Novel Translation Products from Simian Immunodeficiency Virus SIVmac239 Env-Encoding mRNA Contain both Rev and Cryptic T-Cell Epitopes

Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins.The pathway from viral infection to the cellular immune response is not well understood. Despite the importance of T-cell responses in control of AIDS virus replication (1, 3, 8, 22), the sources of the peptides recognized by virus-specific T cells are still being discovered. AIDS virus-specific CD8⁺ T lymphocytes (CD8-TL) recognize complexes of major histocompatibility complex (MHC) class I and virus-derived epitopes presented on the surface of infected cells. These epitopes can be derived from exogenous viral proteins in the infecting virion (19, 20) or from de novo synthesis of viral proteins (9, 21). Additional sources of epitopes are also being explored (4, 6).CD8-TL can also recognize epitopes derived from translation of viral alternate reading frames (ARFs). Though CD8-TL specific for ARF-derived epitopes have been detected in human immunodeficiency virus (HIV) (2), they remain a largely unexplored source of epitopes that might elicit potent antiviral cellular immune responses. We recently showed that SIVmac239-infected rhesus macaques that spontaneously controlled viral replication, termed elite controllers, made immunodominant CD8-TL responses against an epitope (RHLAFKCLW, or cRW9) derived from an ARF of the env gene (15). This response selected for viral escape in vivo and suppressed viral replication in an in vitro assay. These findings imply that CD8-TL specific for ARF-derived epitopes might be an important component of the total AIDS virus-specific cellular immune response.Here, we show that the cRW9 epitope is translated as part of two distinct products that differ in size due to start codon usage. The larger and more frequent product contains both the first 23 amino acids of the Rev protein (exon 1) and 50 amino acids translated from the rev intron. The smaller is produced by translation initiation at a start codon within the rev intron and is a subset of the larger product. Finally, we show that these products are degraded after translation from the mature Env-encoding mRNA.     

A biochemical basis for induction of retina regeneration by antioxidants

The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. Embryonic chicks are able to regenerate their retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye only if a source of exogenous factors, such as FGF2, is present. This study shows that NAC modifies the redox status of the CM, initiates self-renewal of the stem/progenitor cells, and induces regeneration in the absence of FGF2. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with different biochemical structures and modes of action, and found that NAC induces regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway.     

Towards new sources of resistance to the currant-lettuce aphid (<Emphasis Type="Italic">Nasonovia ribisnigri</Emphasis>)

Domesticated lettuce varieties encompass much morphological variation across a range of crop type groups, with large collections of cultivars and landrace accessions maintained in genebanks. Additional variation not captured during domestication, present in ancestral wild relatives, represents a potentially rich source of alleles that can deliver to sustainable crop production. However, these large collections are difficult and costly to screen for many agronomically important traits. In this paper, we describe the generation of a diversity collection of 96 lettuce and wild species accessions that are amenable to routine phenotypic analysis and their genotypic characterization with a panel of 682 newly developed expressed sequence tag (EST)-linked KASP? single nucleotide polymorphism (SNP) markers that are anchored to the draft Lactuca sativa genome assembly. To exemplify the utility of these resources, we screened the collection for putative sources of resistance to currant-lettuce aphid (Nasonovia ribisnigri) and carried out association analyses to look for potential SNPs linked to resistance.     

Isolation and characterization of microsatellite loci from the endangered Mediterranean sponge Spongia agaricina (Demospongiae: Dictyoceratida)

The abundance of the bath sponge Spongia agaricina has decreased drastically in recent years and it is now considered an endangered species under Annex 3 of Bern and Barcelona conventions. We describe eight microsatellite markers and present data on their allelic variation and utility as high resolution genetic markers. We analyzed 36 individuals from two populations and found that the number of alleles per locus ranged between 1 and 7. Observed heterozygosity ranged from 0 to 0.72. We found deviations from Hardy–Weinberg expectations for some loci. We exclusively detected null alleles for those loci that deviated from Hardy–Weinberg expectations. Also, distributions of allele frequencies differed significantly between the two populations, making them suitable for population genetic analyses.     

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation

The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.     

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.     

Plasma visfatin concentrations in severely obese subjects are increased after intestinal bypass

Objective: Visfatin has shown to be increased in obesity and in type 2 diabetes. The aim of this study was to determine the change in plasma visfatin in severely obese (SO) persons after weight loss following bariatric surgery in relation to glucose concentration. Research Methods and Procedures: Visfatin and leptin were studied in 53 SO persons (BMI, 54.4 ± 6.8 kg/m²) before and 7 months after bariatric surgery and in 28 healthy persons (BMI, 26.8 ± 3.8 kg/m²). All of the patients underwent bariatric surgery with biliopancreatic diversion or gastric bypass. Results: The pre‐surgery levels of visfatin in the SO group were greater than in the control group (55.9 ± 39.9 vs. 42.9 ± 16.6 ng/mL, p = 0.024). This increase was significant in the SO group with impaired fasting glucose (63.4 ± 36.6 ng/mL) and diabetes (60.0 ± 46.0 ng/mL). SO patients with normal fasting glucose had similar levels of visfatin to the controls. Seven months after surgery, visfatin levels were significantly increased (84.8 ± 32.8 ng/mL, p < 0.001). This increase was independent of the pre‐surgical glucose levels. The type of bariatric surgery had no influence on visfatin levels. Post‐surgical visfatin was significantly correlated with the post‐surgery plasma concentrations of leptin (r = 0.39, p = 0.014). Discussion: Plasma levels of visfatin in the SO group were increased but only when accompanied by high glucose levels, even in the range of impaired fasting glucose. Bariatric surgery causes an increase in visfatin, which is correlated mainly with the changes produced in the leptin concentration.     

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background

The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings

In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions

Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.