Objective:To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis ( P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods:A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 μg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results:At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1β [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm 2), the thickness of the esophageal wall (median 172.52 μm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1β [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions:P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Objective:To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis ( P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods:A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 μg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results:At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1β [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm 2), the thickness of the esophageal wall (median 172.52 μm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1β [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions:P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Rehabilitation and multidisciplinary team management are very important to improve the prognosis of children with prolonged mechanical ventilation(PMV). The current status of rehabilitation intervention including physical therapy, neuromuscular electrical stimulation and inspiratory muscle training in adult patients with PMV were discussed, aiming to provide some evidence for the implementation of rehabilitation in children with PMV.

Objective:To investigate the effects of Porphyromonas gingivalis ( P. gingivalis) fimbrillin (FimA) on the progression of esophageal squamous cell carcinoma (ESCC). Methods:Wild-type P. gingivalis and fimA gene-deleted P. gingivalis ( fimA-/-P. gingivalis) were used to infect ESCC cells after morphology and PCR identification. Immunofluorescence, CCK-8 and Transwell chamber were used to detect the effects of FimA on the infectivity of P. gingivalis and it influences on cell invasion, proliferation and migration. Western blot was used to detect pSmad2/3 changes. The growth of tumor was detected in a nude mouse model bearing subcutaneous tumor. Results:Deletion of FimA might reduce the interbacterial adhesion of P. gingivalis. Compared with wild-type P. gingivalis, less fimA-/-P. gingivalis could infect NE6-T cells. Moreover, the proliferation, migration and invasion of NE6-T and KYSE30 cells as well as the activation of pSmad2/3 induced by P. gingivalis were inhibited after deletion of FimA. The growth of KYSE30 infected by fimA-/-P. gingivalis in nude mice was significantly slower than that of the wild-type P. gingivalis group. Conclusions:FimA mediated the effects of P. gingivalis on promoting the evolution of ESCC and was a potential target molecule to block the tumor-promoting effect of P. gingivalis.

Objective:To explore the effects of external diaphragm electrical stimulation on the diaphragm thickness and function in mechanically ventilated children.Methods:A randomized controlled trial was conducted in children who were admitted to PICU at Children′s Hospital of Fudan University and received mechanical ventilation between June 2021 and April 2022.The control group was given the routine treatment of mechanical ventilation, and the intervention group was given external diaphragm electrical stimulation in the early stage of mechanical ventilation in addition to routine treatment.Diaphragm thickness was continuously measured by bedside ultrasound every day for one week after mechanical ventilation, and the changing trend of diaphragm thickness was observed, and the diaphragmatic thickening fraction (DTf) and the incidence of ventilator-induced diaphragmtic dysfunction(VIDD) were calculated at the same time.Results:A total of 32 valid samples were included, including 15 cases in intervention group (10 males) and 17 cases in control group (11 males). The median age of the patients was 33 (10, 77) months, and the median duration of mechanical ventilation was 12 (8, 21) days.The reasons for mechanical ventilation in children included respiratory insufficiency in ten cases, brain dysfunction in ten cases, heart failure in eight cases, and postoperative surgery in four cases.The diaphragm end-expiratory thickness (DTe) in intervention group and the control group showed a gradually decreasing trend from the 1st day to the 7th day.The left thickness was reduced by 11% on the 7th day compared to 1st day in intervention group, which was reduced by 18% in control group; the average daily DTe was reduced by 2% per day in intervention group and by 3% per day in control group.The trends on the right and left were similar.The DTe thickness in the intervention group was greater than that in control group, among which, the mean DTe thickness in the left side of the intervention group on the 7th day was (0.110 7±0.023 7)cm, which was greater than that in control group (0.093 5±0.016 9)cm, and the difference was statistically significant ( t=-2.372, P<0.05); On the second day, the mean DTe thickness on the right side in the intervention group was (0.1267±0.0277) cm, which was greater than that in control group (0.104 7±0.018 1)cm, and the difference was statistically significant ( t=-2.688, P<0.05). DTf in the intervention group was lower than that in control group at 7th day, but the difference was not statistically significant(left DTf: adjusted mean difference was -0.117, P=0.088; right DTf: adjusted mean difference was -0.065, P=0.277). The incidence of VIDD in the intervention group was lower than that in control group(33.3% vs.41.2%), but the difference was not statistically significant ( χ2=0.005, P=0.946). Conclusion:External diaphragmatic electrical stimulation may be helpful for alleviating diaphragmatic atrophy in mechanically ventilated children.However, whether the improvement of diaphragm atrophy is beneficial to clinical outcome still needs further study.

Objective:To explore the reproducibility of ultrasound measurements of children′s anterior, middle and posterior diaphragm motions.Methods:Thirty children admitted to a pediatric intensive care unit were positioned supine and a 5MHz ultrasound probe was placed over the intersection of their right midclavicular line with the costal margin. M-mode ultrasound was used to record the excursion and contraction velocity of the anterior, middle and posterior diaphragm during respiration. The observations were duplicated so the repeatability of the measurements could be evaluated using intra-group correlation coefficients calculated for the diaphragm excursions and the contraction velocities. Analysis of variance was used to explore the differences in excursion and contraction velocity among different parts of the diaphragm.Results:The intra-group correlation coefficients calculated for the anterior, middle and posterior diaphragm were 0.89, 0.95 and 0.90 respectively. The corresponding values for the contraction velocities were 0.90, 0.94 and 0.95 respectively. Both variables measured by ultrasound showed high repeatability. The average anterior, middle and posterior diaphragm excursion values (in mm) were 8.1±3.1, 7.4±3.0 and 5.5±2.3, and the corresponding average contraction velocities (in mm/s) were 12.5±4.8, 11.5±6.3 and 8.9±4.0.Conclusions:Measurements of children′s diaphragm motions using ultrasound show high repeatability. The excursions and contraction velocities of the anterior, middle and posterior diaphragm differ in children. The motion of one part of the diaphragm cannot represent the functioning of the entire diaphragm.

Objective: To isolate and identify Prevotella nigrescens (P.nigrescens) in gingival crevicular fluid of patients with chronic periodontitis and to analyze its tumor-promoting role in esophageal squamous cell carcinoma (ESCC). Methods: Samples of gingival crevicular fluid were collected from patients with chronic periodontitis and cultured on GAM agar medium under anaerobic conditions. Black colonies on GAM agar plates were picked for subculture, and then the bacteria were isolated and purified. Bacterial identification was conducted using Gram staining and 16S rDNA sequencing analysis. The isolated bacterial species was used to infect ESCC NE6-T cells and the changes in biological characteristics of the infected cells were evaluated. Results: Multiple bacterial colonies were observed after anaerobic culturing of gingival crevicular fluid samples for 120 h on GAM agar plates. A single bacterial colony with pure black and smooth appearance was obtained from grey black bacterial colonies with streak method. It was a Gram-negative bacterium with bead-like shape. It showed 99.78% 16S rDNA sequence identity with P. nigrescens F0103 and was named P. nigrescens LY01. P. nigrescens LY01 infection promoted the in vitro proliferation, migration and invasion of NE6-T cells and the growth of subcutaneous xenograft in nude mice. In addition, it could also induce the upregulation of Ki67 and activation of p-STAT3. Conclusions P. nigrescens inhabiting in gingival sulcus might promote the progression of ESCC in human with chronic periodontitis.

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.
Clostridium Infections ; diagnosis ; Clostridium difficile ; genetics ; Electrophoresis, Capillary ; Feces ; microbiology ; Genes, Bacterial ; Humans ; Polymerase Chain Reaction ; Ribotyping ; Sensitivity and Specificity

Objective To investigate the prenatal diagnosis and postnatal clinical outcomes of fetal congenital choledochal cyst (CCC) to improve the recognition and treatment of fetal CCC.Methods Clinical data of 23 cases of fetal CCC which were diagnosed during routine prenatal ultrasonic examination in Jinhua Municipal Central Hospital from June 2009 to May 2015 were retrospectively analyzzed. Maternal age, gestational age at diagnosis of CCC, location and size of cyst, postnatal examination, age at operation and follow-up outcomes were recorded and statistically analyzed by Wilcoxon rank-sum test.Results (1) Among the 23 cases, six (26%) were terminated and the rest 17 continued their pregnancies (74%). (2) Results of the prenatal ultrasonography of the 23 cases indicated that hepatic portal cysts were closely related to hepatic portal veins or arteries. Six of the cysts communicated with gall bladder and eight connected to intrahepatic bile duct. The maximum diameter of the cysts in the 23 cases was 16.0-31.0 mm, averagely (24.7±3.7) mm. The maximum diameter of cysts diagnosed in the third trimester was significantly larger than that in the second trimester [ 27.0 (22.0-31.0) vs 23.0 (21.0-25.0) mm,Z=-2.134,P<0.05]. (3) Among the 17 cases of continued pregnancy, one underwent cesarean section at 35+ weeks of gestation and 16 delivered at term with the average gestational age at delivery of (38.2±1.1) weeks. All neonates were re-examined by abdominal ultrasound at 1-2 postnatal weeks and confimed prenatal diagnosed of CCC. (4) The 17 neonates were re-examined by abdominal ultrasound during the second postnatal week and the results showed that cyst size remained the same in four, decreased in one and gradually increased with the gestational age in 12 neonates. Among the 16 cases of confirmed CCC, 12 received surgery, including 11 (Ⅰa, 6;Ⅰc, 3;Ⅳb, 2) within one year-old and one (Ⅰc) around 18 months old. The prognosis was uneventful. Four out of the 16 cases rejected surgical operation and were followed up in outpatient. One neonate was diagnosed with congenital biliary atresia and transferred to Children's Hospital for operation.Conclusions When fetal abdominal cyst presented with hepatic portal cyst which communicates with gallbladder or intra-hepatic duct in ultrasonography, a congenital choledochal cyst should be taken into consideration by excluding the possibility of biliary atresia in the first place. Surgery for CCC infants without symptoms or signs is suggested to be performed around three months after birth. The postoperative prognosis of CCC is favorable, so termination is not recommended for gravidas with fetal CCC in prenatal consultation.

Objective To study the effects of one finger massage on sciatic nerve injury rats. Methods The sciatic nerves were exposed, and the sciatic nerve was held by micro needles to make the sciatic nerve injury model. SD rats were randomly divided into sham-operation group, model group and massage group. In the sham-operation group, the sciatic nerves were exposed but not held. 7 d after the establishment of modeling, rats in massage group received one finger massage for 30 d. After 30 d, SFI and BBB of sciatic nerves were detected. HE staining, transmission electron microscope and immunochemistry assay were used to measure the changes of sciatic nerves. Results Compared with model group, massage group could speed up the recovery of SFI and increase BBB, promote the recovery of sciatic nerve morphology, increased protein level of S-100β, and enhance ultrastructure of newborn nerve growth and recovery. Conclusion One finger massage can effectively promote neurological and functional recovery after sciatic nerve injury in rats.