The effect of surface poly(ethylene glycol) length on in vivo drug delivery behaviors of polymeric nanoparticles.

Engineering nanoparticles of reasonable surface poly(ethylene glycol) (PEG) length is important for designing efficient drug delivery systems. Eliminating the disturbance by other nanoproperties, such as size, PEG density, etc., is crucial for systemically investigating the impact of surface PEG length on the biological behavior of nanoparticles. In the present study, nanoparticles with different surface PEG length but similar other nanoproperties were prepared by using poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) copolymers of different molecular weights and incorporating different contents of PCL3500 homopolymer. The molecular weight of PEG block in PEG-PCL was between 3400 and 8000 Da, the sizes of nanoparticles were around 100 nm, the terminal PEG density was controlled at 0.4 PEG/nm2 (or the frontal PEG density was controlled at 0.16 PEG/nm2). Using these nanoproperties well-designed nanoparticles, we demonstrated PEG length-dependent changes in the biological behaviors of nanoparticles and exhibited nonmonotonic improvements as the PEG molecular weight increased from 3400 to 8000 Da. Moreover, under the experimental conditions, we found nanoparticles with a surface PEG length of 13.8 nm (MW = 5000 Da) significantly decreased the absorption with serum protein and interaction with macrophages, which led to prolonged blood circulation time, enhanced tumor accumulation and improved antitumor efficacy. The present study will help to establish a relatively precise relationship between surface PEG length and the in vivo behavior of nanoparticles.

Heart-type fatty acid binding protein (H-FABP) as a biomarker for acute myocardial injury and long-term post-ischemic prognosis.

Acute myocardial infarction (AMI) is a life-threatening event. Even with timely treatment, acute ischemic myocardial injury and ensuing ischemia reperfusion injury (IRI) can still be difficult issues to tackle. Apart from radiological and other auxiliary examinations, laboratory tests of applicable cardiac biomarkers are also necessary for early diagnosis and close monitoring of this disorder. Heart-type fatty acid binding protein (H-FABP), which mainly exists inside cardiomyocytes, has recently emerged as a potentially promising biomarker for myocardial injury. In this review we discuss the sensitivity and specificity of H-FABP in the assessment of myocardial injury and IRI, especially in the early stage, and its long-term prognostic value in comparison with other commonly used cardiac biomarkers, including myoglobin (Mb), cardiac troponin I (cTnI), creatine kinase MB (CK-MB), C-reactive protein (CRP), glycogen phosphorylase isoenzyme BB (GPBB), and high-sensitivity cardiac troponin T (hs-cTnT). The potential and value of combined application of H-FABP with other biomarkers are also discussed. Finally, the prospect of H-FABP is summarized; several technical issues are discussed to facilitate wider application of H-FABP in clinical practice.

Conformations and molecular interactions of poly-γ-glutamic acid as a soluble microbial product in aqueous solutions.

Soluble microbial products (SMPs) are of significant concern in the natural environment and in engineered systems. In this work, poly-γ-glutamic acid (γ-PGA), which is predominantly produced by Bacillus sp., was investigated in terms of pH-induced conformational changes and molecular interactions in aqueous solutions; accordingly, its sedimentation coefficient distribution and viscosity were also elucidated. Experimental results indicate that pH has a significant impact on the structure and molecular interactions of γ-PGA. The conformation of the γ-PGA acid form (γ-PGA-H) is rod-like while that of the γ-PGA sodium form (γ-PGA-Na) is sphere-like. The transformation from α-helix to random coil in the γ-PGA secondary structure is primarily responsible for this shape variation. The intramolecular hydrogen bonds in the γ-PGA-H structure decrease and intramolecular electrostatic repulsion increases as pH increases; however, the sedimentation coefficient distributions of γ-PGA are dependent on intermolecular interactions rather than intramolecular interactions. Concentration has a more substantial effect on intermolecular electrostatic repulsion and chain entanglement at higher pH values. Consequently, the sedimentation coefficient distributions of γ-PGA shift significantly at pH 8.9 from 0.1 to 1.0 g/L, and the viscosity of γ-PGA (5% w/v) significantly increases as pH increases from 2.3 to 6.0.

Response of extracellular polymeric substances to thermal treatment in sludge dewatering process.

Sludge dewatering is an important process in municipal wastewater treatment and critically influences the subsequent transportation and disposal. Thermal treatment coupled with other chemical processes has been widely used to improve sludge dewaterability. However, information about the response of sludge extracellular polymeric substances (EPS) to thermal treatment and its role in sludge dewatering is still limited. In this work, the effects of thermal treatment on anaerobic and aerobic sludges were investigated with an emphasis on the colloid properties of released EPS in sludge dewatering process. The results indicate that sludge dewaterability became deteriorated with the increased temperature in the range of 30-170 °C, which was ascribed to the disintegration of sludge flocs and change of EPS characteristics. Disintegrated sludge induced the release of the negatively charged EPS, resulting in the weakened bridging interaction and lower compactness. After thermal treatment, the EPS with a higher average molecular weight and stretched coil configuration retained more water. In addition, difference in dewaterability between anaerobic and aerobic sludges was found to be attributed to their different contents and structures of EPS components. These results provide an insight into thermal-dependent sludge dewatering process and are useful to facilitate water-sludge separation.

Quantitative evaluation of noncovalent interactions between polyphosphate and dissolved humic acids in aqueous conditions.

As one kind of phosphorus species, polyphosphate (poly-P) is ubiquitous in natural environments, and the potential interactions between poly-P and humic substances in the sediments or natural waters would influence the fate of poly-P in the environments. However, the mechanism of the interactions has not yet been understood clearly. In this work, the characteristics and mechanisms of the interactions between humic acids (HA) and two model poly-P compounds with various chain lengths have been investigated. Results show that a stable polyphosphate-HA complex would be formed through the noncovalent interactions, and hydrogen bond might be the main driving force for the binding process, which might be formed between the proton-accepting groups of poly-P (e.g., PO and P-O(-)) and the oxygen containing functional groups in HA. Our findings implied that the presence of humic substances in natural waters, soils and sediments would influence the potential transport and/or mobility of environmental poly-P.

Regulating the surface poly(ethylene glycol) density of polymeric nanoparticles and evaluating its role in drug delivery in vivo.

Poly(ethylene glycol) (PEG) is usually used to protect nanoparticles from rapid clearance in blood. The effects are highly dependent on the surface PEG density of nanoparticles. However, there lacks a detailed and informative study in PEG density and in vivo drug delivery due to the critical techniques to precisely control the surface PEG density when maintaining other nano-properties. Here, we regulated the polymeric nanoparticles' size and surface PEG density by incorporating poly(ε-caprolactone) (PCL) homopolymer into poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) and adjusting the mass ratio of PCL to PEG-PCL during the nanoparticles preparation. We further developed a library of polymeric nanoparticles with different but controllable sizes and surface PEG densities by changing the molecular weight of the PCL block in PEG-PCL and tuning the molar ratio of repeating units of PCL (CL) to that of PEG (EG). We thus obtained a group of nanoparticles with variable surface PEG densities but with other nano-properties identical, and investigated the effects of surface PEG densities on the biological behaviors of nanoparticles in mice. We found that, high surface PEG density made the nanoparticles resistant to absorption of serum protein and uptake by macrophages, leading to a greater accumulation of nanoparticles in tumor tissue, which recuperated the defects of decreased internalization by tumor cells, resulting in superior antitumor efficacy when carrying docetaxel.

[Analysis of cost-effectiveness of molluscacide combined with herbicide by spraying].

OBJECTIVE: To explore an economical and efficient molluscicidal method suitable for large area of nursery stock field. METHODS: Two nursery stock fields with Oncomelania hupensis were selected as experimental sites, and an experimental group and a control group were set. In the experimental group, the molluscacide and herbicide were alternately used (a purification molluscicidal method) during the period of May to October, 2011. In the control group, grass shoveling and soil burying combined with molluscacide were used in the same period. The snail control effects of the two groups were observed and the costs of the two methods were analyzed. RESULTS: No living snails were found in both experimental and control groups three consecutive years after the snail control intervention above mentioned. The costs of snail control intervention in the experimental group and control group were 0.90 and 1.80 Yuan/m2, respectively. CONCLUSION: The effect of the purification molluscicidal method in nursery stock field is satisfying, and the cost is lower.

[Observation of molluscicidal effect of black plastic film combined with carbon amide].

OBJECTIVE: To evaluate the molluscicidal effect of black plastic film combined with carbon amide. METHODS: In Jiangdong Town, Jinhua City, the field with Oncomelania hupensis in the history was selected as experimental area and divided into 3 groups: Group One was administered with black plastic film combined with carbon amide; Group Two was administered with simple black plastic film; and Group Three was a control group. RESULTS: On the 3rd, 7th, 15th, 20th and 30th day after the experiment, the mortality rates of 0. hupensis of Group One were 86.0%, 88.0%, 100%, 100% and 100% respectively, which were significantly higher than those of the control group (all P < 0.05). The differences of mortality rates between Group One and Group Two were statistically significant on the 3rd and 7th day after the experiment (Group One was superior to Group Two). CONCLUSION: The bladk plastic film combined with carbon amide can improve the molluscicidal effect.

[Cost-effectiveness analysis of mechanical and manual soil-buried methods for Oncomelania hupensis control].

OBJECTIVE: To explore a high molluscicidal efficient method in special Oncomelania hupensis snail environments. METHODS: In 2005 and 2006, in large special environments (rubble creek beaches and seepage barren hills with snails), the mechanical soil-buried method (excavator digging to bury deep snails) and manual soil-buried method were used respectively, and the results were compared for the cost-effectiveness. RESULTS: With the mechanical soil-buried method in 2006, the investment was 0.78 yuan/m2, and the compression rate of snail areas was 100%; with the manual soil-buried method in 2005, the investment was 1.34 yuan/m2, and the compression rate of snail areas was 20.26%. The former was much better than the latter. CONCLUSION: In the large special environments with snails, the mechanical soil-buried method is superior to manual soil-buried method.

Transcriptomic analyses reveal the adaptive features and biological differences of guts from two invasive whitefly species.

BACKGROUND: The gut of phloem feeding insects is critical for nutrition uptake and xenobiotics degradation. However, partly due to its tiny size, genomic information for the gut of phloem feeding insects is limited. RESULTS: In this study, the gut transcriptomes of two species of invasive whiteflies in the Bemisia tabaci complex, Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED), were analyzed using the Illumina sequencing. A total of 12,879 MEAM1 transcripts and 11,246 MED transcripts were annotated with a significant Blastx hit. In addition, 7,000 and 5,771 gut specific genes were respectively identified for MEAM1 and MED. Functional analyses on these gut specific genes demonstrated the important roles of gut in metabolism of insecticides and secondary plant chemicals. To reveal the molecular difference between guts of MEAM1 and MED, a comparison between gut transcriptomes of the two species was conducted and 3,910 pairs of orthologous genes were identified. Based on the ratio of nonsynonymous and synonymous substitutions, 15 genes were found evolving under positive selection. Many of those genes are predicted to be involved in metabolism and insecticide resistance. Furthermore, many genes related to detoxification were expressed at an elevated level in the gut of MED compared to MEAM1, which might be responsible for the MED's higher resistance to insecticides and environmental stresses. CONCLUSION: The sequencing of MED and MEAM1 gut transcriptomes and extensive comparisons of MEAM1 and MED gut transcripts provide substantial sequence information for revealing the role of gut in whiteflies.

Expression and functional characterisation of a soluble form of Tomato yellow leaf curl virus coat protein.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus within the family Geminiviridae, is an important pathogen of tomato in many tropical, subtropical and temperate regions. TYLCV is exclusively transmitted by the whitefly Bemisia tabaci in a circulative manner. The viral coat protein (CP) has been assumed to play important roles in the entry of TYLCV into the insect midgut cells. RESULTS: Testing the hypothesis that CP plays an important role in TYLCV acquisition by B. tabaci, a soluble form of the CP was expressed and purified. The purified recombinant CP made it possible to examine the function of TYLCV CP without other viral proteins. In an in vivo binding assay, specific binding of TYLCV CP to B. tabaci midguts was detected when purified CP was fed to B. tabaci. In addition, real-time polymerase chain reaction analysis of virus titre revealed that B. tabaci fed with purified CP had reduced the level of virus in their midgut compared with those fed with bovine serum albumin or maltose-binding protein. These results suggest that binding of TYLCV CP to the B. tabaci midgut specifically inhibits virus acquisition. CONCLUSIONS: The findings that TYLCV CP binds to B. tabaci midguts and decreases virus acquisition provide direct evidence that CP mediates the attachment of TYLCV to receptors on the epithelial cells of the B. tabaci midgut.

Hydration interactions and stability of soluble microbial products in aqueous solutions.

Soluble microbial products (SMP) are organic compounds excreted by microorganisms in their metabolism and decay and the main constituents in effluent from biological wastewater treatment systems. They also have an important contribution to the dissolved organic matters in natural aqueous systems. So far the interactions between SMP colloids have not been well explored. In this work, the interactions between SMP colloids in water and salt solutions were studied by using a combination of static and dynamic light scattering, Fourier transform infrared spectra, Zeta potential and acid-base titration techniques. The second osmotic virial coefficient had a larger value in a 750-mM salt solution than that in a 50-mM solution, indicating that repulsion between SMP colloids increased with an increase in salt concentration, which is contrary with the classic Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory. Such a repulsion was attributed to water structuring and enhanced by the accumulation of hydrophilic counter ions around SMP colloids and the formed hydration force. The repulsion and hydration effect led to the dispersing and deeper draining structure, accompanied by a decreased hydrodynamic radius and increased diffusion coefficient. This hydration force was related to so-called ion specific effect, and electrolyte sodium chloride had a more substantial effect on hydration force than KCl, CsCl, NaBr and NaI. Our results provide an experimental approach to explore the SMP structures, inter-colloid interactions and confirm the non-DLVO forces.

Coagulation kinetics of humic aggregates in mono- and di-valent electrolyte solutions.

Coagulation behaviors of humic acids (HAs) aggregates in electrolyte solutions at different pHs, valences and concentrations of electrolyte cations were investigated using dynamic light scattering technique in combination of other analytical tools. For monovalent electrolyte sodium chloride (NaCl) solution, at its low concentrations the average hydrodynamic radius () of aggregates kept nearly constant. However, at high NaCl concentrations, could be scaled to the time t as ∝ t(a), suggesting a diffusion-limited colloid aggregation (DLCA). The coagulation value of NaCl in a buffer at pH 7.1 was calculated to be in a range of 61.3-84.4 mM. Divalent cation Mg(2+) was far more effective in enhancing the HA coagulation, as evidenced by a lower coagulation value (between 1.0 and 1.7 mM) and a more rapid coagulation rate. Such an enhancement could be explained by the combined effects of electrostatic repulsion, complexation and bridging. The highest coagulation rate (d/dt) and coagulation value at different pHs followed the order of: acidic > neutral > alkaline, and alkaline > neutral > acidic, respectively. Such a difference was associated with the extent of hydrogen bond and electrostatic repulsion at different protonation/deprotonation states of carboxyl and phenolic -OH groups. Transmission electron microscopic imaging reveals that HA was predominantly globular aggregates with a rough periphery at pH 5.26, and was changed to smooth spherical particles at pH 10.00. These results are useful for better understanding the coagulation behaviors of HAs in both natural and engineered aqueous systems.

Transcriptomic analysis of the salivary glands of an invasive whitefly.

BACKGROUND: Some species of the whitefly Bemisia tabaci complex cause tremendous losses to crops worldwide through feeding directly and virus transmission indirectly. The primary salivary glands of whiteflies are critical for their feeding and virus transmission. However, partly due to their tiny size, research on whitefly salivary glands is limited and our knowledge on these glands is scarce. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the transcriptome of the primary salivary glands of the Mediterranean species of B. tabaci complex using an effective cDNA amplification method in combination with short read sequencing (Illumina). In a single run, we obtained 13,615 unigenes. The quantity of the unigenes obtained from the salivary glands of the whitefly is at least four folds of the salivary gland genes from other plant-sucking insects. To reveal the functions of the primary glands, sequence similarity search and comparisons with the whole transcriptome of the whitefly were performed. The results demonstrated that the genes related to metabolism and transport were significantly enriched in the primary salivary glands. Furthermore, we found that a number of highly expressed genes in the salivary glands might be involved in secretory protein processing, secretion and virus transmission. To identify potential proteins of whitefly saliva, the translated unigenes were put into secretory protein prediction. Finally, 295 genes were predicted to encode secretory proteins and some of them might play important roles in whitefly feeding. CONCLUSIONS/SIGNIFICANCE: The combined method of cDNA amplification, Illumina sequencing and de novo assembly is suitable for transcriptomic analysis of tiny organs in insects. Through analysis of the transcriptome, genomic features of the primary salivary glands were dissected and biologically important proteins, especially secreted proteins, were predicted. Our findings provide substantial sequence information for the primary salivary glands of whiteflies and will be the basis for future studies on whitefly-plant interactions and virus transmission.

Spatial configuration of extracellular polymeric substances of Bacillus megaterium TF10 in aqueous solution.

Configuration of extracellular polymeric substances (EPS) excreted by microorganisms is related greatly to the inherent properties of EPS, and has a significant effect on the physicochemical characteristics of microbial aggregates, such as activated sludge for wastewater treatment, as well as their interaction with other substances in aqueous systems. In this work, the spatial configuration of microbial EPS is characterized using laser light scattering (LLS) technique, with EPS extracted from Bacillus megaterium TF10 as an example. The combined utilization of static light scanning (SLS) and dynamic light scanning (DLS) offers an effective avenue to explore the EPS configuration in aqueous solution, thus enables a better understanding about the physicochemical properties of EPS. The results show that EPS exist in the form of colloids in neutral aqueous solution (pH 7.0) and that their shape is random coil with incompletely extending chains. The attraction interaction between EPS colloids is related with the high flocculability of B. megaterium TF10. The cryo-electron microscopy image further confirms the spherical shape of EPS colloids. The LLS approach offers a powerful and convenient tool for characterizing microbial EPS configuration and understanding their behaviors in biological wastewater treatment systems.

pH dependence of structure and surface properties of microbial EPS.

The flocculation of microorganisms plays a crucial role in bioreactors, and is substantially affected by pH. However, the mechanism for such an effect remains unclear. In this work, with an integrated approach, the pH dependence of structure and surface property of microbial extracellular polymeric substances (EPS), excreted from Bacillus megaterium TF10, and accordingly its flocculation is elucidated. From the Fourier transform infrared spectra and acid-base titration test results, the main functional groups and buffering zones in the EPS responsible for the microbial flocculation are indentified. The laser light scattering analysis reveals that the deprotonated or protonated states of these functional groups in EPS result in more dense and compact structure at a lower pH because of hydrophobicity and intermolecular hydrogen bonds. The zeta potential measurements identify the isoelectric point and indicate that the electrostatic repulsion action of EPS is controlled by pH. The highest flocculation efficiency is achieved near the isoelectric point (pH 4.8). These results clearly demonstrate that the EPS structure, surface properties, and accordingly the microbial flocculation are dependent heavily on pH in solution.

Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33.

The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b2 to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded ß-sheet flanked by two α-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded ß-sheet surrounded by five α-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.

[Insulin-like growth factor 2 imprinting status and promoter usage in the placenta of macrosomia].

OBJECTIVE: To study the mechanism of marcosomia by investigating insulin-like growth factor 2 (IGF(2))imprinting status, expression level and the promoter usage in the placenta of macrosomia. METHODS: We selected heterozygous cases for Apa I polymorphism in exon 9 of IGF(2) gene and then analyzed its imprinting status in 168 placentas of macrosomia and normal pregnancies. IGF(2) transcription levels and promoter usages in macrosomic and normal placenta were evaluated by using semi-quantitative RT-PCR assay. RESULTS: Thirty specimens of macrosomic placenta and 30 of normal placenta were identified as heterozygous for IGF(2). All of the heterozygous specimens showed maintenance of imprinting. The expression of placental IGF(2) mRNA (2.2 +/- 1.2) was significantly higher in macrosomia than that of normal weight group (1.6 +/- 0.6, P < 0.05). Of four promoters, P4 was the most powerful, P3 was the second, and P2 was weakest. Transcripts from P1 were the fewest, and they were only detected in two specimens. The value of P4 was 2.06 +/- 1.26, P3 0.99 +/- 0.72, P2 0.20 +/- 0.20 in macrosomia group and P4 2.05 +/- 1.27, P3 0.98 +/- 0.80, P2 0.19 +/- 0.17 in normal group. There were no significant differences between two groups (P > 0.05). CONCLUSION: It is possible that over expression of IGF(2) in placenta contributes to macrosomia while the promoter usage and imprinting status are not associated with macrosomia.

[Application of real-time fluorescence quantitative polymerase chain reaction techniques for prenatal diagnosis of Down syndrome].

OBJECTIVE: To investigate the value of real-time fluorescence quantitative PCR in diagnosis of Down syndrome with uncultured amniotic cells. METHODS: The uncultured amniocytes of 80 fetuses who were confirmed disomy 21 by chromosome analysis and 5 fetuses detected trisomy 21 and peripheral blood samples of 7 children diagnosed as Down syndrome were collected to extract gDNA and the real-time fluorescence quantitative PCR method was used to detect the original copies of Down syndrome critical region gene(3) (DSCR(3)) and GAPDH gene and then the ratio of DSCR(3)/GAPDH was calculated. RESULTS: The PCR product ratios of DSCR(3) to GAPDH in trisomy 21 from amniocytes and peripheral blood were ranged from 1.64 to 1.98 while in the normal control the ratio was only from 0.46 to 1.30. CONCLUSION: Real-time fluorescence quantitative PCR is a valuable method for rapid and accurate prenatal diagnosis of Down syndrome in uncultured amniotic cells.