FITC Fluorescence Research Articles

Abstract Or117: JNK2-mediated gut-heart crosstalk in aging-associated AF pathogenesis.

Introduction: Aging is an independent risk factor for atrial fibrillation (AF). We recently revealed a critical role of activated cardiac stress kinase JNK2 in AF risk. Yet, the mechanisms underlying JNK2 activation in the aged heart remain unclear. Emerging evidence suggests that organ crosstalk critically contributes to the development of heart diseases. Interestingly, increased AF episodes were clinically observed in patients with active gastrointestinal (GI) diseases (with a hallmark of enhanced intestinal epithelial barrier permeability (‘leaky gut’), but reduced AF during remission. While both GI dysfunction and enhanced AF risk are seen in aged humans and animal models, whether aging-associated leaky gut leads to AF pathogenesis through activated JNK2 remains unknown. Methods: To assess the leaky gut and AF link, young (Yg, 2 mo) wildtype (WT) and JNK2dn mice (cardiac-specific overexpression of inactive dominant negative (dn) JNK2) were used for aged fecal matter transplant (FMT), while aged (Ag, 24 mo) mice were transplanted with Yg fecal matter to rescue aging-associated AF. Atrial tachycardia/fibrillation (AT/AF) incidence was measured using our well-established protocol, and GI permeability was measured using a serum dextran (4-kDa) FITC fluorescence assay 7 days post-FMT. Results: Aged WT mice had an elevated FITC level (4.9±1.5 µg/ml vs 0.98±0.04 µg/ml; n=11,8; p<0.01) with increased AT/AF inducibility compared to Yg mice (n=7,10; p<0.05). Interestingly, Yg WT recipients subjected to aged FMT (Ag-to-Yg) exhibited an ~3-fold increase in GI permeability (n=14; p<0.01) and significantly enhanced AT/AF inducibility relative to Yg sham controls (8 of 13 vs 0 of 10; p<0.05), while Yg-to-Yg FMT did not alter GI permeability and AT/AF inducibility in Yg recipients (0 of 5, p=NS). Strikingly, cardiac-specific JNK2 inhibition prevented aged-FMT-caused AT/AF in Yg JNK2dn recipient mice (0 of 10; p<0.01), suggesting that activated cardiac JNK2 underlies leaky gut-evoked AF in aging. Further, Yg-to-Ag FMT eliminated aged-associated AT/AF inducibility in aged recipients (0 of 6). Conclusion: Aging-associated GI dysfunction enhances cardiac JNK2 activation, which in turn drives AF risk. Targeting cardiac JNK2 could be a novel anti-AF therapeutic strategy.

Intestinal microbiota dysbiosis contributes to the liver damage in subchronic arsenic-exposed mice.

There is an extensive amount of evidence that links changes in the intestinal microbiota structure to the progression and pathophysiology of many liver diseases. However, comprehensive analysis of gut flora dysbiosis in arsenic-induced hepatotoxicity is lacking. Herein, C57BL/6 mice are exposed to arsenic (1, 2, or 4 mg/kg) for 12 weeks, after which fecal microbiota transplantation (FMT) study is conducted to confirm the roles of the intestinal microbiome in pathology. Treatment with arsenic results in pathological and histological changes in the liver, such as inflammatory cell infiltration and decreased levels of TP and CHE but increased levels of ALP, GGT, TBA, AST, and ALT. Arsenic causes an increase in the relative abundance of Escherichia-Shigella, Klebsiella and Blautia, but a decrease in the relative abundance of Muribaculum and Lactobacillus. In arsenic-exposed mice, protein expressions of Occludin, ZO-1, and MUC2 are significantly decreased, but the level of FITC in serum is increased, and FITC fluorescence is extensively dispersed in the intestinal tract. Importantly, FMT experiments show that mice gavaged with stool from arsenic-treated mice exhibit severe inflammatory cell infiltration in liver tissues. Arsenic-manipulated gut microbiota transplantation markedly facilitates gut flora dysbiosis in the recipient mice, including an up-regulation in Escherichia-Shigella and Bacteroides, and a down-regulation in Lactobacillus and Desulfovibrio. In parallel with the intestinal microbiota wreck, protein expressions of Occludin, ZO-1, and MUC2 are decreased. Our findings suggest that subchronic exposure to arsenic can affect the homeostasis of the intestinal microbiota, induce intestinal barrier dysfunction, increase intestinal permeability, and cause damage to liver tissues in mice.

A FAPI-conjugated FITC fluorescence probe for targeted cancer imaging

The targeted fluorescent probe, FITC-FAPI, can achieve high accumulation at the tumor site and extend the residence time of the fluorescent probe, resulting in clear tumor imaging.

Extraction of polysaccharides from Maca enhances the treatment effect of 5-FU by regulating CD4+T cells

In our previous studies, we used a graded alcohol precipitation method to extract four maca polysaccharide components (MCP1, MCP2, MCP3, and MCP4) from maca with various molecular weights. Compared to other three components, MCP2 had stronger immunoregulatory abilities on CD4+T cells. To avoid the immunosuppressive effect of 5-fluorouracil (5-FU), maca polysaccharides in combination with 5-FU treatment were investigated in this study. The results show that 500 mg/kg and 1000 mg/kg MCP2 could significantly delay the growth of tumor and enhance the anti-tumor effect of 5-FU in vivo. Furthermore, MCP2 can partly recover the proliferation of CD4+T cells after being suppressed by 5-FU in vitro. Additionally, in order to explore the mechanism in which MCP2 acts on CD4+T cells, the MCP2 is marked with FITC fluorescence and synthesis MCP2-Tyr-FITC for the first time. Confocal microscope results show that MCP2-Tyr-FITC can directly bind to the surface of CD4+T cells. Together, our work demonstrates that maca polysaccharides could enhance the anti-tumor effect when combined with 5-FU by regulating CD4+T cells, suggesting a novel potential immunomodulator in tumor therapy.

Spectral Multiplexing of Fluorescent Endoscopy for Simultaneous Imaging with Multiple Fluorophores and Multiple Fields of View

Complex clinical procedures and small-animal research procedures can benefit from dual-site imaging provided by multiple endoscopic devices. Here, an endoscopic system is proposed which enables multiple fluorescence microendoscopes to be spectrally multiplexed on a single microscope base, enabling light sources and optical relays to be shared between endoscopes. The presented system is characterized for resolution using USAF-1951 resolution test charts and for modulation transfer function using the slanted edge method. Imaging is demonstrated both directly and with microendoscopes attached. Imaging of phantoms was demonstrated by targeting USAF charts and fiber tissues dyed for FITC and Texas Red fluorescence. Afterwards, simultaneous liver and kidney imaging was demonstrated in mice expressing mitochondrial Dendra2 and injected with Texas Red-dextran. The results indicate that the system achieves high channel isolation and submicron and subcellular resolution, with resolution limited by the endoscopic probe and by physiological movement during endoscopic imaging. Multi-channel microendoscopy provides a potentially low-cost means of simultaneous multiple endoscopic imaging during biological experiments, resulting in reduced animal harm and potentially increasing insight into temporal connections between connected biological systems.

Ratiometric fluorescent Si-FITC nanoprobe for immunoassay of SARS-CoV-2 nucleocapsid protein.

Coronavirus disease 2019 (COVID-19) highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission. Here, we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles (NPs) for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N protein). The synthesis of Si-FITC NPs did not need post-modification, which addressed the issue of quantum yield reduction during the coupling reaction. Si-FITC NPs showed two distinct peaks, Si fluorescence at λem = 385 nm and FITC fluorescence at λem = 490 nm. In the presence of KMnO4, Si fluorescence was decreased and FITC fluorescence was enhanced. Briefly, in the presence of N protein, catalase (CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates. Subsequently, hydrogen peroxide (H2O2) and Si-FITC NPs/KMnO4 were injected into the microplate together. The decomposition of H2O2 by CAT resulted in remaining of KMnO4, which changed the fluorescence intensity ratio of Si-FITC NPs. The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL, and the detection limit was 0.003 ng/mL, which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL. The N protein concentration can be accurately determined in human serum. Furthermore, the COVID-19 and non-COVID-19 patients were distinguishable by this method. Therefore, the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity.Electronic Supplementary MaterialSupplementary material (characterization of Si-FITC NPs (FTIR, HRXPS); stability investigation of Si-FITC NPs (photostability, pH stability, anti-interference ability); stability investigation of free FITC (pH value, KMnO4); quenching mechanism of KMnO4 (UV-vis absorption spectra, fluorescence lifetime decay curves); reaction condition optimization of biotin-CAT with H2O2 (pH value, temperature, time); detection of N protein using commercial ELISA Kit; selectivity investigation of assays for SARS-CoV-2 N protein detection; determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-5005-z.

Nitric oxide synthase inhibitor is an effective therapy for ischemia‐reperfusion injury in mice

BackgroundInhibition of nitric oxide synthase (NOS) has been shown to paradoxically protect against ischemic brain injury by an unknown mechanism. Recently, we found that NOS inhibitor, N (gamma)‐nitro‐L‐arginine methyl ester (L‐NAME), protects primary cultures of endothelial cells and neurons against oxygen‐glucose deprivation‐reoxygenation injury by preventing NOS uncoupling. Thus, the objective of the present study was to demonstrate the effectiveness of locally administered L‐NAME against ischemia‐reperfusion (I‐R) injury.MethodsTransient focal cerebral ischemia in male mice (C57Bl/6, 2‐4 months) was achieved by filament method of MCAO with a 60 min occlusion. We employed a novel approach to inject L‐NAME (1 mg/Kg dosages at a rate of 60 ug/minute) locally into the ischemic zone by threading a catheter up the middle cerebral artery. This was timed to the onset of reperfusion by rapidly followng the removal of the occlusion with the silicon suture. Following 24‐hour reperfusion, infarct size was determined by 2,3,5‐Triphenyltetrazolium chloride (TTC) staining and neurological function was assessed by employing a standard neurological score (0‐4 range) and Rotarod performance test.ResultsAdmnistration of FITC Dextran 40K and fluorescence microscopy of the brain slices 1 hour post‐ischemia confirmed the effective delivery of injected L‐NAME into the ischemic tissue. Localized injection of L‐NAME was protective against I‐R injury and promoted significant recovery of neurological and motor function (Figure).ConclusionLocal NOS inhibition could serve as an adjuvant for reperfusion therapies of ischemic stroke.

Detection of Mu‐Opioid Receptors in SH‐SY5Y Human Neuroblastoma Cells by Flow Cytometry

PurposePharmacological actions of morphine are mediated through G‐protein‐coupled receptors (GPCRs), specifically mu‐opioid receptors (MORs). Morphine‐induced activation of GPCRs inhibits adenylyl cyclase and decreases 3’,5’‐cyclic adenosine monophosphate (cAMP). Neuroblastoma SH‐SY5Y cell lines endogenously express MORs. Our aim was to develop a flow cytometry (FC) assay for MORs on the surface of SH‐SY5Y cells. This assay will provide in‐depth understanding of opioid‐induced downstream cAMP signaling.MethodsSH‐SY5Y human neuroblastoma cells (ATCC® CRL2266™) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37ºC/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and a stably transfected SH‐SY5Y cell line was established using GloSensor™ ‐23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Cells were washed in FACS buffer (PBS + 2% FBS) and incubated for 1h at room temperature with monoclonal rabbit anti‐MOR antibody (Ab) (Novus Biologicals). Following the wash, cells were incubated with donkey anti‐rabbit secondary Ab (Abcam). We used monoclonal mouse anti‐muscarinic Ab (Novus Biologicals) and donkey anti‐mouse secondary Ab (Jackson Labs). Both secondary Abs are bound to a fluorophore (Alexa Fluor® 488) which emits light when excited by FITC channel frequency (excitation/emission: 495nm/519nm). A Cytoflex flow cytometer (Beckman Coulter) analyzes cells for FITC fluorescence emission. Multiple primary and secondary concentrations were tested; Human TruStain Fc blocker (BioLegend) was used to prevent nonspecific tagging.ResultsRelative fluorescence emission was measured as percent shift increase from the untagged control group. Because MOR Abs have been tested in a limited capacity, we tested for presence of muscarinic receptors (another endogenously expressed GPCR in these cells) to establish the FC assay. Initial trials with low primary and secondary MOR Ab concentrations showed limited shifts (~1‐2%), however, muscarinic receptor Abs bound to their receptors at lower concentrations compared to MOR Abs. Testing muscarinic receptors showed secondary Ab concentration‐dependent fluorescence shifts indicating that the technique was viable. Studies with increased concentration ratios of primary and secondary MOR Abs showed secondary Ab concentration‐dependent response with maximum % shifts reaching up to 16%. Use of Fc blocker eliminated nonspecific binding, increasing the percent shift.ConclusionCompared to untagged controls, antibody‐tagging on SH‐SY5Y cell surface produced greater immunofluorescence indicating presence of MORs in both non‐transfected and 23F cAMP plasmid transfected cells. Direct binding of receptor modulators leads to altered downstream cAMP signaling as shown in previous in vitro signaling studies and behavioral mouse models. Detection of the MOR is essential to ensure that cAMP signaling is regulated by MOR agonists. In future studies, we plan to investigate the expression of endothelin‐A receptor (ETAR) and explore potential interactions between MOR and ETAR in SH‐SY5Y cells.

Dual recognition and highly sensitive detection of Listeria monocytogenes in food by fluorescence enhancement effect based on Fe3O4@ZIF-8-aptamer

A highly sensitive and selective foodborne pathogen detection system is essential to ensure food quality and public health. Therefore, in this study, a dual recognition and highly sensitive detection of Listeria monocytogenes by fluorescence enhancement strategy based on Fe3O4 @ZIF-8 have been developed. Aptamer absorbed on Fe3O4 @ZIF-8 were used for the specific capture of Listeria monocytogenes, and the conjugates of anti-Listeria monocytogenes antibody-biotin and streptavidin-FITC were used as signal amplification and fluorescence recognition probes binding with target bacteria. Simultaneously, the quantitative detection of Listeria monocytogenes was realized by monitoring the increase of FITC fluorescence signal due to hydrophobic environment. The linear range of the detection of pure culture was from 1.4 × 101 to 1.4 × 107 CFU/mL, with the detection limits as low as 0.88 CFU/mL and the coefficient of variation of each concentration no more than 2%. A good specificity of the detection system was also showed in the results, and the detection took about 70 min. Furthermore, the spiked samples of pork and milk were also tested by the detection system, and the recoveries ranged from 88.0% to 103.8%, with the coefficient of variation less than 12.8%. Taking advantages of sensitivity, rapidly and good selectivity, the proposed strategy in this study has great application potential in detection of foodborne pathogens in food.

Impact of Superparamagnetic Iron Oxide Nanoparticles on THP-1 Monocytes and Monocyte-Derived Macrophages.

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently under examination for magnetic particle imaging, which represents a radiation free technology for three-dimensional imaging with high sensitivity, resolution and imaging speed. SPIONs are rapidly taken up by monocytes and other phagocytes which carry them to the site of inflammation. Therefore, the SPION biocompatibility is an essential parameter for a widespread MPI usage. Many improvements are expected from SPION development and its applications for cell visualization, but the impact of MPI optimized dextran coated SPIONs on the cellular characteristics of monocytic cells has been poorly studied up to now. THP-1 monocytes, monocyte-derived macrophages (MDM) as well as peripheral blood monocytes were incubated with MPI-optimized dextran-coated SPIONs of a size between 83.5 and 86 nm. SPION uptake was measured by FITC fluorescence of labeled SPIONs and Prussian blue staining. The activation of monocytes and MDMs was evaluated by CD14, CD11b and CD86 in flow cytometry. The secretion of IL-1β, and IL-10 was analyzed in supernatants. SPIONs were rapidly taken up by monocytes and monocyte-derived macrophages while no decrease in cell viability was observed. Expression patterns of CD11b, CD14, and CD86 were not affected in THP-1 monocytes and MDMs. Monocyte differentiation in macrophages was hindered during SPION uptake. THP-1 monocytes as well as monocyte-derived macrophages showed significantly increased IL-1β and decreased IL-10 secretion by tendency after SPION treatment. Dextran-coated SPIONs showed a low cytotoxicity on monocytes but exert undesirable inflammatory side effects that have to be considered for imaging applications.

Advanced age exacerbates intestinal epithelial permeability after burn injury in mice.

Advanced age is an independent risk factor for morbidity and mortality after burn injury. Following burn, the intestines can become permeable leading to the leakage of bacteria and their products from the lumen of the ileum to the portal and systemic circulation. Here, we sought to determine the effects of advanced age on intestinal permeability post burn injury and assess intestinal inflammatory biomarkers. Young (4-5months) and aged (18-22months) female BALB/cBy mice were subjected to a 12-15% total body surface area (TBSA) sham or burn injury. 24h after injury, mice were euthanized, and organs collected. Colony-forming units (CFU) were counted from plated mesenteric lymph nodes (MLN). Gene expression of ileal tight junctional proteins, occludin and zonula occludens 1 (ZO-1), in addition to ileal damage associated molecular pattern (DAMP) proteins, S100A8 and S100A9, as well as ileal inflammatory markers IL-6 and TNF-α were measured by qPCR. Intestinal cell death was measured by ELISA. Intestinal permeability was determined by FITC fluorescence in serum; 4kD FITC-dextran was given by oral gavage 3h before euthanasia. Aged mice subjected to burn injury had increased intestinal permeability as evidenced by a 5.8-fold higher level of FITC-dextran in their serum when compared to all other groups (p<0.05). In addition, aged burn-injured mice exhibited heightened bacterial accumulation in the MLN with a 15.5-fold increase over all other groups (p<0.05). Histology of ileum failed to show differences in villus length among all groups. Analysis of ileal tight junctional proteins and inflammatory marker gene expression revealed no difference in Ocln, Tjp1, Il6, or Tnf expression among all groups, but 2.3 and 2.9-fold upregulation of S100a8 and S100a9, respectively, in aged burn-injured mice relative to both young groups and aged sham-injured mice (p<0.05). Lastly, cell death in the ileum was elevated more than two-fold in aged burn-injured mice relative to young animals regardless of injury (p<0.05). These data demonstrate that advanced age exacerbates intestinal epithelial permeability after burn injury. Heightened apoptosis may be responsible for the elevated intestinal leakiness and accumulation of bacteria in mesenteric lymph nodes. In addition, S100a8/9 may serve as a biomarker of elevated inflammation within the intestine.

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure.

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P<0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.

A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected by liposuction surgery, was digested with collagenase to separate adipocytes from other tissue cells, comprising blood and lymphatic endothelial cells, fibroblasts, and all vessel- and tissue-resident leukocytes - the stromal vascular fraction (SVF). SVF cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, adding Brefeldin-A to prevent cytokine secretion during the final 4 hours. Cells were incubated with CD11b-FITC and CD40-APC (M1 MØ)‚ or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then incubated with either (1) anti-TNF-PE, (2) anti- IL-1β-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGFβ-PE or (6) isotype-IgG-PE (control), and stained with Hoechst 33342, preserved in permanent mounting media and examined by confocal microscopy. The FITC, PE and APC fluorescence channels were set to achieve minimal cross-channel emission using single-colour controls and voltages set for optimal detection by thresholding on isotype-IgG stained activated cells. Finally, transmission and z-stack images were captured. Cells were analysed as regions of interest (ROI) based on Hoechst-33342 then enumerated as FITC+, FITC+APC+ or FITC+APC+PE+ using an ImageJ script and exported into Excel. This permitted the examination of >9000 SVF cells individually, per LE sample. This method allows for the analysis of a high number of heterogeneous cells defined into any subtype or combination by the investigators' choice of surface and intracellular expression profiles. Fibroblasts, or other cytokine producing cells, can also be analysed by using other antibodies, and the cell count data can be correlated with any clinical or laboratory data.

Peptide modified manganese-doped iron oxide nanoparticles as a sensitive fluorescence nanosensor for non-invasive detection of trypsin activity in vitro and in vivo.

Herein, a fluorescence turn-on nanosensor (MnIO@pep-FITC) has been proposed for detecting trypsin activity in vitro and in vivo through covalently immobilizing an FITC modified peptide substrate of trypsin (pep-FITC) on manganese-doped iron oxide nanoparticle (MnIO NP) surfaces via a polyethylene glycol (PEG) crosslinker. The conjugation of pep-FITC with MnIO NPs results in the quenching of FITC fluorescence. After trypsin cleavage, the FITC moiety is released from the MnIO NP surface, leading to a remarkable recovery of FITC fluorescence signal. Under the optimum experimental conditions, the recovery ratio of FITC fluorescence intensity is linearly dependent on the trypsin concentration in the range of 2 to 100 ng mL−1 in buffer and intracellular trypsin in the lysate of 5 × 102 to 1 × 104 HCT116 cells per mL, respectively. The detection limit of trypsin is 0.6 ng mL−1 in buffer or 359 cells per mL HCT116 cell lysate. The MnIO@pep-FITC is successfully employed to noninvasively monitor trypsin activity in the ultrasmall (ca. 4.9 mm3 in volume) BALB/c nude mouse-bearing HCT116 tumor by in vivo fluorescence imaging with external magnetic field assistance, demonstrating that it has excellent practicability.

Flavor and texture characteristics of microwave-cooked Kung Pao Chicken by different heat conduction effects and further aroma improvement with moderate enzymatic hydrolyzed chicken fat.

Ceramic dish cooking method (CDCM) and microwave absorption dish cooking method (MADCM) were used to obtain one-step microwave-cooked Kung Pao Chicken. Processing the optimization of recipes, steaming time and microwave time was conducted for microwave cooking methods. CDCM showed higher taste scores, better umami and sweet attributes, and better color and aroma than MADCM. The NMR and FITC fluorescence analysis results indicated that free water in chicken cooked by CDCM was lost more and tends to shift to immobilized water during the microwave heating as compared with MADCM. However, the aroma intensity by CDCM was weaker than the traditional cooking method (TCM). Electronic nose analysis also showed difference in the flavor profile from CDCM and TCM. According to the GC-MS analysis, aldehydes, the oxidation products of fats, were higher from TCM than from other cooking methods. Therefore, enzymatic hydrolyzed chicken fat at 5 g per 150 g chicken with a degree of enzymatic hydrolysis of 17.00% was used in CDCM to produce ideal fatty and meaty flavor. Both fatty and meaty flavor have increased by 52% and 60% respectively, with less off-flavor, thus, obtaining a similarity of 92% compared to TCM and with appropriate contents of volatiles such as hexanal, heptanal, (E)-2-octenal, (E)-2-decenal, (E)-2-nonenal and 2,4-decadienal.

Chromogranin A (CGA)-derived polypeptide (CGA47–66) inhibits TNF-α-induced vascular endothelial hyper-permeability through SOC-related Ca2+ signaling

CGA1−78 (Vasostatin-1, VS-1) a N-terminal Chromogranin A (CGA)-derived peptide, has been shown to have a protective effect against TNF-α-induced impairment of endothelial cell integrity. However, the mechanisms of this effect have not yet been clarified. CGA47−66 (Chromofungin, CHR) is an important bioactive fragment of CGA1−78. The present study aims to explore the protective effects of CHR on the vascular endothelial cell barrier response to TNF-α and its related Ca2+ signaling mechanisms. EA.hy926 cells were used as a vascular endothelial culture model. The synthetic peptides CHR and CGA4−16 were assessed for their ability to suppress TNF-α-induced EA.hy926 cells hyper-permeability through Transwell® and TEER assays. Changes in [Ca2+]i were measured through confocal laser scanning microscopy. SOC channel currents (Isoc) were measured via patch-clamp analysis. RT-PCR and western blot were used to analyze mRNA and protein expression of the transient receptor potential channels TRPC1 and TRPC4, respectively. FITC and rhodamine-phalloidin fluorescence were used to assess cell morphology and the distribution of MyPT-1 and F-actin. Compared to untreated cells, TNF-α increased the permeability of EA.hy926 cells that was inhibited by pre-treatment with CHR (10–1000 nM) in concentration-dependent manner, and the effect was most obvious at 100 nM, but CGA4−16 (100 nM) had no effect. TNF-α treatment increased the phosphorylation of MyPT-1 and stress fiber formation. CHR (10–1000 nM) pretreatment inhibited the cytoskeletal rearrangements and increased [Ca2+]i in response to TNF-α treatment. CHR also reduced TRPC1 expression following TNF-α induction. Similar to SOC inhibitor 2-APB, CHR suppressed IP3 mediated SOC activation. These findings suggest that CHR inhibits TNF-α-induced Ca2+ influx and protects the barrier function of vascular endothelial cells, and that these effects are related to the inhibition of SOC and Ca2+ signaling by CHR.

A microfluidic strategy to fabricate ultra-thin polyelectrolyte hollow microfibers as 3D cellular carriers.

Microfluidics-based microfibers have been widely used as bottom-up scaffolds for tissue engineering applications. Different forms of microfibers with certain thickness of shell have been developed during the past decade. Ultra-thin microfiber, as a special and promising carrier of cells, was less explored. In this work, by using the interfacial ionic interaction between sodium alginate (NaA) and chitosan (CS), a novel ultra-thin polyelectrolyte hollow microfiber with the diameter of ~200 μm and the shell thickness of 1.3 ± 0.3 μm was fabricated via a microfluidic device for liver tissue engineering. The fluorescence of FITC labeled CS confirmed the inner CS layer of the fabricated microfiber and the SEM results illustrated its ultra-thin characteristic. Although there are only two layers in the ultra-thin polyelectrolyte hollow microfiber, the following cells encapsulation experiments indicated that it could bear cells loading and the hollow space of the microfibers could encapsulate sufficient number of cells for tissue engineering applications. The presence of inner CS layer in the microfiber promoted cell adhesion and ultra-thin shell characteristic facilitated the exchange of nutrient substance and O2 and thus promoted cell proliferation. HepG2 cells encapsulated in the microfibers maintained favorable viability, proliferation ability and hepatic specific functions during 10 days' culture. These results suggest that the established polyelectrolyte microfibers hold great potential applications in the field of liver tissue engineering. We believe this work will lead to the development of innovative methodologies and materials for both cell culture and biomedical application.

Annona stenophylla aqueous extract stimulate glucose uptake in established C2Cl2 muscle cell lines.

Annona stenophylla is a folk medicine popularly used in Zimbabwe for the treatment of many ailments. This study was carried out to determine some of the possible anti diabetic mechanisms of its action using in vitro cell culturing methods. A. stenophylla's effects on glucose uptake were tested using muscle cells (C2Cl2). Expression of glucose 4 transporters was determined by treating cell lines with plant extract. Total RNA was isolated and using RT-PCR, GLUT 4 expression levels were quantified. Translocation of GLUT 4 was assessed using FITC fluorescence measured by flow cytometry. Treatment of cells with plant extract significantly increased glucose uptake in a concentration dependent manner, with the highest concentration (250 µg/ml) giving 28% increased uptake compared to the negative control. The increase in glucose uptake (2.5 times more than control) was coupled to increase in GLUT 4 mRNA and subsequently GLUT 4 translocation. Wortmannin expunged the A. stenophylla induced increase in GLUT 4 mRNA and glucose uptake. The results suggest that A. stenophylla aqueous extract increases glucose uptake partly through increasing the GLUT 4 mRNA and translocation potentially acting via the PI-3-K pathway. This study confirms the ethnopharmacological uses of A. stenophylla indicating potential for anti-diabetic products formulation.

Cell surface pH imaging using poly(ethylene glycol)-phospholipid: its potential as the core structure of membrane anchored-probes

Various physiological and pathological processes are accompanied with the local acidification of extracellular local pH. However, imaging tools to investigate the spatio-temporal dynamics as well as the functional significance of cell surface pH are limitedly available. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). PEG-lipid, amphiphilic synthetic polymer, is a biomaterial originally synthesized for cell-surface engineering for transplantation therapy. When added into the cell culture medium, FITC-PEG-lipid was spontaneously inserted into the plasma membrane via its phospholipid moiety. FITC-PEG-lipid was retained at the extracellular surface due to the hydrophobic PEG moiety. The ratiometric readout of FITC fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH (pH 5.0-7.5). The pH measurement with FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pH unit for the external solutions at pH 5.9, 6.0 and 6.1, near the inflection point of fluorescence ratio. The response of FITC-PEG-lipid to the extracellular pH was reversible. Continuous alteration of extracellular pH was successfully visualized by time-lapse imaging analysis. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell surface-anchored pH probe. The simple labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.

Sensing ultra-trace dopamine by restoration of fluorescence on locally acidified gold nanoparticles.

An ultra-sensitive sensor was fabricated to measure dopamine through quenching and restoring FITC fluorescence by the competitive binding of dopamine and N-acetylneuraminic acid with mercaptophenylboronic acid anchored on the gold nanoparticles. Assessed by quantifying dopamine in human urine samples, it reached a limit of detection of as low as 50 pM dopamine over a linear range from 10-10 M to 10-7 M. It also features low technical barriers and operation cost, and the strategy proposed could be extendable to other analytes and not restricted to the gold nanoparticles.