Surface-Displayed Amuc_1100 From Akkermansia muciniphila on Lactococcus lactis ZHY1 Improves Hepatic Steatosis and Intestinal Health in High-Fat-Fed Zebrafish.

Fatty liver and intestinal barrier damage were widespread in most farmed fish, which severely restrict the development of aquaculture. Therefore, there was an urgent need to develop green feed additives to maintain host liver and intestinal health. In this study, a probiotic pili-like protein, Amuc_1100 (AM protein), was anchored to the surface of Lactococcus lactis ZHY1, and the effects of the recombinant bacteria AM-ZHY1 on liver fat accumulation and intestinal health were evaluated. Zebrafish were fed a basal diet, high-fat diet, and high-fat diet with AM-ZHY1 (108 cfu/g) or control bacteria ZHY1 for 4 weeks. Treatment with AM-ZHY1 significantly reduced hepatic steatosis in zebrafish. Quantitative PCR (qPCR) detection showed that the expression of the lipogenesis [peroxisome-proliferator-activated receptors (PPARγ), sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1)] and lipid transport genes (CD36 and FABP6) in the liver were significantly downregulated (p < 0.05), indicating that AM-ZHY1 could reduce liver fat accumulation by inhibiting lipid synthesis and absorption. Moreover, supplementing AM-ZHY1 to a high-fat diet could significantly reduce serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, indicating that liver injury caused by high-fat diets was improved. The expression of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the liver decreased significantly (p < 0.05), while IL-1ß and IL-10 did not change significantly in the AM-ZHY1 group. Compared to the high-fat diet-fed group, the AM-ZHY1 group, but not the ZHY1 group, significantly increased the expression of intestinal tight junction (TJ) proteins (TJP1a, claudina, claudin7, claudin7b, claudin11a, claudin12, and claudin15a; p < 0.05). Compared to the high-fat diet group, the Proteobacteria and Fusobacteria were significantly reduced and increased in the AM-ZHY1 group, respectively. In conclusion, the recombinant bacteria AM-ZHY1 has the capacity to maintain intestinal health by protecting intestinal integrity and improving intestinal flora structure and improving fatty liver disease by inhibiting lipid synthesis and absorption. This study will lay a foundation for the application of AM protein in improving abnormal fat deposition and restoring the intestinal barrier in fish.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.

Simultaneous or Staged Decompressions for Patients with Tandem Spinal Stenosis.

OBJECTIVE: To compare the clinical effects of cervical decompression first, lumbar decompression first, or simultaneous decompression of both lesions in the treatment of tandem spinal stenosis (TSS). METHODS: This is a retrospective analysis. From January 2013 to December 2018, 51 TSS patients underwent our surgery and postoperative investigation. Among the 51 subjects, 27 females and 24 males, aged 49-77 years with an average age of 66.3 ± 6.8, were selected. According to the different operation sequences, all patients were divided into three groups. In simultaneous operation group, five patients underwent cervical and lumbar vertebrae surgery at the same time. In first cervical surgery group, 28 patients underwent cervical vertebra surgery first, followed by lumbar spine surgery after a period of recovery. And in first lumbar surgery group, 18 patients underwent lumbar vertebrae surgery first. The choice for neck surgery is posterior cervical single-door vertebroplasty, the surgery of lumber is plate excision and decompression needle-rod system internal fixation. The outcome measures are visual analogue scale (VAS), Japanese Orthopaedic Association cervical (JOA-C) and lumbar (JOA-L) scores, which were assessed at 3 months and 1 year after the operation by telephone interview. In addition, operative time, estimated blood loss, and hospital stay were also recorded. RESULTS: All the patients in the study had surgery performed successfully by the same group of orthopaedic surgeons. The preoperative VAS scores of simultaneous operation group, first cervical surgery group, and first lumbar surgery group were 8.00 ± 1.00, 8.36 ± 0.68, and 8.17 ± 0.71 (P > 0.05). The preoperative JOA-C scores were 7.00 ± 2.35, 6.54 ± 1.53, and 7.83 ± 1.04 (P < 0.05). And the preoperative JOA-L scores were 7.20 ± 2.17, 4.64 ± 2.36, and 5.78 ± 1.22 respectively (P < 0.05). During the final 1-year follow-up, the JOA-C improvement rates of simultaneous operation group, first cervical surgery group, and first lumbar surgery group were 85.68% ± 5.44%, 84.27% ± 5.02%, and 83.34% ± 10.25%, respectively (P > 0.05), and the JOA-L improvement rates were 80.04% ± 3.35%, 81.65% ± 3.74%, and 80.21% ± 4.76% (P > 0.05). The difference among them was not statistically significant. In addition, operation time (OP), blood loss (BL), and hospital stay (HS) in the simultaneous operation group were 245.00 ± 5.00 min, 480.00 ± 27.39 mL, and 16.60 ± 0.55 days, respectively. While those parameters in the first cervical surgery group were 342.50 ± 18.18 min, 528.21 ± 43.97 mL, and 22.75 ± 2.15 days, and in the first lumbar surgery group they were 346.11 ± 24.77 min, 519.44 ± 43.99 mL, and 22.89 ± 1.64 days. The average blood loss in simultaneous operation group was less (P > 0.05); meanwhile, the operation time and hospital stay time were significantly shorter in the simultaneous operation group than in the first cervical surgery group and first lumbar surgery group (P < 0.05). Only one case of fat liquefaction occurred in first cervical surgery group, which healed spontaneously after a regular change of dressing for 1 month. CONCLUSIONS: Under the condition of ensuring the surgical effect, the choice of staged surgery or concurrent surgery according to the patients' own symptoms of cervical and lumbar symptoms could both obtain satisfactory results, and the damage of simultaneous surgery was less than that of staged surgery.

The role of fibroblast activation protein in progression and development of osteosarcoma cells.

To investigate the expression levels of fibroblast activation protein (FAP) in human osteosarcoma tissues and its possible correlations with clinical pathological characteristics of patients with osteosarcoma, and to explore the potential effects of FAP on progression and development of osteosarcoma. Immunohistochemistry (IHC) assay was initially performed to detect the expression levels of FAP in 66 tumor tissues and adjacent non-tumor tissues. Patients were sequentially divided into two groups based on different expression levels of FAP. The correlations between the expression levels of FAP and the clinical pathological characteristics were investigated, and the role of FAP in proliferation, migration, and invasion of osteosarcoma cells was assessed via colony formation, MTT, wound healing, and transwell assays, respectively. The possible effects of FAP on tumor growth and metastasis were evaluated in vivo. We further attempted to reveal the underlying mechanism of FAP involved in tumor growth through bioinformatics and IHC assays. High expression levels of FAP were noted in human osteosarcoma tissues. It also was unveiled that FAP was significantly associated with the tumor size (P = 0.005*) and clinical stage (P = 0.017*). Our data further confirmed that knockdown of FAP remarkably blocked proliferation, migration, and invasion of osteosarcoma cells in vitro, and suppressed tumor growth and metastasis in mice via AKT signaling pathway. The possible role of FAP in progression and development of osteosarcoma could be figured out. Our data may be helpful to develop a novel therapeutic target for the treatment of osteosarcoma.

Bone morphogenetic protein and activin membrane-bound inhibitor suppress bone cancer progression in MG63 and SAOS cells via regulation of the TGF-ß-induced EMT signaling pathway.

Bone cancer is one of the most common tumor types that occurs in bones and their affiliated tissues. The prognosis remains poor due to the limited number of effective therapeutic targets. Downregulation of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) has been observed in human cancer cells and BAMBI reconstitution can inhibit growth and metastasis of human cancer cells. In the present study, a potential mechanism mediated by BAMBI in osteosarcoma cells was investigated. The data demonstrated that BAMBI reconstitution suppressed the cell growth, migration and invasion of the osteosarcoma cell lines SAOS2 and MG63. Alterations to the epithelial-to-mesenchymal transition (EMT) marker expression were observed in BAMBI-treated osteosarcoma SAOS2 and MG63 cells. The apoptosis rate of SAOS2 and MG63 cells induced by cisplatin were increased in BAMBI-treated osteosarcoma SAOS2 and MG63 cells via downregulation of the anti-apoptosis genes P16, P21 and B-cell lymphoma 2. The potential mechanism investigated indicated that BAMBI administration downregulated the transforming growth factor-ß (TGF-ß) signaling pathway, whilst knockdown of BAMBI upregulated the TGF-ß signaling pathway in SAOS2 and MG63 cells. Reconstitution of BAMBI in SAOS2 and MG63 cells resulted in a notable reduction of TGF-ß-induced EMT, cell growth, migration and invasion in vitro. In conclusion, the results demonstrated that BAMBI reconstitution inhibited growth and invasiveness of osteosarcoma, as well as promoted the apoptotic sensibility, which indicated that the TGF-ß-induced EMT signaling pathway may be regarded as a potential target for osteosarcoma therapy.

Pentoxifylline decreases post-operative intra-abdominal adhesion formation in an animal model.

BACKGROUND: Intra-abdominal adhesions develop after nearly every abdominal surgery, commonly causing female infertility, chronic pelvic pain, and small bowel obstruction. Pentoxifylline (PTX) is a methylxanthine compound with immunomodulatory and antifibrotic properties. The aim of this study was to investigate whether PTX can reduce post-operative intra-abdominal adhesion formation via collagen deposition, tissue plasminogen activator (tPA) level, inflammation, angiogenesis, and fibrosis. METHODS: Seventy male BALB/c mice were randomized into one of three groups: (1) sham group without peritoneal adhesion model; (2) peritoneal adhesion model (PA group); (3) peritoneal adhesion model with PTX (100 mg/kg/day i.p.) administration was started on preoperative day 2 and continued daily (PA + PTX group). On postoperative day 3 and day 7, adhesions were assessed using the Lauder scoring system. Parietal peritoneum was obtained for histological evaluation with hematoxylin and eosin (HE) and picrosirius red staining. Fibrinolysis was analyzed by tPA protein levels in the peritoneum by ELISA. Immunohistological analysis was also conducted using markers for angiogenesis (ki67+/CD31+), inflammation (F4/80+) and fibrosis (FSP-1+ and α-SMA+). All the comparisons were made by comparing the PA group with the PTX treated PA group, and p < 0.05 was considered statistically significant. RESULTS: Intra-abdominal adhesions were markedly reduced by PTX treatment. Compared with the PA group, PTX treatment had lower adhesion scores than the PA group on both day 3 and day 7 (p < 0.05). Histological evaluations found that PTX treatment reduced collagen deposition and adhesion thickening. ELISA analysis showed that PTX treatment significantly increased the level of tPA in the peritoneum. In addition, in the immunohistological analysis, PTX treatment was found to significantly decrease the number of ki67+/CD31+ cells at the site of adhesion. Finally, we also observed that in the PTX treated group, there was a reduction in the expression of F4/80+, FSP-1+, and α-SMA+ cells at the site of adhesion. CONCLUSION: PTX may decrease intra-abdominal adhesion formation via increasing peritoneal fibrinolytic activity, suppressing angiogenesis, decreasing collagen synthesis, and reducing peritoneal fibrosis. Our findings suggest that PTX can be used to decrease post-operative intra-abdominal adhesion formation.

Resuscitation Using Liposomal Vasopressin in an Animal Model of Uncontrolled Hemorrhagic Shock.

BACKGROUND: Current research suggests that administration of vasopressin to patients with uncontrolled hemorrhagic shock (UHS) can avoid the detrimental effects associated with aggressive fluid resuscitation. However, vasopressin has a short half-life of 10~35 minutes in in vivo use and precludes its use in the pre-hospital setting. To increase the half-life of vasopressin, we proposed to synthesize liposome-encapsulated vasopressin and test it in a rat model of UHS. METHODS: The film hydration method was used to prepare liposomal vasopressin consisting of: Dipalmitoylphosphatidylcholine, cholesterol, and dipalmitoyl phosphatidylethanolamine (20:20:1 mole ratio). 42 rats were subjected to UHS and randomly received 5 different treatments (vasopressin, liposomal vasopressin, lactate ringer (LR), liposome only and sham). Outcome of UHS were measured using 4 common prognostic tests: mean arterial pressure (MAP), serum lactate level, inflammatory profile and pulmonary edema. RESULTS: The dynamic light scattering results confirmed that we had prepared a successful liposomal vasopressin complex. Comparing the serum vasopressin concentration of liposomal vasopressin and vasopressin treated animals by ELISA, we found that the concentration of vasopressin for the liposomal vasopressin treated group is higher at 60 minutes. However, there was no significant difference between the MAP profile of rats treated with vasopressin and liposomal vasopressin in UHS. We also observed that animals treated with liposomal vasopressin performed indifferently to vasopressin treated rats in serum lactate level, inflammatory profile and edema profile. For most of our assays, the liposome only control behaves similarly to LR resuscitation in UHS rats. CONCLUSION: We have synthesized a liposomal vasopressin complex that can prolong the serum concentration of vasopressin in a rat model of UHS. Although UHS rats treated with either liposomal vasopressin or vasopressin showed no statistical differences, it would be worthwhile to repeat the experiments with different liposomal compositions.

Induction of COX-2/PGE(2)/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation.

Exposure to cigarette smoke extract (CSE) leads to airway and lung inflammation through an oxidant-antioxidant imbalance. Cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) have been shown to play critical roles in respiratory inflammation. Here, we show that COX-2/PGE(2)/IL-6 induction is dependent on Toll-like receptor 4 (TLR4)/NADPH oxidase signaling in human tracheal smooth muscle cells (HTSMCs). CSE induced COX-2 expression in vitro in HTSMCs and in vivo in the airways of mice. CSE also directly caused an increase in TLR4. Moreover, CSE-regulated COX-2, PGE(2), and IL-6 generation was inhibited by pretreatment with TLR4 Ab; inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride and apocynin), p38 MAPK (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin); a ROS scavenger (N-acetyl-l-cysteine); and transfection with siRNA of TLR4, MyD88, TRAF6, Src, p47(phox), p38, p42, JNK2, or p65. CSE-induced leukocyte numbers in BAL fluid were also reduced by pretreatment with these inhibitors. Furthermore, CSE induced p47(phox) translocation and TLR4/MyD88/TRAF6 and c-Src/p47(phox) complex formation. We found that PGE(2) enhanced IL-6 production in HTSMCs and leukocyte count in BAL fluid. In addition, treatment with nicotine could induce COX-2, PGE(2), and IL-6 generation in in vivo and in vitro studies. These results demonstrate that CSE-induced ROS generation was mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiated the activation of MAPKs and NF-kappaB, and ultimately induced COX-2/PGE(2)/IL-6-dependent airway inflammation.

Cigarette smoke extract induces COX-2 expression via a PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells.

Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E2 (PGE2) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKCalpha (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKCalpha, Src, or Akt. Furthermore, CSE-stimulated NF-kappaB p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-kappaB in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-kappaB (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE2 generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE2 generation was mediated through PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-kappaB complex.