Nicotine ameliorates inflammatory mediators in RU486 induced preterm labor model through activating cholinergic anti-inflammatory pathway.

BACKGROUND: Preterm birth is a global public health threat. Inflammatory reaction is thought to mediate preterm birth. The role of nicotine, an anti-inflammatory agent that is mediated by cholinergic anti-inflammatory pathways (CAP), remains unclear in the pathogenesis. METHODS: Pregnant rats were randomly divided into four groups (20 rats each): pregnant control group (P), RU486-treated group (PTL), RU486 and nicotine-treated group (PTL + N), RU486, nicotine and α-BGT treated group (PTL + N + A). Rats were administered RU486 (1.0 mg/kg) by subcutaneous injection on gestational day (GD) 18 to establish PTL model. Subcutaneous injection of nicotine (1 mg/kg) was administered daily from GD 16 to 18. α-BGT (1 µg/kg) was administrated subcutaneously in two sessions and each session was 30 min prior to nicotine. TNF-α, IL-1ß, IL-4, IL-6, IL-10 in myometrium and serum were detected by Luminex. Macrophage infiltration and α7nAChR were detected by IHC. RESULTS: We established a RU486-induced preterm labor rat model. Preterm labor was associated with a striking upregulation inflammatory mediators and increased macrophage infiltration. Nicotine significantly prolonged gestation (P < 0.05) and α-BGT treatment reversed the prolonged interval (P < 0.05). The cytokines all markedly elevated at 12 h, but deceased after delivery (P < 0.05). The IL-1ß and TNF-α in serum were significantly increased in PTL group vs P group (P < 0.05), and decreased after nicotine treatment (P < 0.05). The cytokines IL-1ß, IL-4, IL-6, IL-10 and TNF-α in myometrium increased as the same trend as in serum. Nicotine treatment also downregulated the expression of α7nAChR in pregnant tissue. CONCLUSION: We confirmed the increased inflammation in preterm birth. Nicotine was able to down-regulate the inflammatory mediates and prolong the pregnant duration in PTL model, which might be induced by activating α7nAChR through CAP. This study provides a novel evidence supporting the future development of therapeutic target for preterm birth.

Desloratadine citrate combined with compound glycyrrhizin in the treatment of subacute eczema: A randomized trial.

To investigate the efficacy of desloratadine citrate combined with compound glycyrrhizin in the treatment of subacute eczema. 100 patients with subacute eczema who were admitted in our hospital from June 2019 to June 2020 were selected according to the order of admission, and divided into experimental groups (n=50, using a single compound glycyrrhizin) and control group (n=50, using compound glycyrrhizin combined with desloratadine citrate); the curative effect was compared between the two groups. After treatment, the inflammatory factors in the experimental group were lower than those in the control group [TNF-α (ng/L) (35.16±3.31), IL-2 (pg/ml) (24.39±3.11), IL-4 (pg/ml) (39.82± 4.48) vs TNF-α (ng/L) (44.24±3.87), IL-2 (pg/ml) (41.68±3.89), IL-4 (pg/ml) (49.88±5.74)] (P<0.05). After treatment the adverse reaction rate of the experimental group was lower than that of the control group (P<0.05). After treatment,the experimental group yielded higher total effective rate in relative to the control group (P<0.05). Desloratadine citrate plus compound glycyrrhizinfor might be a preferable option for clinical treatment of patients with subacute eczema, with an ideal effectiveness profile.

Quantitative evaluation of bone marrow infiltration using dual-energy spectral computed tomography in patients with multiple myeloma.

OBJECTIVE: To explore the potential value of quantitative parameters derived from dual-energy spectral computed tomography (DESCT) as comparing to the parameters derived from magnetic resonance imaging (MRI) in detecting bone marrow (BM) infiltration and distinguishing different patterns of BM infiltration in patients diagnosed with Multiple myeloma (MM). METHODS: This study involved 35MM patients and 15 healthy control subjects who had undergone spinal DESCT and MRI. Pattern assignment was based on visual assessment of MR images, and the regions of interest were defined on both DESCT and apparent diffusion coefficient maps. Quantitative values of DESCT parameters were measured and compared between infiltrated and healthy bone marrow. Receiver operating characteristic (ROC) analysis was performed to determine potential utility of DESCT parameters in identifying BM infiltration and different patterns defined by MRI. Sensitivity and specificity under the optimal thresholds determined by the Youden Index were also calculated. RSULTS: Statistical differences were observed between the DESCT parameters including Ca(Water), Water(Ca), HAP(Fat), Fat(HAP) and Effective atomic number (Eff-Z) but not for the 70-keV CT value between the infiltrated and healthy BM (all P < 0.001). The 70keV CT value and Ca(Water), HAP(Fat) and Eff-Z values were also found to be statistically different in comparing different infiltration patterns (all P < 0.05). Performance of the model-based parameter Ca/Water was superior in differentiating between infiltrated and healthy BM in which the area under ROC curve, AUC = 0.856 [95% CI, 81.4-89.1%] with sensitivity = 0.841 and specificity = 0.768, as well as between MM patients and control subjects (AUC = 0.910 [95% CI, 79.5-97.3%], sensitivity = 0.829 and specificity = 1.000). CONCLUSIONS: Analysis of DESCT offers potential as a quantitative method to detect infiltrated BM and evaluate infiltration patterns of BM in patients diagnosed with MM.

Platelet-lymphocyte ratio as a potential prognostic factor in gynecologic cancers: a meta-analysis.

PURPOSE: Cancer-related inflammation plays an important role in tumor development and progression. Platelet-lymphocyte ratio (PLR) has been studied as a biomarker for prognosis in gynecologic cancers. But, the results of previous studies were controversial, so we performed this meta-analysis. METHODS: We searched the scientific database of PubMed, Embase, Web of Science, Wanfang, and China National Knowledge Infrastructure (CNKI) using free text and MeSH keywords. Crude HR (hazard ratio) with 95% confidence interval was used to evaluate the risk association between PLR and overall survival (OS) or progression-free survival (PFS) in gynecologic neoplasms. RESULTS: There totally 23 studies, including 6869 patients who were eligible, most of which are published after 2015 or later. PLR greater than the cut-off was associated with poorer survival prognosis in ovarian cancer [OS: HR 1.80 (95% CI 1.37-2.37), p = 0.000; PFS: HR 1.63 (95% CI 1.38-1.91), p = 0.000] and cervical cancer [OS: HR 1.36 (95% CI 1.10-1.68), p = 0.005; PFS: HR 1.40 (95% CI 1.16-1.70), p = 0.002], but not in endometrial cancer [OS: HR 1.95 (95% CI 0.65-5.84), p = 0.234]. CONCLUSIONS: The current meta-analysis revealed that pretreatment PLR was a simple, promising prognostic indicator for OS and PFS in ovarian and cervical cancers. But, its significance of prognosis did not agree with endometrial neoplasm. However, due to the limited number of original studies, future large-scale studies with more well-designed, high-quality studies are still needed.

Clinical value of high-intensity focused ultrasound in fetal reduction.

Complex twin reduction surgery is a common but challenging procedure that aims to reduce the risks and complications of multiple pregnancies. The search for safer and more effective methods has led to the development of high-intensity focused ultrasound (HIFU) technology in the field of fetal reduction. This technology utilizes high-energy sound waves to focus precisely on specific areas, achieving non-invasive therapeutic effects. This paper discusses the principles and features of HIFU technology, as well as its application in complex twin reduction surgery. The paper aims to elucidate the important role of this technology in improving surgical outcomes and reducing risks, explore the current limitations of the modality, and propose directions for future development. Through these investigations, it is hoped to improve overall understanding of HIFU, and thereby promote the application of this technology in the field of fetal reduction.

Nicotine regulates abnormal macrophage polarization and trophoblast invasion associated with preterm labor via the α7nAChR/SIRT1 axis.

INTRODUCTION: Preterm birth (PTB) frequently results from the syndrome of preterm labor (PTL). PTL is linked to an atypical maternal inflammatory response, as well as intrauterine inflammation and/or infection. In this study, we explored the mechanisms involved in nicotine-mediated abnormal macrophage polarization and trophoblast invasion associated with PTL. METHODS: First, THP-1-M0 macrophages were generated by treating the human monocytic leukemia cell line (THP-1) with phorbol 12-myristate 13-acetate for a duration of 24 h. Afterward, nicotine treatment was administered, followed by coculturing with the HTR-8/SVneo trophoblast cell line (HTR-8) at a ratio of 1:1. Next, we transfected sh-α7nAChR and treated THP-1-M0 macrophages and HTR-8 cells with nicotine. In addition, we transfected THP-1-M0 macrophages with sh-NC or sh-SIRT1 or subjected them to 4 nM nicotinamide adenine dinucleotide (NAD) metabolic inhibitor FK866 treatment. Moreover, HTR-8 cells were treated with nicotine, after which THP-1-M0 macrophages were cocultured with HTR-8 cells. Finally, we constructed an in vivo RU486-induced PTL rat model to verify the effect of nicotine and the mechanisms involved. RESULTS: We found that nicotine affected polarization and α7nAChR expression in HTR-8 cocultured THP-1-M0 macrophages. Knocking down α7nAChR blocked the effect of nicotine on the proliferation and invasion of HTR-8 cells. Furthermore, nicotine activated the α7nAChR/SIRT1 axis to regulate THP-1-M0 macrophage polarization through the cholinergic anti-inflammatory pathway. Additionally, NAD metabolism mediated the role of the α7nAChR/SIRT1 axis in nicotine-induced polarization of HTR-8 cocultured THP-1-M0 macrophages. In vivo experiments demonstrated that nicotine alleviated inflammation in PTL rats, which involved the α7nAChR/SIRT1 axis. CONCLUSION: Nicotine regulated abnormal macrophage polarization and trophoblast invasion associated with PTL via the α7nAChR/SIRT1 axis.

DDIT3 switches osteogenic potential of BMP9 to lipogenic by attenuating Wnt/ß-catenin signaling via up-regulating DKK1 in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) functions as a potent inducer of osteogenic differentiation in mesenchymal stem cells (MSCs), holding promise for bone tissue engineering. However, BMP9 also concurrently triggers lipogenic differentiation in MSCs, potentially compromising its osteogenic potential. In this study, we explored the role of DNA damage inducible transcript 3 (DDIT3) in regulating the balance between BMP9-induced osteogenic and lipogenic differentiation in MSCs. Utilizing techniques such as PCR, Western blot, histochemical staining, and in vivo experiments, we analyzed the osteogenic and lipogenic markers induced by BMP9 and delved into the underlying molecular mechanism. We found a significant upregulation of DDIT3 in C3H10T1/2 cells treated with BMP9. This upregulation led to a reduction in BMP9-induced osteogenic markers but an enhancement in lipogenic markers. Conversely, knocking down DDIT3 produced the opposite effects. Furthermore, BMP9-induced bone formation was decreased in the presence of DDIT3, but adipocyte formation was increased. Further investigations demonstrated that BMP9 increased the phosphorylation level of GSK-3ß and promoted nuclear translocation of ß-catenin, both of which were suppressed by DDIT3. Moreover, DDIT3 decreased the total ß-catenin protein level while BMP9 increased the DKK1 protein level, which was further enhanced by DDIT3. Notably, knocking down DKK1 partially reversed the effect of DDIT3 on reducing BMP9-induced osteogenic markers and increasing lipogenic markers. Our findings indicated that DDIT3 enhances lipogenic differentiation by diminishing BMP9's osteogenic potential, possibly through inhibiting Wnt/ß-catenin signaling via DKK1 upregulation in MSCs.

Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/ß-catenin via HIF-1a repression in 3T3-L1 cells.

BACKGROUND: Bone morphogenetic protein 9 (BMP9) is a promising growth factor in bone tissue engineering, while the detailed molecular mechanism underlying BMP9-oriented osteogenesis remains unclear. In this study, we investigated the effect of lysyl oxidase (Lox) on the BMP9 osteogenic potential via in vivo and in vitro experiments, as well as the underlying mechanism. METHODS: PCR assay, western blot analysis, histochemical staining, and immunofluorescence assay were used to quantify the osteogenic markers level, as well as the possible mechanism. The mouse ectopic osteogenesis assay was used to assess the impact of Lox on BMP9-induced bone formation. RESULTS: Our findings suggested that Lox was obviously upregulated by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN, and mineralization were all enhanced by Lox inhibition or knockdown, while Lox overexpression reduced their expression. Additionally, the BMP9-induced adipogenic makers were repressed by Lox inhibition. Inhibition of Lox resulted in an increase in c-Myc mRNA and ß-catenin protein levels. However, the increase in BMP9-induced osteoblastic biomarkers caused by Lox inhibition was obviously reduced when ß-catenin knockdown. BMP9 upregulated HIF-1α expression, which was further enhanced by Lox inhibition or knockdown, but reversed by Lox overexpression. Lox knockdown or HIF-1α overexpression increased BMP9-induced bone formation, although the enhancement caused by Lox knockdown was largely diminished when HIF-1α was knocked down. Lox inhibition increased ß-catenin levels and decreased SOST levels, which were almost reversed by HIF-1α knockdown. CONCLUSION: Lox may reduce the BMP9 osteoblastic potential by inhibiting Wnt/ß-catenin signaling via repressing the expression HIF-1α partially.

Chest CT Features of Patients with COVID-19 Pneumonia Following Hospital Discharge.

OBJECTIVE: To evaluate chest computed tomographic (CT) findings in patients with coronavirus disease 2019 (COVID-19) pneumonia following hospital discharge. METHODS: 52 patients with confirmed COVID-19 pneumonia underwent follow-up chest CT. The scans were obtained on average 43.1 days after hospital admission and analyzed for parenchymal abnormality (e.g., ground-glass opacities, consolidation, or interstitial thickening) and evidence of fibrosis (e.g., assigned to one of three groups: Group 1 (normal lung), Group 2 (parenchymal abnormality but without evidence of fibrosis), and Group 3 (evidence of fibrosis)). Clinical data and CT manifestations of the patients were compared among the three groups. RESULTS: 30.8% (16/52) of patients with COVID-19 pneumonia showed normal lung and were designated as Group 1. 69.2% (36/52) of patients showed parenchymal abnormality ranging from residual ground-glass opacities, consolidation, or interstitial thickening in Group 2 (51.9%) to fibrosis in Group 3 (17.3%). All patients in Group3 had severe/critical COVID-19, while most patients in Group 2 and Group 1 had common COVID-19. Patients in Group 3 were older (60.9 vs. 40.8 and 36.8 years, p<0.001, there is a significant difference), had a longer hospitalization day (20.2 vs. 15.3 and 12.3 days, p<0.05, there is a significant difference), a higher ratio of patients with comorbidities (88.9%vs14.8% and 25%, p<0.001, there is a significant difference), and higher peak CT scores (13 vs. 6.2 and 3.2, p<0.001, there is a significant difference) than those patients in Group 2 and Group 1. CONCLUSIONS: Elderly severe/critical COVID-19 patients with comorbidities are more prone to develop fibrosis early on following hospital discharge. On the other hand, lung inflammation in younger patients with common COVID-19 can be resolved completely.

MMP13 promotes the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via HIF-1α upregulation in mouse embryonic fibroblasts.

Bone morphogenetic protein 9 (BMP9) has been validated as one of the most potent osteoinduction factors, but its underlying mechanism remains unclear. As a member of the matrix metalloproteinase (MMP) family, MMP13 may be involved in regulating the lineage-specific differentiation of mouse embryonic fibroblasts (MEFs). The goal of this study was to determine whether MMP13 regulates the osteoinduction potential of BMP9 in MEFs, which are multipotent progenitor cells widely used for stem cell biology research. In vitro and in vivo experiments showed that BMP9-induced osteogenic markers and/or bone were enhanced by exogenous MMP13 in MEFs, but were reduced by MMP13 knockdown or inhibition. The expression of hypoxia inducible factor 1 alpha (HIF-1α) was induced by BMP9, which was enhanced by MMP13. The protein expression of ß-catenin and phosphorylation level of glycogen synthase kinase-3 beta (GSK-3ß) were increased by BMP9 in MEFs, as was the translocation of ß-catenin from the cytoplasm to the nucleus; all these effects of BMP9 were enhanced by MMP13. Furthermore, the MMP13 effects of increasing BMP9-induced ß-catenin protein expression and GSK-3ß phosphorylation level were partially reversed by HIF-1α knockdown. These results suggest that MMP13 can enhance the osteoinduction potential of BMP9, which may be mediated, at least in part, through the HIF-1α/ß-catenin axis. Our findings demonstrate a novel role of MMP13 in the lineage decision of progenitor cells and provide a promising strategy to speed up bone regeneration.

Selective conversion of cellobiose and cellulose into gluconic acid in water in the presence of oxygen, catalyzed by polyoxometalate-supported gold nanoparticles.

Gold nanoparticles loaded onto Keggin-type insoluble polyoxometalates (Cs(x)H(3-x)PW(12)O(40)) showed superior catalytic performances for the direct conversion of cellobiose into gluconic acid in water in the presence of O(2). The selectivity of Au/Cs(x)H(3-x)PW(12)O(40) for gluconic acid was significantly higher than those of Au catalysts loaded onto typical metal oxides (e.g., SiO(2), Al(2)O(3), and TiO(2)), carbon nanotubes, and zeolites (H-ZSM-5 and HY). The acidity of polyoxometalates and the mean-size of the Au nanoparticles were the key factors in the catalytic conversion of cellobiose into gluconic acid. The stronger acidity of polyoxometalates not only favored the conversion of cellobiose but also resulted in higher selectivity of gluconic acid by facilitating desorption and inhibiting its further degradation. On the other hand, the smaller Au nanoparticles accelerated the oxidation of glucose (an intermediate) into gluconic acid, thereby leading to increases both in the conversion of cellobiose and in the selectivity of gluconic acid. The Au/Cs(x)H(3-x)PW(12)O(40) system also catalyzed the conversion of cellulose into gluconic acid with good efficiency, but it could not be used repeatedly owing to the leaching of a H(+)-rich hydrophilic moiety over long-term hydrothermal reactions. We have demonstrated that the combination of H(3)PW(12)O(40) and Au/Cs(3.0)PW(12)O(40) afforded excellent yields of gluconic acid (about 85%, 418 K, 11 h), and the deactivation of the recovered H(3)PW(12)O(40)-Au/Cs(3.0)PW(12)O(40) catalyst was not serious during repeated use.

Clinical Characterizations and Radiological Findings of COVID-19: A 4 Case Report.

BACKGROUND: Coronavirus disease 2019 (COVID-19, previously known as novel coronavirus [2019-nCoV]), first reported in China, has now been declared a global health emergency by World Health Organization. The clinical severity ranges from asymptomatic individuals to death. Here, we report clinical features and radiological changes of a cured family cluster infected with COVID-19. CASE PRESENTATION: In this report, we enrolled a family of 4 members who were admitted to our hospital in January 2020. We performed a detailed analysis of each patient's records. All patients underwent chest computed tomography (CT) examination with 120 kilovolts peak and 150 kilovolt-ampere. Realtime polymerase chain reaction (RT-PCR) examinations for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid were done using nasopharyngeal swabs. CONCLUSION: In the family members infected with COVID-19 who were accompanied by other diseases or had low immunity, the pneumonia was prone to be aggravated.

Gd-EOB-DTPA Enhanced MRI Features of Liver Hemangiomatosis Coexistent with GCH.

OBJECTIVES: This study aimed to clarify features of giant cavernous hemangioma (GCH) and liver hemangiomatosis, existing simultaneously on gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI). METHOD: A total of 17 patients with reported hepatic hemangiomatosis between 2015 and 2017 were identified retrospectively. All our patients underwent pre-contrast MRI, triphasic (atrial, portal, venous) Gd-EOB-DTPA dynamic enhancement and hepatobiliary phase (20 minutes delayed). The location, size, morphology and signal characteristics on T1-weighted (T1WI) and T2-weighted images (T2WI), and Gd-EOB-DTPA-enhanced MRI of liver hemangiomatosis were evaluated. RESULTS: Hemangiomatosis involved the liver adjacent to the edge of the GCH with no normal liver tissue found in 13 cases; in the other 4 patients, a small area of normal liver tissue separated GCH from hemangiomatosis was seen. On non-contrast MRI images, hemangionmatosis presented as numerous microcystic lesions, with low signal intensity on T1WI and high signal intensity on T2WI, compared with unaffected liver. After administration of Gd-EOB-DTPA, heterogeneous enhancement was presented in the arterial phase, during portal and venous phase imaging, becoming more homogeneous. 11 cases showed hypointensity in the hepatobiliary phase (6 cases with intratumor necrosis), and 6 cases showed hyper-intensity in the hepatobiliary phase with a remaining unfilled portion. CONCLUSION: Hemangiomatosis is extremely rare in the liver adjacent to a GCH. MRI is of great diagnostic and clinical value for this kind of tumor according to the configuration, size, signal, and style of enhancement, but the final diagnosis depends on pathology. Gd-EOB-DTPA-enhanced MRI may help in diagnosing hemangiomatosis coexistent with GCH.

Cancer-secreted exosomal miR-21-5p induces angiogenesis and vascular permeability by targeting KRIT1.

Cancer-secreted exosomes are critical mediators of cancer-host crosstalk. In the present study, we showed the delivery of miR-21-5p from colorectal cancer (CRC) cells to endothelial cells via exosomes increased the amount of miR-21-5p in recipient cells. MiR-21-5p suppressed Krev interaction trapped protein 1 (KRIT1) in recipient HUVECs and subsequently activated ß-catenin signaling pathway and increased their downstream targets VEGFa and Ccnd1, which consequently promoted angiogenesis and vascular permeability in CRC. A strong inverse correlation between miR-21-5p and KRIT1 expression levels was observed in CRC-adjacent vessels. Furthermore, miR-21-5p expression in circulating exosomes was markedly higher in CRC patients than in healthy donors. Thus, our data suggest that exosomal miR-21-5p is involved in angiogenesis and vascular permeability in CRC and may be used as a potential new therapeutic target.

Radiation Priming Chimeric Antigen Receptor T-Cell Therapy in Relapsed/Refractory Diffuse Large B-Cell Lymphoma With High Tumor Burden.

Chimeric antigen receptor T-cell (CAR-T) therapy demonstrates impressive efficacy in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, CAR-T therapy-related severe cytokine release syndrome and neurological toxicity limit its clinical application in R/R DLBCL patients with high tumor burden. Here, we conducted a phase II clinical trial testing the efficacy and toxicities of CAR-T therapy in R/R non-Hodgkin lymphoma patients (NCT03196830). Among the enrolled patients, 10 R/R DLBCL patients with high tumor burden were analyzed. Before CAR-T therapy, 4 were treated with intensive combined chemotherapy (C-CAR-cohort), and 6 were exposed to radiotherapy (R-CAR-cohort). Patients in the R-CAR-T-cohort showed a higher overall response rate (100% vs. 25%, P=0.033) and less severe cytokine release syndrome (0% vs. 100%, P=0.0048) and neurotoxicity (0% vs. 75%, P=0.033) incidences than patients in the C-CAR-T-cohort. Furthermore, one case who responded to CAR-T therapy initially and who suffered a relapse shortly was exposed to radiation and achieved complete remission, with an increase in the number of CAR-T copies detected. This study demonstrates that radiotherapy is an optimal debulking regimen to managing R/R DLBCL patients before CAR-T therapy and a promising alternative salvage therapy for patients who suffer a relapse after CAR-T therapy by fuelling CAR-T copies.

Cloning of two cDNAs encoding a family of ATP sulfurylase from Camellia sinensis related to selenium or sulfur metabolism and functional expression in Escherichia coli.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.

Activation of the cholinergic anti-inflammatory pathway by nicotine ameliorates lipopolysaccharide-induced preeclampsia-like symptoms in pregnant rats.

INTRODUCTION: Preeclampsia (PE) exerts a more intense systemic inflammatory response than normal pregnancy. Recently, the role of the cholinergic anti-inflammatory pathway (CAP) in regulating inflammation has been extensively studied. The aim of this study was to investigate the effect of nicotine, a selective cholinergic agonist, on lipopolysaccharide (LPS)-induced preeclampsia-like symptoms in pregnant rats and to determine the molecular mechanism underlying it. METHODS: Rats were administered LPS (1.0 µg/kg) via tail vein injection on gestational day 14 to induce preeclampsia-like symptoms. Nicotine (1.0 mg/kg/d) and α-bungarotoxin (1.0 µg/kg/d) were injected subcutaneously into the rats from gestational day 14-19. Clinical symptoms were recorded. Serum and placentas were collected to determine cytokine levels using Luminex. The mRNA and protein expression levels of α7 nicotinic acetylcholine receptor (α7nAChR) were determined using Real time-PCR and Western blot analysis. Immunohistochemistry was performed to determine the level of activation of nuclear factor-κB (NF-κB) in placentas. RESULTS: Nicotine significantly ameliorated LPS-induced preeclampsia-like symptoms in pregnant rats (P < 0.05). Nicotine treatment decreased the levels of LPS-induced pro-inflammatory cytokines in the serum (P < 0.05) and placenta (P < 0.05). Nicotine significantly increased the expression of α7nAChR (P < 0.01) and attenuated the activation of NF-κB p65 in the placenta in LPS-induced preeclampsia (P < 0.01). Meanwhile, these protective effects of nicotine were abolished by the administration of the cholinergic antagonist α-bungarotoxin in preeclampsia rats. DISCUSSION: Our findings suggest that the activation of α7nAChR by nicotine attenuates preeclampsia-like symptoms, and this protective effect is likely the result of the inhibition of inflammation via the NF-κB p65 pathway.

Nicotine increases eclampsia-like seizure threshold and attenuates microglial activity in rat hippocampus through the α7 nicotinic acetylcholine receptor.

OBJECTIVE: A considerable number of studies have demonstrated that nicotine, a α7-nicotinic acetylcholine receptor (α7-nAChR) agonist, can dampen immune response through the cholinergic anti-inflammatory pathway. Evidence suggests that inflammation plays a critical role in eclampsia, which contributes to maternal and fetal morbidity and mortality. In the present study, possible anti-inflammation and neuro-protective effects of nicotine via α7-nAChRs have been investigated after inducing eclampsia-like seizures in rats. METHODS: Rat eclampsia-like models were established by administering lipopolysaccharide (LPS) plus pentylenetetrazol (PTZ) in pregnant rats. Rats were given nicotine from gestation day (GD) 14-19. Then, clinical symptoms were detected. Seizure severity was recorded by behavioral tests, serum levels of inflammatory cytokines were measured by Luminex assays, microglia and astrocyte expressions were detected by immunofluorescence, and changes in neuronal number in the hippocampal CA1 region among different groups were detected by Nissl staining. RESULTS: Our results revealed that nicotine effectively improved fetal outcomes. Furthermore, it significantly decreased systolic blood pressure, and maternal serum levels of Th1 cytokines (TNF-α, IL-1ß, IL-6 and IL-12P70) and an IL-17 cytokine (IL-17A), and dramatically increased eclampsia-like seizure threshold. Moreover, this attenuated neuronal loss and decreased the expression of microglial activation markers of the hippocampal CA1 region in the eclampsia-like group. Additionally, pretreatment with α-bungarotoxin, a selective α7-nAChR antagonist could prevent the protective effects of nicotine in eclampsia-like model rats. CONCLUSION: Our findings indicate that the administration of nicotine may attenuate microglial activity and increase eclampsia-like seizure threshold in rat hippocampus through the α7 nicotinic receptor.

[Effect of the expression of Escherchia coli glutamate-1-semiadhyde aminotransferase on the red fluorescent protein uroporphyrinogen III methyltransferase].

Glutamate-1-semiadhyde aminotransferase (GSAT) is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase (UPMT), a novel red fluorescent protein. In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli, we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU. The expressed UPMT was fused histidine tag at N terminus. We also amplified E. coli hemL gene encoding GSAT by PCR reaction, eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid. The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU. The expressed GSAT has the extra Strep-TagII at N terminus. Compared to overexpression upmt gene alone, coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT, as estimated by western blot analysis, or the constitution of red fluorescent materials, as shown by UV/visible light scanning analysis, but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm. The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine, the GSAT inhibitor, suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen III, and thus enhanced the red fluorescence of the E. coli cells conferred by the recombinant UPMT.

Differentially expression of Tua1, a tubulin-encoding gene, during flowering of tea plant Camellia sinensis (L.) O. Kuntze using cDNA amplified fragment length polymorphism technique.

The complementary DNA (cDNA) amplified fragment length polymorphism technique was used to isolate transcript-derived fragments corresponding to genes involved in the flowering of tea plant. Comparative sequence analysis of an approximately 300 bp differential fragment amplified by primer combination E11M11 revealed 80%-84% similarity to the corresponding part of an a-tubulin gene of other species. The complete cDNA sequence of this a-tubulin was cloned by the rapid amplification of cDNA ends technique; its full length is 1537 bp and contains an open reading frame of 450 amino acid residues with two N-glycosylation sites and four protein kinase C phosphorylation sites. The deduced amino acid sequences did show significant homology to the a-tubulin from other plants that has been reported to be a pollen-specific protein and could be correlated with plant cytoplasm-nucleus-interacted male sterility. We named this complete cDNA Tua1. The nucleotide and amino acid sequence data of Tua1 have been recorded in the GenBank sequence database. This Tua1 gene was cloned into the pET-32a expression system and expressed in Escherichia coli BL21trxB(DE3). The molecular weight of expressed protein was deduced to be approximately 49 kDa. Western blot analysis was used to identify the temporal expression of Tua1 in tea plant. Further studies of the effect of Tua1 protein on pollen tube growth indicated the Tua1 solution obviously promoted the growth of tea pollen tube.