The screening and expression of polysaccharide deacetylase from Caulobacter crescentus and its function analysis.

The bacterium Caulobacter crescentus secretes an adhesive polysaccharide called holdfast, which is the known strongest underwater adhesive in nature. The deacetylase encoded by hfs (holdfast synthesis) H gene is a key factor affecting the adhesion of holdfast. Its structure and function are not yet clear, and whether other polysaccharide deacetylases exist in C. crescentus is still unknown. The screening of both HfsH and its structural analogue as well as their purification from the artificial expression products of Escherichia coli is the first step to clarify these questions. Here, we determined the conserved domains of HfsH via sequence alignment among carbohydrate esterase family 4 enzymes and screened out its structural analogue (CC_2574) in C. crescentus. The recombinant HfsH and CC_2574 were effectively expressed in E. coli. Both of them were purified by chromatography from their corresponding productions in E. coli and were then functionally analyzed. The results indicated that a high deacetylase activity (61.8 U/mg) was observed in recombinant HfsH but not in CC_2574, which suggesting that HfsH might be the irreplaceable gene mediating adhesion of holdfast in C. crescentus. Moreover, the divalent metal ions Zn2+ , Mg2+ , and Mn2+ could promote the activity of recombinant HfsH at the concentration from 0.05 to 1 mM, but inhibit its activity when the concentration exceeds 1 mM. In sum, our study first realized the artificial production of polysaccharide deacetylase HfsH and its structural analogue, and further explored their functions, both of which laid the foundation for the development of new adhesive materials.

Landform and lithospheric development contribute to the assembly of mountain floras in China.

Although it is well documented that mountains tend to exhibit high biodiversity, how geological processes affect the assemblage of montane floras is a matter of ongoing research. Here, we explore landform-specific differences among montane floras based on a dataset comprising 17,576 angiosperm species representing 140 Chinese mountain floras, which we define as the collection of all angiosperm species growing on a specific mountain. Our results show that igneous bedrock (granitic and karst-granitic landforms) is correlated with higher species richness and phylogenetic overdispersion, while the opposite is true for sedimentary bedrock (karst, Danxia, and desert landforms), which is correlated with phylogenetic clustering. Furthermore, we show that landform type was the primary determinant of the assembly of evolutionarily older species within floras, while climate was a greater determinant for younger species. Our study indicates that landform type not only affects montane species richness, but also contributes to the composition of montane floras. To explain the assembly and differentiation of mountain floras, we propose the 'floristic geo-lithology hypothesis', which highlights the role of bedrock and landform processes in montane floristic assembly and provides insights for future research on speciation, migration, and biodiversity in montane regions.

[Study on the diagnostic value of different posterior cruciate ligament index measurement methods for anterior cruciate ligament injury].

OBJECTIVE: To compare the posterior cruciate ligament(PCL) index with six different measurement methods, and analyze and verify its clinical diagnostic value in anterior cruciate ligament (ACL) injury. METHODS: The Magnetic resonance imaging (MRI) data of 225 knee joints in our hospital from May 2018 to March 2022 were retrospectively analyzed, aged from 18 to 60 years old, with a median of 32 years old. On the sagittal MRI images of 114 patients with ACL injury and 111 patients with intact ACL, Measure the straight-line distance (A) between the femoral attachment point and the tibial attachment point of the PCL on the MRI sagittal image and the maximum vertical distance (B) between the straight line and the arcuate mark point of the PCL on the sagittal image, calculate the PCL index and evaluate the diagnostic value of the PCL index for ACL injury. RESULTS: The PCL index of the ACL normal group and the ACL injury group were statistically described. There was no significant difference in PCL index 1, 2, 3 and 6 between the two groups(P>0.05). The difference of PCL index 4 and 5 between the two groups was statistically significant (P<0.001). This study only found that the PCL index 2, 6 in the ACL normal group had a negative correlation with the patient's age (correlation coefficient=-0.213, -0.819;P<0.05), and the PCL index 5 in the ACL injury group was significantly correlated with the patient's body mass index(BMI)had a negative correlation (correlation coefficient=-0.277, P<0.05). CONCLUSION: The change of PCL index is helpful for the diagnosis of ACL injury, PCL index 4 and 5 can be used as effective reference indexes for diagnosing ACL injury in clinic.

Screening and identification of potential hub genes and immune cell infiltration in the synovial tissue of rheumatoid arthritis by bioinformatic approach.

Background: Rheumatoid arthritis (RA) is an autoimmune disease that affects individuals of all ages. The basic pathological manifestations are synovial inflammation, pannus formation, and erosion of articular cartilage, bone destruction will eventually lead to joint deformities and loss of function. However, the specific molecular mechanisms of synovitis tissue in RA are still unclear. Therefore, this study aimed to screen and explore the potential hub genes and immune cell infiltration in RA. Methods: Three microarray datasets (GSE12021, GSE55457, and GSE55235), from the Gene Expression Omnibus (GEO) database, have been analyzed to explore the potential hub genes and immune cell infiltration in RA. First, the LIMMA package was used to screen the differentially expression genes (DEGs) after removing the batch effect. Then the clusterProfiler package was used to perform functional enrichment analyses. Second, through weighted coexpression network analysis (WGCNA), the key module was identified in the coexpression network of the gene set. Third, the protein-protein interaction (PPI) network was constructed through STRING website and the module analysis was performed using Cytoscape software. Fourth, the CIBERSORT and ssGSEA algorithm were used to analyze the immune status of RA and healthy synovial tissue, and the associations between immune cell infiltration and RA-related diagnostic biomarkers were evaluated. Fifth, we used the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate the expression levels of the hub genes, and ROC curve analysis of hub genes for discriminating between RA and healthy tissue. Finally, the gene-drug interaction network was constructed using DrugCentral database, and identification of drug molecules based on hub genes using the Drug Signature Database (DSigDB) by Enrichr. Results: A total of 679 DEGs were identified, containing 270 downregulated genes and 409 upregulated genes. DEGs were primarily enriched in immune response and chemokine signaling pathways, according to functional enrichment analysis of DEGs. WGCNA explored the co-expression network of the gene set and identified key modules, the blue module was selected as the key module associated with RA. Seven hub genes are identified when PPI network and WGCNA core modules are intersected. Immune infiltration analysis using CIBERSORT and ssGSEA algorithms revealed that multiple types of immune infiltration were found to be upregulated in RA tissue compared to normal tissue. Furthermore, the levels of 7 hub genes were closely related to the relative proportions of multiple immune cells in RA. The results of the qRT-PCR demonstrated that the relative expression levels of 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) were up-regulated in RA synovial tissue, compared with normal tissue. Simultaneously, ROC curves indicated that the above 6 hub genes had strong biomarker potential for RA (AUC >0.8). Conclusions: Through bioinformatics analysis and qRT-PCR experiment, our study ultimately discovered 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) that closely related to RA. These findings may provide valuable direction for future RA clinical diagnosis, treatment, and associated research.

[Screening and characterization of aptamers of Cepsilon3-Cepsilon4 protein].

In order to obtain nucleotides aptamers bind to IgE, 80 bp nucleotides single-stranded DNA library containing 40 random nucleotides was designed and synthesized. Oligonucleotides that bind to human Cepsilon3-Cepsilon4 protein were isolated from ssDNA pools by the systematic evolution of ligands by exponential enrichment (SELEX) method using nitrocellulose filters as screening medium. Through the optimization of critical PCR and asymmetric PCR parameters including annealing temperature, cycles, and molar ratios of target protein and ssDNA etc, a suitable screening system was established. The aptamers of Cepsilon3-Cepsilon4 protein with high affinity and high specificity were identified by ELISA with biotin-streptavidin-horseradish peroxidase system, and its primary sequence and second structure were analyzed by DNAMAN package and DNA folding sever after being cloned and sequenced. Moreover, target protein was bound to one aptamer and another aptamer modified with biotion together forming a sandwich-like complex, which was captured in microwell to detect IgE concentration using the optimal combination in the sandwich method named enzyme-linked aptamers sorption assay (ELASA). The method could be used for the quantitative detection of human IgE, and whose sensitivity reached to 120 ng x mL(-1).

Argostemmaehuangzhangense (Rubiaceae), a new species from Guangdong, China.

Argostemmaehuangzhangense, a new Rubiaceae species from E'huangzhang Nature Reserve, Guangdong Province, China, is here described and illustrated. A morphological comparison between the new species and its putative relatives, A.lamxayanum, A.laotica and A.verticillatum, is presented. The new species is mostly similar to A.laotica, but they can be distinguished from each other since Argostemmaehuangzhangense presents solitary flower (vs. 2-flowered inflorescences), flower lobes 4 (vs. 5) and anthers opening by longitudinal slits (vs. apical pores). In a preliminary IUCN Red List status of Argostemmaehuangzhangense this species is assigned as Vulnerable (VU).

[Progress in the study of allergic disease drugs targeting on IgE/FcepsilonRI signaling pathway].

Allergic diseases have become global social health problems. The binding of IgE with its high affinity receptor FcepsilonRI plays a key step in I-type allergy. Recently, more and more key molecules on the IgE/FcepsilonRI signaling transduction pathway were to be the drug candidates against allergic diseases, with in-depth study of FcepsilonRI signal pathway gradually. The main drugs include molecule antibodies, peptides, vaccines, fusion proteins, small molecules, and other drugs related to IgE/FcepsilonRI. The recent progress in the study of mechanisms of representative drugs targeting on IgE/FcepsilonRI signaling pathway was reviewed in this article.

[Comparative study on posterior cruciate ligament reconstruction with autologous hamstring tendon and LARS artificial ligament in the treatment of KD-â ¢-M knee dislocation].

OBJECTIVE: To observe the curative effect of one-stage reconstruction of anterior cruciate ligament(ACL), posterior cruciate ligament (PCL) and medial collateral ligament (MCL) in patients with KD-Ⅲ-M knee injury, and to compare the operation time, hospitalization cost and curative effect after arthroscopic reconstruction of PCL with LARS artificial ligament and autogenous hamstring tendon, ACL reconstruction with autogenous hamstring tendon and MCL repair combined with limited incision. METHODS: From March 2016 to January 2019, a total of 36 patients met the criteria of this study. Twenty patients in group A were treated with autogenous hamstring tendon reconstruction of ACL and PCL and repair of MCL, including 17 males and 3 females, with an average age of (34.7±9.2) years old. Sixteen patients in group B with LARS artificial ligament reconstruction of PCL, with an autogenous hamstring tendon reconstruction of PCL and MCL repair as before as group B, including 15 males and 1 female, with an average age of (36.8±8.6) years old. The operation time, hospitalization time and total hospitalization cost were compared between the two groups. The preoperative and postoperative functions of the two groups were evaluated by Hospital for Sepcial Surgery (HSS) score and Lysholm score respectively, and the curative effects were compared within and between groups. RESULTS: All the patients in the two groups were followed up for at least 1 year. There were no complications such as infection and poor wound healing in both groups. There was significant difference in operation time between (120.25±9.55) min in group A and (106.63±8.85) min in group B (P<0.01). The average hospitalization days in group A was (10.60±1.64) days, while that in group B was (10.38±1.59) days. There was no significant difference between the two groups (P>0.05). The HSS score of group A increased from preoperative 32.95±5.03 to postoperative 84.70±5.72 (P<0.01), and that of group B increased from preoperative 33.81±4.10 to postoperative 85.00±5.25 (P<0.01). The Lyshlom score in group A increased from preoperative 21.10±3.46 to postoperative 80.25±5.75 (P<0.01), and in group B increased from preoperative 21.56±3.01 to postoperative 80.00±4.30(P<0.01). There was no significant difference in preoperative and postoperative scores between the two groups(P>0.05). CONCLUSION: There was no significant difference in the average hospitalization days between the two groups, but the operation time in group A was longerthan that in group B, and the hospitalization cost in group B was higher than that in group A. There was no difference in HSS score and Lysholm score before and follow-up for a certain period of time after operation.

[Establishment of a rat chronic asthma model and its evaluation].

This study is to establish a rat chronic asthma model. Sensitive SD rats were selected through histamine challenge. The asthmatic groups were sensitized by ih and ip with OVA, aluminium hydroxide gel and inactivated bacillus pertussis on day 1 and 14. From day 21, acute asthmatic group was aerosolized 1% OVA for 1 week, chronic asthmatic group was aerosolized 0.1% OVA for 12 weeks. The control groups received saline as the substitution of OVA. Twenty four hours after the last provocation, physiological monitoring equipment was used to detect the pulmonary function, then the rats were sacrificed. Bronchoalveolar lavage fluid (BALF) was collected to calculate the ratio of different inflammatory cells. ELISA was used to detect total IgE and OVA-specific IgE in serum. Microscopy was conducted to observe the histopathology of lung stained with haematoxylin and eosin staining. Collagen fibers were detected using Picric acid-Sirius red staining technique. The optical density at 610 nm of extractive from locus caeruleus was detected by passive cutaneous anaphylaxis (PCA). The results showed that the asthmatic characteristics were significantly developed in model groups, but not in control groups. Chronic asthmatic group had significantly higher indexes than acute asthmatic group, including the thickness of airway smooth muscle and bronchial basement membrane, and goblet cell hyperplasia, the area of collagen in airways, A610 of extractive from locus caeruleus, the concentration of total IgE and OVA-specific IgE in serum. However, inflammatory cell infiltrate in lungs and the percentage of eosinophils of white blood cells in BALF were lower in chronic asthmatic group than those in acute asthmatic group. Respiratory rate and respiratory flow showed no significant difference in both model groups. In conclusion, the rat chronic asthma model is established by the way in this study, which is comparable to the physiopathologic characteristics of human asthma.

New combinations and synonyms in Rehderodendron (Styracaceae).

We demonstrate with morphological characters that the species Pterostyrax burmanicus W.W.Sm. & Farrer and Parastyrax macrophyllus C.Y.Wu & K.M.Feng (Styracaceae) are best placed in the genus Rehderodendron Hu. Rehderodendron burmanicum (W.W.Sm. & Farrer) W.Y.Zhao, P.W.Fritsch & W.B.Liao, comb. nov. and Rehderodendron macrophyllum (C.Y.Wu & K.M.Feng) W.Y.Zhao, P.W.Fritsch & W.B.Liao, comb. nov., are created. We also provide a lectotype for R. macrophyllum. These revisions result in the reduction of Pterostyrax Siebold & Zucc. to three species and this genus is no longer considered to be documented from Myanmar. Further, Parastyrax W.W.Sm. becomes a monotypic genus comprising only P. lacei (W.W.Sm.) W.W.Sm., distributed in Kachin State, northeast Myanmar and Yunnan Province, south-western China.

[Effects of interleukin 1 receptor antagonist on allergy asthma in rat model and its mechanism].

OBJECTIVE: To study the effects of interleukin 1 receptor antagonist (IL-Ira) on allergy asthma and its mechanism. METHODS: Thirty female SD rats underwent intraperitoneal and hypodermic injection of ovalbumin (OVA) on days 1 and 14, and then underwent spraying of OVA aerosol since day 21 for 7 days so as to provoke asthma, and then the rats were randomly divided into 3 equal groups: asthma model group, low dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 6 mg/kg before each provocation (low dose treatment group), and high dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 30 mg/kg before each provocation (high dose treatment group). Another 10 rats were used as normal controls. Twenty-four hours after the last provocation physiological monitoring equipment was used to detect the pulmonary function. Then the rats were killed. Bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the serum IgE content. The ratio of inflammatory cells from the BALF was calculated. Microscopy was conducted to observe the histopathology of lung. RT-PCR was used to examine the mRNA expression of NF-kappaB and signal transducer and activator of the transcription 6 (STAT6). RESULTS: The respiratory rate, expiratory flow, percentage of eosinophils in BALF inflammatory cells, peripheral blood IgE concentration, mRNA expression of STAT6 and NF-kappaB of the asthma group were (206 +/- 11) times/min, (77 +/- 8) microl/s, 24.8% +/- 1.3%, (72.5 +/- 8.1) ng/ml, 0.294 +/- 0.048, and 0.686 +/- 0.052 respectively, all significantly higher than those of the low dose treatment group [(183 +/- 9) times/min, (64 +/- 5) microl/s, 18.5% +/- 3.1%, (63.4 +/- 4.8) ng/ml, 0.229 +/- 0.038, and 0.613 +/- 0.062 respectively, all P < 0.05] and those of the high dose treatment group [(181 +/- 11) times/min, (57 +/- 4) microl/s, 14.7% +/- 2.1%, (41.4 +/- 7.4) ng/ml, 0.194 +/- 0.076, and 0.352 +/- 0.267, all P < 0.05]. The therapeutic effect of high dose treatment group is superior to that of low dose treatment group (P < 0.05). CONCLUSION: IL-1ra is significantly effective in treatment of allergic asthma, and its potential mechanism is through regulating both STAT6 mRNA and NF-kappaB mRNA expression simultaneously.

Cyclosporine A combined with medium/low dose prednisone in progressive IgA nephropathy.

The aim of the present study was to evaluate the efficacy of cyclosporine A (CsA) combined with medium/low dose prednisone in the treatment of progressive immunoglobulin A nephropathy (IgAN). Ninety-six patients who satisfied the inclusion criteria were enrolled in a prospective controlled clinical study. They were assigned into two groups and initially given either 0.6-0.8 mg/kg/day prednisone (maximum 40 mg/day) plus 3 mg/kg/day CsA (CsA group), or 1 mg/kg/day prednisone (maximum 60 mg/day) alone (steroid group). During therapy, the dose of prednisone was reduced in both groups and the dose of CsA was gradually tailed off over the first 3 months and maintained at 2 mg/kg/day in the CsA group. Urinary protein excretion, serum biochemical indexes, clinical efficacy and side effects of CsA were assayed. A significant decline in mean 24-hour urinary protein excretion (p < 0.05) was observed 1 month after treatment in patients in the CsA group, which was observed 2 months after treatment in the steroid group. The decline in mean 24-hour urinary protein excretion in the CsA group was more significant than in the steroid group. Serum albumin level increased significantly in the CsA group 2 months after therapy (p < 0.05). Moreover, at the end of the course, a higher remission rate was observed in patients with Lee's Grade III IgAN after combined treatment with prednisone and CsA (p < 0.05). No significant difference in clinical efficacy was observed in patients with Lee's Grade IV and Grade V IgAN between the two groups (p > 0.05). CsA at a dose of 2-3 mg/kg/day in combination with medium/low dose prednisone was effective in inducing remission of IgAN, especially for patients with Lee's Grade III IgAN, and is a safe and effective choice for short-term treatment of patients with progressive IgAN.

[Construction and identification of the eukaryotic expression vector pIRES2-EGFP-IL-1ra-Fcepsilon].

The cDNA of IgE constant domain of rat was cloned from the spleen of allergy asthma rat by RT-PCR. The IL-1ra segment was obtained from intermediate vector pBV220-IL-1ra. By overlap extension PCR, the fusion gene IL-1ra-Fcepsilon was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL-1ra-Fcepsilon. The recombinant expression plasmid was transfected into 293T cells using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcepsilon was identified by Western blot, RT-PCR, and this protein could inhibit the activity of IL-1 in vitro. Green fluorescent protein could be detected in the transfected 293T cells and the rat lungs at different times. The research paved the way for the gene therapy of allergy asthma.