Establishment of a hydrodynamic delivery system in ducks.

Chronic hepatitis B virus (HBV) poses a significant global health challenge as it can lead to acute or chronic liver disease and hepatocellular carcinoma (HCC). To establish a safety experimental model, a homolog of HBV-duck HBV (DHBV) is often used for HBV research. Hydrodynamic-based gene delivery (HGD) is an efficient method to introduce exogenous genes into the liver, making it suitable for basic research. In this study, a duck HGD system was first constructed by injecting the reporter plasmid pLIVE-SEAP via the ankle vein. The highest expression of SEAP occurred when ducks were injected with 5 µg/mL plasmid pLIVE-SEAP in 10% bodyweight volume of physiological saline for 6 s. To verify the distribution and expression of exogenous genes in multiple tissues, the relative level of foreign gene DNA and ß-galactosidase staining of LacZ were evaluated, which showed the plasmids and their products were located mainly in the liver. Additionally, ß-galactosidase staining and fluorescence imaging indicated the delivered exogenous genes could be expressed in a short time. Further, the application of the duck HGD model on DHBV treatment was investigated by transferring representative anti-HBV genes IFNα and IFNγ into DHBV-infected ducks. Delivery of plasmids expressing IFNα and IFNγ inhibited DHBV infection and we established a novel efficient HGD method in ducks, which could be useful for drug screening of new genes, mRNAs and proteins for anti-HBV treatment.

Transcriptomic and functional characterization reveals CsHAK5;3 as a key player in K+ homeostasis in grafted cucumbers under saline conditions.

Grafting can improve the salt tolerance of many crops. However, critical genes in scions responsive to rootstock under salt stress remain a mystery. We found that pumpkin rootstock decreased the content of Na+ by 70.24 %, increased the content of K+ by 25.9 %, and increased the K+/Na+ ratio by 366.0 % in cucumber scion leaves. RNA-seq analysis showed that ion transport-related genes were the key genes involved in salt stress tolerance in grafted cucumber. The identification and analysis of the expression of K+ transporter proteins in cucumber and pumpkin revealed six and five HAK5 members, respectively. The expression of CsHAK5;3 in cucumber was elevated in different graft combinations under salt stress and most notably in cucumber scion/pumpkin rootstock. CsHAK5;3 was localized to the plasma membrane, and a yeast complementation assay revealed that it can transport K+. CsHAK5;3 knockout in hairy root mutants decreased the K+ content of leaves (45.6 %) and roots (50.3 %), increased the Na+ content of leaves (29.3 %) and roots (34.8 %), and decreased the K+/Na+ ratio of the leaves (57.9 %) and roots (62.9 %) in cucumber. However, CsHAK5;3 overexpression in hairy roots increased the K+ content of the leaves (31.2 %) and roots (38.3 %), decreased the Na+ content of leaves (17.2 %) and roots (14.3 %), and increased the K+/Na+ ratio of leaves (58.9 %) and roots (61.6 %) in cucumber. In conclusion, CsHAK5;3 in cucumber can mediate K+ transport and is one of the key target pumpkin genes that enhance salt tolerance of cucumber grafted.

The structures and applications of microbial chondroitin AC lyase.

As an important glycosaminoglycan hydrolase, chondroitin lyases can hydrolyze chondroitin sulfate (CS) and release disaccharides and oligosaccharides. They are further divided into chondroitin AC, ABC, and B lyases according to their spatial structure and substrate specificity. Chondroitin AC lyase can hydrolyze chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), and hyaluronic acid (HA), making it an essential biocatalyst for the preparation of low molecular weight chondroitin sulfate, analysis of the structure of the chondroitin sulfate, treatment of spinal cord injury, and purification of heparin. This paper provides an overview of reported chondroitin AC lyases, including their properties and the challenges faced in industrial applications. Up to now, although many attempts have been adopted to improve the enzyme properties, the most important factors are still the low activity and stability. The relations between the stability of the enzyme and the spatial structure were also summarized and discussed. Also perspectives for remodeling the enzymes with protein engineering are included.

Katsuwonus pelamis Peptide and its Complexes Protect Zebrafish and Mice From Hyperuricemia Through Promoting Kidney Excretion of Uric Acid and Inhibiting Liver Xanthine Oxidase Activity.

Katsuwonus pelamis peptide and its complexes have the effect of lowering uric acid (UA)-levels. To identify the effect and possible mechanisms, different concentrations of Katsuwonus pelamis peptide and its complexes were administered to the zebrafish and mice hyperuricemia models, and the UA level was measured. Meanwhile, the hyperuricemic mice were treated orally at 0.83, 1.67, and 5.00 mg/g body weight for 7 days with Katsuwonus pelamis peptide and the complexes groups, separately. The levels of serum UA (SUA), urinary UA (UUA), serum creatinine (SCR), blood urine nitrogen (BUN), and xanthine oxidase (XOD) activities were detected in each group. The results showed that the Katsuwonus pelamis peptide (125 µg/ml) and its complexes (83.3 and 250 µg/ml) effectively reduced UA level in zebrafish with hyperuricemia (p < 0.05). The Katsuwonus pelamis peptide at high concentration (5.00 mg/g) decreased the SUA level, SCR level, BUN level, and hepatic XOD activity, and the complexes (1.67 and 5.00 mg/g) significantly reduced the SUA level and hepatic XOD activity (p < 0.05) in the hyperuricemic mice. In addition, in a hyperuricemic mouse model, the UUA level was increased after treatment with Katsuwonus pelamis peptide and its complexes at high concentrations (p < 0.05). The total therapeutic effects in the Katsuwonus pelamis peptide complex group were better than those in the Katsuwonus pelamis peptide group. Thus, Katsuwonus pelamis peptide and its complexes may possibly be used to prevent hyperuricemia via promoting urate secretion and inhibiting XOD activity production.

Comparing the Bridge-Type Zero-Profile Anchored Spacer (ROI-C) Interbody Fusion Cage System and Anterior Cervical Discectomy and Fusion (ACDF) with Plating and Cage System in Cervical Spondylotic Myelopathy.

OBJECTIVE: To compare the clinical efficacy and radioactivity of the bridge-type zero-profile anchored spacer (ROI-C) interbody fusion cage and anterior cervical discectomy and fusion with plating and cage system (ACDF) for cervical spondylotic myelopathy (CSM). METHODS: This is a retrospective contrastive study. We recruited 35 patients who received ROI-C (ROI-C group) and 34 patients who received ACDF (ACDF group), between January 2014 to January 2019, at our treatment center. The ROI-C group comprised of 11 males and 24 females with a mean age of 61.59 ± 8.21 years (range, 51-71 years). The ACDF group comprised of 12 males and 22 females with a mean age of 60.15 ± 7.52 years (range, 52-74 years). Neck Disability Index (NDI), Japanese Orthopaedic Association score (JOA), Odom's score, cervical Cobb angle, fusion rate, adjoining ossification, and dysphagia. RESULTS: A total of 69 patients met the inclusion criteria, and these patients received more than two years of follow-up. There were significant differences in surgical duration (101 ± 22 min vs. 118 ± 29 min) and blood loss (102 ± 46 ml vs. 145 ± 58 ml) between two groups (P < 0.05). The JOA and NDI of these two groups of patients significantly improved, when compared with those before the operation (P < 0.05). Twenty-nine of 35 patients in the ROI-C group and 27 of 34 patients in ACDF group achieved good or excellent outcomes according to Odom's criteria. The cervical lordosis of both two groups significantly increased, when compared with those before the operation (P < 0.05). In the ROI-C group, the postoperative fusion rate was 85.7% at the 3-month follow-up and 100% at the final follow-up. In the ACDF group, the postoperative fusion rate was 82.4% at the 3-month follow-up and 100% at the final follow-up. The dysphagia incidence of the ACDF group was higher than that of the ROI-C group postoperatively and at the one month after surgery (P < 0.05), but no significant difference was found in the incidence of dysphagia at final follow-up (P > 0.05). CONCLUSION: Both ROI-C and ACDF achieved good therapeutic effects. However, ROI-C can reduce the operation time and postoperative complications.

An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops.

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot-root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K+ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock, and by overexpressing the pumpkin tonoplast Na+/H+ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.

Comparison of Clinical Outcomes Following Anterior Cervical Discectomy and Fusion with Zero-Profile Anchored Spacer-ROI-C-Fixation and Combined Intervertebral Cage and Anterior Cervical Discectomy and Fusion: A Retrospective Study from a Single Center.

BACKGROUND The aim of this study was to compare the outcomes following anterior cervical discectomy and fusion with zero-profile anchored spacer-ROI-C-fixation (ROI-C) vs combined intervertebral cage and anterior cervical discectomy and fusion (ACDF). MATERIAL AND METHODS We retrospectively analyzed 87 patients who underwent operations between January 2015 and January 2019, including 42 patients that underwent ROI-C treatment (group A) and 45 that were treated by the ACDF approach (group B). Operative duration, blood loss, dysphagia, Neck Disability Index scores (NDI), Japanese Orthopaedic Association scores (JOA), and other complications were compared between these groups. In addition, implant settlement, fusion, and cervical Cobb angle were assessed via imaging analyses. RESULTS Patients in group A and group B were followed for 22.6±3.3 months and 27.1±3.5 months, respectively (range: 13-30 months). Relative to preoperative values, JOA scores were increased and NDI scores were reduced in both groups following treatment (P<0.05), with comparable outcomes between groups (P>0.05). However, operative duration, intraoperative blood loss, and postoperative complications did differ significantly between these groups (P<0.05). Specifically, rates of short-term dysphagia were lower and recovery time was faster in group A relative to group B (P<0.05). CONCLUSIONS The findings from this study showed that ROI-C fixation achieved satisfactory outcomes, improved cervical curvature, restored intervertebral height, and was associated with shorter operative duration, reduced blood loss, and less dysphagia.

One-step selective affinity purification and immobilization of His-tagged enzyme by recyclable magnetic nanoparticles.

The NiFe2O4 magnetic nanoparticles (NF-MNPs) were prepared for one-step selective affinity purification and immobilization of His-tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier-transform infrared spectrophotometer and microscopy. The immobilization and purification of His-tagged GluDH on NF-MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris-HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 µg mg-1 support, respectively. The immobilized GluDH exhibited high thermostability, pH-stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni-NTA resin, the NF-MNPs displayed a higher specific affinity with His-tagged recombinant GluDH.

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization.

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae.

Potassium (K+) is a critical determinant of salinity tolerance, and H2O2 has been recognized as an important signaling molecule that mediates many physiological responses. However, the details of how H2O2 signaling regulates K+ uptake in the root under salt stress remain elusive. In this study, salt-sensitive cucumber and salt-tolerant pumpkin which belong to the same family, Cucurbitaceae, were used to answer the above question. We show that higher salt tolerance in pumpkin was related to its superior ability for K+ uptake and higher H2O2 accumulation in the root apex. Transcriptome analysis showed that salinity induced 5816 (3005 up- and 2811 down-) and 4679 (3965 up- and 714 down-) differentially expressed genes (DEGs) in cucumber and pumpkin, respectively. DEGs encoding NADPH oxidase (respiratory burst oxidase homolog D; RBOHD), 14-3-3 protein (GRF12), plasma membrane H+-ATPase (AHA1), and potassium transporter (HAK5) showed higher expression in pumpkin than in cucumber under salinity stress. Treatment with the NADPH oxidase inhibitor diphenylene iodonium resulted in lower RBOHD, GRF12, AHA1, and HAK5 expression, reduced plasma membrane H+-ATPase activity, and lower K+ uptake, leading to a loss of the salinity tolerance trait in pumpkin. The opposite results were obtained when the plants were pre-treated with exogenous H2O2. Knocking out of RBOHD in pumpkin by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] editing of coding sequences resulted in lower root apex H2O2 and K+ content and GRF12, AHA1, and HAK5 expression, ultimately resulting in a salt-sensitive phenotype. However, ectopic expression of pumpkin RBOHD in Arabidopsis led to the opposite effect. Taken together, this study shows that RBOHD-dependent H2O2 signaling in the root apex is important for pumpkin salt tolerance and suggests a novel mechanism that confers this trait, namely RBOHD-mediated transcriptional and post-translational activation of plasma membrane H+-ATPase operating upstream of HAK5 K+ uptake transporters.

[Analysis of therapeutic effects of external fixator for the treatment of comminuted fracture of distal radius].

OBJECTIVE: To evaluate the clinical effects of external fixator in treating comminuted fracture of distal radius. METHODS: From Mar.2008 to Dec.2009, 37 patients with comminuted fracture of distal radius were treated with external fixator or assisted with Kirschner wire and T-shape locking compression plate (T-LCP) fixation. There were 14 males and 23 females, ranging in age from 30 to 79 years, with an average of 59.1 years. According to AO typing, type C1 was in 3 cases, type C2 was in 11 cases and type C3 was in 23 cases. Function of wrist joint and X-ray films were observed according to Gratland-Werley system at different months. RESULTS: All patients were followed up from 8 to 24 months with an average of 12 months. All fractures had healing with an average time of 8 weeks. According to Gratland-Werley system, 16 cases obtained excellent result, 17 good, 4 fair, the rate of excellent and good was 89.0%. CONCLUSION: External fixator can obtained satisfactory clinical effect in treating comminuted fracture of distal radius.

Neurovascular musculus obliquus internus abdominis flap free transfer for facial reanimation in a single stage.

A study of the anatomy and transplantation of the musculus obliquus internus abdominis with a neurovascular pedicle transfer for facial reanimation in one stage is presented. Eleven adult cadavers (22 face sides) were dissected to observe the shape, thickness, innervation, and blood supply of the musculus obliquus internus abdominis. The blood supply of this muscle primarily comes from the musculus obliquus internus abdominis branch of the deep circumflex iliac artery (diameter, 1.3 +/- 0.2 mm), but it can also come from the eleventh intercostal artery (diameter, 1.14 +/- 0.3 mm) and the infracostal artery (diameter, 1.5 +/- 0.2 mm). The branch of the deep circumflex iliac artery and its vena comitans, or the infracostal artery and its vena comitans, could be anastomosed for muscle transplantation. The innervation of the musculus obliquus internus abdominis comes from the tenth and eleventh intercostal nerves (length, 12.7 +/- 1.5 cm) and the infracostal nerve (length, 12.9 +/- 1.3 cm). The eleventh intercostal nerve and the infracostal nerve were selected for anastomosis of muscle transplantation. From November of 1995 to November of 1999, 14 patients with long established facial paralysis were treated with transplantation of a musculus obliquus internus abdominis flap in one stage and were followed for 10 months to 6 years. In 13 patients, the dynamic functions of the transplanted muscles were restored, the obliqueness of the mouth and philtrum while static was corrected, and the facial muscle activities while smiling were harmonized. The eyelids of the paralyzed side could be closed postoperatively, indicating that the function of the orbicularis oculi of the paralyzed side was restored. The single-stage transplantation of a free musculus obliquus internus abdominis flap with one vascular, multi-nerve pedicle is a new method for facial reanimation in the treatment of long established facial paralysis. Because of the simplicity of the procedure and the completeness of the functional reanimation of the paralyzed facial muscles, compared with the results of other free muscle flap transfers, it is an ideal procedure for facial reanimation.