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Objective: The study aimed to clarify (1) the effect of the Beneslider distalization appliance on maxillary molars (M1+2), (2) the type of tooth movement, and (3) changes in skeletal parameters., Study Design: Dental distalization paths and transversal changes were measured according to the distance of the pterygoid vertical as well as on the virtualized 3D plaster model., Results: Distalization of the second premolar (P2; T1 - T2) was 2.51 mm (±1.81 mm; P < .001) combined with a distal tipping of 6.50° (±5.09°; P < .001), whereas for M1 a distalization of 3.49 mm (±1.10 mm) occurred with negligible tipping (0.73° ± 5.04°). No significant differences (T1 - T2) on sagittal and vertical skeletal variables were found within or between G1+2. In contrast, a significant increase in N-Sp' in G2 and in Sp'-Gn in G1+2 with consecutive Hasund index reduction was shown. Comparing T1 and T2, the device had an effect on the transversal development., Conclusion: The Beneslider distalization appliance causes an effective distalization of M1+2, with slight second premolar tipping. No modification of skeletal parameters was recorded, but a relevant effect on Hasund index and the transverse was found., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Skeletally anchored devices have been developed to distalize maxillary molars in class II malocclusion. NiTi springs with high pseudoelastic forces are required to overcome friction and concomitantly create ideal translation force for molar distalization. We present a new maxillary molar distalization device (the Longslider) generating forces of up to gf 600 (5.88 N)., Materials and Methods: Six patients with class II malocclusion owing to maxillary excess were treated by molar distalization. The required distalization path was laboratory set and delivered chair side., Results: The device deactivated itself automatically at the end of the distance in all cases without dental tipping or any implant-related complications. The average distal translational movement of the first molar was 0.81 ± 0.02 mm/mo., Conclusion: Clinically, owing to the high pseudoelastic forces, the Longslider appliance was effective for tooth translation, generating constant pseudoelastic forces of 350 gf through the desired distalization path., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 is the causative agent of the endothelial cell-derived tumour Kaposi's sarcoma. Herpesviruses possess large complex genomes which provide many options to regulate cellular physiology during the viral life cycle and in the course of tumourigenicity. Novel techniques of systems biology and reverse genetics are increasingly applied to dissect the complex interaction of KSHV with endothelial cells. This review will outline novel results and pitfalls of these technologies in the elucidation of KSHV pathogenicity.

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma. Activation of the cellular transcription factor nuclear factor-kappa B (NF-kappaB) is essential for latent persistence of HHV-8, survival of HHV-8-infected cells, and disease progression. We used reverse-transfected cell microarrays (RTCM) as an unbiased systems biology approach to systematically analyze the effects of HHV-8 genes on the NF-kappaB signaling pathway. All HHV-8 genes individually (n = 86) and, additionally, all K and latent genes in pairwise combinations (n = 231) were investigated. Statistical analyses of more than 14,000 transfections identified ORF75 as a novel and confirmed K13 as a known HHV-8 activator of NF-kappaB. K13 and ORF75 showed cooperative NF-kappaB activation. Small interfering RNA-mediated knockdown of ORF75 expression demonstrated that this gene contributes significantly to NF-kappaB activation in HHV-8-infected cells. Furthermore, our approach confirmed K10.5 as an NF-kappaB inhibitor and newly identified K1 as an inhibitor of both K13- and ORF75-mediated NF-kappaB activation. All results obtained with RTCM were confirmed with classical transfection experiments. Our work describes the first successful application of RTCM for the systematic analysis of pathofunctions of genes of an infectious agent. With this approach, ORF75 and K1 were identified as novel HHV-8 regulatory molecules on the NF-kappaB signal transduction pathway. The genes identified may be involved in fine-tuning of the balance between latency and lytic replication, since this depends critically on the state of NF-kappaB activity.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.

Reversely transfected cell microarrays (RTCM) have been introduced as a method for parallel high throughput analysis of gene functions in mammalian cells. Hundreds to thousands of different recombinant DNA or RNA molecules can be transfected into different cell clusters at the same time on a single glass slide with this method. This allows either the simultaneous overexpression or--by using the recently developed RNA interference (RNAi) techniques--knockdown of a huge number of target genes. A growing number of sophisticated detection systems have been established to determine quantitatively the effects of the transfected molecules on the cell phenotype. Several different cell types have been successfully used for this procedure. This review summarizes the presently available knowledge on this technique and provides a laboratory protocol.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases.

Guanylate-binding proteins (GBPs) are the most abundant cellular proteins expressed in response to interferon-gamma (IFN-gamma), with seven highly homologous members in humans, termed HuGBP-1 to HuGBP-7. To date, differential features that may indicate differential functions of these proteins have not been described. Here, we investigated the expression and subcellular localization of the different HuGBPs in endothelial cells (EC). IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) induced the expression of HuGBP-1, HuGBP-2, and HuGBP-3 at similar high levels. In contrast, expression of HuGBP-4 and HuGBP-5 was robustly induced only by IFN-gamma and not by TNF-alpha and IL-1beta. Expression of HuGBP-6 and HuGBP-7 was not detected in EC under the various conditions examined. Investigating subcellular localization of the EC-expressed HuGBPs, HuGBP-1, HuGBP-3, and HuGBP-5 were exclusively detected in the cytoplasm, whereas HuGBP-2 and HuGBP-4 displayed a nucleocytoplasmic distribution. Treatment of the cells with IFN-gamma and aluminum fluoride caused rapid enrichment of HuGBP-1 and HuGBP-2 in the Golgi apparatus, as demonstrated by time-lapse microscopy and fluorescence analyses of GFP-tagged HuGBPs. HuGBP-3 and HuGBP-4 were never detected in the Golgi apparatus, whereas HuGBP-5 was constitutively enriched in this cytosolic compartment, irrespective of stimulation. These results assign a characteristic pattern of expression and subcellular localization to each of the HuGBPs, indicating for the first time that these proteins may have different cellular functions.

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.

Herpesviruses occur in two distinct forms of infection, lytic replication and latent persistence. In this study, we investigated the molecular mechanisms that govern the latent-lytic switch in the prototype gamma-2 herpesvirus, herpesvirus saimiri (HVS). We utilized a persistently HVS-infected A549 cell line, in which HVS DNA is stably maintained as nonintegrated circular episomes, to assess the role of the open reading frame 50 (ORF 50) (Rta) proteins in the latent-lytic switch. Northern blot analysis and virus recovery assays determined that the ORF 50a gene product, when expressed under the control of a constitutively active promoter, was sufficient to reactivate the entire lytic replication cycle, producing infectious virus particles. Furthermore, although the ORF 50 proteins of HVS strains A11 and C488 are structurally divergent, they were both capable of inducing the lytic replication cycle in this model of HVS latency.

Herpesvirus saimiri can be used as an efficient gene expression vector for human T lymphocytes and thus may allow applications in experimental leukemia therapy. We constructed recombinant viruses for the functional expression of the thymidine kinase (TK) of herpes simplex virus type 1 (HSV) as a suicide gene. These viruses reliably allowed the targeted elimination of transduced nonpermissive human T cells in vitro after the administration of ganciclovir. To test the reliability of this function under the most stringent permissive conditions, in this study we analyzed the influence of the prodrugs ganciclovir and acyclovir in common marmosets on the acute leukemogenesis induced by either wild-type herpesvirus saimiri C488 or by a recombinant derivative expressing TK of HSV. Antiviral drug treatment did not influence the rapid development of acute disease. In contrast, the presence of the HSV tk gene resulted in a faster disease progression. In addition, HSV TK-expressing viruses showed faster replication than wild-type virus in culture at low serum concentrations. Thus, HSV TK accelerates the replication of herpesvirus saimiri and enhances its pathogenicity. This should be generally considered when HSV TK is applied as a transgene in replication-competent DNA virus vectors for gene therapy., (Copyright 2000 Academic Press.)

The transformation-associated region of herpesvirus saimiri strains is variable, whereas other parts of the virus genome are highly conserved. However, we observed considerable interstrain sequence divergence of the early viral regulatory orf50 gene, which encodes the R transactivator, a homolog of Epstein-Barr virus BRLF1. The orf50 gene of strain C488 was transcribed at low abundance during lytic infection, whereas antisense transcripts were simultaneously expressed at high levels. A spliced variant, orf50a, was detectable by RT-PCR and RNase protection assays in stimulated C488-transformed, nonpermissive human T cells. In contrast to strain A11, the short, unspliced orf50b form of C488 displayed complete transactivation capability on the orf6 and orf57 promoters. In summary, there are unexpected structural and functional differences between the orf50 genes of herpesvirus saimiri strains, which differ in their capability to transform human T lymphocytes., (Copyright 2000 Academic Press.)

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.

The immediate-early gene ie14/vsag of herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with either ie14/vsag deletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viral ie14/vsag gene.