BACKGROUND: The Epstein-Barr virus (EBV) is consistently detected in patients with nasopharyngeal carcinoma. To determine whether EBV infection is an early, initiating event in the development of this malignant tumor, we screened nasopharyngeal-biopsy samples, most of which were archival, for preinvasive lesions, including dysplasia and carcinoma in situ. Preinvasive lesions were found in 11 samples, which were tested for the presence of EBV.
METHODS: EBV infection was detected with in situ hybridization for EBV-encoded RNAs (EBERs) and by immunohistochemical staining for latent membrane protein 1 (LMP-1). The larger samples were also tested for the EBV genome with the use of Southern blotting. The expression of specific EBV RNAs was determined by the amplification of complementary DNA with the polymerase chain reaction.
RESULTS: Evidence of EBV infection was detected in all 11 tissue samples with dysplasia or carcinoma in situ. EBERs were identified in all eight samples tested, and LMP-1 was detected in all six of the tested samples. Six of the seven samples tested for the EBV termini contained clonal EBV DNA: Transcription of the latent EBV gene products, EBV nuclear antigen 1, LMP-1, LMP-2A, and the BamHI-A fragment, was detected in most of the samples. Viral proteins characteristic of lytic lesions were not detected.
CONCLUSIONS: Preinvasive lesions of the nasopharynx are infected with EBV. The EBV DNA is clonal, indicating that the lesions represent a focal cellular growth that arose from a single EBV-infected cell and that EBV infection is an early, possibly initiating event in the development of nasopharyngeal carcinoma. Preinvasive lesions contain EBV RNAs that are characteristic of latent infection but not the viral proteins that are characteristic of lytic infection. The detection of the EBV-transforming gene, LMP-1, in all the neoplastic cells suggests that its expression is essential for preinvasive epithelial proliferations associated with nasopharyngeal carcinoma.
Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1.
Haemorrhagic septicaemia (HS) is a major disease in cattle and buffaloes, caused by certain serotypes of Pasteurella multocida, mainly B and E serotypes. Frequent HS outbreak has a major impact in many Asian countries, including Malaysia, where farmers encounter economic loss due to low milk production as well as death of their livestock. There are four types of vaccines available; broth bacterins, alum precipitated vaccine, aluminium hydroxide gel vaccine and oil adjuvant vaccine (OAV), but these vaccines can only provide short term immunity and therefore need to be administered annually. Hence, the development of a protein vaccine using recombinant antigen can be a potential candidate for the production of HS vaccine that would give longer immunity. We have successfully cloned the ABA392 gene fragment into a protein expression vector, pET-30a. The protein was expressed from our ABA392/pET30a clone and the immunogenicity of the protein has been tested on rats. This vaccine was able to trigger an immune response and therefore has the potential to be tested as suitable vaccine candidate in future studies. It is envisaged that this subunit vaccine will make a significant contribution in the management of HS among livestock in future.
Cardiac c-kit cells show promise in regenerating an injured heart. While heart disease commonly affects elderly patients, it is unclear if autologous cardiac c-kit cells are functionally competent and applicable to these patients. This study characterised cardiac c-kit cells (CCs) from aged mice and studied the effects of human Wharton's Jelly-derived mesenchymal stem cells (MSCs) on the growth kinetics and cardiac differentiation of aged CCs in vitro. CCs were isolated from 4-week- and 18-month-old C57/BL6N mice and were directly co-cultured with MSCs or separated by transwell insert. Clonogenically expanded aged CCs showed comparable telomere length to young CCs. However, these cells showed lower Gata4, Nkx2.5, and Sox2 gene expressions, with changes of 2.4, 3767.0, and 4.9 folds, respectively. Direct co-culture of both cells increased aged CC migration, which repopulated 54.6 ± 4.4% of the gap area as compared to aged CCs with MSCs in transwell (42.9 ± 2.6%) and CCs without MSCs (44.7 ± 2.5%). Both direct and transwell co-culture improved proliferation in aged CCs by 15.0% and 16.4%, respectively, as traced using carboxyfluorescein succinimidyl ester (CFSE) for three days. These data suggest that MSCs can improve the growth kinetics of aged CCs. CCs retaining intact telomere are present in old hearts and could be obtained based on their self-renewing capability. Although these aged CCs with reduced growth kinetics are improved by MSCs via cell-cell contact, the effect is minimal.
The mantled phenotype is an abnormal somaclonal variant arising from the oil palm cloning process and severe phenotypes lead to oil yield losses. Hypomethylation of the Karma retrotransposon within the B-type MADS-box EgDEF1 gene has been associated with this phenotype. While abnormal Karma-EgDEF1 hypomethylation was detected in mantled clones, we examined the methylation state of Karma in ortets that gave rise to high mantling rates in their clones. Small RNAs (sRNAs) were proposed to play a role in Karma hypomethylation as part of the RNA-directed DNA methylation process, hence differential expression analysis of sRNAs between the ortet groups was conducted. While no sRNA was differentially expressed at the Karma-EgDEF1 region, three sRNA clusters were differentially regulated in high-mantling ortets. The first two down-regulated clusters were possibly derived from long non-coding RNAs while the third up-regulated cluster was derived from the intron of a DnaJ chaperone gene. Several predicted mRNA targets for the first two sRNA clusters conversely displayed increased expression in high-mantling relative to low-mantling ortets. These predicted mRNA targets may be associated with defense or pathogenesis response. In addition, several differentially methylated regions (DMRs) were identified in Karma and its surrounding regions, mainly comprising subtle CHH hypomethylation in high-mantling ortets. Four of the 12 DMRs were located in a region corresponding to hypomethylated areas at the 3'end of Karma previously reported in mantled clones. Further investigations on these sRNAs and DMRs may indicate the predisposition of certain ortets towards mantled somaclonal variation.
Goniothalamin, a styryllactone, has been shown to induce cytotoxicity via apoptosis in several tumor cell lines. In this study, we have examined the potential role of several genes, which were stably transfected into T-cell lines and which regulate apoptosis in different ways, on goniothalamin-induced cell death. Overexpression of full-length receptor for activated protein C-kinase 1 (RACK-1) and pc3n3, which up-regulates endogenous RACK-1, in both Jurkat and W7.2 T cells resulted in inhibition of goniothalamin-induced cell death as assessed by MTT and clonogenic assays. However, overexpression of rFau (antisense sequence to Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene) in W7.2 cells did not confer resistance to goniothalamin-induced cell death. Etoposide, a clinically used cytotoxic agent, was equipotent in causing cytotoxicity in all the stable transfectants. Assessment of DNA damage by Comet assay revealed goniothalamin-induced DNA strand breaks as early as 1 h in vector control but this effect was inhibited in RACK-1 and pc3n3 stably transfected W7.2 cells. This data demonstrate that RACK-1 plays a crucial role in regulating cell death signalling pathways induced by goniothalamin.
Despite the importance of methicillin-resistant Staphylococcus aureus (MRSA) as a priority nosocomial pathogen, the genome sequences of Malaysian MRSA isolates are currently limited to a small pool of samples. Here, we present the genome sequence analyses of 88 clinical MRSA isolates obtained from the main tertiary hospital in Terengganu, Malaysia in 2016-2020, to obtain in-depth insights into their characteristics. The EMRSA-15 (ST22-SCCmec IV) clone of the clonal complex 22 (CC22) lineage was predominant with a total of 61 (69.3%) isolates. Earlier reports from other Malaysian hospitals indicated the predominance of the ST239 clone, but only two (2.3%) isolates were identified in this study. Two Indian-origin clones, the Bengal Bay clone ST772-SCCmec V (n = 2) and ST672 (n = 10) were also detected, with most of the ST672 isolates obtained in 2020 (n = 7). Two new STs were found, with one isolate each, and were designated ST7879 and ST7883. From the core genome phylogenetic tree, the HSNZ MRSA isolates could be grouped into seven clades. Antimicrobial phenotype-genotype concordance was high (> 95%), indicating the accuracy of WGS in predicting most resistances. Majority of the MRSA isolates were found to harbor more than 10 virulence genes, demonstrating their pathogenic nature.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.