona Clinic Liver Cancer Stage C Hepatocellular Carcinoma.

PURPOSE: Given its heterogeneity and diverse clinical outcomes, precise subclassification of Barcelona Clinic Liver Cancer stage C (BCLC-C) hepatocellular carcinoma (HCC) is required for appropriately determining patient prognosis and selecting treatment. EXPERIMENTAL DESIGN: We recruited 2,626 patients with BCLC-C HCC from multiple centers, comprising training/test (n = 1,693) and validation cohorts (n = 933). The XGBoost model was chosen for maximum performance among the machine learning (ML) models. Patients were categorized into low-, intermediate-, high-, and very high-risk subgroups based on the estimated prognosis, and this subclassification was named the CLAssification via Machine learning of BCLC-C (CLAM-C). RESULTS: The areas under the receiver operating characteristic curve of the CLAM-C for predicting the 6-, 12-, and 24-month survival of patients with BCLC-C were 0.800, 0.831, and 0.715, respectively-significantly higher than those of the conventional models, which were consistent in the validation cohort. The four subgroups had significantly different median overall survivals, and this difference was maintained among various patient subgroups and treatment modalities. Immune-checkpoint inhibitors and transarterial therapies were associated with significantly better survival than tyrosine kinase inhibitors (TKI) in the low- and intermediate-risk subgroups. In cases with first-line systemic therapy, the CLAM-C identified atezolizumab-bevacizumab as the best therapy, particularly in the high-risk group. In cases with later-line systemic therapy, nivolumab had better survival than TKIs in the low-to-intermediate-risk subgroup, whereas TKIs had better survival in the high- to very high-risk subgroup. CONCLUSIONS: ML modeling effectively subclassified patients with BCLC-C HCC, potentially aiding treatment allocation. Our study underscores the potential utilization of ML modeling in terms of prognostication and treatment allocation in patients with BCLC-C HCC.

Esculetin induces cell cycle arrest and apoptosis in human colon cancer LoVo cells.

We investigated the anti-cancer effects of ESC in human colon cancer LoVo cells. Cell counting assay results showed that ESC inhibited the proliferation of LoVo cells. Cell cycle arrest results showed that cell cycle was arrested during the G0/G1 phase in the ESC-treated LoVo cells. Western blot results showed that the cell cycle inhibitory proteins p53, p27, and p21 were increased, and cyclin D1, the cell cycle progressive protein, was decreased. Sp1 is a transcription factor regulating cell proliferation, was decreased in the ESC-treated LoVo cells. Annexin V/propidium iodide staining results showed that ESC induces apoptosis in LoVo cells. Western blot results showed that Bax, cleaved caspase -3, -7, -9, and poly(ADP-ribose) polymerase, which are proapoptotic proteins, were increased and the antiapoptotic protein Bcl-2 was decreased. Taken together, ESC induced apoptosis and has an anti-cancer effect in LoVo cells.

Comparison of genetic diversity between wild-caught broodstock and hatchery-produced offspring populations of the vulnerable Korean kelp grouper (Epinephelus bruneus) by microsatellites.

The kelp grouper Epinephelus bruneus (Perciformes: Haemulidae), is one of the most economically important fishery resources in Korea. This fish is regarded as a target for prospective aquaculture diversification; therefore, maintenance of stock quality is important. To investigate the effects of current artificial reproduction in a hatchery facility, genetic variation in wild-caught broodstock and hatchery-produced offspring of kelp grouper was analyzed using eight polymorphic nuclear microsatellite DNA loci; 77 alleles were identified. Allelic variability ranged from 2 to 22 in the broodstock and from 1 to 10 in the offspring. The average observed and expected heterozygosities were 0.620 and 0.623 in the broodstock and 0.600 and 0.513 in the offspring, respectively. The possibility of a recent genetic bottleneck was suggested in both populations of E. bruneus. The minor, but significant, genetic differentiation (FST = 0.047, P < 0.05) observed was mainly due to statistically significant reductions in the number of alleles in the offspring compared with the broodstock, suggesting that these genetic changes could be related to genetic drift. Our results demonstrate the usefulness of microsatellite markers to monitor genetic variation and raise concerns about potential harmful genetic effects of inappropriate hatchery procedures. Therefore, genetic variation between broodstock and offspring in a hatchery should be monitored in both breeding and release programs as a routine hatchery operation, and inbreeding should ideally be controlled to improve kelp grouper hatchery management. Our data provide a useful genetic basis for future planning of sustainable culture and management of E. bruneus in fisheries.

Genetic differences between the wild and hatchery-produced populations of Korean short barbeled grunter (Hapalogenys nitens) determined with microsatellite markers.

Short barbeled grunter, Hapalogenys nitens, is an economically important fishery resource. In Korea, this fish is in the early stage of domestication, and it has been regarded as the candidate marine fish species for prospective aquaculture diversification. This study presents a preliminary investigation of the future viability of sustainable fry production from short barbeled grunter. We used 12 polymorphic nuclear microsatellite DNA loci to analyze the possible genetic variability between the wild and hatchery-produced populations of short barbeled grunter from Korea and identified 91 alleles. Compared to the wild population, significant genetic changes including reduced genetic diversity (average allele number: 7.42 vs 3.75; average expected heterozygosity: 0.713 vs 0.598, Wilcoxon signed-rank test; P < 0.05) and differentiation [overall fixation index (FST) = 0.088, P < 0.01] occurred in the hatchery-produced population, as indicated by the observation of allele richness, unique allele, heterozygosity, FST, and results of molecular analysis of variance. These findings indicate that genetic drift may have promoted the differentiation between these 2 populations, which may have negative effects on sustainable fry production. Therefore, genetic variations of the wild and hatchery-produced populations should be monitored and subjected to control inbreeding through a commercial breeding program. The information presented by this paper would provide a useful genetic basis for future sustainable culturing planning and management of H. nitens.

Genetic characterization of hatchery populations of Korean spotted sea bass (Lateolabrax maculatus) using multiplex polymerase chain reaction assays.

The spotted sea bass, Lateolabrax maculatus, is an important commercial and recreational fishery resource in Korea. Aquacultural production of this species has increased because of recent resource declines, growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. In this study, the genetic diversity and structure of three cultured populations in Korea were assessed using multiplex assays with 12 highly polymorphic microsatellite loci; 144 alleles were identified. The number of alleles per locus ranged from 6 to 28, with an average of 13.1. The mean observed and expected heterozygosities were 0.724 and 0.753, respectively. Low levels of inbreeding were detected according to the inbreeding coefficient (mean FIS = 0.003-0.073). All hatchery populations were significantly differentiated from each other (overall fixation index (FST) = 0.027, P < 0.01), and no population formed a separate cluster. Pairwise multilocus FST tests, estimates of genetic distance, mantel test, and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. For optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the spotted sea bass stocks that are being released every year. This genetic information will be useful for the management of both L. maculatus fisheries and the aquaculture industry.

Genetic relationships of Pacific abalone (Haliotidae) species determined using universal rice primer-polymerase chain reaction fingerprinting.

Random amplified polymorphic DNA (RAPD) with universal rice primers (URP) was used to identify species and to determine phylogenetic relationships for the 6 economically important Korean Pacific abalone species: Haliotis discus hannai, H. discus discus, H. madaka, H. gigantea, H. diversicolor supertexta, and H. diversicolor diversicolor, whose morphological differentiation is difficult. Of the 12 URPs used in this study, 7 were effective in producing reproducible RAPD markers for these 6 species. Amplifications with the 7 URP primers yielded 129 reproducible amplified fragments ranging between 100 and 6000 bp in length. The dendrogram generated by the unweighted pair-group method using arithmetic averages showed that the 6 species were divided into 4 groups at 0.44 similarity level, indicating that they were genetically distant from each other and had little internal phylogenetic resolution. One group included H. discus hannai, H. discus discus, H. madaka, and H. gigantea, which were divided into 2 groups at 0.52 similarity level: one group of H. discus hannai, H. discus discus, and H. madaka, and the other of H. gigantea. H. diversicolor supertexta and H. diversicolor diversicolor belonged to the other group. Furthermore, the reproducible pattern of amplified DNA bands by URP primers indicated the possibility of using these as molecular markers for the discrimination of the 6 Pacific abalone species. These results suggest that the URP-PCR approach will be a useful tool for obtaining accurate taxonomic identification and genetic relationship of Korean Pacific abalones, which is one of the first prerequisites in effective conservation programs.

Comparative genetic variability between broodstock and offspring populations of Korean starry flounder used for stock enhancement in a hatchery by using microsatellite DNA analyses.

Korean starry flounder, Platichthys stellatus (Pleuronectidae), is one of the most economically important fishery resources in Korea. We investigated the effect of current artificial reproduction in a hatchery facility, genetic divergence between the broodstock and their offspring populations of starry flounder in a hatchery strain to be stocked into natural sea areas was accessed using 9 polymorphic nuclear microsatellite DNA loci. High levels of polymorphism were observed between the 2 populations. A total of 96 alleles were detected at the loci, with some alleles being unique in the broodstock. Allelic variability ranged from 8 to 17 in the broodstock and from 7 to 12 in the offspring population. Average observed and expected heterozygosities were estimated at 0.565 and 0.741 in the broodstock samples and 0.629 and 0.698 in the offspring population, respectively. Although no statistically significant reductions were found in heterozygosity or allelic diversity in the offspring population, a considerable loss of rare alleles was observed in the offspring population compared with that in the broodstock. Significant genetic difference was detected between the broodstock and offspring populations (FST = 0.021, P < 0.05). These results suggest that more intensive breeding practices for stock enhancement might have resulted in a further decrease of genetic diversity. Thus, genetic variations of broodstock and progeny should ideally be monitored in both breeding and release programs as a routine hatchery operation in order to improve the starry flounder hatchery management. This information might be useful for fishery management and aquaculture industry of P. stellatus.

Comparative genetic diversity of wild and hatchery-produced populations of tongue sole (Cynoglossus semilaevis) using multiplex PCR assays with polymorphic microsatellite markers.

The tongue sole, Cynoglossus semilaevis (Cynoglossidae), is one of the most economically important fishery resources in Korea. This study presents a preliminary investigation of the future viability of the complete aquaculture of tongue sole in Korea. Specifically, possible differences in genetic variability between wild populations of tongue sole from Korea and hatchery-produced populations of tongue sole from China were assessed using multiplex assays with 12 polymorphic nuclear microsatellite DNA loci. High levels of polymorphism were observed between the 2 populations. A total of 135 different alleles were found, varying from 5-15 alleles per locus, with some alleles being unique. These findings indicate a high level of genetic variability in both the wild and hatchery-produced populations. Although a considerable loss of rare alleles was observed in hatchery samples, there were no statistically significant reductions of heterozygosity or allelic diversity in the hatchery population compared to the wild population. Moreover, the inbreeding coefficient was very low (FIS = -0.010-0.052) for both populations. However, significant genetic heterogeneity was found between the 2 populations. These findings indicate that genetic drift has likely promoted differentiation between these 2 populations, and might have negative effects on the reproductive capacity of the stock, because genetic factors are important in the production of high quality seed for complete aquaculture. Therefore, aquaculture management should incorporate basic genetic principles into existing molecular monitoring protocols. The information compiled by this study is anticipated to provide a useful genetic basis for future complete culturing plans and management of C. semilaevis in fisheries.

Mitochondrial and microsatellite DNA analyses showed comparative genetic diversity between parent and offspring populations of Korean black rockfish in a hatchery facility.

The black rockfish, Sebastes inermis (Sebastidae), is an important commercial fishery resource in Korea. As a preliminary investigation into the effect of artificial reproduction in a hatchery facility, the genetic divergence between parent and offspring populations of black rockfish was accessed using 10 polymorphic nuclear microsatellite DNA loci and a mitochondrial (mt) control gene. All loci that were screened showed marked polymorphisms. mtDNA control region sequences were also highly variable. Of approximately 350 base pairs (bp) sequenced, 52 variable sites, comprising 56 base substitutions, were found among 233 individuals. Offspring populations showed less genetic variability than the parent population in terms of numbers of microsatellite alleles and mtDNA haplotypes, as well as mtDNA haplotype diversity. Statistical analysis of the fixation index (ΦST and F(ST)) and analysis of molecular variance using both DNA markers showed significant genetic differences between the parent and offspring populations. These results suggest that random genetic drift and/or inbreeding events, as well as artificial selection and founder effects, occurred when the offspring strain was reproduced in a hatchery facility despite thousands of males and females from different hatcheries being maintained for artificial reproduction. Therefore, it is necessary to improve current hatchery programs by monitoring genetic variation in both the broodstock and progeny and controlling inbreeding within stocks in commercial breeding facilities to maintain the production of high-quality black rockfish. This information will be useful for determining suitable guidelines for establishing and maintaining cultured stocks and the aquaculture industry of S. inermis.

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers.

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.

A polymorphic mutation, c.-3279T>G, in the UGT1A1 promoter is a risk factor for neonatal jaundice in the Malay population.

The uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) gene encodes the enzyme responsible for bilirubin glucuronidation. To evaluate the contribution of UGT1A1 promoter mutations to neonatal jaundice, we determined the genotypes of c.-3279T>G, c.-3156G>A, and A(TA)7TAA in Malay infants with neonatal jaundice (patients) and in infants without neonatal jaundice (controls). In our population study, only c.-3279T>G was associated with neonatal jaundice. The genotype distributions between both groups were significantly different (p = 0.003): the frequency of homozygosity for c.-3279G was much higher in patients than those in controls. Allele frequency of c.-3279G was significantly higher in patients than those in controls (p = 0.006). We then investigated changes in transcriptional activity because of c.-3279T>G. Luciferase reporter assay in HepG2 cells demonstrated that transcriptional activity of the c.-3279G allele was significantly lower than that of the c.-3279T allele in both the absence and presence of bilirubin. Luciferase reporter assay in COS-7 cells elucidated that c.-3279T>G modified the synergistic effects of the nuclear factors associated with transcriptional machinery. In conclusion, the c.-3279T>G mutation in the UGT1A1 promoter is a genetic risk factor for neonatal jaundice.

Germ-line mutation of KCNQ2, p.R213W, in a Japanese family with benign familial neonatal convulsion.

BACKGROUND: Benign familial neonatal convulsion (BFNC) is an autosomal-dominantly inherited epilepsy of neonates. The KCNQ2 and KCNQ3 genes have been cloned as the responsible genes for BFNC. Detection of mutations in these genes is helpful for confirmation of BFNC or differential diagnosis of convulsive disorders in the neonatal period. METHODS: A Japanese family with BFNC was investigated. Two siblings were clinically diagnosed as having BFNC. KCNQ2 and KCNQ3 were screened for mutations using a combination of polymerase chain reaction and denaturing high-performance liquid chromatography. Nucleotide substitutions were confirmed by direct sequencing. RESULTS: In the affected siblings a C-to-T heterozygous substitution was detected at nucleotide 683 (c.683C>T) in KCNQ2, leading to substitution of arginine with tryptophan at amino acid position 213 (p.R213W) in the S4 voltage-sensing domain of the KCNQ2 protein. The detected mutation may disrupt this highly conserved region among potassium channel proteins. The c.683C>T substitution in KCNQ2 was not present in the parents. KCNQ3 was also analyzed and a single nucleotide polymorphism, c.1241A>G (National Center for Biotechnology Information (NCBI), SNP ID: rs2303995), was detected in the index family. CONCLUSIONS: Two siblings with BFNC had a novel heterozygous missense mutation, p.R213W, in KCNQ2. This mutation may affect potassium gating, leading to neuronal excitability or convulsions in the patients. Furthermore, neither of the parents had the p.R213W mutation, indicating that it was a germ-line mutation. The possibility of recurrence of such a germ-line mutation in the next siblings should be explained during genetic counseling.

Identification of defensin alpha6 as a potential biomarker in colon adenocarcinoma.

There is substantial interest in the identification of circulating human tumor-derived proteins in serum for the purposes of early cancer diagnosis. We have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 283 tumors and normal tissues revealed that defensin alpha6 was maximally expressed in colon cancer. These findings were corroborated by reverse transcription-PCR, in which the colon cancer cell lines LoVo, Caco2, HCT-15, SW480, and SW620 showed significantly higher levels of defensin alpha6 expression than did non-colon cancer cell lines. Moreover, our data were concordant with data obtained from the NCI, National Institutes of Health Cancer Genome Anatomy Project. To evaluate defensin alpha6 as a potential biomarker of colon cancer, a preliminary "training" set of serum from 91 healthy donors and 109 colon cancer patients was analyzed by enzyme-linked immunosorbent assay. The data pattern was confirmed by an independent set of 67 masked serum samples: 18 from healthy donors and 49 from colon cancer patients. This result yielded a sensitivity of 69.4% (95% CI 54.6-81.8), specificity of 83.3% (58.6-96.4), and positive predictive value of 91.9% (78.1-98.3). These findings justify a prospective assessment of serum defensin alpha6 protein as a screening tool for colon cancer.

Screening for G71R mutation of the UGT1A1 gene in the Javanese-Indonesian and Malay-Malaysian populations.

BACKGROUND: There are significant differences in the prevalence and severity of neonatal jaundice among various populations. Recently, it has been reported that a mutation of the UGT1A1 gene, glycine to arginine at codon 71 (G71R), is related to the development of neonatal jaundice in East Asian populations. However, whether the G71R mutation contributes to the high incidence of neonatal jaundice in different Asian populations remains unknown. The authors screened for this mutation in the Javanese-Indonesian and Malay-Malaysian populations. METHODS: One hundred and thirty-six subjects were enrolled in this study: 68 Javanese-Indonesian adults and 68 Malay-Malaysian newborns (32 with jaundice and 36 without jaundice). Denaturing high-performance liquid chromatography (DHPLC) was used to screen for the G71R mutation, and the results were confirmed by nucleotide sequencing analysis. RESULTS: With DHPLC, the authors easily and clearly detected seven subjects carrying the G71R mutation: two Javanese-Indonesian adults and five Malay-Malaysian newborns. In the 68 Javanese-Indonesian adults, the genotype distribution for G71R mutation was 66 G/G, two G/R and no R/R genotypes, and the mutated allele frequency was 0.015. In the 68 Malay-Malaysian newborns, genotype distribution for the mutation was 63 G/G, five G/R and no R/R genotypes, and the mutated allele frequency was 0.037. The genotype distributions did not differ significantly between the newborns with jaundice and those without jaundice. CONCLUSION: The G71R mutation is present, but very rare, in Javanese-Indonesians and Malay-Malaysians. Thus, G71R mutation may not contribute to the high incidence of the neonatal jaundice in South-east Asian populations. DHPLC analysis is a very useful method for detecting the G71R mutation.

Molecular profiling of the immune response in colon cancer using protein microarrays: occurrence of autoantibodies to ubiquitin C-terminal hydrolase L3.

We implemented a protein microarray approach to identify proteins that induce a humoral response in colon cancer. Solubilized proteins from the LoVo colon adenocarcinoma cell line were separated into 1760 fractions, arrayed onto nitrocellulose-coated slides, and hybridized with individual sera from 15 newly diagnosed patients with colon cancer, 15 with lung cancer, and 15 healthy subjects. 39/1760 fractions showed enhanced reactivity with sera from patients with colon cancer (p < 0.01) relative to healthy controls. A distinct pattern of reactivity was observed with sera from colon cancer relative to lung cancer. One fraction that exhibited reactivity with 9/15 colon cancer sera was subjected to mass spectrometry leading to the identification of ubiquitin C-terminal hydrolase isozyme 3 (UCH-L3) as a constituent. To validate the occurrence of autoantibodies to UCH-L3, independent analysis was done by means of Western blots. UCH-L3 antibodies were detected in 19/43 sera from patients with colon cancer, and in 0/54 sera from subjects with lung cancer (24), colon adenoma (15) or otherwise healthy (15). Our findings indicate the occurrence of an immune response to a broad set of antigens in colon cancer and the feasibility of identifying the antigenic targets using a combination of protein microarrays and mass spectrometry.