Intraperitoneal administration of timosaponin A-III (TA-III) has therapeutic effects on high-fat diet-induced metabolic dysfunction-associated steatotic liver disease (MASLD), but oral administration has no effect. This suggests that gut microbiota may affect the oral bioavailability of TA-III. Metabolic dysfunction-associated steatohepatitis (MASH) is an inflammatory subtype of MASLD. To investigate the therapeutic effect of different administration modes of TA-III on MASH and its relationship with gut microbiota metabolism. In this study, a MASH mouse model was induced by choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Comparing the therapeutic effect of intraperitoneal injection (10 mg·kg^-1, ip) and intragastric administration (100 mg·kg^-1, ig) of TA-III. The concentration of TA-III in serum of rats under the two administration modes was analyzed. On this basis, the metabolic effect of gut microbiota on TA-III in mice was verified by the experiment of metabolism of gut microbiota in vitro. The pharmacokinetic experiment of combined antibiotic intervention in mice further verified the metabolism of TA-III by gut microbiota in mice. Finally, the concentration of TA-III in serum of mice after the administration of TA-III by intragastric administration under different antibiotic intervention conditions was compared, and 16S rRNA sequencing analysis was combined to find the key bacteria that may participate in the metabolism of TA-III. The animal welfare and experimental procedures in this paper were in accordance with the provisions of the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. The ethics approval number is PZSHUTCM2307030004 and PZSHUTCM2310200003. The results showed that TA-III (10 mg·kg^-1, ip) had definite therapeutic effect on MASH mice, but TA-III (100 mg·kg^-1, ig) was ineffective. The analysis showed that the prototype concentration of TA-III in serum and liver of mice in TA-III (100 mg·kg^-1, ig) was significantly lower than TA-III (10 mg·kg^-1, ip), suggesting that the oral administration of TA-III may be metabolized by gut microbiota. The concentration of TA-III in serum of streptomycin (Str) treated mice was higher than normal mice. Combined with 16S rRNA gene sequencing analysis, it was found that the abundance of Akkermansia_muciniphila (A. muciniphila) was significantly reduced in the Str group. In vitro experiments showed that A. muciniphila could metabolize TA-III. In conclusion, gut microbiota is an important factor affecting the efficacy of TA-III administration through the gastrointestinal tract, in which A. muciniphila may play an important role.

Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5E. The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07. HRP- Ⅱ protein was used as antigenic reagent for panning and screening. Results The total RNA from the spleen cells was isolated, and cDNA obtained and VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. After panning and screening, 8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA. Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be used as reagents in the diagnosis and therapy of malaria.

Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5E. The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07. HRP- Ⅱ protein was used as antigenic reagent for panning and screening. Results The total RNA from the spleen cells was isolated, and cDNA obtained and VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. After panning and screening, 8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA. Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be used as reagents in the diagnosis and therapy of malaria.

OBJECTIVETo investigate the effects of inward rectifier potassium channel blockers (BaCl2, CsCl) on the functions of endothelial progenitor cells (EPCs).

METHODSDensity gradient centrifugation-isolated rat hone marrow mononuclear cells were cultured in vitro. EPCs were harvested and seeded on six culture dish when cells grew to 3-5 passages. Before testing the EPCs were synchronized with M199, which contain 2% fetal calf serum. In the end, EPCs were treated with different intervention. The experiment mainly included two parts: (1) BaCl2 (100 micromol/L) and free BaC2 of Tyrodes solution; (2) CsCl (1 mmol/L) and control. Cell pretreated with blockers above mentioned for 12 h, then the gene expression of stromal cell-derived factor-1 (SDF-1), epoprotenol (PGI2) were assessed, beyond that the ability of adhesion, migration were assayed with different tests. In addition, the medium was collected when EPCs were treated for 3 days. The levels of SDF-1 were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Going even further, EPCs were treated with the signal pathway blockers in advance, after repeat the above steps, in order to analyze the change of SDF-1 and then discuss its mechanism.

RESULTSCompared with control group, BaCl2, CsCl could increase EPC adhesion and migration to same extent. Moreover, the gene expression of SDF-1, PGI2 was significantly up-regulated and the production of SDF-1 increased evidently. Furthermore, the mechanism of SDF-1 secretion increasing mainly was associated with eNOS signaling pathways.

CONCLUSIONBa2+ and Cs+ play important roles in increasing EPCs functions, such as adhesion, migration and secretion.

Animals ; Barium Compounds ; pharmacology ; Cells, Cultured ; Cesium ; pharmacology ; Chemokine CXCL12 ; metabolism ; Chlorides ; pharmacology ; Endothelial Cells ; cytology ; Enzyme-Linked Immunosorbent Assay ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; physiology ; Rats ; Stem Cells ; cytology

A 51-year-old woman presented with metachronous tumor development in bilateral breasts, thyroid, and endometrium. Additional signs and symptoms fulfilled the National Comprehensive Cancer Network criteria for Cowden syndrome. Immunohistochemistry showed loss of PTEN expression in all tumors. Single nucleotide variants, 647 germline variants (including one each in PTEN and MSH3), and 21 somatic mutations within exons were detected in all tumors after whole-exome sequencing. There were 0, 11, and 46 specific somatic mutations in bilateral breasts, thyroid, and endometrial cancers, respectively.Although PTEN mutation is key to the development of Cowden syndrome, DNA repair dysfunction might be the initial driver of mutations. Fewer mutations were required to induce initial bilateral breast carcinomas, with subsequent thyroid and endometrial carcinomas requiring more mutations for induction. When genetic screening is unavailable, breast cancer patients with clinical manifestations of Cowden syndrome must be carefully assessed for secondary malignancies, such as thyroid and endometrial carcinomas.

BACKGROUNDReal-time perfusion imaging (RTPI) using ultrasound contrast agents has shown good "accuracy" in detecting myocardial infarction, however its accuracy in the assessment of peri-infarct ischemia and stress echocardiography are not known. The aim of this study was to determine the accuracy of RTPI in assessment of peri-infarct ischemia during dobutamine and adenosine stress.

METHODSWe employed the RTPI modality (Agilent and ATL Philips) in a canine model (18 dogs) of distal coronary occlusion and proximal coronary stenosis. Using coronary flow probe recordings, the physiologic significance of proximal coronary stenosis was established by confirming abolition of the coronary reserve. The contrast agent Optison was given as a slow bolus injection at baseline, during prolonged distal coronary occlusion, during adenosine bolus stress and during dobutamine stress. Triphenyltetrazolium chloride (TTC) staining was used to verify a distal infarction. RTPI recordings at baseline, the distal coronary occlusion and stress protocols were randomly mixed and reviewed blindly.

RESULTSIn all but one dog, RTPI detected a distal infarct as small as 9% of the left ventricle. The sensitivity, specificity and overall diagnostic accuracy of RTPI in the detection of distal infarcts were: 94%, 89% and 92%, respectively. The sensitivity, specificity, and overall diagnostic accuracy of RTPI in the assessment of peri-infarction ischemia were 83%, 92% and 88% for adenosine stress and 95%, 86% and 91% for dobutamine stress, respectively.

CONCLUSIONSEven small distal infarcts can be detected by RTPI; peri-infarct ischemia can be accurately recognized by RTPI during stress; adenosine and dobutamine stress appear equally reliable in the RTPI evaluation of peri-infarct ischemia.

Adenosine ; toxicity ; Animals ; Coronary Circulation ; drug effects ; Dobutamine ; toxicity ; Dogs ; Echocardiography ; methods ; Female ; Hemodynamics ; drug effects ; Male ; Myocardial Infarction ; diagnostic imaging

Objective To evaluate the effect of comprehensive intervention measures on improving hand hygiene (HH)compliance of health care workers(HCWs)in intensive care units (ICUs)by multicenter study.Methods A total of 47 hospitals in 12 provinces and cities were enrolled in the study,from October 2013 to September 2014, HH compliance among HCWs in ICUs was investigated according to World Health Organization HH investigation method,HH compliance rates before and after intervention were compared.Results HH compliance rate of HCWs in ICUs increased from 73.70%(11 155/15 135)before intervention to 78.70%(18 206/23 132)after intervention (χ2 =128.16,P <0.01);except northeast region ,HH compliance of HCWs in the other provinces increased signif-icantly after intervention(all P <0.05);among all departments,HH compliance of HCWs in general ICUs,respira-tory ICUs,emergency ICUs,and surgical ICUs increased significantly(all P <0.05);HH compliance of HCWs of all kinds of occupations increased significantly(all P <0.05);HH compliance rates before touching a patient,before clean/aseptic procedure,after touching a patient,and after touching patient surroundings increased significantly after intervention(all P <0.01 ).Conclusion Comprehensive interventions are helpful for improving HH compliance of HCWs in ICUs. Objective To evaluate the effect of comprehensive intervention measures on improving hand hygiene (HH)compliance of health care workers(HCWs)in intensive care units (ICUs)by multicenter study.Methods A total of 47 hospitals in 12 provinces and cities were enrolled in the study,from October 2013 to September 2014, HH compliance among HCWs in ICUs was investigated according to World Health Organization HH investigation method,HH compliance rates before and after intervention were compared.Results HH compliance rate of HCWs in ICUs increased from 73.70%(11 155/15 135)before intervention to 78.70%(18 206/23 132)after intervention (χ2 =128.16,P <0.01);except northeast region ,HH compliance of HCWs in the other provinces increased signif-icantly after intervention(all P <0.05);among all departments,HH compliance of HCWs in general ICUs,respira-tory ICUs,emergency ICUs,and surgical ICUs increased significantly(all P <0.05);HH compliance of HCWs of all kinds of occupations increased significantly(all P <0.05);HH compliance rates before touching a patient,before clean/aseptic procedure,after touching a patient,and after touching patient surroundings increased significantly after intervention(all P <0.01 ).Conclusion Comprehensive interventions are helpful for improving HH compliance of HCWs in ICUs.

OBJECTIVETo observe the laryngopharynx manifestation and electromyography characteristics of myasthenia gravis (MG) patients.

METHODSThirty cases of MG were included in this study, their laryngopharynx symptoms and signs, voice acoustic assessment, laryngeal electromyography (LEMG) behaviors and repetitive nerve stimulation test(RNS) were analyzed, and the data was compared with that of normal subjects.

RESULTSAbout 36.7% of MG patients (11/30) had the symptoms of hoarseness, voice fatigue, dysphonia and dysphagia. The vocal folds movements of 16.7% of MG patients(5/30) appeared weaker than normal, and their vocal glottic couldn't close completely, while with a seam during phonation. Voice amplitude (68.3 +/- 14.6) dB (x +/- s, same at below), and maximum phonation time (15.1 +/- 4.0) s, were greatly lower than normal; shimmer(2. 43 +/- 1.19)%, and normalized noise energy (-9.6 +/- 3.3) dB, were greatly higher than normal. The amplitudes of interference patterns in MG patients' LEMG markedly decreased, except introarytenoid muscle, during low, normal and high pitch phonation, the amplitudes of thyoiarytenoid muscle were (215 +/- 69) microV, (298 +/- 113) microV and (380 +/- 153) microV, those of cricoarytenoid muscle were (253 +/- 92) microV, (361 +/- 116) microV and (486 +/- 155) RV. The turns increased but had no statistical difference. In the RNS test, 83.3% MG patients (25/30) showed masculine response. There were about 2.20 +/- 1.32 pieces of laryngeal muscles involved, and the reduction rate in amplitude of the compound muscle action potential for RNS was about (27.9 +/- 19.2)%.

CONCLUSIONSOnly parts of MG patients had laryngopharyngeal symptoms, but the laryngeal muscles of most of them were involved, appearing as the masculine response for RNS, the decreased synchronization of the laryngeal muscles' interference patterns, the decreased capacity of phonation. MG must be differentiated when a patient has the symptoms of voice weakness, hoarseness and dysphonia. Laryngeal RNS test should be used in the early diagnosis of MG.

Adolescent ; Adult ; Aged ; Articulation Disorders ; Case-Control Studies ; Electromyography ; Female ; Humans ; Hypopharynx ; physiopathology ; Laryngeal Muscles ; physiopathology ; Male ; Middle Aged ; Myasthenia Gravis ; physiopathology ; Young Adult

OBJECTIVETo investigate the value of serum cystatin C (Cys-C), urinary Cys-C, urinary retinol binding protein (RBP) and urinary neutrophil gelatinase-associated lipocalin (NGAL) in the early assessment of multiple myeloma (MM) and their characteristic changes in different pathological types of renal impairment.

METHODSAccording to glomerular filtration rate (eGFR), the patients were divided into two groups, of which marked group A with normal renal function, the other marked group B with abnormal renal function. Sixty healthy subjects were chosen as control. Detection of the serum Cys-C, urinary RBP, urinary Cys-C, urinary NGAL, serum creatinine (Scr), urinary microalbumin (MAU) and urinary α1-microglobulin (α1-MG) were performed. Renal biopsy was carried out for patients who had abnormal serum Cys-C, urinary Cys-C, urinary RBP, urinary NGAL and were willing to accept further test.

RESULTSCompared with healthy controls, the serum Cys-C, urinary RBP, urinary Cys-C, urinary NGAL of group A were significantly higher than that of healthy controls. Six group A patients received renal biopsy, and varying degrees of renal damage were discovered. The serum Cys-C, urinary RBP, urinary Cys-C and urinary NGAL positive rate were 66.7%, 66.7%, 66.7% and 83.3%, respectively. Of twenty-four cases received biopsy after abnormal examination results were shown, six turned out to be amyloidosis, twelve cast nephropathy (CN) and 6 monoclonal immunoglobulin deposition disease (MIDD). Compared with MIDD and amyloidosis, the urinary Cys-C and NGAL of the CN group are significantly higher (P < 0.05). Compared with CN and amyloidosis, urinary RBP of MIDD is significantly higher (P = 0.043). Compared with MIDD and CN, the MAU of amyloidosis is significantly higher (P = 0.006).

CONCLUSIONCompared with the conventional indicators, serum Cys-C, urinary Cys-C, RBP and NGAL are more sensitive in early assessment of MM patients with renal damage. The MAU is higher in amyloid, the urinary Cys-C and urinary NGAL are significantly elevated in CN, the urinary RBP is significantly elevated in MIDD.

Acute-Phase Proteins ; urine ; Adult ; Aged ; Case-Control Studies ; Cystatin C ; blood ; urine ; Female ; Humans ; Kidney ; pathology ; Kidney Diseases ; blood ; diagnosis ; urine ; Kidney Function Tests ; Lipocalin-2 ; Lipocalins ; urine ; Male ; Middle Aged ; Multiple Myeloma ; blood ; pathology ; urine ; Proto-Oncogene Proteins ; urine ; Retinol-Binding Proteins ; urine

OBJECTIVETo observe expression pattern of aquaporin 4 (AQP4) in brain tissue of severely scalded rabbit during early stage of brain edema and its relationship with signal changes in magnetic resonance imaging (MRI), and to explore the feature of water transmembrane transportation in brain edema at early stage after severe scald.

METHODSThirty-five healthy New Zealand white rabbits were divided into control group (C, n = 5) and scald group (S, n = 30) according to the random number table. Rabbits in S group were inflicted with 50% TBSA full-thickness scald with brain edema (confirmed by histopathologic examination). At post scald hour (PSH) 1, 2, 3, 4, 5, and 6, signal change in cerebral MRI, as well as dynamic change in apparent diffusion coefficient (ADC) were examined in rabbits of S group. Specimens were harvested from frontal cortex, parietal cortex, temporal lobe cortex, basal ganglia, and cerebellum in rabbits of S group for determination of protein and gene expressions of AQP4 with immunohistochemical method and RT-PCR. Above-mentioned indexes were also determined in C group. Data were processed with one-way analysis of variance and linear correlation analysis (between protein expression of AQP4 and ADC value).

RESULTSThere was no significant change in image signal of MRI at each time point in S group as compared with that in C group, including T1WI, T2WI, and DWI. Compared with those in C group, ADC in S group at PSH 4, 5, and 6 were significantly decreased (with F values from 0.492 to 2.271, P values all below 0.05). The expression of AQP4 protein in each part of brain tissue in S group was obviously increased at PSH 2, 3, 4, 5, and 6 as compared with those in C group (from 0.164 ± 0.022 to 0.247 ± 0.018), and it peaked at PSH 3 or 4 (from 0.237 ± 0.042 to 0.306 ± 0.026), with F values from 2.420 to 11.439, P values all below 0.05. The expressions of AQP4 protein were similar in brain tissue of all regions, and they were negatively correlated with corresponding ADC values (with r values from -0.489 to -0.337, P < 0.05 or P < 0.01). Compared with that in C group, the expression of AQP4 mRNA in each part of brain tissue in S group was obviously increased at each time point, and it reached the peak at PSH 2 (with F values from 39.992 to 238.584, P values all below 0.05). The expressions of AQP4 mRNA were similar in all brain tissue regions.

CONCLUSIONSBrain edema within 6 hours after severe scald was mainly characterized by cytotoxicity, in which AQP4 may play an important role. ADC value may have important reference value for non-invasive and convenient assessment of development of brain edema.

Animals ; Aquaporin 4 ; metabolism ; Brain ; metabolism ; pathology ; Brain Edema ; etiology ; metabolism ; pathology ; Burns ; complications ; metabolism ; pathology ; Diffusion Magnetic Resonance Imaging ; Rabbits