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Background Internal ribosome entry sites (IRESs) allow the translation of a transcript independent of its cap structure. They are distributed in some viruses and cellular RNA. The element is applied in dual gene expression in a single vector. Although it appears the lower efficiency of IRES-mediated translation than that of cap-dependent translation, it is with the crucial needs to know the precise differences in translational efficacy between upstream cistrons (cap-dependent) and downstream cistrons (IRES-mediate, cap-independent) before applying the bicistronic vector in biomedical applications. Material and methods This study aimed to provide real examples and showed the precise differences for translational efficiency dependent upon target gene locations. We generated various bicistronic constructs with quantifiable reporter genes as upstream and downstream cistrons of the encephalomyocarditis virus (EMCV) IRES to precisely evaluate the efficacy of IRES-mediated translation in mammalian cells. Results There was no significant difference in protein production when the reporter gene was cloned as an upstream cistron. However, lower levels of protein production were obtained when the reporter gene was located downstream of the IRES. Moreover, in the presence of an upstream cistron, a markedly reduced level of protein production was observed. Conclusion Our findings demonstrate the version of the EMCV IRES that is provided in many commercial vectors is relatively less efficient than cap-dependent translation and provide valuable information regarding the utilization of IRES to facilitate the expression of more than one protein from a transcript.

The purpose of this study was to use an environmentally friendly steam explosion method to achieve α- and β-chitin structural alterations, pore generation, and deacetylation, enhancing the degree of deacetylation (DD) in chitin and extending its applications. The samples of α- and β-chitin possessing various moisture contents that were exploded at 9 kg/cm(2) exhibited higher DDs, lower densities, lower crystallinity and more porous structures compared to unexploded chitin. After explosion, β-chitin exhibited a larger expansion ratio, lower crystallinity and contained a larger proportion of small-sized particles compared to α-chitin. The highest DD values of exploded α- and β-chitin with 75% moisture content were 42.9% and 43.7%, respectively. The exploded chitin samples with lower moisture content exhibited lower DDs, densities, crystallinity indices, smaller particle sizes, and higher expansion ratios than the chitin samples with higher moisture content. The chitin samples with lower moisture content also contained larger and more numerous pores.

Chitin derivatives, such as those with modified main saccharide chains and deacetylated side chains, exhibit versatile biological functions. The biomedical properties of chitin oligosaccharides depend on their degree of oligomerization. Of the chitin oligosaccharides, chitin hexamers are generally the most potent. In our recent study, N -acetylchitohexaose was obtained by digesting chitin with ASCHI61, a chitinase from Aeromonas schubertii . In this work, the factors involved in the production of chitin hexasaccharide were evaluated experimentally. Using steep map analysis and cross-analysis, the substrate concentration and reaction pH were identified as the key factors in this reaction, and the interactions between these parameters were observed. Using a response surface experimental design, we predicted that a colloidal chitin concentration of 3.4 mg mL −1 and a pH of 6.54 were the optimal conditions for producing hexaoligochitin. These conditions were verified in separate experiments, in which 38.73 mmol L −1 of N -acetylchitohexaose was obtained. The maximum amount of hexamer produced was 42.175 mg L −1 , an increase of only 0.27% from the predicted value.

Superparamagnetic iron oxide nanocrystals (SPION) and their colloidal solutions of magnetic nanoparticles (ferrofluid) appear to contribute to the magnetite imaging applications. Here we present an aqueous ferrofluid in physiological pH and its cell tracking potential. In situ coating method was adapted to synthesize N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC)-coated SPION. Characterization results reveal their features as follows: (1) z -average diameter of 274.1 ± 1.934 nm; (2) polydispersity index (PDI) of 0.135 ± 0.029; (3) zeta potential of 11.4 ± 0.410 mV; (4) iron concentration of 5.4 mg Fe/ml; (5) stable in both water and normal saline; (6) uptake by a variety of cell lines and (7) little cytotoxicity. Finally, islet grafting experiments demonstrated the potential usefulness of this product as a MRI contrast agent for cell tracking.

Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.

We present conditions under which one can bound the probabilistic relationships between random variables in a Bayesian network by exploiting known or induced qualitative relationships. Generic strengthening and weakening operations produce bounds on cumulative distributions, and the directions of these bounds are maintained through qualitative influences. We show how to incorporate these operations in a state-space abstraction method, so that bounds provably tighten as an approximate network is refined. We apply these techniques to qualitative tradeoff resolution demonstrating an ability to identify qualitative relationships among random variables without exhaustively using the probabilistic information encoded in the given network. In an application to path planning, we present an anytime algorithm with run-time computable error bounds.

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.

We investigate state-space abstraction methods for computing approximate probabilities with Bayesian networks. These methods approximate Bayesian networks by aggregating the states of variables. We implement an iterative approximation procedure based on this idea, and the procedure demonstrates the desirable anytime property in experiments. Further theoretical analysis reveals special properties of the approximations, and we exploit these properties to design heuristics for improving performance profiles of the iterative procedure.

Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD(50) = 5 mg/kg versus 20 microg/kg of body weight). Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A. The protein synthesis inhibitory activity of AAG-A (IC(50) = 3.5 nM) was weaker than that of ABRa-A (0.05 nM). Molecular modeling followed by site-directed mutagenesis showed that Pro(199) of AAG-A, located in amphiphilic helix H and corresponding to Asn(200) of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.

Penicillins are effective for treating bacterial infections in many clinical situations, but in certain cases, they cause allergic reactions varying from mild skin rashes to anaphylactic shock and death. These symptoms often resemble those seen in immediate or delayed hypersensitivity reactions, and may be associated with drug specific IgE and/or drug specific T cells. Currently, the laboratory tests include isotope based serum IgE antibody measurement as well as skin test. However, radioisotope allergen specific tests (RAST) are not convenient, and the standardized skin-testing reagents and protocol do not usually exist. In fact, the skin test is often avoided because of the high risks. Therefore, in the current study, we have developed a non-isotope based penicillin-reactive IgE screening platform with high sensitivity and specificity. Using this screening platform to detect IgE of a mouse model sensitized with penicillin reveals that high levels of penicillin specific IgE were found in the penicillin sensitized animals but not in OVA sensitized or untreated control animals. Penicillin specific T cells produce relatively higher levels of IL-4 and IL-5 in the penicillin sensitized group, indicating a Th2-cytokine dominated response of T-cell dependent IgE production against penicillin. This observation and the development of such convenient platform to measure penicillin specific IgE may benefit further study of penicillin allergy.