Characterization of phospholipidome in milk, yogurt and cream, and phospholipid differences related to various dairy processing methods.

Milk phospholipids have multiple health benefits, but the deficiency of detailed phospholipid profiles in dairy products brings obstacles to intake calculation and function evaluation of dairy phospholipids. In present study, 306 phospholipid molecular species were identified and quantified among 207 milk, yogurt and cream products using a HILIC-ESI-Q-TOF MS and a HILIC-ESI-QQQ MS. The phospholipid profiles of five mammals' milk show that camel milk contains the most abundant phosphatidylethanolamine, phosphatidylserine and sphingomyelin; cow, yak and goat milk have similar phospholipidomes, while buffalo milk contains abundant phosphatidylinositol. Fewer plasmalogens but more lyso-glycerolphospholipids were found in ultra-high-temperature (UHT) sterilized milk than in pasteurized milk, and higher proportions of lyso-glycerolphospholipid/total phospholipid were observed in both cream and skimmed/semi-skimmed milk than whole milk, indicating that UHT and skimming processes improve glycerolphospholipid degradation and phospholipid nutrition loss. Meanwhile, more diacyl-glycerolphospholipids and less of their degradation products make yogurt a better phospholipid resource than whole milk.

Development and Immunological Evaluation of a Multiantigen Thermostable Nanovaccine Adjuvanted with T-Cell-Activating Scaffold for African Swine Fever.

African swine fever is an acute and highly contagious infectious disease with a mortality rate of up to 100%. The lack of commercial vaccines and drugs is a serious economic threat to the global pig industry. Cell-mediated immunity plays an essential role in protection against viral infection. We previously reported the rational design of a T-cell-activating thermostable scaffold (RPT) for antigen delivery and improved cellular immunity. We conjugated antigens P30, P54, P72, CD2 V, and CP312R to RPT, using a SpyCatcher/SpyTag covalent attachment strategy to construct nanovaccines (multiantigens-RPT). Multiantigens-RPT exhibited significantly higher thermal, storage, and freeze-thaw stability. The specific antibodies IgG and IgG2a of the multiantigen-RPT-immunized were higher than the antigens cocktail-immunized by approximately 10-100 times. ELISpot demonstrated that more IFN-γ-secreting cells were produced by the multiantigen-RPT-immunized than by the antigens cocktail-immunized. Delivery of the multiantigen nanovaccine by a T-cell-activating scaffold induced strong humoral and cellular immune responses in mice and pigs and is a potentially useful candidate vaccine for the African swine fever virus.

Thermostable T Cell Multiepitope Nanoparticle Antigens Inducing Potent Immune Responses against the Swine Fever Virus.

African swine fever (ASF) is caused by the African swine fever virus (ASFV) and is a highly contagious, acute, febrile disease that has high morbidity and mortality rates in domestic and wild swine. However, a safe and effective vaccine against ASF remains unavailable as single antigens fail to provide sufficient protection. Therefore, a combination of multiple antigens with an efficient delivery system might be an alternative strategy. Herein, a de novo-designed antigen with multiple T-cell epitopes (TEPs) of ASFV was conjugated for surface display on self-assembled nanoparticles (NPs) of Aquifex aeolicus lumazine synthase (AaLS) and Quasibacillus thermotolerans encapsulin (QT) through the SpyCatcher/SpyTag system to construct nanovaccines (TEP-Spy-NPs). TEP-Spy-NPs exhibited significantly more thermal, storage, and freeze-thaw stability in comparison to TEP monomers. TEP-Spy-NPs were highly immunogenic and induced strong polyclonal antibody responses in mice and pigs. The specific antibody titers against the TEP of the TEP-Spy-AaLS and TEP-Spy-QT groups were significantly higher than those of the TEP monomer immune group after the second booster immunization. The antibody titer against TEP of the TEP-Spy-QT group was approximately twice that of the TEP-Spy-AaLS group in mice. ELISpot analysis demonstrated that more IFN-γ- and IL-2-secreting splenic lymphocytes were produced by TEP-Spy-AaLS- and TEP-Spy-QT-immunized mice than by TEP monomer-immunized mice. TEP-Spy-NPs elicited stronger cellular immunity and in vivo immunity in immunized pigs than did TEP monomers. Thus, the TEP nanovaccine successfully induced strong humoral and cellular immune responses in mice and pigs, and TEP-Spy-NPs have the potential as candidate vaccines for ASFV.

[Analysis of soybean phospholipids based on liquid chromatography-ion trap-time of flight-mass spectrometry].

OBJECTIVE: Using liquid chromatography-ion trap-time of flight-mass spectrometry to establish a soybean phospholipids(PL) analysis method, carry out comprehensive structural identification and quantitative analysis of soybean PL molecule species, and obtain soybean PL molecular composition and content data. METHODS: The PL profiles of 10 soybean varieties cultivated in northeast China were determined by a hydrophilic interaction liquid chromatography-ion trap-time of flight-mass spectrometry(HILIC-ESI-IT-TOF-MS) system. The hydrophilic interaction liquid chromatography realized class separation of PLs, and ion trap-time of flight-mass spectrometry realized the estimation of head group type, fatty acyl structure, and substituent position. The identified PL molecule species were quantified using accurate mass extraction and internal standard method. RESULTS: A total of 101 PL molecular species from 11 classes were estimated in soybean seeds, including 20 phosphatidylcholines(PC), 15 phosphatidylethanolamines(PE), 17 phosphatidylinositols(PI), 12 phosphatidylglycerols(PG), 9 phosphatidic acids(PA), 6 phosphatidylserines(PS), 5 lysophosphatidylcholines(LPC), 5 lysophosphatidylethanolamines(LPE), 6 lysophosphatidylinositols(LPI), 4 lysophosphatidylglycerols(LPG) and 2 lysophosphatidicacids(LPA). The limits of detection for the target object were ≤ 60 ng/mL and R~2 were all >0.99. The total concentration of PL ranged from 6873.1 to 12 678.6 µg/g in detected soybeans. Generally, most of the detected soybean cultivars were great resources of PL, especially SN-29, SN-61, HN-40 and DN-690. The concentrations of PC and PE in the tested soybean were higher, followed by PI, PG, PA and PS. Among the lysophospholipids, the concentration of LPC was the highest, followed by LPE, LPI, LPG and LPA. CONCLUSION: The soybean PL component analysis method established based on the HILIC-IT-TOF-MS system can achieve good separation of various types of PL, and can accurately characterize the head group type, fatty acyl structure and substituent position of PL molecule species. The molecular composition and content data of soybean PL were obtained.

[Determination of 21 polyphenols in fruits by high performance liquid chromatography].

OBJECTIVE: To establish a high performance liquid chromatography(HPLC) method for simultaneous determination of flavonoids(epicatechin, isoorientin, eriocitrin, hyperoside, narirutin, naringin, hesperidin, neohesperidin, quercetin, didymin, naringenin and hesperetin), phenolic acids(gallic acid, chlorogenic acid, caffeic acid, ferulic acid, sinapic acid and p-coumaric acid), terpenoids(limonin), stilbenes(piceatannol and resveratrol) in fruits. METHODS: Add ethanol-water(4∶1, V/V) solution to the fruit pulp sample, ultrasonic extraction at 55 ℃ for 10 min, after centrifugation, the supernatant was dried and re-dissolved, and then eluted with ZORBAX Eclipse XDB-C_(18)(4.6 mm×100 mm, 3.5 µm) as chromatographic column, polyphenols are determined by HPLC under switching wavelength, and quantified by external standard method. RESULTS: The 21 polyphenols can be detected within 45 min and well separated from baseline, with linear correlation coefficients greater than 0.999, the detection limit of the method was 0.03-0.31 µg/g, and the limit of quantitation of the method was 0.09-1.03 µg/g. The intra-day precision was 0.5%-9.3%, the inter-day precision was 2.0%-9.6%. The recoveries of more than 90% polyphenols in the three fruits at three levels were between 80.0% and 119.8%. The relative standard deviation of spiked recoveries of three samples was less than 10.8%. The detection result of citrus, mango, blueberry, persimmon and other samples proved that the composition of polyphenols in different fruits was different. CONCLUSION: The method is sensitive, accurate and reproducible, and is suitable for the determination of polyphenols in fruits.

Unilateral facial injection of Botulinum neurotoxin A attenuates bilateral trigeminal neuropathic pain and anxiety-like behaviors through inhibition of TLR2-mediated neuroinflammation in mice.

OBJECTIVES: In this study, we investigated the possible analgesic effects of Botulinum toxin type A (BoNT/A) on trigeminal neuralgia (TN). A modified TN mouse model was established by chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) in mice, and the possible roles of microglia toll-like receptor 2 (TLR2) and neuroinflammation was investigated. METHODS: Male C57BL/6 mice were divided into 3 groups, including sham group, vehicle-treated TN group and BoNT/A-treated TN group. Bilateral mechanical pain hypersensitivity, anxiety-like and depressive-like behaviors were evaluated by using von Frey test, open field, elevated plus-maze testing, and forced swimming test in mice, respectively. The mRNA or protein expression levels of toll-like receptors (TLRs), glia activation markers and proinflammatory factors in the trigeminal nucleus caudalis (TNC) were tested by RT-qPCR, immunofluorescence and Western blotting. We also tested the pain behaviors of TN in Tlr2-/- mice. RESULTS: We found that unilateral subcutaneous injection of BoNT/A into the whisker pad on the ipsilateral side of dIoN-CCI mice significantly attenuated bilateral mechanical pain hypersensitivity and anxiety-like behaviors induced by dIoN-CCI surgery in mice. The dIoN-CCI surgery significantly up-regulated the expression of TLR2, MyD88, CD11b (a microglia marker), IL-1ß, TNF-α and IL-6 in the ipsilateral TNC in mice, and BoNT/A injection significantly inhibited the expression of these factors. Immunostaining results confirmed that BoNT/A injection significantly inhibited the microglia activation in the ipsilateral TNC in dIoN-CCI mice. TLR2 deficiency also alleviated bilateral mechanical pain hypersensitivity and the up-regulation of MyD88 expression in the TNC of dIoN-CCI mice. CONCLUSION: These results indicate that unilateral injection of BoNT/A attenuated bilateral mechanical pain hypersensitivity and anxiety-like behaviors in dIoN-CCI mice, and the analgesic effects of BoNT/A may be associated with the inhibition of TLR2-mediated neuroinflammation in the TNC.