Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 microM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3'-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5' cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5'SL and 3'CS (targeting the 5'-terminal nucleotides and the 3' cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5'SL and 3'CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 microM 3'CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3'CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5'SL and 3'CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5'SL and 3'CS compounds as potential DEN virus therapeutics is warranted.     

Non-Complexed Four Cascade Enzyme Mixture: Simple Purification and Synergetic Co-stabilization

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10⁹ mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.     

Physicochemical Characterization of Efavirenz–Cyclodextrin Inclusion Complexes

Efavirenz (EFV) is an oral antihuman immunodeficiency virus type 1 drug with extremely poor aqueous solubility. Thus, its gastrointestinal absorption is limited by the dissolution rate of the drug. The objective of this study was to characterize the inclusion complexes of EFV with β-cyclodextrin (β-CD), hydroxypropyl β-CD (HPβCD), and randomly methylated β-CD (RMβCD) to improve the solubility and dissolution of EFV. The inclusion complexation of EFV with cyclodextrins in the liquid state was characterized by phase solubility studies. The solid-state characterization of various EFV and CD systems was performed by X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy analyses. Dissolution studies were carried out in distilled water using US Pharmacopeia dissolution rate testing equipment. Phase solubility studies provided an A_L-type solubility diagram for β-CD and A_P-type solubility diagram for HPβCD and RMβCD. The phase solubility data enabled calculating stability constants (K _s) for EFV-βCD, EFV-HPβCD, and EFV-RMβCD systems which were 288, 469, and 1,073 M⁻¹, respectively. The physical and kneaded mixtures of EFV with CDs generally provided higher dissolution of EFV as expected. The dissolution of EFV was substantially higher with HPβCD and RMβCD inclusion complexes prepared by the freeze drying method. Thus, complexation with HPβCD and RMβCD could possibly improve the dissolution rate-limited absorption of EFV.     

Outlook for cellulase improvement: screening and selection strategies

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.     

Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.     

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D(9)-glycine betaine, D(9)-trimethylamine-N-oxide, (13)C(2)-arsenobetaine, D(6)-DMSP, and D(4)-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r(2) values=0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.     

The catalase activity of Nalpha-acetyl-microperoxidase-8.

Nalpha-Acetylated microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for peroxidases. We report here that Ac-MP-8 catalyzes catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The catalase activity of Ac-MP-8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The Km and kcat of the decomposition of hydrogen peroxide (H2O2) catalyzed by Ac-MP-8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (kcat/Km) of Ac-MP-8 in catalase-type reaction of H2O2 is 100.2,/m/s, which is 5- to 12- and 50- to 100-fold less than those of MPs in cytochrome P450-type reaction of aniline/H2O2 and peroxidase-type reaction of o-methoxyphenol/H2O2, respectively. These results indicate that Ac-MP-8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroxidase-type > cytochrome P450-type > catalase-type reactions. Comparisons of the enzyme activities of Ac-MP-8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.     

Silencing of 4-coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production

? The lignin content of feedstock has been proposed as one key agronomic trait impacting biofuel production from lignocellulosic biomass. 4-Coumarate:coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthethic pathway. ? Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass (Panicum virgatum) through phylogenetic analysis. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down-regulated. ? RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. Altered lignification patterns in the stems of RNAi transgenic plants were observed with phloroglucinol-HCl staining. The transgenic plants also had uncompromised biomass yields. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency. ? The results demonstrate that Pv4CL1, but not Pv4CL2, is the key 4CL isozyme involved in lignin biosynthesis, and reducing lignin content in switchgrass biomass by silencing Pv4CL1 can remarkably increase the efficiency of fermentable sugar release for biofuel production.     

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

hTERT is the catalytic subunit of the telomerase complex. Elevated expression of hTERT is associated with the expansion and metastasis of gastric tumor. In this study, we aimed to identify novel tumor suppressor miRNAs that restrain hTERT expression. We began our screen for hTERT-targeting miRNAs with a miRNA microarray. miRNA candidates were further filtered by bioinformatic analysis, general expression pattern in different cell lines, gain-of-function effects on hTERT protein and the potential of these effects to suppress hTERT 3′ untranslated region (3′UTR) luciferase activity. The clinical relevance of two miRNAs (miR-1207-5p and miR-1266) was evaluated by real-time RT-PCR. The effects of these miRNAs on cell growth, cell cycle and invasion of gastric cancer cells were measured with CCK-8, flow cytometry and transwell assays. Finally, the ability of these miRNAs to suppress the transplanted tumors was also investigated. Fourteen miRNAs were identified using a combination of bioinformatics and miRNA microarray analysis. Of these fourteen miRNAs, nine were expressed at significantly lower levels in hTERT-positive cell lines compared with hTERT-negative cell lines and five could downregulate hTERT protein expression. Only miR-1207-5p and miR-1266 interacted with the 3′ UTR of hTERT and the expression levels of these two miRNAs were significantly decreased in gastric cancer tissues. These two miRNAs also inhibited gastric tumor growth in vitro and in vivo. Altogether, miR-1207-5p and miR-1266 were determined to be hTERT suppressors in gastric cancer, and the delivery of these two miRNAs represents a novel therapeutic strategy for gastric cancer treatment.