[Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells].

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

[Clinical characteristics of a case of severe pneumonia caused by coinfection of COVID-19 and Chlamydia Psittaci].

A 69-year-old female patient was admitted to the Department of Respiratory and Critical Care Medicine, Affiliated Hospital of Xuzhou Medical University due to a "cough and fever for eight days". On admission, a coronavirus disease (COVID-19) nucleic acid test was positive, and a chest CT scan showed progressive patchy shadows and consolidation shadows in both lungs. Arterial blood gas analysis showed type Ⅰ respiratory failure. The primary diagnosis was severe community-acquired pneumonia in an older adult without underlying disease. However, oxygen inhalation, steroid, anti-inflammatory, and antibacterial empirical treatment with piperacillin/tazobactam was ineffective. Metagenomic next-generation sequencing of bronchoscopy alveolar lavage fluid showed Chlamydia psittaci(C. psittaci). Severe pneumonia was confirmed, caused by coinfection with severe acute respiratory syndrome coronavirus 2 and C. psittaci. A combination of doxycycline and moxifloxacin significantly improved the targeted and symptomatic treatment of the underlying cause. After discharge, the patient recovered within four weeks of follow-up. Therefore, clinicians should be alert to the possibility of coinfection of C. psittaci in patients already diagnosed with COVID-19.

[Joinpoint regression analysis of the incidence trend of syphilis and gonorrhea among adolescents aged 10-19 in Liaoning Province from 2006 to 2020].

Joinpoint regression was used to analyze the trend of syphilis and gonorrhea incidence rate among 10-19 year old adolescents in Liaoning Province from 2006 to 2020. The syphilis and gonorrhea data in Liaoning Province were reported in the infectious disease monitoring system of China's disease prevention and control information system. From 2006 to 2020, a total of 7 721 cases of syphilis in 10-19 year old adolescents were reported in Liaoning Province, with an incidence rate about 0.90/100 000-22.13/100 000. The incidence rate of syphilis in women was higher than that in men. Adolescents infected with stage Ⅰ and stageⅡ syphilis accounted for 72.6%. There were 2 726 patients with gonorrhea, with an incidence rate about 1.29/100 000-10.74/100 000. The incidence rate of gonorrhea in men was higher than that in women. Joinpoint regression model analysis showed that the incidence of syphilis generally took 2012 as the inflection point. From 2006 to 2012, the average annual growth rate of syphilis incidence rate among adolescents was 67.30% (P<0.001). The average annual growth rate of syphilis incidence rate in adolescents from 2012 to 2020 was -0.02% (P=0.994).The overall incidence of gonorrhea incidence rate took 2015 as the inflection point. From 2006 to 2015, the average annual growth rate of juvenile gonorrhea incidence rate was 23.95% (P<0.001). The average annual growth rate of gonorrhea incidence rate in adolescents from 2015 to 2020 was 4.06% (P=0.492). Overall, from 2006 to 2020, the incidence rate of syphilis and gonorrhea among 10-19 year old adolescents in Liaoning Province increased slowly. The primary and secondary prevention strategies were significantly effective in reducing the risk of sexually transmitted diseases.

Leptin reduces ventilator-induced lung injury in rats by regulating NLRP3, NLRC4 and NLRC3.

Leptin has been linked to acute lung injury (ALI) through its regulation of immune responses. We aimed to scrutinize the effects of leptin on nucleotide oligomerization domain-like receptors containing pyrin domain 3 (NLRP3), nucleotide oligomerization domain-like receptors with caspase activation and recruitment domain 4 (NLRC4), and nucleotide oligomerization domain-like receptors with caspase activation and recruitment domain 3 (NLRC3), as an essential part of the immune system, in ventilator-induced lung injury (VILI) of rats. In the present study, pathogen-free adult male SD rats were given saline or leptin, followed by ventilation. Lung tissue samples, bronchoalveolar lavage fluids (BALF), and blood were collected four hours after installation. Notable acute lung inflammation induced by mechanical ventilation is well-characterized by a massive increase in lung injury score and wet/dry weight (W/D) ratio. We also observed VILI was associated with interleukin (IL-1ß and IL-18). Rats that received ventilation showed a decrease in the levels of NLRP3 and NLRC4, and an increased level of NLRC3. Pre-treatment with leptin could abolish all of these effects induced by VILI. It has been suggested that the regulation of NLRP3, NLRC4, and NLRC3 may underlie the protection observed during VILI by exogenous leptin.

[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.

[Comparison of hereditary breast and ovarian cancer syndrome and sporadic ovarian cancer in ovarian cancer BRCA mutations].

Objective: To study the difference between BRCA gene mutations in hereditary breast and ovarian cancer syndrome (HBOC) and in sporadic ovarian cancer (SOC). Methods: This study was for exploratory research, the inclusion criteria were 284 patients with ovarian cancer admitted at Shanxi Provincial Cancer Hospital from November 2018 to December 2019, with high-throughput DNA sequencing including the full coding regions and exon-intron link regions of BRCA1 and BRCA2 gene. Pathogenic mutations in the BRCA gene of patients with ovarian cancer were collected and mutation site analysis was performed to compare phenotypic differences in pathogenic mutations between HBOC syndrome and SOC patients. Results: (1) Of the 284 ovarian cancer patients, seventy-seven had BRCA pathogenic mutations with a mutation rate of 27.1% (77/284), with BRCA1 mutation rate of 19.7% (56/284), BRCA2 gene 6.7% (19/284) and BRCA1/2 common mutation rate of 0.7% (2/284). Of the 284 patients with ovarian cancer, the pathogenic mutation rate in the BRCA gene in HBOC syndrome patients was 43.8% (32/73), which were significantly higher than that in SOC patients [21.3% (45/211); χ²=13.905, P<0.01]. Among BRCA1 gene mutation, the mutation rate in HBOC syndrome was higher than that of SOC [87.5% (28/32) vs 62.2% (28/45)], the BRCA2 gene mutation rate in patients with HBOC syndrome was lower than that in SOC patients [6.2% (2/32) vs 37.8% (17/45)], and there were statistically significant differences (all P<0.05). Two of the 77 patients with pathogenic mutations in the BRCA gene were multisite mutations, including one simultaneous two site mutation, one simultaneous three site mutation. There were 80 mutation sites with frameshift deletion mutations (55.0%, 44/80) and nonsense mutations (31.2%, 25/80). (2) Of the 73 patients with HBOC syndrome, 32 cases had pathogenic mutations in BRCA gene, including 28 cases in BRCA1, mainly in exon 11 and 24 (9 and 7 cases, respectively), and only two cases in BRCA2, both in exon 11; another two had multiple locus mutations. Of the 211 patients with SOC, 45 cases had pathogenic mutants in BRCA gene, including 28 cases in BRCA1, mainly in exon 11 and 24 (15 and 2 cases, respectively), and 17 cases in BRCA2, mainly in exon 11 (11 cases). (3) Thirty-four pathogenic mutation sites in BRCA gene were found newly, twenty of them were located in the BRCA1 gene, including a locus located on the intron 6, 301+1G>A, and the remaining 19 sites were located on the exons, including 283_286delCTTG, 68_69delAG, 132C>T, 514_547+3del37, 742delA, 1126_1129delAATA, 1196delA, 1352_1364del, 1465G>T, 2171delC, 2341G>T, 3359_3363delTTAAT, 4085_4086ins11, 4161_4162delTC, 4165_4166delAG, 4258G>T, 4338_4339del8insAGAA, 4468G>T, and 4783delA; fourteen sites were located in the BRCA2 gene, including a locus located on the intron 7, 631+1G>A, and the remaining 13 sites were located on the exons, including 2648delT, 2914A>T, 2950_2951insG, 4357+1G>A, 5054C>T, 5257A>T, 5291_5292insTC, 5913delT, 3593delA, 6091_6092insA, 6135_6136delTT, 7452delT, 9097_9098insA. A tal of 28 repeat mutations were located in the BRCA1 gene; among them, the site 5470_5477del8 was repeated 6 times, while 3 times in 981_982delAT. Conclusions: Patients with HBOC syndrome have a significantly higher rate of pathogenic mutation in the BRCA gene than that in patients with SOC. BRCA gene pathogenic mutation sites in HBOC syndrome patients occur commonly in exon 11 and 24 of BRCA 1 gene, while SOC patients occur mainly in exon 11 and 24 of BRCA1 gene and exon 11 of BRCA2 gene. The two loci of BRCA1∶5470_5477del8, BRCA1∶981_982delAT may be ancestor mutations in Chinese ovarian cancer patients, and 34 newly discovered pathogenic mutations in the BRCA gene, enriching the BRCA gene mutation spectrum in the Chinese population.

[The 479th case: cognitive impairment, respiratory failure, colon mass].

A 78-year-old man was admitted to Peking Union Medical College Hospital with fever, weakness of lower extremities, less speech, loss of memory. Fever was relieved after antibiotic treatment, while cognitive impairment and disorder of consciousness progressed rapidly, followed by critical pulmonary infections, respiratory failure, and septic shock. Lab tests showed negative occult blood, normal serum CEA level and positive Anti-nuclear-antibody. PET-CT suggested that strong FDG uptake signals were seen at sigmoid, while bilateral frontal lobe, temporal lobe, parietal lobe, posterior cingulate gyrus showed lower metabolic activity. Colonoscopy biopsy revealed differentiated adenocarcinoma of sigmoid colon. Therefore, paraneoplastic syndrome of nervous system secondary to colon cancer was considered. Rapid and proper diagnosis and treatment were completed by multidisciplinary team including departments of neurology, gastroenterology, general surgery, ICU, rheumatology, clinical nutrition. The laparoscopic sigmoid colectomy was performed under general anesthesia. The patient finally presented with significant improvement of cognition and consciousness. Respiratory function was totally recovered.

[Clinical analysis of golimumab in the treatment of severe/refractory cardiovascular involvement in Behcet syndrome].

OBJECTIVE: To explore the effectiveness and safety of golimumab in the treatment of severe/refractory cardiovascular Behcet syndrome (BS). METHODS: We retrospectively analyzed the clinical data of nine patients diagnosed with severe/refractory cardiovascular BS and treated with golimumab from February 2018 to July 2020 in Peking Union Medical College Hospital. We analyzed levels of erythrocyte sedimentation rate (ESR) and high-sensitivity C-reactive protein (hsCRP), imaging findings, and the doses of glucocorticoids and immunosuppressive agents during the period of combined treatment with golimumab. RESULTS: Nine patients were enrolled, including 8 males and 1 female, with a mean age and median course of (37.0±8.6) years and 120 (60, 132) months, respectively. Seven patients presented with severe aortic regurgitation combined with other cardiovascular involvement secondary to BS. Two patients presented with large vessel involvement, including multiple aneurysms and vein thrombosis. Prior to golimumab treatment, seven patients were treated with glucocorticoids and multiple immunosuppres-sants [with a median number of 3 (1, 3) types] while still experienced disease progression or elevated inflammation biomarkers during postoperative period. Eight patients with disease progression, uncontrolled inflammation and history of severe postoperative complications required effective and fast control of inflammation during perioperative period. One patient had adverse reaction with tocilizumab and switched to golimumab during perioperative period. The patients were treated with golimumab 50 mg every 4 weeks, along with concomitant treatment of glucocorticoid and immunosuppressants. After a median follow-up of (16.3±5.6) months, all the patients achieved clinical improvement. Vascular lesions were radiologically stable and no vascular progressive or newly-onset of vascular lesions was observed. The eight patients who experienced cardiac or vascular operations showed no evidence of postoperative complications. The ESR and hsCRP levels decreased significantly [16.5 (6.8, 52.5) mm/h vs. 4 (2, 7) mm/h, and 21.24 (0.93, 32.51) mg/L vs. 0.58 (0.37, 1.79) mg/L (P < 0.05), respectively]. The dose of prednisone was tapered from 35 (15, 60) mg/d to 10.0 (10.0, 12.5) mg/d. No prominent adverse reactions were observed. CONCLUSION: Our study suggests that golimumab is effective in the treatment of severe/refractory cardiovascular BS. Combination immunosuppression therapy with golimumab contributes to control of inflammation, reduction of postoperative complications and tapering the dose of glucocorticoids or immunosuppressants.

[Clinical features and prognostic factors of parainfluenza virus infections in adult patients].

Objective: To explore the clinical features and prognostic factors of parainfluenza viral lower respiratory tract infections in adults. Methods: A total of 70 patients withpositive nucleic acid of parainfluenza virus (PIV) admitted to China-Japan Friendship Hospital between August 2016 and November 2019 were enrolled. Multiplex real-time polymerase chain reaction (RT-PCR) assays for viral detection were implemented to specimens (nasopharynx swab, sputum or bronchoalveolar lavage) obtained from all the patients, which was consistent with the diagnosis of lower respiratory tract infection. Patients were divided into two groups depending on the status of immune function (immunocompromised group, n=26; immunocompetent group, n=44), and patients were divided into PIV infection group (n=43) and mixed infection group (n=27) according to whether there was mixed infection. Characteristics including age, gender, underlying diseases, symptoms, disease severity, imaging manifestations, etiology, respiratory failure, mechanical ventilation, vasoactive drug, antimicrobial drug and 30-day mortality between the groups were compared, and the prognostic factors of PIV infections were investigated using Cox regression. Results: The peak incidence of PIV infection time was in May, August, September and December, accounting for 58.6% of all cases. The enrolled 70 cases included 43 cases with pneumonia, 16 cases with interstitial lung disease and infection, 7 cases with bronchiectasis and infection, and 4 cases with acute exacerbation of chronic obstructive pulmonary disease. Eight patients (11.4%) had no underlying diseases, 21 patients (30.0%) had respiratory failure, 18 patients (25.7%) were treated with mechanical ventilation, 15 patients (21.4%) died within 30 days after admission. The pneumonia severity index score, percentage of patients with ground-glass opacity according CT scan, with honeycomb or reticular pattern, with mechanical ventilation, with respiratory failure, with ICU admission, and 30-day mortality in immunocompromised group were higher than those of immunocompetent group [(91.5 vs 84.0), (60.0% vs 34.1%), (44.0% vs 11.4%), (42.3% vs 15.9%), (50.0% vs 18.2%), (38.5% vs 22.7%), (34.6% vs 13.6%)] (all P<0.05). There were 27 cases (38.6%) with mixed infection, including 17 viruses (24.3%), 19 bacteria (27.1%), 14 (20.0%) fungi (PCP, aspergillus) and 1 (1.4%) Mycobacterium intracellulare. Sixteen patients (59.3%) in the mixed infection group were immunocompromised patients and 21 patients (77.8%) had chronic lung disease. Cox regression analysis showed that mechanical ventilation and interstitial lung disease were independent predictors of prognosis in all patients, and mechanical ventilation was an independent predictor of prognosis in PIV infection group. Conclusions: Most of the patients with PIV lower respiratory tract infection in adults are complicated with underlying diseases and mixed infection, with a high 30-day mortality. Interstitial lung disease and mechanical ventilation indicate poor prognosis in these patients.

[Long-term oncological outcomes of laparoscopic versus abdominal surgery in stage â a1 （LVSI+）-â b1 cervical cancer patients with different tumor size: a big database in China].

Objective: To compare the long-term oncological outcomes between laparoscopic and abdominal surgery in stage Ⅰa1 （lymph-vascular space invasion-positive, LVSI+）- Ⅰb1 cervical cancer patients with different tumor sizes. Methods: Based on the Big Database of Clinical Diagnosis and Treatment of Cervical Cancer in China (1538 project database), patients with stage Ⅰa1 （LVSI+）-Ⅰb1 cervical cancer who treated by laparoscopic or abdominal surgery were included. The 5-year overall survival (OS) and 5-year disease-free survival (DFS) between the two surgical approaches were compared under 1∶1 propensity score matching (PSM) in different tumor diameter stratification. Results: (1) A total of 4 891 patients with stage Ⅰa1 （LVSI+）-Ⅰb1 cervical cancer who underwent laparoscopy or laparotomy from January 1, 2009 to December 31, 2016 were included in the 1538 project database. Among them, 1 926 cases in the laparoscopic group and 2 965 cases in the abdominal group. There were no difference in 5-year OS and 5-year DFS between the two groups before matching. Cox multivariate analysis suggested that laparoscopic surgery was associated with lower 5-year DFS (HR=1.367, 95%CI: 1.105-1.690, P=0.004). After 1∶1 PSM matching, 1 864 patients were included in each group, and there was no difference in 5-year OS between the two groups （94.1% vs 95.4%, P=0.151）. While, the inferior 5-year DFS was observed in the laparoscopic group (89.0% vs 92.3%, P=0.004). And the laparoscopic surgery was associated with lower 5-year DFS (HR=1.420, 95%CI: 1.109-1.818, P=0.006). (2) In stratification analysis of different tumor sizes, and there were no difference in 5-year OS and 5-year DFS between the laparoscopic group and abdominal group in tumor size ≤1 cm, >1-2 cm and >2-3 cm stratification (all P>0.05). Cox multivariate analysis showed that laparoscopic surgery were not related to 5-year OS and 5-year DFS (P>0.05). In the stratification of tumor size >3-4 cm, there was no difference in 5-year OS between the two groups (P>0.05). The 5-year DFS in the laparoscopic group was worse than that in the abdominal group (75.7% vs 85.8%, P=0.025). Cox multivariate analysis suggested that laparoscopic surgery was associated with lower 5-year DFS (HR=1.705, 95%CI: 1.088-2.674, P=0.020). Conclusions: For patients with stage Ⅰa1 （LVSI+）-Ⅰb1 cervical cancer, laparoscopic surgery is associated with lower 5-year DFS, and the adverse effect of laparoscopic surgery on oncology prognosis is mainly reflected in patients with tumor size >3-4 cm. For patients with tumor sizes ≤1 cm, >1-2 cm and >2-3 cm, there are no difference in oncological prognosis between the two surgical approaches.

[Comparison of clinical and pathological features between severe acute respiratory syndrome and coronavirus disease 2019].

Severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19) shared similar pathogenetic, clinical and pathological features. Fever and cough were the most common symptoms of both diseases, while myalgia and diarrhea were less common in patients with COVID-19. Acute respiratory distress syndrome (ARDS) was the most severe pulmonary complication that caused high mortality rate. Histologically, diffuse alveolar damage (DAD) was the most characteristic finding in non-survivors with either SARS or COVID-19. Cases of patients died less than 10-14 days of disease duration demonstrated acute-phase DAD, while cases beyond 10-14 days of disease duration exhibited organizing-phase DAD in SARS. Meanwhile, organization and fibrosis were usually accompanied by exudation. Coronavirus was mostly detected in pneumocytes, but less in macrophages and bronchiolar epithelial cells. Hemorrhagic necrosis and lymphocyte depletion were found in lymph nodes and spleen in both SARS and COVID-19, indicating a pathological basis of lymphocytopenia. Thrombosis was commonly observed in small vessels and microvasculaturr in lungs accompanying DAD. Microthrombosis was also found in extrapulmonary organs in COVID-19, that was less reported in SARS. Damages in multiple extrapulmonary organs were observed, but coronavirus was not detected in some of those organs, indicating an alternative mechanism beyond viral infection, such as hypoxemia, ischemia and cytokine storm induced immunological injury. DAD due to viral infection and immunological injury, as well as multi-organ dysfunction and extensive microthrombus formation, brought huge challenge to the management of patients with severe SARS or COVID-19.

[The value of assessing femoral vein to femoral artery ratio in the treatment of patients with heart failure].

Objective: To investigate the relationship between ultrasound derived ratio of femoral vein to femoral artery diameter and hemodynamics in patients with heart failure. Methods: This was a case-control study. A total of 61 patients with heart failure and 49 patients with non-heart failure hospitalized in the Department of Critical Care Medicine from September 2017 to September 2018 were included in this study. Doppler ultrasound was used to measure the femoral artery and vein diameter. After deep inhalation, the femoral vein diameter was measured again, and the ratio of femoral vein and artery diameter was calculated. The central venous pressure (CVP) and mean pulmonary wedge pressure (mPAWP) were also measured. Pearson correlation analysis was used to explore the correlation between the ratio of femoral vein diameter to femoral artery diameter and CVP and mPAWP, and linear regression equation was established. Results: The overall CVP and mPAWP levels were significantly higher, and the femoral vein diameter after deep inhalation was bigger in heart failure patients than in non-heart failure patients(all P<0.001). The femoral vein diameter/femoral artery diameter ratio was positively correlated with CVP (r=0.76, P<0.001), and positively correlated with mPAWP (r=0.40, P<0.001) in heart failure group. The linear regression equation established by the femoral vein/femoral artery diameter ratio and CVP in the heart failure group showed that the inner diameter of the femoral vein/the inner diameter of the femoral artery ratio≥1.3 corresponded CVP≥15.518 cmH2O(1 cmH2O=0.098 kPa) in heart failure patients. Conclusions: In patients with heart failure, the inner diameter of the femoral vein/femoral artery ratio is positively correlated with CVP and mPAWP. The ratio of inner diameter of the femoral vein/femoral artery can be used to assess the volumetric load of patients with heart failure and to guide the clinical treatment of heart failure patients.

[Treatment of liver cancer in vitro and in mice by monoclonal antibody targeting epithelial specific antigen-positive liver cancer stem cells in combination with cisplatin].

OBJECTIVE: To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. METHODS: Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody (McAb) 15D2 and epithelial specific antigen (ESA) and PKH26-positive cells in the Bel7402-V3 cells were detected by immunofluorescence assay. Serum-free suspension culture was used to detect the self-renewal ability of 15D2-positive Bel7402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Bel7402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. RESULTS: Single PKH26-positive cells were observed in the Bel7402-V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Bel7402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [(30.4±3.4)% vs. (8.8±1.8)%, P<0.01]. The cisplatin resistance of 15D2-positive cells was obviously higher than that of 15D2-negative cells (IC50: 1.014 µmol/L vs. 0.365 µmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302-V3 cells. The IC50 was 0.211 µg/ml in the 15D2 group and 0.325 µg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91.0%, and that of the cisplatin monotherapy was 56.7%. CONCLUSION: McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell-targeted therapy of liver cancer.

Association analysis of rs2275913G>A and rs763780T>C interleukin 17 polymorphisms in Chinese women with cervical cancer.

We conducted a case-control study with a relatively large sample size, and investigated the association between rs2275913G>A and rs763780T>C and the risk of cervical cancer. Three hundred and six newly diagnosed patients with histologically confirmed cervical cancer and 354 cancer-free control subjects were recruited from the Forestry General Hospital between May 2011 and May 2014. The gene polymorphisms rs2275913G>A and rs763780T>C were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. By unconditional logistic regression analysis, our study found that the AA genotype and the A allele of rs2275913 were associated with a higher risk of cervical cancer compared with the wild-type genotype, and the ORs (95%CIs) were 2.84 (1.57-5.23) and 1.55 (1.22-1.97), respectively. Compared with the G allele, the A allele of rs699947 was associated with a significantly increased risk of cervical cancer in subjects above 20 years and who were positive for human papillomavirus 16 (HPV-16) or HPV-18 infection. Patients with the A allele of rs2275913 had increased risk of cervical cancer, regardless of the number of births they had experienced or their smoking habits. We suggest that rs2275913 may play a role in the etiology of cervical cancer, although further large-sample studies are needed to confirm these observations.

Spontaneous emission modulation of colloidal quantum dots via efficient coupling with hybrid plasmonic photonic crystal.

The spontaneous emission of colloidal CdSe/ZnS quantum dots (CQDs) modified by the hybrid plasmonic-photonic crystal is reported in this paper. By using a spin coater, the spatial overlap between CQDs and the surface resonance modes in this quasi-2D crystal slab is achieved. In this case, the coupling efficiency of them is enhanced greatly and most excited CQDs radiate through the surface modes. Consequently, despite the low refractive index contrast of our hybrid structure, the directionality of spontaneous emission, increased radiative probability and narrowed full width at half maximum of emission peak are all clearly observed by our home-made microscopic angle-resolved spectroscopy and time-resolved photoluminescence system. Our results manifest that the quasi-2D hybrid plasmonic-photonic crystal is an ideal candidate to tailor the radiative properties of CdSe/ZnS CQDs, which might be significant for the applications of light emitting devices.

[The imaging appearances of stapical footplate fistula related to inner ear malformation].

Objective: To summarize the HRCT and MRI appearances of stapical footplate fistula related to inner ear malformation (SFF-Re-IEM). Methods: The HRCT and MRI materials of 48 cases (53 ears) SFF-Re-IEM were retrospectively analyzed. Among them, 25 SFF-Re-IEM ears were confirmed by surgery. Their CT and MRI findings including associated IEM type, internal auditory canal (IAC) malformation, tympanic fluid, its density and signal features, and accompanied labyrinthitis were recorded. Results: Among 48 cases (53 ears) with SFF-Re-IEM, 17 ears with incomplete partition type Ⅰ, accounting for 32.1%, 13 ears with common cavity for 24.5%, 13 ears with cochlear aplasia for 24.5%, 7 ears with cochlear dysplasia Ⅱ for 13.2%, and 3 ears with Mondini for 5.7%,were found respectively. 94.3% of them were associated with a defect or dysplasia in the found of the IAC. They were divided into 4 types according to the intact of the stapical footplate and accompanied CSF otorrhea: 22 ears were diagnosed as the stapical footplate leaking, of them, 2 ears might come from the stapical footplate bony defect, 6 ears were from the stapical footplate hernia. 1 ear belonged to the peristapical footplate leaking. 30 ears with the isolated the stapical footplate hernia were another found. The bony defect in 2 ears with the stapical footplate bony defect were not presented on CT and MRI.The focal bony defect of the affected stapical footplate of 36 ears with the stapical footplate hernia were demonstrated, which presented the hemispherical protruding into the tympana, the soft-tissue density on CT, and CSF-like signal on the MR heaved-T2WI images. Among 22 ears with the stapical footplate leaking, their imaging appearances varied from the different amount of the leaking CSF. Besides the focal bony defects of the affected stapical footplates, there were much more CSF-like density or signal in the ipsilateral tympanic cavity in 17 affected ears connecting with the vestibule through the defect area. In the CSF leaking ears with less CSF leaking in 5 ears, the CSF-like cysts like SFH were shown on the stapical footplate defect area, but their outer edges were irregular, and the CSF-like signal scattering in the tympanic cavity did not connect with the protruding cysts at the stapical area. Conclusion: The variable appearances of the SFF-Re-IEM ears based on the different subtypes are its characteristic HRCT and MRI appearances. This is helpful for the SFF-Re-IEM diagnosing to grasp its imaging features.

Endothelial cells on the proliferation and expression of intercellular adhesion molecule 1 and interleukin 8 of vascular smooth muscle cells.

The aim of this study was to investigate the influence of activated endothelial cells on the proliferation and secretion of vascular smooth muscle cells (VSMCs). Cultured lung microvascular endothelial cells were treated with or without tumor necrosis factor alpha (TNF-α; 10 ng/mL) for 6 h, and the supernatant was collected and filtered. The supernatant with TNF-α was called fluid A, and that without TNF-α was called fluid B. VSMCs were cultured and divided into 3 groups with different media as follows: activated medium [fluid A and Dulbecco's modified Eagle medium (DMEM); activated group], inactivated medium (fluid B and DMEM; inactivated group), and DMEM only (control group) for 24 h. Intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-8, and IL-6 levels in the supernatant of VSMCs were measured with enzyme-linked immunosorbent assay 0 and 24 h after grouping. The proliferation of VSMCs was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ICAM-1 and IL-8 increased above baseline values in the 3 groups; the maximum increase occurred in activated medium. The optical densities in MTT assay of the activated, inactivated, and control groups was 1.35 ± 0.11, 1.01 ± 0.09, and 0.29 ± 0.01, respectively, which correlated positively with the initial IL-6 level in the supernatant of the VSMCs (r = 0.63, P < 0.05). TNF-α-activated endothelial cells promote VSMC proliferation and secretion of ICAM-1 and IL-8 by elevating IL-6 release.

Stability of daphnoretin in vitro.

The stability of daphnoretin in sodium phosphate buffers at different pH and temperature, and in different biological samples at 37 degrees C was investigated using HPLC with UV detector set at 345 nm. Daphnoretin degraded rapidly in alkaline environment and was stable in acidic environment. Daphnoretin was stable in simulated gastrointestinal liquid, stomach contents, gastric mucosa and colon contents; it was unstable in plasma, liver homogenates, small intestine contents, small intestinal mucosa and blind gut contents. The stability of daphnoretin in plasma and other biomaterials could have a significant impact on its absorption.

[Investigation on the growth factor regulatory network of dermal fibroblasts in mouse full-thickness skin defect wounds based on single-cell RNA sequencing].

Objective: To explore the heterogeneity and growth factor regulatory network of dermal fibroblasts (dFbs) in mouse full-thickness skin defect wounds based on single-cell RNA sequencing. Methods: The experimental research methods were adopted. The normal skin tissue from 5 healthy 8-week-old male C57BL/6 mice (the same mouse age, sex, and strain below) was harvested, and the wound tissue of another 5 mice with full-thickness skin defect on the back was harvested on post injury day (PID) 7. The cell suspension was obtained by digesting the tissue with collagenase D and DNase Ⅰ, sequencing library was constructed using 10x Genomics platform, and single-cell RNA sequencing was performed by Illumina Novaseq6000 sequencer. The gene expression matrices of cells in the two kinds of tissue were obtained by analysis of Seurat 3.0 program of software R4.1.1, and two-dimensional tSNE plots classified by cell group, cell source, and gene labeling of major cells in skin were used for visual display. According to the existing literature and the CellMarker database searching, the expression of marker genes in the gene expression matrices of cells in the two kinds of tissue was analyzed, and each cell group was numbered and defined. The gene expression matrices and cell clustering information were introduced into CellChat 1.1.3 program of software R4.1.1 to analyze the intercellular communication in the two kinds of tissue and the intercellular communication involving vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) signal pathways in the wound tissue, the relative contribution of each pair of FGF subtypes and FGF receptor (FGFR) subtypes (hereinafter referred to as FGF ligand receptor pairs) to FGF signal network in the two kinds of tissue, and the intercellular communication in the signal pathway of FGF ligand receptor pairs with the top 2 relative contributions in the two kinds of tissue. The normal skin tissue from one healthy mouse was harvested, and the wound tissue of one mouse with full-thickness skin defect on the back was harvested on PID 7. The multiple immunofluorescence staining was performed to detect the expression and distribution of FGF7 protein and its co-localized expression with dipeptidyl peptidase 4 (DPP4), stem cell antigen 1 (SCA1), smooth muscle actin (SMA), and PDGF receptor α (PDGFRα) protein. Results: Both the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7 contained 25 cell groups, but the numbers of cells in each cell group between the two kinds of tissue were different. Genes PDGFRα, platelet endothelial cell adhesion molecule 1, lymphatic endothelial hyaluronic acid receptor 1, receptor protein tyrosine phosphatase C, keratin 10, and keratin 79 all had distinct distributions on two-dimensional tSNE plots, indicating specific cell groups respectively. The 25 cell groups were numbered by C0-C24 and divided into 9 dFb subgroups and 16 non-dFb groups. dFb subgroups included C0 as interstitial progenitor cells, C5 as adipose precursor cells, and C13 as contractile muscle cells related fibroblasts, etc. Non-dFb group included C3 as neutrophils, C8 as T cells, and C18 as erythrocytes, etc. Compared with that of the normal skin tissue of healthy mice, the intercellular communication in the wound tissue of full-thickness skin defected mice on PID 7 was more and denser, and the top 3 cell groups in intercellular communication intensity were dFb subgroups C0, C1, and C2, of which all communicated with other cell groups in the wound tissue. In the wound tissue of full-thickness skin defected mice on PID 7, VEGF signals were mainly sent by the dFb subgroup C0 and received by vascular related cell groups C19 and C21, PDGF signals were mainly sent by peripheral cells C14 and received by multiple dFb subgroups, EGF signals were mainly sent by keratinocyte subgroups C9 and C11 and received by the dFb subgroup C0, and the main sender and receiver of FGF signals were the dFb subgroup C6. In the relative contribution rank of FGF ligand receptor pairs to FGF signal network in the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7, FGF7-FGFR1 was the top 1, and FGF7-FGFR2 or FGF10-FGFR1 was in the second place, respectively; compared with those in the normal skin tissue, there was more intercellular communication in FGF7-FGFR1 signal pathway, while the intercellular communication in FGF7-FGFR2 and FGF10-FGFR1 signal pathways decreased slightly or did not change significantly in the wound tissue; the intercellular communication in FGF7-FGFR1 signal pathway in the wound tissue was stronger than that in FGF7-FGFR2 or FGF10-FGFR1 signal pathway; in the two kinds of tissue, FGF7 signal was mainly sent by dFb subgroups C0, C1, and C2, and received by dFb subgroups C6 and C7. Compared with that in the normal skin tissue of healthy mouse, the expression of FGF7 protein was higher in the wound tissue of full-thickness skin defected mouse on PID 7; in the normal skin tissue, FGF7 protein was mainly expressed in the skin interstitium and also expressed in the white adipose tissue near the dermis layer; in the two kinds of tissue, FGF7 protein was co-localized with DPP4 and SCA1 proteins and expressed in the skin interstitium, co-localized with PDGFRα protein and expressed in dFbs, but was not co-localized with SMA protein, with more co-localized expression of FGF7 in the wound tissue than that in the normal skin tissue. Conclusions: In the process of wound healing of mouse full-thickness skin defect wound, dFbs are highly heterogeneous, act as potential major secretory or receiving cell populations of a variety of growth factors, and have a close and complex relationship with the growth factor signal pathways. FGF7-FGFR1 signal pathway is the main FGF signal pathway in the process of wound healing, which targets and regulates multiple dFb subgroups.

Hollow fiber liquid-phase microextraction for the determination of nimesulide in human plasma and its application to a pharmacokinetic study.

A hollow fiber liquid-phase microextraction (HF-LPME) method in combination with HPLC-UV for the determination of nimesulide in human plasma was developed and validated. A small volume of dihexyl ether contained within a polypropylene hollow fiber was used for the extraction of nimesulide from acidified plasma solutions. Factors affecting the extraction efficiency were optimized and discussed. With HPLC-UV as the end analysis technique, the procedure was validated for nimesulide in the concentration range of 50-5000 ng/mL. The intra- and inter-assay precisions were less than 9.1%, and accuracy was within 3.2%. The lower limit of quantification (LLOQ) was 50 ng/mL. Enrichment factor from 144-fold to 156-fold was achieved at three quality control (QC) concentrations. The mean extraction recovery was greater than 41.2%. This method was successfully applied for the evaluation of pharmacokinetics of nimesulide after single oral doses of 100 mg nimesulide to six healthy Chinese volunteers.