Prp8 protein is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 snRNPs. Intriguingly, although it is ubiquitously expressed, mutations in the C-terminus of human Prp8p cause the retina-specific disease Retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks a mature U5 snRNP component, Brr2p, and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes, which is disrupted by mutations that cause human RP.Nuclear pre-mRNA splicing is an essential housekeeping process in all eukaryotic cells. It is catalyzed by a large ribonucleoprotein (RNP) complex called the spliceosome, which contains the small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6, as well as many nonsnRNP proteins1, 2. Each snRNP consists of an snRNA, a set of specific proteins, and seven common Sm proteins or, in the case of U6 snRNP, seven Lsm proteins.Unexpectedly, mutations in four human snRNP-associated proteins, PRPF83, PRPF314, PRPF35 and PAP-1/RP96, 7 were found in patients with a dominantly inherited form of retinal degeneration, Retinitis pigmentosa (RP). Here, we investigate the role of Prp8p (the yeast ortholog of PRPF8) in U5 snRNP biogenesis in Saccharomyces cerevisiae, and the effect of RP mutations on this process.Biogenesis of the U snRNPs has been studied extensively in metazoans1, 8. The U1, U2, U4 and U5 snRNAs are produced as precursors in the nucleus by RNA polymerase II then exported to the cytoplasm, facilitated by nuclear cap-binding proteins and the export factors, CRM1 and PHAX8. In the cytoplasm seven Sm proteins bind to the snRNAs, facilitated by the SMN complex9, 10, and the m 7 G cap is hypermethylated to form a 2,2,7-

PurposeStaphylococcus aureus is the most common persistent pathogen in humans, so development of new formulations to combat pathogen invasion is quite necessary.MethodsIn the current study, for the first time, the synergistic activity of recombinant lysostaphin and LL-37 peptide was studied against S. aureus. Moreover, different niosomal formulations of the peptide and protein were prepared and analyzed in terms of size, shape, zeta potential, and entrapment efficiency. Also, a long-term antibacterial activity of the best niosomal formulation and free forms was measured against S. aureus in vitro.ResultsThe optimal niosomal formulation was obtained by mixing the surfactants (span60 and tween60; 2:1 w/w), cholesterol, and dicetylphosphate at a ratio of 47:47:6, respectively. They showed uniform spherical shapes with the size of 565 and 325 nm for lysostaphin and LL-37, respectively. This formulation showed high entrapment efficiency for the peptide, protein, and a slow-release profile over time. Release kinetic was best fitted by Higuchi model indicating a diffusion-based release of the drugs. The lysostaphin/LL-37 niosomal formulation synergistically inhibited growth of S. aureus for up to 72 hours. However, the same amounts of free forms of both anti-microbial agents could not hold the anti-microbial effect and growth was seen in the following 72 hours. Cytotoxicity assay specified that lysostaphin/LL-37 niosomal combination had no deleterious effect on normal fibroblast cells at effective antimicrobial concentrations.ConclusionThis study indicated that the use of lysostaphin in combination with LL-37, either in niosomal or free forms, synergistically inhibited growth of S. aureus in vitro. In addition, niosomal preparation of antimicrobial agents could provide a long-term protection against bacterial infections.

Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

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PRPF8-retinitis pigmentosa is said to be severe but there has been no overview of phenotype across different mutations. We screened RP patients for PRPF8 mutations and identified three new missense mutations, including the first documented mutation outside exon 42 and the first de novo mutation. This brings the known RP-causing mutations in PRPF8 to nineteen. We then collated clinical data from new and published cases to determine an accurate prognosis for PRPF8-RP. Clinical data for 75 PRPF8-RP patients were compared, revealing that while the effect on peripheral retinal function is severe, patients generally retain good visual acuity in at least one eye until the fifth or sixth decade. We also noted that prognosis for PRPF8-RP differs with different mutations, with p.H2309P or p.H2309R having a worse prognosis than p.R2310K. This correlates with the observed difference in growth defect severity in yeast lines carrying the equivalent mutations, though such correlation remains tentative given the limited number of mutations for which information is available. The yeast phenotype is caused by lack of mature spliceosomes in the nucleus, leading to reduced RNA splicing function. Correlation between yeast and human phenotypes suggests that splicing factor RP may also result from an underlying splicing deficit.

The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

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