Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants.

BACKGROUND: Oxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare long-template libraries for long-read TGS, particularly the ONT method for analysis of hemoglobinopathy variants involving complex structures and occurring in GC-rich and/or homologous regions. METHODS: A multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons for HBA2/1, HBG2/1, HBD, and HBB, as well as the allelic amplicons for targeted deletions and special structural variations. Library construction was performed using long-PCR products, and sequencing was conducted on an Oxford Nanopore MinION instrument. Genotypes were identified based on integrative genomics viewer (IGV) plots. RESULTS: This novel long-read TGS method distinguished all single nucleotide variants and structural variants within HBA2/1, HBG2/1, HBD, and HBB based on the whole-gene sequence reads. Targeted deletions and special structural variations were also identified according to the specific allelic reads. The result of 158 α-/ß-thalassemia samples showed 100% concordance with previously known genotypes. CONCLUSIONS: This ONT TGS method is high-throughput, which can be used for molecular screening and genetic diagnosis of hemoglobinopathies. The strategy of multiplex long PCR is an efficient strategy for library preparation, providing a practical reference for TGS assay development.

Effect of ultrasonic degradation on the physicochemical property and bioactivity of polysaccharide produced by Chaetomium globosum CGMCC 6882.

Similar to the enzymatic process, there might also be an active fragment in polysaccharides, how to obtain is important for investigating the bioactivity and pharmacological mechanism of polysaccharides. Presently, a Gynostemma pentaphyllum endophytic fungus Chaetomium globosum CGMCC 6882 polysaccharide [Genistein Combined Polysaccharide (GCP)] was degraded by ultrasonic treatment, two polysaccharide fragments of GCP-F1 and GCP-F2 were obtained. Physicochemical results showed that GCP-F1 and GCP-F2 had the same monosaccharide composition of arabinose, galactose, glucose, xylose, mannose, and glucuronic acid as compared to GCP with slightly different molar ratios. However, weight-average molecular weights of GCP-F1 and GCP-F2 decreased from 8.093 × 104 Da (GCP) to 3.158 × 104 Da and 1.027 × 104 Da, respectively. In vitro scavenging assays illustrated that GCP-F1 and GCP-F2 had higher antioxidant activity against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anions, and hydroxyl radical than GCP, the order was GCP < GCP-F1 < GCP-F2. Meanwhile, antibacterial tests showed that ultrasonic degradation increased the antibacterial activity of GCP-F1 as compared to GCP, but GCP-F2 almost lost its antibacterial activity with further ultrasound treatment. Changes in the antioxidant and antibacterial activities of GCP-F1 and GCP-F2 might be related to the variation of their molecular weights.

Phosphoproteomic analysis of FAC overload-triggered human hepatic cells reveals G2/M phase arrest.

Hepatic iron overload is a universal phenomenon in patients with myelodysplastic syndromes (MDS) who undergo bone marrow transplantation and may experience the toxicity of peri- and post-bone marrow transplantation. To clarify the mechanisms of iron overload-triggered liver injury, we determined the effects of iron overload on changes in protein phosphorylation in human hepatocyte cell line HH4 in vitro by using a phosphoproteomics approach. The hepatocytes were exposed to high concentrations of ferric ammonium citrate (FAC) to build up an iron overload model in vitro. Changes in protein phosphorylation initiated by iron overloading were studied by 2D-LC/MS. We identified 335 differentially expressed phosphorylated proteins under the condition of excess hepatocyte iron, 11% of which were related to cell cycle progression. The results of phosphoproteomics showed that iron overload induced 10.9 times increase in Thr 14/Tyr 15-phosphorylated Cdk1 in HH4 cells. Flow cytometry analysis revealed that FAC-treated HH4 cells showed significant G2/M phase arrest. Our subsequent RT-PCR and Western blot experiments indicated that FAC-induced G2/M phase arrest was related to the activation of p53-p21-Cdk1, p53-14-3-3 sigma-Cdk1, and 14-3-3 gamma pathway. Our findings demonstrate the first evidence that iron overload causes G2/M arrest in HH4 hepatocytes.

Effect of Polysaccharide Extracted From Gynostemma Pentaphyllum on the Body Weight and Gut Microbiota of Mice.

Researchers have investigated the role of polysaccharides in disease treatment via gut microbiota regulation but ignore their function in disease prevention and physique enhancement. In this work, a Gynostemma pentaphyllum polysaccharide (GPP) was tested by methyl thiazolyl tetrazolium (MTT) assay and proved to be safe to Caco-2 cells. Animal experiments showed that the administration of GPP for 3 weeks decreased the body weight gain of mice from 15.4 ± 1.7 to 12.2 ± 1.8 g in a concentration-dependent manner. Analysis of short-chain fatty acids (SCFAs) indicated that GPP increased the levels of acetate, propionate, butyrate, and total SCFAs in the cecum contents of normal mice. Furthermore, GPP improved the species richness and abundance in the gut microbiota but reduced the Firmicutes/Bacteroidetes ratio from 0.8021 to 0.3873. This work provides a basis for incorporating GPP into diet to prevent or mitigate the occurrence of obesity via gut microbiota regulation.

An improved solvent evaporation method to produce poly (lactic acid) microspheres via foam-transfer.

The aim of this work was to study an improved solvent evaporation method to prepare poly (lactic acid) (PLA) microspheres via foam-transfer. Since the foaming process and its transfer were critical to the improved method, they have been studied. Additionally, the delivery capability of foams was studied as a function of the oil/water ratio, the stirring rate, the concentration of polyvinyl alcohol (PVA) and ethanol (EtOH) in the aqueous phase (ωPVA, ωEtOH). It was found that foaming varied during the preparation process and it influenced the properties of PLA microspheres. When the oil/water ratio (w/w) ≥ 3:10, stirring rate ≥ 600 r/min, ωPVA ≥ 1 wt%, and ωEtOH = 0 wt%, solvent evaporation was able to produce enough foams for foam-transfer, which helped to deliver more than 89 wt% PLA microspheres to the receiving vessel. However, ωPVA ≤ 0.3 wt% and ωEtOH = 20 wt% were unfavorable for maintaining the spherical shape of PLA microspheres and caused the aggregation. The methodology was further used to prepare azoxystrobin-loaded PLA microspheres successfully with a high encapsulation efficiency of 86.54%. This work is meaningful since it enables an efficient and continuous route to prepare functional biodegradable polymer microspheres.

[Attenuated total reflection-fourier transform infrared spectroscopic study of dried shark fin products].

Sixty-four pieces of shark fin dried products (including real, fake and artificial shark fin products) and real products coated with gelatin were rapidly and nondestructively analyzed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). The characteristic of IR spectrograms among the above four kinds of samples were systematically studied and comparied, the results showed that the spectrograms of the same kind of samples were repeatable, and different kinds of shark fin products presented significant differences in the spectrograms, which mainly manifested as the specific absorption peaks of amido bonds in protein (1650, 1544 cm(-1)) and skeletal vibration in polysaccharide (1050 cm(-1)). The spectrograms of real shark fins were characterized by the strong absorption peaks of protein characteristic amide I and II absorbent (1650, 1544 cm(-1)) and relatively weak C--O--C vibration absorbent (1050 cm(-1)) owing to the high content of protein and relatively low level of polysaccharide. For fake shark fin products that were molded form by mixing together with the offcut of shark, collagen and other substances, the introduction of non-protein materials leaded to the weaker amido bonds absorbent than real products along with a 30 cm(-1) blue shift of amide I absorbent. Opposite to the real sample, the relatively strong absorption peak of polysaccharide (approximately 1047 cm(-1)) and barely existed amide absorbent were the key features of the spectrogram of artificial samples, which was synthersized by polysaccharide like sodium alginate. Real samples coated with gelatin, the peak strength of protein and polysaccharide were decreased simultaneously when the data collection was taken at the surface of sample, while the spectrogram presented no significant difference to real samples when the data was collected in the section. The results above indicated that by analyzing the characteristic of IR spectrograms and the value range of Apro/Apol collected by ATR-FTIR method could perform the undamaged and rapid identification for shark fins.

Geographical origin of cereal grains based on element analyser-stable isotope ratio mass spectrometry (EA-SIRMS).

The stable carbon and nitrogen isotopic compositions (δ(13)C and δ(13)N) of different cereal grains from different regions were determined, using element analyser-stable isotope ratio mass spectrometry (EA-SIRMS) as the key method. Systematically, δ(13)C and δ(13)N of 5 kinds of cereal grains of different origins, 30 wheat samples from different cultivation areas and 160 rice samples of different cultivars from Guangdong province of China were examined. The results indicated that the δ(13)C values of rice, soybean, millet, wheat and corn were significantly (P < 0.05) different within different origins (Heilongjiang, Shandong and Jiangsu province of China), respectively, while δ(13)N values were not. Interestingly, there exists discrimination between these 5 kinds of cereals grains, no matter C-3 or C-4 plants. Further study showed that the δ(13)C values of wheat from Australia, the USA, Canada, and Jiangsu and Shandong province of China were also significantly (P < 0.01) different. Furthermore, the P-value test for 160 rice samples of 5 cultivars was not significant (P > 0.05), which indicated that the cultivar of cereal grains was not significant based on δ(13)C value. Thus, the comparison of δ(13)C would be potentially useful for rapid and routine discrimination of geographical origin of cereal grains.

Structure, morphology and cell affinity of poly(L-lactide) films surface-functionalized with chitosan nanofibers via a solid-liquid phase separation technique.

Poly(L-lactide) films with a nano-structured surface by immobilizing chitosan nanofibers (CSNFs) for improving the cell affinity were fabricated via a solid-liquid phase separation technique. The successful grafting of CSNFs on the surface of poly(L-lactide) films was confirmed by the binding energy of N1s at 398.0 eV in the X-ray photoelectron spectroscopy and the amide I and II bands of chitosan at 1650 and 1568 cm(-1) in the Fourier transform infrared spectroscopy. Compared with the poly(L-lactide) film, the hydrophilicity was improved with a lower water contact angle of 83.3±1.9° and 75.3±2.5° for the CSNFs-grafted and CSNFs-grafted/anchored poly(L-lactide) films respectively. The scanning electron microscopy and atomic force microscopy analyses showed that the grafted CSNFs with 50-500 nm in diameter were randomly arranged on the film surface and entangled with the anchored CSNFs on the outermost layer. The 3T3 fibroblasts culture indicated cells tended to attach and stretch along the CSNFs on the film surface. The cell viability measurement revealed that among all the samples, the film with both grafted and anchored CSNFs exhibited the highest cell proliferation rate that was twice as much of the poly(L-lactide) film at 7 d. Herein, engineering a nano-structured surface by solid-liquid phase separation will be a promising tool for surface modification of biomaterials.

Preparation, characterization and cytocompatibility of polyurethane/cellulose based liquid crystal composite membranes.

Two types of polyurethane/liquid crystal (PU/LC) composite membranes with different LC contents, namely polyurethane/octyl hydroxypropyl cellulose ester (PU/OPC) and polyurethane/propyl hydroxypropyl cellulose ester (PU/PPC), were prepared and studied. The effects of surface properties on cell compatibility of the membranes were elucidated. PPC tended to assemble to independent phases in the composite membranes, while OPC formed uniformly distributed LC domains. As the introduction of LC, phase separation occurred, and the crystallization of PU was disrupted. The surface of PU/LC composite membranes showed fingerprint texture and two-phase morphology. Hydrophilicity of the two types of composite membranes exhibited a reversal tendency with the increase of LC contents. Cells seeded on the composite membranes presented favorable growth when the content of LC was over 30%, especially on PU/OPC complex. The surface morphology, phase separation between LC and PU as well as the type of LC showed significant effects on the cell behaviors.

Fabrication and in vivo osteogenesis of biomimetic poly(propylene carbonate) scaffold with nanofibrous chitosan network in macropores for bone tissue engineering.

A biomimetic poly(propylene carbonate) (PPC) porous scaffold with nanofibrous chitosan network within macropores (PPC/CSNFs) for bone tissue engineering was fabricated by a dual solid-liquid phase separation technique. PPC scaffold with interconnected solid pore wall structure was prepared by the first phase separation, which showed a high porosity of 91.9% and a good compressive modulus of 14.2 ± 0.56 MPa, respectively. By the second phase separation, nanofibrous chitosan of 50-500 nm in diameter was formed in the macropores with little influence on the pore structure and the mechanical properties of PPC scaffold. The nanofibrous chitosan content was calculated to be 9.78% by elemental analysis. After incubation in SBF for 14 days, more apatite crystals were deposited on the pore surface as well as the nanofibrous chitosan surface of PPC/CSNFs scaffold compared with PPC scaffold. The in vitro culture of bone mesenchymal stem cells showed that PPC/CSNFs scaffold exhibited a better cell viability than PPC scaffold. After implantation in rabbits for 16 weeks, the defect was entirely repaired by PPC/CSNFs scaffold, as opposed to the incomplete healing for PPC scaffold. It indicated that PPC/CSNFs scaffold showed a faster in vivo osteogenesis rate than PPC scaffold. Hereby, PPC/CSNFs scaffold will be a potential candidate for bone tissue engineering.

Preparation and properties of biomimetic porous nanofibrous poly(L-lactide) scaffold with chitosan nanofiber network by a dual thermally induced phase separation technique.

A biomimetic nanofibrous poly(L-lactide) scaffold decorated by chitosan nanofiber network inside the macropores was fabricated using a dual thermally induced phase separation technique. The first phase separation was used to build a nanofibrous poly(L-lactide) scaffold with interconnected macropores, where chitosan nanofibers about 500nm in diameter were incorporated via the second phase separation. The content of nanofibrous chitosan was determined to be 5.76 in weight percentage by elemental analysis. The composite scaffold showed the highest protein adsorption of 7225±116 µg/cm(3) and the most hydroxyapatite crystal deposition in the mineralization. Compared with non-nanofibrous poly(L-lactide) scaffold, nanofibrous poly(L-lactide) scaffold exhibited a much faster degradation, but it could be restrained by the introduced chitosan nanofibers. The bone mesenchymal stem cell culture results indicated that the cells would rather attach and stretch along the chitosan nanofibers in the composite scaffold that showed the highest viability and the best cytocompatibility may be attributed to the biomimetic nanofibrous network and good cell affinity of chitosan nanofibers.