The mechanisms of natural products for eye disorders by targeting mitochondrial dysfunction.

The human eye is susceptible to various disorders that affect its structure or function, including glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy (DR). Mitochondrial dysfunction has been identified as a critical factor in the pathogenesis and progression of eye disorders, making it a potential therapeutic target in the clinic. Natural products have been used in traditional medicine for centuries and continue to play a significant role in modern drug development and clinical therapeutics. Recently, there has been a surge in research exploring the efficacy of natural products in treating eye disorders and their underlying physiological mechanisms. This review aims to discuss the involvement of mitochondrial dysfunction in eye disorders and summarize the recent advances in the application of natural products targeting mitochondria. In addition, we describe the future perspective and challenges in the development of mitochondria-targeting natural products.

Targeted inhibition of branched-chain amino acid metabolism drives apoptosis of glioblastoma by facilitating ubiquitin degradation of Mfn2 and oxidative stress.

Glioblastoma is one of the most challenging malignancies with high aggressiveness and invasiveness and its development and progression of glioblastoma highly depends on branched-chain amino acid (BCAA) metabolism. The study aimed to investigate effects of inhibition of BCAA metabolism with cytosolic branched-chain amino acid transaminase (BCATc) Inhibitor 2 on glioblastoma, elucidate its underlying mechanisms, and explore therapeutic potential of targeting BCAA metabolism. The expression of BCATc was upregulated in glioblastoma and BCATc Inhibitor 2 precipitated apoptosis both in vivo and in vitro with the activation of Bax/Bcl2/Caspase-3/Caspase-9 axis. In addition, BCATc Inhibitor 2 promoted K63-linkage ubiquitination of mitofusin 2 (Mfn2), which subsequently caused lysosomal degradation of Mfn2, and then oxidative stress, mitochondrial fission and loss of mitochondrial membrane potential. Furthermore, BCATc Inhibitor 2 treatment resulted in metabolic reprogramming, and significant inhibition of expression of ATP5A, UQCRC2, SDHB and COX II, indicative of suppressed oxidative phosphorylation. Moreover, Mfn2 overexpression or scavenging mitochondria-originated reactive oxygen species (ROS) with mito-TEMPO ameliorated BCATc Inhibitor 2-induced oxidative stress, mitochondrial membrane potential disruption and mitochondrial fission, and abrogated the inhibitory effect of BCATc Inhibitor 2 on glioblastoma cells through PI3K/AKT/mTOR signaling. All of these findings indicate suppression of BCAA metabolism promotes glioblastoma cell apoptosis via disruption of Mfn2-mediated mitochondrial dynamics and inhibition of PI3K/AKT/mTOR pathway, and suggest that BCAA metabolism can be targeted for developing therapeutic agents to treat glioblastoma.

Natural Products in Liver Fibrosis Management: A Five-year Review.

Liver fibrosis, characterized by the overproduction of extracellular matrix proteins within liver tissue, poses a rising global health concern. However, no approved antifibrotic drugs are currently available, highlighting the critical need for understanding the molecular mechanisms of liver fibrosis. This knowledge could not only aid in developing therapies but also enable early intervention, enhance disease prediction, and improve our understanding of the interaction between various underlying conditions and the liver. Notably, natural products used in traditional medicine systems worldwide and demonstrating diverse biochemical and pharmacological activities are increasingly recognized for their potential in treating liver fibrosis. This review aims to comprehensively understand liver fibrosis, emphasizing the molecular mechanisms and advancements in exploring natural products' antifibrotic potential over the past five years. It also acknowledges the challenges in their development and seeks to underscore their potency in enhancing patient prognosis and reducing the global burden of liver disease.

Metformin protects against retinal ischemia/reperfusion injury through AMPK-mediated mitochondrial fusion.

Retinal ischemia/reperfusion (I/R) injury is a common pathological process responsible for cellular damage in glaucoma, diabetic retinopathy and hypertensive retinopathy. Metformin is a biguanide drug that exerts strong effects on multiple diseases. This study aims to evaluate the protective effect of metformin against retinal I/R injury and its underlying mechanism. I/R induced reduction in retina thickness and cell number in ganglion cell layer, and metformin alleviated I/R-induced retinal injury. Both retinal I/R and simulated ischemia/reperfusion (SIR) in R28 cells down-regulated expression of mitochondrial fusion protein Mfn2 and OPA1, which led to mitochondrial fission. Metformin also alleviated damage in R28 cells, and reversed the alteration in Mfn2 and OPA1, mitochondrial fission and mitochondrial membrane potential (MMP) disruption-induced by I/R or SIR as well. Intriguingly, inhibition of AMPK by compound C or siRNA prevented metformin-mediated up-regulation of Mfn2 and OPA1. Compound C and knockdown of Mfn2 or OPA1 dramatically alleviated the protective effect of metformin against intracellular ROS generation, MMP disruption, mitochondrial fission and loss of RGCs in ganglion cell layer induced by SIR or I/R. Moreover, scavenging mitochondrial ROS (mito-ROS) by mito-TEMPO exerted the similar protection against I/R-induced retinal injury or SIR-induced damage in R28 cells as metformin. Our data show for the first time that metformin protects against retinal I/R injury through AMPK-mediated mitochondrial fusion and the decreased mito-ROS generation. These findings might also repurpose metformin as a therapeutic agent for retinal I/R injury.

BCATc inhibitor 2 ameliorated mitochondrial dysfunction and apoptosis in oleic acid-induced non-alcoholic fatty liver disease model.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent hepatic disease in the world. Disorders of branched chain amino acid (BCAA) metabolism is involved in various diseases. In this study, we aim to explore the role of BCAA metabolism in the development of NAFLD and the protective effect of BCATc Inhibitor 2, an inhibitor of cytosolic branched chain amino acid transaminase, against NAFLD as well as its underlying mechanism. It was found that oleic acid induced lipid accumulation and apoptosis in HepG2 and LO2 cells. Supplementation of BCAAs further aggravated oleic acid-induced lipid accumulation and apoptosis. In contrast, treatment of BCATc Inhibitor 2 ameliorated oleic acid-induced lipid accumulation and apoptosis. Molecularly, supplementation of BCAAs or treatment of BCATc Inhibitor 2 up-regulated or down-regulated the expression of SREBP1 and lipogenesis-related genes without affecting lipolysis-related genes. BCATc Inhibitor 2 maintained mitochondrial function by ameliorating oleic acid-induced mitochondrial ROS generation and mitochondrial membrane potential disruption. In addition, BCATc Inhibitor 2 treatment alleviated oleic acid-induced activation of JNK and AKT signaling pathway and Bcl2/Bax/Caspase axis. In conclusion, our results indicate BCAA metabolism is involved in NAFLD and BCATc Inhibitor 2 protects against oleic acid-induced lipid accumulation and apoptosis. These findings suggest that BCATc Inhibitor 2 is a promising candidate drug for the treatment of NAFLD.

The N6-methyladenosine modification enhances ferroptosis resistance through inhibiting SLC7A11 mRNA deadenylation in hepatoblastoma.

BACKGROUND: Solute carrier family 7 member 11 (SLC7A11) is overexpressed in multiple human tumours and functions as a transporter importing cystine for glutathione biosynthesis. It promotes tumour development in part by suppressing ferroptosis, a newly identified form of cell death that plays a pivotal role in the suppression of tumorigenesis. However, the role and underlying mechanisms of SLC7A11-mediated ferroptosis in hepatoblastoma (HB) remain largely unknown. METHODS: Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting were used to measure SLC7A11 levels. Cell proliferation, colony formation, lipid reactive oxygen species (ROS), MDA concentration, 4-HNE, GSH/GSSG ratio and cell death assays as well as subcutaneous xenograft experiments were used to elucidate the effects of SLC7A11 in HB cell proliferation and ferroptosis. Furthermore, MeRIP-qPCR, dual luciferase reporter, RNA pulldown, RNA immunoprecipitation (RIP) and RACE-PAT assays were performed to elucidate the underlying mechanism through which SLC7A11 was regulated by the m6A modification in HB. RESULTS: SLC7A11 expression was highly upregulated in HB. SLC7A11 upregulation promoted HB cell proliferation in vitro and in vivo, inhibiting HB cell ferroptosis. Mechanistically, SLC7A11 mRNA exhibited abnormal METTL3-mediated m6A modification, which enhanced its stability and expression. IGF2 mRNA-binding protein 1 (IGF2BP1) was identified as the m6A reader of SLC7A11, enhancing SLC7A11 mRNA stability and expression by inhibiting SLC7A11 mRNA deadenylation in an m6A-dependent manner. Moreover, IGF2BP1 was found to block BTG2/CCR4-NOT complex recruitment via competitively binding to PABPC1, thereby suppressing SLC7A11 mRNA deadenylation. CONCLUSIONS: Our findings demonstrated that the METTL3-mediated SLC7A11 m6A modification enhances HB ferroptosis resistance. The METTL3/IGF2BP1/m6A modification promotes SLC7A11 mRNA stability and upregulates its expression by inhibiting the deadenylation process. Our study highlights a critical role of the m6A modification in SLC7A11-mediated ferroptosis, providing a potential strategy for HB therapy through blockade of the m6A-SLC7A11 axis.

Comprehensive Analysis of Glycolysis-Related Genes for Prognosis, Immune Features, and Candidate Drug Development in Colon Cancer.

The dysregulated expression of glycolysis-related genes (GRGs) is closely related to the occurrence of diverse tumors and regarded as a novel target of tumor therapy. However, the role of GRGs in colon cancer is unclear. We obtained 226 differential GRGs (DE-GRGs) from The Cancer Genome Atlas (TCGA) database. Cox regression analysis was used to construct a DE-GRG prognostic model, including P4HA1, PMM2, PGM2, PPARGC1A, PPP2CB, STC2, ENO3, and CHPF2. The model could accurately predict the overall survival rate of TCGA and GSE17536 patient cohorts. The risk score of the model was closely related to a variety of clinical traits and was an independent risk factor for prognosis. Enrichment analysis revealed the activation of a variety of glycolysis metabolism and immune-related signaling pathways in the high-risk group. High-risk patients displayed low expression of CD4+ memory resting T cells and resting dendritic cells and high expression of macrophages M0 compared with the expression levels in the low-risk patients. Furthermore, patients in the high-risk group had a higher tumor mutation load and tumor stem cell index and were less sensitive to a variety of chemotherapeutic drugs. Quantitative reverse transcription polymerase chain reaction and immunohistochemistry analyses validated the expression of eight GRGs in 43 paired clinical samples. This is the first multi-omics study on the GRGs of colon cancer. The establishment of the risk model may benefit the prognosis and drug treatment of patients.

Correction to: m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.

After the publication of this work [1], the authors noticed that the affiliations were incorrectly provided. Updated affiliation section is provided in this paper.

Choice of Treatment for Stage IA Non-small Cell Lung Cancer Patients Ineligible for Surgery: Ablation or Stereotactic Body Radiotherapy?

Purpose: To compare the survival outcomes of ablation and stereotactic body radiotherapy (SBRT) in inoperable patients with stage IA non-small cell lung cancer (NSCLC). Patients and Methods: Using the Surveillance, Epidemiology, and End Results (SEER) database, we identified 6,395 patients with stage IA NSCLC who had complete clinical information from 2004 to 2015. Kaplan-Meier analysis was performed to determine the propensity score based on the clinical characteristics of patients with stage IA NSCLC. Overall survival (OS) was compared between patients with stage IA NSCLC who were treated with ablation and SBRT after adjusting, stratifying, or matching. Results: Kaplan-Meier analysis demonstrated no significant difference in survival curves (log-rank, p>0.05) between the ablation and SBRT groups. Compared with the SBRT group, the hazard ratio (HR) (95% confidence interval [CI]) of OS was 0.930 (0.817-1.058, p=0.269) in the ablation group on univariate analysis. On multivariate analysis, similar effects on OS (HR: 0.974, 95% CI: 0.858-1.105, p=0.680) were seen in patients with stage IA NSCLC in both the groups. Conclusions: This study suggests that survival does not differ significantly between patients with stage IA NSCLC treated with ablation and SBRT. These results will be helpful for patients with stage IA NSCLC who are ineligible for surgery.

m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.

BACKGROUND: N6-Methyladenosine (m6A) modification has been implicated in many biological processes. It is important for the regulation of messenger RNA (mRNA) stability, splicing, and translation. However, its role in cancer has not been studied in detail. Here we investigated the biological role and underlying mechanism of m6A modification in hepatoblastoma (HB). METHODS: We used Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting to determine the expression of m6A related factors. And we clarified the effects of these factors on HB cells using cell proliferation assay, colony formation, apoptotic assay. Then we investigated of methyltransferase-like 13 (METTL3) and its correlation with clinicopathological features and used xenograft experiment to check METTL3 effect in vivo. m6A-Seq was used to profiled m6A transcriptome-wide in hepatoblastoma tumor tissue and normal tissue. Finally, methylated RNA immunoprecipitation (MeRIP) assay, RNA remaining assay to perform the regulator mechanism of MEETL3 on the target CTNNB1 in HB. RESULTS: In this research, we discovered that m6A modifications are increased in hepatoblastoma, and METTL3 is the main factor involved with aberrant m6A modification. We also profiled m6A across the whole transcriptome in hepatoblastoma tumor tissues and normal tissues. Our findings suggest that m6A is highly expressed in hepatoblastoma tumors. Also, m6A is enriched not only around the stop codon, but also around the coding sequence (CDS) region. Gene ontology analysis indicates that m6A mRNA methylation contributes significantly to regulate the Wnt/ß-catenin pathway. Reduced m6A methylation can lead to a decrease in expression and stability of the CTNNB1. CONCLUSION: Overall our findings suggest enhanced m6A mRNA methylation as an oncogenic mechanism in hepatoblastoma, METTL3 is significantly up-regulated in HB and promotes HB development. And identify CTNNB1 as a regulator of METTL3 guided m6A modification in HB.

Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Assessment of the role in protection and pathogenesis of the Chlamydia muridarum V-type ATP synthase subunit A (AtpA) (TC0582).

A novel Chlamydia muridarum antigen (TC0582) was used to vaccinate BALB/c mice. Mice were also immunized with other components of the ATP synthase complex (TC0580, TC0581, and TC0584), or with the major outer membrane protein (MOMP). TC0582 was also formulated in combination with TC0580, TC0581 or MOMP. TC0582 alone, or in combination with the other antigens, elicited strong Chlamydia-specific humoral and cellular immune responses. Vaccinated animals were challenged intranasally and the course of the infection was followed for 10 days. Based on percentage change in body weight, lung weight, and number of Chlamydia inclusion forming units recovered from the lungs, mice immunized with TC0582, TC0581 or MOMP, as single antigens, showed significant protection. Mice immunized with combinations of two antigens were also protected but the level of protection was not additive. TC0582 has sequence homology with the eukaryotic ATP synthase subunit A (AtpA). Therefore, to determine if immunization with TC0582, or with Chlamydia, elicited antibodies that cross-reacted with the mouse AtpA, the two proteins were printed on a microarray. Sera from mice immunized with TC0582 and/or live Chlamydia, strongly reacted with TC0582 but did not recognize the mouse AtpA. In conclusion, TC0582 may be considered as a Chlamydia vaccine candidate.

Proteomic identification of immunodominant chlamydial antigens in a mouse model.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen in the world. To identify new vaccine candidates a protein microarray was constructed by expressing the open reading frames (ORFs) from Chlamydia mouse pneumonitis (MoPn). C57BL/6, C3H/HeN and BALB/c mice were immunized either intranasally or intravaginally with live MoPn elementary bodies (EB). Two additional groups were immunized by the intramuscular plus subcutaneous routes with UV-treated EB, using CpG and Montanide as adjuvants to favor a Th1 response, or Alum, to elicit a Th2 response. Serum samples collected from the three strains of mice were tested in the microarray. The array included the expression of 909 proteins from the 921 ORFs of the MoPn genome and plasmid. A total of 530 ORFs were recognized by at least one serum sample. Of these, 36 reacted with sera from the three strains of mice immunized with live EB. These antigens included proteins that were previously described as immunogenic such as MOMP and HSP60. In addition, we uncovered new immunogens, including 11 hypothetical proteins. In summary, we have identified new immunodominant chlamydial proteins that can be tested for their ability to induce protection in animal models and subsequently in humans.

Patient satisfaction and masticatory efficiency of single implant-retained mandibular overdentures using the stud and magnetic attachments.

OBJECTIVES: To study patient satisfaction and masticatory efficiency of single implant-retained mandibular overdentures using the stud and magnetic attachments in a randomized clinical trial with a crossover design. METHODS: Patients received a single implant placed in the midline of the mandible and either a stud (Locator) or a magnetic (Magfit) attachment, assigned at random. Patient satisfaction, including patient comfort, speech, chewing ability and retention, and masticatory efficiency measured by chewing peanuts, were assessed before and 3 months after attachment insertion. Patient satisfaction and masticatory efficiency were evaluated again 3 months after insertion of the alternate attachment bodies. The outcomes were compared before and after insertion of the attachments and between the two types of attachments using Wilcoxon signed rank tests. RESULTS: Patient overall satisfaction, comfort, speech, chewing ability, and retention improved significantly after insertion of both types of attachment bodies (p<0.05). Masticatory efficiencies also increased in both the Locator and the Magfit groups (p<0.05). There were no statistically significant differences in patient overall satisfaction, comfort, speech, and retention between the two types of attachments (p>0.05). The Locator attachments performed better in perceived chewing ability than the Magfit (p<0.05), but there was no statistically significant difference in masticatory efficiency between the two attachment types (p>0.05). CONCLUSIONS: Clinical outcomes were significantly improved in single implant-retained mandibular overdentures using either the Locator or the Magfit magnetic attachments. There was no difference in masticatory efficiency between the two attachment types.

Amphipols stabilize the Chlamydia major outer membrane protein and enhance its protective ability as a vaccine.

The native major outer membrane protein (nMOMP) from Chlamydia was purified in its trimeric form using the zwitterionic detergent Z3-14. In aliquots from this preparation, Z3-14 was exchanged for amphipol (APol) A8-35. CD analysis showed that trapping with A8-35 improved the thermostability of nMOMP without affecting its secondary structure. Recombinant MOMP (rMOMP) was also formulated with Z3-14 or A8-35. Four groups of mice were vaccinated with nMOMP/Z3-14, nMOMP/A8-35, rMOMP/Z3-14 or rMOMP/A8-35 using CpG and Montanide as adjuvants. A positive control group was inoculated intranasally with live Chlamydia and a negative control group with culture medium. Mice were challenged intranasally with live Chlamydia and protection was assessed based on changes in body weight, the weight of the lungs and the number of chlamydial inclusion forming units recovered from the lungs 10 days after the challenge. Overall, vaccines formulated with nMOMP elicited better protection than those using rMOMP. Furthermore, the protection afforded by nMOMP/A8-35 was more robust than that achieved with nMOMP/Z3-14. In contrast, no differences in protection were observed between rMOMP/Z3-14 and rMOMP/A8-35 preparations. These findings suggest that the higher protection conferred by nMOMP/A8-35 complexes most likely results from a better preservation of the native structure of MOMP and/or from a more efficient presentation of the antigen to the immune system, rather than from an adjuvant effect of the amphipol. Thus, amphipols can be used in vaccine formulations to stabilize a membrane-protein component and enhance its immunogenicity.

Identification of immunodominant antigens by probing a whole Chlamydia trachomatis open reading frame proteome microarray using sera from immunized mice.

Chlamydia trachomatis infections can lead to severe chronic complications, including trachoma, ectopic pregnancy, and infertility. The only effective approach to disease control is vaccination. The goal of this work was to identify new potential vaccine candidates through a proteomics approach. We constructed a protein chip array (Antigen Discovery, Inc.) by expressing the open reading frames (ORFs) from C. trachomatis mouse pneumonitis (MoPn) genomic and plasmid DNA and tested it with serum samples from MoPn-immunized mice. Two groups of BALB/c female mice were immunized either intranasally or intravaginally with live elementary bodies (EB). Another two groups were immunized by a combination of the intramuscular and subcutaneous routes with UV-treated EB (UV-EB), using either CpG and Montanide as adjuvants to favor a Th1 response or alum to elicit a Th2 response. Serum samples collected at regular intervals postimmunization were tested in the proteome array. The microarray included the expression products of 909 proteins from a total of 921 ORFs of the Chlamydia MoPn genome and plasmid. A total of 185 immunodominant proteins elicited an early and sustained antibody response in the mice immunized with live EB, and of these, 71 were also recognized by the sera from mice immunized with UV-EB. The reactive antigens included some proteins that were previously described as immunogenic, such as the major outer membrane protein, OmpB, Hsp60, and IncA and proteins from the type III secretion system. In addition, we identified in mice several new immunogens, including 75 hypothetical proteins. In summary, we have identified a new group of immunodominant chlamydial proteins that can be tested for their ability to induce protection.

Protection against an intranasal challenge by vaccines formulated with native and recombinant preparations of the Chlamydia trachomatis major outer membrane protein.

To compare the ability of a native and a recombinant preparation of the major outer membrane protein of Chlamydia trachomatis mouse pneumonitis (MoPn; Ct-nMOMP and Ct-rMOMP) to protect against an intranasal (i.n.) challenge, BALB/c mice were vaccinated by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-1826 and Montanide ISA 720 as adjuvants. Animals inoculated i.n. with live elementary bodies (EB) of Chlamydia served as a positive control. Negative control groups were immunized with either Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or with minimal essential medium (MEM-0). Mice immunized with Ct-rMOMP, Ct-nMOMP and EB developed a strong immune response as shown by high levels of Chlamydia specific antibodies in serum and a strong T-cell lymphoproliferative response. Following the i.n. challenge with 10(4) inclusion forming units (IFU) of C. trachomatis, mice immunized with Ct-nMOMP or Ct-rMOMP lost significantly less weight than the negative control animals immunized with Ng-rPorB or MEM-0 (P<0.05). However, mice vaccinated with the Ct-nMOMP lost less weight than those immunized with the Ct-rMOMP (P<0.05). Mice were euthanized at 10 days following the challenge, their lungs weighed and the number of IFU of Chlamydia determined. Based on the lung weight and number of IFU recovered, significant protection was observed in the groups of mice immunized with both Ct-nMOMP and the Ct-rMOMP (P<0.05). Nevertheless, significantly better protection was achieved with the Ct-nMOMP in comparison with the Ct-rMOMP (P<0.05). In conclusion, vaccination with a preparation of the nMOMP elicited a more robust protection than immunization with rMOMP, suggesting that the conformational structure of MOMP is critical for inducing strong protection.

Structural and functional analyses of the major outer membrane protein of Chlamydia trachomatis.

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of beta-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37 degrees C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a beta-sheet structure and porin function.

Association of the GLI gene with ventricular septal defect after the susceptibility gene being narrowed to 3.56 cM in 12q13.

BACKGROUND: Our previous research has suggested that genes around D12S1056 in 12q13 may confer susceptibility to ventricular septal defect (VSD) in humans. The present study was to define the chromosome region assignment by transmission disequilibrium test (TDT), and to identify the important candidate gene by family-based association study and haplotype analysis. METHODS: Surrounding D12S1056, ten microsatellite markers including D12S329, D12S305, D12S1662, D12S1056, D12S1293, D12S334, D12S102, D12S83, D12S1655 and D12S1691 were chosen, and TDT was performed in 62 nuclear family trios each consisting of an affected child and two healty parents. Subsequently, the GLI gene, a positional candidate gene that maps to the target region, was selected for further analysis. Three single nucleotide polymorphisms (SNPs), G11888C, G11388A, and G11625T, were selected for family-based association study and haplotype analysis. RESULTS: VSD was significantly associated with all selected markers except D12S1691 [72.2 centi morgen (cM)] and D12S1700 (75.76 cM). VSD was also significantly associated with G11888C (chi(2) = 5.918, P = 0.015), G11388A (chi(2) = 8.067, P = 0.005), and G11625T (chi(2) = 11.842, P = 0.001). Haplotype analysis showed a strong linkage disequilibrium between G11888C and G11388A (D' = 0.999), but in significant (chi(2) = 1.035, df = 2, P > 0.05). CONCLUSIONS: The susceptibility gene of VSD was mapped to 3.56 cM in 12q13 by TDT, and the GLI gene, an important candidate in the target region, was associated with VSD.

TBX5, a gene mutated in Holt-Oram syndrome, is regulated through a GC box and T-box binding elements (TBEs).

TBX5 is a member of the T-box gene family and encodes a transcription factor that regulates the expression of other gene(s) in the developing heart and limbs. Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant condition characterized by congenital heart defects and limb anomalies. How TBX5 gene expression is regulated is still largely unknown. In order to identify transcription factors regulating TBX5 expression, we examined the 5'-flanking region of the human TBX5 gene. We determined that up to 300 bp of the 5'-flanking region of the TBX5 gene was necessary for promoter activity in mouse cardiomyocyte ECL2 cells. One GC box, three potential T-box-like binding elements (TBE-A, -B, and -C), and one NKX2.5 binding site were identified. Site-directed mutagenesis of the potential binding sites revealed that the GC box, TBE-B, TBE-C, and NKX2.5 are functionally positive for the expression of TBX5. DNA footprint analysis showed that these binding regions are resistant to DNaseI digestion. Electrophoretic mobility shift assays (EMSAs) further demonstrated the protein-DNA interactions at the GC box and the potential TBE-B, TBE-C, and NKX2.5 sites in a sequence-specific manner. The ability of TBX5 to regulate its own promoter was demonstrated by the ability of ectopically expressed human TBX5 to increase reporter expression. We conclude that the GC box, T-box-like binding elements, and NKX2.5 binding site play important roles in the regulation of TBX5 expression, and that TBX5 is likely to be autoregulated as part of the mechanism of its transcription.