Uncovering sample heterogeneity in gaming and social withdrawal behaviors in adolescent and young adult gamers in Hong Kong.

OBJECTIVE: The current study aimed to examine the latent heterogeneity of gaming and social withdrawal behaviors in internet gamers and their associations with help-seeking behaviors. METHOD: The present study recruited 3430 young people (1874 adolescents and 1556 young adults) in Hong Kong in 2019. The participants completed the Internet Gaming Disorder (IGD) Scale, Hikikomori Questionnaire, and measures on gaming characteristics, depression, help-seeking, and suicidality. Factor mixture analysis was used to classify the participants into latent classes based on their latent factors of IGD and hikikomori in separate age groups. Latent class regressions examined the associations between help-seeking and suicidality. RESULTS: Both adolescents and young adults supported a 4-class, 2-factor model on gaming and social withdrawal behaviors. Over two-third of the sample were classified as healthy or low-risk gamers with low IGD factor means and low prevalence of hikikomori. Around one-fourth was moderate-risk gamers with elevated prevalence of hikikomori, higher IGD symptoms and psychological distress. A minority of the sample (3.8%-5.8%) belonged to high-risk gamers with the highest IGD symptoms and prevalence of hikikomori and heightened suicidal risks. Help-seeking in low-risk and moderate-risk gamers was positively associated with depressive symptoms and negatively associated with suicidal ideation. Perceived usefulness of help-seeking was significantly linked with lower likelihoods of suicidal ideation in the moderate-risk gamers and suicide attempt in the high-risk gamers. CONCLUSIONS: The present findings explicate the latent heterogeneity of gaming and social withdrawal behaviors and associated factors on help-seeking and suicidality among internet gamers in Hong Kong.

Effects of oxidant concentration and temperature on decolorization of azo dye: comparisons of UV/Fenton and UV/Fenton-like systems.

This study applies photo-Fenton and photo-Fenton-like systems to decolorize C.I. Reactive Red 2 (RR2). The oxidants were H(2)O(2) and Na(2)S(2)O(8); Fe(2+), Fe(3+), and Co(2+) were used to activate these two oxidants. The effects of oxidant concentration (0.3-2 mmol/L) and temperature (25-55 °C) on decolorization efficiency of the photo-Fenton and photo-Fenton-like systems were determined. The decolorization rate constants (k) of RR2 in the tested systems are consistent with pseudo-first-order kinetics. The rate constant increased as oxidant concentration and temperature increased. Activation energies of RR2 decolorization in the UV/H(2)O(2)/Fe(2+), UV/H(2)O(2)/Fe(3+), UV/Na(2)S(2)O(8)/Fe(2+) and UV/Na(2)S(2)O(8)/Fe(3+) systems were 32.20, 39.54, 35.54, and 51.75 kJ/mol, respectively.

Disease-Induced Anomalous Human Microsatellite DNA - Implication in Forensic DNA Typing.

Short tandem repeat (STR) markers adopted for forensic applications are generally stable and have been proved effective for the analysis of many archival pathology specimens and postmortem tissues. However, recent studies on STR typing of cancerous samples have shown misinterpreted profiles caused by two types of genetic alterations in tumor materials: microsatellite instability (MSI, contractions or expansions of a heterozygous allele) and loss of heterozygosity (LOH). Forensic STR typing of tumor material is unlikely; however, under rare circumstances, these may be the only samples available. This article reviews literature information in the following areas: unusual sample types encountered in casework; possible causes of STR alterations; genetic diseases associated with CODIS 13 markers; and forensic evaluation of commercial STR markers used in solid tumor tissues. Literature information suggested that STR-associated events observed in unusual cases may not be readily explained by known biological and disease processes. In actual practice, false profiles would only arise from complete LOH (rather than allelic imbalance) and MSI. Adoption of different analysis methods and various sets of genetic markers in profiling may lead to inconsistent results. When comparing profiling data reported by different laboratories, one should also note whether the same methodology was applied to the same set of genetic markers used for the evaluation of the disease tissues. To avoid misinterpretations, each tumor type should be evaluated individually, as various disease types may exhibit distinct behavior nature.

Extraction of human DNA for PCR from chewed residues of betel quid using a novel "PVP/CTAB" method.

Residues of chewed betel quid (BQ) are often found on crime scenes in Taiwan and possibly some of the Southeast Asian countries. Although these residues are important biological evidences relating to the suspects, the forensic analysis of BQ evidence has been hindered by failures in extraction of human DNA for PCR analysis. Therefore, it is a prerequisite for relevant forensic casework to establish a reliable method for extracting DNA from chewed BQ residues. Three conventional methods (salt/chloroform, 5% Chelex-100 resin, and QIAamp) were first tested for extraction of human DNA from 33 mock BQ samples, which had been stored for less than two months, and 50 four-year-old forensic BQ samples. PCR amplifications from the HLA-DQA1&PM and the STR loci were then used to test the quality of the extracted DNA. For the mock samples, three observations were made. First, PCR amplification of DNA extracted by using these conventional methods had low success rate. Second, the addition of extra Taq DNA polymerase could compensate the lost enzyme activities due to putative inhibitors and, thus, increase the yield. Third, using the Centricon-100 column to remove putative inhibitors substantially improved the efficiency of PCR. However, for the four-year-old forensic BQ samples, none of the attempts for PCR were successful. In order to solve the problem in PCR analysis of DNA from old BQ samples, we developed a DNA extraction method based on the use of polyvinyl pyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), which bind to two common classes of PCR inhibitors in plants, polyphenols, and polysaccharides, respectively. The result showed that this "PVP/CTAB" method is completely successful for the mock BQ samples, and 92% (46 out of 50) successful for the four-year-old forensic BQ samples. To our best knowledge, this is the first report of a reliable method for the extraction of human DNA for PCR from chewed BQ residues. This method should provide a useful means for forensic identification in countries where betel chewing is common.

Biomechanics of slips.

The biomechanics of slips are an important component in the prevention of fall-related injuries. The purpose of this paper is to review the available literature on the biomechanics of gait relevant to slips. This knowledge can be used to develop slip resistance testing methodologies and to determine critical differences in human behaviour between slips leading to recovery and those resulting in falls. Ground reaction forces at the shoe-floor interface have been extensively studied and are probably the most critical biomechanical factor in slips. The ratio of the shear to normal foot forces generated during gait, known as the required coefficient of friction (RCOF) during normal locomotion on dry surfaces or 'friction used/achievable' during slips, has been one biomechanical variable most closely associated with the measured frictional properties of the shoe/floor interface (usually the coefficient of friction or COF). Other biomechanical factors that also play an important role are the kinematics of the foot at heel contact and human responses to slipping perturbations, often evident in the moments generated at the lower extremity joints and postural adaptations. In addition, it must be realized that the biomechanics are dependent upon the capabilities of the postural control system, the mental set of the individual, and the perception of the environment, particularly, the danger of slipping. The focus of this paper is to review what is known regarding the kinematics and kinetics of walking on surfaces under a variety of environmental conditions. Finally, we discuss future biomechanical research needs to help to improve walkway-friction measurements and safety.

Human-centred approaches in slipperiness measurement.

A number of human-centred methodologies--subjective, objective, and combined--are used for slipperiness measurement. They comprise a variety of approaches from biomechanically-oriented experiments to psychophysical tests and subjective evaluations. The objective of this paper is to review some of the research done in the field, including such topics as awareness and perception of slipperiness, postural and balance control, rating scales for balance, adaptation to slippery conditions, measurement of unexpected movements, kinematics of slipping, and protective movements during falling. The role of human factors in slips and falls will be discussed. Strengths and weaknesses of human-centred approaches in relation to mechanical slip test methodologies are considered. Current friction-based criteria and thresholds for walking without slipping are reviewed for a number of work tasks. These include activities such as walking on a level or an inclined surface, running, stopping and jumping, as well as stair ascent and descent, manual exertion (pushing and pulling, load carrying, lifting) and particular concerns of the elderly and mobility disabled persons. Some future directions for slipperiness measurement and research in the field of slips and falls are outlined. Human-centred approaches for slipperiness measurement do have many applications. First, they are utilized to develop research hypotheses and models to predict workplace risks caused by slipping. Second, they are important alternatives to apparatus-based friction measurements and are used to validate such methodologies. Third, they are used as practical tools for evaluating and monitoring slip resistance properties of footwear, anti-skid devices and floor surfaces.

HLA-DQB1 genotyping with simple automated DNA sequencing and single-strand conformation polymorphism analysis.

BACKGROUND AND PURPOSE: Accurate human leukocyte antigen (HLA) typing is important for matching donors and recipients of bone marrow transplantation. Because HLA is highly polymorphic, HLA genotyping is also a valuable tool in forensic identification of humans. The primary objective of this study was to establish a simple, rapid, and economic HLA analysis system suitable for use in forensic applications. METHOD: We used the primer pair DB130 and CH29 to amplify the HLA class II DQB1 gene by polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were analyzed with an ABI Prism 377 automatic DNA sequencer, with the aid of its systematic analytical procedure. Some genotypes were confirmed by single-strand conformation polymorphism (SSCP) analysis. RESULTS: We identified 15 alleles and 37 genotypes from 86 Taiwanese subjects. The most frequent allele was 03011 (27.9%) and the most frequent genotype was 03011/03011 (15.1%). Statistical analysis showed that the allelic diversity was 0.862 and the power of discrimination was 0.948. CONCLUSIONS: The results of this study indicate that the combined use of automated sequencing with only one primer pair and SSCP provides a simple, rapid, and economic tool for analyzing the DQB1 gene. Compared with other sequencing methods that use a set of multiple primers, this method has two advantages. First, it is simpler and faster, because the HLAB1 genotype can be determined in a single PCR reaction. Second, the use of only one primer set obviates the need for preferential annealing of any one primer set.

Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function.

The Drosophila Homothorax (HTH) and Extradenticle (EXD) are two homeoproteins required in a number of developmental processes. EXD can function as a cofactor to Hox proteins. Its nuclear localization is dependent on HTH. In this study we present evidence of in vivo physical interaction between HTH and EXD, mediated primarily through an evolutionarily conserved MH domain in HTH. This interaction is essential for the mutual stabilization of both proteins, for EXD nuclear localization, and for the cooperative DNA binding of the EXD-HTH heterodimer. Some in vivo functions require both EXD and HTH in the nucleus, suggesting that the EXD-HTH complex may function as a transcriptional regulator.

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

A common mechanism for antenna-to-Leg transformation in Drosophila: suppression of homothorax transcription by four HOM-C genes.

The Drosophila HOM-C genes encode transcription factors containing the DNA-binding homeodomain. Mutations in the HOM-C genes can cause specific homeotic transformation, suggesting that the HOM-C genes determine segmental identities by acting on different target genes. However, misexpression of several HOM-C genes in the antenna disc causes similar antenna-to-leg transformations. Here we show that the Scr, Antp, Ubx, and abd-A HOM-C genes all exert their effects through a common mechanism: suppressing the transcription of the homothorax (hth) homeobox gene and thereby preventing the nuclear localization of the Extradenticle homeodomain protein. We also show that ectopic hth expression can cause duplication of the proximodistal axis of the antenna, suggesting that it is involved in proximodistal development of the antenna.

Dorsotonals/homothorax, the Drosophila homologue of meis1, interacts with extradenticle in patterning of the embryonic PNS.

The homeotic genes of the bithorax complex are required, among other things, for establishing the patterns of sensory organs in the embryonic peripheral nervous system (PNS). However, the molecular mechanisms by which these genes affect pattern formation in the PNS are not understood and other genes that function in this pathway are not characterized. Here we report the phenotypic and molecular analysis of one such gene, homothorax (hth; also named dorsotonals). Mutations in the hth gene seem to alter the identity of the abdominal chordotonal neurons, which depend on Abd-A for their normal development. However, these mutations do not alter the expression of the abd-A gene, suggesting that hth may be involved in modulating abd-A activity. We have generated multiple mutations in the hth locus and cloned the hth gene. hth encodes a homeodomain-containing protein that is most similar to the murine proto-oncogene meis1. The hth gene is expressed throughout embryonic development in a spatially restricted pattern, which is modulated in abdominal segments by abd-A and Ubx. The spatial distribution of the HTH protein during embryonic development is very similar to the distribution of the Extradenticle (EXD) protein, a known modulator of homeotic gene activity. Here we show that the PNS phenotype of exd mutant embryos is virtually indistinguishable from that of hth mutant embryos and does not simply follow the homeotic transformations observed in the epidermis. We also show that the HTH protein is present in extremely low levels in embryos lacking exd activity as compared to wild-type embryos. In contrast, the EXD protein is present in fairly normal levels in hth mutant embryos, but fails to accumulate in nuclei and remains cytoplasmic. Ectopic expression of hth can drive ectopic nuclear localization of EXD. Based on our observations we propose that the genetic interactions between hth and exd serve as a novel mechanism for regulating homeotic protein activity in embryonic PNS development.

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, extradenticle, and suppresses eye development in Drosophila.

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development.

Haplotyping of mitochondrial DNA in the D-loop region by PCR: forensic application.

A rapid and simple method using restriction enzymes to detect the restriction fragment length polymorphism (RFLP) pattern of hypervariable segment 1 in the D-loop region of human mitochondrial DNA (mtDNA) was developed. We first focused on the investigation of variations of DNA sequence in the D-loop region among Chinese subjects, as well as on the determination of RFLP patterns of each restriction enzyme. Seven restriction enzymes were used to digest a 618 bp polymerase chain (PCR) reaction product of the D-loop region of mtDNA. Frequency distribution of RFLP patterns of each restriction enzyme among 145 unrelated Chinese subjects in Taiwan was also established. For the purposes of practical forensic application, a routine typing system was designed on the basis of the RFLP data. Two short hypervariable, mtDNA fragments, which were contained within the 618 bp region, were selected for this purpose. In this haplotyping system, a 281 bp PCR-amplified DNA product was analyzed by five restriction enzymes: Mnl I, Nla III, Rsa l, Mse I and Hinf I, and a 237 bp fragment was analyzed by Kpn I. The RFLP patterns were determined by agarose gel electrophoresis of the restriction enzyme-digested DNA fragments. Six restriction enzymes. Mul I, Nla III, Rsa I. Msc I, Hinf I and Kpn 1, defined eight, four, four, five, two and four polymorphic patterns, respectively among the 145 Chinese subjects. The RFLP patterns of restriction fragments for each individual were systematically analyzed and the mtDNAs of the 145 Chinese subjects were grouped into 52 haplotypes. This PCR-RFLP haplotyping system revealed a high degree of variability and diversity of segment I in the D-loop region of human mtDNA. The power of discrimination and allelic diversity values were 0.923 and 0.929, respectively. Successful application of this haplotyping system in a murder case is also discussed.

Functional analysis of C-terminal deletion mutants of Epstein-Barr virus thymidine kinase.

Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein-Barr virus (EBV) using the pET expression plasmid and E. coli BL21 (DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.

Cell proliferation-dependent expression of two isoforms of the nucleolar phosphoprotein p130.

A highly phosphorylated human nucleolar protein p130 (130-kDa) was found to be expressed in synchrony with cell-growth activation. It was not detectable in the resting lymphocytes, but its expression was increased rapidly after mitogenic stimulation. During the terminal differentiation-coupled growth-arrest of HL60 cells, the mRNA and protein of p130 reduced significantly within 24 h after the induction. In addition to the previously identified form (now referred to as p130 alpha ), a novel isoform p 130 beta was found. It contains an insert of ten amino acids located within a region corresponding to the fourth proline-rich basic domain of p130 alpha. Both isoforms were coexpressed in cell lines of different origins, with the beta-transcript exhibiting much less compared to the alpha-transcript. Furthermore, both alpha- and beta-transcripts diminished when cells returned to the quiescent stage. cDNA transfection experiments demonstrated that the two p130 isoforms existed stably in the nucleoli and showed the same nucleolar distribution pattern. It implies that the ten-amino-acid-insert does not alter significantly the interactions of p130 with other nucleolar components in interphase cells.

Prevention of false results from preferential PCR amplification or VNTR alleles.

In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework.

Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.

We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluorescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early G1-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.

Frequency distribution of hypervariable VNTRs in Apo B, HVR-Ig and COL2A1 loci in Taiwan: forensic application.

By use of a simple, rapid and reliable polymerase chain reaction (PCR)-based method, we analyzed three hypervariable tandem repeats in the 3'-Apo B, 5'-HVR-Ig and 3'-COL2A1 loci. As accurate data of allele frequency of genetic markers is a prerequisite for forensic application, the allele frequency distribution of the three variable number of tandem repeats (VNTR) among the Chinese population in Taiwan were studied. In a total of 123 unrelated Chinese subjects, the Apo B VNTR demonstrated a heterozygosity of 68.2% with 9 alleles, 0.85 of the power of discrimination (PD) value and 0.74 of the allelic diversity (h) value. In a sample of 103 unrelated Chinese subjects, the COL2A1 VNTR showed 49.0% heterozygosity with six alleles, 0.79 of the PD value and 0.74 of the h value. In 106 unrelated subjects, the HVR-Ig VNTR showed 47.4% heterozygosity with seven alleles, 0.79 of the PD value and 0.59 of the h value. The data obtained in this study are not only useful for forensic identification, but will also be helpful for paternity testing, genetic linkage studies and the identification of the three VNTR loci associated with human genetic diseases. Some verifying examinations for the validity and reliability of the three VNTR were performed. The high sensitivity and inexpensive nature of this approach make it superior to the traditional method of DNA fingerprinting for forensic typing. With the use of this PCR-VNTR system, many forensic cases have been successfully identified. The value of this system is illustrated in the investigation of a rape and murder case.

Flow chart HLA-DQA1 genotyping and its application to a forensic case.

The detection of genetic polymorphism has become increasingly important in forensic science as well as in medical genetics. In this report, we describe a systematic flow chart system for HLA-DQA1 genotyping by an improved PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method coupled with the PRSM (PCR-mediated restriction site modification) method. This flow chart typing system can easily discriminate between a total of eight reported DQA1 alleles commonly found in Chinese. We have applied this flow chart typing system in a forensic case as well as in the determination of the frequencies of the eight DQA1 alleles in 121 unrelated Taiwan Chinese subjects. Our results show that the flow chart DQA1 genotyping is a simple, fast, and accurate system which, in the future, may be considered as an alternative method for routine individual identification in forensic casework, and for paternity testing and tissue typing in medical genetics.

Use of antigen expressed in bacteria for detection of EBV-specific thymidine kinase antibodies in sera from patients with nasopharyngeal carcinoma.

Two cDNA clones covering the N- and C-terminal portions of the EBV BXLF1 open reading frame were selected from a cDNA library derived from P3HR1 cells. The two clones were ligated, the N-terminal untranslated region truncated, and the product inserted into an E. coli expression vector, pET3CP*. The fusion protein was expressed under control of the T7 phage phi 10 gene promoter and shown to possess thymidine kinase activity. The protein was then used as an antigen to detect antibody reactivities in serum samples of nasopharyngeal carcinoma patients and healthy blood donors. Using a 1:400 dilution of serum samples in Western blot analyses, it was possible to differentiate the reactivities of serum IgA of NPC patients and healthy donors. The prevalence of positive reactivity to EBV TK in NPC was around 84%. The test was compared to others used for early diagnosis of NPC and was able to detect some patients who were negative in those tests.