Computational Prediction of Usutu Virus E Protein B Cell and T Cell Epitopes for Potential Vaccine Development.

Usutu virus (family Flaviviridae), once confined to Africa, has emerged in Europe a decade ago. The virus has been spreading throughout Europe at a greater pace mostly affecting avian species. While most bird species remain asymptomatic carriers of this virus, few bird species are highly susceptible. Lately, Usutu virus (USUV) infections in humans were reported sporadically with severe neuroinvasive symptoms like meningoencephalitis. As so much is unknown about this virus, which potentially may cause severe diseases in humans, there is a need for more studies of this virus. In this study, we have used computational tools to predict potential B cell and T cell epitopes of USUV envelope (E) protein. We found that amino acids between positions 68 and 84 could be a potential B cell epitope, while amino acids between positions 53 and 69 could be a potential major histocompatibility complex (MHC) class I- and class II-restricted T cell epitope. By homology 3D modeling of USUV E protein, we found that the predicted B cell epitope was predominantly located in the coil region, while T cell epitope was located in the beta-strand region of the E protein. Additionally, the potential MHC class I T cell epitope (LAEVRSYCYL) was predicted to bind to nearly 24 human leucocyte antigens (HLAs) (IC50 ≤5000 nm) covering nearly 86.44% of the Black population and 96.90% of the Caucasoid population. Further in vivo studies are needed to validate the predicted epitopes.

MicroRNA-101 regulated transcriptional modulator SUB1 plays a role in prostate cancer.

MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate cancer.

Protective effects of Asparagus racemosus on oxidative damage in isoniazid-induced hepatotoxic rats: an in vivo study.

To investigate the hepatoprotective activity of Asparagus racemosus against isoniazid-induced hepatotoxicity in male albino rats. Rats (n = 6 per group)were divided into four groups: saline-treated control, saline-treated control with A. racemosus extract (50 mg/kg), isoniazid treatment alone (100 mg/kg, intraperitoneal [i.p.]), and isoniazid-A. racemosus extract (50 mg/kg)administered orally as cotreatment. Animals were treated for 21 days and euthanized 1 h after the last drug administration. Evaluated body weight, serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, g-glutamyl transferase, total protein, albumin, hepatic malondialdehyde content, superoxide dismutase, catalase, cytochrome P450 2E1 (CYP2E1)activity and glutathione (GSH). A. racemosus extract prevented isoniazid-induced hepatotoxicity, indicated by both diagnostic indicators of liver damage, liver functional profile, significantly (p < 0.05)inhibited CYP2E1 activity, markedly attenuated oxidative stress by improved enzymatic, non-enzymatic antioxidants levels and mitigate malondialdehyde, lipid hydroperoxide significantly (p < 0.05). These results suggest that A. racemosus extract exerts its hepatoprotective activity by inhibiting the production of free radicals and acts as a scavenger, reducing the free radical generation via inhibition of hepatic CYP2E1 activity, increasing the removal of free radicals through the induction of antioxidant enzymes and improving non-enzymatic thiol antioxidant GSH.

The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.

The neuronal repellent SLIT2 is a target for repression by EZH2 in prostate cancer.

The neuronal repellent SLIT2 is repressed in a number of cancer types primarily through promoter hypermethylation. SLIT2, however, has not been studied in prostate cancer. Through genome-wide location analysis we identified SLIT2 as a target of polycomb group (PcG) protein EZH2. The EZH2-containing polycomb repressive complexes bound to the SLIT2 promoter inhibiting its expression. SLIT2 was downregulated in a majority of metastatic prostate tumors, showing a negative correlation with EZH2. This repressed expression could be restored by methylation inhibitors or EZH2-suppressing compounds. In addition, a low level of SLIT2 expression was associated with aggressive prostate, breast and lung cancers. Functional assays showed that SLIT2 inhibited prostate cancer cell proliferation and invasion. Thus, this study showed for the first time the epigenetic silencing of SLIT2 in prostate tumors, and supported SLIT2 as a potential biomarker for aggressive solid tumors. Importantly, PcG-mediated repression may serve as a precursor for the silencing of SLIT2 by DNA methylation in cancer.

Increase in nitric oxide and reductions in blood pressure, protein kinase C beta II and oxidative stress by L-carnitine: a study in the fructose-fed hypertensive rat.

Recently we showed that the administration of intraperitoneal L-carnitine (CA) has insulin-sensitizing effects in the high fructose-fed Wistar rat, a widely used model of metabolic syndrome. The present study was conducted to examine the regulatory effects of CA on blood pressure (BP) and related pressor mechanisms. Fructose-fed rats (FFR) showed elevated BP, cardiac hypertrophy, glucose intolerance, and increases in plasma glucose, insulin, free fatty acids (FFA), and angiotensin-converting enzyme (ACE) activity. They also showed increased protein kinase C betaII (PKC betaII) expression and oxidative stress in cardiac tissue. In plasma, decreased kallikrein enzyme activity and nitric oxide metabolites were observed, compared to control. Simultaneous treatment with CA (300 mg/Kg) mitigated these alterations. PKC betaII expression was similar to that of control; the rats displayed normal BP and ACE activity, enhanced antioxidant protection, and close to normal values of metabolic parameters. The BP-lowering effect of CA was abolished when CA-treated rats were administered L-nitroarginyl methyl ester (L-NAME 6g/Kg). These observations suggest that the BP-lowering action of CA in this model could be attributed to multiple and interrelated mechanisms, such as an increase in NO and kinin availability, reduction in PKC action, and antioxidant protection.

Deregulation of FCGR2B expression by 1q21 rearrangements in follicular lymphomas.

We report here the molecular cloning and characterization of a t(1;14)(q21;q32) in a follicular lymphoma (FL) with an unusual BCL2 aberration. Fluorescence in situ hybridization (FISH) and Southern blot analysis of tumor cells identified the translocation breakpoint within the 5' switch region of IGHG (Sgamma). We cloned the chimeric breakpoint region approximately 1.5 kbp downstream from the HindIII site of 5'Sgamma2 on chromosome 14q32 and identified a 360-bp novel segment with homology to the CpG island clone 11h8. Two BAC clones containing this sequence were isolated and mapped to 1q21 by FISH. BAC 342/P13 contained sequences homologous to Fcgamma receptors 2A, 3A, 2B, 3B, and a heat shock protein gene HSP70B. The translocation brought the Sgamma2 region of a productive IGH allele 20 approximately 30 kbp upstream of FCGR2B. As a result of the translocation, the b2 isoform of FCGR2B was overexpressed in the tumor. Screening of a panel of 76 B-cell lymphomas with 1q21-23 cytogenetic aberrations by Southern blot analysis using breakpoint probes identified an additional FL with a t(14;18)(q32;q21) and a breakpoint in the FCGR2B region. These results suggest that FCGR2B may be deregulated by 1q21 aberration in BCL2 rearranged FLs and possibly play a role in their progression.

IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy.

Abnormalities of chromosome 1q21 are common in B cell malignancies, but their target genes are largely unknown. By cloning the breakpoints of a (1;14) (q21;q32) chromosomal translocation in a myeloma cell line, we have identified two novel genes, IRTA1 and IRTA2, encoding cell surface receptors homologous to the Fc and inhibitory receptor families. Both genes are selectively expressed in mature B cells: IRTA1 in marginal zone B cells and IRTA2 in centrocytes, marginal zone B cells, and immunoblasts. As a result of the t(1;14), IRTA1 is fused to the immunoglobulin Calpha domain to produce a chimeric IRTA1/Calpha fusion protein. In tumor cell lines with 1q21 abnormalities, IRTA2 expression is deregulated. Thus, IRTA1 and IRTA2 are novel immunoreceptors implicated in B cell development and lymphomagenesis.

Vitamin D receptor as a candidate tumor-suppressor gene in severe hyperparathyroidism of uremia.

Most chronic renal failure patients with severe refractory hyperparathyroidism harbor at least one monoclonal parathyroid tumor, but the specific acquired genetic defects that confer this clonal selective advantage remain poorly understood. Somatic inactivation of the vitamin D receptor (VDR) gene could contribute to clonal outgrowth, because a parathyroid cell containing this lesion would have an impaired response to the antiproliferative influence of 1,25-dihydroxyvitamin D3. Furthermore, diminished expression of VDR protein has been described in uremia-associated parathyroid tumors. Therefore, to assess VDR gene inactivation's potential pathogenetic role in this disease, we rigorously analyzed the VDR gene in 59 parathyroid tumors surgically resected from uremic patients. First, Southern blotting and/or PCR analyses of 29 tumor samples from 14 genetically informative patients revealed no allelic losses at the VDR locus. Next, direct DNA sequencing of all VDR splice junctions, associated intronic sequences, and virtually the entire VDR-coding region for all 59 tumors revealed no acquired mutations. Last, 37 tumor DNA samples were subjected to comparative genomic hybridization, and no chromosomal losses in the VDR region (12cen-q12) were observed. These observations suggest that inactivating defects within the VDR gene do not commonly contribute to the primary pathogenesis of severe refractory hyperparathyroidism in uremia.

MUC1 is activated in a B-cell lymphoma by the t(1;14)(q21;q32) translocation and is rearranged and amplified in B-cell lymphoma subsets.

The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non-Hodgkin's lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG (Cgamma) probe. The translocation linked the IGHG4 switch (Sgamma4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1 transcriptional unit intact. MUC1 was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably, MUC1 transcription is aberrantly regulated by the IGHA (Calpha) 3' enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3' MUC1 breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1 activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis. (Blood. 2000;95:2666-2671)

A common molecular basis for rearrangement disorders on chromosome 22q11.

The chromosome 22q11 region is susceptible to rearrangements that are associated with congenital anomaly disorders and malignant tumors. Three congenital anomaly disorders, cat-eye syndrome, der() syndrome and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) are associated with tetrasomy, trisomy or monosomy, respectively, for part of chromosome 22q11. VCFS/DGS is the most common syndrome associated with 22q11 rearrangements. In order to determine whether there are particular regions on 22q11 that are prone to rearrangements, the deletion end-points in a large number of VCFS/DGS patients were defined by haplotype analysis. Most VCFS/DGS patients have a similar 3 Mb deletion, some have a nested distal deletion breakpoint resulting in a 1.5 Mb deletion and a few rare patients have unique deletions or translocations. The high prevalence of the disorder in the population and the fact that most cases occur sporadically suggest that sequences at or near the breakpoints confer susceptibility to chromosome rearrangements. To investigate this hypothesis, we developed hamster-human somatic hybrid cell lines from VCFS/DGS patients with all three classes of deletions and we now show that the breakpoints occur within similar low copy repeats, termed LCR22s. To support this idea further, we identified a family that carries an interstitial duplication of the same 3 Mb region that is deleted in VCFS/DGS patients. We present models to explain how the LCR22s can mediate different homologous recombination events, thereby generating a number of rearrangements that are associated with congenital anomaly disorders. We identified five additional copies of the LCR22 on 22q11 that may mediate other rearrangements leading to disease.

Chromosomal amplification is associated with cisplatin resistance of human male germ cell tumors.

Chemotherapy resistance of tumors is an important biological and clinical problem. Studies from many tumor types have indicated that resistance may be based on multiple genetic pathways. Human male germa cell tumors (GCTs) are an especially good model system to study the genetic basis of tumor sensitivity and resistance to chemotherapy. GCTs are exquisitely sensitive to treatment with DNA-damaging drugs such as cisplatin, rarely exhibit TP53 gene mutations, express normal p53 protein, and undergo p53-mediated apoptosis upon drug treatment. A small proportion of tumors (20-30% of metastatic lesions) escape the apoptotic response and result in treatment resistance. We have recently shown (J. Houldsworth, et al., Oncogene, 16: 2345-2359, 1998) that in a subset of such tumors, resistance is linked to TP53 gene mutations. In a further search for genetic mechanisms underlying resistance, we subjected a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemotherapy-resistant tumors to comparative genomic hybridization analysis to identify possible amplified regions (implying amplified/overexpressed genes) associated with resistance. With the exception of 12p11.2-12, high level amplification was not detected in any of the sensitive tumors. We have identified eight amplified regions (1q31-32, 2p23-24, 7q21, 7q31, 9q22, 9q32-34, 15q23-24, and 20q11.2-12) in five resistant tumors, which suggests that chromosomal and, hence, gene amplification may comprise a pathway to drug resistance. Identification of amplified/overexpressed genes at these sites may elucidate new genetic pathways of chemotherapy resistance in GCTs and possibly also in other tumors.

Novel chromosomal abnormalities identified by comparative genomic hybridization in parathyroid adenomas.

The molecular basis of parathyroid adenomatosis includes defects in the cyclin D1/PRAD1 and MEN1 genes but is, in large part, unknown. To identify new locations of parathyroid oncogenes or tumor suppressor genes, and to further establish the importance of DNA losses described by molecular allelotyping, we performed comparative genomic hybridization (CGH) on a panel of 53 typical sporadic (nonfamilial) parathyroid adenomas. CGH is a new molecular cytogenetic technique in which the entire tumor genome is screened for chromosomal gains and/or losses. Two abnormalities, not previously described, were found recurrently: gain of chromosome 16p (6 of 53 tumors, or 11%) and gain of chromosome 19p (5 of 53, or 9%). Losses were found frequently on 11p (14 of 53, or 26%), as well as 11q (18 of 53, or 34%). Recurrent losses were also seen on chromosomes 1p, 1q, 6q, 9p, 9q, 13q, and 15q, with frequencies ranging from 8-19%. Twenty-four of the 53 adenomas were also extensively analyzed with polymorphic microsatellite markers for allelic losses, either in this study (11 cases) or previously (13 cases). Molecular allelotyping results were highly concordant with CGH results in these tumors (concordance level of 97.5% for all informative markers/chromosome arms examined). In conclusion, CGH has identified the first two known chromosomal gain defects in parathyroid adenomas, suggesting the existence of direct-acting parathyroid oncogenes on chromosomes 16 and 19. CGH has confirmed the locations of putative parathyroid tumor suppressor genes, also defined by molecular allelotyping, on chromosomes 1p, 6q, 9p, 11q, 13q, and 15q. Finally, CGH has provided new evidence favoring the possibility that distinct parathyroid tumor suppressors exist on 1p and 1q, and has raised the possibility of a parathyroid tumor suppressor gene on 11p, distinct from the MEN1 gene on 11q. CGH can identify recurrent genetic abnormalities in hyperparathyroidism, especially chromosomal gains, that other methods to not detect.

Genomic organization and allelic polymorphism of the human killer cell inhibitory receptor gene KIR103.

Killer cell inhibitory receptors (KIR) belong to the immunoglobulin superfamily of molecules and are expressed on natural killer (NK) cells. The KIRs of the p58/p50 family have two immunoglobulin domains and are ligands for HLA-Cw antigens, whereas the p70/p70 delta family has three immunoglobulin domains and comprises ligands for HLA-B antigens and possibly some HLA-A antigens. Members of a third KIR family, KIR103, have two immunoglobulin domains but have highest nucleotide sequence homology to the p70 family. The ligands for KIR103 on target cells are currently unknown. We here report the complete genomic organization of KIR103. It spans about 12 kb of DNA and consists of eight exons of which exon 1 and exon 2 encode the leader sequence. Exon 3 encodes the first immunoglobulin domain (gamma 1), and exon 4 encodes the main part of the second immunoglobulin domain (gamma 3), which also contains sequences contributed by exon 5 and exon 6. Exon 6 encodes the transmembrane domain, whereas exons 7 and 8 encode most of the cytoplasmic domain. KIR103 is polymorphic, and two alleles, 103AS and 103LP, are defined in this study. Additional full-length cDNA clones for KIR103 have been isolated and are shown to be formed by alternative mRNA splicing with exon skipping. Some of these truncated KIR103 cDNA could encode shorter transmembrane molecules, whereas others lack the transmembrane domain and are candidate genes for secreted KIR products. KIR103 is localized to the KIR genetic region on chromosome 19q13.4.