Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice.

OBJECTIVE: Currently, there is no cure for chronic pancreatitis (CP). Germline loss-of-function variants in SPINK1 (encoding trypsin inhibitor) are common in patients with CP and are associated with acute attacks and progression of the disease. This preclinical study was conducted to explore the potential of adeno-associated virus type 8 (AAV8)-mediated overexpression of human SPINK1 (hSPINK1) for pancreatitis therapy in mice. DESIGN: A capsid-optimised AAV8-mediated hSPINK1 expression vector (AAV8-hSPINK1) to target the pancreas was constructed. Mice were treated with AAV8-hSPINK1 by intraperitoneal injection. Pancreatic transduction efficiency and safety of AAV8-hSPINK1 were dynamically evaluated in infected mice. The effectiveness of AAV8-hSPINK1 on pancreatitis prevention and treatment was studied in three mouse models (caerulein-induced pancreatitis, pancreatic duct ligation and Spink1 c.194+2T>C mouse models). RESULTS: The constructed AAV8-hSPINK1 vector specifically and safely targeted the pancreas, had low organ tropism for the heart, lungs, spleen, liver and kidneys and had a high transduction efficiency (the optimal expression dose was 2×1011 vg/animal). The expression and efficacy of hSPINK1 peaked at 4 weeks after injection and remained at significant level for up to at least 8 weeks. In all three mouse models, a single dose of AAV8-hSPINK1 before disease onset significantly alleviated the severity of pancreatitis, reduced the progression of fibrosis, decreased the levels of apoptosis and autophagy in the pancreas and accelerated the pancreatitis recovery process. CONCLUSION: One-time injection of AAV8-hSPINK1 safely targets the pancreas with high transduction efficiency and effectively ameliorates pancreatitis phenotypes in mice. This approach is promising for the prevention and treatment of CP.

Effect of LRRC15 on autophagy in A549 cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and irreversible interstitial lung disease with unknown cause. To explore the role and regulatory mechanism of leucine-rich repeat-containing protein 15 (LRRC15) in IPF, bleomycin (BLM)-induced pulmonary fibrosis in mouse and A549 cells were constructed, and the expression of LRRC15 were detected. Then, MTT, GFP-RFP-LC3 dual fluorescent labeling system and Western blotting were used to investigate the effects of LRRC15 on cell activity and autophagy after transfection of siLRRC15, respectively. The results indicated that the expression of LRRC15 was significantly increased after the BLM treatment in mouse lung tissue and A549 cells. The designed and synthesized siLRRC15 followed by transfection into A549 cells resulted in a dramatic reduction in LRRC15 expression and partially restored the cell damage induced by BLM. Moreover, the expression of LC3-II and P62 were up-regulated, the amount of autophagosome were increased by GFP-RFP-LC3 dual fluorescent labeling assay after BLM treatment. Meanwhile, this study also showed that the key autophagy proteins LC3-II, ATG5 and ATG7 were up-regulated, P62 was down-regulated and autophagic flux were enhanced after further treatment of A549 cells with siLRRC15. The above findings suggest that LRRC15 is an indicator of epithelial cell damage and may participate in the regulation of fibrosis through autophagy mechanism in IPF. This study provides necessary theoretical basis for further elucidating the mechanism of IPF.

Programmable Biomolecule-Mediated Processors.

Programmable biomolecule-mediated computing is a new computing paradigm as compared to contemporary electronic computing. It employs nucleic acids and analogous biomolecular structures as information-storing and -processing substrates to tackle computational problems. It is of great significance to investigate the various issues of programmable biomolecule-mediated processors that are capable of automatically processing, storing, and displaying information. This Perspective provides several conceptual designs of programmable biomolecule-mediated processors and provides some insights into potential future research directions for programmable biomolecule-mediated processors.

Neo-adjuvant radiation therapy provides a survival advantage in T3-T4 nodal positive gastric and gastroesophageal junction adenocarcinoma: a SEER database analysis.

BACKGROUND: Due to negative results in clinical trials of postoperative chemoradiation for gastric cancer, at present, there is a tendency to move chemoradiation therapy forward in gastric and gastroesophageal junction (GEJ) adenocarcinoma. Several randomized controlled trials (RCTs) are currently recruiting subjects to investigate the effect of neo-adjuvant radiotherapy (NRT) in gastric and GEJ cancer. Large retrospective studies may be beneficial in clarifying the potential benefit of NRT, providing implications for RCTs. METHODS: We retrieved the clinicopathological and treatment data of gastric and GEJ adenocarcinoma patients who underwent surgical resection and chemotherapy between 2004 and 2015 from Surveillance, Epidemiology, and End Results (SEER) database. We compared survival between NRT and non-NRT patients among four clinical subgroups (T1-2N-, T1-2N+, T3-4N-, and T3-4N+). RESULTS: Overall, 5272 patients were identified, among which 1984 patients received NRT. After adjusting confounding variables, significantly improved survival between patients with and without NRT was only observed in T3-4N+ subgroup [hazard ratio (HR) 0.79, 95% confidence interval (CI): 0.66-0.95; P = 0.01]. Besides, Kaplan-Meier plots showed significant cause-specific survival advantage of NRT in intestinal type (P <  0.001), but not in diffuse type (P = 0.11) for T3-4N+ patients. In the multivariate competing risk model, NRT still showed survival advantage only in T3-4 N+ patients (subdistribution HR: 0.77; 95% CI: 0.64-0.93; P = 0.006), but not in other subgroups. CONCLUSIONS: NRT might benefit resectable gastric and GEJ cancer patients of T3-4 stages with positive lymph nodes, particularly for intestinal-type. Nevertheless, these results should be interpreted with caution, and more data from ongoing RCTs are warranted.

Poorly differentiated is more significant than signet ring cell component for lymph node metastasis in mixed-type early gastric cancer: a retrospective study from a large-volume hospital.

OBJECTIVE: The purpose of this study was to explore the role of different undifferentiated components in the lymph node metastasis (LNM) of early mixed gastric cancer. METHODS: A total of 1596 patients with EGC who underwent gastrectomy were divided into four types: pure differentiated (PD), pure poorly differentiated (Poorly D), pure signet ring cell carcinoma (SRC), and mixed. Mixed type was classified into four subtypes: differentiated-predominant type mixed with poorly differentiated component (MD-P), poorly differentiated-predominant type mixed with differentiated component (MP-D), differentiated-predominant type mixed with SRC component (MD-S), and poorly differentiated-predominant type mixed with SRC component (MP-S). We analyzed the clinicopathological differences between all types and the rates of LNM of patients fulfilling endoscopic submucosal dissection (ESD) criteria. RESULTS: LNM was more common in mixed (21.9%) than in PD (7.5%, P < 0.001) or SRC (11.3%, P < 0.001). When analyzed the subgroups of mixed type, LNM was more frequent in MD-P (15.4%) than in PD (7.5%, P = 0.003). LNM in MD-S (7.4%, P = 1.000) was not higher than in PD. MP-S (24.5%) showed a higher rate of LNM than SRC (11.3%, P < 0.001) rather than Poorly-D (22.7%, P = 0.681). For lesions satisfying ESD criteria, MD-S (0%, P = 1.000), and MD-P (5.9%, P = 0.12) did not have higher rates of LNM than PD (1.4%). CONCLUSION: The presence of poorly differentiated component but not SRC increases the LNM rate of mixed type. ESD is recommended for the treatment of MD-S and MD-P consistent with ESD criteria.

Identification and validation of tumour microenvironment-based immune molecular subgroups for gastric cancer: immunotherapeutic implications.

BACKGROUND: Immunotherapy could trigger durable response in advanced gastric cancer, but it only benefits a minority of patients. We aimed to propose a robust molecular classification of gastric cancer microenvironment to identify ideal candidates for tailoring effective immunotherapy. METHODS: A training cohort of 375 gastric cancer samples with RNA sequencing data was analysed. We virtually microdissected tumour, stromal, and immune cell gene expression patterns employing a non-negative matrix factorization algorithm. These expression patterns were annotated using immune- and stromal-related gene signatures. Validation of immunogenomic classification was performed across six microarray datasets of 1406 samples. RESULTS: We found approximately half of gastric cancer samples to have higher immune cell infiltrates, PD-L1 expression, markers of cytolytic activity, and fewer copy number aberrations (all P < 0.05). We termed this group of tumours the Immune Class, which incorporated two components, namely Immune Activation and Immunosuppressive Subtype, according to immunosuppressive or activated microenvironment. Immune Activation Subtype was associated with improved survival in multivariate survival analysis and shared similar genomic characteristics with responders of anti-PD-1 therapy. Immunosuppressive Subtype featured high immune infiltration, stromal enrichment, and transforming growth factor (TGF)-ß signalling pathway activation and correlated with non-responsiveness signature of checkpoint blockade therapy, which might be suitable for anti-PD-L1 and anti-TGF-ß combined therapy. CONCLUSIONS: We proposed and independently validated three reproducible immune molecular subtypes of gastric cancer, which may provide implications for patient selection of immunotherapy.

Marital status, an independent predictor for survival of gastric neuroendocrine neoplasm patients: a SEER database analysis.

BACKGROUND: Marital status proves to be an independent prognostic factor in a variety of cancers. However, its prognostic impact on gastric neuroendocrine neoplasms (G-NEN) has not been investigated. METHODS: We identified 3947 G-NEN patients from the Surveillance, Epidemiology, and End Results (SEER) database. Meanwhile, propensity scores for marital status were used to match 506 unmarried patients with 506 married patients. We used Kaplan-Meier method and multivariate Cox regression to analyse the association between marital status and the overall survival (OS) and G-NEN cause-specific survival (CSS) before matching and after matching. RESULTS: Married patients enjoyed better OS and CSS, compared with divorced/separated, single, and widowed patients. Multivariate Cox regression analysis indicated that unmarried status was associated with higher mortality hazards for both OS and CSS among G-NEN patients. Additionally, widowed individuals had the highest risks of overall (adjusted hazard ratio (HR): 1.56, 95% confidence interval (CI): 1.35-1.81, P < 0.001) and cancer-specific mortality (adjusted HR: 1.33, 95% CI: 1.05-1.68, P = 0.02) compared to other unmarried groups in both males and females. Furthermore, unmarried status remained an independent prognostic and risk factor for both OS (HR 1.51, 95% CI 1.19-1.90, P = 0.001) and CSS (HR 1.50, 95% CI 1.10-2.05, P = 0.01) in 1:1 propensity score-matched analysis. CONCLUSION: Marital status was an independent prognostic factor for G-NEN. Meanwhile, widowed patients with G-NEN had the highest risk of death compared with single, married, and divorced/separated patients.

Anti-arthritic activity of ethanol extract of Claoxylon indicum on Freund's complete adjuvant-induced arthritis in mice.

BACKGROUND: Claoxylon indicum Hassk. (Euphorbiaceae), named Diu Le Bang, have functions of dehumidification and relieving swelling pain, and is used as a folk medicine to treat Rheumatoid arthritis (RA), lumbocrural pain and foot edema in the south of China. The aim of the present study was to investigate the anti-arthritic activity of the ethanol extract of Claoxylon indicum (CIE) on mice with adjuvant induced joint arthritis. METHODS: Adjuvant arthritis was induced in mice by subcutaneous injection of complete Freund's adjuvant into the plantar surface of right hind paw. Arthritis severity was evaluated by arthritic score, hind paws oedema and spleen index, and histological examinations. Serum samples were collected for determination of malondialdehyde (MDA) and alkaline phosphatase (ALP) levels. The expression of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in the specimens of knee joints was determined by standard immunohistochemical techniques. RESULTS: CIE administration (0.4 and 0.8 g/kg) suppressed the inflammatory responses in the joints of adjuvant-induced arthritis (AIA) mice, suggested by the modulatory effects on paw swelling, hyperplasia of lymphatic tissues and synovial membrane. It also decreased the levels of MDA and ALP in serum and downregulated the expression of IL-1ß and TNF-α in the arthritic joints of AIA mice. CONCLUSION: These results suggested that CIE possessed substantial anti-arthritic activity due to immumodepression and regulation of cytokines. CIE may be a potential candidate for the treatment of RA.

[Xanthones from Rhizoma of Securidaca inappendiculata].

Objective: To study the chemical constituents of Securidaca inappendiculata. Methods: The chemical constituents were isolated and purified by chromatography on silica gel, Sephadex LH-20 columns. Their structures were elucidated by means of physicochemical properties and spectroscopic analysis. Results: Ten compounds were identified from the dichloromethane fraction of Securidaca inappendiculata, and identified as 1,7-dihydroxyxanthone (1), 1,3,8-trihydroxy-2-methoxyxanthone (2), 1,7-dihydroxy-3,4-dimethoxyxanthone (3), 1,3,8-trihydroxy-4-methoxyxanthone (4), 7-hydroxy-1,2-dimethoxyxanthone (5), 1,3,6-trihydroxy-2,7-dimethoxyxanthone (6), 1,4, 8-trihydroxyxanthone (7), 1,7-dihydroxy-3-methoxyxanthone (8), 1,6-dihydroxy-7-methoxyxanthone (9) and 1,8-dihydroxy-3,4-dimethoxyxanthone (10). Conclusion: Compounds 7­10 are isolated from this plant for the first time, and compounds 7­10 are isolated from this genus for the first time.

[Regulation of autophagy on dendritic cells during rat liver regeneration by IPA].

To understand the mechanism underlying autophagy in regulating dendritic cells during rat liver regeneration, we used the method of percoll density gradient centrifugation combined with immunomagnetic bead to isolate dendritic cells, the Rat Genome 230 2.0 Array to determine the expression changes of autophagy-related genes, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the autophagy activities. The results indicated that LC3, BECN1, ATG7 and SQSTM1 genes had significant expression changes during rat liver regeneration. There were 593 genes related to autophagy, among which 210 genes were identified as significant. We also showed that the activity of autophagy was enhanced in the priming phase and teminal phase of liver regeneration, weakened in the proliferative stage by comparative analysis method of IPA. The autophagy-related physiological activities mainly included RNA expression, RNA transcription, cell differentiation and proliferation, involving in PPARα/RXRα activation, acute phase response signaling, TREM1 signaling, IL-6 signaling, IL-8 signaling and IL-1 signaling, whose activities were increased or decreased in liver regeneration. Cluster analysis found that P53 and AMPK signaling participated in the regulation of dendritic cells autophagy, with AMPK signaling in the priming phase of liver regeneration, and both signaling pathways in the terminal phase. We conclude that dendritic cells autophagy played an important role in initiation of the immune response in priming phase and depletion of dendritic cells in late phase during rat liver regeneration.

[Effect of autophagy on liver regeneration].

Autophagy is a lysosome-mediated degradation pathway, which plays an important role in hepatic physiological and pathological processes, in eukaryotic cells. The liver has a remarkable regenerative capacity. After acute or chronic injury, the residual hepatic cells can be activated to enter the cell-cycle for proliferation, in order to compensate for lost liver tissue and recover liver function. In this review, we summarize the relationship between liver regeneration (LR) after various types of injury and autophagy. For example, autophagy is activated to accelerate LR after physically, alcohol and food borne induced liver injury, while the role of autophagy in animal models of LR after chemical injury remains controversial. Autophagy can also be used to promote the replication of virus particles by some hepatotropic viruses (e.g., HBV, HCV) and inhibit LR after viral infection. Studies on mechanisms of autophagy and LR will contribute to clarify the regenerative process and provide new methods for the treatment of liver disease.

Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo.

Sodium fluoride (NaF) is associated with embryonic and fetal development abnormalities, but the mechanism by which this occurs is unclear. DNA methylation, an important epigenetic reprogramming mechanism, is essential for normal embryonic development. Thus, we investigated the effect of NaF on DNA methylation in early mouse embryos, as well as mouse sperm and liver using bisulfite sequencing and ELISA. Data indicate that H19, a paternally imprinted gene, compared to control embryos, was less methylated in 8-cell embryos from pregnant mice treated with NaF (100 mg/l) in drinking water for 48 h. Peg3, a maternally imprinted gene, and the Line1 repeated sequence were similarly methylated in NaF-treated and control embryos. Oral ingestion of NaF for 35 days did not significantly change Line1 and genomic global DNA methylation in the liver. H19, Rasgrf1, Line1, and genomic global DNA methylation were also similar in NaF-treated and control sperm. Female mice mated with NaF-treated male mice (35 days) had less methylated H19, but Peg3 was significantly more methylated. Line1 was similarly methylated in treated 8-cell embryos, compared to control embryos. NaF treatment of male mice before copulation significantly increased the expression of H19 in blastocysts, whereas H19 expression was not detected in 8-cell embryos. Data suggest that NaF may interact directly with the embryo to disrupt the maintenance of normal gene imprinting during pregnancy. Long-term NaF exposure of males may not directly affect DNA methylation of the sperm and liver, but the sperm may signal to early embryos with abnormal gene imprinting.

[Enhanced anticancer effects of 5'-DFUR on colorectal cancer cell lines SW480 and LOVO by transfection with thymidine phosphorylase cDNA].

OBJECTIVES: To study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells. METHODS: TP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay. RESULTS: The colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01). CONCLUSIONS: The stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.

Lactobacillus Intestinalis Primes Epithelial Cells to Suppress Colitis-Related Th17 Response by Host-Microbe Retinoic Acid Biosynthesis.

Gut microbiome is integral to the pathogenesis of ulcerative colitis. A novel probiotic Lactobacillus intestinalis (L. intestinalis) exerts a protective effect against dextran sodium sulfate-induced colitis in mice. Based on flow cytometry, colitis-associated Th17 cells are the target of L. intestinalis, which is supported by the lack of protective effects of L. intestinalis in T cell-null Rag1-/- mice or upon anti-IL-17-A antibody-treated mice. Although L. intestinalis exerts no direct effect on T cell differentiation, it decreases C/EBPA-driven gut epithelial SAA1 and SAA2 production, which in turn impairs Th17 cell differentiation. Cometabolism of L. intestinalis ALDH and host ALDH1A2 contributed to elevated biosynthesis of retinoic acid (RA), which accounts for the anti-colitis effect in RAR-α -mediated way. In a cohort of ulcerative colitis patients, it is observed that fecal abundance of L. intestinalis is negatively associated with the C/EBPA-SAA1/2-Th17 axis. Finally, L. intestinalis has a synergistic effect with mesalazine in alleviating murine colitis. In conclusion, L. intestinalis and associated metabolites, RA, have potential therapeutic effects for suppressing colonic inflammation by modulating the crosstalk between intestinal epithelia and immunity.

[Synthesis of a supermolecular nanoparticle γ-hy-PC/Ada-Dox and its antitumor activity].

OBJECTIVE: To synthesize a (2-Hydroxypropyl)-γ-cyclodextrin-polyethylenimine/adamantane-conjugated doxorubicin (γ-hy-PC/Ada-Dox) based supramolecular nanoparticle with host-guest interaction and to identify its physicochemical characterizations and antitumor effect. METHODS: A novel non-viral gene delivery vector γ-hy-PC/Ada-Dox was synthesized based on host-guest interaction. 1H-NMR, NOESY, UV-Vis, XRD and TGA were used to confirm the structure of the vector. The DNA condensing ability of complexes was investigated by particle size, zeta potential and gel retardation assay. Cytotoxicity of complexes was determined by MTT assay in BEL-7402 and SMMC-7721 cells. Cell wound healing assay was performed in HEK293 and BEL-7404 cells. The transfection efficiency was investigated in HEK293 cells. H/E staining and cell uptake assay was performed in BEL-7402 cells. RESULTS: The structure of γ-hy-PC/Ada-Dox was characterized by 1H-NMR, NOESY, UV-Vis, XRD, TGA. The drug loading was 0.5% and 5.5%. Gel retardation assay showed that γ-hy-PC was able to completely condense DNA at N/P ratio of 2; 0.5% and 5.5% γ-hy-PC/Ada-Dox was able to completely condense DNA at N/P ratio of 3 and 4,respectively. The cytotoxicity of polymers was lower than that of PEI25KDa. The transfection efficiency of γ-hy-PC was higher than that of γ-hy-PC/Ada-Dox at N/P ratio of 30 in HEK293 cells; and the transfection efficiency was decreasing when Ada-Dox loading was increasing. Cell uptake assay showed that γ-hy-PC/Ada-Dox was able to carry drug and FAM-siRNA into cells. CONCLUSION: The novel vector γ-hy-PC/Ada-Dox has been developed successfully, which has certain transfection efficiency and antitumor activity.

[Anticancer effect of triptolide-polyethylenimine-cyclodextrin in vitro].

OBJECTIVE: To develop a drug delivery system triptolide-polyethylenimine-cyclodextrin and to evaluate its anticancer activity in vitro. METHODS: Triptolide was conjugated to polyethylenimine-cyclodextrin by N, N'-carbonyldiimidazole to form triptolide-polyethylenimine-cyclodextrin. (1)H-NMR, FT-IR and XRD were used to confirm its structure. The anticancer effect of the polymer was assessed by MTT assay, erasion trace test and hematoxylin-eosin staining. The potential to condense siRNA and to delivery siRNA into cytoplasm was demonstrated by gel retardation assay, zeta-potential determination and fluorescence staining. RESULTS: Triptolide was successfully conjugated to polyethylenimine-cyclodextrin and the conjugation rate of triptolide was 10% (w/w). siRNA was effectively condensed by the polymer at the N/P ratio of 5, and its particle size was 300 ±15 nm and zeta potential was 8 ±2.5 mV. MTT assay, erasion trace test and hematoxylin-eosin staining revealed that triptolide-polyethylenimine-cyclodextrin had anticancer effect and low cytotoxicity to normal cells. The polymer was able to deliver siRNA to the cytoplasm effectively as demonstrated by fluorescence staining. CONCLUSION: Triptolide-polyethylenimine-cyclodextrin is able to inhibit the growth and migration of cancer cells in vitro and to carry siRNA into cells effectively. It is potential to be used as a novel prodrug for co-delivery of gene and drug in cancer treatment.

[Characteristics of cationic polymers PEI-CyD, PEI-PHPA, PEE-PHPA and PEI25kD in vitro and in vivo].

OBJECTIVE: To study the characteristics of cationic polymers polyethylenimine-ß-cyclodextrin (PEI-CyD), polyethylenimine-poly-(3-hydroxypropyl)-aspartamide (PEI-PHPA), N,N-Dimethyldipropylenetriamine-Bis(3-aminopropyl)amine-aspartamide (PEE-PHPA) in vitro and in vivo. METHODS: PEI-PHPA, PEI-CyD and PEE-PHPA were synthesized and the chemistry structure of PEI-PHPA, PEI-CyD and PEE-PHPA was confirmed by (1)H-NMR. The particle size and zeta potential of these polymers were measured, and capacity of plasmid DNA condensation was tested. The inhibition of COS-7, A549, HEK293 and C6 cells was measured by MTT assay. The transfection efficiency was determined in HEK293 cell lines. The toxicity, tissue distribution and transfection efficiency of cationic polymers were tested in vivo. RESULTS: When the N/P of polymers/DNA at 30, the particle sizes were close 250 nm and the zeta-potential were near 35 mv. They were able to condense DNA at N/P ratio < 5. The MTT assay showed that the IC(50) of PEE-PHPA was 21.5, 20.2, 7.30 and 37.1 µg/ml, and that of PEI25kD was 15.8, 18.3, 11.4 and 36.7 µg/ml in C6, COS-7, A549 and HEK293cell lines, respectively. The cell viability of PEI-CyD and PEI-PHPA in above cell lines was over 60%. They had high transfection efficiency in HEK293 cell lines. The LD(50) of PEI25Kd, PEI-CyD, PEI-PHPA and PEE-PHPA in vivo was 19.50, 100.4, 521.2 and 630.0, respectively by intraperitoneal (ip) injection. The contractions of these polymers were higher in kidney than in other organs and tissues.PEE-PHPA had slight effect on kidney and liver function. CONCLUSION: PEE and PEI25kD have higher transfection efficiency and higher toxicity; while PC and PHPA-PEI have lower toxicity and higher transfection efficiency to be used as non-viral gene vector.

[Modified montmorillonite as multifunction gene and drug delivery system].

OBJECTIVE: To develop polyethylenimine-Doxorubicin-montmorillonite (PEI-Dox-MTT) as a novel multifunction delivery system. METHODS: Dox was intercalated into montmorillonite, PEI covered to the surface of Dox/MMT to make the nano-particle. XRD, FT-IR and TGA were used to confirm chemical property of the nano-particle. SEM was used to observe the morphology. The capability of drug release was investigated by PBS buffer solution (pH 7.4). The DNA binding ability of nano-particle was detected by gel electrophoresis retardation assay. The cell viability in COS-7 and SKOV3 cell lines was tested using MTT assay. The gastric mucosa protection was evaluated in vitro. RESULTS: XRD image showed that Dox was intercalated into montmorillonite, inter space of which increased to 31.3Å; the FT-IR spectra showed the vibration bands of PEI at 1 560 cm(-1) and 2 850 cm(-1), the vibration band of Dox at 1 350 cm(-1). Size analysis and SEM revealed that the size of nano-particle was 600 nm, and the zeta-potential was 30 mV. Drug release experiment explored that the nano-particle stably released drug in range of 6 X10(-4) ≊ 8 X10(-4) mg/ml within 72 h. MTT assay showed that the cell viability was over 80% in experiment condition in COS-7 and SKOV3 cell lines. 0.3 mg PEI-MMT nano-particle was able to protect gastric mucosa from alcohol. CONCLUSION: Multifunction system of PEI/Dox/MMT has been prepared successfully.

ERBB2 Mutations as Potential Predictors for Recurrence in Colorectal Serrated Polyps by Targeted Next-Generation Sequencing.

Background: Follow-up guidelines for serrated polyps (SPs) are mainly based on factors such as histology and size with limited evidence. The underlying genomic mechanism of SPs in relation to recurrence risks is utterly unknown. Methods: We applied targeted next-generation sequencing (NGS) approach on two groups of SPs [polyp-relapsed SPs (PRSPs) vs. polyp-free SPs (PFSPs)] based on the surveillance outcomes to compare differences of DNA variants in 71 colorectal cancer-associated genes. A multicenter validation cohort was established longitudinally from 2016 to 2019 to confirm the relevant results. Results: Among the 96 NGS samples, at least one mutant after filtration was detected in 90 samples (94%). Molecular profiling presented BRAF, KRAS, and APC as top 3 mutated genes. FBXW7, MSH2, and ERBB2 might be recurrence-relevant, while DMD, BRCA1, and BRCA2 might be negatively correlated with recurrence. Notably, ERBB2 mutants (R678Q and V842I) (n = 5) had higher risks of polyp recurrence than the wild types (n = 85), with a median polyp-free interval of 15 months compared to 26 months [P < 0.001; hazard ratio (HR) = 4.9; 95% confidence interval (CI) = 1.9-12.8]. Furthermore, a multicenter cohort composed by 321 SPs verified that ERBB2-mutated SPs had increased risks of polyp recurrence (P < 0.001; HR = 3.7; 95% CI = 2.3-6.0) and advanced neoplastic lesion (ANL) recurrence (P < 0.001; HR = 10.0; 95% CI = 2.7-36.9) compared with wild-type SPs, respectively. Conclusions: Our results are emphasizing that SP individuals with ERBB2 mutants are at higher risks of subsequent colorectal neoplasms. ERBB2 mutants might work as facilitated markers for prediction of high-risk SPs and might implicate a potential mechanism in the serrated pathway to colorectal carcinoma (CRC).

Lactobacillus johnsonii alleviates colitis by TLR1/2-STAT3 mediated CD206+ macrophagesIL-10 activation.

Imbalance of gut microbiota homeostasis is related to the occurrence of ulcerative colitis (UC), and probiotics are thought to modulate immune microenvironment and repair barrier function. Here, in order to reveal the interaction between UC and gut microbiota, we screened a new probiotic strain by 16S rRNA sequencing from Dextran Sulfate Sodium (DSS)-induced colitis mice, and explored the mechanism and clinical relevance. Lactobacillus johnsonii (L. johnsonii), as a potential anti-inflammatory bacterium was decreased colonization in colitis mice. Gavage L. johnsonii could alleviate colitis by specifically increasing the proportion of intestinal macrophages and the secretion of Il-10 with macrophages depleted model and in Il10-/- mice. We identified this subset of immune cells activated by L. johnsonii as CD206+ macrophagesIL-10. Mechanistically, L. johnsonii supplementation enhanced the mobilization of CD206+ macrophagesIL-10 through the activation of STAT3 in vivo and in vitro. In addition, we revealed that TLR1/2 was essential for the activation of STAT3 and the recognition of L. johnsonii by macrophages. Clinically, there was positive correlation between the abundance of L. johnsonii and the expression level of MRC1, IL10 and TLR1/2 in UC tissues. L. johnsonii could activate native macrophages into CD206+ macrophages and release IL-10 through TLR1/2-STAT3 pathway to relieve experimental colitis. L. johnsonii may serve as an immunomodulator and anti-inflammatory therapeutic target for UC.