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Controlling chronic pain in humans is a major unresolved problem. Spinal cord astrocytes and microglia are critically involved in the creation and maintenance of diverse enhanced pain states via the release of proinflammatory cytokines. Interluekin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production and activity. Control of chronic neuropathic pain such as sensitivity to light touch (allodynia) requires chronic spinal delivery of IL-10. Chronic pain can be surgically induced in rats by loose ligature of chromic cat gut around the sciatic nerve in four places. This creates a chronic constriction injury (CCI) as the nerve bundle swells against the irritating ligature. The CCI model produces an enhanced pain phenotype for 3 months. We have demonstrated that this pain is reversed transiently (4hrs.) through intrathecal injection of recombinant IL-10 protein and that two temporally spaced injections of an IL-10 expression vector containing inverted terminal repeats (ITRs) reverses this pain long term (3+ months, Milligan et al., these proceedings). The role of ITRs in gene expression is not well known, however there is evidence that they possess transcription promoter and enhancer activity (Flotte et al., 1992 Am J Respir Cell Mol Biol. 7:p349-56 and 1993 J Biol Chem. 268: p3781-90). Based on this and other evidence in the literature we sought to determine if the ITRs in our expression vector, which are located 5′ of the CMV enhancer and Chicken β-actin promoter (5′ ITR) and 3′ of the SV 40 poly A tail sequence (3′ ITR), influence reversal of pain in the CCI model. To this end we deleted both ITR's from the expression vector and found that it was incapable of producing pain reversal. We next asked if one or both ITRs are necessary for pain reversal. We found that plasmids containing just the 3′ or 5′ ITR are not as robust in reversing chronic pain. We have also found that ITRs are required on the first injection but not on a second injection for long-term pain relief. In other words, initial injection of a plasmid containing ITRs seems to “prime” the system making the second injection of the expression vector vastly more efficacious. Ongoing studies seek to further characterize the role of ITRs on gene expression and pain reversal in the CCI model.Molecular Therapy (2006) 13, S100–S100; doi: 10.1016/j.ymthe.2006.08.287 [ABSTRACT FROM AUTHOR]

Transgender and gender diverse (TGD) people have a variety of ways of embodying their gender. We present preliminary work on The Gender Embodiment Scale for trans masculine individuals as a collaborative product from a trans-identified community-engaged team. This scale provides researchers and clinicians a survey to diversify ways gender is understood and counteracts assumptions of a singular gender experience for TGD people. This scale reflects gender embodiment as individually unique and inclusive of the body, behavior, and social treatment. Use of the scale can enhance discussion and enable assessments regarding relative importance and satisfaction across items in these domains., Competing Interests: None of the authors have any conflicts of interest to disclose., (Copyright 2022, Mary Ann Liebert, Inc., publishers.)

The presence of adenoviral cis-elements interfering with the activity of tissue-specific promoters has seriously impaired the use of transcriptional targeting adenoviruses for gene therapy purposes. As an approach to overcome this limitation, transcription terminators were previously employed in cultured cells to insulate a transgene promoter from viral activation. To extend these studies in vivo, we have injected into heart and skeletal muscle, adenoviruses containing the human growth hormone terminator and the cardiac-specific alpha-myosin heavy chain promoter (alphaMyHC) driving the chloramphenicol acetyltransferase (CAT) reporter gene. Promoterless CAT constructs were also tested to study interfering viral transcription and terminator activity. Here we demonstrate that the presence of a terminator can produce undesirable effects on the activity of heterologous promoters. Our analysis shows that in particular conditions, a terminator can reduce the tissue specificity of the transgene promoter. By RNAse protection assay performed on cardiac myocytes, we also show that adenoviral elements can direct high levels of autonomous transcription within the E1A enhancer region. This finding supports the model that passive readthrough of the transgene promoter is responsible for loss of selective expression.

An adenovirus type 5 mutant deleted for the preterminal protein (pTP) gene was constructed using cell lines that express pTP. The pTP deletion mutant virus is incapable of replicating in the absence of complementation and does not express detectable levels of viral mRNAs that are expressed only after the onset of replication. Accumulation of early-region mRNAs, including that for E1A, exhibits a lag relative to that observed from the wild-type virus. However, E1A mRNA accumulation attains a steady-state level similar to the level of expression during the early phase of infection with the wild-type virus. In 293-pTP cells (human embryonic kidney cells that express pTP in addition to high levels of adenovirus E1A and E1B proteins), the pTP deletion mutant virus replicates efficiently and yields infectious titers within 5-fold of that of the wild-type virus. The deletion of 1.2 kb of pTP-encoding sequence increases the size of foreign DNA that can be introduced into the virus and, with an absolute block to replication, makes this virus an important tool for gene therapy.

293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein (pTP) under the control of a tetracycline-dependent promoter were constructed. To construct the cell lines expressing pTP, 293 cells were stably transfected with a plasmid encoding the tetracycline repressor/VP16 transactivator protein (tTA) using selection with hygromycin. Cell lines that expressed high levels of tTA activity were then stably transfected with plasmids in which pTP expression is directed by the tTA-dependent promoter from either a cDNA or a modified genomic construct using selection with G418. Cell lines that expressed high, inducible levels of pTP efficiently complemented a temperature-sensitive pTP mutant virus for growth and plaque formation at the nonpermissive temperature.

Oncogene products not localized to the nucleus regulate the expression of a diverse group of genes. The identities of genes regulated by non-nuclear oncogenes can supply insights into the changes at the cellular level that accompany the altered expression of such genes during the multi-step process of carcinogenesis. For example, one set of genes whose expression is affected by non-nuclear oncogenes are genes encoding extracellular proteases and components of the extracellular matrix. The expression of these genes during tumorigenesis could have important consequences for tumor invasiveness and metastasis. Genes regulated by non-nuclear oncogenes also define signal transduction pathways that allow communication between the plasma membrane and the nucleus. Oncogene-regulated nuclear targets provide a tool to approach the problem of cellular signal transduction and may contribute a clearer view of intracellular signaling pathways and the interactions between these pathways during cell growth and differentiation. Studying the regulation of these genes has revealed that c-jun and members of the ets family of transcription factors are important nuclear targets for the action of several non-nuclear oncogenes. This approach has also indicated that ras p21 is necessary for selective signal transduction events mediated by receptor and nonreceptor tyrosine kinases, including the colony-stimulating factor 1 (CSF-1) receptor, the product of the c-fms gene.

The practice of liaison psychiatry has from its inception been hampered by an inadequate or non-existing funding base. A model is presented for funding an authentic liaison training program, fully supported by consultation-generated revenue. A specific description of the liaison teaching unit is given, which illustrates how the following objectives of the program were successfully met: provision of comprehensive biopsychosocial care, dissemination of psychological skills and knowledge to nonpsychiatrist staff, training of the liaison fellow, and generation of sufficient revenue to offset its costs.

A nucleotide sequence for the mRNA of the major nucleocapsid (N) protein gene of respiratory syncytial virus was reported previously (N. Elango and S. Venkatesen, 1983, Nucleic Acids Res. 11, 5941-5951). However, we have been unable to confirm part of this sequence as N mRNA-specific and suggest that the published sequence represents that of an aberrant chimeric transcript. Here we present an alternative sequence for the N mRNA and provide data supporting its authenticity. The corrected N mRNA sequence contains 1197 rather than 1427 nucleotides exclusive of poly(A), and encodes a protein of 391 rather than 467 amino acids. The calculated molecular weight for the 391-amino acid protein described by the sequence presented here is 42,600, in agreement with the molecular weight of 42,000 determined for the RS viral N protein by gel electrophoresis. In addition, we present sequence data from dicistronic RNAs that span the junction between the 1B protein and N cistrons, and the junction between the N and phosphoprotein (P) cistrons.