[Recent advances in early diagnosis of head and neck cancer in precision medicine era].

Head and neck squamous cell carcinoma, HNSCC, has high morbidity and mortality. Even in America, more than 1/2 to 2/3 patients have been diagnosed at advanced stage. So, it's urgent to find ways to diagnose HNSCC earlier and foresee the curative effect. With the achievement of Next-Generation Sequencing, researchers are trying to develop early diagnostic technology from a new perspective, such as personal genomics, proteomics, metabolomics and other related personal information. Here we reviewed the recent research in early diagnosis of HNSCC.

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction.

Vibrio anguillarum, an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum. PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum. Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA, or from six colony-forming units (CFU) mL(-1) of cultured V. anguillarum. However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g(-1) tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.

Recombinant polioviruses expressing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes.

T-cell immune response to recombinant poliovirus expressing foreign antigens has not been elucidated well. In order to investigate the potential use of poliovirus as a T-cell vaccine vector, we constructed recombinant polioviruses expressing HBV-derived CTL epitopes, HBsAg(28-39) (L(d)-restricted; IPQSLDSWWTSL) and HBc(93-100) (K(b)-restricted; MGLKFRQL) at the junction between the P2 and P3 regions, designated V3CDs and V3CDc, respectively. The V3CDs and V3CDc recombinant viruses replicated efficiently in HeLa cells and showed a similar infection profile to that of the wild-type Mahoney strain in one-step growth kinetics. Genetic stability analysis showed that V3CDc retained the foreign insert over twelve successive passages examined, whereas V3CDs lost part of the foreign insert after four passages. Our results indicated that the stability of the inserted foreign sequences was rather affected by their nucleotide sequence than by their length when located between the P2 and P3 regions. The junction between these nonstructural protein-coding regions is a novel site for the construction of replication-competent recombinant poliovirus. Immunization of BALB/c (H-2(d)) and C57BL/6 mice (H-2(b)) with V3CDs and V3CDc, respectively, elicited significant antigen-specific CTL responses to HBsAg(28-39) but not to HBcAg(93-100).

Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene.

Although the mechanism of carcinogenesis by hepatitis C virus (HCV) is not clearly known, core and NS3P protein have been shown to form tumors in specific cell lines. In this study, on the basis of the fact that the core and NS4B proteins of Kunjin virus translocate into the nucleus, we were prompted to investigate whether the HCV nonstructural protein NS4B has any function in tumor formation. First, we examined the location of the NS4B protein of HCV in transfected cells and then its oncogenic activity by transfection of NIH3T3 cells with the NS4B gene in the presence or absence of the Ha-ras gene. The NS4B protein was present only in the cytoplasm, particularly in the perinuclear region, different from the case of the Kunjun virus. The cells expressing HCV NS4B cooperatively with the Ha-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent growth. These biological activities were confirmed by the transcription activation of the reporter gene from the AP1 promoter, by the NS4B protein in association with Ha-ras. Our results demonstrated that HCV NS4B protein in association with the Ha-ras gene played an important role in the malignant transformation of cells by HCV.

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.

Induction of V3-specific cytotoxic T lymphocyte responses by HIV gag particles carrying multiple immunodominant V3 epitopes of gp120.

Effort to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimeric gag-env virus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2 gag precursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1IIIB, HIV-1MN, HIV-1RF, and HIV-1SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purified gag-env VLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than the gag VLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1IIIB, HIV-1MN, HIV-1RF, and the gp120 antiserum against HIV-1SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2 gag can be used as a potential HIV vaccine against various HIVs.

Transcriptional analyses of baculovirus polyhedrin and foreign gene expression relative to baculovirus p10 mRNA levels.

Comparisons have been made between the p10 and polyhedrin mRNA levels recovered from Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV). In molar terms and from 18 h post-infection (p.i.), the polyhedrin mRNA species increased to levels one and a half times to twice as high as the p10 levels. The influence of the polyhedrin leader sequence on the expression of a foreign gene under the control of the polyhedrin promoter was investigated using a series of four recombinant baculoviruses expressing the lymphocytic choriomeningitis virus nucleocapsid (N) protein gene. The different recombinants varied in the length and composition of the upstream polyhedrin mRNA leader sequence. The recombinant containing the full-length polyhedrin leader sequence gave levels of N mRNA comparable to those of AcNPV polyhedrin mRNA. These levels were either equal to (12 h p.i.) or higher (18 to 42 h p.i.) than the p10 levels at corresponding times. Three other recombinants, with different lengths of leader sequence, accumulated significantly lower quantities of N mRNA in comparison to the p10 mRNA levels. However the mRNA levels for the three recombinants were similar (20 to 50% of the p10 level) and did not correspond to their N protein expression levels. By comparing the mRNA and protein levels, it is concluded that the sequence between -8 to +1 of the AcNPV polyhedrin translation-initiating ATG has an important function for mRNA transcription (or accumulation), while the sequences between -32 to -8 affect the overall translation efficiencies.

Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus.

The M RNA species of a candidate vaccine strain of Rift Valley fever virus (RVFV ZH-548M12), derived by consecutive high level mutagenesis using 5-fluorouracil (H. Caplen, C. J. Peters, and D. H. L. Bishop, J. Gen. Virol., 66, 2271-2277, 1985), has been cloned and the cDNA sequenced. The data have been compared to those obtained for the parent virus strain RVFV ZH-548 as well as the previously published data for RVFV ZH-501 (M. S. Collett, A. F. Purchio, K. Keegan, S. Frazier, W. Hays, D. K. Anderson, M. D. Parker, C. Schmaljohn, J. Schmidt, and J. M. Dalrymple, Virology, 144, 228-245, 1985). Some eight nucleotide and three amino acid differences were identified between the M RNAs of ZH-501 and ZH-548. Between the M RNAs of ZH-548 and that of the M12 mutant there were 12 nucleotide and 7 amino acid changes. Unique to the mutant virus is a new AUG codon upstream of that which initiates the open reading frame of the RVFV M gene product (the viral glycoprotein precursor). The significance of this and other differences in the mutant RNA with regard to the derivation and potential attenuation of the candidate vaccine is discussed.