Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of P. pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold. DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect. Cultures grown to 1.15-2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10(10)/mL gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with Sacl gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 microF, and 186 omega. The transformation efficiency at optimal conditions reached 4 x 10(6) transformants/microgram DNA with pPIC9K. A maximum of 2.6 x 10(5) transformants was produced when 1 microgram of pPIC9K DNA was used.

Flockhouse virus (FHV) is a member of the nodavirus group ofpositive-strand RNA viruses. In the absence of additional compounds, a template-dependent RNAdependent RNA polymerase extracted from FHV-infected cells synthesizes complementary (-)-strand copies of added FHV RNA to yield a double-stranded RNA product. Upon in of glycerophosphoilpid (GPL) Complete replication of FHV RNA by the membraneassociated FHV replicase depended on the absence of membrane-disrupting detergents and the presentation of template RNA in combination with Lipofectin (BRL), a reagent commonly used to introduce RNA and DNA into membrane compartments (4, 7). In the presence of detergent or absence of Lipofectin, added FHV RNA was copied to give (-)-strand RNA, but no new (+)-strands were synthesized.Lipofectin and its analogs are mixtures of a specific glycerophospholipid (GPL) and a cationic lipid. We have now analyzed the contribution of each component and found that only the GPL is required for complete FHV RNA replication in vitro. We further show that a number of GPLs support complete FHV RNA replication and that the activity of effective GPLs is dependent on the presence and structure of both the polar head group and the acyl chains. The amount of GPL required to support (+)-strand synthesis increases as more FHV replicase extract is added to the reaction. This result, the dispensability of the cationic lipid, and other observations suggest that the role of GPL in facilitating (+)-strand RNA synthesis is independent of delivery of the initial template RNA. Since the RNA replication complexes of most or all (+)-strand RNA viruses are membraneassociated and (+)-strand RNA synthesis has generally been the block to complete RNA replication in vitro, the effects of GPL on FHV RNA replication might have general implications for many viruses. MATERIALS AND METHODSPhospbolrps and Related Compounds. The following compounds were obtained from Sigma in the highest purity grades available: dimethyldioctadecylammonium bromide (DDAB); L-a-phosphatidic acid (PA, from egg yolk); L-a-phosphatidylcholine (PC, from egg yolk); L-a-phosphatidylethanolamine, dioleoyl (PE, synthetic); L-a-phosphatidyl-racglycerol, dioleoyl (PG, synthetic); L-a-phosphatidylinositol (PI, from soybean); L-a-phosphatidyl-L-serine (PS, from bovine brain); sphingomyelin (SPM, from bovine brain); cholesterol; 1,2-dimyristoyl-rac-glycerol [14:0 (i.e., carbons: double bonds in each acyl chain = 14:0)]; 1,2-dioleoyl-racglycerol [18:1 (double bond position = cis-9)

Alteration of lysosome acidity has been implicated in many biological events ranging from apoptosis to cancer metastasis, etc. Mesoporous silica nanoparticles doped with acid activable rhodamine-lactam and fluorescein isothiocyanate (FITC) were developed for ratiometric sensing of lysosomal pH changes in live cells with flow cytometry.

Reliable long-term in vivo tracking of stem cells is of great importance in stem cell-based therapy and research. Fluorescence imaging with in situ excitation has significant autofluorescence background, which results in poor signal-to-noise ratio (SNR). Here we report TAT penetrating peptide-bioconjugated long persistent luminescence nanoparticles (LPLNP-TAT) for long-term tracking of adipose-derived stem cells (ASC) without constant external excitation. LPLNP-TAT exhibits near-infrared emitting, red light renewable capability, and superior in vivo imaging depth and SNR compared with conventional organic dye and quantum dots. Our findings show that LPLNP-TAT can successfully label ASC without impairing their proliferation and differentiation and can effectively track ASC in skin-regeneration and tumor-homing models. We believe that LPLNP-TAT represents a new generation of cell tracking probes and will have broad application in diagnosis and therapy.

Persistent luminescence phosphors are a novel group of promising luminescent materials with afterglow properties after the stoppage of excitation. In the past decade, persistent luminescence nanoparticles (PLNPs) with intriguing optical properties have attracted a wide range of attention in various areas. Especially in recent years, the development and applications in biomedical fields have been widely explored. Owing to the efficient elimination of the autofluorescence interferences from biotissues and the ultra-long near-infrared afterglow emission, many researches have focused on the manipulation of PLNPs in biosensing, cell tracking, bioimaging and cancer therapy. These achievements stimulated the growing interest in designing new types of PLNPs with desired superior characteristics and multiple functions. In this review, we summarize the works on synthesis methods, bioapplications, biomembrane modification and biosafety of PLNPs and highlight the recent advances in biosensing, imaging and imaging-guided therapy. We further discuss the new types of PLNPs as a newly emerged class of functional biomaterials for multiple applications. Finally, the remaining problems and challenges are discussed with suggestions and prospects for potential future directions in the biomedical applications.