Supernatant Medium Research Articles

Dihydromyricetin inhibits the M1 polarization of macrophage induced by oxidized low-density lipoprotein through JAK3/STAT5 signaling pathway

Objective To observe the effect of dihydromyricetin(DMY) on the M1 polarization of macrophages induced by oxidized low-density lipoprotein(Ox-LDL) and to explore its mechanism. Methods RAW 264.7 cells were incubated in RPMI1640 medium containing Ox-LDL(50 mg/L) and DMY(40 μmol/L) for 24 h. The effect of JAK/STAT signaling pathway inhibitor AG-490 on DMY was observed.The levels of tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), transforming growth factor-β(TGF-β) and interleukin-10(IL-10) in the supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA). The protein expressions of inducible nitric oxide synthase(iNOS) and arginase 1(Arg1) were tested by Western blot. Results Compared with the control group, the Ox-LDL group showed that the protein expression in macrophage of iNOS and levels of TNF-α and IFN-γ in the supernatant of culture medium were significantly increased(t=4.12, 2.89 and 3.44, P=0.023, 0.036 and 0.031), while the protein expression of Arg1 in cells and levels of TGF-β and IL-10 in the supernatant of culture medium were significantly decreased(t=3.71, 2.31 and 2.67, P=0.034, 0.038 and 0.034), and the ratios of p-JAK3/JAK3 and p-STAT5/STAT5 in cells were significantly increased(t=3.93 and 2.65 and P=0.007 and 0.021). Compared with the Ox-LDL group, the Ox-LDL+ DMY group showed that the protein expression of iNOS in cells and levels of TNF-α and IFN-γ in the supernatant of culture medium were significantly decreased(t=5.74, 2.37 and 3.07, P=0.022, 0.031 and 0.033), while the protein expression of Arg1 in cells and levels of TGF-β and IL-10 in the supernatant of culture medium were significantly increased(t=3.94, 2.19 and 2.44, P=0.025, 0.047 and 0.035), and the ratios of p-JAK3/JAK3 and p-STAT5/STAT5 in cells were significantly decreased(t=3.41 and 2.32, P=0.015 and 0.023). Compared with the Ox-LDL+ DMY group, the Ox-LDL+ DMY+ AG-490 group showed that the protein expression of iNOS and levels of TNF-α and IFN-γ in supernatant of culture medium were significantly increased(t=3.14, 3.41 and 3.74, P=0.017, 0.028 and 0.023), while the protein expression of Arg1 and levels of TGF-β and IL-10 were significantly decreased(t=2.87, 2.85 and 3.47, P=0.025, 0.035 and 0.028). Conclusions Dihydromyricetin inhibits the M1 polarization of macrophages induced by Ox-LDL, and the mechanism may be related with the activation of JAK3/STAT5 signaling pathway. Key words: Atherosclerosis; Lipoprteins, LDL; Macrophage activation

MicroRNA-146a inhibits NF-κB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.

MicroRNA (miR)-146a levels are reduced in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE); however, its function is not well understood. The present study investigated the role of miR-146a in the regulation of lipopolysaccharide (LPS)-induced inflammation in THP-1 cells. A miR-146a mimic and an inhibitor were used to overexpress and downregulate miR-146a expression, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to evaluate interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) expression, and western blot analysis was applied to assess nuclear factor-κB activation by analyzing p65 subunit levels in the nucleus. To investigate the effects of miR-146a on LPS-induced inflammation, IL-6 and tumor necrosis factor-α (TNF-α) levels were also measured using ELISA. The results of the present study revealed thatmiR-146a overexpression significantly reduced IRAK1 expression, reduced p65 levels in the nucleus and reduced IL-6 and TNF-α levels in the supernatant of the cell culture medium of THP-1 cells following LPS treatment. Luciferase assays confirmed IRAK1 to be a direct target of miR-146a in THP-1 cells. In conclusion, miR-146a may regulate IRAK1 expression and inhibit the activation of inflammatory signals and secretion of pro-inflammatory cytokines. The present study revealed, at least in part, the mechanisms by which miR-146a regulate the inflammatory response in SLE.

Characterizing the SLC39A14 zinc transporter and collagen products of the chondrocytes from the human umbilical cord-derived mesenchymal stromal cell

Background: The SLC39A14 zinc transporter showed a role in the growth of the mouse model. There was no report regarding SLC39A14 protein expression in human chondrocyte, which might prevent further identification of SLC39A14’s function in human growth. This study aims to describe the SLC39A14 protein expression in chondrocyte using human umbilical cord (UC)-derived mesenchymal stromal cell (MSC) isolation and differentiation. Methods: A cross sectional study was conducted at the Faculty of Medicine, Universitas Padjajaran Bandung, Indonesia, in 2019. The UC was derived after the respondents signed informed consents were documented. Specimens transferred, UC tissue digestion, cell isolation, cultures, and passages were done under the laboratory standard protocols. The immuno-phenotyping and differentiation toward chondrocyte procedure were done after third passage. The SLC39A14 expression of the chondrocytes was observed under immunofluorescence microscopy after staining using polyclonal anti SLC39A14 antibody. The collagen 2A and 10A production was measured using the medium supernatant and analyzed using GraphPad Prism 8 edition.Results: The adherent fibroblast-like cells appeared on day 5th, which continued to proliferate and showed MSC characteristic with positive markers for CD 73, CD90 and CD105, but absent for negative lineage markers. Upon differentiation, cells positive for Alcian blue staining denoted the chondrocyte which shown capacity for further proliferation on Methyl Tetra Toluene assay. Zinc transporter SLC39A14 was expressed on cell membrane. The Collagen 2A produced at a mean level 0.097 ng/ml (SE 0.011;95%CI 0.073-0.12) and Collagen 10A at mean level 31.078 ng/ml (SE 3.792;95%CI 22.870-39.287). Conclusion: Zinc transporter SLC39A14 was expressed in monolayer human chondrocyte from UC-derived MSC. The chondrocyte shown capacity for proliferation and collagen-producing enables future utility to identify the role of SLC39A14 in chondrocyte pathology.

86 Impact of steeping corn DDGS over 72 hours without or with fiber degrading enzymes and protease alone or in combination on concentration of sugars and organic acids and pH in the liquid medium

Abstract We investigated the impact of steeping corn DDGS over 72 h with or without fiber degrading enzymes (FDE) and protease (PRO) on concentration of sugar, acetic and lactic acid, and pH. The concentration of crude fat, CP, NDF and ADF in DDGS sample was 8.6, 30.8, 36.1, 12.2% DM, respectively. Treatments were: 1) DDGS steeped without enzymes (Control), 2) DDGS steeped with FDE (FDE), 3) DDGS steeped with protease (PRO), 4) DDGS steeped with combination (FDEPRO). Enzymes were added at 1% of DDGS. Briefly, 50g of DDGS was mixed with 500-mL water with or without enzymes in sterile plastic bottles and steeped for 0, 12, 24, 48 and 72-h at 37℃ with continuous agitation. Samples were aliquoted to individual bottle for each timepoint. At each time point, bottles were pulled out from the incubator for pH measurements and liquid medium supernatant for sugars and acids. Highest arabinose, xylose and glucose was observed at 12 and 24 h, followed by decrease at 48 and 72 h. At 12 h, arabinose was higher (P < 0.05) for FDE and FDEPRO than control. The highest xylose and glucose was seen at 12 h for PRO, FDE and FDEPRO while control was highest at 24 h. Acetic acid and lactic acid increased over time. At 72 h, FEDPRO had highest acetic acid compared to control and PRO, while lactic acid was highest for FDE and FDPRO compared with (P < 0.0001) control and PRO. The pH decreased (P < 0.05) over time for all treatments. At 72 h, FDE had lowest pH followed by FDEPRO, PRO and control (P < .0001). Overall, arabinose, xylose and glucose increased to a certain timepoint and decreased, while lactic and acetic acid increased over time, subsequently leading to lowest pH at 72 h. In conclusion, FDE increased concentration of sugars and acids in steeped DDGS whereas PRO had no effect.

Effects of blood-derived products and sodium hyaluronate on equine synovial fluid cells and on synovial fluid from osteochondrotic joints of horses after arthroscopy and administration of treatment.

To compare effects of platelet-rich plasma (PRP), interleukin-1 receptor antagonist protein (IRAP), autologous processed plasma (APP), and sodium hyaluronate treatments on synovial fluid cells in vitro and on synovial fluid obtained from osteochondrotic joints of horses. Synovial fluid cells from 8 healthy equine tibiotarsal joints (in vitro experiment) and synovial fluid samples from 40 tibiotarsal joints of 25 horses with osteochondrosis dissecans (in vivo experiment). Effects of various treatments on concentrations of prostaglandin (PG) E2, interleukin (IL)-1β, tumor necrosis factor-α, IL-10, and IL-1 receptor antagonist (IL-1ra) were analyzed in cell medium supernatant, and production of reactive oxygen species was analyzed by use of flow cytometry. In an in vivo experiment, synovial fluid samples were collected before and 48 hours after arthroscopy and treatment administration (8 joints/treatment) and evaluated to determine concentrations of hyaluronic acid, chondroitin sulfate, PGE2, tumor necrosis factor-α, IL-1, IL-10, and IL-1ra. All in vitro treatments reduced reactive oxygen species production, PRP increased PGE2 concentrations, and PRP, IRAP, and APP increased IL-1ra concentrations. Only IRAP and APP increased IL-1 concentrations. For the in vivo experiment, PRP increased and IRAP decreased PGE2 concentrations in synovial fluid after arthroscopy. All treatments increased IL-1ra concentrations, but only sodium hyaluronate resulted in a significant increase in concentration, compared with the concentration for untreated joints. Also, IRAP reduced hyaluronic acid breakdown in synovial fluid. PRP should be used with caution in the period immediately after arthroscopy and treatment of osteochondrotic joints of horses. All treatments had antioxidant effects. Sodium hyaluronate, APP, and IRAP might help ameliorate joint inflammation.

Effects of pH and sugar supplements on bacteriocin-like inhibitory substance production by Pediococcus pentosaceus.

To improve bacteriocin-like inhibitory substance (BLIS) production by Pediococcus pentosaceus ATCC 43200, the influence of pH as well as the addition of sugars-either prebiotic (inulin) or not (sucrose)-on its metabolism were investigated. This strain was grown at pH 5.0 or 6.0 either in glucose-based MRS medium (control) or after addition of 0.5, 1.0 or 1.5% (w/w) sucrose and inulin (GSI-MRS) in the same percentages. In the control medium at pH 5.0, cell mass concentration after 48h of fermentation (Xmax = 2.26g/L), maximum specific growth rate (µmax = 0.180h-1) and generation time (Tg = 3.84h) were statistically coincident with those obtained in supplemented media. At pH 6.0 some variations occurred in these parameters between the control medium (Xmax = 2.68g/L; µmax = 0.32h-1; Tg = 2.17h) and the above supplemented media (Xmax = 1.90, 2.52 and 1.86g/L; µmax = 0.26, 0.33 and 0.32h-1; Tg = 2.62, 2.06 and 2.11h, respectively). Lactate production was remarkable at both pH values (13 and 16g/L) and improved in all supplemented media, being 34 and 54% higher than in their respective control media, regardless of the concentration of these ingredients. Cell-free supernatant of the fermented control medium at pH 5.0 displayed an antimicrobial activity against Enterococcus 101 5.3% higher than that at pH 6.0 and even 20% higher than those of all supplemented media, regardless of the concentration of supplements. BLIS production was favored either at pH 5.0 or in the absence of any additional supplements, which were able, instead, to stimulate growth and lactate production by P. pentosaceus.

Interaction of zero valent copper nanoparticles with algal cells under simulated natural conditions: Particle dissolution kinetics, uptake and heteroaggregation

Some metal-based engineered nanoparticles (ENPs) undergo fast dissolution and/or aggregation when they are released in the environment. The underlying processes are controlled by psychochemical/biological parameters of the environment and the properties of the particles. In this study, we investigated the interaction between algal cells and zero valent copper nanoparticles (Cu0-ENPs) to elucidate how the cells influence the dissolution and aggregation kinetics of the particles and how these kinetics influence the cellular uptake of Cu. Our finding showed that the concentration of dissolved Cu ([Cu]dissolved) in the supernatant of the culture media without algal cells was higher than the [Cu]dissolved in the media with algal cells. In the absence of the cells, dissolved organic matter (DOC) increased the dissolution of the particle due to increasing the stability of the particles against aggregation, thus increasing the available surface area. In the presence of algae, Cu0-ENPs heteroaggregated with the cells. Thus, the available surface area decreased over time and this resulted in a low dissolution rate of the particles. The DOC corona on the surface of the particles increased the heteroaggregation of the particles with the cells and decreases the uptake of the particles. Our findings showed that microorganisms influence the fate of ENPs in the environment, and they do so by modifying the dissolution and aggregation kinetics of the Cu0-ENPs.

Identification and Phenotypic Evaluation of Microbes Isolated from Breast and Formula-fed Infants Delivered Either Vaginally or by Cesarean Section (P11-075-19)

ObjectivesMany factors influence the development of the infant microbiota. Based on 16S rDNA sequencing information, the type of feeding, i.e., breast vs formula, has been shown to be the most important factor, with route of delivery having transient effects in the early postpartum period. However, few recent studies have isolated individual strains from human infants or have investigated the functional properties of the infant gut microbes. We used a 2 × 2 experimental design with the goal of determining how diet and delivery interact to shape infant microbiota composition and metabolic activity. MethodsFresh fecal samples were collected from 3-month-old infants who were either exclusively breast- (BF) or formula-fed (FF) and cesarean- (CD) or vaginally-delivered (VD). Samples were diluted and plated on two types of media: Gut Microbiota Medium (GMM) or MRS medium. Individual colonies were picked, purified and their 16S rRNA gene sequenced for identification. These isolates were screened for growth on various carbon substrates, including human milk oligosaccharides, prebiotics and volatile fatty acids for 24–36h. Medium supernatant was collected and metabolic profiles assessed by HPLC. ResultsMore than 150 bacterial strains have been isolated and identified from infant fecal samples to date. Isolates from BF/VD fecal samples were predominantly from the phylum Actinobacteria (dominated with Bifidobacterium) followed by Firmicutes and Proteobacteria. In contrast, isolates from FF/VD or FF/CD infants were primarily from the phylum Firmicutes with only a few Actinobacterial and Proteobacterial isolates. Quantitatively, most isolates represented the genera Bifidobacterium, followed by Enterococcus, and Ruminococcus. On-going phenotypic evaluation of the isolated strains for the growth and utilization of various carbon sources and metabolic profiles will provide insight into species- and strain-specific utilization of common prebiotics in human milk and infant formula. ConclusionsThis collective data will provide novel insights on how the physiological and metabolic function of the infant gut microbiota is influenced by route of delivery and early infant nutrition. Funding SourcesNIH R01DK107561.

Effects of IL-34 on the secretion of RANKL/OPG by fibroblast-like synoviocytes and peripheral blood mononuclear cells in rheumatoid arthritis.

To detect the effect of interleukin (IL)-34 on the secretion of Receptor activator of nuclear factor kappa-B ligand (RANKL)/Osteoprotegerin (OPG) and Matrix metalloproteinase (MMP)-3 by fibroblast-like synoviocytes (FLS) and peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients and to investigate whether the effect is mediated by IL-17. RA-FLS and RA-PBMCs were stimulated with recombinant human (rh) IL-34, with or without the IL-17 inhibitor Plumbagin. The supernatant of the culture medium was collected and the levels of RANKL, OPG, and MMP-3 were detected by enzyme-linked immunosorbent assay (ELISA). RhIL-34 promoted RANKL secretion and inhibited OPG secretion in RA-FLS. The effect was weakened by the addition of the IL-17 inhibitor. In contrast, rhIL-34 had no significant effect on MMP-3 secretion by FLS. RhIL-34 elevated the secretion of RANKL by RA-PBMCs but not by healthy-PBMCs. Furthermore, the secretion of RANKL by RA-PBMCs reduced after the addition of the IL-17 inhibitor. OPG secretion by both RA-FLS and FLS from healthy controls was inhibited by rhIL-34, but were elevated after the addition of the IL-17 inhibitor. RhIL-34 had no significant effect on MMP-3 secretion by both RA-PBMCs and healthy-PBMCs. IL-34 enhances RANKL/OPG expression by RA-FLS and RA-PBMCs, and this effect is, indirectly, mediated by IL-17. This cytokine is therefore likely to to play an important role in local joint destruction and systemic osteoporosis in RA, and is therefore a potential therapeutic target for the treatment of this disease.

Transduction of retinoic acid-inducible gene 1 by Ebola virus-like particles enhances antigen-presentation

Abstract Ebola virus (EBOV), a filovirus family member, is a highly pathogenic virus that causes Ebola hemorrhagic fever resulting in documented mortality rates in humans as high as 50%. EBOV virus-like particles (VLPs) are morphologically and biochemically similar to parental virus, yet because they lack a genome and cannot replicate, are safe enough to be used as vaccines. We hypothesize that addition of a constitutionally active retinoic acid-inducible gene 1 (RIG-I) would enhance the ability of the EBOV VLPs to induce antigen-presentation from infected antigen-presenting cells. Expression of EBOV VP40 in 293T cells induces the spontaneous production of VLPs into the media supernatant and if expressed with EBOV glycoprotein (GP), will produce VLPs studded with the attachment GP. Recombinant chimeric constitutively active (ca)RIG-I-VP40 matrix and a nonfunctional mutant L58A (mu)RIG-I-VP40 matrix genes were constructed to produce VLPs containing constitutively active and nonfunctional RIG-I, respectively. Supernatant from 293Ts transfected with caRIG-I-VP40, muRIG-I-VP40 or VP40 along with GP expression plasmids were tested for the presence of VLPs. Western blotting of purified VLPs confirmed the presence of RIG-I in caRIG-I-VP40 and muRIG-I-VP40, but not VP40 containing VLPs. Monocyte-like THP-1 reporter cells were treated with nothing, VP40+GP, caRIG-I-VP40+GP and muRIG-I-VP40+GP VLPs as well as LPS control and tested for induction of interferon signaling. CaRIG-I containing, but not muRIG-I containing VLPs induced interferon signaling as well as greater MHC I upregulation.

The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells

Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.

CD47 deficiency protects cardiomyocytes against hypoxia/reoxygenation injury by rescuing autophagic clearance.

To assess the effect of cluster of differentiation(CD47) downregulation on autophagy in hypoxia/reoxygenation(H/R)‑treated H9c2 cardiomyocytes. H9c2cells were maintained in normoxic conditions (95% air, 5%CO2, 37˚C) without CD47antibodies, Si‑CD47 or chloroquine(CQ) treatment; H9c2cells in the H/R group were subjected to 24h of hypoxia (1%O2, 94%N2, 5%CO2, 37˚C) followed by 12h of reoxygenation (95% air, 5%CO2, 37˚C). All assays were controlled, triplicated and repeated on three separately initiated cultures. The biochemical parameters in the medium supernatant were measured to evaluate the oxidative stress in cardiomyocytes. The AnnexinV‑fluorescein isothiocyanate assay was used to detect the apoptotic rate in the H9c2cells. Transmission electron microscope, immunofluorescent staining and western blot analysis were performed to detect the effect of the CD47 antibody on autophagic flux in H/R‑treated H9c2 cardiomyocytes. The cardiomyocytic oxidative stress and apoptotic rate decreased and autophagic clearance increased after CD47 downregulation. H/R triggered cell autophagy, autophagosome accumulation and apoptosis in H9c2 cell lines. However, these effects can be attenuated by CD47 downregulation. This study demonstrates its clinical implications in ischemia/reperfusion injury treatment.

ADAM17 Cleaves the Insulin Receptor α‐Subunit on Endothelial Cells and Induces Vascular Insulin Resistance in Type 2 Diabetes

Microvascular insulin resistance is a hallmark of type 2 diabetes (T2D) and an underlying event in the pathophysiology of endothelial dysfunction and cardiovascular disease. However, the molecular mechanisms by which T2D causes vascular insulin resistance remain largely unknown. Hyperglycemia and the proinflammatory state associated with T2D are characterized, in part, by an increased activity of ADAM17, an enzyme that cleaves the ectodomain of various transmembrane proteins. Here, we hypothesized that increased expression and activation of ADAM17 sheds the insulin receptor α‐subunit (IRα) from endothelial cells causing vascular insulin resistance in T2D. We tested this hypothesis in isolated resistance arteries from T2D and non‐T2D subjects undergoing bariatric surgery and in cultured human umbilical vein endothelial cells (HUVECs). Resistance arteries from T2D subjects exhibited increased ADAM17 expression, reduced presence of endothelial IRα as well as the endogenous ADAM17 inhibitor, TIMP3, and impaired insulin‐induced vasodilation, relative to arteries from non‐T2D subjects (P<0.05). Exposure of HUVECs to high glucose (30mM, 24h) increased the expression and activity of ADAM17 (P<0.05), led to increased shedding of IRα detected in the cell culture media supernatant (P<0.05) and decreased IRα on cell surfaces as observed using super‐resolution microscopy. Moreover, pharmacological inhibition of ADAM17 led to reduced shedding of IRα and improved insulin signaling in cells exposed to high glucose or the ADAM17 activator, PMA (P<0.05). Collectively, these findings suggest that T2D, and in particular hyperglycemia, leads to ADAM17 shedding of IRα from the endothelial surface and reduced insulin‐dependent vasodilation, all of which supports ADAM17 activity as a new therapeutic target to ameliorate vascular insulin resistance in T2D.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protection of chickens against fowl cholera by supernatant proteins of Pasteurella multocida cultured in an iron-restricted medium

ABSTRACTPasteurella multocida (P. multocida), a causative agent of fowl cholera, is an important pathogen in the poultry industry. In the present study, we found that the inactivated vaccine of P. multocida grown in an iron-restricted medium provided better protection than that grown in normal medium. Thus, we adopted a comparative proteomics approach, by using two-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS), to profile the supernatant proteins associated with P. multocida under both conditions. Eleven upregulated proteins were identified, including aspartate ammonia-lyase (AspA), diacylglycerol kinase (DgK), 30S ribosomal protein S6 (RpsF), and eight outer membrane proteins (OMPs). To further characterize the three novel supernatant proteins identified under iron-restricted conditions, the AspA, DgK and RpsF proteins were expressed and purified, and used as immunogens to vaccinate chickens. The results showed that AspA, DgK and RpsF proteins induced 80.0%, 66.7%, and 80.0% immunity, respectively. These data indicate that the three novel proteins identified in the supernatant of the culture media might play important roles in the survival of bacteria under iron-restricted conditions, and thus protect chickens against P. multocida. These findings also suggest that the proteins identified can be used as subunit vaccines.

Rat Hippocampal Neural Stem Cell Modulation Using PDGF, VEGF, PDGF/VEGF, and BDNF

Neural stem cells have become the focus of many studies as they have the potential to differentiate into all three neural lineages. This may be utilised to develop new and novel ways to treat neurological conditions such as spinal cord and brain injuries, especially if the stem cells can be modulated in vivo without additional invasive surgical procedures. This research is aimed at investigating the effects of the growth factors vascular endothelial growth factor, platelet-derived growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor/platelet-derived growth factor on hippocampal-derived neural stem cells. Cell growth and differentiation were assessed using immunohistochemistry and glutaminase enzyme assay. Cells were cultured for 14 days and treated with different growth factors at two different concentrations 20 ng/mL and 100 ng/mL. At 2 weeks, cells were fixed, and immunohistochemistry was conducted to determine cellular differentiation using antibodies against GFAP, nestin, OSP, and NF200. The cell medium supernatant was also collected during treatment to determine glutaminase levels secreted by the cells as an indicator of neural differentiation. VEGF/PDGF at 100 ng/mL had the greatest influence on cellular proliferation of HNSC, which also stained positively for nestin, OSP, and NF200. In comparison, HNSC in other treatments had poorer cell health and adhesion. HNSC in all treatment groups displayed some differentiation markers and morphology, but this is most significant in the 100 ng/ml VEGF/PDGF treatment. VEGF/PDGF combination produced the optimal effect on the HNSCs inducing the differentiation pathway exhibiting oligodendrocytic and neuronal markers. This is a promising finding that should be further investigated in the brain and spinal cord injury.

The role and mechanism of 2-deoxyglucose in reversing osimertinib-acquired resistance of non-small cell lung cancer cell line

Objective: To explore the role and mechanism of 2-deoxyglucose (2-dg) in reversing osimertinib- acquired resistance of non-small cell lung cancer(NSCLC)cell line. Methods: The NSCLC line H1975 (purchased from the American Type Culture Collection) was conducted by induction method in vitro to construct the osimertinib-resistance NSCLC cell line H1975-OR. The osimertinib-resistance of H1975-OR cell line was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony-formation assay, Ki67 incorporation assay and the expression of apoptosis-related protein. The glycolysis level was assayed by the lactic acid production measured in the culture medium supernatant of H1975 and H1975-OR. The expression of glycolysis key enzymes (HK2, GLUT1, P-PKM2) and apoptosis-related protein (BIM, Bcl-2) were detected by Western blot. The cells were divided into control group, 2-deoxyglucose (4 mmol/L) monotherapy group, osimertinib (3 μmol/L) monotherapy group and 2-deoxyglucose (4 mmol/L)+ osimertinib (3 μmol/L) combination therapy group, then the apoptosis rate of cells was measured by flow cytometry to evaluate the pro-apoptotic ability of drugs. Date were analyzed by Independent-Samples t-test using SPSS 16.0 statistical software. Results: The glycolysis level of osimertinib-sensitive cell line H1975 was lower than that of osimertinib-resistance cell line H1975-OR [the yield of lactic acid, respectively, was (21.0±0.9) and (26.5±2.8) mmol·L(-1)·10(4)cells(-1), P<0.05]. The osimertinib- acquired resistance of H1975-OR could be reversed by 4 mmol/L 2-deoxyglucose(the IC(50) value of osimertinib in H1975-OR cell line decreased from (7.0±1.9) μmol/L to (1.4±0.1) μmol/L, which was close to the IC(50) value of osimertinib in H1975 cell line (1.0±0.2) μmol/L. The apoptosis rate of H1975-OR was significantly higher in 2-deoxyglucose + osimertinib combination therapy group (26.7±2.4)%, compared to control group (5.1±0.7)%, 2-deoxyglucose monotherapy group (6.1±2.5)% and osimertinib monotherapy group (11.4±2.7)%(all P<0.05). The expression of pro-apoptotic protein BIM in H1975-OR was significantly higher in 2-deoxyglucose+ osimertinib combination therapy group (177.8±28.1)% and the expression of anti-apoptotic protein Bcl-2 in H1975-OR was significantly lower in 2-deoxyglucose+ osimertinib combination therapy group (24.6±5.2)%, compared to control group (100±0)%, all P<0.05. Conclusion: 2-deoxyglucose can reverse the acquired resistance of NSCLC cell line to osimertinib, which may be related to the inhibition of cell glycolysis and the induction of apoptosis.

Esculetin enhances the inhibitory effect of 5-Fluorouracil on the proliferation, migration and epithelial-mesenchymal transition of colorectal cancer.

Colorectal cancer (CRC) is the most common malignant disease worldwide and thus new therapeutic approaches are needed. 5-Fluorouracil (5-FU) remains the most widely used agent to treat colorectal cancer (CRC). However, its clinical efficacy is currently limited by the development of drug resistance. Esculetin (EST), a coumarin, was found to have anti-proliferative and anti-migration activity in cancer. This research aims to evaluated the influence and possible mechanism of EST on the proliferation, migration and epithelial-mesenchymal transition of CRC cell lines. Human CRC cell lines HT-29, SW480, HCT-116, and Caco-2 were treated with various concentrations of EST (0.2, 2, 20, 200, 2000 μg/ml) or 5-FU (0.1, 1, 10, 100, 1000 μg/ml) for 48 h, and cell viability was determined by the MTT and CCK-8 assay. The motility of HCT-116 cells was detected by scratch assay. Western blot was applied to detect the protein expression. Besides, levels of Wnt3a and VEGF in HCT-116 cell culture medium supernatant were analyzed by ELISA. The anti-tumor effect was detected with HCT-116 subcutaneous tumor bearing tumor model by monitoring the tumor vomume in vivo. Finally, the tumoral expression of VEGF was measured by immunohistochemistry, and the expression of Ki67, PCNA, β-catenin, c-Myc, Cyclin D1, MMP2 and MMP7 was measured by Western blot analysis. EST inhibited HCT-116 cell proliferation in a dose-dependent manner. Western blot analysis revealed that EST decreased the expression of Ki67, PCNA, N-cadherin, E-cadherin, vimentin, fibronectin, β-catenin, c-Myc, Cyclin D1, MMP2 and MMP7. Furthermore, EST reduced the release of Wnt3a and VEGF into HCT-116 cells culture medium. After EST treatment, the tumor volume was significant smaller than that of the control group, and the tumoral levels of VEGF were decreased. Moreover, western blot analysis indicated that the expression of Ki67, PCNA, β-catenin, c-Myc, Cyclin D1, MMP2 and MMP7 were also significantly decreased after treated with EST. In addition, in vitro and in vivo anti-tumor results demonstrated that EST combined with 5-FU could increase the inhibitory effect of 5-FU on HCT-116 cells proliferation, migration and epithelial-mesenchymal transition. EST enhances the inhibitory effect of 5-FU on the proliferation, migration and epithelial-mesenchymal transition of CRC.

Knocking down forkhead box M1 reduces glycolytic level in osteosarcoma cells

Objective To study the effects of forkhead box M1 (FOXM1) on glycolysis of osteosarcoma cells. Methods FOXM1 small interfering RNA (siRNA) lentivirus and control siRNA lentivirus infect osteosarcoma cells, named as FOXM1 siRNA and siRNA control, the cells that were not infected with lentivirus were used as Control. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) detection of cell proliferation ability, the colony forming ability of cells was detected by plate cloning test, the content of lactic acid and the content of adenosine-triphosphate (ATP) in the supernatant of the culture medium were detected by the kit, Western blotting was used to detect the level of HK2 and M2-pyruvate kinase (PKM2) protein in the cells. Results The levels of FOXM1 mRNA and protein in FOXM1 siRNA cells were lower than those in siRNA control (FOXM1 mRNA: 0.31±0.03 vs. 0.98±0.13, 1.00±0.09; FOXM1 protein: 0.21±0.02 vs. 0.63±0.08, 0.62±0.06, P<0.05). Compared with Control, siRNA control, the survival rate of FOXM1 siRNA cells was reduced [(64.17±3.25)% vs. (97.56±10.48)%, (100.00±10.25)%, P<0.05], the cell clone formation rate decreased [(21.26±8.66)% vs. (49.89±7.36)%, (45.16±9.23)%, P<0.05], the content of lactic acid in the supernatant decreased [(2.18±0.27) mmol/L vs. (4.62±0.68) mmol/L, (4.56±0.51) mmol/L, P<0.05], the content of ATP in cells also decreased [(16.20±1.65) mmol/L vs. (33.58±4.15) mmol/L, (31.57±2.63) mmol/L, P<0.05], the level of HK2 and PKM2 protein in the cells decreased [HK2: 0.20±0.02 vs. 0.38±0.05, 0.35±0.03; PKM2: 0.32±0.04 vs. 0.78±0.09, 0.75±0.08, P<0.05]. Conclusion Knocking down FOXM1 lowers the ability of proliferate and clone in osteosarcoma cells, the glycolysis level of osteosarcoma cells was reduced. Key words: Osteosarcoma; Glycolysis; Forkhead box M1; Proliferation

A Lactic Acid Bacteria (LAB)-Based Vaccine Candidate for Human Norovirus.

Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the United States. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model for pathogenesis study. In this study, we developed lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. A LAB strain (Lactococcus lactis) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector and was secreted into media supernatants. To test whether LAB-based HuNoV vaccine candidate is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. LAB DNA and VP1 were detected in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces respectively at day 13 post-inoculation, and further increased at later time points. After being challenged with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less, or no, HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that a LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.

Promotion of lipopeptide antibiotic production by Bacillus sp. IA in the presence of rice husk biochar.

The purpose of this study is to isolate the beneficial microorganisms whose growth is promoted in the presence of charcoal materials. We successfully isolated strain IA, whose growth is promoted on an agar plate with charcoal materials, and identified it as a novel strain of the Bacillus sp. The growth of strain IA in the liquid medium was promoted by the addition of both activated charcoal (AC) and rice husk biochar (RHB). Moreover, the sporulation of strain IA in the RHB medium and the antifungal activity of the culture supernatant of the RHB medium were much higher than those with AC. HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A in the RHB medium was 8 times higher than that in the medium without RHB. This is the first paper to describe the positive effect of RHB on microbial metabolisms.