Objective: To develop an approach for simultaneous detection of multi-mycotoxins in fresh fruits, so as to provide technical supports for mycotoxins surveillance in fresh fruits. Methods: Fresh fruits were collected from markets and homogenized. Then, 2 g of fresh fruits were added with 10 mL of 0.1% formic acid ( 99∶1, v/v ) in acetonitrile and wortexed for 10 min. Following extraction with 1 g of sodium chloride and 4 g of anhydrous sodium sulfate, samples were centrifuged and 5 mL of the supernatant was cleaned up with 25 mg C18. Following centrifugation, the supernatant was dried under nitrogen. The residue was dissolved in 300 μL of methanol-acetonitrile mixture solution ( 1∶1, v/v ), and mixed evenly in 700 μL of the distilled water. Samples were then eluted in gradient series of 0.1% formic acid and 5 mmol ammonium formate and methanol-acetonitrile mixture solution ( 1∶1, v/v ). The 15 mycotoxins were determined using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) with electrospray ion source (ESI+/ESI-) under multiple reaction monitoring. In addition, a matrix-matched standard curve was employed for quantitative analysis. Results: There was a good linear relationship for 15 mycotoxins at concentrations of 0.25 to 10 ng/mL ( R2>0.992 ), the LC-MS/MS method showed the detection limits of 0.1-1.0 μg/kg, the spiked recovery rates of 71.68%-117.50%, and the relative standard deviations ( RSDs ) of 0.01%-13.60%. The detection rate of mycotoxins was 27.09% in 203 fresh fruits sold in markets. Conclusions The optimized LC-MS/MS method can be used for simultaneous determination of multi-mycotoxins in fresh fruits.

Objective To test the protective immunity in mice induced by recombinant Schistosoma japonicum Sj14FABP through several adjuvant formulations. Methods The recombinant Schistosoma japonicum Sj14FABP was prepared by expression in E. coli as a GST fusion protein (rSj14/GST) and used to vaccinate outbred Kunming mice by using complete Freund's adjuvant (FCA)/incomplete Freund's adjuvant (FIA), Bacillus Calmette-Guerin (BCG) and the immunostimulating complex (ISCOM) as adjuvant respectively. Results The purified recombinant protein rSj14/GST was immunogenic in mice, and 34.3% and 36.0% worm reduction rates were obtained in outbred Kunming mice immunized intradermally with BCG adjuvant and immunized subcutaneously with ISCOM adjuvant respectively, compared with non-vaccinated control group. However, intramuscularly vaccination with rSj14/GST in FCA/FIA was not protective, although the high level of IgG antibody was induced. Conclusion Both BCG and ISCOM are suitable adjuvants for rSj14/GST.

The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RT-PCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S.japonicum, the corresponding mDNA fragment of Chinese strain as template and then the 5' and 3' ends of this gene cDNA were amplified with 5' RACE and 3' RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame (ORF) of gene encoding the egg-shell precursor protein of S.japonicum.(Chinese strain). As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E.coli after cloning into the expression vector pET28c(+). The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S.japonicum;indicating the good antigenicity of this expressed product.

Objective : To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events. Methods : The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS. Results : The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS. Conclusion The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.

Objective To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum.Methods Total RNA was extracted from adult worms of S.japonicum by Trizol reagent and mRNA was isolated from the total RNA.The ds cDNA was synthesized by reverse transcription using random primer.Directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoRⅠand HindⅢ,which resulted in ds cDNA with EcoRⅠand HindⅢ adhering ends.The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector.After packaging in vitro,the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library.Plaque assay and PCR were used to evaluate the library.Seven known objective genes of S.japonicum were screened by PCR to detect the representation of the library.Result Primary library capacity was 4.98?106 pfu,and the titer of amplified library was 3.85?1011 pfu/mL.The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%,in which 95.6% inserted cDNA fragments were longer than 300 bp in length.All the seven known objective genes of S.japonicum were amplified from the library.Conclusion The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.

Objective:To investigate the effect of iodine nutrition status and sorts of indexes on homocysteine (HCY) in the second trimester of pregnancy under conditions with normal thyroid function.Methods:481 second-trimester pregnant (13th to 27th week of pregnancy) women with normal thyroid function were selected for the study. Urinary iodine concentration (UIC), thyroid auto-antibody, HCY and biochemical indexes were measured. The HCY levels were compared among subjects with different iodine nutritional status, and factors related to HCY level were analyzed.Results:Patients were stratified into iodine deficiency, iodine adequate, iodine more than adequate and iodine excess groups and the proportion were 57.0% ( n=274), 29.7% ( n=143), 10.8% ( n=52) and 2.5% ( n=12), respectively. The overall median UIC was 134.1 μg/L. There was significant difference in HCY levels between iodine excess group and iodine adequate group(1.83 μmol/L vs. 2.46 μmol/L, P=0.036). Spearman correlation analysis showed that HCY was negatively correlated with iodine excess, free triiodothyronine (FT3), thyroglobulin antibody, and fasting blood glucose( r=-0.101, P=0.026; r=-0.099, P=0.03; r=-0.192, P<0.01; r=-0.099, P=0.03), and was positively correlated with TPOAb ( r=0.177, P<0.01). Multiple linear regression analysis showed that HCY was independently negatively correlated with iodine excess (β=-1.505, P=0.043) and FT3 (β=-0.661, P=0.008). Conclusion:Up to 57% women in the second trimester of pregnancy are with iodine deficiency and normal thyroid function. Moderate iodine excess and elevated FT3 levels are beneficial to the decrease of HCY levels and thus reduce the risk of vascular complications in pregnant women.

Objective:To modify the blepharoplasty through palpebral margin incision with preservaton of the pretarsal orbicularis oculi muscle, and to discuss the clinical application effects.Methods:The clinical data of patients who underwent double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle were retrospectively analyzed from August 2021 to August 2023 in Changsha Aist Medical Cosmetology Hospital. After the skin peeling rang was designed, the skin was removed, tissue was separated under the orbicularis oculi muscle. The pretarsal orbicularis oculi muscle was thinned-shaped at the upper edge of tarsus as wedge-shaped, and was detached from the upper edge of tarsus. The pretarsal fascia or levator aponeurosis was fixed internally with the orbicularis oculi muscle at the position of the double-eyelid crease. After confirming the shape and symmetry of the double-eyelid, the skin was sutured. Incision healing, eyelid swelling and complications were observed, and double-eyelid morphology was followed up. Six months after surgery, the patients’ satisfaction with the surgical effect (satisfied, somewhat satisfied, dissatisfied) was investigated, and the upper eyelid scar status (including pigmentation, thickness, vascularity, and pliability) was scored using the Vancouver scar scale (VSS). The higher the score, the more serious the scar was.Results:A total of 78 patients with single eyelid or subtle double eyelid were included, including 6 males and 72 females, aged 18-40 years old. All the patients underwent modified double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle, 29 patients underwent ptosis correction simultaneously, and 47 patients underwent epicanthus correction simultaneously. The incision healed by first intention after the operation. 78 cases were followed up for 1 to 6 months, and slight upper eyelid swelling was eliminated in 71 cases at 10-15 days and in 7 cases at 25-30 days. Among them, 61 cases were followed up for 6 months, the creases of double-eyelid was natural and smooth, bilateral basic symmetry, and there were no adverse manifestations such as double-eyelid shallowing and pretarsal tissue pilling, and no complications such as incompleted eye-closure. 57 cases were satisfied with the results and 4 cases were somewhat satisfied. The incision scar was not obvious when the eyes were closed in 61 patients. The VSS score was close to 0, and the average score of pigmentation, thickness, vascularity, and pliability were 0.29, 0, 0.31 and 0 points, respectively.Conclusion:This operation method preserves almost all the pretarsal orbicularis oculi muscle to promote the healling of lymph circulation. After the orbicularis oculi muscle is choosed for internal fixation, this more simple operation method can not only guarantee the efficacy of double-eyelid blepharoplasty through palpebral margin incision, but also accelerate the healling of the eyelid after surgery and reduce the incidence of incompleted eye-closure.

Objective:To modify the blepharoplasty through palpebral margin incision with preservaton of the pretarsal orbicularis oculi muscle, and to discuss the clinical application effects.Methods:The clinical data of patients who underwent double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle were retrospectively analyzed from August 2021 to August 2023 in Changsha Aist Medical Cosmetology Hospital. After the skin peeling rang was designed, the skin was removed, tissue was separated under the orbicularis oculi muscle. The pretarsal orbicularis oculi muscle was thinned-shaped at the upper edge of tarsus as wedge-shaped, and was detached from the upper edge of tarsus. The pretarsal fascia or levator aponeurosis was fixed internally with the orbicularis oculi muscle at the position of the double-eyelid crease. After confirming the shape and symmetry of the double-eyelid, the skin was sutured. Incision healing, eyelid swelling and complications were observed, and double-eyelid morphology was followed up. Six months after surgery, the patients’ satisfaction with the surgical effect (satisfied, somewhat satisfied, dissatisfied) was investigated, and the upper eyelid scar status (including pigmentation, thickness, vascularity, and pliability) was scored using the Vancouver scar scale (VSS). The higher the score, the more serious the scar was.Results:A total of 78 patients with single eyelid or subtle double eyelid were included, including 6 males and 72 females, aged 18-40 years old. All the patients underwent modified double-eyelid blepharoplasty through palpebral margin incision with preservation of the pretarsal orbicularis oculi muscle, 29 patients underwent ptosis correction simultaneously, and 47 patients underwent epicanthus correction simultaneously. The incision healed by first intention after the operation. 78 cases were followed up for 1 to 6 months, and slight upper eyelid swelling was eliminated in 71 cases at 10-15 days and in 7 cases at 25-30 days. Among them, 61 cases were followed up for 6 months, the creases of double-eyelid was natural and smooth, bilateral basic symmetry, and there were no adverse manifestations such as double-eyelid shallowing and pretarsal tissue pilling, and no complications such as incompleted eye-closure. 57 cases were satisfied with the results and 4 cases were somewhat satisfied. The incision scar was not obvious when the eyes were closed in 61 patients. The VSS score was close to 0, and the average score of pigmentation, thickness, vascularity, and pliability were 0.29, 0, 0.31 and 0 points, respectively.Conclusion:This operation method preserves almost all the pretarsal orbicularis oculi muscle to promote the healling of lymph circulation. After the orbicularis oculi muscle is choosed for internal fixation, this more simple operation method can not only guarantee the efficacy of double-eyelid blepharoplasty through palpebral margin incision, but also accelerate the healling of the eyelid after surgery and reduce the incidence of incompleted eye-closure.

Objective To investigate the effects of calcitriol intervention on PI3K/AKT signaling pathway and wound healing in rats with diabetic foot ulcer(DFU).Methods Thirty-two male SD rats were divided into normal control(Con)group,DFU group,calcitriol low dose(L)group and calcitriol high dose(H)group.A circular wound of 5 mm in diameter and deep to the fascia was made on the dorsum of the left foot of rats in each group.HE staining was used to evaluate the histopathological changes of the wounds.Immunohistochemical method was selected to compare the distribution of CD34-positive cells and the expression of p-PI3K and p-AKT in traumatic tissues of each group.ELISA was adopted in the detection of serum IL-6,IFN-γ,TNF-α and IL-7.RT-PCR was used to detect the mRNA expression of PI3K and AKT in each group,and western blot was used to detect the protein expression of PI3K,p-PI3K,AKT,p-AKT and VEGF.Results Compared with Con group,the expressions of IL-6,IFN-γ,TNF-α,IL-7,CD34,PI3K mRNA,AKT mRNA,p-AKT protein,p-PI3K protein,p-PI3K/PI3K,p-AKT/AKT increased,while PI3K protein expression decreased in DFU,L and H groups(P<0.05),VEGF and AKT protein expression decreased in DFV and L groups(P<0.05).Compared with DFU group,the expressions of VEGF,AKT and PI3K protein increased(P<0.05),while the expressions of p-PI3K/PI3K,p-AKT/AKT decreased in L and H groups(P<0.05),IL-6 decreased in L group(P<0.05),and CD34 expression increased in H group(P<0.05),while IL-6,IFN-γ,TNF-α and IL-7,PI3K mRNA,AKT mRNA,p-AKT protein and p-PI3K protein expression decreased(P<0.05).Compared with L group,the expressions of CD34,VEGF,AKT and PI3K protein increased(P<0.05),while IL-6,PI3K mRNA,AKT mRNA,p-AKT protein,p-PI3K protein,p-PI3K/PI3K and p-AKT/AKT decreased in H group(P<0.05).Conclusions Calcitriol intervention may reduce the activity of the PI3K/AKT signaling pathway,inhibit inflammation,promote neovascularization,and facilitate wound healing in rats with DFU.