Establishment of a novel obesity mouse model: the induction of intestinal microbiota dysbiosis.

To establish and evaluate an intestinal microbiota dysbiosis-induced obesity mouse model. 50 C57BL/6 J male healthy mice were randomly divided into an obesity model group and the control group. The body weight, body length, and Lee's index of the two groups of mice at week 1 and week 10 were compared. Serum glucose (GLU), total cholesterol (TC) and triglyceride (TG) were measured by enzyme-labeled colorimetric methods. Illumina HiSeq 16S rDNA high-throughput sequencing technology was used to characterize intestinal microbiota in feces. The success rate of model establishment in obese mice was 52%. The body weight, body length, Lee's index, and abdominal fat (wet weight) in the obese model group were all higher than those in the control group, and the differences were statistically significant (P < 0.01). Serum GLU and TC levels in the obesity model group were higher than those in the control group (P < 0.05), and there was no difference in TG levels between the two groups (P > 0.05). The control group contained more abundant intestinal microbiota phyla and genera than did the obesity model group; the differences between the two groups were significant (FDR ≤ 0.05, P ≤ 0.05). Intestinal microbiota dysbiosis can be used to generate an obesity model in mice.

NIR-II-absorbing diimmonium polymer agent achieves excellent photothermal therapy with induction of tumor immunogenic cell death.

Photothermal therapy has shown great promise for cancer treatment and second near-infrared (NIR-II) -absorbing particles could further improve its precision and applicability due to its superior penetration depth and new imaging ability. Herein, high NIR-II-absorbing polymer particles were prepared by using soluble isobutyl-substituted diammonium borates (P-IDI). The P-IDI showed stronger absorption at 1000-1100 nm, which exhibited excellent photostability, strong photoacoustic imaging ability and high photothermal conversion efficiency (34.7%). The investigations in vitro and in vivo demonstrated that the excellent photothermal effect facilitated complete tumor ablation and also triggered immunogenic cell death in activation of the immune response. The high solubility and excellent photothermal conversion ability demonstrated that polymer IDI particles were promising theranostic agents for treatment of tumors with minor side effects.

G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation.

CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++-GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.

NIR-II emissive AIEgen photosensitizers enable ultrasensitive imaging-guided surgery and phototherapy to fully inhibit orthotopic hepatic tumors.

Accurate diagnosis and effective treatment of primary liver tumors are of great significance, and optical imaging has been widely employed in clinical imaging-guided surgery for liver tumors. The second near-infrared window (NIR-II) emissive AIEgen photosensitizers have attracted a lot of attention with higher-resolution bioimaging and deeper penetration. NIR-II aggregation-induced emission-based luminogen (AIEgen) photosensitizers have better phototherapeutic effects and accuracy of the image-guided surgery/phototherapy. Herein, an NIR-II AIEgen phototheranostic dot was proposed for NIR-II imaging-guided resection surgery and phototherapy for orthotopic hepatic tumors. Compared with indocyanine green (ICG), the AIEgen dots showed bright and sharp NIR-II emission at 1250 nm, which extended to 1600 nm with high photostability. Moreover, the AIEgen dots efficiently generated reactive oxygen species (ROS) for photodynamic therapy. Investigations of orthotopic liver tumors in vitro and in vivo demonstrated that AIEgen dots could be employed both for imaging-guided tumor surgery of early-stage tumors and for 'downstaging' intention to reduce the size. Moreover, the therapeutic strategy induced complete inhibition of orthotopic tumors without recurrence and with few side effects.

A new insight into acute lymphoblastic leukemia in children: influences of changed intestinal microfloras.

BACKGROUND: Previous studies have shown that changes in intestinal microfloras are associated with both gastrointestinal (GI) and non-GI tumors. It is not clear whether there is an association between GI microflora changes and hematological malignancies. METHODS: In the current study, we used 16S rDNA gene sequencing techniques to profile the GI microbiome in children with lymphoblastic leukemia (ALL, n = 18) and matched healthy control (n = 18). Using multiple specialized software [Heatmap, Principal coordinates analysis (PCoA), Claster and Metastates], we analyzed the sequencing data for microfloral species classification, abundance and diversity. RESULTS: A total of 27 genera between the ALL and control groups (FDR ≤ 0.05 and/or P ≤ 0.05) showed significantly different abundance between ALL patients and healthy controls: 12 of them were predominant in healthy group and other 15 species were significantly higher in ALL group. In addition, we compared the abundance and diversity of microfloral species in ALL patients prior to and during remission stage after chemotherapy, and no significant difference was detected. CONCLUSIONS: Compared to healthy controls, ALL patient showed significant changes of GI microfloras. Further explorations of the intestinal micro-ecology in ALL patients may provide important information to understand relationship between microfloras and ALL.

A pH-sensitive near-infrared fluorescent probe with alkaline pKa for chronic wound monitoring in diabetic mice.

A pH-sensitive near-infrared fluorescent probe with alkaline pKa, AlkaP-1, was developed by incorporating a benzoyl hydrazine group into a cyanine dye. The significant fluorescence changes in the alkaline regions enable the probe to monitor the alkalization process from acute wounds to chronic wounds in diabetic mice.

Effect of Montmorillonite powder on intestinal mucosal barrier in children with abdominal Henoch-Schonlein purpura: A randomized controlled study.

BACKGROUND: Our previous studies found that intestinal barrier function has been changed in children with abdominal Henoch-Schonlein purpura (HSP). Montmorillonite has been shown to be protective for digestive tract mucosa. OBJECTIVE: The present study aimed to investigate whether Montmorillonite powder could improve the intestinal mucosal barrier function in children with abdominal HSP. METHODS: Using a randomized controlled study design, we compared plasma levels of diamine oxidase (DAO), D-lactate, and endotoxin in children with abdominal HSP before and after Montmorillonite powder treatment. RESULTS: Among 28 patients in experimental group and 30 in control group, there was no significant difference in age, sex, height, weight, and course of disease between 2 groups (P > .05). Before treatment, there was no statistical difference in DAO, D-lactic acid, and endotoxin between experimental group and the control group (P > .05). However, significant differences were detected for DAO and D-lactate after treatment in comparison to before treatment in the Montmorillonite experimental group (P < .05). Such differences were not found in the control group (P > .05). CONCLUSION: Montmorillonite powder is effective in the treatment of HSP via maintaining intestinal mucosal barrier function.

A study of the correlation between obesity and intestinal flora in school-age children.

With the improvement of living standards and dietary changes, childhood obesity has increased worldwide. This study aimed to understand the differences of intestinal flora structure between obese and normal children at school-age. Using the next generation sequencing platform, Illumina Miseq, 16S rDNA high-throughput sequencing technology, we analyzed the diversity and relative abundance of intestinal flora in 39 obese and 38 normal control school-age children. First, we categorized gut bacteria on the basis of their Operational taxonomic units (OTUs) using the RDP 16s rRNA database in RDP classifier. The alpha (α) diversity was used to measure the diversity within a sample and is calculated as a value for each sample. The beta (ß) diversity was used to compare different samples and to measure the dissimilarity between each other sample. Our results indicated that intestinal flora in obese children showed lower diversity than normal controls. Significant differences of relative abundance of intestinal flora were detected at multiple levels of classifications. Identification of intestinal flora with significant difference between obese and normal children may provide important information to uncover the roles of these specific bacteria in the development of obesity and find new strategy to prevent and treat obesity through intervening the intestinal flora.

Self-Assembled Fluorescent Nanoprobe Based on Forster Resonance Energy Transfer for Carbon Monoxide in Living Cells and Animals via Ligand Exchange.

Carbon monoxide (CO) is recognized as a biologically essential gaseous neurotransmitter that modulates many physiological processes in living subjects. Currently reported fluorescent probes for CO imaging in cells basically utilize palladium related chemistry which requires complicated synthetic work. Herein we provide a new strategy to construct a fluorescent nanoprobe, NanoCO-1, based on the Forster resonance energy transfer (FRET) mechanism by entrapping the existing dirhodium complex as the energy acceptor and the CO recognition part, and a commonly used nitrobenzoxadiazole (NBD) dye as energy donor into a micelle formed by self-assembly. The exchange of ligands in the dirhodium complex by CO in the nanoprobe disrupts the FRET and leads to the turn-on of fluorescence. The merits of NanoCO-1 including good biocompatibility, selectivity, photostability, and low cytotoxity, render this nanoprobe ability to track CO in living cells, zebrafish embryo, and larvae. Our straightforward approach can be extended to establish the CO fluorescent probes based on adsorption of CO on a variety of metal derivatives.

Respiratory Syncytial Virus Aggravates Renal Injury through Cytokines and Direct Renal Injury.

The purpose of this study was to investigate the relationship between renal injury and reinfection that is caused by respiratory syncytial virus (RSV) and to analyze the mechanism of renal injury. Rats were repeatedly infected with RSV on days 4, 8, 14, and 28, then sacrificed and examined on day 56 after the primary infection. Renal injury was examined by transmission electron microscopy and histopathology. The F protein of RSV was detected in the renal tissue by indirect immunofluorescence. Proteinuria and urinary glycosaminoglycans (GAGs), serum levels of albumin, urea nitrogen, and creatinine, secretion of cytokines, T lymphocyte population and subsets, and dendritic cell (DC) activation state were examined. The results showed that renal injury was more serious in the reinfection group than in the primary infection group. At a higher infection dose, 6 × 106 PFU, the renal injury was more severe, accompanied by higher levels of proteinuria and urinary GAGs excretion, and lower levels of serum albumin. Podocyte foot effacement was more extensive, and hyperplasia of mesangial cells and proliferation of mesangial matrix were observed. The maturation state of DCs was specific, compared with the primary infection. There was also a decrease in the ratio of CD4+ to CD8+ T lymphocytes, due to an increase in the percentage of CD8+ T lymphocytes and a decrease in the percentage of CD4+ T lymphocytes, and a dramatic increase in the levels of IL-6 and IL-17. In terms of the different reinfection times, the day 14 reinfection group yielded the most serious renal injury and the most significant change in immune function. RSV F protein was still expressed in the glomeruli 56 days after RSV infection. Altogether, these results reveal that RSV infection could aggravate renal injury, which might be due to direct renal injury caused by RSV and the inflammatory lesions caused by the anti-virus response induced by RSV.

[Preparing Bifidobacteria for Quantitative Detections].

OBJECTIVES: To compare different preparation methods for quantitative real-time PCR (qPCR) detection of Bifidobacteria. METHODS: Standard strains of Bifidobacteria were prepared with concentration gradients using strain DNA, PCR product amplification and purification, and plasmid DNA methods. The concentrations of Bifidobacteria were determined with ultraviolet spectrophotometer and real-time quantitative PCR. RESULTS: Greater than 0.99 R 2 in values of standard curves were achieved by all three preparation methods. The plasmid DNA method obtained a higher level of concentration and purity of Bifidobacteria than the other two methods ( P<0.01). CONCLUSIONS: The plasmid DNA method produces high quality preparations and is more suitable for real-time quantitative PCR, which can provide a reference for the molecular biological detection of Bifidobacteria.

Obesity in school-aged children and its correlation with gut E.coli and Bifidobacteria: a case-control study.

BACKGROUND: To determine the correlation between obesity in school-aged children and imbalance of gut microbes by examining the ratio change of intestinal Bifidobacteria and E.coli in obese children compared to non-obese controls. METHODS: A hospital-based 1:1 case-control study was performed. Fecal samples of the subjects were collected for DNA extraction and analyzed by quantitative real-time PCR (qPCR) to determine the copy number of Bifidobacteria and E.coli. The ratio of two microbes (B/E) was then calculated and statistically analyzed. RESULTS: Subjects of the obesity group and control group showed no significant difference in age, gender or height (P > 0.05); whereas they had significant differences in body weight and BMI. Copy numbers of Bifidobacteria and E.coli per gram of wet fecal samples were first determined using qPCR in both obese and normal groups, which were further used for the calculation of B/E ratio. We found that B/E ration in the two groups showed significant difference (P < 0.05). Corrected χ(2) test was performed for the two groups against B/E < 1, and it was found that there was a positive correlation (OR = 719.2, OR 95% C.I. = 81.57-6341.18) between B/E ratio decrease with childhood obesity. CONCLUSIONS: The obese children have a lower amount of Bifidobacteria and higher amount of E.coli (smaller B/E ratio) compared to normal non-obese children. It was suggested that obesity in children may be associated with the imbalance of gut microbes.

Increased serum levels of soluble vascular endothelial-cadherin in patients with systemic vasculitis.

Henoch-Schönlein purpura (HSP) is a commonest systemic vasculitis (SV) in childhood characterized by an inflammatory reaction directed at vessels. Endothelial damage and perivascular leukocyte infiltrates are vital in the development of HSP. Vascular endothelial (VE)-cadherin is an endothelial cell-specific adhesion molecule, which plays critical roles in angiogenesis and endothelial integrity. Herein, we investigated the serum levels of soluble VE-cadherin (sVE-cadherin) in patients with HSP and other forms of SV. The serum levels of sVE-cadherin in 30 patients with HSP, together with patients with urticarial vasculitis, allergic vasculitis, Behcet disease, psoriasis vulgaris (PV) and atopic dermatitis (AD) and 26 health controls were measured by enzyme-linked immunosorbent assay. Serum levels of sVE-cadherin were significantly increased in patients with HSP in acute stage and patients with other forms of SV but not in patients with PV or AD. Moreover, Serum sVE-cadherin levels in HSP patients were correlated with the severity of this disease and serum concentrations of IgA anticardiolipin antibodies and vascular endothelial growth factor. Taken together, we show firstly that serum sVE-cadherin is abnormally increased in HSP patients. Increased serum levels of sVE-cadherin might be a novel biomarker for evaluating the severity of HSP and useful for identifying the presence of SV in inflammatory skin conditions.

Paeoniflorin suppresses vascular damage and the expression of E-selectin and ICAM-1 in a mouse model of cutaneous Arthus reaction.

Paeoniflorin (PF) extracted from the root of Paeonia lactiflora pall, displays anti-inflammation properties in several animal models. Adhesion molecules are important for the recruitment of leucocyte to the vessel wall and involved in the pathogenesis of various autoimmune and inflammatory diseases. Herein, we investigate the effects of PF on adhesion molecule expression in a mouse model of cutaneous Arthus reaction and cultured human dermal microvascular endothelial cells (HDMECs). We showed that PF significantly ameliorated the immune complex (IC) induced vascular damage, leucocyte infiltrates and adhesion molecules expression. Furthermore, PF markedly blocked tumor necrosis factor-α (TNF-α)-induced E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression in HDMECs at both mRNA and protein levels. PF also suppressed TNF-α-induced adhesion of polymorphonuclear leucocytes (PMNs) to HDMECs. Finally, western blot data revealed that PF can inhibit the phosphorylation of p38, JNK in TNF-α-treated HDMECs. These data suggest that PF, as an anti-inflammatory agent, can downregulate adhesion molecules expression. PF may be a candidate medicine for the treatment of IC-induced inflammatory response.

TWEAK enhances E-selectin and ICAM-1 expression, and may contribute to the development of cutaneous vasculitis.

Our previous work indicated that TWEAK is associated with various types of cutaneous vasculitis (CV). Herein, we investigate the effects of TWEAK on vascular injury and adhesion molecule expression in CV mice. We showed that TWEAK priming in mice induced a local CV. Furthermore, TWEAK priming also increased the extravasation of FITC-BSA, myeloperoxidase activity and the expression of E-selectin and ICAM-1. Conversely, TWEAK blockade ameliorated the LPS-induced vascular damage, leukocyte infiltrates and adhesion molecules expression in LPS-induced CV. In addition, TWEAK treatment of HDMECs up-regulated E-selectin and ICAM-1 expression at both mRNA and protein levels. TWEAK also enhanced the adhesion of PMNs to HDMECs. Finally, western blot data revealed that TWEAK can induce phosphorylation of p38, JNK and ERK in HDMECs. These data suggest that TWEAK acted as an inducer of E-selectin and ICAM-1 expression in CV mice and HDMECs, may contribute to the development of CV.

Increased HMGB1 serum levels and altered HMGB1 expression in patients with psoriasis vulgaris.

High mobility group box-1 (HMGB1) has been implicated as a pro-inflammatory cytokine in the pathogenesis of various inflammatory and autoimmune diseases. However, information about HMGB1 in inflammatory skin diseases is unknown. Herein, we investigated the serum HMGB1 levels and tissue HMGB1 expression in patients with psoriasis vulgaris (PV) and atopic dermatitis (AD). Serum levels of HMGB1 in patients with PV and AD were detected by enzyme-linked immunosorbent assay (ELISA). The expression of HMGB1 in lesional skin was evaluated by immunohistochemistry and immunofluorescence. Protein levels of HMGB1 in the nuclear fraction and cytoplasmic fraction were determined by western blot. Serum levels of HMGB1 in patients with PV but not AD were significantly higher than those in nornal controls. Moreover, serum HMGB1 levels were correlated with the severity of PV according to PASI socres. Furthermore, by immunohistochemistry and immunofluorescence, we showed that the expression of HMGB1 in normal skin was almost completely restricted to the nucleus. However, abundant cytoplasmic expression of HMGB1 was observed in the epidermis in lesional skin of PV patients. In addition, western blot data indicated that HMGB1 expression was in the nucleus protein and was absent in the cytoplasm protein in control group. In contrast, HMGB1 expression in the cytoplasmic fraction was detectable in AD patients and more distinct in PV patients. Taken together, this study provides first observations on the association of HMGB1 with PV, and showed the elevated HMGB1 serum levels and altered HMGB1 distribution in lesional skin in patients with PV. We suggest that HMGB1 might be involved in the pathogenesis of PV.

Elevated serum interleukin-33 levels in patients with Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is the most common systemic vasculitis and is known as an immunoglobulin (Ig) A related immune complex-mediated disease. However, the molecular mechanisms in the development of HSP are not yet fully understood. Herein, we investigated the serum levels of Interleukin (IL)-33 and soluble ST2 (sST2) in HSP patients and their association with disease severity and IgA autoantibodies production. The serum levels of IL-33 and sST2 were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) in the serum of 33 patients with HSP and 22 controls. Serum levels of IgA anti-endothelial cell antibodies (AECA) and IgA anticardiolipin antibodies (ACA) in HSP patients were detected by double antigen sandwich ELISA. Our results indicated that serum levels of IL-33 but not sST2 were significantly elevated in patients with HSP in acute stage and restored to normal levels in convalescent stage. Moreover, serum IL-33 levels were correlated with the severity of HSP and serum concentrations of AECA-IgA and ACA-IgA. Taken together, we show firstly that serum IL-33 is abnormally elevated in HSP patients. IL-33 might be associated with the IgA autoantibodies production in the pathogenesis of HSP.

Suppression of inflammation by tumor-derived exosomes: a kind of natural liposome packaged with multifunctional proteins.

Exosomes are small-membrane vesicles secreted by hematopoietic and malignant epithelial cells as well as trophoblasts. The composition of cancerous exosomes has been proven to play pivotal roles in the maintenance of the microenvironment that is beneficial for the progression of cancer, such as Fas-ligand-triggered lymphocyte apoptosis. We supposed that the immunosuppressive effect of cancerous exosomes might be helpful in the treatment of diseases characterized by overactivation of the immune system and subsequent tissue injury. The aim of this study was to evaluate the protective effect of tumor-derived exosomes in the mice model of lipopolysaccharide (LPS)-induced inflammation. Tetrazolium (MTT) and DNA electrophoresis were used to measure the cytotoxicity of exosomes on lymphocytes. Pathologic observation of tissue sections, serologic analysis of aspartate aminotransferase/alanine aminotransferase (AST/ALT), and urinary analysis of protein were used to assess the protection effect of exosomes in LPS-induced multiorgan damage. In vitro outcomes of MTT and DNA electrophoresis showed the cytotoxicity of exosomes on lymphocytes. Together with the alleviation of organ damages evaluated by urine protein, serum AST/ALT, and pathologic analysis, we confirmed the possibility that pretreatment of mice with exosomes, produced by H22 hepatic tumor cells, resulted in protection against LPS-induced tissue damage, which is caused by overactivation of the immune system and inflammation response. This therapeutic strategy will raise an interesting way to search new therapeutics in pairs of diseases with complementarities in etiology and pathology, namely, a strategy of taking advantage of the mutual complementarities between diseases.