BK virus-associated urinary bladder carcinoma in transplant recipients: report of 2 cases, review of the literature, and proposed pathogenetic model.

Despite strong experimental evidence, BK polyomavirus involvement in human cancers has been controversial. We report 2 cases of kidney ± pancreas transplant recipients with evidence of BK polyomavirus reactivation, who developed aggressive urinary bladder urothelial carcinomas with adenocarcinomatous and/or micropapillary differentiation. Diffuse strong nuclear positivity for viral T antigen, p53, Ki-67, and p16 was observed in both malignancies. The BK polyomavirus role in promoting urothelial neoplasia in transplant recipients may be partly indirect, based on the demonstration by polymerase chain reaction in both tumors of BK polyomavirus with intact open reading frames and close phylogenetic clustering with known replication-competent strains, and viral capsid protein VP1 messenger RNA and intranuclear virions by electron microscopy in 1 tumor. No unique cancer-associated mutations were found, but some viral T antigen mutations were potentially associated with increased rate of viral replication and risk for "rare" carcinogenic events. The BK polyomavirus-induced profound effects on cell activation, cell cycle shift to proliferation, and apoptosis inhibition, in the context of marked immunosuppression, constitute a potentially ideal background for malignant transformation. The long time lapse between transplantation and tumor manifestation, 7 and 11 years, respectively, further supports the concept of multistep carcinogenesis cascade and long-term risk for these patients. We propose a model of changes ranging from viral reactivation to dysplasia to invasive carcinoma. Clinical vigilance is warranted for early diagnosis of BK polyomavirus-related urothelial malignancies in transplant recipients.

FH535 inhibited migration and growth of breast cancer cells.

There is substantial evidence indicating that the WNT signaling pathway is activated in various cancer cell types including breast cancer. Previous studies reported that FH535, a small molecule inhibitor of the WNT signaling pathway, decreased growth of cancer cells but not normal fibroblasts, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we tested FH535 as a potential inhibitor for malignant phenotypes of breast cancer cells including migration, invasion, and growth. FH535 significantly inhibited growth, migration, and invasion of triple negative (TN) breast cancer cell lines (MDA-MB231 and HCC38) in vitro. We demonstrate that FH535 was a potent growth inhibitor for TN breast cancer cell lines (HCC38 and MDA-MB-231) but not for other, non-TN breast cancer cell lines (MCF-7, T47D or SK-Br3) when cultured in three dimensional (3D) type I collagen gels. Western blotting analyses suggest that treatment of MDA-MB-231 cells with FH535 markedly inhibited the expression of NEDD9 but not activations of FAK, Src, or downstream targets such as p38 and Erk1/2. We demonstrated that NEDD9 was specifically associated with CSPG4 but not with ß1 integrin or CD44 in MDA-MB-231 cells. Analyses of gene expression profiles in breast cancer tissues suggest that CSPG4 expression is higher in Basal-type breast cancers, many of which are TN, than any other subtypes. These results suggest not only a mechanism for migration and invasion involving the canonical WNT-signaling pathways but also novel strategies for treating patients who develop TN breast cancer.

VP-1 quasispecies in human infection with polyomavirus BK.

Polyomavirus BK is a recognized cause of nephropathy and hemorrhagic cystitis in kidney or allogeneic hematopoietic stem cell transplant recipients. This study explored a role of genetic variations in capsid protein VP-1 gene as a factor in viral pathogenesis. VP-1 was amplified from 7 healthy subjects with viruria, 7 transplant patients with viruria, and 11 patients with viremia or nephropathy. PCR products were cloned and a total of 558 clonal sequences were subjected to phylogenetic analysis using standard methods. VP-1 quasispecies were found in 25/25 and coinfection with different genotypes in 12/25 subjects. Genotype II was found as an unexpected minority species in 5/25 individuals. Recombinant strains of uncertain biologic significance, which frequently contained genotype II and IV sequences were identified in 9/25 subjects. Viremia/nephropathy group was characterized by (a) greater sequence complexity in whole VP-1 versus BC loop and BC loop compared to the HI loop, (b) greater intra-strain genetic diversity in the BC loop compared to whole VP-1 protein and HI loop, (c) more non-synonymous substitutions (dN) in the BC loop compared to whole VP-1 and HI loop, (e) fewer synonymous substitutions (dS) compared to healthy-viruria group, and (f) selection pressure (dN/dS >1.0) exerted on VP-1. In conclusion, this study documents frequent occurrence of quasispecies in a host DNA polymerase dependent virus, which is theoretically expected to show high replication fidelity. Quasispecies occur even in healthy subjects with viruria, but evolutionary selection pressure directed at the viral capsid protein (VP-1) is seen only in patients with viremia or nephropathy.

The polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response.

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4(+) T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4(+) T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

Genotyping schemes for polyomavirus BK, using gene-specific phylogenetic trees and single nucleotide polymorphism analysis.

BK virus (BKV) genotyping has been historically based on nucleotides 1744 to 1812 in the VP1 gene. We reevaluated this practice by making BKV whole-genome and gene-specific phylogenetic trees as well as performing single nucleotide polymorphism (SNP) analysis of 162 sequences available in the public domain. It was found that currently known BKV subtypes and subgroups can no longer be reliably determined by sequencing certain partial gene sequences. Phylogenetic trees based on large T-antigen (LTA) allow separation of subtype I into subgroups Ia, Ib1, Ib2, and Ic, with bootstrap values of 100%, which are better than bootstraps obtained using VP1 sequences (bootstrap values of 71 to 97%). Subtype IV can be subdivided into subgroups, but LTA bootstrap values (33 to 80%) are lower than those obtained by whole-genome analysis (68 to 87%). Subtypes V and VI provisionally identified earlier on the basis of more limited sequence data are better classified as subgroups Ib2 and Ib1, respectively. LTA positions 3634, 3772, 3934, and 4339 can serve as a minimal SNP set to distinguish between the four major BKV subtypes. No subtype II-, IVa-, or IVb-defining SNPs are available in the VP1 gene. However, the overall congruence of viral strain classification based on either VP1 or LTA phylogenetic analysis indicates that these two areas of the viral genome are genetically linked. Interstrain genetic recombination between distant loci in the VP1 and LTA areas is not a common event.

Validation of BKV large T-antigen ATP-binding site as a target for drug discovery.

BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. Pilot screening of 300 compounds from the Tim Tec ActiTarg K library identified a compound, STO18584, with selectivity index of 19.2.

Rapid evolution of a recently retroposed transcription factor YY2 in mammalian genomes.

YY2 was originally identified due to its unusual similarity to the evolutionarily well-conserved zinc finger gene YY1. In this study, we have determined the evolutionary origin and conservation of YY2 using comparative genomic approaches. Our results indicate that YY2 is a retroposed copy of YY1 that has been inserted into another gene locus named Mbtps2 (membrane-bound transcription factor protease site 2). This retroposition is estimated to have occurred after the divergence of placental mammals from other vertebrates based on the detection of YY2 only in the placental mammals. The N- and C-terminal regions of YY2 have evolved under different selection pressures. The N-terminal region has evolved at a very fast pace with very limited functional constraints, whereas the DNA-binding, C-terminal region still maintains a sequence structure very similar to that of YY1 and is also well conserved among placental mammals. In situ hybridizations using different adult mouse tissues indicate that mouse YY2 is expressed at relatively low levels in Purkinje and granular cells of cerebellum and in neuronal cells of cerebrum, but at very high levels in testis. The expression levels of YY2 are much lower than those of YY1, but the overall spatial expression patterns are similar to those of Mbtps2, suggesting a possible shared transcriptional control between YY2 and Mbtps2. Taken together, the formation and evolution of YY2 represent a very unusual case where a transcription factor was first retroposed into another gene locus encoding a protease and survived with different selection schemes and expression patterns.

DNA sequence comparative analysis of the 3pter-p26 region of human genome.

Most proterminal regions of human chromosomes are GC-rich and gene-rich. Chromosome 3p is an exception. Its proterminal region is GC-poor, and likely to lose heterozygosity, thus causing a number of fatal diseases. Except one gap left in the telomeric position, the proterminal region of human chromosome 3p has been completely sequenced. The detailed sequence analysis showed: (i) the GC content of this region was 38.5%, being the lowest among all the human proterminal regions; (ii) this region contained 20 known genes and 22 predicted genes, with an average gene size of 97.5 kb. The previously mapped gene Cntn3 was not found in this region, but instead located in the 74 Mb position of human chromosome 3p; (iii) the interspersed repeats of this region were more active than the average level of the whole human genome, especially (TA)n, the content of which was twice the genome average; (iv) this region had a conserved synteny extending from 104.1 Mb to 112.4 Mb on the mouse chromosome 6, which was 8% larger in size, not in accordance with the whole genome comparison, probably because the 3pter-p26 region was more likely to lose nucleotides and its mouse synteny had more active interspersed repeats.

High throughput SNP genotyping with two mini-sequencing assays.

Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 24 to 30 bp long, whereas MassArray assay prefers to be as short as only 16 bp. Altogether 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals, 4792 sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.

Structure-based preliminary analysis of immunity and virulence of SARS coronavirus.

The research on SARS-associated coronavirus (SARS-CoV) has not stopped since its discovery, but the pathogenesis of SARS is still unclear. To explore the possible molecular mechanisms of the invasion and virulence of SARS-CoV, we investigated the structural basis of the viral proteins using computational biology. Forty-five motifs relating to superantigens, toxins and other bioactive molecules were detected in the proteins of SARS-CoV. The results showed that the distribution of the motifs varied in different proteins. Enzyme-like motifs were located in the R protein, while ICAM- 1-like and toxin-like molecules were located in the spike, envelop, nucleocapsid, PUP1, PUP 2 and PUP 4 proteins. Comparison of SARS-CoV with other viruses (OC43, PEDV, HRSV, HHerpV and HAdenoV) showed that each group of motifs was different for each type of virus. Data suggest that the proteins of SARS-CoV with toxic motifs might play crucial roles in targeting host cells and interfering with the immune system. This study provides new information for drug and vaccine design, as well as therapeutic strategies against SARS.

The structural characterization and antigenicity of the S protein of SARS-CoV.

The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phylogenetic analysis reveals several conserved regions that might be potent drug targets.

[Whole microbial genome shotgun sequencing].

Two strategies introduced for whole genome sequencing, one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient. In this article we discussed the library construction, long-to-short-ratio of insert, total number of reads should be sequenced, assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.