Three-dimensional manganese cobaltate: a highly conductive electrocatalyst for paraoxon-ethyl detection.

The synthesis of manganese cobaltate (MnCo2O4) with the hybrid three-dimensional architecture has been developed as an electrocatalyst for the electrochemical sensing of paraoxon-ethyl (PEL). The detailed physicochemical and structural characterization of MnCo2O4 is meticulously examined. The MnCo2O4-modified screen-printed carbon electrode (SPCE) exhibits good electrocatalytic activity for the reduction of PEL compared with the bare SPCE due to numerous unique properties. By profiting from these advantages, the proposed MnCo2O4/SPCE shows superior sensing performance toward the determination of PEL, including low cathodic peak potential (- 0.64 V), wide detection range (0.015-435 µM), low limit of detection (0.002 µM), high detection sensitivity (2.30 µA µM-1 cm-2), excellent selectivity, and good reproducibility. Notably, the electrochemical performance of the MnCo2O4-based electrocatalyst is superior to those previously reported in the literatures. The practical application of the MnCo2O4/SPCE is effectively assessed in the analysis of food and water samples with satisfied recoveries of 96.00-99.35%. The superior performance of the proposed MnCo2O4 electrocatalyst holds considerable potential for future development of electrochemical sensing platforms.

Two-Dimensional Mechano-thermoelectric Heterojunctions for Self-Powered Strain Sensors.

We here demonstrate the multifunctional properties of atomically thin heterojunctions that are enabled by their strong interfacial interactions and their application toward self-powered sensors with unprecedented performance. Bonding between tin diselenide and graphene produces thermoelectric and mechanoelectric properties beyond the ability of either component. A record-breaking ZT of 2.43 originated from the synergistic combination of graphene's high carrier conductivity and SnSe2-mediated thermal conductivity lowering. Moreover, spatially varying interaction at the SnSe2/graphene interface produces stress localization that results in a novel 2D-crack-assisted strain sensing mechanism whose sensitivity (GF = 450) is superior to all other 2D materials. Finally, a graphene-assisted growth process permits the formation of high-quality heterojunctions directly on polymeric substrates for flexible and transparent sensors that achieve self-powered strain sensing from a small temperature gradient. Our work enhances the fundamental understanding of multifunctionality at the atomic scale and provides a route toward structural health monitoring through ubiquitous and smart devices.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L L-leucine in shake flask fermentation.

Metabolic engineering of l-leucine production in Escherichia coli and Corynebacterium glutamicum: a review.

l-Leucine, as an essential branched-chain amino acid for humans and animals, has recently been attracting much attention because of its potential for a fast-growing market demand. The applicability ranges from flavor enhancers, animal feed additives and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. Microbial fermentation is the major method for producing l-leucine by using Escherichia coli and Corynebacterium glutamicum as host bacteria. This review gives an overview of the metabolic pathway of l-leucine (i.e. production, import and export systems) and highlights the main regulatory mechanisms of operons in E. coli and C. glutamicum l-leucine biosynthesis. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating l-leucine producing strains. Finally, future perspectives to construct industrially advantageous strains are considered with respect to recent advances in biology.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L-1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L-1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L-1 to 2.72 ± 0.11 g·L-1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L-1 l-leucine with a glucose conversion efficiency of 0.191 g·g-1.

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L-1 ), carbon yield (from 0.35 to 0.44 g [g glucose]-1 ) and productivity (from 2.07 to 2.93 g L-1 hr-1 ) of l-lysine in JL-6 ΔdapB::Ec-dapBC115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapBC115G,G116C , followed by JL-6 Cg-dapBC37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapBC46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity.

[Congenital Fâ ¦ Deficiency Associated with a Novel Mutation in F7 Gene].

OBJECTIVE: To identify the genetic mutation of coagulation factor Ⅶ ( F7) gene in a pedigree with coagulation factor Ⅶ (FⅦ) deficiency and explore the molecular pathogenesis. METHODS: The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), D-dimer (DD), fibrin degradation products (FDP) and coagulation factor Ⅶ activity (FⅦ:C) of the proband and her family members were detected by Sysmex-CS5100 analyzer. All exons and exon-intron boundaries of the F7 gene were amplified by PCR followed by direct sequencing. The detected mutation was confirmed by reverse sequencing. The ClustalW software was used to analyze the conservatism of the mutant site. Pathogenicity of the mutation was assessed with Mutation Taster and PolyPhen-2 online bioinformatics software. Structure of the mutant protein was analyzed using Swiss-PdbViewer software. RESULTS: The results of routine coagulation tests showed that PT of the proband was markedly extended to 42.5 s, and her FⅦ:C significantly reduced to 2%. The FⅦ:C of her grandmother, mother and sister had slightly reduced to 49%, 51%, and 42%, respectively. These coagulant parameters of her father were within the normal range. Genetic analysis reveled a heterozygous G>A change at cDNA 646 in exon 6 of F7 gene in the proband, resulting in a replacement of glycine at 156 of FⅦ catalytic region with serine (p.Gly156Ser). The sequencing results of other exons and exon-intron boundaries of her F7 gene were normal. The proband's grandmother, mother and sister were all the carriers of this missense mutation except her father. Bioinformatics analysis showed that the p.Gly156Ser mutation caused polarity change of the amino acid at this site and formation of side chains, leading to increase of protein instability, which may affect catalytic activity of structural domain. Meanwhile, both Mutation Taster and PolyPhen-2 online bioinformatics software also predicted the pathogenicity of this missense mutation with high scores. CONCLUSION: The heterozygous p.Gly156Ser mutation is the direct cause of the reduced FⅦ in this proband.

Gualou xiebai decoction ameliorates cardiorenal syndrome type II by regulation of PI3K/AKT/NF-κB signalling pathway.

BACKGROUND: Cardiorenal syndromes type II (CRS2) is a multi-organ ailment that manifests as a combination of cardiac and renal dysfunction, resulting in chronic kidney disease due to chronic cardiac insufficiency. It affects at least 26 million people worldwide, and its prevalence is increasing. Gualou Xiebai Decoction (GXD), a traditional Chinese medicine (TCM) with a rich history of application in the management of coronary artery disease, has been explored for its potential therapeutic benefits in CRS2. Nevertheless, the mechanism by which GXD alleviates CRS2 remains obscure, necessitating further investigation. PURPOSE: The aim of this study was to assess the effects of the ethanolic extract of GXD on CRS2 and to elucidate the underlying mechanism in a rat model of myocardial infarction, offering a potential target for clinical treatment for CRS2. STUDY DESIGN AND METHODS: A rat model of CRS2 was induced by surgical myocardial infarction and treated with GXD for 10 weeks. Cardiac function was assessed using echocardiography, while serum and urine biochemistry were analyzed to evaluate potential cardiac and renal damage. Furthermore, tissue samples were obtained for histological, protein, and genetic investigations. In addition, network pharmacology analysis and molecular docking were utilized to predict the primary active compounds, potential therapeutic targets, and interventional pathways through which GXD could potentially exert its effects on CRS2. Subsequently, these predictions were confirmed in vivo and vitro through various analyses. RESULTS: The current investigation employed echocardiography to exhibit the apparent cardiac remodeling following the induction of myocardial infarction. Damage to the heart and kidneys of CRS2 rats was effectively ameliorated by administration of GXD. The outcomes derived from the analyses of HE and Masson staining indicated that the pathological damage to the heart and kidney tissues of rats in the GXD groups was considerably alleviated. Using network pharmacology analysis, AKT1, IL-6, and TNF-α were identified as plausible therapeutic targets for the treatment of CRS with GXD. Subsequent functional and pathway enrichment analysis of the underlying targets disclosed that the PI3K/AKT/NF-κB signaling pathway may be involved in the mechanism of GXD in the treatment of CRS2. Immunohistochemical, western blot, RT-PCR and immunofluorescence staining were employed to demonstrate that GXD can regulate the PI3K/AKT/NF-κB signaling pathway in the CRS2 rat model. Ultimately, administration of the PI3K/AKT agonist 740Y-P counteracted the effect of diosmetin, which was one of the potential active components of GXD analysed by compound-target-disease network, on p-PI3K and p-AKT in vitro. CONCLUSIONS: The findings of this study suggest that GXD improves cardiac and renal function in CRS2 rats and that the underlying mechanism involves inhibition of the PI3K/AKT/NF-κB pathway.

Insights into optimal surgical fixation for posterior malleolar fractures.

Aims: The optimal management of posterior malleolar ankle fractures, a prevalent type of ankle trauma, is essential for improved prognosis. However, there remains a debate over the most effective surgical approach, particularly between screw and plate fixation methods. This study aims to investigate the differences in outcomes associated with these fixation techniques. Methods: We conducted a comprehensive review of clinical trials comparing anteroposterior (A-P) screws, posteroanterior (P-A) screws, and plate fixation. Two investigators validated the data sourced from multiple databases (MEDLINE, EMBASE, and Web of Science). Following PRISMA guidelines, we carried out a network meta-analysis (NMA) using visual analogue scale and American Orthopaedic Foot and Ankle Score (AOFAS) as primary outcomes. Secondary outcomes included range of motion limitations, radiological outcomes, and complication rates. Results: The NMA encompassed 13 studies, consisting of four randomized trials and eight retrospective ones. According to the surface under the cumulative ranking curve-based ranking, the A-P screw was ranked highest for improvements in AOFAS and exhibited lowest in infection and peroneal nerve injury incidence. The P-A screws, on the other hand, excelled in terms of VAS score improvements. Conversely, posterior buttress plate fixation showed the least incidence of osteoarthritis grade progression, postoperative articular step-off ≥ 2 mm, nonunions, and loss of ankle dorsiflexion ≥ 5°, though it underperformed in most other clinical outcomes. Conclusion: The NMA suggests that open plating is more likely to provide better radiological outcomes, while screw fixation may have a greater potential for superior functional and pain results. Nevertheless, clinicians should still consider the fragment size and fracture pattern, weighing the advantages of rigid biomechanical fixation against the possibility of soft-tissue damage, to optimize treatment results.

Determining the Optimal Treatment for Idiopathic Clubfoot: A Network Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Clubfoot, or congenital talipes equinovarus deformity, is a common anomaly affecting the foot in infants. However, clinical equipoise remains between different interventions, especially those based on the Ponseti method. The aim of this study was to examine the clinical outcomes of the various interventions for treating idiopathic clubfoot. METHODS: Searches of the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, Scopus, and CINAHL were conducted. Randomized controlled trials comparing different interventions, including the Ponseti method, accelerated Ponseti method, Ponseti method with botulinum toxin type A (Botox) injection, Ponseti method with early tibialis anterior tendon transfer (TATT), Kite method, and surgical treatment, were included. Network meta-analyses (NMAs) were conducted according to the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) reporting guidelines. The primary outcomes were the change in total Pirani score and maximal ankle dorsiflexion. Secondary outcomes were the number of casts, time in casts, and rates of tenotomy, total complications, relapse, adverse events, and additional required major surgery. RESULTS: Eleven randomized controlled trials involving 740 feet were included. According to the SUCRA (surface under the cumulative ranking curve)-based relative ranking, the Ponseti method was associated with the best outcomes in terms of Pirani score changes, maximal ankle dorsiflexion, number of casts, adverse events, and total complications, whereas the accelerated Ponseti method was associated with the best outcomes in terms of time in casts and tenotomy rate. Early TATT ranked best in terms of relapse rate. The Ponseti method with Botox injection was associated with the best outcomes in terms of the need for additional major surgery. CONCLUSIONS: The NMAs suggest that the Ponseti method is the optimal treatment overall, despite potential drawbacks such as longer time in casts and higher rates of tenotomy, relapse, and the need for additional surgery compared with other modified approaches. Therefore, clinicians should consider how treatments can be tailored individually. LEVEL OF EVIDENCE: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

Is implant choice associated with fixation strength for displaced radial neck fracture: a network meta-analysis of biomechanical studies.

The multitude of fixation options for radial neck fractures, such as pins, screws, biodegradable pins and screws, locking plates, and blade plates, has led to a lack of consensus on the optimal implant choice and associated biomechanical properties. This study aims to evaluate the biomechanical strength of various fixation constructs in axial, sagittal, and torsional loading directions. We included biomechanical studies comparing different interventions, such as cross/parallel screws, nonlocking plates with or without augmented screws, fixed angle devices (T or anatomic locking plates or blade plates), and cross pins. A systematic search of MEDLINE (Ovid), Embase, Scopus, and CINAHL EBSCO databases was conducted on September 26th, 2022. Data extraction was carried out by one author and verified by another. A network meta-analysis (NMA) was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Primary outcomes encompassed axial, bending, and torsional stiffness, while the secondary outcome was bending load to failure. Effect sizes were calculated for continuous outcomes, and relative treatment ranking was measured using the surface under the cumulative ranking curve (SUCRA). Our analysis encompassed eight studies, incorporating 172 specimens. The findings indicated that fixed angle constructs, specifically the anatomic locking plate, demonstrated superior axial stiffness (mean difference [MD]: 23.59 N/mm; 95% CI 8.12-39.06) in comparison to the cross screw. Additionally, the blade plate construct excelled in bending stiffness (MD: 32.37 N/mm; 95% CI - 47.37 to 112.11) relative to the cross screw construct, while the cross-screw construct proved to be the most robust in terms of bending load failure. The parallel screw construct performed optimally in torsional stiffness (MD: 139.39 Nm/degree; 95% CI 0.79-277.98) when compared to the cross screw construct. Lastly, the nonlocking plate, locking T plate, and cross-pin constructs were found to be inferior in most respects to alternative interventions. The NMA indicated that fixed angle devices (blade plate and anatomic locking plate) and screw fixations may exhibit enhanced biomechanical strength in axial and bending directions, whereas cross screws demonstrated reduced torsional stability in comparison to parallel screws. It is imperative for clinicians to consider the application of these findings in constraining forces across various directions during early range of motion exercises, taking into account the distinct biomechanical properties of the respective implants.

[Microscopic and polariscopic characteristics of 30 medicinal plants of Polygonum].

Polygonum L. s. str., belonging to Polygonaceae family, is a big genus with abundant medicinal plants. More than 10 plants are specified in Chinese Pharmacopoeia and many local medicinal standards and over 50 species are used as folk medicines. Owing to the similar morphologies and very small flowers and fruits, they are uneasily identified and often confused with each other and misused clinically. In order to provide a basis for identification of Polygonum s. str. plants, a histological study on stems and leaves of 30 species from Polygonum was undertaken by a routine/polarized light microscopy for the first time. The results showed that: (1) the transverse sections of stems of Polygonum are relatively similar, sclerenchyma such as xylem and fibres with strong polarization effects; (2) the surface views of leaves of Polygonum are distinguishable on distributions and types of stomata, with or without attachments (such as glandular hairs/scales or non-glandular hairs) and the polariscopic features of epidermal cell walls, stomata and cell contents. Observed under polarized light, it was found for the first time that stomata on leaf surface of some plants have a Maltese-cross effect with the arms of the cross intersecting at the stomatal opening. As a result, a key combining the microscopic and polariscopic characteristics of the stems as well as leaves was provided for identifying the 30 medicinal plants of Polygonum. The polarized light microscopic method was proven to be one of the quick, simple and effective techniques for the identification of medicinal plants and botanic crude materials.

The synthesis of puerarin derivatives and their protective effect on the myocardial ischemia and reperfusion injury.

Puerarin is a naturally occurring isoflavone and is frequently used for the treatment of cardiovascular symptoms in China. By the structural modification of the puerarin molecule at different positions, seven new puerarin derivatives were obtained, and their cardioprotective activities (in vitro and in vivo) were respectively evaluated. The finding that the activities of 3 and 8 markedly exceeded puerarin suggested that the acylated modification of phenolic hydroxyl at C-7 in the puerarin molecule may improve the cardioprotective activity, which will be an important reference for further structural optimization.

Glutathione depletion upregulates P-glycoprotein expression at the blood-brain barrier in rats.

OBJECTIVES: Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier are subjected to chronic oxidative stress. Using an in-vivo system, we have investigated whether glutathione depletion changed expression of P-glycoprotein at the blood-brain barrier in rats. METHODS: Diethyl maleate was intraperitoneally injected to induce GSH depletion in rats. P-glycoprotein expression at the blood-brain barrier was examined by Western blotting and RT-PCR, and its function was assessed by measuring the brain-to-plasma concentration ratios (Kp values) of rhodamine 123 (Rh123). Evans Blue dye was used as a blood-brain barrier indicator for examining the extravasation from the blood to the brain. KEY FINDINGS: Four hours after treatment of rats with diethyl maleate, the brain GSH content significantly reduced. The mdr1a mRNA expression at the blood-brain barrier was upregulated, whereas no significant change in mdr1b mRNA expression was found. The P-glycoprotein level was significantly increased compared with control rats. At the same time, the Kp values of Rh123 suggested that function of P-glycoprotein was significantly enhanced at the blood-brain barrier in rats with GSH depletion induced by diethyl maleate. No significant difference of the Evans Blue dye concentration in the brain cortex was found between GSH depletion rats and control rats. Treatment of rats with N-acetylcysteine decreased P-glycoprotein upregulation induced by diethyl maleate. CONCLUSIONS: The oxidative stress induced by GSH depletion played a positive role in the regulation of function and expression of P-glycoprotein at the blood-brain barrier in rats.

Novel conductive polypyrrole/silk fibroin scaffold for neural tissue repair.

Three dimensional (3D) bioprinting, which involves depositing bioinks (mixed biomaterials) layer by layer to form computer-aided designs, is an ideal method for fabricating complex 3D biological structures. However, it remains challenging to prepare biomaterials with micro-nanostructures that accurately mimic the nanostructural features of natural tissues. A novel nanotechnological tool, electrospinning, permits the processing and modification of proper nanoscale biomaterials to enhance neural cell adhesion, migration, proliferation, differentiation, and subsequent nerve regeneration. The composite scaffold was prepared by combining 3D bioprinting with subsequent electrochemical deposition of polypyrrole and electrospinning of silk fibroin to form a composite polypyrrole/silk fibroin scaffold. Fourier transform infrared spectroscopy was used to analyze scaffold composition. The surface morphology of the scaffold was observed by light microscopy and scanning electron microscopy. A digital multimeter was used to measure the resistivity of prepared scaffolds. Light microscopy was applied to observe the surface morphology of scaffolds immersed in water or Dulbecco's Modified Eagle's Medium at 37°C for 30 days to assess stability. Results showed characteristic peaks of polypyrrole and silk fibroin in the synthesized conductive polypyrrole/silk fibroin scaffold, as well as the structure of the electrospun nanofiber layer on the surface. The electrical conductivity was 1 × 10-5-1 × 10-3 S/cm, while stability was 66.67%. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was employed to measure scaffold cytotoxicity in vitro. Fluorescence microscopy was used to observe EdU-labeled Schwann cells to quantify cell proliferation. Immunohistochemistry was utilized to detect S100ß immunoreactivity, while scanning electron microscopy was applied to observe the morphology of adherent Schwann cells. Results demonstrated that the polypyrrole/silk fibroin scaffold was not cytotoxic and did not affect Schwann cell proliferation. Moreover, filopodia formed on the scaffold and Schwann cells were regularly arranged. Our findings verified that the composite polypyrrole/silk fibroin scaffold has good biocompatibility and may be a suitable material for neural tissue engineering.

[Anti-mesangial cell autoantibodies and their target antigens in lupus nephritis].

OBJECTIVE: Anti-mesangial cell autoantibodies (anti-MC) have been reported in patients with IgA nephropathy and Henoch-Schoenlein purpura. The aim of the current study was to identify anti-MC autoantibodies in sera from patients with lupus nephritis (LN). METHODS: 96 serum samples were collected from 96 patients with renal biopsy proven LN. Anti-MC autoantibodies were examined by cellular ELISA and Western blot analysis. In vitro cultured human mesangial cells were used as cell substrate in ELISA and the their soluble proteins in non-reducing conditions were used as antigens in Western-blot analysis. RESULTS: 37.5% and 97.9% of the serum samples from patients with LN had anti-MC autoantibodies in cellular ELISA and Western blot analysis respectively, and 12 protein bands could be blotted by the LN sera in Western blot analysis. The percentage of anti-63 000 autoantibody positive samples was significant higher in female patients than that in the male (59.8% vs 21.4%, P < 0.01). The prevalence of anti-74 000, 46 000, and 36 000 autoantibodies were significantly higher in patients with hematuria than that in patients without hematuria (36.8% vs 5.0%, P < 0.01; 34.2% vs 10.0%, P < 0.05; 31.6% vs 5.0%, P < 0.05; respectively). The prevalence of anti-63 000 autoantibodies was also significantly higher in patients with ANA than that in patients without (67.6% vs 16.7%, P < 0.001). The prevalence of anti-18 000 autoantibodies was significantly higher in patients with anti-dsDNA antibodies than that in patients without (61.5% vs 34.4%, P < 0.05). CONCLUSIONS: There are heterogeneous anti-MC autoantibodies in sera from patients with LN. Anti-MC autoantibodies may have a pathogenic role in lupus nephritis and some anti-MC autoantibodies may be associated with different clinical manifestations.

[Characterization of target antigens of the novel anti-mesangial cell antibodies in sera from patients with lupus nephritis].

OBJECTIVE: Novel anti-mesangial cell antibodies have been identified in sera of majority patients with lupus nephritis in our previous study. The aims of current study were to investigate the association of anti-mesangial cell antibodies and anti-DNA antibodies, and whether the target antigens were on mesangial cell membrane. METHODS: Affinity parified anti-DNA antibodies and sera with depletion of anti-DNA antibodies from three patients with lupus nephritis were used in Western-blot analysis to identify antigens in culture human glomerular mesangial cell line. Cell membrane from in vitro cultured human mesagial cell line was separated by gradient centrifugation technique, and the target antigens were identified by Western-blot analysis using 1% Triton X-100 solublized membrane protein as antigens and four known anti-mesangial cell antibody positive sera from patients with lupus nephritis as probes. RESULTS: Soluble proteins from in vitro cultured human mesangial cells could be blotted by both affinity purified anti-DNA antibodies (molecular weight at 63,000, 91,000 and 125,000) and sera with depletion of anti-DNA antibodies (molecular weight at 74,000 and 36,000). Proteins with molecular weight at 101,000, 91,000, 74,000 and 31,000 in mesangial cell membrane could be blotted by the four known anti-mesangial cell antibody positive sera. CONCLUSION: Some anti-mesangial cell antibodies were not associated with anti-DNA antibodies and anti-mesiangial cell antibodies could direct interact with antigens located on mesangial cell membrane. Anti-mesangial cell antibodies may play an important role in the pathogenesis of lupus nephritis.

[Analysis of a hereditary protein C deficient consanguineous pedigree caused by Phe139Val homozygous mutation].

OBJECTIVE: To analyze genetic mutation and explore its molecular pathogenesis for an hereditary protein C (PC) deficient consanguineous pedigree. METHODS: The pedigree included three generations and contained eight members. PC activity (PC:A), PC antigen (PC:Ag) and other coagulant parameters were detected for all family members. Protein C gene (PROC) include all the exons and intron exon boundaries were amplified by PCR for the proband, then analyzed by direct sequencing. Mutation sites were detected for the other family members. RESULTS: The PC:A and PC:Ag in the proband plasma were 20% (normal range 70% -140%) and 13.2% (normal range 70%-130%). A homozygous missense mutation g.6128T>G in exon 7 resulting in Phe139Val was identified in the proband. The PC:A and PC:Ag in her younger brother were 31% and 18.90%, Phe139Val homozygous was also found. The left family members were heterozygous for Phe139Val. CONCLUSION: Phe139Val homozygous missense mutation in exon 7 of PROC caused serious hereditary protein C deficiency. We speculated that homozygous mutation might be resulted from this consanguineous marriage.

Promotion of immunity of mice to Pasteurella multocida and hog cholera vaccine by pig interleukin-6 gene and CpG motifs.

A novel oligodeoxynuleotides containing 11 CpG motifs was synthesized and inserted into the VR1020 plasmid containing pig interleukin-6 (IL-6) gene (VPIL6) to construct recombinant plasmid, VPIL6C. The chitosan nanoparticles (CNP) were prepared by ionic cross linkage to entrap the VPIL6C (VPIL6C-CNP), VPIL6 (VPIL6-CNP) and CpG (CpG-CNP). 42-Day old female mice were divided into four groups and intramuscularly injected respectively with 6 pmol VPIL6C-CNP, VPIL6-CNP, CpG-CNP and VR1020-CNP along with the bivalent vaccines against the Pasteurellosis and hog cholera. The blood was weekly collected from mice after vaccination to detect the changes of immunoglobulins, specific antibodies, IL-2, IL-4, IL-6 and immune cells. 28 days after vaccination, the mice were orally challenged with virulent Pasteurella multocida. The results showed that in comparison with those of the control VR1020 group, the content of immunoglobulins, specific antibodies and interleukins significantly increased in the sera from the treated two groups (P<0.05). Meanwhile, the number of lymphocytes and monocytes also remarkably elevated in the treated groups (P<0.05). The immune responses of VPIL6C mice were notably stronger than those of VPIL6 and CpG group. The challenge results proved that the overall immunity was further promoted in the treated mice which resisted the challenge infection; while the control mice manifested evident symptoms and lesions, and died of infection. These suggested that VPIL6C-CNP could better promote the immunity and resistance of mice against Pasteurellosis than VPIL6-CNP and CpG-CNP, and facilitate the development of effective adjuvant to enhance the immunity of animal against infection.

Downregulation of LRP1 [correction of LPR1] at the blood-brain barrier in streptozotocin-induced diabetic mice.

Deposition of amyloid-beta peptide (Abeta) in the diabetic brain is poorly understood. Low-density lipoprotein receptor related protein 1(LRP1) at the blood-brain barrier (BBB) is critical for regulation of Abeta homeostasis in the brain. In this study, we used streptozotocin-induced diabetic mice to observe the expression of LRP1 at the BBB by Western blot and immunocytochemical analysis, and to study in vivo brain-to-blood efflux transport of 125I-Abeta1-40 using brain clearance studies. In the diabetic mice with hyperglycemia (>16.0 mmol/l) at 6 weeks, LRP1 expression at the BBB was significantly downregulated; no significant changes of LRP1 levels were found at 1 and 3 weeks after diabetes induction. The data of brain clearance studies for Abeta showed significant decrease in LRP1-dependent transport of Abeta across the BBB at 6 weeks after diabetes induction, while no significant changes of LRP1-dependent transport of Abeta across the BBB at 1 or 3 weeks after diabetes induction were apparent. We conclude that the downregulation of LRP1 at the BBB contributes to cerebral Abeta deposition in diabetes mellitus.