Fabrication and characterization of soy ß-conglycinin-dextran-polyphenol nanocomplexes: Improvement on the antioxidant activity and sustained-release property of curcumin.

In this study, glycated soy ß-conglycinin (ß-CG) stabilized curcumin (Cur) composites were fabricated by a unique reversible self-assembly character of ß-conglycinin-dextran conjugates (ß-CG-DEX). Intrinsic fluorescence and far-UV CD spectra revealed that glycation did not affect the self-assembly property of ß-CG in the pH-shifting treatment. The structure of ß-CG-DEX could be unfolded at pH 12.0 and reassembled during acidification (from pH 12.0 to 7.0). Meanwhile, ß-CG-DEX-3d, which was incubated at 60 °C for 3 days, exhibited a high loading capacity (123.4 mg/g) for curcumin, which far exceeds that (74.90 mg/g) of ß-CG-Cur. Moreover, the reassembled ß-CG-DEX-3d-Cur showed eminent antioxidant activity of approximately 1.5 times higher than that of free curcumin. During the simulated gastrointestinal condition, compared with ß-CG-Cur, ß-CG-DEX-3d-Cur nanoparticles showed a more stable and sustained release of curcumin. Thus, ß-CG-DEX has immense potential to become a new delivery carrier for hydrophobic food components by means of a self-assembly strategy.

Common mtDNA variations at C5178a and A249d/T6392C/G10310A decrease the risk of severe COVID-19 in a Han Chinese population from Central China.

BACKGROUND: Mitochondria have been shown to play vital roles during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) development. Currently, it is unclear whether mitochondrial DNA (mtDNA) variants, which define mtDNA haplogroups and determine oxidative phosphorylation performance and reactive oxygen species production, are associated with COVID-19 risk. METHODS: A population-based case-control study was conducted to compare the distribution of mtDNA variations defining mtDNA haplogroups between healthy controls (n = 615) and COVID-19 patients (n = 536). COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning. The exclusion criteria for the healthy controls were any history of disease in the month preceding the study assessment. MtDNA variants defining mtDNA haplogroups were identified by PCR-RFLPs and HVS-I sequencing and determined based on mtDNA phylogenetic analysis using Mitomap Phylogeny. Student's t-test was used for continuous variables, and Pearson's chi-squared test or Fisher's exact test was used for categorical variables. To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for possible confounding factors of age, sex, smoking and diseases (including cardiopulmonary diseases, diabetes, obesity and hypertension) as determined through clinical and radiographic examinations. RESULTS: Multivariate logistic regression analyses revealed that the most common investigated mtDNA variations (> 10% in the control population) at C5178a (in NADH dehydrogenase subunit 2 gene, ND2) and A249d (in the displacement loop region, D-loop)/T6392C (in cytochrome c oxidase I gene, CO1)/G10310A (in ND3) were associated with a reduced risk of severe COVID-19 (OR = 0.590, 95% CI 0.428-0.814, P = 0.001; and OR = 0.654, 95% CI 0.457-0.936, P = 0.020, respectively), while A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) were related to an increased risk of severe COVID-19 (OR = 2.336, 95% CI 1.179-4.608, P = 0.015; OR = 2.033, 95% CI 1.242-3.322, P = 0.005; OR = 3.040, 95% CI 1.522-6.061, P = 0.002; and OR = 2.890, 95% CI 1.199-6.993, P = 0.018, respectively). CONCLUSIONS: This is the first study to explore the association of mtDNA variants with individual's risk of developing severe COVID-19. Based on the case-control study, we concluded that the common mtDNA variants at C5178a and A249d/T6392C/G10310A might contribute to an individual's resistance to developing severe COVID-19, whereas A4833G, A4715G, T3394C and G5417A/C16257a/C16261T might increase an individual's risk of developing severe COVID-19.

Predictive Value of Combined LIPS and ANG-2 Level in Critically Ill Patients with ARDS Risk Factors.

To investigate the predictive value of the acute physiology and chronic health evaluation 2 (APACHE2) score and lung injury prediction score (LIPS) for acute respiratory distress syndrome (ARDS) when combined with biomarkers for this condition in patients with ARDS risk factors. In total, 158 Han Chinese patients with ARDS risk factors were recruited from the Respiratory and Emergency Intensive Care Units. The LIPS, APACHE2 score, primary diagnosis at admission, and ARDS risk factors were determined within 6 h of admission, and PaO2/FiO2 was determined on the day of admission. Blood was collected within 24 h of admission for the measurement of angiopoietin-2 (ANG-2), sE-selectin, interleukin-6 (IL-6), and interleukin-8 (IL-8) levels. ARDS was monitored for the next 7 days. Univariate and multivariate analyses and receiver operating characteristic (ROC) analyses were employed to construct a model for ARDS prediction. Forty-eight patients developed ARDS within 7 days of admission. Plasma ANG-2 level, sE-selectin level, LIPS, and APACHE2 score in ARDS patients were significantly higher than those in non-ARDS patients. ANG-2 level, LIPS, and APACHE2 score were correlated with ARDS (P < 0.001, P < 0.006, and P < 0.042, resp.). When the APACHE2 score was used in combination with the LIPS and ANG-2 level to predict ARDS, the area under the ROC curve (AUC) was not significantly increased. Compared to LIPS or ANG-2 alone, LIPS in combination with ANG-2 had significantly increased positive predictive value (PPV) and AUC for the prediction of ARDS. In conclusion, plasma ANG-2 level, LIPS, and APACHE2 score are correlated with ARDS. Combined LIPS and ANG-2 level displays favorable sensitivity, specificity, and AUC for the prediction of ARDS.

Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity.

BACKGROUND: LPS-binding protein (LBP) and its ligand CD14 are located upstream of the signaling pathway for LPS-induced inflammation. Blocking LBP and CD14 binding might prevent LPS-induced inflammation. In previous studies, we obtained a peptide analog (MP12) for the LBP/CD14 binding site and showed that this peptide analog had anti-endotoxin activity. In this study, we used in vitro directed evolution for this peptide analog to improve its in vivo and in vitro anti-endotoxin activity. METHODS: We used error-prone PCR (ep-PCR) and induced mutations in the C-terminus of LBP and attached the PCR products to T7 phages to establish a mutant phage display library. The positive clones that competed with LBP for CD14 binding was obtained by screening. We used both in vivo and in vitro experiments to compare the anti-endotoxin activities of a polypeptide designated P1 contained in a positive clone and MP12. RESULTS: 11 positive clones were obtained from among target phages. Sequencing showed that 9 positive clones had a threonine (T) to methionine (M) mutation in amino acid 287 of LBP. Compared to polypeptide MP12, polypeptide P1 significantly inhibited LPS-induced TNF-α expression and NF-κB activity in U937 cells (P<0.05). Compared to MP12, P1 significantly improved arterial oxygen pressure, an oxygenation index, and lung pathology scores in LPS-induced ARDS rats (P<0.05). CONCLUSION: By in vitro directed evolution of peptide analogs for the LBP/CD14 binding site, we established a new polypeptide (P1) with a threonine (T)-to-methionine (M) mutation in amino acid 287 of LBP. This polypeptide had high anti-endotoxin activity in vitro and in vivo, which suggested that amino acid 287 in the C-terminus of LBP may play an important role in LBP binding with CD14.

Role of Angptl4 in vascular permeability and inflammation.

BACKGROUND: Angptl4 is a secreted protein involved in the regulation of vascular permeability, angiogenesis, and inflammatory responses in different kinds of tissues. Increases of vascular permeability and abnormality changes in angiogenesis contribute to the pathogenesis of tumor metastasis, ischemic-reperfusion injury. Inflammatory response associated with Angptl4 also leads to minimal change glomerulonephritis, wound healing. However, the role of Angptl4 in vascular permeability, angiogenesis, and inflammation is controversy. Hence, an underlying mechanism of Angptl4 in different kind of tissues needs to be further clarified. METHODS: Keywords such as angptl4, vascular permeability, angiogenesis, inflammation, and endothelial cells were used in search tool of PUBMED, and then the literatures associated with Angptl4 were founded and read. RESULTS: Data have established Angptl4 as the key modulator of both vascular permeability and angiogenesis; furthermore, it may also be related to the progression of metastatic tumors, cardiovascular events, and inflammatory diseases. This view focuses on the recent advances in our understanding of the role of Angptl4 in vascular permeability, angiogenesis, inflammatory signaling and the link between Angptl4 and multiple diseases such as cancer, cardiovascular diseases, diabetic retinopathy, and kidney diseases. CONCLUSIONS: Taken together, Angptl4 modulates vascular permeability, angiogenesis, inflammatory signaling, and associated diseases. The use of Angptl4-modulating agents such as certain drugs, food constituents (such as fatty acids), nuclear factor (such as PPARα), and bacteria may treat associated diseases such as tumor metastasis, ischemic-reperfusion injury, inflammation, and chronic low-grade inflammation. However, the diverse physiological functions of Angptl4 in different tissues can lead to potentially deleterious side effects when used as a therapeutic target. In this regard, a better understanding of the underlying mechanisms for Angptl4 in different tissues is necessary.

MtDNA haplogroups M7 and B in southwestern Han Chinese at risk for acute mountain sickness.

We conducted a case-control study to investigate the association of mitochondrial DNA (mtDNA) haplogroups with acute mountain sickness (AMS) in Han Chinese from southwestern (SW) China. Pearson's chi-square test or Fisher's exact test revealed significant reduction of mtDNA haplogroups D and M9, while a significant increase of haplogroup M7 in AMS subjects compared with non-AMS subjects. The multivariate logistic regression analysis after adjustment for body mass index (BMI), a risk factor of AMS in the present study, showed that both D and M9 were associated with significantly decreased risk of AMS, while M7 was associated with a significantly increased risk of AMS (OR=0.605, p=0.000; OR=0.037, p=0.001, and OR=2.419, p=0.001, respectively). In addition, further analysis stratified by the AMS severities indicated that haplogroup B was correlated with a 2.41-folds increased risk of developing severe AMS (95%C.I=1.288-4.514, p=0.006). Our findings provide evidence that, in SW Han Chinese, mtDNA haplogroups D and M9 are related to individual tolerance to AMS, while haplogroups M7 and B are risk factors for AMS.

[Effect of nuclear factor-kappaB on peroxisome proliferator activated receptor gamma expression in Ana-1 cells].

OBJECTIVE: To determine the effects of nuclear factor-alpha B (NF-alpha B) on peroxisome proliferator activated receptor gamma (PPAR gamma) expression in the murine macrophage cell line Ana-1, based on the investigation of the PPAR gamma expression stimulated by lipopolysaccharide (LPS). METHODS: Ana-1 cells were divided randomly into seven groups: control group, LPS groups (cells were activated by 0.1 mg/L LPS for 1, 2, 4, 8 hours respectively), SN50 group (cells were stimulated by 50 mg/L SN50 for 4 hours) and NF-alpha B high expression plasmid transfected group. PPAR gamma and tumor necrosis factor-alpha(TNF-alpha) mRNA levels were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and the TNF-alpha protein was measured by enzyme linked immunosorbent assay (ELISA) after being activated by LPS. The eukaryotic expression vector of murine NF-alpha B p65 gene was constructed and stably transfected into Ana-1 cells with DOTAP liposome . The PPAR gamma expression and NF-alpha B p65 translocation as stimulated by LPS and SN50 were assayed by Western blotting. RESULTS: Expressions of both PPAR gamma mRNA and protein were downregulated when cells were stimulated by LPS, which were accompanied with the activity of NF-alpha B and TNF-alpha in Ana-1 cells (all P<0.05). The eukaryotic expression vectors containing murine p65 gene were successfully constructed and stably transfected into Ana-1 cells. LPS stimulation and NF-alpha B p65 gene overexpression resulting in upregulation of p65 could downregulate PPAR gamma expression in Ana-1 cells (r=-0.913, P<0.05).But downregulation of NF-alpha B by SN50 could upregulate PPAR gamma expression in the nucleus (r=-0.959, P<0.05). CONCLUSION: LPS can markedly decrease the expression of PPAR gamma in Ana-1 cells, which may be related to its activity of enhancing NF-alpha B. NF-alpha B p65 gene can control PPAR gamma expression in the reverse direction in Ana-1 cells.

[Involvement of bcl-2 in multidrug resistance in human small cell lung cancer cell subline H446/DDP].

OBJECTIVE: To study the mechanism of bcl-2 involvement in multidrug resistance in human small cell lung cancer (SCLC) cell subline H446/DDP. METHODS: After the construction of pLXSN-bcl-2, in which the full length of bcl-2 cDNA amplified from total RNA of H446/DDP cells was integrated into the mammalian expression vector pLXSN, allowing transcription of a bicistronic mRNA, and the synthesis in vitro of 20-mer ODNs targeting the coding region of bcl-2 mRNA and the scrambled ODNs used as the control, the cationic lipid DOTAP was used to transfect them into H446 and H446/DDP cells, respectively. When H446 cells were transfected with mammalian expression vector pLXSN, the cells were divided into 3 groups, including cells transfected with the recombinant pLXSN-bcl-2, cells transfected with the vector pLXSN and cells untransfected (control); when H446 cells and H446/DDP cells were transfected with PS-ODNs, the cells were divided into 3 groups, including cells transfected with AS-PS-ODNs, cells transfected with NS-PS-ODNs and cells untransfected (control), respectively. After transfection, Western blot were carried out to detect the expression level of bcl-2 in the control cells and the transfected cells, respectively. Meanwhile flow cytometry (FCM) was used to detect cell apoptosis in the control cells and the transfected cells. RESULTS: (1) The data from Western blot showed that compared with the control H446 cells (gray-scale value 0.103 +/- 0.005), the expression level of bcl-2 in the pLXSN-bcl-2 transfected H446 cells (gray-scale value 0.854 +/- 0.016) was increased significantly (P < 0.01); and the apoptosis from DNA content analysis decreased significantly in the pLXSN-bcl-2 transfected H446 cells [(20.9 +/- 0.2)%] compared with that of the control H446 cells [(31.1 +/- 0.2)%] by DNA content analysis (P < 0.01). (2) The results from Western blot showed that bcl-2 expression in the AS-PS-ODNs transfected H446/DDP cells (gray-scale value 0.695 +/- 0.014) was significantly reduced compared with that of the control H446/DDP cells (gray-scale value 0.942 +/- 0.018), although not totally reduced to the level of the control H446 cells (P < 0.01); and the data from DNA content analysis indicated that the sensitivity of the AS-PS-ODNs transfected H446/DDP cells to 5 microg/ml DDP-induced apoptosis was strongly increased as compared with that of the control H446/DDP cells (P < 0.01). CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression may be one of the important therapeutic approaches to overcoming chemoresistance in human SCLC.

[Research advance on molecular and cellular biology of small cell lung cancer].

Various genetic mutations and biologic alterations occur during cancerization, which promote the malignant development of tumors. Multiple genes are involved in and cooperated with the occurrence and development of small cell lung cancer (SCLC). SCLC manifests early and frequent metastasis as well as ultimate poor response to chemotherapy. New therapies for SCLC based on understanding of molecular and cellular mechanism of transformation are needed. SCLC is associated with multiple chromosomal abnormalities, the most common of which is chromosome 3p deletion, as well as abnormal oncogenes and tumor suppressor genes. Along with the genetic alterations, SCLC overexpresses various cell surface receptors, such as receptor tyrosine kinase (RTKs), G-protein-coupled receptors, integrins, and so on. Some activated downstream molecules, such as phosphatidylinositol 3'kinase, would serve as good candidates for therapeutic targets.

Isolation and chromosomal localization of ZFX homologue in genome of rice field eel (Monopterus albus Zuiew).

When used as a probe in rice field eel (Monopterus albus Zuiew) genomic Southern blotting hybridization, the giant panda Zfx gene hybridized strongly to a fragment of about 9.5 kb. A 512 bp long DNA fragment has been isolated by polymerase chain reaction from rice field eel genomic DNA using the primers for amplifying zinc finger repeats 7 to 13 of mammalian and reptilian ZFX-related genes. Cloned in pBS, four recombinant plasmids were selected randomly from male and female specimens and sequenced. The nucleotide sequences in these clones were identical and showed 88% and 87% identity to human ZFX and ZFY respectively. But its extent of homology was greater with American alligator Zfc (90%). And the amino acid sequences of the putative protein showed 95.9%, 95.9% and 93.5% identity to human ZFX and ZFY and American alligator Zfc respectively. Thus, the cloned sequence encodes a homologue of mammalian ZFX/ZFY and was named Zfa for rice field eel zinc finger domain gene. It appears that the mammalian and reptilian ZFX-related genes evolved from fish ancestors with a considerable degree of conservation. By fluorescence in situ hybridization, the Zfa has been mapped to rice field eel chromosome 1 and at the position of 60.1 +/- 0.38 from the centromere. Chromosomal mapping of fish genes related to mammalian X-linked genes might lead to further understanding of the evolution of vertebrate sex chromosomes.

[Chromosomal localization of the hormone-sensitive lipase gene (Hsl) in rice field eel].

Adipose tissue triacylglycerols are the quantitatively most important source of stored energy in animals. Hormone-sensitive lipase encoded by hormone-sensitive lipase gene (Hsl) is a multifunctional enzyme that catalyzes the hydrolysis of triacylglycerol stored in adipose tissue and cholesterol esters in the adrenals, ovaries, testes and macrophages. Using pig Hsl gene inserted into pBS labeled by the radioactive isotope and the digoxigenin as the probes respectively one band, 11.5kb, has been shown to hybridized with total DNA of rice field eel digested with Pst I by Southern blotting and Hsl gene has been assigned to metaphase chromosome 5, at the position of 78.35+/-1.26 from the centromere in rice field eel by fluorescent in situ hybridization (FISH). The mapping results are corresponding to that of "specific-chromosomal DNA pool" obtained by chromosome microisolation used to map gene and the mapping result is more accurate. The results of the study further illustrate the importance of the presence of Hsl gene in rice field eel genome and provide the first FISH mapping data for rice field eel chromosome 5. The current studies would advance the addition of known genetic markers and the construction of high resolution genetic map in rice field eel genome.

[Construction and homologous detection of a DNA plasmid library specific for Z chromosome of quail].

A simple method was used to adapt a standard light microscope for the collection of quail Z chromosomes from mitotic-metaphase spreads. The microisolated chromosomes were subjected to proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed PCR (DOP-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis, and smears of DNA revealed that ranged in size from 200-1 400 bp, without any evidence of preferential amplification. The second-round PCR products were cloned into pBluescript plasmids to construct a Z chromosome-specific DNA library. The size range of the cloned inserts was 200-1 400 bp. Using inserted fragments from the library as probes, chromosome painting was performed on quail chromosomes. The results showed that Z chromosomes of quail were completely covered by strong signals and there were little signals on other chromosomes. It was indicated that inserted DNA of the library was specific to the Z chromosome of quail. The library can be used as chromosome painting probe to detect conserved syntenic groups on the chromosomes of other related species and study mechanisms of sex-chromosomes evolution in birds.

[Chromosomal localization of rice field eel Hox genes by PRINS].

The genome duplication and chromosome rearrangement are two kinds of evolution models at the chromosome level during the evolution of vertebrate genome. And Hox genes are the powerful proves to support the evolution theory of genome duplication, which has been found recently. In this study, the chromosomal localization of rice field eel Hox genes has been carried out by PRINS. The mapping results indicated that 6 Hox clusters might exist in rice field eel genome, and these clusters were localized on chromosome 1, 2, 3, 6, 8, 10 and at the position of 28.24 +/- 2.88, 4.55 +/- 1.39, 13.89 +/- 2.03, 74.32 +/- 1.86, 38.03 +/- 2.41, 58.18 +/- 2.05 from the centromere respectively. The mapping results that Hox genes were localized on chromosome 1, 3, 6 and 10 in the study are corresponding to that by chromosome microdissection. The chromosomal localization of rice field eel Hox genes will help us to discover the origin and evolution of rice field eel chromosomes, and provide cellular genetic proves of this special species to support the evolution theory of genome duplication.