[Investigation of the inhibitory potential of caffeic acid phenethyl ester on prion replication, amplification, and fibril formation in vitro].

Objective: To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrPSc). Methods: The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrPSc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrPSc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results: CCK8 cell viability assay results demonstrated that treatment with 1 µmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 µmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 µmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrPSc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrPSc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10-6 mol/L. Conclusions: CAPE inhibits PrPSc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.

Periodontitis and the risk of oral, gastric and esophageal cancers: a two-sample Mendelian randomization study.

BACKGROUND: Periodontitis is a common oral disease and the chronic inflammation caused by it may influence the development of cancers in the upper gastrointestinal tract. Many observational studies have established a relationship between the two, but the results are not entirely consistent. METHODS: Two-sample MR was performed using publicly available genome-wide association studies data for periodontitis, oral, gastric and oesophagal cancers. The Inverse Variance Weighting (IVW) method serves as the primary method, with MR Egger, Weighted Median, Simple Model and Weighted Model Algorithm methods as complementary methods to assess genetic causal associations. Cochran Q-test, MR-Egger regression and MR polytropic residuals and outliers were used to assess heterogeneity and horizontal pleiotropy. RESULTS: IVW results did not support a causal association between periodontitis and oral (OR = 1.00, 95% CI: 1.00, 1.00) and oesophagal cancer (OR = 1.00, 95% CI: 1.00, 1.00). Similarly, there was again no causal association between periodontitis and gastric cancer, which was integrated with an OR of 1.04 (95% CI: 0.97, 1.12). Complementary method results were consistent with IVW and heterogeneity and horizontal pleiotropy were not found in most studies. CONCLUSIONS: The findings of our MR study do not support a causal relationship between periodontitis and oral, gastric and oesophagal cancers.

New primers for sex identification in the Chukar partridges (Alectoris chukar).

1. Male and female Chukar partridges are difficult to differentiate based on their morphology or by the Chromobox-Helicase-DNA binding (CHD) during early growth.2. The current study developed a novel, simple, low-cost and rapid sexing protocol for Chukar partridges based on the newly defined sexing gene ubiquitin-associated protein 2 (UBAP2).3. The length of polymorphism between UBAP2-W and UBAP2-Z homologous genes allows for easy sex discrimination in this species. Molecular sexing analysis was based on the simultaneous amplification of both genes, resulting in two distinct amplicons (947 bp and 535 bp) in heterogametic females and only a single band (535 bp) in homogametic males, which is easy to detect with agarose gel electrophoresis.4. This technique is simple and convenient for genetic sex determination in Chukar partridges.

A new method for molecular sex identification in the emu (Dromaius novaehollandiae).

1. Sex chromosomes of emus are largely homomorphic. Therefore, the standard methodology for molecular sexing based on screening intron length variations in sex-linked genes is not applicable. However, emu sexing requires costly and time-consuming PCR-RFLP or multiplex PCR methods.2. This experiment used a directed PCR amplification and capillary electrophoresis sexing protocol. Two distinct peaks were observed in females (ZW), while only one peak was observed in males (ZZ).3. This sexing technique proved to be rapid, non-invasive, and highly sensitive and may be useful for verifying the sex ratio and breeding management of emus.

[Comparative analysis on prevalence of tobacco and e-cigarettes uses in junior middle school students in Shanghai, 2013 and 2019].

Objective: To examine the prevalence and trend of tobacco and e-cigarettes uses and identify the influencing factors for smoking behavior in junior middle school students in Shanghai, and provide data support and scientific basis for the development of tobacco control intervention strategy in adolescents. Methods: Multi-stage stratified random sampling method was used to select junior middle school students in 8 districts and 10 districts in Shanghai in 2013 and in 2019 respectively. Information about tobacco and e-cigarettes uses in the students were collected by using self-administrated questionnaire. The prevalence of tobacco and e-cigarettes uses were calculated, the difference between two years was compared with χ2 test. The influencing factors were identified by multivariate logistic regression analysis. Results: In 2019, the current smoking rate was 0.6% in junior middle school students in Shanghai, and the smoking attempt rate was 2.9%, both were lower than the levels in 2013 (0.7% and 6.9%). The current use rate of e-cigarettes was 0.6% in 2019,with no significant change compared with 2013 (0.6%). The proportion of the students who had heard of e-cigarettes in 2019 (78.4%) was higher than that in 2013 (47.2%). In 2019, the second-hand smoke (SHS) exposure rate at home, in both indoor and outdoor public places and on public transportations was 72.5%, which was slightly lower than the level in 2013 (73.0%), the differences were all significant (P<0.05). In 2019, the students seeing close friend smoking (OR=27.381, 95%CI: 12.037-62.287), seeing someone smoking in school (OR=2.477, 95%CI: 1.155-5.312), believing that SHS may not be harmful (OR=8.471, 95%CI: 1.464-49.005) had higher possibility of smoking. Being aged ≥15 years (compared with being aged ≤12 years, OR=8.688, 95%CI: 1.922-39.266), exposure to SHS in outdoor public place (OR=8.608, 95%CI: 1.048-70.692), close friend smoking (OR=8.115, 95%CI: 1.754-37.545) were positively associated with e-cigarettes use, and believing that smoking results in uncomfortable social contact [compared with believing that smoking results in comfortable social contact (OR=0.105,95%CI: 0.018-0.615)] were negatively associated with e-cigarettes use, the difference was significant (P<0.05). Conclusion: The prevalence of tobacco and e-cigarette uses in junior middle school students in Shanghai remained at a low level in recent years. The SHS exposure rate in junior middle school students is high. Smoking behavior of junior middle school students is closely related to personal attitude and awareness of tobacco, exposure to SHS, peer smoking and the situation of tobacco control in schools. Prevention and intervention should be carried out from multi-dimensions to effectively protect teenagers from tobacco hazards.

[Effect of ectodysplasin-A1 on proliferation and cell cycle of ameloblast-like cell].

Objective: To investigate the effects of ectodysplasin-A1 (EDA1) on the proliferation and cell cycle of ameloblast-like epithelial cells (LS8 cells). Methods: Wild EDA1 plasmid pCR3-Flag-EDA1-W (wild group), syndrome mutant EDA1 plasmid pCR3-Flag-EDA1-H252L (mutant group) and empty vector plasmid pCR3-Flag (control group) were transfected into LS8 cells. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay and cell cycle was detected by flow cytometry. All tests were repeated three times. Results: Compared with the control group (0.105±0.032), the proliferation activity of the wild group (0.201±0.009) was significantly higher after 72 h (P<0.05). Compared with the control group (0.168±0.054) and the mutant group (0.194±0.059), the proliferation activity of the wild group (0.386±0.066) was significantly higher after 96 h (P<0.05). There was no significant difference between the mutant group and the control group at all time points (P>0.05). In the G0/G1 phase, compared with the control group (65.4%±2.1%) and the mutant group (66.6%±3.1%), the cell distribution ratio of the wild group (51.2%±1.1%) was significantly lower (P<0.01). In the S phase, compared with the control group (23.1%±2.0%) and the mutant group (21.9%±1.8%), the cell distribution ratio of the wild type group (37.3%±2.4%) was significantly higher (P<0.01). There was no significant difference in cell cycle distribution between the mutant group and the control group (P<0.05). Conclusions: Wild EDA1 promotes the proliferation of LS8 cells and the transformation from G0/G1 to S phase. The syndrome mutant EDA1 (EDA1-H252L) loses its function of regulating the cell proliferation and cell cycle of LS8 cells.

Genetic diversity of Jiangsu native chicken breeds assessed with the mitochondrial DNA D-loop region.

1. The objective of this study was to determine the origin and evolution of chickens from 5 native breeds that are traditionally raised in Jiangsu Province. 2. To address this question, the complete mitochondrial DNA D-loop sequence of 149 chickens from 5 native breeds of Jiangsu Province was analysed. 3. Sequence read lengths of the native breeds were 1231 to 1232 bp, with a single-base deletion from the 859 bp site in the 1231 bp haplotype. A total of 33 variable sites that defined 19 haplotypes were identified. The average haplotype diversity and nucleotide diversity were 0.862 ± 0.017 and 0.00591 ± 0.00135. 4. Phylogenetic analysis showed that genetic structure of the mtDNA haplotypes of Jiangsu chickens are distributed across 5 clades (haplogroups): Clades A, B, C, D, and E. However, most of the individuals characterised in this study belonged to clades A and B. 5. The results of this study indicate that Jiangsu chicken populations have relatively low nucleotide and haplotype diversity and likely share 5 common maternal lineages.

[Effect of allogenic mesenchymal stem cells transplantation on the expression of interleukin-22 and matrix metalloproteinase-3 in rats with collagen induced arthritis].

Objective: To study the effect of allogeneic bone marrow mesenchymal stem cells transplantation on the expression of interleukin -22 (IL-22), matrix metalloproteinase -3 (MMP-3) in serum and synovial of rats with collagen induced arthritis. Methods: Type Ⅱ collagen were injected twice to establish the collagen induced arthritis (CIA) rat model. Forty-eight rats were randomly divided into 3 groups: control group, CIA control group, CIA experiment group. Rat bone marrow mesenchymal stem cells were cultured by bone marrow method combined with adherent culture method. After identify, the remaining cells were injected in the CIA experimental group. Enzyme linked immunosorbent assay (ELISA) and immunohistochemistry were used to detect the expression of IL-22 and MMP-3 in serum and anklebone joint's synovium of rats, respectively. Synovial cells were isolated and cultured, and were treated with different concentrations of IL-22. MMP-3 protein and mRNA were detected before and after stimulation by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). Results: After MSC transplantation, arthritis index, X-ray, HE staining of CIA rat showed that joint damage significantly reduced compared with the control group. The ELISA results showed that the expression of MMP-3 and IL-22 in CIA control group was higher than those in the control group (125.79±9.12 vs 102.00±7.63 ng/ml, P<0.05), (292.35±31.23 vs 257.27±13.99 ng/ml, P<0.05) and CIA experiment group (125.79±9.12 vs 97.94±9.50 ng/ml, P<0.05), (292.35±31.23 vs 262.16±22.02 ng/ml, P<0.05) with statistically significant difference (P<0.05). There was no significant difference between the control group and CIA experimental group. Immunohistochemical showed similar results with ELISA. Western blotting and RT-qPCR showed that MMP-3 protein and mRNA expression was increased after IL-22 stimulation in a dose-dependent manner. Conclusion: IL-22 and MMP-3 play an important role in the pathogenesis of rheumatoid arthritis. IL-22 could regulate the expression of MMP-3, and bone marrow mesenchymal stem cells could reduce the expression of MMP-3 in the treatment of rheumatoid arthritis by reducing the expression of IL-22.

miR-133 inhibits pituitary tumor cell migration and invasion via down-regulating FOXC1 expression.

Many studies have shown that microRNA (miR)-133 functions as a tumor suppressor in a variety of metastatic cancers, including breast cancer, gastric cancer, and liver fibrosis. However, the influence of miR-133 on pituitary tumor malignancy has not yet been reported. The purpose of this study was to explore the role of miR-133 in pituitary tumor cell migration and invasive ability and the molecular mechanisms involved. Our findings suggest that in pituitary adenoma cell lines, through direct targeting and negative control of forkhead box C1 (FOXC1), miR-133 can inhibit pituitary adenoma cell migration and invasion. In addition, epithelial-to-mesenchymal transition can be induced by miR-133. Additionally, a negative correlation was found between FOXC1 and miR-133 expression when comparing their expression levels between cancerous tissue and adjacent normal tissue. This suggests that miR-133 can inhibit cell migration and invasion by directly targeting FOXC1, implying that miR-133 could be a potential therapeutic target for treatment of invasive pituitary adenoma.

Peroxynitrite-induced protein nitration is responsible for renal mitochondrial damage in diabetic rat.

Oxidative stress, especially mediated by peroxynitrite (ONOO-), plays a key role in diabetes. Mitochondria, as the generating source of ONOO-, may also be the major damaging target of ONOO-. Whether ONOO--induced protein nitration is responsible for renal mitochondrial damage in diabetes is not fully known. This study was aimed to clarify the relationship between nitration of entire mitochondrial proteins induced by ONOO- and the renal mitochondrial damage in diabetes. Sprague-Dawley male rats were injected ip with streptozotocin to induce diabetes. After 10 weeks, inducible nitric oxide synthase (iNOS) mRNA expression and protein content in renal cortex were detected. Distribution of nitrotyrosine (NT), a specific marker of ONOO-, in renal cortex and NT content in mitochondrial proteins were detected. The ultrastructure of glomerulus was observed. Aminoguanidine was used as a selective inhibitor of iNOS to reduce the derivation of ONOO-. In diabetic rat, increasing levels of iNOS mRNA and protein content, and NT content were observed, in accord with the pathological alterations of glomerulus. In aminoguanidine group, these alterations were attenuated significantly. In conclusion, ONOO- could induce entire mitochondrial proteins nitration, responsible for the damage of renal mitochondria in diabetes.

Pattern of expression of engrailed in relation to gamma-aminobutyric acid immunoreactivity in the central nervous system of the adult grasshopper.

Engrailed (En) protein expression in neurons of the mesothoracic and metathoracic ganglia of the adult grasshopper, Schistocerca americana, was examined by immunohistochemistry. Each neuromere had a dorsally located cluster of En-positive neurons within the dorsal unpaired median (DUM) group, comprising one cluster in the mesothoracic ganglion (T2) and four clusters in the metathoracic ganglion, one for each component neuromere (T3, A1, A2, A3). Ventrally, En-positive neurons occurred in the posterior one-third of each neuromere. In T2 and T3, three ventral groups of neurons were labeled bilaterally. In the abdominal neuromeres, many fewer ventral neurons were En-positive. These also were bilaterally symmetrical, but did not occur in patterns that allowed assignment of homology with the T2 and T3 groups. Altogether, En-positive neurons comprised roughly 10% of the ganglionic populations. In the bilateral groups, as in the DUM groups, En expression was restricted to interneurons, consistent with the suggestion that En expression contributes to some aspect of interneuronal phenotype. En-positive neurons in the DUM groups also expressed gamma-aminobutyric acid (GABA) immunoreactivity. Further study showed that all neurons in one En-positive bilateral group and some neurons in another bilateral group were GABA immunoreactive, but that neurons in a third bilateral group were En-positive only. Additionally, several discrete clusters of neurons were GABA-immunoreactive but En-negative. A provisional morphological scheme is presented, which relates the several neuronal clusters to their likely neuroblasts of origin, as a basis for further research into the composition of neuronal lineages.

The gene mod(mdg4) affects synapse specificity and structure in Drosophila.

The mechanisms by which synapse assembly and maturation are orchestrated during development are largely unknown. We used P-element mutagenesis and a larval anatomical screen to isolate mutants in which synapse structure was altered. Here, we describe a mutation isolated with this screen, branch point disrupted (bpd), in which both synapse specificity and synapse morphology were altered. Synaptic terminals in bpd mutants developed abnormally, forming multiple branch points, overgrowing to inappropriate neighboring muscles, and establishing aberrant folding of postsynaptic membranes. Ultrastructural characterization of synaptic boutons in bpd demonstrated abnormal layering of the postsynaptic specialization or subsynaptic reticulum (SSR). Genetic and molecular analyses revealed that bpd is an allele of mod(mdg4), a gene coding for a protein with many similarities to transcription factors, which has been implicated in the regulation of chromatin insulation. Our results suggest that mod(mdg4) may regulate a gene(s) essential to normal synapse formation.

Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system.

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity.

The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure.

The Drosophila tumor suppressor gene lethal (1) discs large (dlg) encodes a protein necessary for normal cell growth in epithelial and brain tissue. It shares high sequence identity to the mammalian synaptic proteins PSD-95 and SAP-70, whose functions are unknown. To determine the localization and role of dlg at synapses, we investigated its distribution and the effects of dlg mutations on Drosophila neuromuscular junctions. We show that dlg immunoreactivity is expressed at one type of glutamatergic synapse and is associated with both presynaptic and postsynaptic membranes. Mutations in dlg alter the expression of dlg and cause striking changes in the structure of the subsynaptic reticulum, a postsynaptic specialization at these synapses. These results indicate that dlg is required for normal synaptic structure and offer insights regarding the role of dlg homologs at vertebrate synapses.

Ultrastructure of neuromuscular junctions in Drosophila: comparison of wild type and mutants with increased excitability.

The ventral longitudinal muscles of the Drosophila larval body wall are innervated by at least four types of synaptic terminals that can be distinguished on morphological grounds at the light microscopical level. The innervation of these muscles has been previously shown to be regulated by neuronal activity. In this report we investigate the ultrastructural basis for synaptic bouton differences by using serial sections, and examine the structure of synaptic terminals in mutants with increased excitability. We report that individual identifiable muscle fibers are innervated by terminals containing two to three types of synaptic boutons that can be distinguished in terms of synaptic vesicle population, presynaptic and postsynaptic specialization, and general shape. We propose a model to account for the bouton types observed at the light microscopical level. We find that in the hyperexcitable mutant eag Sh, there are dramatic ultrastructural alterations at synaptic boutons. These alterations include a partial depletion of two types of synaptic vesicles and a change in appearance of a third type, changes in number and appearance of synaptic densities, and the presence of multivesicular bodies. Our results show that an increase in neuronal excitability produces profound effects in synaptic terminal structure.