Objectve To investigate the application value of ultraifltration with millipore ifltration method test of old biological samples DNA. Methods 23 old blood samples separately were cut blood piece equally suitable size of three groups, labeled A, B and C group. DNA was extracted by magnetic bead methed and get DNA template with 80μL, 80μL, 20μL elution solution, respectively, then the DNA template in group A and C take right amount directly ampliifed, group B with milipore ultraifltration ifltration condensed amplication. PCR products were detected by ABI3130xl genetic analyzer and analyzed by GeneMapperID V3.2 software. The date were analyzed by using SPSS software. Results No samples were ampliifed for all STR loci in group A. There were 18 cases in group B samples amplified all STR loci and 11 samples in group C. Conclusion Application of millipore ultrafiltration method can signiifcantly improve the old biological samples DNA classiifcation success rate.

Objective To demonstrate the histology in acellular dermal matrix(ADM)after being transplanted in vivo over time. Methods Forty male SD rats were recruited for the experiment. Subcutaneous implantation of an 1 cm × 1 cm ADM was given in the left sides on the back of the rat for the experimental group, while only dissection and suturing were performed in right side of the back for the control group. All the animals will be sacrificed at appointed time after operation, Five ADM samples were harvested in each time point. The content and proportion of collagen type were examined with HE staining, Picrosirius staining, Masson′s trichrome staining, and Immunohistochemical staining (targets: pan macrophage, M1 macrophage and M2 macrophage). Results All rats survived after operative without any complications. Significant differences of thickness were not observed at the end of 5 months; HE scores suggested that ADM increased in cell infiltration scores in 2 weeks before the plateau , vascularity also showed a similar trend; Collagen trichrome staining showed a substantial increase in density of collagen bundles with time. The comparison of the proportion of collagen among days showed significant differences (P < 0.05). Immunohistochemical staining of M1 and M2 showed that macrophages had distinct polarization profiles in materials. Furthermore, the comparison of M1 vs M2 response associated with different materials showed significant differences in all time points (P < 0.05). Conclusions The chemically cross-linked ADM could keep long time in the body; ADM significantly stimulated proinflammatory of M2 differentiation from M1 in constructive remodeling.

Animals ; Antioxidants ; metabolism ; Fatigue ; blood ; Female ; Hypericum ; chemistry ; Male ; Mice ; Mice, Inbred ICR ; Physical Conditioning, Animal ; Plant Extracts ; pharmacology

Aim To investigate the role of p38 mitogen-activated protein kinases(MAPKs) in genistein-induced osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells(BMSCs).Methods Mouse BMSCs cultured in phenol red-free ?-MEM containing 10% V/V FBS,were added ?-glycerophosphate and ascorbic acid for inducing osteoblastic differentiation,and treated with 0.01~1 ?mol?L~(-1) genistein and/or SB203580 and PD98059.The temporal sequence of osteoblastic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity(ALP) and calcium deposition.The MAPK phosphorylation level was detected by Western-blot.Results Genistein(0.01~1 ?mol?L~(-1)) showed a dose-dependent effect on osteoblastic differentiation as evidenced by increased alkaline phosphatase(ALP) activity and calcium deposition in mouse BMSCs cultures.Genistein(1 ?mol?L~(-1))-induced osteoblastic differentiation of BMSCs was diminished by p38 MAPK inhibitor but not the p44/42 MAPK inhibitor.The effects of Genistein were associated with rapid and sustained activation of p38 MAPK in the BMSCs cultures,which was blocked by SB203580(1 ?mol?L~(-1)).In contrast,Genistein treatment resulted in inactivation of p42/44MAPK,which was further attenuated by PD98059(25 ?mol?L~(-1)).Conclusion p38 MAPK plays an important role in genistein-induced osteoblastic differentiation of mouse BMSCs cultures.

Objective To study the enhancement patterns of hilar bile duct wall of ischemic-type biliary lesion (ITBL) on contrast-enhanced ultrasound (CEUS).Methods Eighteen healthy subjects,18 orthotropic liver transplantation (OLT) recipients without complications,and 36 patients,which were subdivided into 2 groups according to the final diagnosis:patients with (n =24) and without (n =12)ITBL,were enrolled in this study.The patients without ITBL had anastomotic biliary stricture (n =3),cholangitis (n =4),biliary sludge (n =1),and acute rejection (n =4),respectively.The images of baseline sonography and CEUS were retrospectively analyzed in consensus by 2 readers.The enhancement time and level of hilar bile duct wall,hepatic artery and liver parenchyma were recorded.Results Hilar bile duct wall became enhancing earlier than liver parenchyma in all of 4 groups.During arterial phase,hyper-or isoenhancing bile duct walls were present in most cases in the groups of healthy subjects,OLT recipients without complications and patients without ITBL.However,non-or hypo-enhancement of hilar bile duct wall were present in 16 (66.7％) ITBL patients,which is different from the other groups (P ＜0.05).Conclusions The main features of ITBL differing from the other groups were non-or hypo-enhancement of hilar bile duct wall in arterial phase.It may be a diagnostic index to apply in detecting ITBL with CEUS.

Abstract： Objective　To analyze the characteristics and corresponding drug resistance of pathogenic bacterial spectrum in eight major infection sites of hospitalized patients, and to provide epidemiological data for the rational selection of antibiotics in clinical practice. Methods　A total of 396 bacterial strains isolated from clinical specimens of hospitalized patients in member institutions of the Hainan Provincial Bacterial Resistance Monitoring Network from September 1, 2020, to September 30, 2022, were included in this study. Data were screened and filtered from the database of MH120 Microbial Identification and Drug Sensitivity Analysis System based on the technical scheme of the National Bacterial Drug Resistance Surveillance Network and Science and Technology Basic Resources Investigation Project research plan in 2020. The testing data were integrated, summarized, and analyzed using EXCEL and WHONET 5.6 software, and statistical analysis was conducted using SPSS 26.0 software. Results　Among of 396 strains of bacteria, 78 (19.7%) were isolated from respiratory tract specimens, 74 (18.7%) from urinary tract specimens, 72 (18.2%) from blood specimens, 54 (13.6%) from abdominal cavity specimens, 48 (12.1%) from skin and soft tissue specimens 48 strains (12.1%), 30 (7.6%) from reproductive tract specimens, 22 (5.6%) from central nervous system specimens, 18 (4.5%) from digestive tract specimens. Gram-negative bacteria accounted for 69.4% of the isolates, while gram-positive bacteria accounted for 30.6%. The top five gram-negative bacteria isolated were Klebsiella pneumoniae (14.9%), Escherichia coli (14.4%), Pseudomonas aeruginosa (10.4%), Acinetobacter baumannii (5.3%), and Salmonella species (4.5%). The top five gram-positive bacteria were Staphylococcus aureus (11.1%), Streptococcus agalactis (7.8%), Enterococcus faecalis (3.0%), Enterococcus faecium (2.8%), and Streptococcus suis (1.8%). Respiratory failure and bloodstream infection were independent influencing factors of treatment response (P<0.01). The resistance rate of Escherichia coli to ampicillin was 81.4%, and the resistance rate of Staphylococcus aureus to gentamicin and levofloxacin were both below 7%. Conclusions　The pathogen spectra vary with different infection sites of patients, and rational selection of antibiotics based on drug susceptibility testing is crucial to shorten the treatment time of patients and avoid the unnecessary emergence of drug-resistant strains caused by drug abuse.

OBJECTIVETo find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol (TCP) and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied.

METHODSVero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane.

RESULTS0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was <1.0 mg/L. After exposure to leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Vero cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis.

CONCLUSIONThese results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to indicate the composite bio-toxicity of trace chemicals than proliferation inhibition, inhibition on bioluminescence and necrosis. Nevertheless, the quantification of morphological change should be studied further.

Animals ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cercopithecus aethiops ; Vero Cells ; Water Pollutants, Chemical ; toxicity

Objective To investigate the application value of deep learning optical coherence tomography ( OCT) on wet age-related macular degeneration ( wAMD) assisted diagnosis. Methods Weakly supervised deep learning algorithms was applied on the premise that only disease or not can be provided as a marker. The OCT image automatically assisted in the diagnosis of diseased areas of wAMD,and thermograms were applied to provide a basis for doctors to detect disease areas. Based on the deep learning of weak supervision,a new network algorithm structure was proposed for detecting disease area in ophthalmic OCT images. At the same time, thermograms were adopted to improve the accuracy of the lesion map,which is the location of the lesion area. This study followed the Declaration of Helsinki. This study protocol was approved by Ethic Committee of Ningbo Eye Hospital ( No. 2018-YJ05 ) . Written informed consent was obtained from each subject before entering study cohort. Results Resnet-based deep learning algorithm gave a diagnostic accuracy rate of 94. 9％ for the disease, which was much higher than that of AlexNet 85. 3％,VGG 88. 7％, and Google-Net 89. 2％. The thermograms with different colors provided a more convenient auxiliary diagnosis basis for doctors. Conclusions Compared with the original classification network,which needs disease area markers as empirical knowledge, deep learning algorithm model not only provides better results in the classification of retinal diseases,but also marks potential disease areas. The lesion area provides a basis for judging the area of the lesion for the diagnosis of wAMD.

OBJECTIVETo observe the effect of recombinant interleukin-6 (IL-6) and osteoprotegerin (OPG) on inhibiting bone absorption induced by receptor activator for nuclear factor-kB ligand (RANKL) in murine osteoclast precursor cells (OCPs) model.

METHODSRAW 264.7 cells were solely treated with 50 ng/ml RANKL for 1 day, and then they were divided into three groups: RANKL (control group), RANKL+IL-6 (IL-6 group) and RANKL+IL-6+OPG (combination group). These cells were harvested and investigated by means of HE staining under light microscope after consecutive 9 days. Furthermore, staining tartrate-resistant acid phosphatase(TRAP)-positive multinucleated cells were detected by inverted phase contrast microscope. The absorption pits of bone slices were observed under scanning electron microscope.

RESULTSThe number of mature osteoclast cells in control group was more than that in IL-6 alone or IL-6 combined with OPG group (P less than 0.05). Interestingly, this experiment has also demonstrated that there was a large number of TRAP-positive multinucleated osteoclasts (more than 3 nuclei) and several bone absorption formation in the control group, whereas the outcome was completely different in both IL-6 group and IL-6+OPG group (P less than 0.05).

CONCLUSIONIL-6 can suppress the differentiation of mature osteoclasts as directly adding it into the RAW 264.7 cells induced by 50 ng/ml RANKL, and further the effect of osteolysis is remarkably reduced. When treatment with IL-6 combined with OPG, a more effective strategy for the treatment of osteoporosis is reached.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Interleukin-6 ; administration & dosage ; pharmacology ; Mice ; Osteoclasts ; cytology ; drug effects ; Osteoprotegerin ; administration & dosage ; pharmacology ; RANK Ligand ; pharmacology ; Recombinant Proteins ; administration & dosage ; pharmacology

OBJECTIVETo investigate the prevalence of periodontal pathogens from saliva of patients with type 2 diabetes mellitus (T2DM), and to characterize the association between the glucose status and periodontal pathogens in oral cavity.

METHODSAll the subjects were hypertension patients under regular care at Beijing hypertension prevention and management institute. Whole unstimulated saliva samples were collected from 45 non-diabetic subjects (non-DM group), 80 well-controlled diabetic patients (DM-well group) and 100 poor-controlled diabetic patients (DM-poor group). DNA was extracted from the salivary deposition, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and Treponema denticola (Td) were detected by polymerase chain reaction (PCR) method based on 16SrRNA. Prevalence and quantity of the pathogens under different glucose states were compared and logistic regression model was set to analyze the factors related to each bacterium.

RESULTSThe prevalence of Tf in DM-well group and DM-poor group was significantly lower than that of non-DM group [81% (65/80), 80% (80/100) vs 91% (41/45), P = 0.048], meanwhile the quantity of Tf was also lower than that of non-DM group [1.9(2.6), 2.1(5.3) vs 3.4(6.4)] (P > 0.05). With the worsening of glucose control, the quantity of Tf was declining (P = 0.032). However, the prevalence and the quantity of Pg, Td in 3 groups had no statistical differences (P > 0.05). After adjusting age, gender, number of missing teeth and other periodontal parameters, OR of having Tf in saliva from DM-well group and DM-poor group was 0.58 and 0.53, respectively.

CONCLUSIONSAbnormal blood glucose state may affect the colonization of Tf in oral cavity.

Aged ; China ; epidemiology ; Diabetes Mellitus, Type 2 ; epidemiology ; microbiology ; Female ; Humans ; Male ; Middle Aged ; Periodontitis ; microbiology ; Porphyromonas gingivalis ; isolation & purification ; Saliva ; microbiology ; Treponema denticola ; isolation & purification