Ultrahigh-energy photons up to 1.4 petaelectronvolts from 12 γ-ray Galactic sources.

The extension of the cosmic-ray spectrum beyond 1 petaelectronvolt (PeV; 1015 electronvolts) indicates the existence of the so-called PeVatrons-cosmic-ray factories that accelerate particles to PeV energies. We need to locate and identify such objects to find the origin of Galactic cosmic rays1. The principal signature of both electron and proton PeVatrons is ultrahigh-energy (exceeding 100 TeV) γ radiation. Evidence of the presence of a proton PeVatron has been found in the Galactic Centre, according to the detection of a hard-spectrum radiation extending to 0.04 PeV (ref. 2). Although γ-rays with energies slightly higher than 0.1 PeV have been reported from a few objects in the Galactic plane3-6, unbiased identification and in-depth exploration of PeVatrons requires detection of γ-rays with energies well above 0.1 PeV. Here we report the detection of more than 530 photons at energies above 100 teraelectronvolts and up to 1.4 PeV from 12 ultrahigh-energy γ-ray sources with a statistical significance greater than seven standard deviations. Despite having several potential counterparts in their proximity, including pulsar wind nebulae, supernova remnants and star-forming regions, the PeVatrons responsible for the ultrahigh-energy γ-rays have not yet been firmly localized and identified (except for the Crab Nebula), leaving open the origin of these extreme accelerators.

[Molecular mechanisms of quinolone resistance in non-typhoidal Salmonella].

By conducting retrospective analysis, this study aim to investigate the resistance mechanism of quinolones in non-typhoidal Salmonella (NTS). A total of 105 strains of NTS isolated from clinical specimens from the Fifth Affiliated Hospital of Southern Medical University from May 2020 to February 2021 were used as research objects. VITEK2 Compact automatic identification drug sensitivity analysis system and serological test were used to identify the strains. The sensitivity of the strains to ciprofloxacin, levofloxacin and nalidixic acid was detected by AGAR dilution method. The whole genome of 105 strains of NTS was sequenced. Abricate and other softwares were used to analyze drug-resistant genes, including plasmid-mediated quinolone resistance gene (PMQR) and Quinolone resistance determination region (QRDR). Serotypes and ST types were analyzed using SISTR and MLST, and phylogenetic trees were constructed. The results showed that the NTS isolated in this region were mainly ST34 Salmonella typhimurium (53.3%). The drug sensitivity results showed that the drug resistance rates of NTS to ciprofloxacin, levofloxacin and nalidixic acid were 30.4%, 1.9% and 22.0%, respectively, and the intermediate rates of ciprofloxacin and levofloxacin were 27.6% and 54.2%.A total of 46 (74.2%) of the 62 quinolone non-susceptible strains carried the PMQR gene, mainly qnrS1 (80.4%), followed by aac(6')-Ib-cr(15.2%); there were 14 NTS and 8 NTS had gyrA and parC gene mutations, respectively. The gyrA was mutations at the amino acid position 87, Asp87Tyr, Asp87Asn, Asp87Gly, and Thr57Ser mutations were detected in parC. In conclusion, this study found that NTS had relatively high resistance to quinolones, carrying qnrS1 gene mainly resulted in decreased sensitivity of NTS to ciprofloxacin and levofloxacin, and gyrA:87 mutation mainly resulted in NTS resistance to Nalidixic acid; Salmonella typhimurium in clinical isolates showed clonal transmission and required further epidemiological surveillance.

Measurement of Ultra-High-Energy Diffuse Gamma-Ray Emission of the Galactic Plane from 10 TeV to 1 PeV with LHAASO-KM2A.

The diffuse Galactic γ-ray emission, mainly produced via interactions between cosmic rays and the interstellar medium and/or radiation field, is a very important probe of the distribution, propagation, and interaction of cosmic rays in the Milky Way. In this Letter, we report the measurements of diffuse γ rays from the Galactic plane between 10 TeV and 1 PeV energies, with the square kilometer array of the Large High Altitude Air Shower Observatory (LHAASO). Diffuse emissions from the inner (15°10 TeV). The energy spectrum in the inner Galaxy regions can be described by a power-law function with an index of -2.99±0.04, which is different from the curved spectrum as expected from hadronic interactions between locally measured cosmic rays and the line-of-sight integrated gas content. Furthermore, the measured flux is higher by a factor of ∼3 than the prediction. A similar spectrum with an index of -2.99±0.07 is found in the outer Galaxy region, and the absolute flux for 10≲E≲60 TeV is again higher than the prediction for hadronic cosmic ray interactions. The latitude distributions of the diffuse emission are consistent with the gas distribution, while the longitude distributions show clear deviation from the gas distribution. The LHAASO measurements imply that either additional emission sources exist or cosmic ray intensities have spatial variations.

Exploring Lorentz Invariance Violation from Ultrahigh-Energy γ Rays Observed by LHAASO.

Recently, the LHAASO Collaboration published the detection of 12 ultrahigh-energy γ-ray sources above 100 TeV, with the highest energy photon reaching 1.4 PeV. The first detection of PeV γ rays from astrophysical sources may provide a very sensitive probe of the effect of the Lorentz invariance violation (LIV), which results in decay of high-energy γ rays in the superluminal scenario and hence a sharp cutoff of the energy spectrum. Two highest energy sources are studied in this work. No signature of the existence of the LIV is found in their energy spectra, and the lower limits on the LIV energy scale are derived. Our results show that the first-order LIV energy scale should be higher than about 10^{5} times the Planck scale M_{Pl} and that the second-order LIV scale is >10^{-3}M_{Pl}. Both limits improve by at least one order of magnitude the previous results.

Constraints on Heavy Decaying Dark Matter from 570 Days of LHAASO Observations.

The kilometer square array (KM2A) of the large high altitude air shower observatory (LHAASO) aims at surveying the northern γ-ray sky at energies above 10 TeV with unprecedented sensitivity. γ-ray observations have long been one of the most powerful tools for dark matter searches, as, e.g., high-energy γ rays could be produced by the decays of heavy dark matter particles. In this Letter, we present the first dark matter analysis with LHAASO-KM2A, using the first 340 days of data from 1/2-KM2A and 230 days of data from 3/4-KM2A. Several regions of interest are used to search for a signal and account for the residual cosmic-ray background after γ/hadron separation. We find no excess of dark matter signals, and thus place some of the strongest γ-ray constraints on the lifetime of heavy dark matter particles with mass between 10^{5} and 10^{9} GeV. Our results with LHAASO are robust, and have important implications for dark matter interpretations of the diffuse astrophysical high-energy neutrino emission.

[The 494th case: acute pancreatitis, acute acalculous cholecystitis and anaphylaxis].

A young male patient with abdominal pain and fever was diagnosed as acute hyper-triglyceridemicpancreatitis is clear. During the recovery of pancreatitis, the patient developed acute acalculous cholecystitis, as well as carbapenem-resistant Enterobacter infection and Cytomegaloviremia, and had anaphylaxis for several times after the use of antibiotics, which cannot be completely explained by drug allergy. This paper analyzes the possible causes of multiple diseases in the same patient in detail.

[Methylation of p16 gene and reduced expression of p16 protein in insulinoma associated with clinicopathological features].

Objective: To study the alterations of p16 gene and its expression in insulinoma and to correlate the findings with clinicopathological characteristics. Methods: Expression of p16 protein was detected in 72 insulinomas and 49 para-tumoral or normal pancreatic tissues by immunohistochemical staining. Genomic DNA was isolated from 32 tumor tissue and 17 paired pancreatic tissues and bisulfite-modified. Promoter methylation status of p16 gene was detected in 32 tumor tissue and 17 paired pancreatic tissues by methylation specific PCR. The findings were correlated with the clinicopathological features. Results: There were 30 males and 42 females in all 72 patients, aged (46.5±14.0) years. Loss or reduced expression of p16 protein was found in 42 of 72 insulinomas (58.3%) while loss or reduced expression of p16 was seen in only 34.7% (17/49) of para-tumoral or normal pancreatic tissues (χ²=6.52, P=0.011). Promoter methylation of p16 gene was found in 13 of 32 insulinomas (40.6%) and only 2 of 17 (11.8%) para-tumoral tissues (χ²=4.35, P=0.037). The expression of p16 protein in insulinoma was not associated with clinicopathological features such as gender, age, tumor size and tumor grade. Conclusions: Loss or reduced expression of p16 protein was found in insulinomas, and associated with p16 gene promoter methylation.

[The effect of maternal HBV DNA levels on HBV intrauterine transmission and fetal distress].

Aim: To identify the key risk factors of intrauterine hepatitis B virus transmission (HBV) and its effect on the placenta and fetus. Methods: 425 infants born to hepatitis B surface antigen (HBsAg)-positive pregnant women who received combined immunization with hepatitis B immunoglobulin and hepatitis B vaccine between 2009 to 2015 were prospectively enrolled in this study. The intrauterine transmission situation was assessed by dynamic monitoring of infants HBV DNA load and quantitative HBsAg. Univariate and multivariate regression analysis was used to determine the high risk factors for intrauterine transmission. Stratified analysis was used to determine the relationship between maternal HBV DNA load and fetal distress. Transmission electron microscopy was used to observe HBV Effects on placental tissue. Results: HBV intrauterine infection rate was 2.6% (11/425). Multivariate analysis result showed that the maternal HBV DNA load was an independent risk factor for intrauterine infection among infants (P=0.011). Intrauterine infection and distress rate was significantly higher in infants with with maternal HBV DNA>106 IU/ml than those with HBV DNA <106 IU/ml (12.2% vs. 1.8%; χ2=11.275, P=0.006), and (24.4% vs. 16.0%, χ2=3.993, P=0.046). Transmission electron microscopy showed that mitochondrial edema, endoplasmic reticulum expansion and thicker basement membrane were apparent when the maternal HBV DNA>106 IU/ml than that of maternal HBV DNA<106 IU/ml (960 nm vs. 214 nm, Z=-2.782, P=0.005) in the placental tissue. Conclusion: Maternal HBV DNA>106 IU/ml is associated not only with intrauterine infection, but also with increased incidence of intrauterine distress and placental sub-microstructural changes, providing strong clinical and histological evidence for pregnancy avoidance and treatment in this population.

Extended Very-High-Energy Gamma-Ray Emission Surrounding PSR J0622+3749 Observed by LHAASO-KM2A.

We report the discovery of an extended very-high-energy (VHE) gamma-ray source around the location of the middle-aged (207.8 kyr) pulsar PSR J0622+3749 with the Large High-Altitude Air Shower Observatory (LHAASO). The source is detected with a significance of 8.2σ for E>25 TeV assuming a Gaussian template. The best-fit location is (right ascension, declination) =(95.47°±0.11°,37.92°±0.09°), and the extension is 0.40°±0.07°. The energy spectrum can be described by a power-law spectrum with an index of -2.92±0.17_{stat}±0.02_{sys}. No clear extended multiwavelength counterpart of the LHAASO source has been found from the radio to sub-TeV bands. The LHAASO observations are consistent with the scenario that VHE electrons escaped from the pulsar, diffused in the interstellar medium, and scattered the interstellar radiation field. If interpreted as the pulsar halo scenario, the diffusion coefficient, inferred for electrons with median energies of ∼160 TeV, is consistent with those obtained from the extended halos around Geminga and Monogem and much smaller than that derived from cosmic ray secondaries. The LHAASO discovery of this source thus likely enriches the class of so-called pulsar halos and confirms that high-energy particles generally diffuse very slowly in the disturbed medium around pulsars.

[The 488th case: chronic diarrhea and abnormal liver function].

A 56-year-old female was admitted to Department of Gastroenterology at Peking Union Medical College Hospital with diarrhea for seven months, and abnormal liver function for six months. She had a history of type 1 diabetes. The main clinical manifestations were recurrent fatty diarrhea and abnormal liver function, accompanied by abdominal and retroperitoneal lymphadenopathy, elevated CA19-9 and CEA. Progressive impairment of hepatic synthetic function and shrinkage of liver developed in a short period of time. The pathology of liver biopsy suggested that nodular regeneration of hepatocytes was followed by hyperplasia of thin bile ducts after submassive necrosis. Intestinal mucosa biopsies were performed twice. The pathology showed that the intestinal villi were completely blunt, accompanied with crypt hyperplasia. Goblet cells disappeared with reduced mucin. Paneth cells were barely seen without intraepithelial infiltration of lymphocytes. Rifaximin was not effective, while glucocorticoids improved clinical situation. The diagnosis of autoimmune enteropathy was finally confirmed by multidisciplinary team including departments of gastroenterology, pathology, endocrinology, hematology, infectious diseases, and rheumatology. With the administration of glucocorticoid and sirolimus, diarrhea relieved and liver function returned to normal.

Transplantation of adipose tissue-derived stem cells ameliorates autoimmune pathogenesis in MRL/lpr mice : Modulation of the balance between Th17 and Treg.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of autoimmune diseases. In this study, we aimed to investigate whether transplantation of adipose tissue-derived stem cells (ADSCs) affects the autoimmune pathogenesis in MRL/lpr mice. METHODS: Fifteen 12-week-old MRL/lpr mice were randomly divided into three groups: ADSC, cyclophosphamide (CTX), and control groups, with five mice in each group. ADSC and control groups were injected with 1â€¯× 106 ADSCs or PBS, respectively, via the tail vein, once a week for 8 weeks. The CTX group was injected with CTX at a dose of 15 mg/kg body weight, once a week for 2 weeks, and this was repeated after 2 weeks rest. Proteinuria, anti-double-stranded DNA (anti-dsDNA) antibody, and serum creatinine levels were then measured. The populations of Th17 and Treg cells in the spleen were detected by flow cytometry. All statistical analyses were performed using least square difference. RESULTS: Eight weeks after treatment, the 24 h proteinuria, anti-dsDNA antibody levels, and serum creatinine were decreased significantly with transplantation of mouse ADSCs. ADSCs markedly reduced the number of TH17 cells, increased Treg cells, and improved renal pathology. CONCLUSION: Our results indicate that transplantation of ADSCs could significantly inhibit autoimmune progression in MRL/lpr mice and the efficacy of ADSCs was comparable to that of CTX.

[The 470th case: recurrent vomiting].

Patients with cyclic vomiting syndrome usually have comorbid psychological disorders, the trigger and aggravating factors of gastrointestinal symptoms. It may decrease the quality of life and the response to the conventional therapies, bring the patients to seek medical care frequently and perform unnecessary examinations, result in the waste of medical resources. We reported a 20-year-old woman with complaint of recurrent vomiting since infant, and less response to symptomatic and supportive treatment. Her vomiting relieved with antidepressant after consultation with gastroenterological specialist and psychologist. Physicians should pay more attention to recognize the comorbid psychological disorders in patients with functional gastrointestinal disorders (FGIDs), including cyclic vomiting syndrome. It is important to refer the patients with refractory symptoms to the psychologists for further professional evaluation and antidepressants.

[Detection of axillary nerve multisegment motor nerve conduction using magnetic stimulation].

Objective: To establish a method for detecting multi-segmental motor nerve conduction of axillary nerve using magnetic stimulation technique, and to preliminarily discuss its clinical application value. Methods: From April 2017 to February 2018, 80 healthy volunteers, 40 male and 40 female, were included and divided into five groups according to age and gender (groupⅠwere <35 years old; groupⅡwere 35-44 years old; group Ⅲ were 45-54 years old; groups Ⅳ were 55-64 years old; groups Ⅴ aged 65-74). Axillary nerve recording electrodes were placed in the deltoid muscle belly, reference electrode was placed in the deltoid muscle check point. Using the magnetic stimulation coil, stimulation was released at spots which were, respectively, 2 fingers from the lateral side of subscapular fossa, 2 fingers from the lateral side of infraclavicular fossae, and Erb's point. The latency, amplitude, detection distance (stimulation to recording electrode) were recorded at each spot, and the height and arm length were also measured. Results: The detection rate of axillary nerve waveform in healthy volunteers was 97.2%. There were no statistically significant differences in latency and amplitude in the different sides, gender (P>0.05).The latency corresponding to different detection distances was compared, and the difference was statistically significant (P<0.05).Height and arm length in different age groups were significantly increased in groupsⅠ, Ⅱ and Ⅲ compared with groups Ⅳ and Ⅴ, with statistical significance (P<0.05). When compared among different age groups, the latency and amplitude of group Ⅳand Ⅴ (more than 55 years old and over) were significantly prolonged and decreased, with statistically significant differences (P<0.05). Conclusions: The method of detecting multi-segmental magnetic stimulation of axillary nerve is stable and the result is reliable, which makes the evaluation of the proximal nerve of upper limbs be possible. However, the biological effects of age on nerve conduction should be carefully considered.

[Comparison of intraocular pressure reduction in eyes with different intraocular tamponades after pars plana vitrectomy by 20% mannitol].

Objective: To investigate the efficacy of 20% mannitol in reducing intraocular pressure (IOP) in eyes with different intraocular tamponades after pars plana vitrectomy (PPV). Methods: Retrospective case-control study. Sixty-eight patients were administered with 20% mannitol and IOP was noted at regular intervals after simple PPV with ocular hypertension, including 24 males (26 eyes) and 44 females (46 eyes), aged (45.6±19.3) years. These cases were divided into three groups according to different tamponades: silicon-oil tamponade, 23 eyes; gas tamponade, 30 eyes; balanced salt solution (BSS), 19 eyes. The data were analyzed using the t test, variance and q test. Results: There was a significant decrease in IOP in all patients after using 20% mannitol. The IOP in the group of silicon-oil decreased from (33.25±2.56) mmHg (1 mmHg=0.133 kPa) to (23.21±1.85) mmHg, with a maximum decrease of 30.10%; the reduction in the group of C(3)F(8) was from (33.25±2.84) mmHg to (12.15±1.12) mmHg, with a maximum decrease of 33.44%. The IOP of the two groups dropped to a minimum both at 75 minutes. In the group of BSS, the IOP decreased from (32.95±2.33) mmHg to (17.50±1.35) mmHg, and the maximum extent of the decrease was 45.82% at 45 minutes. The difference in the IOP among the three groups at 20 min, 30 min, 45 min and 60 min was statistically significant (F=34.02, 112.68, 122.07, 34.83, all P=0.00). There were significant differences between the BSS group and the silicone-oil group (q=6.44, 13.04, 15.00, 17.11, all P=0.00), and between the BSS group and the C(3)F(8) group (q=7.68, 12.56, 12.93, 13.61, all P=0.00). Conclusion: In eyes with different intraocular tamponades, 20% mannitol was useful for short-term IOP reduction after vitrectomy, especially in those with BSS within one hour. But after 75 minutes, there was no statistically significant difference between groups. (Chin J Ophthalmol, 2019, 55:289-293).

[The colonoscopic characteristics of colorectal endometriosis: a single-centered retrospective study].

Objective: To reinforce the awareness of colorectal endometriosis (EM) in colonoscopy examination. Methods: Patients diagnosed as colorectal EM at Peking Union Medical College Hospital between February 2002 and February 2017 were enrolled in this study. The clinical characteristics and endoscopic features of EM lesions were summarized and compared between pathologically positive group and negative group. Results: A total of 34 cases were included with average age of (38.3±8.9) years old. All EM lesions located within rectum and sigmoid colon. The endoscopic lesions manifested as protrusion in 21 cases (61.8%) and protrusion-depression in 13 cases (38.2%), local stenosis in 8 cases (23.5%); erosive surface in 33 cases (97.1%) with local spontaneous hemorrhage in 4 cases (11.8%); nodal surface in 23 cases (67.6%), and lymphangiectasis base in 9 cases (26.4%). Endoscopic biopsy specimens were obtained in all cases with average 3 (2, 4) pieces. Positive results were found only in 4 patients (11.8%) with 3 endometriosis and one (endometrial) adenosarcoma. Compared with negative group, spontaneous hemorrhage was more frequent in positive group (2/4 vs. 2/30, P=0.013). Mean biopsy sample number was significantly larger in positive group (5 vs. 3, P=0.004). Conclusions: Colorectal endometriosis is mostly located within rectosigmoid region. Endoscopic features mainly include protrusion or protrusion-depression lesions with erosive and nodular surface, or local stenosis. Spontaneous hemorrhage under colonoscopy yields higher positive rate for biopsy, thus increasing biopsy sample numbers may improve pathology results.

[Assessment of renal function with intravoxel incoherent motion and diffusion tensor imaging in type 2 diabetic patients].

Objective: To evaluate the application value of intravoxel incoherent motion (IVIM) and diffusion tensor imaging (DTI) in detecting early-stage diabetic nephropathy and to assess the damage of ralated renal function. Methods: A total of 52 patients with type 2 diabetes diagnosed in Zhongda Hospital were collected from April 2016 to May 2017 and were assigned to DM group (diabetes without nephropathy, n=32) and DN group (diabetes with nephropathy, n=20) according to detection of microalbuminuria, a cohort of healthy recipients were included as control group (n=27) in the meantime. All of the subjects underwent IVIM and DTI examination. The cortical and medullary parameters[IVIM: perfusion fraction f, tissue diffusivity D, pseudodiffuvisity D(*;) DTI: fractional anisotropy FA, apparent diffusion coefficient ADC, principal diffusivities (λ1, λ2, λ3)]were obtained respectively and were compared among groups. The relationship between MRI related parameters and estimated glomerular filtration rate (eGFR) were statistically investigated; and diagnostic performance of IVIM and DTI in discriminating DM and DN group was evaluated by receiver operating characteristic analysis. Results: The cortical and medullary f, D values in DN group were lower than those in DM group and control group (F=17.32, 15.69, 6.71, 10.94, all P<0.05). D values of all subjects showed positive correlations with eGFR (cortex r=0.518, medulla r=0.538, both P<0.05). The diagnostic efficiency of cortical f values to discriminate diabetes and diabetic nephropathy was 0.817, the cut-off value was 0.205. The medullary FA value in DM group was lower than that in control group ((0.371±0.051 vs 0.423±0.043, t=4.188, P<0.05); and the medullary FA value in DN group (0.315±0.062) was lower than that in control and DM group (F=25.08, P<0.05). The medullary λ3 values in DM group and DN group were all significantly higher than that in control group (F=7.86, P<0.05). The diagnostic efficiency of medullary FA values to discriminate diabetes and diabetic nephropathy was 0.763, the cut-off value was 0.344. Conclusion: IVIM and DTI can reflect the abnormal perfusion and diffusion during early-stage diabetic nephropathy and have the potential value to assess the damage of ralated renal function.

Multidie LED combined with homogenizing optics to improve frequency response and intensity for FLIM applications.

Application of light-emitting diodes (LEDs) in frequency-domain fluorescence lifetime imaging microscopy (FLIM) has been limited by the trade-off between modulation frequency and illumination intensity of LEDs, which affects the signal-to-noise ratio in fluorescence lifetime measurements. To increase modulation frequency without sacrificing output power of LEDs, we propose to use LEDs with multiple dice connected in series. The LED capacitance was reduced with series connection; therefore, the frequency response of multidie LED was significantly increased. LEDs in visible light, including blue, green, amber and red, were all applicable in FLIM. We also present a homogenizing optics design, so that multidie LEDs produced uniform illumination on the same focal spot. When the homogenizing optics was combined with multicolour emitters, it provides multiple colour selection in a compact and convenient design.

[The efficacy of cyclosporine A as salvage therapy for severe active ulcerative colitis refractory to glucocorticoid].

Objective: To clarify the efficacy and safety of cyclosporine A (CsA) as salvage therapy in patients with severe active ulcerative colitis (UC) and refractory to steroids. Methods: A total of 24 severe active UC patients refractory to steroids and hospitalized from 2006 to 2012, were retrospectively enrolled.Data including demographic features, clinical manifestations, laboratory tests and medications were collected. Results: CsA was effective in 15(62.5%) patients, who did not receive colectomy during 12-week administration. This regimen was tolerable in most patients.Twelve (50.0%) patients reported 16 adverse events, but only one patient withdrew CsA due to intolerance.The rates of adverse events in initial intravenous CsA including 4 mg·kg(-1)·d(-1,) 3 mg·kg(-1)·d(-1) and 2 mg·kg(-1)·d(-1) were 2/2, 9/17 and 1/5 respectively.Responders had higher white blood cell count compared with non-responders (P= 0.045). Conclusions: CsA could be an effective alternative regimen to colectomy in severe active UC patients who are refractory to steroids.

Facilitating the measurement of circulatory hydrogen sulfide with fluorescence probe-coated microplates.

OBJECTIVE: The hydrogen sulfide (H2S) role in pathogenesis of various diseases were wildly addressed in recent decade. The circulatory (plasma or serum) and biological fluid H2S measurement is still an enormous issues due to the technical limitation. This paper aimed to develop a novel measurement method based on fluorescence probe. METHODS: Firstly, 20 µL ethanol was used to dissolve 100 pmol fluorescence probe, then added in a 96-well plate. An equal volume of ethanol was also added to the blank well of the plate. The plate was placed in a dark room for about 1 h until the fluorescence probe was evenly coated in the 96-well microplate and dried. The plate was frozen at -20 °C for later use. Secondly, the plasma or serum sample was added with saturated ammonium sulfate buffer (pH 7.8) and then centrifuged to remove the proteins. The equal volume supernatant liquid was added to the probe-coated well and the probe-uncoated well. The plate was incubated in a dark environment at 37 °C for 2 h. Finally, after incubation, the fluorescence density was acquired at ΛEx/ΛEm 340/445 nm in a microplate reader. The differences of the fluorescence density values between the probe-coated well and probe-uncoated well were counted and H2S concentration of plasma/serum was calculated by standard curve with NaHS. RESULTS: The method had high sensitivity (from 0.3 to 100 µmol/L) and specificity for measuring H2S as compared with other biologically relevant reactive sulfur species and sulfur-containing amino acid. Serum H2S concentrations were assayed in 188 health volunteers using this method [(12.1±3.5) µmol/L, 95%CI: 4.6-19.8 µmol/L], and the frequency distribution showed a normal tendency(one-sample Kolmogorov-Smirnov test, P>0.1). The serum H2S concentrations in 30 hypertension patients were decreased compared with 22 age- and gender-matched health individuals (paired-samples t test, t=9.937, P<0.001). There were no differences of H2S concentration in serum [(19.66±2.32) µmol/L] or plasma [(18.67±2.07) µmol/L], between the samples acquired from artery [(19.34±0.51) µmol/L] or vein [(18.99±0.50) µmol/L] of male Wistar rats (repeated measurement of ANOVA, P=0.38). One week frozen samples did not affect the detection. The values of the repeated measurement did not differ (two-way ANOVA, P>0.05). CONCLUSION: The present method is easily performed with high sensitivity, specificity and repeatability for circulatory H2S. It is also quick and may apply for large samples.

Rapid and sensitive LC-MS/MS method for the determination of auraptene in rat plasma and its application in a pharmacokinetic and bioavailability study in rats.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of auraptene, a constituent isolated from Fructus aurantii with potential to combat Alzheimer's disease, in rat plasma. Rat plasma samples were pretreated by protein precipitation with methanol. The analytes were separated by a Waters Sun Fire C18 column (50 mm x 2 mm, 5 µm) and eluted with 1:1000 methanol and formic acid/water (v/v) mobile phase with a flow rate of 0.5 mL/min. Multiple reaction monitoring was used to monitor the transition of the deprotonated auraptene molecule with an m/z of 299.3 [M+H](+), to the product ion with an m/z of 162.9 [M+H](+). Progesterone, with an m/z of 315.2→ 96.9 was used as an internal standard. The limits of detection and of quantification of auraptene in the rat plasma were 1 and 5 ng/mL, respectively. The method was linear in the concentration range of 20- 2000 ng/mL with coefficient correlation of 0.9956. After auraptene (100 mg/kg, p.o.) administration, the maximum plasma concentration and the time taken to reach maximum concentration were 1719.5 ± 384.3 g/mL and 108.0 ± 25.3 min, respectively. The elimination half-life was 108.0 ± 25.3 for auraptene (100 mg/kg, p.o.) and 3.0 ± 0 min for auraptene (2 mg/kg, i.v.). The oral bioavailability was about 8.5%.