AMBRA1 promotes intestinal inflammation by antagonizing PP4R1/PP4c mediated IKK dephosphorylation in an autophagy-independent manner.

IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/µL to 1000 copies/µL (R2  = 0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.

LAMB3 Promotes Intestinal Inflammation Through SERPINA3 and Is Directly Transcriptionally Regulated by P65 in Inflammatory Bowel Disease.

BACKGROUND: Various extracellular matrix (ECM) reshaping events are involved in inflammatory bowel disease (IBD). LAMB3 is a vital subunit of laminin-332, an important ECM component. Data on the biological function of LAMB3 in intestinal inflammation are lacking. Our aim is to discuss the effect of LAMB3 in IBD. METHODS: LAMB3 expression was assessed in cultured intestinal epithelial cells, inflamed mucosal tissues of patients and mouse colitis models. RNA sequencing, quantitative real-time polymerase chain reaction and Western blotting were used to detect the LAMB3 expression distribution and potential downstream target genes. Dual-luciferase assays and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to determine whether P65 could transcriptionally activate LAMB3 under tumor necrosis factor α stimulation. RESULTS: LAMB3 expression was increased in inflammatory states in intestinal epithelial cells and colonoids and was associated with adverse clinical outcomes in Crohn's disease. Knockdown of LAMB3 inhibited the expression of proinflammatory cytokines. Mechanistically, LAMB3 expression was directly transcriptionally activated by P65 and was inhibited by nuclear factor kappa B inhibitors under tumor necrosis factor α stimulation. Furthermore, RNA sequencing and replenishment experiments revealed that LAMB3 upregulated SERPINA3 to promote intestinal inflammation via the integrin α3ß1/FAK pathway. CONCLUSION: We propose that LAMB3 could serve as a potential therapeutic target of IBD and a predictor of intestinal stenosis of Crohn's disease. Our findings demonstrate the important role of ECM in the progression of IBD and offer an experimental basis for the treatment and prognosis of IBD.

The Effects of a Blood-Brain Barrier Penetrating Erythropoietin in a Mouse Model of Tauopathy.

Erythropoietin (EPO), a hematopoietic neurotrophin, is a potential therapeutic for Alzheimer's disease (AD) but has limited blood-brain barrier (BBB) permeability. EPO fused to a chimeric transferrin receptor monoclonal antibody (cTfRMAb) enters the brain via TfR-mediated transcytosis across the BBB. We previously showed that cTfRMAb-EPO is protective in a mouse model of amyloidosis, but its effects on tauopathy are not known. Given that amyloid and tau pathology are characteristics of AD, the effects of cTfRMAb-EPO were studied in a tauopathy mouse model (PS19). Six-month-old PS19 mice were injected intraperitoneally with either saline (PS19-Saline; n = 9) or cTfRMAb-EPO (PS19-cTfRMAb-EPO, 10 mg/kg; n = 10); every two or three days on alternate weeks for 8 weeks. Age-matched, saline-treated, wildtype littermates (WT-Saline; n = 12) were injected using the same protocol. After 8 weeks, locomotion, hyperactivity, and anxiety were assessed via the open-field test, and brains were harvested and sectioned. Cerebral cortex, hippocampus, amygdala, and entorhinal cortex sections were analyzed for phospho-tau (AT8) and microgliosis (Iba1). Hippocampal cellular density (H&E) was also assessed. PS19-Saline mice were hyperactive and less anxious compared to WT-Saline mice, and these behavioral phenotypes were significantly reduced in the PS19-cTfRMAb-EPO mice compared to the PS19-Saline mice. cTfRMAb-EPO significantly reduced AT8 load by ≥50% in all of the brain regions analyzed and microgliosis in the entorhinal cortex and amygdala compared to the PS19-Saline mice. Hippocampal pyramidal and granule cell layer density did not differ significantly between the PS19-cTfRMAb-EPO and PS19-Saline mice. This proof-of-concept study demonstrates the therapeutic effects of the BBB-penetrating cTfRMAb-EPO in PS19 mice.

Genetic compensation response could exist in colorectal cancer: UPF3A upregulates the oncogenic homologue gene SRSF3 expression corresponding to SRSF6 to promote colorectal cancer metastasis.

BACKGROUND: Genetic compensation response (GCR) is a mechanism that maintains the robustness of functional genes, which has been recently identified. Whether GCR exists in tumors and its effects on tumor progression remains unknown. METHODS: Whole exome sequencing was performed to identify premature termination codon (PTC) gene mutations in colorectal cancer (CRC) tissues. RNA sequencing, Cancer Cell Line Encyclopedia database analysis, and high-throughput output of homologous genes using the Ensemble genome database were performed to further identify homologous genes of target PTC gene mutations. RESULTS: Serine and arginine-rich splicing factor 3 (SRSF3) increased the invasion ability in CRC cells and could be the target gene of up-frameshift 3A (UPF3A). The deletion of the 660th base A in the coding sequence region of SRSF6 caused a frameshift mutation of serine at position 220 (s220fs), which contributed to a PTC UAA termination of translation in HCT116 cells. We further found that SRSF3 was the only homologue of SRSF6 with a frameshift mutation. The transfection of s220fs of SRSF6 into HCT116 cells led to upregulation of its corresponding oncogenic homologue gene SRSF3 expression to promote CRC metastasis. SRSF3 was highly expressed in CRC liver metastases and was positively correlated with UPF3A expression and contributed to poor prognosis. CONCLUSION: GCR may exist in CRC and exert effects on the progression of CRC. Targeted inhibition of UPF3A could reduce the GCR effects and suppress the expression of oncogenic homologue genes corresponding to PTC mutations, indicating a novel therapeutic strategy for treatment of CRC metastasis.

Efficacy and Safety of a Brain-Penetrant Biologic TNF-α Inhibitor in Aged APP/PS1 Mice.

Tumor necrosis factor alpha (TNF-α) plays a vital role in Alzheimer's disease (AD) pathology, and TNF-α inhibitors (TNFIs) modulate AD pathology. We fused the TNF-α receptor (TNFR), a biologic TNFI that sequesters TNF-α, to a transferrin receptor antibody (TfRMAb) to deliver the TNFI into the brain across the blood-brain barrier (BBB). TfRMAb-TNFR was protective in 6-month-old transgenic APP/PS1 mice in our previous work. However, the effects and safety following delayed chronic TfRMAb-TNFR treatment are unknown. Herein, we initiated the treatment when the male APP/PS1 mice were 10.7 months old (delayed treatment). Mice were injected intraperitoneally with saline, TfRMAb-TNFR, etanercept (non-BBB-penetrating TNFI), or TfRMAb for ten weeks. Biologic TNFIs did not alter hematology indices or tissue iron homeostasis; however, TfRMAb altered hematology indices, increased splenic iron transporter expression, and increased spleen and liver iron. TfRMAb-TNFR and etanercept reduced brain insoluble-amyloid beta (Aß) 1-42, soluble-oligomeric Aß, and microgliosis; however, only TfRMAb-TNFR reduced Aß peptides, Thioflavin-S-positive Aß plaques, and insoluble-oligomeric Aß and increased plaque-associated phagocytic microglia. Accordingly, TfRMAb-TNFR improved spatial reference memory and increased BBB-tight junction protein expression, whereas etanercept did not. Overall, despite delayed treatment, TfRMAb-TNFR resulted in a better therapeutic response than etanercept without any TfRMAb-related hematology- or iron-dysregulation in aged APP/PS1 mice.

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections.

Extracellular accumulation of amyloid-beta (Aß) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral Aß plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the Aß load in brain tissues derived from AD transgenic mice. Though the methods for Aß detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on Aß load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the Aß load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of Aß load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on Aß-positive area quantification, and show a strong correlation between the Aß-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread Aß deposition.

Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport.

Brain microvascular endothelial cells, forming the anatomical site of the blood-brain barrier (BBB), are widely used as in vitro complements to in vivo BBB studies. Among the immortalized cells used as in vitro BBB models, the murine-derived bEnd.3 cells offer culturing consistency and low cost and are well characterized for functional and transport assays, but result in low transendothelial electrical resistance (TEER). Human-induced pluripotent stem cells differentiated into brain microvascular endothelial cells (ihBMECs) have superior barrier properties, but the process of differentiation is time-consuming and can result in mixed endothelial-epithelial gene expression. Here we performed a side-by-side comparison of the ihBMECs and bEnd.3 cells for key paracellular diffusional transport characteristics. The TEER across the ihBMECs was 45- to 68-fold higher than the bEnd.3 monolayer. The ihBMECs had significantly lower tracer permeability than the bEnd.3 cells. Both, however, could discriminate between the paracellular permeabilities of two tracers: sodium fluorescein (MW: 376 Da) and fluorescein isothiocyanate (FITC)-dextran (MW: 70 kDa). FITC-dextran permeability was a strong inverse-correlate of TEER in the bEnd.3 cells, whereas sodium fluorescein permeability was a strong inverse-correlate of TEER in the ihBMECs. Both bEnd.3 cells and ihBMECs showed the typical cobblestone morphology with robust uptake of acetylated LDL and strong immuno-positivity for vWF. Both models showed strong claudin-5 expression, albeit with differences in expression location. We further confirmed the vascular endothelial- (CD31 and tube-like formation) and erythrophagocytic-phenotypes and the response to inflammatory stimuli of ihBMECs. Overall, both bEnd.3 cells and ihBMECs express key brain endothelial phenotypic markers, and despite differential TEER measurements, these in vitro models can discriminate between the passage of different molecular weight tracers. Our results highlight the need to corroborate TEER measurements with different molecular weight tracers and that the bEnd.3 cells may be suitable for large molecule transport studies despite their low TEER.

Cut-off value of ulcerative colitis endoscopic index of severity (UCEIS) score for predicting the need for pouch construction in ulcerative colitis: results of a multicenter study with long-term follow-up.

BACKGROUND: Total proctocolectomy with ileal pouch-anal anastomosis (IPAA) was the first choice for the surgical treatment of the ulcerative colitis (UC) patients. The data on the predictive value of the ulcerative colitis endoscopic index of severity (UCEIS) for the need for IPAA in UC patients is scarce. We aimed to establish the UCEIS cut-off value to further analyse whether the UCEIS cut-off was suitable for predicting the need for IPAA in UC patients. METHODS: The clinical data of UC patients from June 1986 to March 2020 at our institute were retrospectively assessed. The UCEIS scores recorded at the time of the first colonoscopy after hospitalization were used in the study. Receiver operating characteristic curve analysis was performed to determine the UCEIS cut-off value for predicting the need for IPAA. RESULTS: A total of 283 UC patients were included in the study, with a median UCEIS of 4. During a median follow-up of 13 years, 80 patients (28.3%) received surgery invention, among whom 75 (93.8%) underwent IPAA surgery and 5 (6.2%) received subtotal colectomy with permanent ostomy. A UCEIS cut-off of 6 had the most significant area under the curve of 0.769 for predicting the need for IPAA (P < 0.001), with a sensitivity of 72.0% and specificity of 81.8%. UCEIS ≥6 was an independent predictive factor for the need for IPAA (P < 0.001) and malignant transformation (P = 0.010). Patients with UCEIS ≥6 had a significantly shorter IPAA-free survival time than those with UCEIS <6 (P < 0.001). CONCLUSIONS: UCEIS ≥6 may be a threshold value for decision-making for IPAA and should be recommended for UC patients for reducing the incidence of malignant transformation.

Increased expression of yes-associated protein/YAP and transcriptional coactivator with PDZ-binding motif/TAZ activates intestinal fibroblasts to promote intestinal obstruction in Crohn's disease.

BACKGROUND: Intestinal obstruction caused by intestinal fibrosis is a common and serious complication of Crohn's disease (CD). Intestinal fibroblasts, the main effector cells mediating gastrointestinal fibrosis, are activated during chronic inflammation. However, the mechanism of fibroblast activation in CD has not been well elucidated. METHODS: Fibroblasts isolated from stenotic and nonstenotic intestines of CD patients were used for RNA sequencing. Immunohistochemical and immunofluorescent staining was performed to evaluate the correlation between intestinal fibrosis and YAP/TAZ expression in our CD cohort and a DSS-induced chronic colitis murine model. A Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) inhibitor was used to explore the ROCK1-YAP/TAZ axis in intestinal fibroblasts in vitro and DSS-induced chronic colitis murine model in vivo. FINDINGS: The expression of YAP/TAZ was significantly upregulated in stenotic fibroblasts, which was associated with the YAP/TAZ target gene signature. YAP/TAZ knockdown suppressed the activation of intestinal fibroblasts. In intestinal fibroblasts, YAP/TAZ were activated by the Rho-ROCK1 signalling pathway. High YAP/TAZ expression was positively correlated with ROCK1 expression, which is a prognostic marker for intestinal obstruction in CD patients. INTERPRETATION: YAP/TAZ activation can lead to fibroblast activation and intestinal obstruction in CD. The effect of ROCK1 inhibitor on alleviating intestinal fibrosis is associated with YAP/TAZ inhibition. Targeted inhibition of YAP/TAZ in fibroblasts may be a potential therapeutic strategy to suppress intestinal fibrosis in CD. FUNDING: This work was supported by the National Key R&D Program of China (2019YFC1316002), the NSFC (81873547, 82073201, 81874177, 82000481) and the Shanghai Sailing Program (20YF1429400).

Increased AOC1 Expression Promotes Cancer Progression in Colorectal Cancer.

BACKGROUND: Amine oxidase copper containing 1 (AOC1) is a gene whose biological function in colorectal cancer (CRC) has not been elucidated. Therefore, the purpose of this study was to investigate the clinical significance of AOC1 expression in CRC and its biological function in CRC cell lines. MATERIALS AND METHODS: AOC1 expression levels were examined in paired CRC and peritumoral tissues, and distant liver metastatic tissues were examined using quantitative real-time PCR, western blotting, and immunohistochemistry staining. The log-rank test and Cox regression model were used to analyze the relationship between AOC1 expression and prognosis. Proliferation assays (Cell Counting Kit-8 and colony formation assays), migration assays (Transwell and wound healing assays) and xenograft tumor formation in nude mice were performed to assess the biological role of AOC1 in CRC cells. RESULTS: AOC1 expression significantly increased in human CRC tissues, especially in liver metastases, and was associated with a worse prognosis. In addition, AOC1 had higher expression in tumor organoids than in normal organoids, suggesting that it was highly expressed in the tumor epithelium. Functional analysis demonstrated that AOC1 knockdown inhibited the proliferation and migration of CRC cells by inducing EMT in vitro. Xenograft tumor formation in nude mice showed that knockdown of AOC1 inhibited the tumor xenografts growth in vivo. CONCLUSION: High expression of AOC1 was significantly associated with worse clinical outcomes, was an independent risk factor for poor prognosis, and promoted aggressive CRC cell phenotypes. AOC1 is expected to become a novel biomarker for predicting the prognosis of patients with CRC and an effective therapeutic target in clinical practice.

Biologic TNF-α inhibitors reduce microgliosis, neuronal loss, and tau phosphorylation in a transgenic mouse model of tauopathy.

BACKGROUND: Tumor necrosis factor-α (TNF-α) plays a central role in Alzheimer's disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aß deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aß-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood-brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy. METHODS: Six-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks. RESULTS: Etanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept. CONCLUSION: Both TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy.

Acute and Chronic Dosing of a High-Affinity Rat/Mouse Chimeric Transferrin Receptor Antibody in Mice.

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mice.

LAMB3 promotes tumour progression through the AKT-FOXO3/4 axis and is transcriptionally regulated by the BRD2/acetylated ELK4 complex in colorectal cancer.

Aberrant expression of laminin-332 promotes tumour growth and metastasis in multiple cancers. However, the dysregulated expression and mechanism of action of LAMB3, which encodes the ß3 subunit of laminin-332, and the mechanism underlying dysregulated LAMB3 expression in CRC remain obscure. Here, we show that LAMB3 is overexpressed in CRC and that this overexpression is correlated with tumour metastasis and poor prognosis. Overexpression of LAMB3 promoted cell proliferation and cell migration in vitro and tumour growth and metastasis in vivo, while knockdown of LAMB3 elicited opposing effects. LAMB3 inhibited the tumour suppressive function of FOXO3/4 by activating AKT in CRC. Both the BET inhibitor JQ1 and the MEK inhibitor U0126 decreased the mRNA level of LAMB3 in multiple CRC cells. Mechanistically, ELK4 cooperated with BRD2 to regulate the transcription of LAMB3 in CRC by directly binding to the ETS binding motifs in the LAMB3 promoter. ELK4 was as acetylated at K125, which enhanced the interaction between ELK4 and BRD2. JQ1 disrupted the interaction between ELK4 and BRD2, resulting in decreased binding of BRD2 to the LAMB3 promoter and downregulation of LAMB3 transcription. Both ELK4 and BRD2 expression was associated with LAMB3 expression in CRC. LAMB3 expression was also negatively correlated with FOXO3/4 in CRC. Our study reveals the pro-tumorigenic role of LAMB3 through the AKT-FOXO3/4 axis and the transcriptional mechanism of LAMB3 in CRC, demonstrating that LAMB3 is a potential therapeutic target that can be targeted by BET inhibitors and MEK inhibitors.

Clinical outcomes and risk factors of secondary extraintestinal manifestation in ulcerative colitis: results of a multicenter and long-term follow-up retrospective study.

BACKGROUND: Extraintestinal manifestations (EIM) are common in ulcerative colitis (UC). In Shanghai, China, data on the incidence rate and risk factors of EIM in UC patients remain scarce. METHODS: The study population consisted of UC patients who were identified from a prospectively maintained, institutional review board-approved database at our institutes from June 1986 to December 2018. The demographic and clinical characteristics of the study participants were analyzed. The study included secondary EIM in UC patients and follow-up, while primary EIM was excluded. The diagnosis of EIM was based on clinical, radiological, endoscopic, and immunologic examination and histological findings. RESULTS: In total, 271 eligible patients were included in the current study, with a median follow-up time of 13.0 years (interquartile range, 9.0-17.0), and including 31 cases (11.4%) that developed EIM. EIM was associated with clinical outcomes in UC patients and the following factors were identified as contributing factors for the development of EIM: a disease duration of >5 years (odds ratio (OR), 3.721; 95% confidence interval (CI) [1.209-11.456]), age at diagnosis >40 years (OR, 2.924, 95% CI [1.165-7.340]), refractory clinical symptoms (OR, 4.119; 95% CI [1.758-9.650]), and moderate or severe anemia (OR, 2.592; 95% CI [1.047-6.413]). CONCLUSION: In this study, approximately 11.4% UC patients go on to develop at least one EIM. Clinicians should prioritize early control of the disease and treatment of anemia in UC in order to prevent the development of EIM and improve disease prognosis.

In vitro effects of N-acetylcysteine alone and combined with tigecycline on planktonic cells and biofilms of Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii (A. baumannii), as a common opportunistic pathogen, has strong ability to form biofilms, which has led to drug resistance and chronic infections. The combination of N-acetylcysteine (NAC) and tigecycline (TGC) was demonstrated to synergistically inhibit biofilm-associated bacterial infections, including methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. The purpose of this study is to investigate the effect of NAC and TGC on planktonic cells and biofilms of A. baumannii. METHODS: Minimum inhibitory concentrations (MICs) of NAC were determined by broth microdilution method. Biofilm susceptibility was assessed by crystal violet stain. Interactive effects of NAC and TGC on planktonic cells were determined by checkerboard MIC assay. Viable cell count was used to evaluate the combined effect of NAC and TGC on biofilm-embedded bacteria. RESULTS: MICs of NAC against 25 A. baumannii isolates ranged from 16 to 128 mg/mL. NAC alone (0.5-128 mg/mL) significantly inhibited biofilm formation and disrupted preformed biofilms. The combination of NAC and TGC induced a partial synergistic effect (60%) and additive effect (28%) on planktonic bacteria. For biofilm-embedded bacteria, treatment with 16 mg/mL NAC alone or 2 µg/mL TGC alone resulted in significant bactericidal effects (P<0.01 and P<0.05, respectively); synergistic bactericidal effect was found at 4 mg/mL NAC combined with 0.5 µg/mL TGC (P<0.01). CONCLUSIONS: NAC alone significantly inhibited biofilm formation of A. baumannii. The combination of NAC and TGC induced partial synergistic effect against planktonic cells and synergistic effect against biofilm-embedded A. baumannii, which might be a therapeutic option for biofilm-related infections of A. baumannii.

Restoration of KLF4 Inhibits Invasion and Metastases of Lung Adenocarcinoma through Suppressing MMP2.

BACKGROUND: KLF4 is a zin-finger transcription factor that plays roles in differentiation, development, and proliferation. Recent studies show that KLF4 is involved in tumorigenesis and somatic cells reprogramming. Metastasis is the primary cause of death in patients with lung cancer, and its biological mechanisms are poorly understood. GOALS: In this study, we aim to explore the expression pattern and biological function of KLF4 in lung adenocarcinoma. METHODS: We determined KLF4 in lung adenocarcinoma tissue and cell lines, using immunohistochemistry and western blotting. And we further analyzed the correlation between KLF4 expression and clinicopathologic parameters. We restored KLF4 expression and studied its effect on lung adenocarcinoma cells in vivo and in vitro. Luciferase assay was used to study impact of KLF4 on activity of MMP2 promoter. RESULTS: KLF4 is dramatically down-regulated in lung adenocarcinoma tissue and cell lines. Promoter methylation contributes to the down-regulation of KLF4. Down-regulation of KLF4 in lung adenocarcinoma tissue is significantly associated with reduced survival time. Restoration of KLF4 inhibits migration and invasion of lung adenocarcinoma cells in vitro. Metastases to lungs significantly decrease in mice intravenously injected with tumor cells overexpressing KLF4. KLF4 inhibits invasion and metastasis via suppressing MMP2 promoter activity. CONCLUSION: The ability of KLF4 to inhibit migration, invasion, and metastasis of lung tumor cells indicates a potential role of KLF4 as therapeutic target in lung adenocarcinoma. KLF4 might be utilized as a favorable biomarker for prognosis of lung adenocarcinoma patients.

Low-density lipoprotein docosahexaenoic acid nanoparticles induce ferroptotic cell death in hepatocellular carcinoma.

Low-density lipoprotein nanoparticles reconstituted with the natural omega-3 fatty acid, docosahexaenoic acid (LDL-DHA), have been reported to selectively kill hepatoma cells and reduce the growth of orthotopic liver tumors in the rat. To date, little is known about the cell death pathways by which LDL-DHA nanoparticles kill tumor cells. Here we show that the LDL-DHA nanoparticles are cytotoxic to both rat hepatoma and human hepatocellular carcinoma (HCC) cell lines. Following LDL-DHA treatment both rat and human HCC cells experience pronounced lipid peroxidation, depletion of glutathione and inactivation of the lipid antioxidant glutathione peroxidase-4 (GPX4) prior to cell death. Inhibitor studies revealed that the treated HCC cells die independent of apoptotic, necroptotic or autophagic pathways, but require the presence of cellular iron. These hallmark features are consistent and were later confirmed to reflect ferroptosis, a novel form of nonapoptotic iron-dependent cell death. In keeping with the mechanisms of ferroptosis cell death, GPX4 was also found to be a central regulator of LDL-DHA induced tumor cell killing. We also investigated the effects of LDL-DHA treatments in mice bearing human HCC tumor xenografts. Intratumoral injections of LDL-DHA severely inhibited the growth of HCC xenografts long term. Consistent with our in vitro findings, the LDL-DHA treated HCC tumors experienced ferroptotic cell death characterized by increased levels of tissue lipid hydroperoxides and suppression of GPX4 expression. CONCLUSION: LDL-DHA induces cell death in HCC cells through the ferroptosis pathway, this represents a novel molecular mechanism of anticancer activity for LDL-DHA nanoparticles.

PTEN expression is associated with the outcome of lung cancer: evidence from a meta-analysis.

INTRODUCTION: Various studies examined the relationship between the expression of phosphatase and tensin homolog (PTEN) with the clinical outcome in patients with lung cancer, but yielded conflicting results. EVIDENCE ACQUISITION: Electronic databases updated to January 2016 were searched to find relevant studies. A meta-analysis was conducted with eligible studies which quantitatively evaluated the relationship between the expression of PTEN and clinical outcomes of patients with lung cancer. Subgroup and sensitivity analysis were conducted. EVIDENCE SYNTHESIS: A total of 13 studies were included. When compared with high PTEN expression group, the lung cancer patients with reduced PTEN expression exhibited shorter overall survival (hazard ratio=0.40, 95%CI=0.32~0.52, P<0.001) and shorter progression-free survival (hazard ratio=0.53, 95%CI=0.38~0.74, P<0.001). In subgroup analysis, among lung cancer patients whose treatments including EGFR TKIs, we observed significant benefits of OS in high PTEN expression group. CONCLUSIONS: Reduced PTEN expression may be an indicator for poor prognosis in patients with lung cancer. It could have the same influence for patients whose treatments include EGFR-TKIs. The presence of PTEN expression may define a subset of patients with lung cancer appropriate for investigational therapeutic strategies.

TP53 mutation is associated with a poor clinical outcome for non-small cell lung cancer: Evidence from a meta-analysis.

A number of studies have examined the association between tumor protein 53 (TP53) mutations and the clinical outcome in patients with non-small-cell lung cancer (NSCLC), although these have yielded conflicting results. In the present study, electronic databases updated to September 2015 were searched to find relevant studies. A meta-analysis was performed on the eligible studies, which quantitatively evaluated the association between the TP53 mutations and the survival of patients with NSCLC. Subgroup and sensitivity analyses were performed. A total of 19 studies that involved a total of 6,084 patients with NSCLC were included. When the TP53 mutation group (n=1,406) was compared with the wild-type group (lacking TP53 mutations; n=1,965), the wild-type group was associated with a significantly higher overall survival rate [hazard ratio (HR), 1.26; 95% confidence interval (CI) 1.12-1.41, P<0.0001]. Significant benefits of overall survival in the wild-type group were found in the subgroup involving patients with NSCLC in the early stages, including the I/II phases (HR, 1.93, 95% CI, 1.17-3.19, P=0.01; heterogeneity, I2=0.0%, P=0.976) and patients with adenocarcinoma (HR, 3.06; 95% CI, 1.66-5.62, P<0.0001; heterogeneity: I2=0.0%, P=0.976). This meta-analysis has indicated that TP53 gene alteration may be an indicator of a poor prognosis in patients with NSCLC. Furthermore, the results also suggested that the role of TP53 mutations may differ according to different pathological types and clinical stages. The presence of these mutations may define a subset of patients with NSCLC appropriate for investigational therapeutic strategies.