Reciprocal Effects of Splicing and Polyadenylation on Human Immunodeficiency Virus Type 1 Pre-mRNA Processing

AbstractInsertion of a functional splicing cassette into a construct containing the HIV-1 poly(A) site followed by the adenovirus L3 poly(A) site results in both specific stimulation of 3′ end processing at the HIV-1 site and an increase in the steady-state levels of RNA processed at both sites. To further evaluate this influence of splicing on processing of the HIV-1 poly(A) site, defined mutations which alter or abolish splicing of the intron were made and analyzed for their effects on polyadenylation and steady-state levels of RNA. The data show that a point mutation at the 3′ splice site caused activation of a cryptic splice acceptor that is as efficient as the wild-type acceptor. Substitution of this mutant intron for the wild-type intron resulted in stimulation of the HIV-1 poly(A) site to levels equivalent to those caused by the wild-type intron. This mutant did not, however, have as great an effect on steady-state RNA levels as the wild-type intron. A second construct containing a mutated branch point and polypyrimidine tract resulted in abolishment of splicing and a decrease in both poly(A) site use and steady-state levels of RNA. These data demonstrate that the enhanced use of the HIV-1 poly(A) site is a direct result of the splicing reaction, and not merely due to the sequences that were inserted. The effect that poly(A) site strength has on splicing was also addressed. Using activation of the cryptic splice acceptor to indicate changes in splicing efficiency resulting from alterations in poly(A) site strength, it was determined that poly(A) site strength does have an effect on the efficiency of the splicing reaction

Redaction of sensitive data in the publication of dual use research of concern

Editorial. The publication of scientific information that derives from dual use research of concern (DURC) poses major problems for journals because it brings into conflict the benefits of free access to data and the need to prevent misuse of that information by others. Recently, a group of authors and a major scientific journal addressed the issue of publishing information on a newly discovered, highly lethal toxin that can be delivered to large populations and for which there are no available countermeasures. The journal addressed this conflict by permitting the redaction of information that is normally considered essential for publication. This action establishes a precedent for redaction of sensitive data that also provides an example of responsible scientific publishing. However, this precedent leaves many questions unanswered and suggests a need for a discussion by all stakeholders of scientific information so as to derive normative standards for the publication of DURC

The Nuclearization of Biology Is a Threat to Health and Security

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78142/1/bsp.2009.0047.pd

Biodefense Research: A Win-Win Challenge

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63273/1/bsp.2008.1114.pd

Promoter Attenuation in Gene Therapy: Interferon-γ and Tumor Necrosis Factor-α Inhibit Transgene Expression

Overview summary Transgene expression can be eliminated even in the presence of substantial amounts of vector DNA in the transduced cells, which suggests that mechanisms other than the antigen-specific immune response may mediate non-cytodestructive events that determine the presence of transgene expression. Our data indicate that the cytokines interferon-γ) (IFN-γ) and tumor necrosis factor-α (TNF-α) inhibit transgene expression from certain widely used viral promoters/enhancers (human cytomegalovirus immediate early, Rous sarcoma virus long terminal repeat, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral, or plasmid vectors in vivo. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Thus, cytokine-regulated promoter function rather than specific immune destruction could limit transgene expression. These results have significant implications for the construction of transfer vectors for human gene therapy because gene transfer vectors could be exposed to a cytokine-rich environment when they are administered in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63157/1/hum.1997.8.17-2019.pd

Resilience Assessment : International Best Practice Principles

PURPOSE This document sets out international best-practice principles for resilience assessment being undertaken within an impact assessment (IA) of some project, plan, programme, or policy (in this context, its function may be different to that of a self-standing resilience assessment). Resilience assessment can contribute to impact assessment by defining specific disturbances that can lead to failure of natural, social, and engineered systems. The disturbance can be caused either by the proposed action, factors beyond the influence of proposed action, or combination of both. The impact assessment can consider all these factors within one coherent framework. It can identify synergies and knock-on effects that can cause potential system failures, and advise on interventions that avoid failures in the critical functions of the system BACKGROUND Resilience assessment evaluates the structure and function of a system of focus (hereafter ‘focal system’) and, in the context of an impact assessment, focuses on the effects of the proposed action on the resilience of that focal system. The focal system can include: socio-ecological, biophysical, engineering, technological, or other components. Resilience assessment should ideally examine the consequences of the proposed action in combination with internal or external factors that may collectively influence the resilience of the focal system (e.g., biophysical system change caused by global warming on engineered structures)

A safety-modified SV40 Tag developed for human cancer immunotherapy

Simian virus 40 (SV40)-like DNA sequences have been found in a variety of human tumors, raising the possibility that strategies targeting SV40 may provide a potential avenue for immunotherapy directed against SV40 large T Antigen (Tag)-expressing tumors. We generated a recombinant vaccinia (vac-mTag) expressing mTag and herein assessed the ability of mTag to transform cells and to interact with anti-oncoproteins, as well as screened for the presence of potential HLA-A2.1-restricted epitopes within mTag. We found that transfection of cells with mTag did not lead to their transformation. Also, we demonstrated that mTag protein is degraded rapidly in cells. In addition, our work revealed that mTag did not physically interact with certain anti-oncoproteins. Finally, two potential HLA-A2.1-restricted functional epitopes within mTag sequence were identified. Our results show that mTag lacks the oncogenecity of full-length Tag and harbors potential HLA-A2.1-restricted immunogenic epitopes, hence suggesting the safety of vac-mTag for use in cancer immunotherapy

Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the human malignancy Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. KSHV establishes lytic infection of monocytes in vivo , which may represent an important cellular reservoir during KS disease progression. KS tumors consist of latently infected endothelial cells; however, lytic phase gene products are important for KS onset. Early KS lesion progression is driven by proinflammatory cytokines supplied by immune cell infiltrates including T cells and monocytes. KSHV-infected monocytes may supply the lytic viral products and the inflammatory milieu conducive to KS tumor progression. To establish successful infection, KSHV extensively modulates the host immune system. KSHV antigens activate both innate and adaptive immune responses including KSHV-specific T cells, but lifelong infection is still established. Programmed death ligand 1 (PD-L1) is a prosurvival cell surface protein that suppresses T-cell-mediated killing. PD-L1 is variably present on various tumor cells and is a targetable marker for cancer treatment. We show that KSHV infection of human monocytes increases PD-L1 expression and transcription in a dose-dependent manner. We also saw evidence of lytic gene expression in the KSHV-infected monocytes. Intact KSHV is needed for full PD-L1 response in human monocytes. KSHV induces a general proinflammatory cytokine milieu including interleukins 1α, 1β, and 6, which have been implicated in early KS lesion progression. KSHV-mediated PD-L1 increase may represent a novel mechanism of KSHV-mediated immune modulation to allow for virus survival and eventually malignant progression. IMPORTANCE KSHV is the etiologic agent of Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. Programmed death ligand 1 (PD-L1) is an immunosuppressive cell surface marker that inhibits T cell activation. We report that KSHV infection of primary human monocytes upregulates PD-L1 transcription and protein expression. Analysis of the cytokine and chemokine milieu following KSHV infection of monocytes revealed that KSHV induces interleukins 1α, 1β, and 6, all of which have been implicated in KS development. Our work has identified another potential immune evasion strategy for KSHV and a potential target for immunotherapy of KSHV-derived disease

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26708/1/0000258.pd

Fecal DNA versus Fecal Occult Blood for Colorectal-Cancer Screening in an Average-Risk Population

BACKGROUND Although fecal occult-blood testing is the only available noninvasive screening method that reduces the risk of death from colorectal cancer, it has limited sensitivity. We compared an approach that identifies abnormal DNA in stool samples with the Hemoccult II fecal occult-blood test in average-risk, asymptomatic persons 50 years of age or older. METHODS Eligible subjects submitted one stool specimen for DNA analysis, underwent standard Hemoccult II testing, and then underwent colonoscopy. Of 5486 subjects enrolled, 4404 completed all aspects ofthe study. A subgroup of 2507 subjects was analyzed, including all those with a diagnosis of invasive adenocarcinoma or advanced adenoma plus randomly chosen subjects with no polyps or minor polyps. The fecal DNA panel consisted of 21 mutations. RESULTS The fecal DNA panel detected 16 of 31 invasive cancers, whereas Hemoccult II identified 4 of 31 (51.6 percent vs. 12.9 percent, P=0.003). The DNA panel detected 29 of 71 invasive cancers plus adenomas with high-grade dysplasia, whereas Hemoccult II identified 10 of 71 (40.8 percent vs. 14.1 percent, P<0.001). Among 418 subjects with advanced neoplasia (defined as a tubular adenoma at least 1 cm in diameter, a polyp with a villous histologic appearance, a polyp with high-grade dysplasia, or cancer), the DNA panel was positive in 76 (18.2 percent), whereas Hemoccult II was positive in 45 (10.8 percent). Specificity in subjects with negative findings on colonoscopy was 94.4 percent for the fecal DNA panel and 95.2 percent for Hemoccult II. CONCLUSIONS Although the majority of neoplastic lesions identified by colonoscopy were not detected by either noninvasive test, the multitarget analysis of fecal DNA detected a greater proportion of important colorectal neoplasia than did Hemoccult II without compromising specificity