Manipulating Diastereomeric Bicyclononynes to Sensitively Determine Enzyme Activity and Facilitate Macromolecule Conjugations.

Bicyclo[6.1.0]nonyne (BCN) is one of the most commonly used cycloalkynes in strain-promoted azide-alkyne cycloaddition (SPAAC). The synthesis of BCN produces two diastereomers, exo-BCN and endo-BCN. The potential significance of the different steric structures of the tricyclic fused rings in SPAAC products synthesized from the BCN diastereomers has not been previously studied. We first demonstrated that only endo-BCN could reduce the level of fluorescence quenching in SPAAC reaction products. The reduction was likely due to the presence of extended tricyclic fused ring systems. This hypothesis was supported by the synthesis of a fluorescence always-on construct by substituting endo-BCN for exo-BCN in a previously reported chemical probe that was characterized with good contact fluorescence quenching. We also synthesized bis-BCN derivatives to enhance the steric structural differences in the corresponding SPAAC products. A constitutional isomer of the azido-derivatized 5(6)-carboxyfluorescein [5(6)-FAM] was reacted with both bis-exo-BCN and bis-endo-BCN compounds. However, one form of the bis-exo-BCN-based product did not augment contact fluorescence quenching, while a second bis-exo-BCN product could not further reduce contact fluorescence quenching. Nevertheless, a new fluorescence turn-on chemical probe was employed to determine the activities of two serum biomarkers, butyrylcholinesterase and paraoxonase 1. Moreover, bis-endo-BCN was exploited to successfully conjugate BSA with a 5-FAM derivative compound.

Evaluation of computed tomography images under deep learning in the diagnosis of severe pulmonary infection.

This work aimed to explore the diagnostic value of a deep convolutional neural network (CNN) combined with computed tomography (CT) images in patients with severe pneumonia complicated with pulmonary infection. A total of 120 patients with severe pneumonia complicated by pulmonary infection admitted to the hospital were selected as research subjects and underwent CT imaging scans. The empty convolution (EC) and U-net phase were combined to construct an EC-U-net, which was applied to process the CT images. The results showed that the learning rate of the EC-U-net model decreased substantially with increasing training times until it stabilized and reached zero after 40 training times. The segmentation result of the EC-U-net model for the CT image was very similar to that of the mask image, except for some deviations in edge segmentation. The EC-U-net model exhibited a significantly smaller cross-entropy loss function (CELF) and a higher Dice coefficient than the CNN algorithm. The diagnostic accuracy of CT images based on the EC-U-net model for severe pneumonia complicated with pulmonary infection was substantially higher than that of CT images alone, while the false negative rate (FNR) and false positive rate (FPR) were substantially lower (P < 0.05). Moreover, the true positive rates (TPRs) of CT images based on the EC-U-net model for patchy high-density shadows, diffuse ground glass density shadows, pleural effusion, and lung consolidation were obviously higher than those of the original CT images (P < 0.05). In short, the EC-U-net model was superior to the traditional algorithm regarding the overall performance of CT image segmentation, which can be clinically applied. CT images based on the EC-U-net model can clearly display pulmonary infection lesions, improve the clinical diagnosis of severe pneumonia complicated with pulmonary infection, and help to screen early pulmonary infection and carry out symptomatic treatment.

Time-dependent effects of storage at -80 °C on the stability of butyrylcholinesterase activity in human serum.

Objectives: Butyrylcholinesterase (BChE) is an important biomarker in serum, and aberrant BChE activity indicates onset and progression of human diseases. The duration of serum storage at -80 °C may introduce variability into and compromise the reproducibility of BChE activity measurements. Design and Methods: We collected serum samples from eight healthy volunteers and determined serum BChE activity in these samples using a sensitive fluorescence assay at various time points during a six-month storage period at -80 °C. Changes in averaged BChE activity over storage time were assessed by repeated measures analysis of variance (ANOVA). Sidak multiple comparisons test was also used to perform post-hoc analysis. Results: Almost all determined BChE activity values lay within the normal physiological range of BChE activity. However, repeated measures ANOVA using mean BChE activity vs. storage time showed that BChE activity values from two time points were significantly different. Analysis by Sidak multiple comparisons test provided no substantial change of BChE activity during the first 90 days of storage, but BChE activity noticeably decreased after 90 days. Conclusions: Serum samples stored in -80 °C for up to 90 days can be exploited to accurately determine BChE activity.

Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes.

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes 16a and 16b (16) able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide-alkyne cycloaddition. Fluorescence quenching in 16 was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of 16b confirmed the outstanding reactivity and specificity of 16b with thiols in the presence of general base catalysts. The 16b-based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8-232.1 U L-1 and detection limit (LOD) of 10.8 U L-1, to quantify serum PON1 activity in human sera, and to determine the Ki of 20.9 µM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing 16b to develop high-throughput assays for PON1 lactonase activity.

Efficacy and tolerability of intravenous iron for patients with restless legs syndrome: evidence from randomized trials and observational studies.

OBJECTIVE: Restless legs syndrome (RLS) is a common neurological disorder of unclear pathophysiology that appears to involve an iron deficiency in the brain. Some studies, but not others, suggest that intravenous injection of iron can reduce RLS severity. METHOD: The databases Web of Science, PubMed, Embase, Chinese National Knowledge Infrastructure, Wanfang, and SinoMed were searched for randomized controlled trials, cohort studies and case-control studies of intravenous iron therapy to treat RLS. Eligible studies were meta-analyzed using Stata 12.0. RESULTS: This analysis indicated that IV iron was more efficacious than placebo in treating RLS (OR: 4.71,95%CI 4.21-5.21,p < 0.0001). According to sub-group analysis, either IV ferric carboxymaltose (FCM) or iron sucrose was more efficacious than placebo in treating RLS. Adverse events did not differ significantly between patients receiving intravenous iron or placebo (OR 1.68, 95%CI 0.92-3.07, p = 0.093). The present study also indicated after accepting IV iron treatment the IRLS score in RLS patients decreased (OR = 6.75,95%CI 4.02-9.49, p < 0.0001). The subgroup analysis showed that IV iron dextran, iron sucrose, and FCM could alleviate the IRLS score. CONCLUSION: The available evidence suggests that intravenous iron is effective and tolerable for patients with RLS regardless of peripheral iron status.

pH-sensitive charge-conversional and NIR responsive bubble-generating liposomal system for synergetic thermo-chemotherapy.

A charge-conversional and NIR responsive rapid release liposomal system (PSD/DOX/Cypate-BTSL) was developed to enhance therapeutic efficacy of cancer therapy. The cationic liposomes containing Cypate, doxorubicin (DOX) and NH4HCO3 were shielded by pH-sensitive poly(methacryloyl sulfadimethoxine) (PSD) through electrostatic interaction at pH 7.4. At the tumor site (pH 6.5), PSD was deshielded and the liposomes displayed pH-sensitive charge reversal capability. The DOX released from PSD/DOX/Cypate-BTSL with irradiation was markedly higher than the other groups, indicating NIR irradiation and NH4HCO3 had a significant effect on the drug release. After irradiation, the hyperthermia induced by Cypate could produce CO2 bubbles quickly on account of the decomposition of NH4HCO3, achieving the rapid drug release. In 4T1 cells, PSD/DOX/Cypate-BTSL improved cellular uptake and cytotoxicity with irradiation at pH 6.5. In vivo results implied that the liposomes with irradiation could efficiently enhance the tumor accumulation and antitumor efficacy, and reduce systemic side effects of DOX. In conclusion, PSD/DOX/Cypate-BTSL is a promising candidate as a carrier for synergistic effects of PTT and chemotherapy.

NIR responsive liposomal system for rapid release of drugs in cancer therapy.

To design a rapid release liposomal system for cancer therapy, a NIR responsive bubble-generating thermosensitive liposome (BTSL) system combined with photothermal agent (Cypate), doxorubicin (DOX), and NH4HCO3 was developed. Cypate/DOX-BTSL exhibited a good aqueous stability, photostability, and photothermal effect. In vitro release suggested that the amounts of DOX released from BTSL were obviously higher than that of (NH4)2SO4 liposomes at 42°C. After NIR irradiation, the hyperthermic temperature induced by Cypate led to the decomposition of NH4HCO3 and the generation of a large number of CO2 bubbles, triggering a rapid release of drugs. Confocal laser scanning microscope and acridine orange staining indicated that Cypate/DOX-BTSL upon irradiation could facilitate to disrupt the lysosomal membranes and realize endolysosomal escape into cytosol, improving the intracellular uptake of DOX clearly. MTT and trypan blue staining implied that the cell damage of Cypate/DOX-BTSL with NIR irradiation was more severe than that in the groups without irradiation. In vivo results indicated that Cypate/DOX-BTSL with irradiation could dramatically increase the accumulation of DOX in tumor, inhibit tumor growth, and reduce systemic side effects of DOX. These data demonstrated that Cypate/DOX-BTSL has the potential to be used as a NIR responsive liposomal system for a rapid release of drugs in thermochemotherapy.

Sequential delivery of chlorhexidine acetate and bFGF from PLGA-glycol chitosan core-shell microspheres.

Wound treatment should meet the challenge both of preventing infection and promoting wound healing. To design a sequential delivery system for wound healing, PLGA-glycol chitosan (GC) core-shell microspheres containing chlorhexidine acetate (CHA) at the GC shell and bFGF in the core of PLGA microspheres were fabricated using emulsion-solvent evaporation method. SEM showed that the microspheres were all spherical in shape with a smooth surface. The average size of PLGA-GC microspheres increased due to the GC coating on the surface. The results of release profiles and fluorescence images indicated that PLGA-GC microspheres had an ability to deliver drugs in sequence. The CHA was rapidly released, whereas the proteins presented a sustained release. The release behavior could be modulated by changing the GC amount. Antibacterial assay and cell proliferation tests suggested that the released CHA and bFGF retained their antimicrobial activity and bioactivity during preparation. The microspheres exhibited non-cytotoxicity against 3T3 cells and had a good biocompatibility. These results demonstrated that PLGA-GC core-shell microspheres could be a promising controlled release system of delivering drugs and proteins in sequence for wound healing.

Fish collagen-based scaffold containing PLGA microspheres for controlled growth factor delivery in skin tissue engineering.

To design a scaffold controlled release system for skin tissue engineering, fish collagen/chitosan/chondroitin sulfate scaffolds were fabricated by freeze-drying and incorporated with bFGF-loaded PLGA microspheres (MPs). SEM showed that the scaffolds exhibited an interconnected porous structure, and the spherical MPs were uniformly distributed into the scaffolds. The higher swelling and degradation rate of scaffolds/MPs could lead to a higher diffusion rate of MPs from the scaffolds, causing an increase in the protein release. The release rate of proteins could be adjusted by the size of MPs and the ratio of collagen to chitosan of scaffolds. Circular dichroism spectroscopy and MTT of bFGF after release indicated that the released bFGF retained its structural integrity and bioactivity during preparation. Cell proliferation and in vivo evaluation results suggested that the scaffolds/MPs had a good biocompatibility and an ability to promote fibroblast cell proliferation and skin tissue regeneration. These results demonstrated that this scaffold/MP controlled release system has the potential for skin tissue engineering.

Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery.

The objective of this study was to design a drug delivery system consisting of biotinylated cholesterol-modified glycol chitosan (Bio-CHGC) nanoparticles and fish collagen/chitosan (Col/Ch) film for localized chemotherapy. Bio-CHGC was synthesized, and then its self-assembled nanoparticles were prepared by probe sonication. Doxorubicin (DOX)-loaded Bio-CHGC (DBC) nanoparticles prepared by dialysis had spherical shape, and their sizes were in the range of 330-397 nm. Col/Ch/DBC nanoparticle films were fabricated by freeze-drying. SEM showed that the DBC nanoparticles were uniformly distributed into the films, and the films retained their structural integrity. A higher degradation and swelling rate of the drug films led to a higher diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from nanoparticles. The release of DOX from the films or Bio-CHGC nanoparticles was sensitive to the pH value of the release medium. In addition, the DOX release ratio of the drug films was lower than that of the nanoparticles alone, suggesting that the drug films had a double-sustained effect on the drug release. MTT assay implied that the DBC nanoparticle film showed a higher inhibitory ratio than the film containing nanoparticles without biotin, indicating that biotin moieties in the nanoparticles played an important role in exerting a cytotoxic effect. These data demonstrate that Col/Ch/DBC nanoparticle film has the potential to be used as a localized delivery system for hydrophobic antitumor drugs.

A community-based sero-epidemiological study of hepatitis B infection in Lianyungang, China, 2010.

INTRODUCTION: The 2010 targets of the China Hepatitis B Prevention Programme were a prevalence of hepatitis B surface antigen (HBsAg) less than 1.0% for children less than five years old and less than 6.0% for the total population. This survey assessed the prevalence of Hepatitis B infection in Lianyungang, Jiangsu province, China in 2009-2010. METHODS: Multistage sampling was used with 2372 subjects among 17 selected villages. Blood specimen collection and testing by enzyme-linked immunosorbnet assay (ELISA) were completed using the following markers for hepatitis infection: HBsAg and antibody to HBsAg (anti-HBs); hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe); and hepatitis B core antibody (total anti-HBc). The data were analysed with Epi Info, version 3.3.2. RESULTS: The prevalence of HBsAg was 2.4% (95% Confidence Interval [CI]: 1.8-3.0; Adjusted Prevalence [AP] 2.9%); anti-HBs prevalence was 51.1% (95% CI: 49.1-53.1; AP 49.2%) and total anti-HBc prevalence was 41.7% (95% CI: 39.8-43.7; AP 45.5%). The prevalence of HBsAg and total anti-HBc positivity increased from young to older age groups, yet the prevalence of anti-HBs positivity decreased from young to older age groups (P < 0.001 for all). There was no difference in the prevalences of HBsAg and anti-HBs among females and males (P = 0.108 and 0.089), but females had a higher prevalence than males for total anti-HBc positivity (P < 0.001). DISCUSSION: This survey showed that in 2010 the prevalence of HBsAg among children aged less than five years was lower than the national target of 1.0% and that the prevalence of HBsAg for the total population was lower than the national target of 6.0%.

Molecular aspect of good eating quality formation in Japonica rice.

The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In molecular biology level, the fine structure of amylopectin is determined by relative activities of starch branching enzyme (SBE), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) in rice grain under the same ADP-Glucose level. But the underlying mechanism of eating quality in molecular biology level remains unclear. This paper reports the differences on major parameters such as SNP and insertion-deletion sites, RNA expressions, and enzyme activities associated with eating quality of japonica varieties. Eight japonica rice varieties with significant differences in various eating quality parameters such as palatability and protein content were used in this experiment. Association analysis between nucleotide polymorphism and eating quality showed that S12 and S13 loci in SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity, consistency viscosity, pasting temperature, which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity; and explained almost all variations in alkali digestion value and pasting temperature. Thirty-five SNPs and insertion-deletions from SBE1, SBE3, GBSS1, SSS1, and SSS2A differentiated high or intermediate palatability rice varieties from low palatability rice varieties. Correlation analysis between enzyme activities and eating quality properties revealed that SBE25 and SSS15/W15 were positively correlated with palatability, whereas GBSS10 and GBSS15 were negatively correlated. Gene expressions showed that SBE1 and SBE3 expressions in high palatability varieties tended to be higher than middle and low palatability varieties. Collectively, SBE1, SBE3, SSS1, and SSS2A, especially SBE1 and SBE3 could improve eating quality, but GBSS1 decreased eating quality. The results indicated the possibility of developing high palatability cultivars through modification of key genes related to japonica rice eating quality formation in starch biosynthesis.

An oligonucleotide microarray bait for isolation of target gene fragments.

A new molecular-baiting method was studied by retrieving targeted gene fragments from an oligonucleotide microarray bait after hybridization. To make the microarray bait, 70-mer oligonucleotides that were designed to specifically represent the SSA1 gene of Saccharomyces cerevisiae were printed on the slide. Samples of the Saccharomyces cerevisiae mRNA were extracted and labeled by the RD-PCR (Restriction Display PCR) method using the Cy5-labelled universal primer, then applied for hybridization. The sample fragments that hybridized to the microarray were stripped, and the eluted cDNAs were retrieved and cloned into the pMD 18-T vector for transformation, plasmid preparation, and sequencing. BLAST searching of the GenBank database identified the retrieved fragments as being identical to the SSA1 gene (from 2057-2541bp). A new method is being established that can retrieve the sample fragments using an oligo-microarray-bait.

Monitoring gene expression profile changes in bladder transitional cell carcinoma using cDNA microarray.

PURPOSE: Differential gene expression profiles between normal bladder mucosas and bladder transitional cell carcinomas TCC were detected. MATERIALS AND METHODS: cDNA microarrays were prepared by spotting PCR products of 12,800 human genes onto specially treated glass slides. The cDNA probes were prepared by labeling normal bladder mucosa mRNA and TCC tissue mRNA with Cy3-dUTP and Cy5-dUTP respectively through reverse transcription. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The ratios of Cy5/Cy3 were computed. Northern analysis was used to confirm the results of microarray hybridization. RESULTS: Eighty-three genes (0.65%), whose ratios of Cy5/Cy3 were greater than 4.0 or less than 0.25, were screened out after 10 groups of hybridization. In the cancerous tissues 28 of them showed higher expression and 55 lower. Twenty-three genes are unregistered in GenBank. These differentially expressed genes are always involved in the physiological processes such as signal transduction, apoptosis and cell cycle, etc. CONCLUSIONS: This technique provides a powerful method for quantitative analysis of the expression levels of thousands of genes in parallel, and is used to identify genes involved in TCC carcinogenesis. The data obtained by this means are comparable to those obtained by other methods. Using cDNA microarrays to define alterations in gene expression associated with a specific cancer may be an efficient way to uncover the clues to specific molecular derangements that account for its pathogenesis and thus identify potential targets for therapeutic intervention.