Metformin is a potential therapeutic for COVID-19/LUAD by regulating glucose metabolism.

Lung adenocarcinoma (LUAD) is the most common and aggressive subtype of lung cancer, and coronavirus disease 2019 (COVID-19) has become a serious public health threat worldwide. Patients with LUAD and COVID-19 have a poor prognosis. Therefore, finding medications that can be used to treat COVID-19/LUAD patients is essential. Bioinformatics analysis was used to identify 20 possible metformin target genes for the treatment of COVID-19/LUAD. PTEN and mTOR may serve as hub target genes of metformin. Metformin may be able to cure COVID-19/LUAD comorbidity through energy metabolism, oxidoreductase NADH activity, FoxO signalling pathway, AMPK signalling system, and mTOR signalling pathway, among other pathways, according to the results of bioinformatic research. Metformin has ability to inhibit the proliferation of A549 cells, according to the results of colony formation and proliferation assays. In A549 cells, metformin increased glucose uptake and lactate generation, while decreasing ATP synthesis and the NAD+/NADH ratio. In summary, PTEN and mTOR may be potential targets of metformin for the treatment of COVID-19/LUAD. The mechanism by which metformin inhibits lung adenocarcinoma cell proliferation may be related to glucose metabolism regulated by PI3K/AKT signalling and mTOR signalling pathways. Our study provides a new theoretical basis for the treatment of COVID-19/LUAD.

Down-regulation of interleukin-2 predicts poor prognosis and associated with immune escape in lung adenocarcinoma.

Background and Objective: Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and has a poor prognosis. Interleukin-2 (IL2) is a cytokine that stimulates lymphocyte proliferation. However, its role in LUAD remains unclear. Methods: The UALCAN, human protein atlas (HPA), and tumor immune estimation resource (TIMER) databases were used to investigate IL2 expression in samples from patients with LUAD. The HPA, PrognoScan, and Kaplan-Meier plotter databases were used to examine the prognostic value of IL2 in LUAD. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to analyze IL2-interacting genes identified through the GeneMANIA database. TIMER was used to analyze the correlation of IL2 expression with immune cell infiltration and immune checkpoint expression levels in LUAD. Results: Bioinformatic analysis using the TIMER, The University of Alabama at Birmingham Cancer data analysis Portal (UALCAN), and HPA public databases showed that IL2 expression was lower in patients with LUAD than in the normal control group. Moreover, patients with low IL2 expression exhibited poor overall survival. Furthermore, IL2 expression was significantly positively correlated with various immune cells, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells, in patients with LUAD. Additionally, IL2 expression was markedly positively associated with the above-mentioned immune cells. Furthermore, IL2 expression was positively correlated with PD-1, PD-L1, and CTLA-4 expression. Conclusion: Our results indicate that down-regulation of IL2 predicts poor prognosis and is associated with immune escape in LUAD, and IL2 could serve as a potential novel prognostic biomarker of LUAD.

Diagnostic value of Serum Amyloid A, Interleukin-6 in gravidas with spontaneous preterm birth.

PURPOSE: Spontaneous preterm birth (SPB) can't be predicted accurately nowadays. We aim to investigate the value of serum amyloid A(SAA) and interleukin-6(IL-6) for forecasting the risk of SPB. METHODS: A total of 302 pregnant women who completed delivery in our hospital from January 2019 to December 2021 were included. According to gestational days, they were divided into the case group (28-33+6 weeks, 41 cases; 34-36+6 weeks, 96 cases) and the control group (37-42 weeks, 165 cases). The general data of the two groups were analyzed and the values of SAA and IL-6 in speculating the risk of SPB were studied in this study. RESULTS: The levels of SAA and IL-6 in the case group were higher than those in the control group(P < 0.05), and the most practical value of SAA and IL-6 access SPB risk were 17.35 mg/L, 112.41 pg/mL respectively. The area under the ROC curve of diagnosis to predict SPB were 0.8849, 0.8664. CONCLUSIONS: The assessment of SPB risk by SAA and IL-6 bearscertain clinical value, which could assist clinicians in recognizing and evaluating the potential dangers of SPB.

Effects of 630 nm Red and 460 nm Blue Light Emitting Diode Irradiation on Healing of the Skin Wound in Japanese Big-ear White Rabbit.

Objective To observe the effects of 630 nm red light and 460 nm blue light emitting diode irradiation on the healing of skin wounds in Japanese big-ear white rabbits. Methods The skin wound model was established with 8 Japanese big-ear white rabbits. Three parts of vulnus in each rabbit were used:two parts of vulnus were irradiated vertically by red and blue LED light,respectively(15 min/time),and the distance between lights and wounds was 15 cm;the 3rd part of the wound was used as a control. On the 21st day of the wounds exposure to light,the number of healing wounds and the percentage of healing area were recorded and the treatment effect of these two light sources was compared. HE staining was used to analyze the newborn tissue structure. Masson staining was used to observe the proliferation of skin collagen fibers. Immuohistochemical staining was used to analyze fibroblast growth factor(FGF),epidermal growth factor(EGF),endothelial growth factor(CD31),proliferating cell nuclear antigen(Ki-67),and inflammatory cytokines(CD68)infiltration in the skin. Results The healing rate in the red light,blue light,and control groups was 50.0%(4/8),25.0%(2/8),and 12.5%(1/8),respectively. Since the 12th day after modeling,the healing area percentage in the red light group was significantly higher than those in the blue light and control groups(P<0.05,P<0.01). On the 21st day after modeling,the skin thickness of the red light group was(2.95±0.34)mm,which was significantly higher than that in control group [(2.52±0.42)mm;F=3.182,P=0.016)]. The average optical density of collagen fibers was 0.15±0.03 in red light group,which was significantly higher than that of the blue light group(0.09±0.01;F=7.316,P=0.012)and control(0.07±0.01;F=7.316,P=0.003). The results of immunohistochemistry showed the expression levels of EGF,FGF,CD31 antigen,and Ki-67 in the red light group were significantly higher than those in the blue light and control groups,whereas the CD68 expression was significantly lower(P<0.05 or P<0.01). Conclusion LED red light irradiation can promote the healing of skin wounds in Japanese big-ear white rabbits,which may be achieved by the effect of red light irradiation in stimulating the proliferation of skin epidermal cells,vascular endothelial cells,and fiberous tissue.

Histopathological and immunological changes during the acute and recovery phase in Henoch-Schönlein purpura rabbit model.

Henoch-Schönlein purpura (HSP) is a systemic vasculitis mediated by autologous immune complex. Animal models of HSP are scarce. Here, we describe the characteristics of HSP rabbit model in the acute and recovery phase. First, we constructed the HSP rabbit models, and then assessed immunologic indicators of models by enzyme-linked immunosorbent assay and immunoturbidimetry. Histomorphological characteristics were analyzed by haematoxylin-eosin, immunofluorescence and special staining. In the acute stage (24 h) after antigen challenge, the model group rabbits featured skin ecchymosis and abnormal laboratory examination results. Three weeks following the allergic reaction, purple spots improved markedly, and edema and blood seeping decreased, but obvious inflammation was present in the skin, kidneys, joints, gastrointestinal, lung and liver. Serological results of CD4, CD/CD8, IL-2, IL-4, and TNF-α, IgA, IgG, TropI, Alb and T were still abnormal. IgA and C3 expressed in skin and kidney and eosinophils expressed in skin and lungs were increased. The rabbit model can mimic human HSP lesions in symptoms, pathology, and immunology and may provide valuable insight into the pathogenesis of HSP and serve as a tool for future therapeutic development targeting HSP.

Gorab Is Required for Dermal Condensate Cells to Respond to Hedgehog Signals during Hair Follicle Morphogenesis.

GORAB is a golgin that localizes predominantly at the Golgi apparatus and physically interacts with small guanosine triphosphatases. GORAB is ubiquitously expressed in mammalian tissues, including the skin. However, the biological function of this golgin in skin is unknown. Here, we report that disrupting the expression of the Gorab gene in mice results in hair follicle morphogenesis defects that were characterized by impaired follicular keratinocyte differentiation. This hair follicle phenotype was associated with markedly suppressed hedgehog (Hh) signaling pathway in dermal condensates in vivo. Gorab-deficient dermal mesenchymal cells also displayed a significantly reduced capability to respond to Hh pathway activation in vitro. Furthermore, we found that the formation of the primary cilium, a cellular organelle that is essential for the Hh pathway, was impaired in mutant dermal condensate cells, suggesting that Gorab may be required for the Hh pathway through facilitating the formation of primary cilia. Thus, data obtained from this study provided insight into the biological functions of Gorab during embryonic morphogenesis of the skin in which Hh signaling and primary cilia exert important functions.

Quantitative protein expression profiling of 14-3-3 isoforms in human renal carcinoma shows 14-3-3 epsilon is involved in limitedly increasing renal cell proliferation.

14-3-3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14-3-3 isoforms have different expression level and isoform-specific roles in different tumors. However, the biological functions of 14-3-3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14-3-3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14-3-3epsilon was up-regulated with 1.44-fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14-3-3sigma was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT-PCR, Western blot and immunohistochemistry analysis for specific 14-3-3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14-3-3epsilon in vitro can limitedly prompt the abnormal growth of renal tumor cells.

Application of the SILAC (stable isotope labelling with amino acids in cell culture) technique in quantitative comparisons for tissue proteome expression.

Stable isotope labelling has recently become a popular tool for the quantitative profiling of the proteome, especially the emergence and development of the SILAC (stable isotope labelling with amino acids in cell culture) technique. Here we have expanded the application of SILAC to comparison of the relative protein expression levels between two different states of tissues based on cultured cells with [2H]leucine labelling as an internal standard in mass spectra. The SILAC ratio of tissue proteins versus labelled cells was determined by the calculation of peak intensity of the pair of labelled and unlabelled peptide fragment ions from the mass spectra, and the relative expression level of proteins in two groups of tissues was estimated by calculating the ratio of their SILAC ratio. To validate our [2H]leucine-based differential proteome analysis for tissues, we successfully compared two known proteins, one up-regulated vimentin and one down-regulated enoyl-CoA hydratase in human renal cancerous tissues versus human normal kidney tissues, which was previously confirmed by other groups using conventional two-dimensional PAGE analysis. Furthermore, we identified a previously unknown down-regulated protein, COX4I1 (cytochrome c oxidase subunit 4 isoform 1), in renal carcinoma tissues by this [2H]leucine-based quantitative proteomics method, which was also validated by immunohistochemistry and Western-blot analysis. In conclusion, the application of the [2H]leucine-based quantitative technique can be effectively expanded to comparison of the expression levels for the tissue proteome at different states, which would help us to identify new candidate biomarkers for tumours.

Quantitative proteomic analysis of HepG2 cells treated with quercetin suggests IQGAP1 involved in quercetin-induced regulation of cell proliferation and migration.

Quercetin, a wild distributed bioflavonoid, exhibits antitumor effects on murine models by inducing apoptosis and inhibiting growth of many cancer cell lines, while proteins involved in antitumor effects at proteomic level are still unclear. In our study, we used a quantitative proteomic strategy termed stable isotope labeling by amino acids in cell culture (SILAC)-mass spectrometry (MS) to study the differential proteomic profiling of HepG2 cells treated by quercetin. In all, there were 70 changed proteins among those quantified proteins in HepG2 cells treated by 50 microM quercetin for 48 h, and 14 proteins showed significant upregulation, whereas 56 proteins were downregulated. The functional classification of changed proteins includes signaling protein, protein synthesis, cytoskeleton, metabolism, etc. Of these, Ras GTPase-activating-like protein (IQGAP1) and beta-tubulin were found to be reduced at a large degree. The migration inhibition of HepG2 cells can be induced by quercetin, and the RNA and protein expression level of IQGAP1 and beta-tubulin were respectively decreased obviously in HepG2 cells exposed to quercetin for 48 h in the scratch migration assay. The downregulated expression of IQGAP1 and beta-tubulin by quercetin treatment correlated with cell migration ability, and quercetin probably inhibits HepG2 proliferation and migration through IQGAP1 and beta-tubulin expression changes and their interactions with other proteins.

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis.

Human 14-3-3 proteins have isoform-specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14-3-3 proteins and isoform-specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14-3-3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture-based quantitative proteomic strategy. As a result, except unexpressed 14-3-3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para-cancerous brain tissues (PBTs). 14-3-3ß and η were upregulated, whereas 14-3-3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14-3-3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14-3-3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation-specific PCR analysis. These results indicated that the four isoforms, including 14-3-3ß, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.

Prokaryotic expression, purification of a new tumor-relative protein FAM92A1-289 and its characterization in renal cell carcinoma.

In order to study the characterization of a new tumor-relative FAM92A1-289 protein, we first constructed plasmid FAM92A1-pQE30 for fusion expression in Escherichia coli. The recombinant protein FAM92A1-289 was affinity-purified by Ni2+-charged resin and separated by HPLC chromatography with high purity, and it was further identified by electrospray ionization-mass spectrometry. Furthermore, the expression and cell localization of FAM92A1-289 by immunohistochemistry using our self-prepared polyclonal antibody showed it was expressed in cytoplasm of renal carcinoma. FAM92A1-289 mRNA was expressed in 2 of 10 kidney tissues and in 6 of 12 primary renal tumors. FAM92A1-289 can promote cell growth in vitro and in vivo by colony formation and mouse xenograft assay. Our present data indicated FAM92A1-289 is a new tumor-related gene with oncogenic potentials to probably play roles in renal carcinogenesis.

Gene expression and methylation status of 14-3-3sigma in human renal carcinoma tissues.

Loss of 14-3-3sigma expression mainly by methylation-mediated silencing has been reported in several human cancers, but the methylation status of 14-3-3sigma in human renal carcinoma is rarely studied so far. In this report, 14-3-3sigma expression was first examined by RT-PCR and immunohistochemistry, and further we investigated the methylation status by methylation-specific PCR and the correlation between 14-3-3sigma expression and its methylation. We found 14-3-3sigma expression was lost in 27 of 31 renal tissues including 16 renal carcinoma tissues, eight para-cancerous kidney tissues and seven normal kidney tissues. Among 16 renal carcinoma tissues, 14 cases had complete hypermethylation of 14-3-3sigma. Eight para-cancerous kidney tissues were almost completely methylated except one case had both methylation and unmethylation. Among seven normal kidney tissues, five cases had partial methylation, and the other two cases were completely methylated. In addition, 14-3-3sigma mRNA had weak expression in OS-RC-2 cells, but it increased with gradual demethylation after treatment by a demethylation agent, 5-aza-2'-deoxycytidine. In general, 14-3-3sigma mRNA was mostly unexpressed, and its DNA frequently hypermethylated within 14-3-3sigma coding region was closely associated with the gene silencing in cancerous and para-cancerous kidney tissues. 14-3-3sigma was also frequently methylated and almost silencing in normal kidney tissues. However, the methylation frequency was gradually reinforced with the extent of malignancy from normal to para-cancerous and cancerous kidney tissues.