HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days—Implications for HIV Remission

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to ‘purge’ the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different ‘reactivation founder’ viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.     

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP⁺-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

PRKX critically regulates endothelial cell proliferation, migration, and vascular-like structure formation

Angiogenesis is a fundamental step in several important physiological events and pathological conditions including embryonic development, wound repair, tumor growth and metastasis. PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX has also been shown to be phylogenetically and functionally distinct from PKA. This study presents the first evidence that PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation, which are the three essential processes for angiogenesis. In contrast, classic PKA demonstrated an inhibitory effect on endothelia vascular-like structure formation. Our findings suggest that PRKX is an important protein kinase engaged in the regulation of angiogenesis and could play critical roles in various physiological and pathological conditions involving angiogenesis. PRKX binds to Pin-1, Magi-1 and Bag-3, which regulate cell proliferation, apoptosis, differentiation and tumorigenesis. The interaction of PRKX with Pin-1, Magi-1 and Bag-3 could contribute to the stimulating role of PRKX in angiogenesis.     

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.     

Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.     

Porcine reproductive and respiratory syndrome (PRRS) virus in wild boar and Iberian pigs in south-central Spain

Porcine reproductive and respiratory syndrome (PRRS) is a swine infectious disease causing major economic problems on the intensive pig industry. This virus has been reported worldwide in domestic pigs and there is evidence of PRRS virus (PRRSV) infection in wild boar (Sus scrofa). Nonetheless, the epidemiological role of wild boar and extensively kept domestic pigs remains unclear. The aim of this study was to determine the occurrence of PRRS in wild boar and Iberian pigs in the dehesa ecosystem of the Castile-La Mancha region of Spain, which boasts one of the most important free-roaming porcine livestock and hunting industries in the country. Using geo-spatial analysis of literature data, we first explored the relationship between domestic pig density and PRRS occurrence in wild boar in Europe. Results revealed that PRRS occurrence in wild boar may be influenced, albeit not significantly, by domestic pig density. Next, we analyzed sera from 294 wild boar and 80 Iberian pigs by indirect enzyme-linked immunosorbent assay for PRRSV antibodies. The sera and 27 wild boar tissue samples were analyzed by two real-time RT-PCR assays, targeting the most conserved genes of the PRRSV genome, ORF1 and ORF7. Seven wild boar (2.4 %) and one Iberian pig (1.3 %) were seropositive, while none of the animals tested positive for PRRSV by RT-PCR. Our results confirm the limited spread of PRRSV in free-roaming Iberian pigs and wild boar living in mutual contact. Further studies would be necessary to address whether this low seroprevalence found in these animals reflects transmission from intensively kept pigs or the independent circulation of specific strains in free-roaming pigs.