[circ_BACH2 affects the malignant biological behavior of papillary thyroid cancer by regulating miR-370-3p].

Objective: To investigate the influence of circ_BACH2 on the malignant biological behavior of papillary thyroid cancer and its molecular mechanism. Methods: Cancer tissues and paracancer tissues of 51 patients with papillary thyroid carcinoma from the Fourth Central Hospital of Tianjin between 2017 and 2019 were collected. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_BACH2, miR-370-3p and G protein coupled receptor kinase interacting factor 1 (GIT1) mRNA in tissues and cells; flow cytometry to detect cell apoptosis and cell cycle; plate clone formation experiment to detect the number of cell clones; cell counting kit 8 (CCK-8) to detect cell proliferation; Transwell array to detect cell migration and invasion; western blot to detect protein expressions; dual luciferase report experiment to detect the targeting relationship between circ_BACH2, miR-370-3p and GIT1; the nude mouse tumor formation experiment to detect the effect of circ_BACH2 on tumors in mice. Results: Compared with adjacent tissues, the expressions of circ_BACH2 and GIT1 in papillary thyroid cancer tissues was increased, while the expression of miR-370-3p was decreased. Compared with Nthy-ori3-1 cells, the expressions of circ_BACH2 in papillary thyroid cancer cells TPC-1 and SW579 were increased, the mRNA and protein levels of GIT1 were increased, miR-370-3p expression was decreased. The expression level of GIT1 mRNA was negatively correlated with that of miR-370-3p (r=-0.634), and the expression level of circ_BACH2 was positively correlated with that of GIT1 (r=0.635). The expression level of circ_BACH2 was negatively correlated with that of miR-370-3p (r=-0.394, P＜0.05). Circ_BACH2 and miR-370-3p has a binding site at the 3' UTR of GIT1. After knocking down circ_BACH2, the proportion of G0/G1 cells in papillary thyroid cancer cells TPC-1 and SW579 was increased, the proportion of S-phase cells was decreased and the proportion of G2/M-phase cells did not change significantly. The cell absorbance value was lower than that in si-NC group. The number of cell clone formation was decreased (43±5 vs 100±6, 54±8 vs 100±9); the cell apoptosis rate was increased [(19.60±2.40)% vs (4.30±0.20)%, (18.10±2.10)% vs (5.10±0.23)%]; cell migration number was decreased (61±7 vs 134±15, 58±6 vs 112±11), the invasion number was also decreased (45±6 vs 113±11, 47±4 vs 92±9); the expressions of Snail and Twist1 were decreased, and the expression of E-cadherin was increased (P＜0.000). Inhibition of miR-370-3p expression reversed the effect of circ_BACH2 knockdown on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Overexpression of GIT1 reversed the effects of overexpression of miR-370-3p on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Mice injected with TPC-1 cells stably transfected with sh-circ_BACH2 showed a reduction in tumor volume [(535±91) mm3 vs (857±114) mm3] after 35 days of culture; tumor weight was decreased [(0.62±0.13) mg vs (1.06±0.15) mg, P＜0.05]; the expressions of circ_BACH2 and GIT1 were decreased, and the expression of miR-370-3p was increased in nude mouse tumor tissue. Conclusion: Silencing circ_BACH2 may inhibit the proliferation, migration and invasion of papillary thyroid cancer cells in vitro, promote cell apoptosis, and inhibit tumor growth in vivo through targeted regulation of miR-370-3p/GIT1.

[Research progress of sarcopenic obesity and chronic liver disease].

Sarcopenia has attracted increasing attention with the study of nutrition in patients with liver disease. Sarcopenia is an independent risk factor for a poor prognosis of liver disease and is becoming increasingly common in patients with liver disease. Studies have shown that patients with liver disease and sarcopenic obesity have a worse prognosis than patients with liver disease and simple sarcopenia or obesity. In clinical practice, it is easy to recognize patients with malnutrition and decreased muscle mass, but we often ignore those patients with normal body weight or even obesity who will likewise experience muscle mass loss. Simply relying on the monitoring of body mass and body mass index to assess the nutritional and muscle status of patients with liver disease is not accurate. At present, our understanding of the relationship between chronic liver disease and sarcopenic obesity is still poorly understood. In this paper, the research progress on chronic liver disease, sarcopenia, and sarcopenic obesity in recent years is reviewed so as to provide a theoretical basis for improving the clinical prognosis of patients with liver disease.

[Graphite furnace atomic absorption spectrometry for the determination of trace gallium in whole blood].

Objective: To establish a graphite furnace atomic absorption spectrometry method for the determination of trace gallium in whole blood. Methods: From January to May 2021, the five factors of ashing temperature, ashing time, atomization temperature, atomization time and matrix modifier concentration in the determination of gallium in whole blood by graphite furnace atomic absorption spectrometry were optimized by using L(16) (4(5)) orthogonal test design. At the same time, within-run, between-run, spiking recovery test and other methodological indicators were tested. Results: Under the optimized detection conditions, the linear range of determination of gallium in whole blood by graphite furnace atomic absorption spectrometry was 0.29-100.00 µg/L (r=0.9991) . The within-run and between-run relative standard deviations (RSD) of repetitive measurement at 10.0, 50.0, 80.0 µg/L concentration levels were 2.3%-4.4% and 1.5%-3.6%, the recovery rate of spiking was 98.1%-103.8%, and the detection limit of the method was 0.13 µg/L. Conclusion: Graphite furnace atomic absorption spectrometry for the determination of trace gallium in whole blood is easy to operate, has a wide linear range, low detection limit, accurate and reliable results, which is suitable for occupational health examinations and the determination of acute gallium poisoning.

FFAR4 promotes cell proliferation and migration and servers as a potential biomarker for clinicopathological characteristics and prognosis in laryngocarcinoma.

OBJECTIVE: The poor prognosis of advanced laryngocarcinoma was associated with the epithelial-mesenchymal transformation (EMT), which was related to the dysregulated expression of free fatty acids receptor 4 (FFAR4). By detecting the expression of FFAR4 in laryngocarcinoma and its relation with the clinicopathological characteristics and prognosis of laryngocarcinoma, as well as conducting in vitro experiments, our aim is to explore the role of FFAR4 in laryngocarcinoma biological and clinical process. PATIENTS AND METHODS: The protein expression level of FFAR4 in 54 cases of laryngocarcinoma and 30 cases of laryngocarcinoma adjacent tissues was detected by immunohistochemistry. Combined with clinical follow-up data, the Kaplan-Meier survival curve and log-rank test were conducted to compare the relation between the expression of FFAR4, the clinicopathological characteristics, and the 5-year survival rate in laryngocarcinoma. Multivariable Cox regression analysis revealed the independent predictors for the prognosis of laryngocarcinoma. CCK-8 and migration assay were used to test cell proliferation and migration abilities. RESULTS: FFAR4 was upregulated in laryngocarcinoma tissues and influenced cell proliferation and migration abilities. The FFAR4 expression was related to the age and lymph node metastasis in laryngocarcinoma patients and indicated a reduced 5-year survival rate and increased lymph node metastasis. CONCLUSIONS: The upregulation FFAR4 expression was associated with the lymph node metastasis and the prognosis. FFAR4 can significantly promote laryngocarcinoma cell proliferation and migration in vitro.

[Clinical characteristics and management of cervical necrotizing fasciitis].

Objective:To investigate the clinical characteristics and treatment of cervical necrotizing fasciitis. Method:Clinical data of 61 patients were analyzed retrospectively. All patients were underwent surgical debridement and treated with broad-spectrum antibiotics after diagnose. Result:Complications occurred in 14 patients. Fifty-nine patients were cured while 2 patients died. After 3 months or more follow-up, 3 patients accompanied with sequelae of vocal hoarseness, and no patient recurred or died. Conclusion:Early surgical debridement and the use of antibiotics should be taken as soon as possible after diagnosis of cervical necrotizing fasciitis, as well as control of comorbidities and systemic support treatment in order to prevent complications and deaths.

Isolation of Desulfovibrio spp. from human gut microbiota using a next-generation sequencing directed culture method.

Desulfovibrio spp. is predominant member of sulphate-reducing bacteria in human gut microbiota. Previous studies indicated that the isolation of Desulfovibrio strains from human faecal samples is very important to study the roles of human intestinal Desulfovibrio spp. in maintaining healthy states or causing diseases, as well as defining their biological characteristics. However, there are very few reports describing the isolation of Desulfovibrio spp. from human faecal samples. In this study, faecal samples were inoculated into various media containing different components. The enriched culture communities were identified using 16S rRNA gene high-throughput sequencing analysis, enabling us to identify the specific components that enable the enrichment of Desulfovibrio. Using this information, we developed five specific media and identified an effective enrichment medium that produced the highest relative abundance of Desulfovibrio in communities cultured from four faecal samples (26·5, 73·5, 44·7 and 77·6% respectively). In addition, the major non-Desulfovibrio genera were identified. Finally, three species of Desulfovibrio, D. desulfuricans, D. piger and D. legallii were isolated, representing the first time that has D. legallii been isolated from a human gastrointestinal source. SIGNIFICANCE AND IMPACT OF THE STUDY: ost of the human intestinal bacteria have not been cultured because of lack of appropriate culture method and appropriate media. Desulfovibrio spp. is associated with several clinical conditions like inflammatory bowel disease, but until now there are very few reports describing the isolation of Desulfovibrio spp. from human faecal samples. In this study, 16S rRNA gene high-throughput sequencing analysis was applied to screen appropriate enrichment media and selective cultivation of Desulfovibrio. This sequencing-based directed culture method described here can be used for the selective cultivation of gut bacteria of interest.

[Determination of arsenic species compound in blood by high performance liquid chromatographyatomic fluorescence spectrometry].

Objective: To establish a method for determination of arsenic species in blood with high performance liquid chromatography-atomic fluorescence spectrometry (HPLC-AFS) . Methods: The effect of mobile phase about chromatography separation and sample pretreatment conditions and atomic fluorescence spectrometry detection parameters has been optimized to reliably measure the following four kinds of species arsenic compound including arsenic[As (III) ]、dimethylarsinic acid (DMA) 、monomethylarsonic acid (MMA) and arsenate[As (V) ] in acute intoxication human blood. The method of technical standard about within-run, between-run and recoveries of standard were optimized. Results: The method showed As (III) linear relationship was 2.63-100.00 µg/L, The detection limit was 2.63 µg/L. The relative coefficient (r) was 0.9999; DMA linear relationship was 3.21-100.00 µg/L, The detection limit was 3.21 µg/L. The r was 0.9992; MMA linear relationship was 3.41-100.00 µg/L, The detection limit was 3.41 µg/L. The r was 0.9998; As (V) linear relationship was 3.90-100.00 µg/L, The detection limit was 3.90 µg/L. The r was 0.9996. The average recovery of four species arsenic in tested samples ranged from 91.3%-99.8% with the relative standard deviation (RSD) from 2.39% to 4.05%. The within-run and between-run relative standard deviations (RSD) of repetitive measurement at 10.00, 40.00, 80.00 µg/L concentration levels were 1.99%-4.59% and 2.72%-4.53%. Conclusion: This method is low detection limit, good accurate and high sensitivity, proposed method had been applied to the analysis of arsenic species in blood samples those who acute intoxication or poisoning diagnosis.

[The surgical management of upper parapharyngeal regions].

Objective: To find proper the surgical approval and evaluate clinical efficacy to treat the tumor of upper parapharyngeal space involving the base of skull and intracranial skull. Method: The data of 9 cases from June 2013 and June 2018 were analyzed retrospectively including schwannoma in 6 cases, pleomorphic adenoma in 2 cases and hemangioma in 1 case. All cases received preoperative high resolution CT and MRI, some cases also did the DSA examination. Tumor invaded top of nasopharyngeal in 4 cases, the base of skull in 3 cases, and intraskull in 2 cases. 9 cases were treated with surgery alone. Surgical approach: transcervical approach (n=1), transcervical approach and mandibular fracture surgery(n=2), transoral approach(n=3), transnasal transpterygoid approach(n=2), transparotid gland approach(n=1). Result: Tumors in 8 cases were completely removed, and 1 case was performed by partial excision. Hemorrhage(>500 ml) occurred in 2 cases, tongue deflection and cerebrospinal fluid leakage occurred in 1 case. No death, tumor recurrence and wound infection was found. Conclusion: The position of benign upper parapharyngeal space tumors is deep and tumor often invade in the base of the skull and brain tissue. It is close to the important nerve, vessels of the skull base and meninges. The appropriate surgical approach should be selected according to the individual situation. The main point of the operation is complete the tumor resection with preserving or reconstructing the important function of the blood vessel and nerve.

[Determination of trace gallium by graphite furnace atomic absorption spectrometry in urine].

OBJECTIVE: To establish a method for determination trace gallium in urine by graphite furnace atomic absorption spectrometry (GFAAS). METHODS: The ammonium dihydrogen phosphate was matrix modifier. The temperature effect about pyrolysis (Tpyr) and atomization temperature were optimized for determination of trace gallium. The method of technical standard about within-run, between-run and recoveries of standard were optimized. RESULTS: The method showed a linear relationship within the range of 0.20~80.00 µg/L (r=0.998). The within-run and between-run relative standard deviations (RSD) of repetitive measurement at 5.0, 10.0, 20.0 µg/L concentration levels were 2.1%~5.5% and 2.3%~3.0%. The detection limit was 0.06 µg/L. The recoveries of gallium were 98.2%~101.1%. CONCLUSION: This method is simple, low detection limit, accurate, reliable and reproducible. It has been applied for determination of trace gallium in urine samples those who need occupation health examination or poisoning diagnosis.

[Determination of trace bismuth by atomic fluorescence spectrometry in blood].

Objective: To establish a method for determination trace bismuth in blood by atomic fluorescence spectrometry (AFS) . Methods: 4.0 ml nitric acid and 1.0 ml perchloric acid was added into 1.0 ml blood sample then through automation graphite digestion instrument digested, after that 1.0 ml thiocarbamide-vitamin (10%) was injected, 8% HCl constant volume to 10.0 ml, the bismuth was detected by atomic fluorescence spectrometry with 5.0 ml digestive sample. Results: The method showed a linear relationship within the range of 0.4-50.0 µg/L (r=0.999 7) . The within-run and between-run relative standard deviations (RSD) of repetitive measurement at 10.0, 20.0, 40.0 µg/L concentration levels were 2.2%-4.9% and 3.0%-4.0%. The detection limit was 0.032 µg/L. The recoveries of bismuth were 93.0%-103.9%. Conclusion: This method is low detection limit, good accurate and high sensitivity. It has been applied for determination of trace bismuth in blood samples those who need occupation health examination or poisoning diagnosis.

Synthesis and molecular characterization of acrylate liquid crystalline resin monomers (ALCRM).

A novel biocompatible resin monomer 4—3—(acryloyloxy)—2—hydroxypropoxy) phenyl 4—(3—(acryloyloxy)—2—hydroxypropoxy) benzoate, as an oral restorative — acrylate liquid crystalline resin monomer (ALCRM) was synthesized. The intermediate product and the final product were characterized by differential scanning calorimetry (DSC), polarized optical microscope (POM), and nuclear magnetic resonance (NMR). A resin matrix which has a potential application in dental composites was prepared by photopolymerizing ALCRM and triethylene glycol dimethacrylate (TEGDMA) as a primary and diluted monomer with a photosensitizer of camphorquinone (CQ) and 2—(Dimethylamino)ethyl methacrylate (DMAEMA) mixture. The molar ratio of ALCRM and TEGDMA was 7:3. The properties such as the curing depth, curing time, and the volumetric shrinkage of the resin matrix were investigated and compared with a traditional composite resin matrix Bis—GMA. After photocuring polymerization, the conversion degree of the resin matrix is 68.06%, higher than Bis—GMA/TEGDMA; the curing time is 4.08±0.20min, the curing depth is 2.10±0.17mm, and the volumetric shrinkage is 3.62%±0.26%. All the properties exhibit a better performance of the prepared resin matrix than Bis—GMA.

Self-assembling of DNA and PAMAM dendrimer.

The self-assembling of DNA and cationic polymers are of interest for their biological applications. In this paper, the interaction of DNA with PAMAM was studied by agarose gel electrophoresis, size and zeta-potential measurements and circular dichroism (CD) spectroscopy. DNA was shown to form stable complexes with PAMAM by gel electrophoresis. The mean particle size of the complexes was in the range of 150-180 nm. The zeta-potential was positive and increased with an increase of N/P ratios. Compared to naked DNA, CD spectra of complexed DNA was changed and had a decreased intensity at 270nm. High salts and excessively low or high pH values could also result in decreased CD signals of PAMAM complexed DNA. These results provide some instructions for assembling of DNA as well as gene therapy applications.

NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells.

The ability to induce cellular defense mechanisms in response to environmental challenges is a fundamental property of eukaryotic and prokaryotic cells. We have previously shown that oxidative challenges lead to an increase in antioxidant enzymes, particularly glutathione peroxidase (GPx) and catalase (CAT), in mouse skeletal muscle. The focus of the current studies is the transcriptional regulatory mechanisms responsible for these increases. Sequence analysis of the mouse GPx and CAT genes revealed putative binding motifs for NF kappa B and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues. To test whether NF kappa B or AP-1 might be mediating the induction of GPx and CAT in muscle cells subjected to oxidative stress, we first characterized their activation by pro-oxidants. Electrophoretic mobility shift assays showed that oxidative stress led to increases in the DNA binding of NF kappa B in differentiated muscle cells. The NF kappa B complexes included a p50/p65 heterodimer, a p50 homodimer, and a p50/RelB heterodimer. AP-1 was also activated, but with slower kinetics than that of NF kappa B. The major component of the AP-1 complexes was a heterodimer composed of c-jun/fos. To test for redox regulation of NF kappa B- or AP-1-dependent transcriptional activation, muscle cells expressing either kappa B/luciferase or TRE/luciferase reporter constructs were subjected to oxidative stress. Pro-oxidant treatment resulted in increased luciferase activity in cells expressing either construct. To test whether NF kappa B mediates oxidant-induced increases of GPx and CAT expression, we transfected cells with either a transdominant inhibitor (I kappa B alpha) or a dominant-negative inhibitor (Delta SP) of NF kappa B. Both inhibitors blocked the induction of antioxidant gene expression by more than 50%. In summary, our results suggest that NF kappa B and AP-1 are important mediators of redox-responsive gene expression in skeletal muscle, and that at least NF kappa B is actively involved in the upregulation of the GPx and CAT in response to oxidative stress.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides ("chimeraplasts") have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated "MDX1") designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were "revertant fibers." Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

p48/STAT-1alpha-containing complexes play a predominant role in induction of IFN-gamma-inducible protein, 10 kDa (IP-10) by IFN-gamma alone or in synergy with TNF-alpha.

Human IFN-gamma-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-gamma alone, and synergistically induced by TNF-alpha and IFN-gamma. Upstream regions of the human and murine genes contain conserved regulatory motifs, including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-gamma. Trans-acting factors mediating the IFN-gamma response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-gamma, with or without TNF-alpha, was demonstrated using p48-deficient U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed IFN-gamma/TNF-alpha-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1. Induction of hIP-10 by TNF-alpha/IFN-gamma also required NFkappaB binding sites, which were protected in vivo and bound p65 homodimeric NFkappaB in vitro. These results documented the essential role of p48 (complexed with STAT-1alpha) for induction and sustained transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory cytokines.

Regulation of human IP-10 gene expression in astrocytoma cells by inflammatory cytokines.

Because of its prominent expression in central nervous system inflammatory pathology by astrocytes, we examined the mechanism of human IP-10 (hIP-10) gene induction by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in astrocytoma cells. When present together, IFN-gamma and TNF-alpha induced robust accumulation of hIP-10 mRNA, but hIP-10 mRNA was minimally induced when astrocytoma cells were treated with individual cytokines. This pattern of expression resembled that previously described for murine IP-10 (mIP-10) gene induction in fibroblasts and in rat astroglia. Nuclear run-on experiments showed that the synergistic effect of the cytokines resulted from an increased rate of IP-10 transcriptional initiation. Functional analysis of the hIP-10 promoter after deletion and substitution mutagenesis indicated that an interferon-stimulated response element (ISRE) governed both simple response to IFN-gamma and synergy with TNF-alpha. Synergistic induction of hIP-10 also required an ISRE-proximal nuclear factor kappa-B (NFkappaB) binding site. TNF-alpha-induced NFkappaB binding activity at this site was composed of RelA (p65) homodimers. Our results document that cis-elements through which cytokines mediate synergistic induction of IP-10 in mouse and human are strictly conserved despite divergence elsewhere within the proximal 5'-flanking region.