Role of interferon alpha-inducible protein 6 in modulating the proliferation, apoptosis and senescence of oesophageal squamous cell carcinoma cells.

Oesophageal cancer is one of the most malignant tumors worldwide. Dysfunction of interferon alpha-inducible protein 6 (IFI6) has been implicated in numerous human diseases, including cancer. We performed the study to investigate the function and potential molecular pathways of IFI6 in oesophageal squamous cell carcinoma (ESCC) cells. IFI6 expression was analysed using databases-derived data and paraffin-embedded tissue samples. CCK-8-based analyses and EdU staining, colony formation, ß-galactosidase staining and Annexin V/PI double-staining assays were used to determine the influence of IFI6 on cell growth, senescence and apoptosis. Tumor growth in vivo was investigated in mouse xenograft models. RNA sequencing (RNA-seq) was performed to identify the transcripts and pathways affected by IFI6. The results showed that IFI6 expression was elevated in ESCC and correlated with poor clinical prognosis (P<0.05). IFI6 was overexpressed and silenced in TE-1 and TE-10 cells using lentiviruses. Upregulation of IFI6 promoted cell growth both in vitro and in vivo, whereas downregulation induced opposite effects. IFI6 overexpression inhibited cell senescence and apoptosis but did not influence cell cycle progression, while IFI6 downregulation increased cell senescence and apoptosis. RNA-seq revealed that 3 mRNAs (EPHA5, CLIP1 and GTF2F2) were consistently associated with both IFI6 overexpression and silencing. IFI6 appeared to modulate TE-1 cells via complex mechanisms. In conclusion, IFI6 plays a positive role in the proliferation of ESCC cells both in vitro and in vivo, which could be a novel therapeutic target for treating ESCC.

[Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses].

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

Efficient electrochemical generation of ferrate(VI) by iron coil anode imposed with square alternating current and treatment of antibiotics.

Anode passivation is still a main challenge for the electrochemical generation of ferrate(VI, Fe(VI)), leading to the reduction of Fe(VI) production efficiency. In this study, cyclic voltammetry, scanning electronic microscopy, and electrochemical impedance spectroscopy were used to select better anode electrode configurations (iron wire, iron gauze, and iron coil). The results indicate that iron coil had the least degree of passivation. Different imposed current waveforms during the electrochemical generation of Fe(VI) were also investigated, and the iron coil imposed with square alternating current (AC) wave can mitigate the anode passivation, resulting in higher Fe(VI) production efficiency. The optimum conditions for the electrochemical generation of Fe(VI) were evaluated and the optimum temperature (40 ℃), current density (10 mA/cm2), AC cycle period (15 s) and electrolyte concentrations (14 M NaOH) were identified. As a result, 0.12 mol/L Fe(VI) concentration and over 50% of current efficiency can be achieved after 3 h electrolysis. The generated Fe(VI) solution was further applied to oxidize doxycycline(DOX) and sulfadiazine(SDZ) as typical antibiotics. Over 80% of DOX can be removed at a Fe(VI) to DOX molar ratio of 5:1 (pH = 4-9), whilst a higher Fe(VI) to SDZ molar ratio of 20:1 (pH = 7) was needed to obtain 75% SDZ removal.

Krebs von den Lungen-6 associated with chest high-resolution CT score in evaluation severity of patients with interstitial lung disease.

AIMS: The purpose of this prospective, single-center study was to measure the value of Krebs von den Lungen-6 (KL-6), a kind of transmembrane mucoprotein, in diagnosing interstitial lung disease (ILD) and in assessing the severity of ILD. METHODS: We enrolled 184 patients and 30 healthy controls. Ninety-eight patients were diagnosed with ILD, 47 with pneumonia, 19 with non-small cell lung cancer without ILD (NSCLC/non-ILD) and 20 with other lung diseases. Serum KL-6 levels, CT scores of high-resolution computerised tomography (HRCT) and pulmonary function in ILD patients were assessed. RESULTS: The mean value of serum KL-6 in patients with ILD, pneumonia, NSCLC/non-ILD, other lung diseases and healthy controls were 1000.67±882.73U/ml, 234.11±91.02U/ml, 269.95±149.23U/ml, 234.85±83.51U/ml and 189.03±55.50U/ml, respectively. Serum KL-6 levels of patients with ILD were significantly higher than that of other groups (P<0.000). The level of serum KL-6 in patients with pneumonia, NSCLC/non-ILD and other lung diseases was also statistically higher than healthy controls (P<0.05). When the cut-off value was 312U/ml, the sensitivity and specificity of KL-6 for the diagnosis of ILD was 84.7% and 85.3% respectively (AUC: 0.936, 95% CI: 0.906-0.965). The serum KL-6 levels in patients with ILD were significantly positively correlated with the CT scores (r=0.539, P=0.000) and negatively correlated with DLCO (r=-0.513, P=0.000). CONCLUSION: Serum KL-6 might be useful in the diagnosis of ILD, especially in the hard-to-diagnose cases, with high sensitivity and specificity. Furthermore, KL-6 might be a valuable marker for evaluation of ILD severity.

EGFL7 silencing inactivates the Notch signaling pathway; enhancing cell apoptosis and suppressing cell proliferation in human cutaneous melanoma.

Melanoma is the main cause of death in patients with skin cancer. While the pathogenesis of cutaneous melanoma is poorly understood, increasing evidence shows that epidermal growth factor (EGF) may be involved. Herein, we tested the hypothesis that down-regulation of EGFL7 inhibits development and progression of human cutaneous melanoma (CM). Initially, we performed immunohistochemical analysis of EGFL7 in 130 specimens and the findings indicated that EGFL7 was highly expressed in CM. The expressions of EGFL7 and Notch signaling pathway-related genes in CM were then measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot assay. In order to assess biological functions of EGFL7 in CM we up-regulated or down-regulated endogenous EGFL7 using EGFL7-OE or shRNA against EGFL7 in the A375 CM cell line. To better understand the pivotal role of Notch signaling pathway in CM, we blocked this pathway in A375 cells by inhibitor treatment. Finally, tumor xenograft in nude mice was performed to test the in vivo tumorigenesis of the transfected A375 cells. While EGFL7 activated the Notch signaling pathway in CM, gain- and loss-of-function studies established that decreased EGFL7 inhibited cell proliferation and promoted apoptosis in A375 cells. Moreover, down-regulated EGFL7 suppressed in vivo tumorigenesis. Most importantly, we determined that down-regulating EGFL7 inhibited CM development by suppressing the Notch signaling pathway. The combined findings define potential roles of decreased EGFL7 as inhibitors of CM development by suppressing the Notch signaling pathway, and EGFL7 may therefore be a novel therapeutic target in cutaneous melanoma patients.

[Preparation of chaperone-antigen peptide vaccine derived from human gastric cancer stem cells and its immune function].

Objective: To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function. Methods: Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes. Results: The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 µg and 1.00 µg HSP70-antigen peptide and 1.00 µg HSP90-antigen peptide activated lymphocytes significantly. Their A(490) values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 µg HSP70-antigen peptide and 1.00 µg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 µg HSP90-antigen peptide and 1.00 µg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048). Conclusions: The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.

Reversal of cisplatin resistance in non-small cell lung cancer stem cells by Taxus chinensis var.

Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR. The molecular basis of T. chinensis var.-mediated drug resistance was established by real-time PCR analysis of ABCC1, ABCB1, and lung resistance-related protein (LRP) mRNA, and western blot analysis of MRP1, MDR1, and LRP. Our results revealed that stem cells treated with higher doses of T. chinensis var. showed significantly lower growth inhibition rates than did H460 cells (P < 0.05). The growth of stem and H460 cells treated with a combination of T. chinensis var. and cisplatin was also significantly inhibited (P < 0.05). Rh-123 was significantly accumulated in the intracellular region and showed delayed efflux in stem cells treated with T. chinensis var. (P < 0.05), compared to those treated with verapamil. T. chinensis var.-treated stem cells showed significant downregulation of the ABCC1, ABCB1, and LRP mRNA and MRP1, MDR1, and LRP (P < 0.05) compared to H460 cells. Thus, T. chinensis var.-mediated downregulation of MRP1, MDR1, and LRP might contribute to the reversal of drug resistance in non-small cell lung cancer stem cells.

[The expression of beta-tubulin gene in myelodysplastic syndrome evoluting to leukemia].

OBJECTIVE: Based on our previous established cohort of myelodysplastic syndrome (MDS), we investigated the potential effect of beta-tubulin(TUBB) gene in the transformation of MDS into acute leukemia. METHODS: From our nested case-control study cohort of MDS patients, we chose 11 paired transformed and non-transformed MDS patients. TUBB gene expression was tested by quantitative real-time PCR. TUBB-siRNA transfection was used to down-regulate TUBB gene expression in SKM-1 cell line. The function of TUBB gene in SKM-1 cell line was evaluated by cell proliferation, soft agar clone formation and electron microscope. RESULTS: TUBB gene expression in MDS patients in transformed group were significantly higher than that in control group (2.91±0.41 vs 0.90±0.23, P<0.01). After TUBB-siRNA transfection, A450/630nm of SKM-1 cells at 24 h, 48 h and 72 h were 0.299±0.045, 0.526±0.034 and 0.652±0.035, respectively, which were significantly decreased than those in negative-siRNA group(0.438±0.074, 0.858±0.064 and 0.974±0.044)(P<0.05). Soft agar clone formation in TUBB-siRNA group was (7.0±0.2)%, which was significantly reduced than that of negative-siRNA group (25.0±0.2)% (P<0.01). Electron microscope showed significant apoptotic signs in TUBB-siRNA group, including vacuoles in cytoplasm and karyorrhexis. CONCLUSION: Our results indicate that TUBB gene may play a role in the transformation of MDS into acute leukemia by affecting the proliferation of malignant clones.

Association of Resting Heart Rate with Infrarenal Aortic Diameter: A Cross Sectional Study in Chinese Hypertensive Adults.

OBJECTIVE: Elevated resting heart rate (RHR) has been shown to be a risk marker for cardiovascular disease. Results from studies on the effects of RHR in large arteries are limited to the functional changes of those arteries, while the association between RHR and aortic diameter remains largely understudied. METHODS: This was a cross sectional study of hypertensive Chinese adults from rural areas. The maximum infrarenal aortic diameter (maxIAD) from renal arteries to the iliac bifurcation was obtained by ultrasound. MaxIADs in different RHR groups were compared in males and females separately because of the significant differences between sexes. Multiple regression analysis was used to determinate the correlation between RHR and maxIAD. Further interactions between three factors (BMI, smoking, and anti-hypertensive regimens) and RHR for maxIAD were examined using subgroup analysis. RESULTS: 19,200 subjects were enrolled in the study, with an average age of 64.8±7.4 years and 61.6% females. Only 22 cases (0.11%) were detected with AAA, with males (n = 17) presenting a higher AAA incidence than females (n = 5). In subjects ≥65 years, there were 18 (0.19%) AAA, and 15 (83.3%) had a history of smoking. In the total subjects, the mean maxIAD ranged from 15.7±2.1 mm to 15.2±2.2 mm as RHR changed from the lowest quartile to the highest (≥84 bpm) in males, with a similar tendency observed in females. The correlation coefficient of RHR on maxIAD was -0.17 in males and -0.12 in females. Further subgroup analysis revealed that smoking exaggerated the correlation between RHR and maxIAD, but only in females. CONCLUSIONS: A low AAA incidence was observed in this hypertensive Chinese population. There was a negative association between RHR and maxIAD, potentially exaggerated by smoking, especially in females.

Three-dimensional structure of Bax-mediated pores in membrane bilayers.

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ~3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.

Repeated intrathecal administration of ropivacaine causes neurotoxicity in rats.

Previous studies have shown that ropivacaine is the least neurotoxic local anaesthetic. Most of the data derive from short-term ropivacaine injection into the subarachnoid space. Intrathecal administration for a prolonged period, and the histological changes and behavioural effects of repeated intrathecal administration, have not previously been investigated. We studied the possible neurotoxicity of intrathecal injection of ropivacaine in a rat model. Rats received 0.12 ml/kg body weight of ropivacaine at concentrations of 0.5 or 1%, or normal saline only, via an implanted intrathecal catheter at 90-minute intervals for 12 hours. On days 1, 3, 5, 7, 14 and 28, the spinal cord was examined by light and electron microscopy at the L3 level. We assessed sensory thresholds to noxious stimulation, behavioural change and protein kinase B immunoreactivity for possible neuronal injury within the spinal cord. Ropivacaine 1% induced thermal hyperalgesia and mechanical allodynia, neuronal injury characterised by tissue oedema, proliferation of glial cells, neuronal morphology changes and degeneration and protein kinase B expression. There were no significant differences in motor function as a result of different concentrations of ropivacaine. Repeated intrathecal injection of ropivacaine 1% can induce neurotoxicity in rats. Our data suggests that expression of protein kinase B might be involved in this neurotoxicity.

Prevalence and prognostic significance of FLT3 gene mutations in patients with acute leukaemia: analysis of patients from the Shanghai Leukaemia Co-operative Group.

This study was designed to evaluate the prevalence of fms-like tyrosine kinase-3 (FLT3) gene mutations in the World Health Organization classified subtypes of acute leukaemia (AL), and their prognostic significance in terms of complete remission (CR), leukaemia-free survival (LFS) and overall survival (OS). Of 468 patients, 374 (79.9%) had acute myeloid leukaemia (AML) and 83 (17.7%) had acute lymphoblastic leukaemia (ALL). Among the AML patients, a FLT3 internal tandem duplication (FLT3/ITD) mutation was present in 59 cases (15.8%), whereas a FLT3/D835 mutation was detected in 15 cases (4.0%). Conversely, in the ALL patients, no FLT3/ITD mutations were detected and a FLT3/D835 mutation was found in only two cases (2.4%). The FLT3/ITD mutation was associated with a lower CR rate compared with those with no mutations (52.3% versus 71.1%) and with a shorter median OS (9 versus 18 months) in AML patients. In conclusion, the FLT3/ITD mutation occurred frequently in AML and was associated with a lower CR and shorter median OS. In contrast, FLT3/D835 mutations were not of prognostic value.

Effect of therapeutic immunization using Ad5/35 and MVA vectors on SIV infection of rhesus monkeys undergoing antiretroviral therapy.

Antiretroviral therapy (ART) effectively slows the progression of AIDS. However, drug resistance and/or toxicity can limit the utility of ART in many patients. In this study, we assessed whether a viral vector-based vaccine can be used as a therapeutic vaccine in simian immunodeficiency virus (SIV)-infected monkeys. The effect of vaccinating SIVmac239-infected rhesus monkeys with an SIV gag and gp120-expressing adenovirus (Ad) vector vaccine and a modified vaccinia Ankara (MVA) vaccine was explored while being treated with ART. Rhesus monkeys were intravenously infected with 10 and 1000 TCID(50) (50% tissue culture infectious dose) of SIVmac239. Two months after SIV infection, the monkeys received a 4-month treatment with ART. Some of the monkeys were immunized with adenovirus-based vaccine and MVA-based vaccine with 2 months interval during ART. Viral load, CD4 count and SIV-specific immune responses were observed for 7 months after interruption of ART. The vaccinated animals had higher (i) CD4 counts, (ii) SIV-specific cell-mediated immune responses and (iii) anti-SIV-neutralizing antibody (Ab) titers than monkeys treated with ART alone. More importantly, the vaccination significantly reduced the SIV RNA load from animals infected with a low dose of SIV (10 TCID(50)). The anti-SIV cell-mediated and humoral responses induced by the vaccination was inversely correlated with a reduction in SIV viral load and positively correlated with an increase in CD4(+) T cell counts. These results suggest that vaccination can improve antiviral cell-mediated and humoral immunity, which may contribute to controlling viral replication.

Intranasal delivery of T-bet modulates the profile of helper T cell immune responses in experimental asthma.

BACKGROUND: Allergic asthma is caused by aberrant helper T (T(H)) type 2 immune responses in susceptible individuals, characterized by airway hyperresponsiveness, chronic airway inflammation, and mucus hypersecretion. Its prevalence continues to increase, but optimal treatment remains a challenge. The transcription factor T-bet is a master regulator of T(H)1 lineage commitment and strongly promotes interferon gamma expression during T(H)1 cell differentiation. OBJECTIVE: The aim of this study was to explore the role of intranasal delivery of T-bet on the differentiation of T(H) cell subsets and airway inflammation in the ovalbumin (OVA)-induced mouse model of allergic airway inflammation. METHODS: BALB/c mice were sensitized by intraperitoneal injection of OVA and challenged with nebulized OVA. Four days before the inhalation challenge, the sensitized mice were subjected to intranasal delivery of a recombinant adeno-associated virus vector carrying murine T-bet gene (AAV-T-bet). Expression of the transcription factors T-bet, GATA3, and Foxp3 was then assayed in the lungs, and airway histology was analyzed along with other inflammatory parameters, such as eosinophils and cytokines in bronchoalveolar lavage (BAL) fluid, and total and OVA-specific immunoglobulin (Ig) E in serum. RESULTS: Intranasal administration of AAV-T-bet efficiently balanced the T(H)1/T(H)2 transcription factor and cytokine profile and significantly decreased the number of eosinophils in BAL fluid. It also resulted in a reduction of peribronchial inflammation scores and serum IgE levels in OVA-sensitized and challenged mice during the effector phase. CONCLUSIONS: Our data show that intranasal delivery of T-bet can promote a T(H)1 immune response, restore a balanced Th immune response, and inhibit airway inflammation during the challenge phase in a mouse model of allergic airway inflammation.

Platelet-induced expression of tissue factor procoagulant activity in freshly isolated human mononuclear cells: implications for experimental use.

Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte-platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF-PCA) was quantitated by a one-stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P-Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF-PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 +/- 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 +/- 17% (P < 0.001). After isolation, 52 +/- 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62-positive monocytes (CD14+ particles) increased from baseline 5% to 13 +/- 6% in ADP-stimulated samples to 53 +/- 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF-PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet-monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.

Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL.

BACKGROUND: Inflammation is implicated in atherogenesis and plaque disruption. Toll-like receptor 2 (TLR-2) and TLR-4, a human homologue of drosophila Toll, play an important role in the innate and inflammatory signaling responses to microbial agents. To investigate a potential role of these receptors in atherosclerosis, we assessed the expression of TLR-2 and TLR-4 in murine and human atherosclerotic plaques. METHODS AND RESULTS: Aortic root lesions of high-fat diet-fed apoE-deficient mice (n=5) and human coronary atherosclerotic plaques (n=9) obtained at autopsy were examined for TLR-4 and TLR-2 expression by immunohistochemistry. Aortic atherosclerotic lesions in all apoE-deficient mice expressed TLR-4, whereas aortic tissue obtained from control C57BL/6J mice showed no TLR-4 expression. All 5 lipid-rich human plaques expressed TRL-4, whereas the 4 fibrous plaques and 4 normal human arteries showed no or minimal expression. Serial sections and double immunostaining showed TLR-4 colocalizing with macrophages both in murine atherosclerotic lesions and at the shoulder region of human coronary artery plaques. In contrast to TLR-4, none of the plaques expressed TLR-2. Furthermore, basal TLR-4 mRNA expression by human monocyte-derived macrophages was upregulated by ox-LDL in vitro. CONCLUSIONS: Our study demonstrates that TLR-4 is preferentially expressed by macrophages in murine and human lipid-rich atherosclerotic lesions, where it may play a role to enhance and sustain the innate immune and inflammatory responses. Moreover, upregulation of TLR-4 in macrophages by oxidized LDL suggests that TLR-4 may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis.

Automated prediction of 15N, 13Calpha, 13Cbeta and 13C' chemical shifts in proteins using a density functional database.

A database of peptide chemical shifts, computed at the density functional level, has been used to develop an algorithm for prediction of 15N and 13C shifts in proteins from their structure; the method is incorporated into a program called SHIFTS (version 4.0). The database was built from the calculated chemical shift patterns of 1335 peptides whose backbone torsion angles are limited to areas of the Ramachandran map around helical and sheet configurations. For each tripeptide in these regions of regular secondary structure (which constitute about 40% of residues in globular proteins) SHIFTS also consults the database for information about sidechain torsion angle effects for the residue of interest and for the preceding residue, and estimates hydrogen bonding effects through an empirical formula that is also based on density functional calculations on peptides. The program optionally searches for alternate side-chain torsion angles that could significantly improve agreement between calculated and observed shifts. The application of the program on 20 proteins shows good consistency with experimental data, with correlation coefficients of 0.92, 0.98, 0.99 and 0.90 and r.m.s. deviations of 1.94, 0.97, 1.05, and 1.08 ppm for 15N, 13Calpha, 13Cbeta and 13C', respectively. Reference shifts fit to protein data are in good agreement with 'random-coil' values derived from experimental measurements on peptides. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.

The role of cellular trace elements in oral carcinogenesis induced by 4-nitroquinoline 1-oxide(4NQO).

The aim of this study was to define the changes of cellular trace element concentration during the carcinogenesis process of Wistar rat palatine mucosa squamous epithelial cell induced by 4-nitroquinoline-1-oxide (4NQO). 4NQO was painted three times weekly for nineteen weeks on the palatine mucosae of 28 Wistar rats. Histologically normal, precancerous and squamous epithelial cell carcinoma tissues were obtained, and were studied by electron probe X-ray microanalysis. The measured elements were copper (Cu), zinc (Zn), selenium (Se), and molybdenum (Mo). The results were that both copper and zinc in the cellular nucleus and cytoplasm of the squamous epithelial carcinoma cells were significantly decreased. The concentration of cytoplasmic molybdenum significantly decreased in precancerous cells and significantly increased in squamous epithelial carcinoma cells. Minor changes in the concentration of selenium were observed in the process of normal to precancerous and then to cancerous cells. Cu/Zn increased in squamous epithelial carcinoma cells and Cu/Se and Zn/Se decreased in squamous epithelial carcinoma cells. These results suggest that the changes in intracellular copper, zinc, molybdenum are distinctly related to experimental oral carcinogenesis.

[The electron probe microanalysis on oral precancer treated by garlic].

OBJECTIVE: To evaluate the effect of 4 kinds of trace elements on experimental oral precancer treated by garlic. METHODS: The palatal mucosae of 42 Wistar rats were painted with 0.5% of 4-nitroquinololine-1-oxide(4NQO) three times weekly for 7 weeks by coating method. Then the animals were divided randomly into two groups. The treatment group was treated three times weekly with garlic solution at the posterior hard palatal mucosae by coating method, and in the control group, the vehicle-distilled water was used instead of garlic solution. At the 5th and 8th weeks of the treatment and the 7th week after the treatment was stopped, some animals were killed. The palatal epithelial cells were prepared and surveyed by electron probe microanalysis. RESULTS: During the treating period, garlic improved the levels of epithelial cells' nuclei copper, selenium, molybdenum and extranuclei selenium, molybdenum(P < 0.01), but it decreased the contents of epithelial cells' extranuclei copper and extra- and intranuclei zinc(P < 0.01). CONCLUSIONS: Garlic can treat the oral precancer by improving the levels of epithelial cells' nuclei copper, selenium, and molybdenum and extranuclei selenium and molybdenum.

[Study on trace elements in epithelial cells during oral carcinogenesis prevented by garlic].

Sixty-two Wistar rats were divided randomly into two groups, thirty-one for each group. The posterior hard palatal mucosae of all animals were painted thrice weekly with 0.5% 4-nitroquinoline 1-oxide(dissolved in dimethyl sulfoxide). Before that, the garlic injection solution and the distilled water were painted at the same place of the experimental and control group animals, respectively. All animals were killed in turn from the beginning of the experiment at random at the 10th, 13th, and 19th week. Then, trace elements of intranuclear and cytoplasm of epithelial cells or cancer cells at the mentioned weeks were surveyed by electron probe microanalysis. The results were that garlic decreased the levels of intranuclear and cytoplasm copper(P < 0.05); the levels of intranuclear and cytoplasm selenium at the 10th week and the 13th week(P < 0.05) and those of zinc at the 19th week (P < 0.01) increased. So, garlic inhibits oral carcinogenesis by changing concentrations of intranuclear and cytoplasm trace elements that is copper, zinc, selenium, and the ratio of the three elements.