Bivalent Aptasensor Based on Silver-Enhanced Fluorescence Polarization for Rapid Detection of Lactoferrin in Milk.

Here we report a novel type of bivalent aptasensor based on silver-enhanced fluorescence polarization (FP) for detection of lactoferrin (Lac) in milk powder with high sensitivity and specificity. The novel two split aptamers were obtained from the aptamer reported in our previous SELEX (systematic evolution of ligands by exponential enrichment) selection, and their minimal structural units were optimized on the basis of their affinity and specificity. Also, dual binding sites of split aptamers were verified. The bivalent aptamers were modified to be linked with signal-molecule fluorescein isothiocyanate (FITC) and enhancer silver decahedral nanoparticles (Ag10NPs). The split aptamers could bind to different sites of Lac and assemble into a split-aptamers-target complex, narrowing the distance between Ag10NPs and FITC dye. As a result, Ag10NPs could produce a mass-augmented and metal-enhanced fluorescence (MEF) effect. In general, ternary amplification based on Ag10NPs, split aptamers, and the MEF effect all contributed to the significant increase of FP values. It was proved that the sensitivity of this assay was about 3 orders of magnitude over traditional aptamer-based homogeneous assays with a detection limit of 1.25 pM. Furthermore, this design was examined by actual milk powder with rapid and high-throughout detection.

Simultaneous detection of α-Lactoalbumin, ß-Lactoglobulin and Lactoferrin in milk by Visualized Microarray.

BACKGROUND: α-Lactalbumin (a-LA), ß-lactoglobulin (ß-LG) and lactoferrin (LF) are of high nutritional value which have made ingredients of choice in the formulation of modern foods and beverages. There remains an urgent need to develop novel biosensing methods for quantification featuring reduced cost, improved sensitivity, selectivity and more rapid response, especially for simultaneous detection of multiple whey proteins. RESULTS: A novel visualized microarray method was developed for the determination of a-LA, ß-LG and LF in milk samples without the need for complex or time-consuming pre-treatment steps. The measurement principle was based on the competitive immunological reaction and silver enhancement technique. In this case, a visible array dots as the detectable signals were further amplified and developed by the silver enhancement reagents. The microarray could be assayed by the microarray scanner. The detection limits (S/N = 3) were estimated to be 40 ng/mL (α-LA), 50 ng/mL (ß-LG), 30 ng/mL (LF) (n = 6). CONCLUSIONS: The method could be used to simultaneously analyze the whey protein contents of various raw milk samples and ultra-high temperature treated (UHT) milk samples including skimmed milk and high calcium milk. The analytical results were in good agreement with that of the high performance liquid chromatography. The presented visualized microarray has showed its advantages such as high-throughput, specificity, sensitivity and cost-effective for analysis of various milk samples.

Tear trough deformity: different types of anatomy and treatment options.

AIM: To explore the efficacy of tear trough deformity treatment with the use of hyaluronic acid gel or autologous fat for soft tissue augmentation and fat repositioning via arcus marginalis release. MATERIAL AND METHODS: Seventy-eight patients with the tear trough were divided into three groups. Class I has tear trough without bulging orbital fat or excess of the lower eyelid skin. Class II is associated with mild to moderate orbital fat bulging, without excess of the lower eyelid skin. Class III is associated with severe orbital fat bulging and excess of the lower eyelid skin. Class I or II was treated using hyaluronic acid gel or autologous fat injections. Class III was treated with fat repositioning via arcus marginalis release. The patients with a deep nasojugal groove of class III were treated with injecting autologous fat into the tear trough during fat repositioning lower blepharoplasty as a way of supplementing the volume added by the repositioned fat. RESULTS: Seventy-eight patients with tear trough deformity were confirmed from photographs taken before and after surgery. There were some complications, but all had complete resolution. CONCLUSIONS: Patients with mild to moderate peri-orbital volume loss without severe orbital fat bulging may be good candidates for hyaluronic acid filler or fat grafting alone. However, patients with more pronounced deformities, severe orbital fat bulging and excess of the lower eyelid skin are often better served by fat repositioning via arcus marginalis release and fat grafting.

DAPT in the control of human hair follicle stem cell proliferation and differentiation.

INTRODUCTION: Stem cells from hair follicle have great therapeutic applications in regenerative medicine as sources of cells for transplantation. The differentiation pathway selected by hair follicle stem cells (HFSC) is largely determined by local microenvironmental signals. In this study, human hair follicle stem cells were treated with Notch signaling blocker to explore a new approach to modulate human hair follicle stem cell proliferation and differentiation in vitro. AIM: To define the functional consequences of blocking the Notch signaling pathway on the proliferation and differentiation of human HSCs. MATERIAL AND METHODS: The human hair follicle stem cells were treated with various concentrations of Notch signaling blocker DAPT (24-diamino-5-phenylthiazole). The viability of the cells was investigated with clonogenicity assays. The expression of stem cell markers, cell cycle and cell apoptosis were analysed by flow cytometry. RESULTS: Notch blocking leads to promotion of human hair follicle stem cell proliferation and inhibition of differentiation in response to DAPT. The maximum effect of DAPT on the viability of human HFSC was observed at a concentration of 20 µM. We found that DAPT treatment results in downregulation of Hes1 and p21 and upregulation of Wnt10b. CONCLUSIONS: γ-Secretase inhibitor DAPT has a modulatory effect on the human HFSC. The DAPT may modulate human hair follicle stem cell proliferation and differentiation through regulation of p21 and Wnt-10b.

Promotional effect of platelet-rich plasma on hair follicle reconstitution in vivo.

BACKGROUND: Platelet-rich plasma (PRP) containing various growth factors has attracted attention in various medical fields. PRP has recently been used during hair transplantation to increase hair density. OBJECTIVE: To investigate the effects of PRP on hair follicle (HF) reconstitution. METHODS AND MATERIALS: Freshly isolated epidermal cells and cultured dermal papilla cells (DPCs) were mixed with various concentrations of activated PRP and transferred to a grafting chamber that was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting, and HF formation was monitored for 4 weeks. RESULTS: We observed a significant difference (p < .05) in the number of newly formed follicles in the area of reconstituted skin (344 ± 27 with 10% PRP vs 288 ± 35 without PRP). PRP also shortened the time of hair formation significantly; the first hairs were observed in 18 ± 1 days using 10% PRP, versus 20 ± 1 days without PRP. CONCLUSION: A considerable effect of PRP on the time of hair formation and the yield of HF reconstitution was observed in this study. Considering the limited evidence available to judge its efficacy, further studies are required to investigate the mechanism of action of PRP.

A syndrome differentiation model of TCM based on multi-label deep forest using biomedical text mining.

Syndrome differentiation and treatment is the basic principle of traditional Chinese medicine (TCM) to recognize and treat diseases. Accurate syndrome differentiation can provide a reliable basis for treatment, therefore, establishing a scientific intelligent syndrome differentiation method is of great significance to the modernization of TCM. With the development of biomdical text mining technology, TCM has entered the era of intelligence that based on data, and model training increasingly relies on the large-scale labeled data. However, it is difficult to form a large standard data set in the field of TCM due to the low degree of standardization of TCM data collection and the privacy protection of patients' medical records. To solve the above problem, a multi-label deep forest model based on an improved multi-label ReliefF feature selection algorithm, ML-PRDF, is proposed to enhance the representativeness of features within the model, express the original information with fewer features, and achieve optimal classification accuracy, while alleviating the problem of high data processing cost of deep forest models and achieving effective TCM discriminative analysis under small samples. The results show that the proposed model finally outperforms other multi-label classification models in terms of multi-label evaluation criteria, and has higher accuracy in the TCM syndrome differentiation problem compared with the traditional multi-label deep forest, and the comparative study shows that the use of PCC-MLRF algorithm for feature selection can better select representative features.

Comparison of autologous bone marrow mononuclear cells transplantation and mobilization by granulocyte colony-stimulating factor in experimental spinal injury.

BACKGROUND: Recent studies have shown that autologous adult stem cells may be a protocol of treatment for spinal cord injury (SCI) in humans. The purpose of this study was to compare the effect of an injection of autologous bone marrow mononuclear cells (BMMCs) and bone-marrow cell mobilization induced by granulocyte colony stimulating factor (G-CSF) in rats with SCI. METHODS: Adult rats were assigned into three groups: control group (SCI only), SCI + BMMCs, and SCI + G - CSF. Neurological scores and electrophysiological testing were done in all rats before SCI, and at 1, 7, 14, 28, 56 days post-SCI. Simultaneously, immunohistochemical labeling and TUNEL assay were performed at the given time. RESULTS: From 1 week post-SCI onward, animals treated with BMMCs or G-CSF had higher BBB scores than control group. Motor and somatosensory evoked potentials (MEPs and SEPs, respectively) of the treated group were significantly better than those in control group at 2 weeks. After sacrifice, compared with the control, animals treated with BMMCs and G-CSF significantly increased expressions of Brdu, NSE, GFAP, Factor VIII, BDNF, and reduced expression of apoptosis cells around the lesion site. Our results indicate that administration of BMMCs and G-CSF in a SCI model achieves similarly positive effect on functional and histologic recovery. CONCLUSIONS: The use of G-CSF may be a viable alternative to BMMCs for autograft in patients with SCI.

Phenotypic and Cellular Characteristics of a Stromal Vascular Fraction/Extracellular Matrix Gel Prepared Using Mechanical Shear Force on Human Fat.

The retention of fat-derived grafts remains a challenge for regenerative medicine. Fat aspirates from patients undergoing liposuction were prepared into standard Coleman fat grafts or further isolated using mechanical shear force to prepare a stromal vascular fraction (SVF)/extracellular matrix (ECM) gel. The retention rate of the SVF/ECM gel was significantly higher than that of the Coleman fat at 3, 14, 28, and 60 days following transplantation on the backs of nude mice. The viscosity of the fat was directly proportional to the shearing force. Although the mechanical isolation did not affect the total number of cells, it significantly decreased the number of living cells. Flow cytometry showed a greater number of mesenchymal stem cells, supra-adventitial (SA)-adipose stromal cells (ASCs), and adipose-derived stem cells but a lower number of endothelial progenitor cells in the SVF/ECM gel than in the Coleman fat. Thus, mechanical isolation of fat can increase the pluripotency of adipocytes, which can improve graft retention in cell therapy.

Prevalence and genomic investigation of Salmonella isolates recovered from animal food-chain in Xinjiang, China.

Salmonella is a major foodborne pathogen worldwide, causing serious cases of morbidity and mortality due to the consumption of contaminated foods. Animal-borne foods were considered the main source of transferring Salmonella to humans; however, route surveillance by genomic platforms along the food-chain is limited in China. Here, we proceeded to the application of whole genome sequencing in the epidemiological analysis of Salmonella isolated along the food-chain in Xinjiang, China. A total of 2408 samples were collected from farms, slaughterhouses, and markets, and subjected to the isolation of Salmonella strains. 314 (13.04%) of the samples were positive for Salmonella. Phenotypic antimicrobial resistance was conducted by the broth dilution method using 14 antimicrobial agents belonging to ten classes for all 314 isolates. A selection of representative 103 isolates was subjected to whole-genome sequencing for understanding the Salmonella diversity, including serovars, antimicrobial and virulence genes, plasmid types, multi-locus sequence types, and allelic types. We found that S. Agona was the dominant serovar and O:4(B) was the dominant serogroup. The dominant genotype was ST13 and each serovar has a unique MLST pattern. Plasmids prediction reported Col(MGD2)_1 and Col(Ye4449)_1 as the dominant plasmids, in addition to the detection of IncFII(S)_1 and IncFIB(S)_1 carried by all S. Enteritidis isolates. Importantly, virulence genes prediction showed the presence of cdtB gene encoding typhoid toxins, spv genes, and pef gene cluster encoding fimbriae in the genomes of S. Indiana and S. Enteritidis. Phenotypic antimicrobial resistance identified 92.04% of the sampled isolates as multi-drug resistance (MDR), with high resistance to tetracycline (78.03%; 245/314), amoxicillin/ clavulanic acid (75.80%; 238/314), and ampicillin (70.70%; 222/314). Together, we firstly reported the prevalence of MDR Salmonella isolates harboring critical virulence factors transmission via animal-borne food-chain in Xinjiang, hence route surveillance by whole-genome sequencing platform could facilitate recognition and project early warning for the emerging MDR clones along the food-chain.

Smartphone-based visualized microarray detection for multiplexed harmful substances in milk.

In this paper, we report a sensitive, simple and inexpensive analytical method for the immunoassay microarray based on a smartphone in which various harmful substances in milk could be assayed. Tetracyclines (TCs) and Quinolones (QNs) were selected as the model targets in this study. TCs and QNs antigens were immobilized in the microarray and then samples containing free of antibiotics and corresponding antibodies as well as AgNPs labeled secondary antibodies were added to the microarray. The signal of this competitive format was further amplified by silver enhancement technique based on the development reagents and achieved a visual dots in the array. The resulting microarray could be detected by the smartphone placed in the minicartridge. The limit of detection (LOD) of this novel detection platform was 1.51ngmL-1 (TCs) and 1.74ngmL-1 (QNs). To achieve one-well quantitative analysis, a series of gradient concentration mouse IgG was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentrations of mouse IgG. The results showed that a quantitative detection of TCs and QNs established were consistent with external standard curve. Compared to other methods, this method was superior in terms of detection limit, time saving, and one-well quantitative detected with smartphone which were simple sample-preparation.

Neonatal murine skin-derived cells transplanted using a mini-chamber model produce robust and normal hair.

Hair follicle reconstitution models are useful tools for investigating signalling and cytokines during hair follicle morphogenesis and cycling. The chamber model is one of the most established methods available for the study of hair follicle reconstitution and appears to be the most reproducible. However, the chamber model has several deficiencies: infection of skin wounds and subsequent animal death commonly occur, a large number of cells are required and only one chamber can be transplanted onto each animal. We modified these deficiencies by using a mini-chamber method, which has the advantages of having a high graft take rate, requiring fewer cells and allowing several mini-chambers to be transplanted onto each animal. In our study, cultured dermal cells at different passages (0 to high) lost the ability to reconstruct hair follicles, but dermal cells cultured overnight (12 h) retained this ability. Using the assay, newborn mice dermal cells that were freshly isolated and cultured overnight (12 h), as well as cultured dermal papilla cells from mice vibrissa follicles, all reconstructed hair follicles. However, cultured dermal papilla cells from human scalp follicles could not reconstruct hair follicles. Copyright © 2013 John Wiley & Sons, Ltd.

Quantitative multiresidue analysis of antibiotics in milk and milk powder by ultra-performance liquid chromatography coupled to tandem quadrupole mass spectrometry.

A simple and fast multiresidue extraction and purification method was developed for the determination of 61 veterinary drugs, belonging to seven classes, in milk and milk powder. The extraction depends on the acetonitrile solvent, followed by a single step to remove lipids with fatty acid chains using a new reversed phase SPE without traditional pre-equilibration and washing steps before eluting SPE. The purifying lipid effect of the present preparation method was evaluated by comparing the response changes of ion peak areas of the milk endogenous metabolites before and after SPE treatment using ultra-fast LC coupled to tandem quadrupole and TOF MS. Subsequently, UPLC coupled to tandem quadrupole MS was performed for the quantitative analysis of milk and milk powder samples spiked with 61 veterinary drugs, including ß-lactam, macrolide, amide alcohol, forest amine, sulfanilamide, tetracyclines, and quinolones antibiotics. This method is very simple, fast, sensitive, and selective, and allows the good recoveries of all compounds, with a recovery range of 61.5-118.6%, and coefficients of variation of less than 11.6%. The 61 compounds behave in the dynamic range 0.01-200µgkg(-1), with correlation coefficient >0.99. The limits of quantification for the analytes are in the range 0.01-5.18µgkg(-1). Finally, this method has been successfully applied to the screening of veterinary drugs in 50 commercial bovine milk and milk powder samples, and ceftiofur and ciprofloxacin were detected in some brand samples.

[Effect of PRP on the proliferation of dermal papilla cells and hair follicle regeneration in mice].

OBJECTIVE: To investigate the effects of platelet-rich plasma (PRP) on the proliferation of dermal papilla cells (DPCs) and hair follicle regeneration. METHODS: PRP was prepared using the double-spin method and applied to DPCs. The proliferative effect of activated PRP on DPCs was measured using MTT assay. To understand the influence of activated PRP on the hair-inductive capacity of DPCs, freshly isolated epidermal cells and DPCs of passage 4 were resuspended, mixed with various concentrations of a PRP (0%, 5% or 10%) and were then transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting and HF formation was monitored for 4 weeks; the graft site was harvested and processed for histological examination. RESULTS: Activated PRP increased the proliferation benefited the aggregative growth of DPCs. There are significant difference in the yield of hair follicles compared with 10% PRP (344 +/- 27) with 0% PRP (288 +/- 35) in the area of reconstituted skin (P < 0.05). The areas treated with PRP demonstrated an increase in hair follicles density of 19.4%. Ten percent PRP (18 +/- 1) d also can significantly shorten the time of hair formation, compared with 0% PRP (20 +/- 1) d (P < 0.05). CONCLUSIONS: There is a considerable effect of PRP on the time of hair formation and the yield of hair follicles reconstitution.

[Inhibition effect of 6-gingerol on hair growth].

OBJECTIVE: To investigate the effect of 6-gingerol, the main active component of ginger, on hair shaft elongation in vitro and hair growth in vivo. METHODS: Firstly, Hair follicles were co-cultured with 3 different concentration of 6-gingerol for 5 days and hair elongation in three groups was measured. Secondly, The proliferative effect of 6-gingerol on DPCs was measured using MTT assay. Thirdly, the expression of Bcl-2 and Bax in DPCs were measured using Western blotting. In vivo study, the influence of 6-gingerol on hair growth in C57BL/6 rats was measured through topical application of 6-gingerol on the dorsal skin of each animal. RESULTS: The length of hair shaft in 20 microg/ml 6-Gingerol group (0.50 +/- 0.08 mm) is less than 0 microg/ml (0.66 +/- 0.19) mm and 10 microg/ml (0.64 +/- 0.03) mm 6-Gingerol group (P < 0.05). In cell culture, compared to 0 microg/ml and 5 microg/ml 6-Gingerol, 10 microg/ml 6-Gingerol can significantly inhibited the proliferation of DPCs (P < 0.05). Along with the growth inhibition of DPCs by 6-gingerol, the Bax/Bcl-2 ratio increased obviously. In vivo study, the hair length and density decreased a lot after using 1 mg/ml 6-gingerol. CONCLUSIONS: 6-Gingerol can suppress human hair shaft elongation because it has pro-apoptotic effects on DPCs via increasing Bax/Bcl-2 ratio. It might inhibit hair growth by prolonging the telogen stage in vivo.

Enhanced neuroprotective effects of co-administration of G-CSF with simvastatin on intracerebral hemorrhage in rats.

AIM: Based on the neuroprotective effect of either G-CSF or statins in various neurological disease models, the purpose of this study was to evaluate the superiority of combined therapy G-CSF with simvastatin in experimental intracerebral hemorrhage (ICH). MATERIAL AND METHODS: Primary ICH was induced in male Sprague Dawley rats. G-CSF (50 µg/kg), simvastatin (2 mg/kg), combined G-CSF and simvastatin, or phosphate buffered saline was given at 24 hours post-ICH. Neurobehavioral outcomes were assessed in all rats. The pathological changes of neuronal ultrastructure were examined with transmission electron microscopy at the given time. Simultaneously, immunohistochemical labeling and TUNEL assay were performed. RESULTS: Co-administration of G-CSF with simvastatin significantly promoted functional recovery and expedited the recovery time. Transmission electron microscopy revealed that combination treatment significantly improved ultrastructural outcomes. Histological examination showed that the expressions of Brdu co-labeled with NSE and GFAP, Factor VIII were higher in combined treatment than in control group. Additionally, the number of cell apoptosis was higher in control group than in experimental groups and lowest in combination group. CONCLUSION: Our results indicated that combination treatment of stroke with G-CSF and simvastatin augments the neuroprotective effect in rats after ICH.

Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury.

Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.

[Simultaneous determination of various aseptics and sweeteners in milk and dairy products].

A method for simultaneous determination of acesulfame, benzoic acid, sodium saccharin, sorbic acid, and aspartame in milk and dairy products using high performance liquid chromatography (HPLC) was developed. The proteins in milk and dairy products were mostly eliminated by the precipitators. Three aseptics and two sweeteners were separated on a C18 column with the mobile phase of methanol-0.05 mol/L potassium dihydrogen phosphate under gradient elution. With a diode array detector, acesulfame, benzoic acid, and sorbic acid were detected at 230 nm and sodium saccharin and aspartame were detected at 210 nm. The recoveries were 96.0% - 103.5% with the relative standard deviations (RSDs) in the range of 1.93% - 2.76%. The detection limits of acesulfame, benzoic acid, sodium saccharin, sorbic acid and aspartame were 1.0, 1.0, 0.5, 1.0, and 1.5 microg/g, respectively. This method can be used for the routine analysis of these additives in milk and dairy products.