Long Noncoding RNA TPRG1-AS1 Suppresses Migration of Vascular Smooth Muscle Cells and Attenuates Atherogenesis via Interacting With MYH9 Protein.

BACKGROUND: Migration of human aortic smooth muscle cells (HASMCs) contributes to the pathogenesis of atherosclerosis. This study aims to functionally characterize long noncoding RNA TPRG1-AS1 (tumor protein p63 regulated 1, antisense 1) in HASMCs and reveal the underlying mechanism of TPRG1-AS1 in HASMCs migration, neointima formation, and subsequent atherosclerosis. METHODS: The expression of TPRG1-AS1 in atherosclerotic plaques was verified a series of in silico analysis and quantitative real-time polymerase chain reaction analysis. Northern blot, rapid amplification of cDNA ends and Sanger sequencing were used to determine its full length. In vitro transcription-translation assay was used to investigate the protein-coding capacity of TPRG1-AS1. RNA fluorescent in situ hybridization was used to confirm its subcellular localization. Loss- and gain-of-function studies were used to investigate the function of TPRG1-AS1. Furthermore, the effect of TPRG1-AS1 on the pathological response was evaluated in carotid balloon injury model, wire injury model, and atherosclerosis model, respectively. RESULTS: TPRG1-AS1 was significantly increased in atherosclerotic plaques. TPRG1-AS1 did not encode any proteins and its full length was 1279nt, which was bona fide a long noncoding RNA. TPRG1-AS1 was mainly localized in cytoplasmic and perinuclear regions in HASMCs. TPRG1-AS1 directly interacted with MYH9 (myosin heavy chain 9) protein in HASMCs, promoted MYH9 protein degradation through the proteasome pathway, hindered F-actin stress fiber formation, and finally inhibited HASMCs migration. Vascular smooth muscle cell-specific transgenic overexpression of TPRG1-AS1 significantly reduced neointima formation, and attenuated atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. CONCLUSIONS: This study demonstrated that TPRG1-AS1 inhibited HASMCs migration through interacting with MYH9 protein and consequently suppressed neointima formation and atherosclerosis.

Renoprotection by Inhibiting Connexin 43 Expression in a Mouse Model of Obesity-Related Renal Injury.

Introduction: Our previous study conducted in an obesity-related renal injury rat model have established a connection between increased connexin 43 (Cx43) expression and renal injury. In this study, we investigated whether inhibiting Cx43 expression could provide renoprotection in a mouse model of obesity-related renal injury. Methods: Five-week-old C57BL/6J mice were fed with a high-fat diet for 12 weeks to establish an obesity-related renal injury model, then they were treated with Cx43 antisense oligodeoxynucleotide (AS) or scrambled oligodeoxynucleotide (SCR) by an implanted osmotic pump for 4 weeks. Finally, the glomerular filtration function, the histological change in the glomeruli, and the markers of podocyte injury (WT-1, Nephrin) and inflammatory infiltration of renal tissue (CD68, F4/80 and VCAM-1) were examined respectively. Results: The results showed that inhibiting Cx43 expression by AS in this mouse model of obesity-related renal injury can effectively improve glomerular filtration function, alleviate glomerular expansion and podocyte injury, and attenuate the inflammatory infiltration of renal tissue. Conclusion: Our results demonstrated that inhibiting Cx43 expression by AS could provide renoprotection for the mouse model of obesity-related renal injury.

Cost-Effectiveness of Secukinumab Versus Other Biologics in the Treatment of Moderate-to-Severe Plaque Psoriasis: The Chinese Healthcare System Perspective.

INTRODUCTION: This study assessed the cost-effectiveness of secukinumab compared with other biologics (adalimumab, infliximab, ustekinumab, ixekizumab, guselkumab, and Yisaipu [etanercept biosimilar]) for moderate-to-severe plaque psoriasis from the Chinese healthcare system perspective. METHODS: A decision-tree (first year)/Markov model (subsequent years), with an annual cycle, was implemented over a lifetime horizon. The Psoriasis Area and Severity Index (PASI) response rate at week 16 was used for treatment response. Efficacy inputs were obtained from a mixed-treatment comparison conducted using data from randomized controlled trials. Other clinical inputs (adverse events, dropout, and mortality rates), utility weights, and costs were derived from published literature and local Chinese sources. Both costs and outcomes were discounted at 5% per annum. Model outcomes included quality-adjusted life years (QALYs) and incremental cost-effectiveness ratio (ICER). One-way and probabilistic sensitivity analyses were conducted to test the robustness of results. RESULTS: For patients with moderate-to-severe psoriasis, secukinumab generated the highest QALYs (12.334) against all comparators at a lifetime cost of ¥231,477. Secukinumab dominated (higher QALYs at lower costs) all other biologics except ixekizumab in this population. Compared with secukinumab, ixekizumab incurred slightly lower costs (¥228,320) but gained lesser QALYs (12.284). Thus, secukinumab was a cost-effective treatment than ixekizumab at a willingness-to-pay (WTP) threshold of ¥257,094 per QALY gained. In the one-way sensitivity analysis, base-case results were most sensitive to changes in the PASI response at 16 weeks and year 2+ dropout rates. CONCLUSION: Secukinumab is the most cost-effective treatment option for patients with moderate-to-severe psoriasis compared with other commonly used biologics from the Chinese healthcare system perspective.

(Fluoro)alkylation of alkenes promoted by photolysis of alkylzirconocenes.

Difluoroalkylated compounds have important applications in pharmaceutical, agrochemical, and materials science. However, efficient methods to construct the alkylCF2-alkyl bond are very limited, and the site-selective introduction of a difluoromethylene (CF2) group into an aliphatic chain at the desired position remains challenging. Here, we report an unprecedented example of alkylzirconocene promoted difluoroalkylation of alkyl- and silyl-alkenes with a variety of unactivated difluoroalkyl iodides and bromides under the irradiation of visible light without a catalyst. The resulting difluoroalkylated compounds can serve as versatile synthons in organic synthesis. The reaction can also be applied to activated difluoroalkyl, trifluoromethyl, perfluoroalkyl, monofluoroalkyl, and nonfluorinated alkyl halides, providing a general method to controllably access fluorinated compounds. Preliminary mechanistic studies reveal that a single electron transfer (SET) pathway induced by a Zr(iii) species is involved in the reaction, in which the Zr(iii) species is generated by the photolysis of alkylzirconocene with blue light.

The mediating role of decision-making conflict in the association between patient's participation satisfaction and distress during medical decision-making among Chinese patients with pulmonary nodules.

OBJECTIVE: When diagnosed as having pulmonary nodules, patients may be mired in the conflict of medical decision-making and suffered from distress. The purpose of this study was to investigate the mediating role of decision-making conflict in the relationship between participation satisfaction in medical decision-making (PSMD) and distress among Chinese patients with incidental pulmonary nodules. METHODS: A total of 163 outpatients with incidental pulmonary nodules detected in a tertiary hospital were recruited and investigated by Impact of Event Scale (IES), Decision Conflict Scale (DCS), participation satisfaction in medical decision-making Scale (PSMDS), and demographic questionnaire. RESULTS: The mean IES score was 37.35 ± 16.65, representing a moderate level. PSMD was negatively associated with distress, while decision-making conflict was positively associated with distress. The final regression model contained three factors: having a first-degree relative diagnosed with lung cancer, worrying about getting lung cancer someday, and decision-making conflict. These three factors explained 49.4 % of the variance of distress. The total effect of PSMD on distress and indirect effect of SPMD on distress caused-by decision-making conflict were significant (P < 0.05). However, the direct effect of PSMD on distress was not significant. CONCLUSIONS: Participation of patients in medical decision-making can lower their distress by reducing patient's decision-making conflict. PRACTICE IMPLICATIONS: Interventions targeting at the decision-making conflict will help alleviate the distress level of patients with pulmonary nodules. DATA AVAILABILITY: The data that support the findings of this study are available on request from the corresponding author.

Diagnostic performance of Neutrophil CD64 index, procalcitonin, and C-reactive protein for early sepsis in hematological patients.

BACKGROUND: Patients with hematological diseases are immunosuppressed due to various factors, including the disease itself and treatments, such as chemotherapy and immunotherapy, and are susceptible to infection. Infections in these patients often progress rapidly to sepsis, which is life-threatening. AIM: To evaluate the diagnostic efficacy of the neutrophil CD64 (nCD64) index, compared to procalcitonin (PCT) and high-sensitivity C-reactive protein (hs-CRP), for the identification of early sepsis in patients with hematological diseases. METHODS: This was a prospective analysis of patients with hematological diseases treated at the Fuxing Hospital affiliated with Capital Medical University, between March 2014 and December 2018. The nCD64 index was quantified by flow cytometry and the Leuko64 assay software. The factors which may affect the nCD64 index levels were compared between patients with different infection statuses (local infection, sepsis, and no infection), and the control group and the nCD64 index levels were compared among the groups. The diagnostic efficacy of the nCD64 index, PCT, and hs-CRP for early sepsis was evaluated among patients with hematological diseases. RESULTS: A total of 207 patients with hematological diseases (non-infected group, n = 50; locally infected group, n = 67; sepsis group, n = 90) and 26 healthy volunteers were analyzed. According to the absolute neutrophil count (ANC), patients with hematological diseases without infection were divided into the normal ANC, ANC reduced, and ANC deficiency groups. There was no statistically significant difference in the nCD64 index between these three groups (P = 0.586). However, there was a difference in the nCD64 index among the non-infected (0.74 ± 0.26), locally infected (1.47 ± 1.10), and sepsis (2.62 ± 1.60) groups (P < 0.001). The area under the diagnosis curve of the nCD64 index, evaluated as the difference between the sepsis and locally infected group, 0.777, which was higher than for PCT (0.735) and hs-CRP (0.670). The positive and negative likelihood ratios were also better for the nCD64 index than either PCT and hs-CRP. CONCLUSION: Our results indicate the usefulness of the nCD64 index as an inflammatory marker of early sepsis in hematological patients.

Flavonoid glycosides from the nectar of Camellia reticulata Lindl.

The nectar of Camellia reticulata Lindl. contains sugar, amino acids and other nutritional components, suggesting that it could be developed for food and food additives. To understand the effects of the nectar on human health, we investigated its chemical constituents. Two new flavonoid glycosides, cameretiins A and B (1 and 2), and two known flavonoid glycosides, kaempferol 3-O-(2''-O-E-p-coumaroyl)-ß-D-glucopyranoside (3) and tiliroside (4) were obtained from the nectar of Camellia reticulata Lindl. Their structures were determined based on analysis of their spectroscopic data and by comparison with 1D NMR spectroscopic data of known compounds reported in the literature. Compounds (1-4) were first isolated from the nectar of Camellia reticulata Lindl.

Transcriptome-wide N6-methyladenosine methylation landscape of coronary artery disease.

Aim: To reveal transcriptome-wide N6-methyladenosine (m6A) methylome of coronary artery disease (CAD). Materials & methods: The m6A levels of RNA from peripheral blood mononuclear cells measured by colorimetry were significantly decreased in CAD cases. Transcriptome-wide m6A methylome profiled by methylated RNA immunoprecipitation sequencing (MeRIP-seq) identified differentially methylated m6A sites within both mRNAs and lncRNAs between CAD and control group. Results: Bioinformatic analysis indicated that differentially methylated genes were involved in the pathogenesis of atherosclerosis. MeRIP-quantitative real-time PCR assay confirmed the reliability of MeRIP-seq data. Finally, the rat carotid artery balloon injury model was performed to confirm the role of m6A demethylase FTO in neointima formation. Conclusion: Our study provided a resource of differentially methylated m6A profile for uncovering m6A biological functions in the pathogenesis of CAD.

High-fat diet-induced adipose tissue expansion occurs prior to insulin resistance in C57BL/6J mice.

BACKGROUND: To date, there is only scare evidence characterizing the temporal features and progression of metabolic dysfunction in high-fat diet (HFD)-fed obese mice. Hence, its specific pathogenesis remains unclear. METHODS: Sixty 6-week-old male C57BL/6J mice were randomly divided into HFD and control diet (CD) groups and sacrificed at 1, 5, 9, 13, 17, and 21 weeks, respectively. At weekly intervals, intraperitoneal glucose tolerance testing (IPGTT) and intraperitoneal insulin tolerance testing (IPITT) were performed in both groups. A detailed time course in HFD-fed mice was investigated by evaluating the initiation of glucose homeostasis impairment, dyslipidemia, systemic insulin sensitivity, monocyte chemoattractant protein-1 (MCP-1) levels, epididymal white adipose tissue (eWAT) expansion, macrophage content changes, pro-inflammatory (M1)/anti-inflammatory (M2) macrophage imbalance, lipid accumulation in the liver, and ß-cell morphometry in the pancreas. RESULTS: In the HFD group, progressive weight gain and impairments in glucose metabolism (elevated fasting blood glucose and area under the curve (AUC) of IPGTT) were observed from the 3rd week, and a significantly elevated AUC of IPITT was first detected after week 7 of HFD feeding. As for dyslipidemia, after 9 weeks of feeding, the low-density lipoprotein cholesterol level and total cholesterol level in HFD group were significantly higher than those in the CD group (all P < 0.05), whereas no significant differences were shown in triglyceride level. Adipocyte size increased significantly in the HFD group in the 1st week, a phenotypic switch in eWAT from anti-inflammatory (M2) to pro-inflammatory (M1) macrophages was observed in the 5th week, and the metabolic inflammation was distinct in eWAT in the 9th week. Additionally, liver steatosis was considerably obvious at the 17th week and pancreatic ß-cell morphometry did not change during 21 weeks of HFD feeding. CONCLUSION: The eWAT expansion was detected early in HFD-induced obese mice, which occurred prior to obvious insulin resistance.

Recombinant murine interleukin 4 protein therapy for psoriasis in a transgenic VEGF mouse model.

BACKGROUND: Psoriasis is a typical autoimmune disease caused by a deregulation of the Th1/Th2 balance, and immunotherapy for psoriasis has been shown to be clinically efficacious. Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis. Evidence suggests that the chronic delivery of VEGF to the skin can result in a profound inflammatory condition with many of the cellular and molecular hallmarks of human psoriasis. In this study, we investigated whether the transgenic VEGF mouse is a suitable model for antipsoriatic studies. AIM: To determine the effect of a recombinant murine interleukin 4 (rmIL-4) in the transgenic VEGF mouse model. METHODS: Fifteen homozygous K14-VEGF transgenic mice were injected subcutaneously with rmIL-4 protein for 30 consecutive days with a prospective dose escalation of 0.5, 2 or 5 microg/kg. Hematoxylin-eosin staining, immunohistochemistry and real-time polymerase chain reaction analyses were performed with ear samples. RESULTS: The rmIL-4 protein therapy was well tolerated. Tissue sections from treated skin showed improvements upon morphological and histological examinations: diminution of erythematous appearance and regression of epidermal thickness were observed, and T lymphocyte infiltration decreased significantly. The expressions of adhesion molecules, such as vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, were found reduced. The level of IL-4 mRNA also increased while the level of gamma-interferon mRNA decreased, resulting in a 10-fold increase in the ratio of Th1/Th2. CONCLUSIONS: Our results reveal that rmIL-4 has clinical efficacy for the treatment of K14-VEGF transgenic mice. Angiogenesis and inflammation were ameliorated by therapy with rmIL-4.

[The construction of recombinant mouse interleukin 4 prokaryotic expressing plasmid and the expression and purification of target protein].

OBJECTIVE: To construct Recombinant Mouse Interleukin 4 prokaryotic expressing plasmid, express it in E. coli strain BL21 (DE3), purify and identify the expressed cytokine. METHODS: The optimized mIL-4 cDNA fragment was cloned into the prokaryotic expressing vector pET-32a (+) to generate pET32/rmIL-4 and transformed into BL21 (DE3) cells. After induction, the expressed protein wasfound to be in the inclusion of E. coli cells. The induced product was purified through Q Sepharose Fast Flow Column and Gel Filtration Column under renaturing condition. The purified protein was identified by Western blot analysis, and the biologic activity was identified by the generation of mIL-4 dependence cell CTLL-2 and in vivo experiment of mouse psoriasis model. RESULTS: The recombinant plasmid pET32/rmIL-4 has been constructed correctly. The inclusion body was washed with 3 mol/L guanidine hydrochloride and denaturized in 7 mol/L guanidine hydrochloride. Then, the denaturized protein was gradient dialysis in the condition of pH 9. 5. The protein we purified has the right immunology specificity and biologic activity. CONCLUSION: The recombinant mouse interleukin-4 with high purity and biologic activity was prepared in this study,which will become the basis for the further study of the biologic activity of IL-4.

[Improvement of Method for Detecting Peripheral Blood Neutrophil CD64 Index in Patients with Hematologic Malignancies].

OBJECTIVE: To improve the method for detecting the neutrophil CD64 (nCD64) index and to enhance the detection rate and accuracy of nCD64 index in patients with hematologic malignancies. METHODS: The nCD64 index in peripheral blood of patients with hematologic malignancies combined with suspicious bacterial infection (255 cases-time) was detected by using array method. When the detection of nCD64 index in samples was interfered with abnormal cells in detection process of enrolled patients, the antibodies CD45, CD15 and CD10 were added into samples on the basis of routine detection by using the primary detection kit, in order to more accurately distinguish the neutrphils and obtain the nCD64 index. The nCD64 index as well as the efficiency of nCD64 index, PCT and CRP for diagnosis of sepsis before and after the improvement of deteation method were compared. RESULTS: The samples of 60 cases were interfered with abnormal cells in detection process, out of which the samples of 18 cases failed in detection, but these samples of 18 cases all got the effective results of detection after the detection method was improved. The detection showed that the nCD64 index before and after the improvement as well as the PCT and CRP levels in sepsis group were higher than those in non-sepsis group(P＜0.05). After improvement of method, the efficiency of nCD64 index for diagnosis of sepsis was suprior to the PCT and CRP, the nCD64 index obtained after the improvement of method possessed the diagnosis efficiency same as the efficiency obtained before improvement of method, moreover the detection results were more reliable. CONCLUSION: For the samples of patients with hematologic malignancies interfered with abnormal cells in the process of detecting the nCD64 index, the corresponding antibodies are added into detected samples according to the kinds of hemotologic diseases of patients, in order to more accurately gate the neutrophils in paripheral blood of patients, there by the detection rate and accuracy for detecting the nCD64 index are enhanced by accurately distinguishing the neutrophils.

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells.

AIM: To construct functional human full-thickness corneal replacements. METHODS: Acellular porcine corneal matrix (APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM (AAPCM) and posterior-APCM (PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations. RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman's membrane, and Descemet's membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM. There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman's membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7d after surgery. CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.

Pd-Catalyzed Alkylation of (Iso)quinolines and Arenes: 2-Acylpyridine Compounds as Alkylation Reagents.

The first Pd-catalyzed alkylation of (iso)quinolines and arenes is reported. The readily available and bench-stable 2-acylpyridine compounds were used as an alkylation reagent to form the structurally versatile alkylated (iso)quinolines and arenes. The method affords a convenient pathway for the introduction of alkyl groups into organic molecules.

A potential link between TSLP/TSLPR/STAT5 and TLR2/MyD88/NFκB-p65 in human corneal epithelial cells for Aspergillus fumigatus tolerance.

Our previous studies reported that pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors (NLRs), recognize pathogen-associated molecular patterns (PAMPS) to initiate downstream signal cascades to active immunity responses. Thymic stromal lymphopoietin (TSLP) has recently emerged as a key cytokine in the development of type 2 adaptive immune responses. However, the crosstalk between PRRs and TSLP has not been well elucidated in Aspergillus fumigates keratitis. Our studies demonstrated that HCECs not only respond to TSLP, but also initiate immunological regulation through TSLP/TSLPR/STAT5 signaling pathway. In addition, we revealed that zymosan TLR2 agonist enhanced the expression of TSLP and TSLPR and phosphorylation of STAT5. Furthermore, neutralization of TLR2 with monoclonal Ab prevented the production of TSLP and TSLPR and phosphorylation of STAT5 from increasing which induced by A. fumigatus hyphae. Interestingly, we also found that human recombinant TSLP induced the increase of TLR2 downstream signal molecules, and TSLP knockdown could reduce the increase of TLR2 downstream signaling molecules(MyD88 and NF-κB-p65) induced by A. fumigatus hyphae. These studies indicated that HCECs represent a novel target of TSLP, TSLP/TSLPR/STAT5 signaling plays an important role in response to A. fumigatus infection in HCECs, and TLR2 downstream signaling molecules up regulate TSLP/TSLPR/STAT5 signaling as well as TSLP downstream signaling molecules up regulate TLR2/MyD88/NFκB-p65 signaling in this phenomenon.