Inhibition and Allosteric Signaling of Transcription Activation by Bacterial AraC Family Activator Proteins

AraC family transcriptional activators are defined by a 100-amino acid DNA-binding domain (DBD) containing two DNA binding helix-turn-helix (HTH) motifs. My research focused on three AraC family proteins: RhaR (activator of the E. coli L-rhamnose catabolic regulon), VirF (activator of expression of the Shigella flexneri type three secretion system), and ToxT (activator of Vibrio cholera virulence gene). By using fluorescence-based thermal shift assay, and intrinsic tryptophan fluorescence assay, I have shown that small molecule inhibitor SE-1 directly binds to VirF and RhaS-DBD. Mutagenesis studies of residues in RhaS support the prediction of docking that SE-1 is likely bound to a small pocket between the two HTH motifs. With a final goal to provide direct evidence of the position on the ToxT protein that serves as the SE-1 binding site, ToxT was crystalized under solution conditions different than the previously published ones, and the structure of ToxT was determined to a higher resolution than the published structure. A region that was missing from the previously determined structure now can be traced entirely. Using Electrophoretic Mobility Shift Assay (EMSA), SE-1 was found to inhibit DNA binding by ToxT. Co-crystallization trials of SE-1 and ToxT were carried out. Unfortunately, I was not able to obtain any crystals of ToxT-SE-1. In collaboration with Dr. Jeff Aubé, I have tested SE-1 analogues with the ultimate goal of optimizing the potency and specificity of SE-1. Unfortunately, no analogs were found with increased VirF inhibition potency relative to SE-1. In the process of synthesizing analogs, we found that SE-1 and its analogues converted to corresponding quaternary salts in aqueous solution, and the quinolinium salt was responsible for the observed inhibition by SE-1. In this study, I also investigated the mechanism by which RhaR responds to L-rhamnose. During these studies, I discovered that the RhaR start codon was previously annotated 30 codons upstream of the true start codon. The equilibrium binding affinity of RhaR to its full and half DNA binding sites, of the isolated RhaR-DBD to the half-site, and the rhamnose dependence of DNA bending by RhaR were measured. The findings support a model in which RhaR NTD increases the DNA binding affinity of each RhaR protomer DBD, independent of rhamnose; and that rhamnose signalling primarily increases the positive cooperativity of DNA binding by the two DBDs in a RhaR dimer. These findings suggest a model for the mechanism of allosteric rhamnose signalling in RhaR. To provide more structural information about RhaR protein, the structures of RhaR-NTD were determined in the presence and absence of L-rhamnose. The 2.05Å rhamnose-bound RhaR-NTD structure showed that the protein forms an antiparallel dimer, and shared a fold that was similar to the AraC-NTD, binding its respective sugar L-rhamnose within a β-barrel. In addition, a Ni2+ ion, which has not been seen in other AraC family protein structures, was present in the sugar-binding, cupin superfamily, motif of RhaR. A rhamnose-free structure was also solved to 1.73 Å, and in this structure, a loop region that is involved in rhamnose binding was completely disordered. A second loop region also has minor structural changes. Each of the two regions with rhamnose-dependent structural changes is predicted to be at the interface between the RhaR NTD and DNA-binding domain, suggesting their potential involvement in rhamnose allosteric signaling. No differences were observed in the RhaR N-terminal arm region in the fully and partially rhamnose-occupied structures, suggesting that RhaR rhamnose-dependent allosteric signaling shares some features with the ‘light switch’ model of AraC, but differs in other features

Auricle shaping using 3D printing and autologous diced cartilage.

ObjectiveTo reconstruct the auricle using a porous, hollow, three-dimensional (3D)-printed mold and autologous diced cartilage mixed with platelet-rich plasma (PRP).MethodsMaterialise Magics v20.03 was used to design a 3D, porous, hollow auricle mold. Ten molds were printed by selective laser sintering with polyamide. Cartilage grafts were harvested from one ear of a New Zealand rabbit, and PRP was prepared using 10 mL of auricular blood from the same animal. Ear cartilage was diced into 0.5- to 2.0-mm pieces, weighed, mixed with PRP, and then placed inside the hollow mold. Composite grafts were then implanted into the backs of respective rabbits (n = 10) for 4 months. The shape and composition of the diced cartilage were assessed histologically, and biomechanical testing was used to determine stiffness.ResultsThe 3D-printed auricle molds were 0.6-mm thick and showed connectivity between the internal and external surfaces, with round pores of 0.1 to 0.3 cm. After 4 months, the diced cartilage pieces had fused into an auricular shape with high fidelity to the anthropotomy. The weight of the diced cartilage was 5.157 ± 0.230 g (P > 0.05, compared with preoperative). Histological staining showed high chondrocyte viability and the production of collagen II, glycosaminoglycans, and other cartilaginous matrix components. In unrestricted compression tests, auricle stiffness was 0.158 ± 0.187 N/mm, similar to that in humans.ConclusionAuricle grafts were constructed successfully through packing a 3D-printed, porous, hollow auricle mold with diced cartilage mixed with PRP. The auricle cartilage contained viable chondrocytes, appropriate extracellular matrix components, and good mechanical properties.Levels of evidenceNA. Laryngoscope, 129:2467-2474, 2019

Larval nutrition-induced plasticity affects reproduction and gene expression of the ladybeetle, Cryptolaemus montrouzieri

Background: Organisms may develop into multiple phenotypes under different nutritional environments by developmental plasticity, whereas the potential costs and mechanisms of such plasticity are poorly understood. Here we examined the fitness and gene expression of nutrition-induced phenotypes in the ladybeetle, Cryptolaemus montrouzieri after having experienced varying larval food regimes. Results: We found that C. montrouzieri adults undergoing a variable larval food regime achieved a similar developmental time, survival, body mass and egg production as those undergoing a high larval food regime. The survival, developmental time, body mass and fecundity of the adults from a restricted larval food regime were inferior to those from the high and variable larval food regimes. However, the adults from this restricted larval food regime had a higher expression level of genes encoding immune-and antioxidant-related enzymes than those from the high and variable larval food regimes when exposed to starvation and pesticide conditions in adult life. Conclusions: These results suggest that larval food availability in C. montrouzieri not only triggers adult phenotypic differences but also affects reproduction and expression level of genes in adult life, indicating that the larval nutritional conditions can affect adult fitness and resistance to stressful conditions through developmental plasticity

PO-105 Exercise regulates HMGB1 / TLR4 / NF- κ B pathway by H2S to improve OJ intestinal injury

Objective To study the effect of aerobic exercise on the damage of intestinal mucosal barrier function caused by obstructive jaundice（OJ）and to explore its mechanism of action. Methods  50 male KM mice were randomly divided into 5 groups: sham operation group (S), model group (M), exercise group (TM), DL-Propargylglycine + exercise (PT) group and sodium hydrosulfide + exercise (NT) group.In addition to the S group which are in the common bile duct to the abdominal wall hanging 48 hours to build mouse obstructive jaundice model. In the PT group, PAG (40 mg/kg) was intraperitoneally injected 7 days after surgery; NaHS (50 μmol/kg) was intraperitoneally injected in the NT group 7 days after surgery; TM group, NT group and PT group were graded at 0%, and the speed was 10m/min no weight training (30min/day).After 6 weeks, HE staining was used to observe the morphological changes of the intestinal mucosa.Biochemical analysis was used to detect the concentration of hydrogen sulfide (H2S) in blood and ileum, and total bilirubin (TBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) Liver function, diamine oxidase (DAO), D-lactic acid intestinal barrier function biochemical index; qRT-PCR and immunohistochemical staining were used to observe the expression changes of H2S-mediated related channel mRNA and protein（HMGB1, TLR4 and NF-Kbp6）in intestinal tissues. Results HE staining showed that the intestinal mucosa of group M was atrophied and the villus was broken.Compared with M group, the intestinal mucosa arrangement in TM group was relatively regular. Compared with TM group, intestinal mucosa atrophy in PT group, fluff hair loss, sparseness and disorder, partial mucosa The layer was separated from the lamina propria and the gland was severely damaged. The intestinal mucosa of the NT group was relatively regular, and the changes of intestinal mucosa atrophy were restored. Serum test results showed that H2S levels were higher in the TM group than in the M group; compared with the TM group, the PT group decreased and the NT group increased. DAO level: The TM group was lower than the M group; compared with the TM group, the PT group was elevated and the NT group was decreased.  Changes in serum D-lactic acid levels were similar to DAO. The results of qRT-PCR and immunohistochemical staining showed that the expressions of HMGB1, TLR4 and NF-Kbp6 mRNA and protein in the intestinal tissues of mice in TM group were significantly lower than those in M group and PT group, and the mRNA and protein expression levels in NT group were the lowest. Conclusions Aerobic exercise inhibits the HMGB1 / TLR4 / NF-κB signaling pathway through the H2S / CSE system, thereby exerting a protective effect on the intestinal mucosal barrier

Understanding, Diagnosing, and Treating Pancreatic Cancer From the Perspective of Telomeres and Telomerase

Telomerase is associated with cellular aging, and its presence limits cellular lifespan. Telomerase by preventing telomere shortening can extend the number of cell divisions for cancer cells. In adult pancreatic cells, telomeres gradually shorten, while in precancerous lesions of cancer, telomeres in cells are usually significantly shortened. At this time, telomerase is still in an inactive state, and it is not until before and after the onset of cancer that telomerase is reactivated, causing cancer cells to proliferate. Methylation of the telomerase reverse transcriptase (TERT) promoter and regulation of telomerase by lactate dehydrogenase B (LDHB) is the mechanism of telomerase reactivation in pancreatic cancer. Understanding the role of telomeres and telomerase in pancreatic cancer will help to diagnose and initiate targeted therapy as early as possible. This article reviews the role of telomeres and telomerase as biomarkers in the development of pancreatic cancer and the progress of research on telomeres and telomerase as targets for therapeutic intervention

PO-106 Anisochronous aerobic exercise improves glucose and lipid metabolism of obstructive jaundice by activating Akt signaling pathway

Objective To observe the effect of moderate intensity and different interval aerobic exercise on hepatic injury and AKT signaling pathway induced by severe obstructive jaundice in mice. Methods 40 male KM mice were randomly divided into 4 groups: sham operation (OJ) group, model (OM) group, 3-week exercise (ST) group and 6-week exercise (SU) group. Mice in OM group, ST group and SU group all adopted the orthotopic hanging choledochotomy method modified by this study group to construct the animal model of obstructive jaundice. The slope of group ST and group SU were 0% and the speed was 10m/min. After the above 6 weeks of intervention, HE staining was used to observe the morphological changes of hepatocytes. Methods of automatic biochemical analyzer test serum total bilirubin (TBIL), alanine aminotransferase (ALT), aspertate aminotransferase (AST), liver function and fasting plasma glucose (GLU), glycosylated hemoglobin (HbA1c), glycosylated serum protein (GSP), total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL - C) of sugar, lipid metabolism and biochemical indexes such as detection; Immunohistochemical staining and qrt-pcr technology were used to observe the expression changes of AKT related molecules such as SREBP-1c, LDL-C, gsk-3β, GCK and G6Pase in liver tissues. Results HE staining showed that the liver cell cords of the normal group were orderly. In the model group, the hepatocytes of the rats were fibrosed in large amounts, which showed degeneration, necrosis and even disordered structure of hepatocytes. In the 3-week exercise group, a small number of hepatic cells were found with patchy necrosis, hepatic lobule structure was changed, and hepatic cords were not well arranged. There was no obvious tissue necrosis in the 6-week exercise group, the hepatic lobule structure was basically normal, and the liver cord was arranged in order. Serological results showed that the levels of TG, TC, LDL-C, HDL - C and GLU in the 6-week exercise group were significantly lower than those in the model group (P < 0.01), and the levels of TG and TC in the 3-week group were also significantly decreased (P < 0.05). In liver tissues, the mRNA and protein expression of related molecules of AKT pathway such as SREBP-1c, LDL-C, gsk-3β, GCK and G6Pase were significantly decreased.  Conclusions Moderate intensity aerobic exercise can regulate glucose and lipid metabolism in mice with severe liver injury caused by obstructive jaundice. The underlying mechanism may be related to regulating the AKT pathway

Risk Prediction of Second Primary Malignancies in Primary Early-Stage Ovarian Cancer Survivors: A SEER-Based National Population-Based Cohort Study

Purpose: This study aimed to characterize the clinical features of early-stage ovarian cancer (OC) survivors with second primary malignancies (SPMs) and provided a prediction tool for individualized risk of developing SPMs. Methods: Data were obtained from the Surveillance, Epidemiology and End Results (SEER) database during 1998–2013. Considering non-SPM death as a competing event, the Fine and Gray model and the corresponding nomogram were used to identify the risk factors for SPMs and predict the SPM probabilities after the initial OC diagnosis. The decision curve analysis (DCA) was performed to evaluate the clinical utility of our proposed model. Results: A total of 14,314 qualified patients were enrolled. The diagnosis rate and the cumulative incidence of SPMs were 7.9% and 13.6% [95% confidence interval (CI) = 13.5% to 13.6%], respectively, during the median follow-up of 8.6 years. The multivariable competing risk analysis suggested that older age at initial cancer diagnosis, white race, epithelial histologic subtypes of OC (serous, endometrioid, mucinous, and Brenner tumor), number of lymph nodes examined (<12), and radiotherapy were significantly associated with an elevated SPM risk. The DCA revealed that the net benefit obtained by our proposed model was higher than the all-screening or no-screening scenarios within a wide range of risk thresholds (1% to 23%). Conclusion: The competing risk nomogram can be potentially helpful for assisting physicians in identifying patients with different risks of SPMs and scheduling risk-adapted clinical management. More comprehensive data on treatment regimens and patient characteristics may help improve the predictability of the risk model for SPMs

OR-012 The Regulation of NF-κB-TNF-α/IDO/5-HT Axis by Aerobic Exercise against Hippocampal Neuroinflammation in CUMS Depressive Mice

Objective To study the effect of aerobic exercise on the anti-chronic stress depression and the key metabolic enzymes indoleamine 2,3 peroxidase (IDO) of tryptophan and kynurenine pathway. Methods &nbsp;Adopt the method of random numbers to make depression modelling for mice with 1 or 2 kinds of stimulating factors for 28 days in view of the 13 kinds of chronic stress stimulation. Collect and analyse motionless time for FST and TST of mice by using the Noldus EthoVision XT9 system. Test the serum factor level of laboratory mice with Cusabio imported IDO, NF-ƙB and TNF-α kit. Make real-time fluorescent quantitative PCR verification of the mRNA expression and protein expression level of IDO, 5-HT, NF-ƙB and TNF-α in hippocampus. Results After 4 weeks of chronic stress stimulation, the motionless time for FST and TST of mice in the Model Group obviously prolonged (p&lt;0.05). The bioactivity of IDO, NF-ƙB and TNF-α in hippocampus increased. The mRNA expression of IDO, NF-ƙB and TNF-α in hippocampus increased, while the mRNA expression of 5-HT decreased (p&lt;0.01). Aerobic exercise can shorten the motionless time of mice, inhibit the activity of IDO, NF-ƙB and TNF-α, reduce the mRNA expression quantity of IDO, NF-ƙB and TNF-α and enhance the expression of 5-HT. Conclusions Aerobic exercise has an antidepressant effect on mice for chronic stress depression, which is related to the IDO activation induced by inhibit inflammatory cytokines. Aerobic exercise may inhibit the NF-ƙB to reduce the pathway of tryptophan and kynurenine, affect the direct and indirect induced effect of IDO, and adjust its activity and expression

Selective modes affect gene feature and function differentiation of tetraploid Brassica species in their evolution and domestication

The genus Brassica contains a diverse group of important vegetables and oilseed crops. Genome sequencing has been completed for the six species (B. rapa, B. oleracea, B. nigra, B. carinata, B. napus, and B. juncea) in U’s triangle model. The purpose of the study is to investigate whether positively and negatively selected genes (PSGs and NSGs) affect gene feature and function differentiation of Brassica tetraploids in their evolution and domestication. A total of 9,701 PSGs were found in the A, B and C subgenomes of the three tetraploids, of which, a higher number of PSGs were identified in the C subgenome as comparing to the A and B subgenomes. The PSGs of the three tetraploids had more tandem duplicated genes, higher single copy, lower multi-copy, shorter exon length and fewer exon number than the NSGs, suggesting that the selective modes affected the gene feature of Brassica tetraploids. The PSGs of all the three tetraploids enriched in a few common KEGG pathways relating to environmental adaption (such as Phenylpropanoid biosynthesis, Riboflavin metabolism, Isoflavonoid biosynthesis, Plant-pathogen interaction and Tropane, piperidine and pyridine alkaloid biosynthesis) and reproduction (Homologous recombination). Whereas, the NSGs of the three tetraploids significantly enriched in dozens of biologic processes and pathways without clear relationships with evolution. Moreover, the PSGs of B. carinata were found specifically enriched in lipid biosynthesis and metabolism which possibly contributed to the domestication of B. carinata as an oil crop. Our data suggest that selective modes affected the gene feature of Brassica tetraploids, and PSGs contributed in not only the evolution but also the domestication of Brassica tetraploids