We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs. [J Natl Cancer Inst 82:1477–1484, 1990]

Infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in a demyelinating encephalomyelitis characterized by mononuclear cell infiltration and white matter destruction similar to the pathology of the human demyelinating disease multiple sclerosis. The contributions of CD4⁺and CD8⁺T cells in the pathogenesis of the disease were investigated. Significantly less severe inflammation and demyelination were observed in CD4^−/−mice than in CD8^−/−and C57BL/6 mice (P≤ 0.002 andP≤ 0.001, respectively). Immunophenotyping of central nervous system (CNS) infiltrates revealed that CD4^−/−mice had a significant reduction in numbers of activated macrophages/microglial cells in the brain compared to the numbers in CD8^−/−and C57BL/6 mice, indicating a role for these cells in myelin destruction. Furthermore, CD4^−/−mice displayed lower levels of RANTES (a C-C chemokine) mRNA transcripts and protein, suggesting a role for this molecule in the pathogenesis of MHV-induced neurologic disease. Administration of RANTES antisera to MHV-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination (P≤ 0.001) compared to those in control mice. These data indicate that CD4⁺T cells have a pivotal role in accelerating CNS inflammation and demyelination within infected mice, possibly by regulating RANTES expression, which in turn coordinates the trafficking of macrophages into the CNS, leading to myelin destruction.

The rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses. We report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies. The MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates. This article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova.

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3′-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys⁶⁴ strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.