Analyzing polysorbate 20(PS20)composition and the impact of each component on stability and safety is crucial due to formulation variations and individual tolerance.The similar structures and polarities of PS20 components make accurate separation,identification,and quantification challenging.In this work,a high-resolution quantitative method was developed using single-dimensional high-performance liquid chromatography(HPLC)with charged aerosol detection(CAD)to separate 18 key components with multiple esters.The separated components were characterized by ultra-high-performance liquid chro-matography-quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS)with an identical gradient as the HPLC-CAD analysis.The polysorbate compound database and library were expanded over 7-time compared to the commercial database.The method investigated differences in PS20 samples from various origins and grades for different dosage forms to evaluate the composition-process relationship.UHPLC-Q-TOF-MS identified 1329 to 1511 compounds in 4 batches of PS20 from different sources.The method observed the impact of 4 degradation conditions on peak components,identifying stable components and their tendencies to change.HPLC-CAD and UHPLC-Q-TOF-MS results provided insights into fingerprint differences,distinguishing quasi products.

The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer(CRC).In this study,we identified reduced microvessel density(MVD)and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance.We focused on the effect of metformin on MVD,vascular maturity,and endothelial apoptosis of CRCs with a non-angiogenic phenotype,and further investigated its effect in overcoming chemoresistance.In situ transplanted cancer models were established to compare MVD,endothelial apoptosis and vascular maturity,and function in tumors from metformin-and vehicle-treated mice.An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis.Transcriptome sequencing was performed for genetic screening.Non-angiogenic CRC developed inde-pendently of angiogenesis and was characterized by vascular leakage,immaturity,reduced MVD,and non-hypoxia.This phenomenon had also been observed in human CRC.Furthermore,non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro.By suppressing endo-thelial apoptosis,metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity.Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling,which was abrogated by metformin administration.These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC.By suppressing endothelial apoptosis,metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.

OBJECTIVES: To study the clinical efficacy of ultrasound-guided endoscopic retrograde appendicitis therapy in children with appendix-related chronic abdominal pain. METHODS: A retrospective analysis was performed on the medical data of 30 children with the chief complaint of chronic abdominal pain who were admitted from August 2019 to May 2021. All the children were found to have inflammation of the appendix or intracavitary stool and fecalith by ultrasound and underwent ultrasound-guided endoscopic retrograde appendicitis therapy. The medical data for analysis included clinical manifestations, endoscopic findings, white blood cell count, neutrophil percentage, length of hospital stay, and cure rate. RESULTS: Among the 30 children with chronic abdominal pain, there were 13 boys (43%) and 17 girls (57%), with a mean age of (9±3) years (range 3-15 years) at diagnosis. The median duration of the disease was 12 months, and the median length of hospital stay was 3 days. The children had a median white blood cell count of 6.7×10⁹/L and a neutrophil percentage of 50%±13%. Fecalith and a large amount of feces were flushed out of the appendix cavity for 21 children (70%) during surgery. The follow-up rate was 97% (29/30), and the median follow-up time was 11 months (range 5-26 months). Of the 29 children, abdominal pain completely disappeared in 27 children (93%). CONCLUSIONS Ultrasound-guided endoscopic retrograde appendicitis therapy is effective in children with chronic abdominal pain caused by feces or fecalith in the appendix cavity.
Abdominal Pain/etiology* ; Adolescent ; Appendicitis/surgery* ; Appendix/surgery* ; Child ; Child, Preschool ; Fecal Impaction ; Female ; Humans ; Male ; Retrospective Studies ; Ultrasonography, Interventional

Objective To analyze the differences in the expression levels of the lncRNA MALAT1, NEAT, NEAT2 in peripheral blood mononuclear cell (PBMC) from tuberculosis patients and healthy controls. Methods We detected the lncRNA expression levels in PBMC from 79 tuberculosis patients and 82 healthy controls by quantitative reverse transcription polymerase chain reaction, and analyzed the correlation between lncRNA expression levels and some clinical features and laboratory indicators in tuberculosis patients. Results The expression levels of MALAT1, NEAT1 in PBMC of tuberculosis patients were significantly higher than healthy controls (Z=-4.386, P<0.001; Z=-10.175, P<0.001). There was no significant difference in the expression of NEAT2 between tuberculosis patients and healthy controls (Z=-0.203，P=0.839). The correlation results of lncRNA levels and some clinical features, laboratory indicators in tuberculosis patients suggested that the NEAT2 level in PBMC of newly treated tuberculosis patients was higher than recurrent tuberculosis patients, while the NEAT2 level in PBMC of sputum smear positive tuberculosis patients was lower than that of sputum smear negative tuberculosis patients (all P<0.05). There was a negative correlation between MALAT1 level and erythrocyte sedimentation rate (rs=-0.256, P=0.034). Conclusion MALAT1 and NEAT1 are abnormally expressed in PBMC of tuberculosis patients, and may be involved in the pathogenesis of pulmonary tuberculosis.

OBJECTIVE: To evaluate the association between rotator cuff tear and the proximal migration of humeral head. METHODS: In this research, we retrospectively selected 30 patients with unilateral rotator cuff tear in Peking University People's Hospital from September 2015 to May 2016, who received magnetic resonance imaging (MRI) and X-ray of the painful shoulder before enrollment in this study, the duration between the two examinations was no longer than 1 week, and also there was no past history of surgery in the selected shoulders. There was no other exclusion criteria. Upward migration index (UMI) was the ratio between the distance of humeral head center to the lower surface of acromion, and the radius of humeral head circle, which could help to minimize the effect of anatomy difference and imaging magnification, compared with the traditional acromiohumeral distance (AHD). Then we introduced this index to stratify the selected 30 patients into 3 groups, and each group contained 10 patients, UMI of group 1 was >1 and ≤1.2, UMI of group 2 was >1.2 and ≤1.4, UMI of group 3 was >1.4. As the supraspinatus was most commonly affected by pathological change among the four rotator cuff tendons, we took it as the research object. Then we used the Spearman correlation analysis to evaluate the relationship between UMI and fatty degeneration, rotator cuff tear size and the thickness of ruptured supraspinatus tendon from X-ray and MRI. RESULTS: In the A-P view, the average UMI was 1.33 (1.02-1.51, SD: ±0.22). UMI and the tear size had a significant negative correlation (R=-0.584, P<0.01), and also there was a negative correlation between the fatty degeneration of the supraspinatus (R=-0.312, P=0.033). However, there was no correlation between UMI and the thickness of ruptured supraspinatus (R=0.127, P=0.071). CONCLUSION UMI is related with the fatty degeneration of supraspinatus and the tear size. The reduction of UMI is a predictable and reliable mark of rotator cuff tear and degeneration in clinic. Physicians can use physical examination and X-ray first when facing the patients with shoulder pain, which is convenient and helpful for evaluating rotator cuff tears.
Humans ; Humeral Head ; Magnetic Resonance Imaging ; Retrospective Studies ; Rotator Cuff ; Rotator Cuff Injuries ; Shoulder Joint

OBJECTIVE: To evaluate the efficacy and safety of proximal femoral nail antirotation (PFNA) and dynamic hip screw (DHS) for unstable intertrochanteric fractures using meta-analysis. METHODS: The PubMed, Embase, Cocharane Central Register of Controlled Trials, Google Scholar, China Science and Technology Papers and Citation Database (CSTPCD) and China Journal Full-text Database (CNKI) were searched for published randomized controlled trials before January 1, 2019. Two researchers independently screened the literature in the light of the inclusion and exclusion criteria, evaluated the quality of the studies and extracted the data which were consisted of clinical efficacy indexes, such as incision length, operation time,intraoperative blood loss, weight-bearing time,fracture-healing time, Harris hip score and safety indicators like complications. Meta-analysis was performed with the Revman 5.3 software provided by Cochrane Community in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) standard. RESULTS: Nine randomized controlled trials met the requirement with a total of 779 patients, of whom 383 were fixed with PFNA and 396 with DHS. Meta-analysis demonstrated that PFNA was associated with smaller surgical incision length [MD=-7.43, 95%CI (-9.31, -5.55), P<0.05], shorter operation time [MD=-22.76, 95%CI (-29.57, -11.95), P<0.05], less intraoperative blood loss [MD=-216.34, 95%CI (-275.18, - 157.49), P<0.05], earlier weight bearing after surgery [MD=-12.34, 95%CI (-17.71, -6.97), P<0.05], shorter fracture healing time [MD=-5.00, 95%CI (-7.73, -2.26), P<0.05], higher postoperative Harris hip score [MD=12.22, 95%CI (3.88, 20.55), P<0.05], higher rate of excellent Harris hip score [OR=3.56, 95%CI (1.44, 8.81), P<0.05] and lower incidence rate of postoperative complications [OR=0.48, 95%CI (0.33, 0.70), P<0.05], such as hip varus, wound infection, urinary tract infection, pulmonary infection, pressure sore, deep vein thrombosis, pulmonary embolism, heart failure and cerebral infraction when compared with DHS. No statistical difference was shown between the groups when it came to subgroup analysis by age. However, there was no significant difference (P>0.05) in the duration of hospitalization and the complications resulting in the occurrences of internal fixation loosening, such as femoral shaft fracture (during or post operation), internal fixation fracture, cut-out, displacement or retraction. CONCLUSION Current published evidence supports the superiority of PFNA to DHS for unstable intertrochanteric fractures in terms of clinical efficacy. The conclusion was limited because of the relatively low quality of evidence with low strength of confidence. Large scale and high-quality randomized controlled trials are required to validate the safety of PFNA and DHS for unstable intertrochanteric fractures.
Bone Nails ; Bone Screws ; China ; Femoral Fractures ; Femur ; Fracture Fixation, Internal ; Hip Fractures ; Humans

OBJECTIVE: To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function. METHODS: The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group. RESULTS: The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (P<0.05), and at the same time, the P131R group had a significantly greater increase in cell membrane permeability after the induction with 100 ng/mL TNF-α (P<0.05). Western blot showed that compared with the normal control group, the P131R group had a significant reduction in the expression of SLC26A3 protein (P=0.001). CONCLUSIONS SLC26A3 c.392C>G (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.
Caco-2 Cells ; Chloride-Bicarbonate Antiporters ; genetics ; Diarrhea ; congenital ; genetics ; Humans ; Intestinal Mucosa ; Metabolism, Inborn Errors ; genetics ; Polymorphism, Single Nucleotide ; Sulfate Transporters ; genetics ; Tight Junctions ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.

METHODSThe Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.

RESULTSAfter EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).

CONCLUSIONSEPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Apoptosis ; drug effects ; Caco-2 Cells ; Eicosapentaenoic Acid ; pharmacology ; Escherichia coli ; pathogenicity ; Humans ; Intestinal Mucosa ; metabolism ; microbiology ; RNA, Messenger ; analysis ; Tight Junctions ; drug effects ; Tumor Necrosis Factor-alpha ; secretion ; Zonula Occludens-1 Protein ; genetics