Publications

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3' untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.

An association between chronic pain conditions and alcohol dependence has been revealed in numerous studies with episodes of alcohol abuse antedating chronic pain in some people and alcohol dependence emerging after the onset of chronic pain in others. Alcohol dependence and chronic pain share common neural circuits giving rise to the possibility that chronic pain states could significantly affect alcohol use patterns and that alcohol dependence could influence pain sensitivity. The reward and emotional pathways that regulate drug/alcohol addiction also mediate chronic pain. For example, pain-evoked activation of brain learning and brain reward circuitry may modulate cortical processing of pain and central sensitization mediated by mesocorticolimbic circuitry. Imbalance and reorganization of amygdala-mPFC interactions may not only be important for persistent pain, but also for disorders characterized by the abnormal persistence of emotional-affective states such as drug and alcohol addiction. Further studies are necessary to understand how these neural circuits are regulated in comorbid conditions of alcoholism and chronic pain. In addition, long term alcohol use could induce pain symptoms and may exacerbate chronic pain arising from other sources. While prior studies have established a role of neuroendocrine stress axis mediators in alcohol abuse and neurotoxic effects, these studies have not explored the distinction between the individual impact of alcohol and stress hormones. Future studies should explore the mechanisms mediating the contribution of alcohol and stress axis hormones on pain, an important question in our understanding of the neurobiology of alcohol abuse and chronic pain.

Determining how somatic copy number alterations (SCNAs) promote cancer is an important goal. We characterized SCNA patterns in 4,934 cancers from The Cancer Genome Atlas Pan-Cancer data set. Whole-genome doubling, observed in 37% of cancers, was associated with higher rates of every other type of SCNA, TP53 mutations, CCNE1 amplifications and alterations of the PPP2R complex. SCNAs that were internal to chromosomes tended to be shorter than telomere-bounded SCNAs, suggesting different mechanisms underlying their generation. Significantly recurrent focal SCNAs were observed in 140 regions, including 102 without known oncogene or tumor suppressor gene targets and 50 with significantly mutated genes. Amplified regions without known oncogenes were enriched for genes involved in epigenetic regulation. When levels of genomic disruption were accounted for, 7% of region pairs were anticorrelated, and these regions tended to encompass genes whose proteins physically interact, suggesting related functions. These results provide insights into mechanisms of generation and functional consequences of cancer-related SCNAs.

PURPOSE

Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for , the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for splice variants in mouse.

MATERIALS AND METHODS

Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-length cDNA or a cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots.

RESULTS

PCR products for transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length transcript was significantly excluded using the exon exclusion method, no bands corresponding to splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein.

CONCLUSIONS

The data provide evidence for the existence of a single, full-length transcript that could give rise to a single protein product that undergoes posttranslational processing.

BACKGROUND AND AIMS

The Centers for Disease Control and Prevention recommend screening for hepatitis C virus (HCV) in patients with injection drug use, blood transfusion before 1992, stigmata of liver disease, or born between 1945 and 1965. The purpose of this study was to examine risk factors for HCV acquisition in Asian Americans.

METHODS

This was a case-controlled study, with 471 consecutive patients testing positive for anti-HCV between January 2001 and December 2008. Controls included 471 patients with negative HCV matched at a one-to-one ratio for sex, age (±5 years), and ethnicity.

RESULTS

For Asian patients, the most common risk factors were blood transfusion and acupuncture or exposure to dirty needles (27 and 20 %, respectively). On multiple logistic regression, potential predictors for a positive anti-HCV test in Asians were acupuncture or exposure to dirty needles (OR = 12.9, P < 0.0001), body tattoo (OR = 12.0, P = 0.001), and history of blood transfusion (OR = 5.7, P < 0.0001).

DISCUSSION

Acupuncture and exposure to dirty needles are independent risk factors of HCV infection. Asians coming from endemic areas should be screened for HCV even when commonly-known risk factors for Western patients are not present.

The neural crest is a population of mesenchymal cells that after migrating from the neural tube gives rise to structure and cell types: the jaw, part of the peripheral ganglia, and melanocytes. Although much is known about neural crest development in jawed vertebrates, a clear picture of trunk neural crest development for elasmobranchs is yet to be developed. Here we present a detailed study of trunk neural crest development in the bamboo shark, Chiloscyllium punctatum. Vital labeling with dioctadecyl tetramethylindocarbocyanine perchlorate (DiI) and in situ hybridization using cloned Sox8 and Sox9 probes demonstrated that trunk neural crest cells follow a pattern similar to the migratory paths already described in zebrafish and amphibians. We found shark trunk neural crest along the rostral side of the somites, the ventromedial pathway, the branchial arches, the gut, the sensory ganglia, and the nerves. Interestingly, C. punctatum Sox8 and Sox9 sequences aligned with vertebrate SoxE genes, but appeared to be more ancient than the corresponding vertebrate paralogs. The expression of these two SoxE genes in trunk neural crest cells, especially Sox9, matched the Sox10 migratory patterns observed in teleosts. Also of interest, we observed DiI cells and Sox9 labeling along the lateral line, suggesting that in C. punctatum, glial cells in the lateral line are likely of neural crest origin. Although this has been observed in other vertebrates, we are the first to show that the pattern is present in cartilaginous fishes. These findings demonstrate that trunk neural crest cell development in C. punctatum follows the same highly conserved migratory pattern observed in jawed vertebrates.

Cardiovascular disease is a leading cause of death of women around the world. Diet, exercise, smoking cessation, and blood pressure control are all recognized as key elements of preventing cardiovascular disease. Infant feeding has received less attention, but the studies reviewed here indicate that lactation may also play an important role in determining women's future risk of heart disease. A growing body of literature indicates that mothers who prematurely discontinue lactation face increased risk of visceral adiposity, hypertension, hyperlipidemia, diabetes, and subclinical cardiovascular disease, as well as cardiovascular morbidity and mortality. Breastfeeding is not always easy, but neither is dieting, exercise, smoking cessation, or treating hypertension. In order to effectively fight heart disease, efforts are needed to promote all aspects of a healthy lifestyle, which for women includes breastfeeding their babies.