SIRT1 Asn346 sugar chain promoting collagen deacetylation protective effect on osteoblasts under stress.

Silencing type information regulator homolog 1 (SIRT1) is a class of nicotinamide adenine dinucleotide (NAD+) dependent deacetylases, which is the convergence point of important physiological processes in vivo, namely, osteoblast aging, energy metabolism, and bone remodeling. To verify whether the O-acetylglucosamine (O-GlcNAc) modification of SIRT1 in the nucleus of osteoblasts enhances its deacetylase activity under stress and protects osteoblasts through the RANK/RANKL signaling pathway by collagen deacetylation. The R language and online data research identified SIRT1 as being involved in bone metabolism. Enrichment analysis showed that SIRT1 is involved in osteoblast transcription, apoptosis, and deacetylation pathways. Interactive Immuno-blotting and immunofluorescence experiments revealed that SIRT1 and O-glycosylation catalytic enzyme (OGT) were localized in the nucleus. Mass Spectrometry analysis showed that O-glycosylation occurred on the asparagine at the 346th position of SIRT1, and N346th was located in the central domain of SIRT1. Furthermore, the protein structure analysis of PyMol also proved that the OGT binding region was in the central domain of SIRT1. Under physiological conditions, both wtSIRT1 and SIRT1N346R can inhibit RANKL-mediated transcriptional activation. The RT-PCR detection results showed that wtSIRT1 reduced RANKL transcription under the conditions of apoptotic agent treatment. The finding that SIRT1 can regulate the physiological process of bone remodeling through the RANK/RANKL signaling pathway in osteoblasts under stress. The O-glycosylation and deacetylation activity of SIRT1 significantly increased, regulating the balance between osteoblast survival and apoptosis by deacetylation of key proteins such as RANKL.

Da vinci robotic-assisted treatment of pediatric choledochal cyst.

Objective: To evaluate the advantages and disadvantages of da Vinci robot and laparoscopy in treating pediatric choledochal cysts. Methods: We retrospectively analyzed clinical data from forty-two children diagnosed with choledochal cysts in our hospital from January 2018 to January 2021. Twenty children underwent da Vinci robotic surgery, and the others underwent traditional laparoscopy. We compared differences in general information and preoperative, intraoperative, and postoperative differences between the two surgical groups. Results: There was no statistically significant difference in age, gender, weight, type, maximum cyst diameter, preoperative C-reactive protein (CRP) value, postoperative complication rate, and postoperative pain score between the two surgical groups (P > 0.05). The average age of the robot-assisted group was 3.62 ± 0.71 years old (range = 1-12 years). There were nineteen cases of Todani type I, one patients of other types, and the maximum cyst diameter was 35.45 ± 9.32 mm (range = 12-56 mm). In the laparoscopic group, the average age was 3.08 ± 0.82 years old (range = 3-10 years). Twenty-one patients had Todani type I, and one had other types. The maximum cyst diameter was 31.94 ± 8.64 mm (range = 10-82 mm). The robot-assisted group had better abdominal drainage, postoperative CRP value, fasting time, and discharge time than the laparoscopic group (P < 0.05). Conclusion: Compared with laparoscopy, the da Vinci system has the advantages of less tissue damage, faster recovery, and better healing in the treatment of children with congenital choledochal cysts. With technological advancements and an increased number of experienced surgeons, robotic surgery may become a new trend in surgery.

House dust mite exposure enhances immune responses to ovalbumin-induced intestinal allergy.

House dust mites (HDM) are one of the important factors of airway allergic diseases, HDM allergens can be detected in the human gut mucosa, which induces local inflammation and increases intestinal epithelial permeability. This study tests a hypothesis that HDM contribute to the development of OVA (ovalbumin)-induced intestinal allergy. The serum levels of IgE against HDM in patients with food allergy were detected with UniCAP100 (Pharmacia, Uppsala, Sweden); a mouse model of food allergy was developed with OVA and HDM as the specific antigens. Compared to healthy controls, patients with food allergy have higher levels of serum HDM-specific IgE. Compared to food allergy alone groups, the levels of HDM-specific IgE in patients with food allergy and asthma or allergic rhinitis were significantly higher. In mouse models, we found that HDM/OVA induced allergy-like symptoms, lower body temperature, and lower body weight. The levels of IgE, IgG1, mMCP-1 (mouse mast cell protease-1), IL-4 and IL-5 in the HDM and HDM + CT (cholera toxin) groups were higher than the control groups, and the levels of IgE, IgG1, IL-4 and IL-5 in the HDM, OVA and HDM + OVA groups were higher than the control groups. The pathological changes of intestinal tissues in the HDM and HDM + CT/the HDM, OVA and HDM + OVA groups were more severe, more eosinophil infiltration than the control groups. Moreover, exposure to HDM induced intestinal barrier dysfunction, and facilitated the development of intestinal allergy in mice. In conclusion, HDM exposure enhances immune responses to OVA-induced food allergy.

Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis.

Rationale: Corticosteroid resistance (CR) is a serious drawback to steroid therapy in patients with ulcerative colitis (UC); the underlying mechanism is incompletely understood. Twist1 protein (TW1) is an apoptosis inhibitor and has immune regulatory functions. This study aims to elucidate the roles of TW1 in inducing and sustaining the CR status in UC. Methods: Surgically removed colon tissues of patients with ulcerative colitis (UC) were collected, from which neutrophils were isolated by flow cytometry. The inflammation-related gene activities in neutrophils were analyzed by RNA sequencing. A CR colitis mouse model was developed with the dextran sulfate sodium approach in a hypoxia environment. Results: Higher TW1 gene expression was detected in neutrophils isolated from the colon tissues of UC patients with CR and the CR mouse colon tissues. TW1 physically interacted with glucocorticoid receptor (GR)α in CR neutrophils that prevented GRα from interacting with steroids; which consequently abrogated the effects of steroids on regulating the cellular activities of neutrophils. STAT3 (Signal Transducer and Activator of Transcription-3) interacted with Ras protein activator like 1 to sustain the high TW1 expression in colon mucosal neutrophils of CR patients and CR mice. Inhibition of TW1 restored the sensitivity to corticosteroid of neutrophils in the colon tissues of a CR murine model. Conclusions: UC patients at CR status showed high TW1 expression in neutrophils. TW1 prevented steroids from regulating neutrophil activities. Inhibition of TW1 restored the sensitivity to corticosteroids in the colon tissues at the CR status.

Cross-reactive antibodies against dust mite-derived enolase induce neutrophilic airway inflammation.

BACKGROUND AND AIMS: Neutrophilic inflammation is a hallmark of some specific asthma phenotypes; its aetiology is not yet fully understood. House dust mite (HDM) is the most common factor in the pathogenesis of airway inflammation. This study aims to elucidate the role of cross-antibodies against HDM-derived factors in the development of neutrophilic inflammation in the airway. METHODS: Blood samples were collected from asthma patients with chronic neutrophilic asthma for analysis of HDM-specific cross-reactive antibodies. The role of an antibody against HDM-derived enolase (EnoAb) in the impairment of airway epithelial barrier function and induction of airway inflammation was assessed in a cell culture model and an animal model. RESULTS: High similarity (72%) of the enolase gene sequences was identified between HDM and human. Serum EnoAb was detected in patients with chronic neutrophilic asthma. The EnoAb bound to airway epithelial cells to form complexes with enolase, which activated complement, impaired airway epithelial barrier functions and induced neutrophilic inflammation in the airway tissues. CONCLUSIONS: HDM-derived enolase can induce specific cross-antibodies in humans, which induce neutrophilic inflammation in the airway.

Enolase-specific cross antibodies induce neutrophilic inflammation in the intestine.

The pathogenesis of ulcerative colitis (UC) is to be further investigated. House dust mites (HDM) are highly associated with the pathogenesis of immune inflammation in the body. This study aims to investigate the role of enolase (one of the HDM-derived proteins)-specific cross Abs in the induction of UC-like inflammation. The enolase specific IgG (EsIgG) was identified in UC patients by mass spectrometry. Mice were treated with EsIgG to induce inflammation in the colon mucosa. EsIgG was detected in the serum and the colon tissues of UC patients, which was positively correlated with the polymorphonuclear neutrophil (PMN) counts in the blood and colon tissues of UC patients. EsIgG formed immune complexes with the constitutive enolase in the UC colon epithelium that activated complement, induced epithelial cell apoptosis, compromised epithelial barrier functions, and resulted in UC-like inflammation in the mouse colon. In summary, UC patients have high serum levels of Abs against HDM-derived enolase and intestinal epithelial cell-derived enolase. These Abs attack the colonic epithelium to induce UC-like inflammation.

Gene synthesizing, expression and immunogenicity characterization of recombinant translation elongation factor 2 from Dermatophagoides farinae.

House dust mite (HDM) hypersensitivity increasingly affects millions of individuals worldwide. Although numerous major allergens produced by HDM species have been characterized, some of the less potent allergens remain to be studied. The present study aimed to obtain the recombinant allergen of Translation Elongation Factor 2 (TEF 2) from the HDM Dermatophagoides farinae by synthesizing, and then expressing the recombinant TEF 2 to identify its immunogenicity. In the present study, the D. farinae TEF 2 (Der f TEF 2) was synthesized, expressed and purified. The molecular characteristics of Der f TEF 2 were analyzed using bioinformatics approaches. The recombinant protein was purified via affinity chromatography, and the allergenicity was assessed using immunoblotting, ELISAs and skin prick tests. The gene for TEF 2 consists of 2,535 bp and encodes an 844­amino acid protein. A positive response to recombinant Der f TEF 2 was detected in 16.2% of 37 patients with HDM allergies using skin prick tests. In addition, the immunoblotting indicated that the protein showed a high ability to bind serum IgE from patients allergic to HDMs, and that the recombinant TEF 2 was highly immunogenic. Bioinformatics analysis predicted 17 peptides as B cell epitopes (amino acids 29­35, 55­64, 92­99, 173­200, 259­272, 311­318, 360­365, 388­395, 422­428, 496­502, 512­518, 567­572, 580­586, 602­617, 785­790, 811­817 and 827­836) and 14 peptides as T cell epitopes (amino acids 1­15, 65­79, 120­134, 144­159, 236­250, 275­289, 404­418, 426­440, 463­477, 510­524, 644­658, 684­698, 716­730 and 816­830). The software DNAStar predicted the secondary structure of TEF 2, and showed that 27 α­helices and five ß­sheets were found in the protein. In conclusion, the present study cloned and expressed the Der f TEF 2 gene, and the recombinant protein exhibited immunogenicity, providing a theoretical bases, and references, for the diagnosis and treatment of allergic disease.

Optimal treatment for spermatogenesis in male patients with hypogonadotropic hypogonadism.

BACKGROUND: To compare the efficacies of gonadotropin-releasing hormone (GnRH) pulse subcutaneous infusion with combined human chorionic gonadotropin and human menopausal gonadotropin (HCG/HMG) intramuscular injection have been performed to treat male hypogonadotropic hypogonadism (HH) spermatogenesis. METHODS: In total, 220 idiopathic/isolated HH patients were divided into the GnRH pulse therapy and HCG/HMG combined treatment groups (n = 103 and n = 117, respectively). The luteinizing hormone and follicle-stimulating hormone levels were monitored in the groups for the 1st week and monthly, as were the serum total testosterone level, testicular volume and spermatogenesis rate in monthly follow-up sessions. RESULTS: In the GnRH group and HCG/HMG group, the testosterone level and testicular volume at the 6-month follow-up session were significantly higher than were those before treatment. There were 62 patients (62/117, 52.99%) in the GnRH group and 26 patients in the HCG/HMG (26/103, 25.24%) group who produced sperm following treatment. The GnRH group (6.2 ±â€Š3.8 months) had a shorter sperm initial time than did the HCG/HMG group (10.9 ±â€Š3.5 months). The testosterone levels in the GnRH and HCG/HMG groups were 9.8 ±â€Š3.3 nmol/L and 14.8 ±â€Š8.8 nmol/L, respectively. CONCLUSION: The GnRH pulse subcutaneous infusion successfully treated male patients with HH, leading to earlier sperm production than that in the HCG/HMG-treated patients. GnRH pulse subcutaneous infusion is a preferred method.

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae.

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.

A pyrene-modified cobalt salophen complex immobilized on multiwalled carbon nanotubes acting as a precursor for efficient electrocatalytic water oxidation.

Immobilization of earth-abundant water oxidation catalysts (WOCs) on carbon supports to produce functional electrodes for electrochemical water splitting is a crucial approach for future clean energy production. Herein we report the non-covalent immobilization of a pyrene-bearing cobalt(ii) Schiff base complex (2) on the surface of multiwalled carbon nanotubes (MWCNTs) to form a hybrid anode for electrocatalytic water oxidation. The 2/MWCNT anode displayed excellent catalytic activity and durability in neutral aqueous solution, and a catalytic current density of 1.0 mA cm-2 was achieved at 1.15 V vs. the normal hydrogen electrode (NHE), corresponding to a low overpotential of 330 mV. A Tafel slope of 96 mV per decade was obtained. The Faradaic efficiency of oxygen evolution was more than 90% by bulk electrolysis measurement. After bulk electrolysis, the hybrid anode characterization using X-ray photoelectron spectroscopy (XPS) confirmed that complex 2 decomposed to form heterogeneous cobalt hydroxides and the cobalt hydroxides should be true catalytic active species, which are responsible for electrocatalytic oxygen evolution.

Der f 31, a novel allergen from Dermatophagoides farinae, activates epithelial cells and enhances lung-resident group 2 innate lymphoid cells.

Airway epithelial cell-derived thymic stromal lymphopoietin (TSLP) and IL-33 can enhance lung-resident group 2 innate lymphoid cells (ILC2s), and they play an important role in the development of allergic diseases. This study tests the hypothesis that Der f 31 (Dermatophagoides farinae-31), an allergen, modulates airway epithelial cell functions and increases the frequency of lung ILC2s. Our previous research identified cofilin (Der f 31) as a novel allergen. In this study, we found that recombinant Der f 31 (r-Der f 31) upregulated the expression of co-stimulatory molecules in DCs and promoted Th2-skewed polarization. The levels of TSLP and IL-33 in epithelial cells were upregulated by r-Der f 31 via the activation of Toll-like receptor 2. Furthermore, in in vivo studies, r-Der f 31 induced eosinophil-like airway allergy and increased the number of lung-resident ILC2s. In summary, Der f 31 can modulate the functions of airway epithelial cells and increase levels of lung-resident ILC2s.

Erratum: Expression, purification and characterization of Der f 27, a new allergen from dermatophagoides farinae.

[This corrects the article on p. 1260 in vol. 7, PMID: 26328010.].

Characterization and analysis of a cDNA coding for the group 29b (Der f 29b) allergen of Dermatophagoides farinae.

This study aims to acquire a recombinant allergen of Der f 29b by cloning and expression, and to identify its immunogenicity. In this study, the total RNA of D. farinae was extracted, cloned and expressed based on the Der f 29b gene. The molecular characteristics of Der f 29b was analyzed by the procedures of Bioinformatics. The allergenicity of recombinant Der f 29b protein was examined by western-blotting, ELISA, Immune inhibitory assays and skin prick test. The gene of Der f 29b consisted of 495 bases, derived from its nucleic acid sequence and encoded 164 amino acids. Positive responses to r-Der f 29b were shown in 24.3% by means of skin prick testing with 37 DM-allergic patients. The immunoblotting assays demonstrated that serum IgE from allergic patients reacted to r-Der f 29b protein. The IgE reactivity of r-Der f 29b in the serum from r-Der f 29b allergic patients was increased by more than 2 folds compared with healthy subjects. Immune inhibition assays showed that the IgE cross-reactivity was between r-Der f 29b and DME. Bioinformatics analysis predicted four peptides (13-17, 67-71, 104-109 and 147-155) as the B cell epitopes and five peptides (5-14, 16-31, 35-43, 52-63 and 87-97) as the T cell epitopes. Secondary structure prediction of Der f 29b with software PSIPRED identified two α-helices and seven ß-sheets in Der f 29b. In conclusion, Derf 29b protein was identified as a novel subtype of dust mite allergen.

Identification of α-tubulin, Der f 33, as a novel allergen from Dermatophagoides farinae.

BACKGROUND: House dust mites are an important source of indoor allergens. More than 30 allergens of Dermatophagoides farinae (D. farinae) have been identified. Yet there may be many other allergens in mites remain to be characterized. METHODS: α-Tubulin (also named Der f 33) was cloned, expressed and purified. Reaction to specific-IgE, skin prick test and a mouse asthma model were employed to determine the allergenicity of Der f 33. RESULTS: The recombinant Der f 33 reacted to the serum of patients with mite allergy. The positive rate of skin prick test (SPT) was 23.5%. In an asthma mouse model, Der f 33 induced the airway allergy-like responses. Moreover, serum specific IgE and IgG1, interleukin-4 (IL-4) from bronchoalveolar lavage fluid (BALF) and spleen cell culture supernatant were markedly increased. In addition, Der f 33 upregulated the CD80 and TNF-α levels in dendritic cells (DCs). CONCLUSIONS: Der f 33 is a novel allergen of D. farinae. It modulates the functions of DCs and induces airway allergy.

Der f 21, a novel allergen from dermatophagoides farina.

The Dermatophagoides farina (D. farina) allergens are an important factor contributing to allergic disease. To identify new allergens is important for diagnosis and treatment of allergic diseases. In this study, we sought to characterize the biological activity of Der f 21 of D. farina. The recombinant Der f 21 protein was characterized by western-blot, ELISA and Skin prick test using clinic patient's serum.An allergic asthma mouse model was established with the rDer f 21 as a specific antigen. The results showed that the sera from 28.9% in 38 dust mite allergic children displayed positive results in response to rDer f 21, and 42% in 98 dust mite allergic patients displayed positive response in skin prick test. In addition, Immune inhibition assays showed there was IgE cross-reactivity between rDer f 21 and rDer f 5. Moreover, an allergic asthma mouse model was established. Airway hyperresponsiveness, serum specific IgE, IgG1, eosinophil infiltration in the allergic mice, interleukin-4(IL-4) and interferon-γ (INF-γ) from spleen cells were markedly increased in the allergic mice. The results demonstrate that Der f 21 is a novel allergen.

Characterization of a new subtype of allergen in dermatophagoides farinae-Der f 28.

BACKGROUND: House dust mites (HDMs) are the major sources of indoor allergens which induce asthma, dermatitis, rhinitis, and some other allergic diseases. Close to 30 sub-allergens have been identified. METHODS: Through analyzing the full genome sequence of dust mite, a new allergen whose primary structure belongs to the heat shock protein family was identified. The sequence of this allergen was determined by cDNA cloning. The allergenicity was assayed by skin prick test, Western-blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: r-Der f 28 bound to serum IgE from mite allergic patients. Positive responses to r-Der f 28 were shown in 11.5% by skin prick testing from 26 DM-allergic patients. Airway hyperresponsiveness, serum specific IgE and IL-4 were significantly increased in allergic asthma mouse model sensitized to r-Der f 28. CONCLUSIONS: Der f 28 is a new subtype of allergen in dermatophagoides farinae.

Expression, purification and characterization of Der f 27, a new allergen from dermatophagoides farinae.

The house dust mite (HDM), Dermatophagoidesfarinae (D. farina), is one of the most important indoor allergen sources and a major elicitor of allergic asthma; itscharacterization is important in the diagnosis and immunotherapy of mite allergen-relevant diseases. This study aims to characterize a novel allergen, the D. farinae-derived serpin (Der f 27). In this study, the total RNA of D. farinae was extracted, and the Der f 27 gene was cloned and expressed. The allergenicity of recombinant Der f 27 protein was determined by enzyme-linked immunosorbent assay, and Western-blotting with the sera of asthma patients, and skin prick test (SPT) in allergic human subjects. A r-Der f 27 allergic asthma mouse model was established. The cloned Der f 27 gene has been presented at the Gene Bank with an accession number of KM009995. The IgE levels of r-Der f 27 in the serum from r-Der f 27 SPT positive allergic patients were 3 folds more than healthy subjects. The Der f 27 SPT positive ratewas 42.1% in 19 DM-SPT positive patients. Airway hyperresponsiveness, serum specific IgE, and levels of interleukin-4 in the spleen cell culture supernatant were significantly increased in allergic asthma mice sensitized to r-Der f 27. In conclusion, Der f 27 is a new subtype of house mite allergen.

Immunohistochemical identification of osteoclasts and multinucleated macrophages.

Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells which are specialised to carry out lacunar bone resorption. The immunophenotype of giant cell-containing bone lesions in a wide range of osteoclast-like giant cells was similarly assessed. Both multinucleated macrophages and osteoclasts were found to express CD68. Multinucleated macrophages, but not osteoclasts, expressed GrB and Ki67. CD13+/CD14+/CD68+/GrB-/Ki67-/CD56- all giant-cell lesions noted in giant cells of bone. Giant cells have an osteoclast phenotype in most giant cell-rich lesions of bone, which do not express the macrophage-associated antigens GrB and Ki67. Our results indicate that they are formed from osteoclast precursors of mononuclear phagocyte.

A two-dimensional hydrogen-bonded water layer in the structure of a cobalt(III) cubane complex.

A tetranuclear Co(III) oxide complex with cubane topology, tetrakis(2,2'-bipyridine-κ(2)N,N')di-µ2-carbonato-κ(4)O:O'-tetra-µ3-oxido-tetracobalt(III) pentadecahydrate, [Co4(CO3)2O4(C10H8N2)4]·15H2O, with an unbounded hydrogen-bonded water layer, has been synthesized by reaction of CoCO3 and 2,2'-bipyridine. The solvent water molecules form a hydrogen-bonded net with tetrameric and pentameric water clusters as subunits. The Co4O4 cubane-like cores are sandwiched between the water layers, which are further stacked into a three-dimensional metallo-supramolecular network.