Network pharmacology integrated with experimental validation revealed potential molecular mechanisms of Camellia nitidissima C. W. Chi in the treatment of lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Camellia nitidissima C.W.Chi (CNC), an ethnomedicine mainly distributed in Southern China's Guangxi Zhuang Autonomous Region, is known as "Panda in plants" and "Camellias Queen" due to its golden blossom. CNC has been applied as a traditional folk medicine in cancer therapy. AIM OF THE STUDY: This study utilized network pharmacology analysis combined with experimental validation to identify the substance basis and potential molecular mechanism of CNC against lung cancer. MATERIALS AND METHODS: The active ingredients of CNC were identified based on published literature. The associated potential targets of CNC in lung cancer treatment were predicted using integrated network pharmacology analysis and molecular docking. The underlying molecular mechanism of CNC in lung cancer were validated in human lung cancer cell lines. RESULTS: A total of 30 active ingredients and 53 targets of CNC were screened. An enrichment analysis of Gene Ontology (GO) revealed that the effects of CNC in lung cancer mainly involve protein binding, regulation of cell proliferation and apoptosis, and signal transduction. KEGG pathways analysis suggested that CNC might exert cancer suppression effects mainly through pathways in cancer, PI3K/AKT signaling pathway. Molecular docking revealed that CNC has high affinity for binding of EGFR, SRC, AKT1, and CCND1 to the key active ingredients including luteolin, kaempferol, quercetin, eriodictyol and 3'4-O-dimethylcedrusin. In in vitro experiments, CNC played the inhibitory roles in lung cancer cells by inducing cell apoptosis, causing G0/G1 and S cell cycle arrest, increasing intracellular ROS levels, and promoting the apoptotic proteins Bax and Caspase-3. Meanwhile, CNC also regulated the expression of core proteins EGFR, SRC, and AKT. CONCLUSION: These results comprehensively clarified the associated substance basis and underlying molecular mechanism of CNC against lung cancer, which would be contributed to develop promising anti-cancer pharmaceuticals or therapeutic approaches for lung cancer therapy.

Examining Health Care Access for Refugee Children and Families in the North Carolina Triangle Area.

BACKGROUND Resettled refugees are at increased risk of poor health outcomes due to acculturation challenges, logistical barriers, experiences of trauma, and other barriers to care that are poorly understood. Refugee children may be particularly vulnerable due to disruptions in health, well-being, education, and nutrition during the resettlement process.METHOD To describe the health care barriers facing refugees in the North Carolina Triangle area (comprised of Durham, Chapel Hill, Raleigh, and their surrounding areas), we conducted three focus group interviews (in Arabic, French, and Swahili) with 25 refugee parents from Syria, Iraq, Central African Republic, the Democratic Republic of the Congo, and Chad. We also administered a survey to nine organizations that provide services for refugees.RESULTS Focus group responses highlighted the multidimensional nature of health care barriers for refugee families and children, encompassing challenges with acculturation, communication, transportation, finances, and health literacy. Organizations emphasized similar challenges and described their efforts to improve access to services through increased communication, coordination, and seeking new financial support for programs.LIMITATIONS Given the geographic focus of the study, results may not be generalizable to other populations and settings. Men spoke more than women in some focus groups, and participants may have been influenced by more vocal contributors. Furthermore, this study is limited by a lack of health outcomes data.CONCLUSIONS This study suggests that the health care needs of refugees living in the North Carolina Triangle area can be better met by providing comprehensive, coordinated, and culturally relevant care. This could include minimizing the number of visits by integrating multiple services under one roof, providing trauma-informed interpreters, and offering accessible transportation services.

Study on the Release Potential of BPA and Steroid Estrogens in the Sediments of Erhai Lake, a Typical Plateau Lake of China.

On-site sampling analysis and laboratory-scale experiments were conducted to study the pollution status and release potential of EDCs in Erhai Lake. We found that nitrogen and phosphorus pollution in Erhai Lake sediment were both at a high level, as well as EDCs pollution. The concentrations of BPA, E2α, E1, E2ß, EE2, and E3 were 36.84 ng/g(DW), 13.04 ng/g(DW), 128.97 ng/g(DW), 52.57 ng/g(DW), 18.48 ng/g(DW) and 5.36 ng/g(DW), respectively. The concentrations of E2α, E1, E2ß and EE2 in the bottom water were higher than the surface water due to the impact of sediment release. The results of the 20 days release test indicated that BPA release from the sediment had a greater correlation with the original concentration and the particle size of sediment, while the steroid EDCs had no obvious correlation with these two factors, probably due to the difference in hydrophobicity between them. Under hydraulic disturbance and aerobic conditions, the release process of EDCs was accompanied by a large amount of microbial degradation, and degradation amount > released amount. BPA was released quickly, 9.56% was released in 20 days, but only 3.37% of steroid EDCs released. In comparison, the release process of steroids was longer and posed a greater threat to aquatic ecology.

Leveraging the genetic basis of Rett syndrome to ascertain pathophysiology.

Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause Rett syndrome (RTT), a progressive X-linked neurological disorder characterized by loss of developmental milestones, intellectual disability and breathing abnormality. Despite being a monogenic disorder, the pathogenic mechanisms by which mutations in MeCP2 impair neuronal function and underlie the RTT symptoms have been challenging to elucidate. The seemingly simple genetic root and the availability of genetic data from RTT patients have led to the generation and characterization of a series of mouse models recapitulating RTT-associated genetic mutations. This review focuses on the studies of RTT mouse models and describe newly obtained pathogenic insights from these studies. We also highlight the potential of studying pathophysiology using genetics-based modeling approaches in rodents and suggest a future direction to tackle the pathophysiology of intellectual disability with known or complex genetic causes.

[Effect of oxymatrine on apoptosis of hippocampal neurons by p38/JNK signaling pathway].

To investigate the effect and mechanism of oxymatrine(OMT) on hippocampal neurons apoptosis. Effect of OMT on survival of hippocampal neurons was measured by MTT.Effect of OMT on LPS-induced lactate dehydrogenase(LDH) release rate in hippocampal neurons was measured by biochemical methods. Hoechst 33342 staining was used to observe the apoptotic morphology of hippocampal neurons.The mRNA expression levels of Bax, Bcl-2, and Caspase-3 were detected by Real-time quantitative PCR(RT-qPCR), and the protein expression levels of p38, p-p38, JNK, p-JNK, Bax, Bcl-2 and Caspase-3 were detected by Western blot.The results showed that, hippocampal neurons all grew well after treatment by different doses (0.37-6.0 g•L⁻¹) of OMT for 24 h. Stimulation from LPS increased the release of LDH(P<0.01), improved the JNK and p38 phosphorylation levels(P<0.01), increased the proportion of Bax/Bcl-2 and the expression of Caspase-3(P<0.01), and promoted the apoptosis of hippocampal neurons. OMT pretreatment could significantly reduce the release of LDH induced by LPS stimulation(P<0.05 or P<0.01), reduce the p38 and JNK phosphorylation, decrease the expression of Caspase-3 and Bax/Bcl-2(P<0.01), and diminish the apoptosis of hippocampal neurons.In conclusion, OMT could reduce the LPS-induced phosphorylation of p38 and JNK, down-regulate the Bax/Bcl-2 ratio and expression of Caspase-3, thus inhibiting apoptosis of hippocampal neurons. The mechanism may be associated with p38/JNK signaling pathway.

Molecular and genetic insights into an infantile epileptic encephalopathy - CDKL5 disorder.

BACKGROUND: The discovery that mutations in cyclin-dependent kinase-like 5 (CDKL5) gene are associated with infantile epileptic encephalopathy has stimulated world-wide research effort to understand the molecular and genetic basis of CDKL5 disorder. Given the large number of literature published thus far, this review aims to summarize current genetic studies, draw a consensus on proposed molecular functions, and point to gaps of knowledge in CDKL5 research. METHODS: A systematic review process was conducted using the PubMed search engine focusing on CDKL5 studies in the recent ten years. We analyzed these publications and summarized the findings into four sections: genetic studies, CDKL5 expression patterns, molecular functions, and animal models. We also discussed challenges and future directions in each section. RESULTS: On the clinical side, CDKL5 disorder is characterized by early onset epileptic seizures, intellectual disability, and stereotypical behaviors. On the research side, a series of molecular and genetic studies in human patients, cell cultures and animal models have established the causality of CDKL5 to the infantile epileptic encephalopathy, and pointed to a key role for CDKL5 in regulating neuronal function in the brain. Mouse models of CDKL5 disorder have also been developed, and notably, manifest behavioral phenotypes, mimicking numerous clinical symptoms of CDKL5 disorder and advancing CDKL5 research to the preclinical stage. CONCLUSIONS: Given what we have learned thus far, future identification of robust, quantitative, and sensitive outcome measures would be the key in animal model studies, particularly in heterozygous females. In the meantime, molecular and cellular studies of CDKL5 should focus on mechanism-based investigation and aim to uncover druggable targets that offer the potential to rescue or ameliorate CDKL5 disorder-related phenotypes.

Inhibitory effect of oxymatrine on hepatocyte apoptosis via TLR4/PI3K/Akt/GSK-3ß signaling pathway.

AIM: To evaluate the effect of oxymatrine (OMT) on hepatocyte apoptosis in rats with lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver failure (ALF). METHODS: LPS/D-GalN was used to establish a model of ALF in rats. To evaluate the effect of OMT, we assessed apoptosis by transmission electron microscopy, and the pathological changes in the liver by light microscopy with hematoxylin and eosin staining. An automated biochemical analyzer was used to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Enzyme-linked immunosorbent assay was used to determine the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. Western blotting was used to detect protein levels in liver tissues. Streptavidin peroxidase immunohistochemistry was used to observe expression of Toll-like receptor (TLR)4, active caspase-3, Bax and Bcl-2. RESULTS: All rats in the normal control and OMT-pretreated groups survived. The mortality rate in the model group was 30%. OMT preconditioning down-regulated apoptosis of hepatocytes and ameliorated pathological changes in liver tissue. The levels of AST, ALT, TNF-α and IL-1ß in the model group increased significantly, and were significantly reduced by OMT pretreatment. OMT pretreatment down-regulated expression of TLR4 and active caspase-3 and the Bax/Bcl-2 ratio, and up-regulated expression of P-AktSer473 (Akt phosphorylated at serine 473) and P-GSK3ßSer9 (glycogen synthase kinase 3ß phosphorylated at serine 9) induced by LPS/D-GalN. CONCLUSION: OMT inhibits hepatocyte apoptosis by suppressing the TLR4/PI3K/Akt/GSK-3ß signaling pathway, which suggests that OMT is an effective candidate for ameliorating acute liver failure.

Total Laparoscopic Hysterectomy in Patients with Large Uteri: Comparison of Uterine Removal by Transvaginal and Uterine Morcellation Approaches.

The aim of this study was to compare the clinical results of total laparoscopic hysterectomy (TLH) for large uterus with uterus size of 12 gestational weeks (g.w.) or greater through transvaginal or uterine morcellation approaches. We retrospectively collected the clinical data of those undergoing total laparoscopic hysterectomies between January 2004 and June 2012. Intraoperative and postoperative outcomes were compared between patients whose large uterus was removed through transvaginal or morcellation approaches. The morcellation group has significantly shorter mean operation time and uterus removal time and smaller incidence of intraoperative complications than the transvaginal group (all P < 0.05). No statistical significant difference regarding the mean blood loss, uterine weight, and length of hospital stay was noted in the morcellation and transvaginal groups (all P > 0.05). In two groups, there was one patient in each group who underwent conversion to laparotomy due to huge uterus size. With regard to postoperative complications, there was no statistical significant difference regarding the frequencies of pelvic hematoma, vaginal stump infection, and lower limb venous thrombosis in two groups (all P > 0.05). TLH through uterine morcellation can reduce the operation time, uterus removal time, and the intraoperative complications and provide comparable postoperative outcomes compared to that through the transvaginal approaches.

Analysis of Bone Height Changes after Maxillary Sinus Augmentation with Simultaneous and Delayed Placement of Dental Implants: A Clinical and Radiographic Study.

PURPOSE: To retrospectively assess the changes of the vertical height of the maxillary sinus floor after augmentation with simultaneous and delayed placement of implants. MATERIALS AND METHODS: In total, 38 patients with 76 implants were involved; vertical bone height of the sinus floor was radiographically measured at different stages including preoperation, immediately postsurgery, 6 and 12 months postsurgery, and 6 and 24 months postfunctional loading. RESULTS: Sinus augmentation significantly increased vertical bone height of the sinus floor for both the simultaneous and delayed groups. The survival rate was 100% in the simultaneous group and 95.46% in the delayed group. For simultaneous placement, the vertical bone height of the sinus floor at 6 and 12 months postsurgery was significantly less than that immediately postsurgery. For both groups, augmented bone height of the sinus floor showed significant decrease from 6 months to 24 months postfunctional loading. The mean value of final bone augmentation was 5.85 mm for simultaneous placement and 5.80 mm for delayed placements. CONCLUSION: Sinus augmentation with simultaneous and delayed placement of implants led to similar survival rates and bone augmentation. Resorption of augmentative bone was evident at 24 months postfunctional loading in both cases.

[The role of TLR4/P38/JNK signaling pathway in apoptosis of hippocampal neurons].

OBJECTIVE: To investigate the effects of the Toll like receptor 4 (TLR4) -P38-JNK signaling pathway in the apoptosis of hippocampal neurons in rats and its mechanisms. And to provide new experimental evidences for the pathogenesis research, prevention and treatment of neurodegenerative diseases (ND). METHODS: The hippocampal neurons derived from newborn rat were cultured for 7 d in vitro. The purity of hippocampal neurons was identified by immunofluorescence method. In order to activate or block the action of TLR4, the hippocampal neurons were pretreated with TLR4 ligand lipopolysaccharide (LPS) or TLR4 antibody. In the experiment 1, the hippocampal neurons were divided into normal control group, LPS group and TLR4 antibody+LPS group. The expressions of P-P38 and P-JNK were deteced by immunofluorescence. In the experiment 2, the hippocampal neurons were divided into 6 groups:normal control group, LPS group, TLR4 antibody +LPS group, SB202190(inhibitor P38)+LPS group, SP600125(inhibitor JNK)+LPS group, PD98059(inhibitor ERK)+LPS group. The cells in above mentioned groups were pretreated with TLR4 antibody, the inhibitors of P38, JNK or ERK for 2 h respectively. Then, all the six groups were stimulated by LPS for 24 h. The expressions of Bcl-2, Bax and Active-caspase-3 were detected by Western blot. The hippocampal neuronal apoptosis rate were tested with flow cytometry. RESULTS: The expressions of P-P38 and P-JNK of hippocampal neurons in LPS group were higher than those in normal control group (P<0.01). Compared with LPS group, the expressions of P-P38 and P-JNK were decreased significantly in TLR4 antibody +LPS group (P<0.01). Compared with the normal control group, the expressions of Bcl-2/Bax were decreased, while the expression of Active-caspase-3 was increased in the hippocampal neurons after LPS stimulation (P<0.01). The apoptotic rate of hippocampal neurons was higher in LPS group than that in the control group (P<0.01). Compared with LPS group, the expressions of Bcl-2/Bax were increasd and the expression of Active-caspase-3 was decreased in TLR4 antibody+LPS group, SB202190+LPS group, and SP600125+LPS group. The apoptotic rate of hippocampal neurons was significantly lower than that in the LPS group (P<0.05, P<0.01). The cell apoptosis rate had no significant differences between PD98059+LPS group and LPS group. CONCLUSIONS: ①TLR4-mediated P38/JNK signaling pathway exists in hippocampal neurons. ②After TLR4 activation in hippocampal neurons, the expressions of P-P38 and P-JNK are up-regulated, the expressions of Bcl-2/Bax are decreased and the expression of Active-caspase-3 is increased, which promote apoptosis of hippocampal neurons. TLR4/P38/JNK signaling pathway is involved in apoptosis of hippocampal neurons.

The role of the TLR4/NF-κB signaling pathway in Aß accumulation in primary hippocampal neurons.

The present study aimed to investigate the role of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway in the accumulation of amyloid ß protein (Aß) in primary hippocampal neurons of rats. The purity of these cultured neurons was determined by using immunofluorescence techniques. Lipopolysaccharide (LPS, a TLR4 ligand) or CLI-095 (a TLR4 inhibitor) was used to activate or inhibit TLR4 signaling, respectively. Pyrrolidine dithiocarbamate (PDTC), on the other hand, was used to inhibit NF-κB, a downstream effector of the TLR4 signaling pathway. The contents of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and Aß1-42 in the supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1ß, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), ß-site APP cleaving enzyme 1 (BACE-1), Presenilin-1 (PS-1), and ß-amyloid precursor protein (ß-APP) were examined by real-time quantitative PCR (RT-qPCR). The protein levels of ADAM10, BACE-1, PS-1 and ß-APP were examined by Western blotting. Meanwhile, the levels of TLR4 mRNA and protein in hippocampal neurons were tested by RT-qPCR and Western blotting, respectively, after stimulation with Aß1-42 at different concentrations. We observed that the purity of cultured hippocampal neurons after being cultured for 7 days was above 95%. Compared with untreated neurons, LPS-treated neurons showed higher expression levels of TNF-α, IL-1ß, BACE-1, PS-1, ß-APP, and Aß1-42, but a lower expression level of ADAM10. These effects were reversed upon pre-treatment with CLI-095 or PDTC. Furthermore, TLR4 expression was upregulated in the presence of Aß1-42. Taken together, these results provide evidence that elevation in the level of inflammatory cytokines accompanies the activation of TLR4 signaling, and that the consequent downregulation of ADAM10 and upregulation of BACE-1/PS-1 are likely responsible for the accumulation of ß-APP and Aß, which in turn increases TLR4 level to create a positive feedback loop that may constitute the basis for the progression of Alzheimer's disease.

Application of laparoscopy in the combined surgical procedures of gynecological and digestive disorders in obese women: a retrospective cohort study.

BACKGROUND: Laparoscopy surgery has been widely used for many decades and combined laparoscopic procedures have become favorable choices for concomitant pathologies in the abdomen. However, the type of combination procedures and their safety in obese women have not been well elucidated in obese women. METHODS: Here we retrospectively reported 147 obese women underwent combined laparoscopic gynecological surgery and cholecystectomy/appendicectomy in our hospital from January 2003 to December 2011. Of the total number of patients (n = 147), various laparoscopic gynecological surgeries were combined with laparoscopic cholecystectomy in 93 patients, and were combined with laparoscopic appendectomy in the rest 54 patients. Patients' ages ranged from 24 to 55 years with an average of 33 years. RESULTS: Our results showed that combined procedures caused various operative time and blood loss, with no difference considering the time to resume oral intake and length of hospital stay. Intraoperative complications occurred in a total of 7 patients (4.8%). None of the patients suffered from major complications after laparoscopic surgery, and minor postoperative complications occurred in 30 patients (20.4%). The follow-up period ranged from 6 to 24 months (average, 18.5 months). None of the patients developed complications during follow-up, except that one patient suffered from colporrhagia. CONCLUSIONS: Our results further suggest that the combined abdominal laparoscopic procedures of gynecologic and general surgery are safe and economic choices for obese women, and benefit patients in many ways including lesser pain, shorter hospital stays and earlier recovery.

The role of Toll-like receptor 4 on inflammation and Aß formation in cortex astrocytes.

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid ß-protein (Aß) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1ß, ß-amyloid precursor protein (ß-APP) and ß-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1ß and Aß in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1ß, Aß contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aß formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.

[Involvement of Toll-like receptor 4 in apoptosis of hippocampal neurons through Akt/FoxO3a/Bim signaling pathways].

The present study was to investigate whether Toll-like receptor 4 (TLR4)-mediated Akt/FoxO3a/Bim signaling pathway participated in lipopolysaccharide (LPS)-induced apoptosis in hippocampal neurons. The primarily cultured rat hippocampal neurons were treated with LPS, TLR4 antibody+LPS, and LY294002+LPS, respectively. Cell vitality was assayed by CCK-8. Expressions of p-Akt, Akt, p-FoxO3a, FoxO3a, Bim and active-Caspase-3 of each group were detected by Western blot analysis; the mRNA expression of Bim was detected by real-time quantitative PCR; FoxO3a nuclear translocation was detected by fluorescence microscope. The rate of cell apoptosis was assayed by flow cytometry. The results showed that cell vitality of hippocampal neurons decreased after being treated with LPS in a time-dependent way. Compared with the control group, the expressions of p-Akt and p-FoxO3a decreased significantly, FoxO3a translocated into the nucleus, meanwhile, the expression of Bim and active-Caspase-3, and the apoptotic ratio of hippocampal neurons increased in LPS treated neurons. Pretreatment with TLR4 antibody significantly blocked, while PI3K antagonist LY294002 further strengthened these changes induced by LPS. In conclusion, the present study suggests that Akt/FoxO3a/Bim signaling pathways mediated by TLR4 participate in the apoptotic processes of primarily cultured hippocampal neurons treated with LPS, and the activation of TLR4 causes neuronal apoptosis.

Distribution and quantitative detection of GABAA receptor in Carassius auratus gibelio.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in brain, is synthesized from glutamate and metabolized to succinic semialdehyde by glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T), respectively. The fast inhibitory effect of GABA is mediated by GABA type A (GABAA) receptors that are associated with several neurological disorders, and GABAA receptors are targets of several therapeutic agents. To date, information on the distribution and quantity of GABAA receptors in Carassius auratus gibelio is still limited. We investigated for the first time, the tissue-specific distribution of GABAARß2a and GABAARß2b, the two subunits of the predominant GABAA receptor subtype (α1ß2γ2), and then, the expression of GABAARß2a, GABAARß2b, GAD, and quantified GABA-T genes in different tissues by quantitative real-time PCR method and compared different expressions between two developmental stages of C. auratus gibelio. Results showed that GABAARß2a and GABAARß2b genes expressed in both brain and peripheral organs using reverse transcription-polymerase chain reaction. In addition, the majority of GABAARß2a, GABAARß2b, GAD, and GABA-T were mainly synthesized in brain; however, a considerable amount of GABA-T was secreted from the peripheral tissues, especially in the liver. Moreover, the expression of GABAARß2a and GABAARß2b genes in different tissues varied with body weight change. This study provides a reference for further studies on GABA and GABAA receptors subunits and an insight on the possible pharmacological properties of the GABAA receptor in C. auratus gibelio.

Carotenoid cleavage dioxygenase4 is a negative regulator of ß-carotene content in Arabidopsis seeds.

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed ß-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for ß-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID cleavage dioxygenase4 (CCD4) as a major negative regulator of seed carotenoid content, especially ß-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing ß-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with ß-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in ß-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.

[The role of TLR4-mediated MyD88-dependent pathway in neuroinflammation in hippocampal neurons of rats].

OBJECTIVE: To investigate weather there is a toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent pathway in hippocampal neurons of rats and the probable role of the pathway in neuroinflammation. METHODS: To establish the proper model, primarily cultured hippocampal neurons were treated with lipopolysaccharides (LPS), or pretreated with TLR4 antibody then co-treated with LPS. The expression of mRNA of MyD88 and TNF-alpha receptor associated factor 6 (TRAF6) were tested by RT-qPCR. The content of MyD88 and TRAF6 were tested by Western blot. The nuclear translocation of nuclear factor-kappaB/P65 (NF-kappaB/p65) was tested by immunofluorescence. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and nitric oxide (NO) were tested by ELISA. RESULTS: LPS could increase MyD88 and TRAF6 mRNA, upregulate protein level of MyD88 and TRAF6 and increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. LPS also could promote NF-kappa B/p65 translation to the nucleus. The pretreatment with TLR4 antibody reduced the translocation to nucleus for NF-kappaB/P65 and the contents of TNF-alpha, IL-1beta and NO in the culture supernatant. CONCLUSION: There is a TLR4-mediated MyD88-dependent pathway in hippocampal neurons. The activation of this pathway can increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. TLR4-mediated MyD88-dependent pathway in hippocampal neurons participate in neuroinflammation, that means neurons are not passive in inflammation.

Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons.

Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined. In this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-1ß and tumor necrosis factor-α were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B α inhibitor in the cytosol and increased nuclear factor-κB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-κB pathway in hippocampal neurons, which may be both "passive victims" and "activators" of neuroinflammation.

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons.

The phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway is considered important for cell survival and has been shown to mediate various anti-apoptotic biological effects. This study explored the role of the Toll-like receptor 4 (TLR4)-mediated PI3K/AKT-glycogen syn-thase kinase 3ß (GSK-3ß) signaling pathways in lipopolysaccharide-induced apoptosis in a primary culture of hippocampal neurons. Results demonstrated that the apoptotic ratio of hippocampal neurons stimulated by lipopolysaccharide was significantly higher compared with the control group. Both the expression of P-AKT(Ser473) and P-GSK-3ß(Ser9) in hippocampal neurons stimulated by lipopo-polysaccharide decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. The level of active Caspase-3 and the ratio of Bax/Bcl-2 in hippocampal neurons treated with TLR4 antibody or the GSK-3ß inhibitor, LiCl, creased before intervention with lipopolysaccharide, but increased after treatment with the AKT hibitor, LY294002. These findings suggest that the TLR4-PI3K/AKT-GSK3ß signaling pathway may be involved in lipopolysaccharide-induced apoptosis of hippocampal neurons.

Reduction of hexavalent chromium using naturally-derived flavonoids.

Quercetin is a naturally occurring flavonoid that is known to form complexes with metals; a process that reduces the environmental availability of toxic metals such as chromium. We hereby report the first evidence of the removal of Cr(VI) from environmental samples using quercetin (QCR) and two synthetic derivatives: namely quercetin pentaphosphate (QPP) and quercetin sulfonic acid (QSA). We successfully synthesized both QPP and QSA using simple procedures while characterizing them with UV-vis spectroscopy, H(1)-NMR, (13)C NMR, (31)P-NMR, and LC-MS techniques. The solubility of QPP was found to be 840 mg/mL and aqueous solutions of both QPP and QSA were stable for over a period of 1 year. Quercetin and these derivatives were subsequently utilized for the reduction of Cr(VI) and QCR was found to have a higher reduction efficiency of 99.8% (30 min), followed by QPP/palladium nanoparticles mixture (PdNPs) at 96.5% (60 min), and finally QSA/PdNPs mixtures at 91.7% (60 min). PdNPs catalyst increased the efficiency by ∼36.5% while a change in operating temperature from 25 to 45 °C improved the efficiency by ∼46.8%. Electron paramagnetic resonance spectroscopy was used to confirm the presence of Cr (III) in the reaction products. This reduction approach was validated in environmental (Binghamton University) BU and standard reference material (BRS) soil samples. Results showed that the analysis could be completed within one hour and the efficiency was higher in BU soil than in BRS soil by 16.1%. QPP registered the highest % atom economy of 94.6%. This indicates enhanced performance compared to bioremediation approach that requires several months to achieve about 90% reduction efficiency.