Dietary Berberine and Ellagic Acid Supplementation Improve Growth Performance and Intestinal Damage by Regulating the Structural Function of Gut Microbiota and SCFAs in Weaned Piglets.

Early weaning is an effective method for improving the utilization rate of sows in intensive pig farms. However, weaning stress induces diarrhea and intestinal damage in piglets. Berberine (BBR) is known for its anti-diarrhea properties and ellagic acid (EA) is known for its antioxidant properties, however, whether their combination improves diarrhea and intestinal damage in piglets has not been studied, and the mechanism remains unclear. To explore the combined effects in this experiment, a total of 63 weaned piglets (Landrace × Yorkshire) were divided into three groups at 21 days. Piglets in the Ctrl group were treated with a basal diet and 2 mL saline orally, while those in the BE group were treated with a basal diet supplemented with 10 mg/kg (BW) BBR, 10 mg/kg (BW) EA, and 2 mL saline orally. Piglets in the FBE group were treated with a basal diet and 2 mL fecal microbiota suspension from the BE group orally, respectively, for 14 days. Compared with the Ctrl group, dietary supplementation with BE improved growth performance by increasing the average daily gain and average daily food intake and reducing the fecal score in weaned piglets. Dietary supplementation with BE also improved intestinal morphology and cell apoptosis by increasing the ratio of villus height to crypt depth and decreasing the average optical density of apoptotic cells; meanwhile, improvements also involved attenuating oxidative stress and intestinal barrier dysfunction by increasing the total antioxidant capacity, glutathione, and catalase, and upregulating the mRNA expressions of Occludin, Claudin-1, and ZO-1. Interestingly, the oral administration of a fecal microbiota suspension to piglets fed BE had similar effects to those of the BE group. According to 16S rDNA sequencing analysis, dietary supplementation with BE altered the composition of the microbiota, including firmicutes, bacteroidetes, lactobacillus, phascolarctobacterium, and parabacteroides, and increased the metabolites of propionate and butyrate. In addition, Spearman analysis revealed that improvements in growth performance and intestinal damage were significantly correlated with differential bacteria and short-chain fatty acids (SCFAs). In brief, dietary supplementation with BE improved the growth performance and intestinal damage by altering the gut microbiota composition and SCFAs in weaned piglets.

Rice bran oil supplementation protects swine weanlings against diarrhea and lipopolysaccharide challenge. / æ—¥ç²®æ·»åŠ ç±³ç³ æ²¹å¯å¢žå¼ºæ–­å¥¶ä»”çŒªæŠµæŠ—è ¹æ³»å’Œè„‚å¤šç³–åº”æ¿€.

Early weaned piglets suffer from oxidative stress and enteral infection, which usually results in gut microbial dysbiosis, serve diarrhea, and even death. Rice bran oil (RBO), a polyphenol-enriched by-product of rice processing, has been shown to have antioxidant and anti-inflammatory properties both in vivo and in vitro. Here, we ascertained the proper RBO supplementation level, and subsequently determined its effects on lipopolysaccharide (LPS)-induced intestinal dysfunction in weaned piglets. A total of 168 piglets were randomly allocated into four groups of seven replicates (42 piglets each group, (21±1) d of age, body weight (7.60±0.04) kg, and half males and half females) and were given basal diet (Ctrl) or basal diet supplemented with 0.01% (mass fraction) RBO (RBO1), 0.02% RBO (RBO2), or 0.03% RBO (RBO3) for 21 d. Then, seven piglets from the Ctrl and the RBO were treated with LPS (100 µg/kg body weight (BW)) as LPS group and RBO+LPS group, respectively. Meanwhile, seven piglets from the Ctrl were treated with the saline vehicle (Ctrl group). Four hours later, all treated piglets were sacrificed for taking samples of plasma, jejunum tissues, and feces. The results showed that 0.02% was the optimal dose of dietary RBO supplementation based on diarrhea, average daily gain, and average daily feed intake indices in early weaning piglets. Furthermore, RBO protected piglets against LPS-induced jejunal epithelium damage, which was indicated by the increases in villus height, villus height/crypt depth ratio, and Claudin-1 levels, as well as a decreased level of jejunal epithelium apoptosis. RBO also improved the antioxidant ability of LPS-challenged piglets, which was indicated by the elevated concentrations of catalase and superoxide dismutase, and increased total antioxidant capacity, as well as the decreased concentrations of diamine oxidase and malondialdehyde in plasma. Meanwhile, RBO improved the immune function of LPS-challenged weaned piglets, which was indicated by elevated immunoglobulin A (IgA), IgM, ß||-defensin-1, and lysozyme levels in the plasma. In addition, RBO supplementation improved the LPS challenge-induced dysbiosis of gut microbiota. Particularly, the indices of antioxidant capacity, intestinal damage, and immunity were significantly associated with the RBO-regulated gut microbiota. These findings suggested that 0.02% RBO is a suitable dose to protect against LPS-induced intestinal damage, oxidative stress, and jejunal microbiota dysbiosis in early weaned piglets.

Multi-omics analysis reveals gut microbiota-ovary axis contributed to the follicular development difference between Meishan and Landrace × Yorkshire sows.

BACKGROUND: The mechanism by which Meishan (MS) sows are superior to white crossbred sows in ovarian follicle development remains unclear. Given gut microbiota could regulate female ovarian function and reproductive capacity, this study aimed to determine the role of gut microbiota-ovary axis on follicular development in sows. METHODS: We compared the ovarian follicular development, gut microbiota, plasma metabolome, and follicular fluid metabolome between MS and Landrace × Yorkshire (L × Y) sows. A H2O2-induced cell apoptosis model was used to evaluate the effects of multi-omics identified metabolites on the apoptosis of porcine ovarian granulosa cells in vitro. RESULTS: Compared with L × Y sows, MS sows have greater ovary weight and improved follicular development, including the greater counts of large follicles of diameter ≥ 5 mm, secondary follicles, and antral follicles, but lesser atretic follicles. The ovarian granulosa cells in MS sows had alleviated apoptosis, which was indicated by the increased BCL-2, decreased caspases-3, and decreased cleaved caspases-3 than in L × Y sows. The ovarian follicular fluid of MS sows had higher concentrations of estradiol, progesterone, follicle-stimulating hormone, luteinizing hormone, and insulin like growth factor 1 than L × Y sows. Gut microbiota of MS sows formed a distinct cluster and had improved alpha diversity, including increased Shannon and decreased Simpson than those of L × Y sows. Corresponding to the enhanced function of carbohydrate metabolism and elevated short-chain fatty acids (SCFAs) in feces, the differential metabolites in plasma between MS and L × Y sows are also mainly enriched in pathways of fatty acid metabolism. There were significant correlations among SCFAs with follicular development, ovarian granulosa cells apoptosis, and follicular fluid hormones, respectively. Noteworthily, compared with L × Y sows, MS sows had higher follicular fluid SCFAs concentrations which could ameliorate H2O2-induced porcine granulosa cells apoptosis in vitro. CONCLUSION: MS sows have more secondary and antral follicles, but fewer atretic follicles and apoptotic ovarian granulosa cells, as well as harbored a distinctive gut microbiota than L × Y sows. Gut microbiota may participate in regulating ovarian follicular development via SCFAs affecting granulosa cells apoptosis in sows.

Dietary ellagic acid supplementation attenuates intestinal damage and oxidative stress by regulating gut microbiota in weanling piglets.

Intestinal oxidative stress triggers gut microbiota dysbiosis, which is involved in the etiology of post-weaning diarrhea and enteric infections. Ellagic acid (EA) can potentially serve as an antioxidant supplement to facilitate weaning transition by improving intestinal oxidative stress and gut microbiota dysbiosis. Therefore, we aimed to investigate the effects of dietary EA supplementation on the attenuation of intestinal damage, oxidative stress, and dysbiosis of gut microbiota in weanling piglets. A total of 126 piglets were randomly assigned into 3 groups and treated with a basal diet and 2 mL saline orally (Ctrl group), or the basal diet supplemented with 0.1% EA and 2 mL saline orally (EA group), or the basal diet and 2 mL fecal microbiota suspension from the EA group orally (FEA group), respectively, for 14 d. Compared with the Ctrl group, EA group improved growth performance by increasing average daily feed intake and average daily weight gain (P < 0.05) and decreasing fecal scores (P < 0.05). EA group also alleviated intestinal damage by increasing the tight junction protein occludin (P < 0.05), villus height, and villus height-to-crypt depth ratio (P < 0.05), while decreasing intestinal epithelial apoptosis (P < 0.05). Additionally, EA group enhanced the jejunum antioxidant capacity by increasing the total antioxidant capacity (P < 0.01), catalase (P < 0.05), and glutathione/oxidized glutathione (P < 0.05), but decreased the oxidative metabolite malondialdehyde (P < 0.05) compared to the Ctrl group. Compared with the Ctrl group, EA and FEA groups increased alpha diversity (P < 0.05), enriched beneficial bacteria (Ruminococcaceae and Clostridium ramosum), and increased metabolites short-chain fatty acids (P < 0.05). Correspondingly, FEA group gained effects comparable to those of EA group on growth performance, intestinal damage, and intestinal antioxidant capacity. In addition, the relative abundance of bacteria shifted in EA and FEA groups was significantly related to the examined indices (P < 0.05). Overall, dietary EA supplementation could improve growth performance and attenuate intestinal damage and oxidative stress by regulating the gut microbiota in weanling piglets.

Berberine Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis and Inhibits the Secretion of Gut Lysozyme via Promoting Autophagy.

Ulcerative colitis (UC) is one of the primary types of inflammatory bowel disease, the occurrence of which has been increasing worldwide. Research in recent years has found that the level of lysozyme in the feces and blood of UC patients is abnormally elevated, and the bacterial product after the action of lysozyme can be used as an agonist to recognize different cell pattern receptors, thus regulating the process of intestinal inflammation. Berberine (BBR), as a clinical anti-diarrhea and anti-inflammatory drug, has been used in China for hundreds of years. In this study, results showed that BBR can significantly inhibit the expression and secretion of lysozyme in mice. Therefore, we try to investigate the mechanism behind it and elucidate the new anti-inflammatory mechanism of BBR. In vitro, lipopolysaccharide (LPS) was used to establish an inflammatory cell model, and transcriptomic was used to analyze the differentially expressed genes (DEGs) between the LPS group and the LPS + BBR treatment group. In vivo, dextran sulfate sodium salt (DSS) was used to establish a UC mice model, and histologic section and immunofluorescence trails were used to estimate the effect of BBR on UC mice and the expression of lysozyme in Paneth cells. Research results showed that BBR can inhibit the expression and secretion of lysozyme by promoting autophagy via the AMPK/MTOR/ULK1 pathway, and BBR promotes the maturation and expression of lysosomes. Accordingly, we conclude that inhibiting the expression and secretion of intestinal lysozyme is a new anti-inflammatory mechanism of BBR.

Dietary Quercetin Supplementation Attenuates Diarrhea and Intestinal Damage by Regulating Gut Microbiota in Weanling Piglets.

Antioxidant polyphenols from plants are potential dietary supplementation to alleviate early weaning-induced intestinal disorders in piglets. Recent evidences showed polyphenol quercetin could reshape gut microbiota when it functioned as anti-inflammation or antioxidation agents in rodent models. However, the effect of dietary quercetin supplementation on intestinal disorders and gut microbiota of weanling piglets, along with the role of gut microbiota in this effect, both remain unclear. Here, we determined the quercetin's effect on attenuating diarrhea, intestinal damage, and redox imbalance, as well as the role of gut microbiota by transferring the quercetin-treated fecal microbiota to the recipient piglets. The results showed that dietary quercetin supplementation decreased piglets' fecal scores improved intestinal damage by increasing tight junction protein occludin, villus height, and villus height/crypt depth ratio but decreased crypt depth and intestinal epithelial apoptosis (TUNEL staining). Quercetin also increased antioxidant capacity indices, including total antioxidant capacity, catalase, and glutathione/oxidized glutathione disulfide but decreased oxidative metabolite malondialdehyde in the jejunum tissue. Fecal microbiota transplantation (FMT) from quercetin-treated piglets had comparable effects on improving intestinal damage and antioxidative capacity than dietary quercetin supplementation. Further analysis of gut microbiota using 16S rDNA sequencing showed that dietary quercetin supplementation or FMT shifted the structure and increased the diversity of gut microbiota. Especially, anaerobic trait and carbohydrate metabolism functions of gut microbiota were enriched after dietary quercetin supplementation and FMT, which may owe to the increased antioxidative capacity of intestine. Quercetin increased the relative abundances of Fibrobacteres, Akkermansia muciniphila, Clostridium butyricum, Clostridium celatum, and Prevotella copri but decreased the relative abundances of Proteobacteria, Lactobacillus coleohominis, and Ruminococcus bromii. Besides, quercetin-shifted bacteria and carbohydrate metabolites short chain fatty acids were significantly related to the indices of antioxidant capacity and intestinal integrity. Overall, dietary quercetin supplementation attenuated diarrhea and intestinal damage by enhancing the antioxidant capacity and regulating gut microbial structure and metabolism in piglets.

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature-supporting protein LITAF.

Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes. Here, we identify the monotopic membrane protein LPS-induced TNF-activating factor (LITAF) and the related protein cell death involved p53 target 1 (CDIP1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC class I and CD59. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. Meanwhile, co-depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF and CDIP1 in membrane scission. Consistent with this, co-depletion of LITAF and CDIP1 impairs integrin recycling and cell migration.

Gut microbiota contributes to the development of endometrial glands in gilts during the ovary-dependent period.

BACKGROUND: The hyper-prolificacy Meishan gilts achieved a superior endometrial gland development (EGD) than white crossbred gilts during the ovary-independent period (before 60 d of age). Then, the EGD continues under the management of ovary-derived steroid hormones that regulated by gut microbiota (after 60 d of age). However, whether Meishan gilts' superiority in EGD lasting to the ovary-dependent period (after 60 d of age) and the role of gut microbiota in this period both remain unclear. METHODS: Meishan gilts and Landrace x Yorkshire (LxY) gilts were raised under the same housing and feeding conditions until sexual maturity and then we compared their EGD and gut microbiota. Meanwhile, we transplanted fecal microbiota from Meishan gilts to L×Y gilts to explore the role of gut microbiota in EGD. We sampled plasma every 3 weeks and collected the uterus, ovary, liver, and rectal feces after the sacrifice. We then determined the hormone concentrations and expressions of the EGD-related genes. We also profiled the gut microbiota using 16S rDNA sequencing and metabolites of plasma and liver tissue using untargeted metabolomics. Finally, the correlation analysis and significant test was conducted between FMT-shifted gut microbes and EGD-related indices. RESULTS: Meishan gilts have larger endometrial gland area (P < 0.001), longer uterine horn length (P < 0.01) but lighter uterine horn weight (P < 0.05), a distinctive gut microbiota compared with L×Y gilts. Fecal microbiota transplantation (FMT) increased endometrial gland area (P < 0.01). FMT markedly shifted the metabolite profiles of both liver and plasma, and these differential metabolites enriched in steroid hormone biosynthesis pathway. FMT increased estradiol and insulin-like growth factor 1 but decreased progesterone dynamically. FMT also increased the expression of the EGD-related genes estrogen receptor 1 gene, epithelial cadherin, and forkhead box protein A2. There is a significant correlation between FMT-shifted gut microbes and EGD-related indices. CONCLUSION: Sexually matured Meishan gilts achieved a superior EGD than LxY gilts. Meanwhile, gut microbiota contribute to the EGD potentially via regulating of steroid hormones during the ovary-dependent period.

Dietary glycyl-glutamine supplementation ameliorates intestinal integrity, inflammatory response, and oxidative status in association with the gut microbiota in LPS-challenged piglets.

During weaning transition, mammalian newborns suffer severe enteric infections and thus induced gut microbiota dysbiosis, which in turn aggravates enteric disorder. The synthetic dipeptide glycyl-glutamine (GlyGln) has been used as a diet supplement to improve the weaning transition of newborns. However, the effect of dietary GlyGln supplementation on the gut microbiota of piglets with enteric infection remains unclear. Here, weaned piglets received a basal diet or a basal diet supplemented with 0.25% GlyGln for 3 weeks. Five piglets in each group received an intraperitoneal injection of lipopolysaccharide (LPS) (100 µg per kg BW) (LPS and GlyGln + LPS groups) and meanwhile five piglets in a control group received an intraperitoneal injection of saline (Ctrl group). The results showed that dietary GlyGln supplementation improved the LPS induced inflammation response and damage to the ileum morphology by increasing interleukin 10, tight junction proteins, villus height, and the ratio villus height/crypt depth, but decreasing the crypt depth. For the oxidative status, dietary GlyGln supplementation increased the ileal superoxide dismutase and meanwhile reduced the malondialdehyde and nitric oxide synthase activity (NOS) (total NOS and inducible NOS), compared with that in the LPS group. LPS challenge reduced the diversity of gut microbiota and enriched the facultative anaerobic Escherichia coli. The GlyGln restored alpha diversity and the structure of the gut microbiota by enriching obligate anaerobes and short-chain fatty acid (SCFA)-producing bacteria, including Clostridium, Lachnospira, Phascolarctobacterium, Roseburia, Lachnospiraceae, and Synergistetes. GlyGln enriched the gut microbiota function of carbohydrate metabolism and elevated the ileal SCFA concentrations of propionic acid and butyric acid that had been decreased by the LPS challenge. The beneficial effects of dietary GlyGln supplementation are closely associated with its enriched bacteria and SCFAs. Taken together, dietary GlyGln supplementation improved the gut microbiota dysbiosis induced by LPS challenge and enriched obligate anaerobes and SCFA-producing bacteria, which contributed to the amelioration of intestinal integrity, inflammatory responses, and oxidative status.

Protective Effects of Sodium Para-aminosalicylic Acid on Manganese-Induced Damage in Rat Pancreas.

Sodium p-aminosalicylic acid (PAS-Na) has been previously shown to protect the brain from manganese (Mn)-induced toxicity. However, the efficacy of PAS-Na in protecting other organs from Mn toxicity and the mechanisms associated with this protection have yet to be addressed. Therefore, here, we assessed pancreatic damage in response to Mn treatment and the efficacy of PAS-Na in limiting this effect, along with specific mechanisms that mediate PAS-Na's protection. Mn exposure led to increased blood Mn content in dose- and time-dependent manner. Furthermore, subchronic Mn exposure (20 mg/kg for 8 weeks) led to pancreatic damage in a dose-dependent manner. In addition, the elevated Mn levels increased iron and decreased zinc and magnesium content in the pancreas. These effects were noted even 8 weeks after Mn exposure cessation. Mn exposure also affected the levels of amylase, lipase, and inflammatory factors such as tumor necrosis factor (TNF-α) and interleukin-1 ß (IL-1ß). PAS-Na significantly inhibited the increase in Mn concentration in both blood and pancreas, restored Mn-induced pancreatic damage, reversed the Mn-induced alterations in metal levels, and restored amylase and lipase concentrations. Taken together, we conclude that in rats, PAS-Na shows pharmacological efficacy in protecting the pancreas from Mn-induced damage.

Anti­inflammatory mechanism of berberine on lipopolysaccharide­induced IEC­18 models based on comparative transcriptomics.

Intestinal surface epithelial cells (IECs) have long been considered as an effective barrier for maintaining water and electrolyte balance, and are involved in the mechanism of nutrient absorption. When intestinal inflammation occurs, it is often accompanied by IEC malfunction. Berberine (BBR) is an isoquinoline alkaloid found in numerous types of medicinal plants, which has been clinically used in China to treat symptoms of gastrointestinal pathogenic bacterial infection, especially bacteria­induced diarrhea and inflammation. In the present study, IEC­18 rat intestinal epithelial cells were treated with lipopolysaccharide (LPS) to establish an in vitro model of epithelial cell inflammation, and the cells were subsequently treated with BBR in order to elucidate the anti­inflammatory mechanism. Transcriptome data were then searched to find the differentially expressed genes (DEGs) compared between two of the treatment groups (namely, the LPS and LPS+BBR groups), and DEGs were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Weighted Gene Correlation Network Analysis and Interactive Pathways Explorer to identify the functions and pathways enriched with DEGs. Finally, reverse transcription­quantitative PCR was used to verify the transcriptome data. These experiments revealed that, comparing between the LPS and LPS+BBR groups, the functions and pathways enriched in DEGs were 'DNA replication', 'cell cycle', 'apoptosis', 'leukocyte migration' and the 'NF­κB and AP­1 pathways'. The results revealed that BBR is able to restrict DNA replication, inhibit the cell cycle and promote apoptosis. It can also inhibit the classic inflammatory pathways, such as those mediated by NF­κB and AP­1, and the expression of various chemokines to prevent the migration of leukocytes. According to transcriptomic data, BBR can exert its anti­inflammatory effects by regulating a variety of cellular physiological activities, including cell cycle, apoptosis, inflammatory pathways and leukocyte migration.

Correction: Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma.

Glypican-3 (GPC3) is an attractive target for chimeric antigen receptor (CAR)-T cell therapy, as it is overexpressed in most hepatocellular carcinoma (HCC) tissues but shows restricted expression in healthy adult tissues. Herein, we generated GPC3-specific CAR-T cells for HCC therapy by electroporation with plasmid DNA encoding the piggyBac (PB) transposon and the hyperactive piggyBac transposase simultaneously instead of by commonly-used viral vectors. Our results demonstrated that GPC3CAR gene was efficiently integrated into the genome of T cells utilizing the PB transposon system. Upon stimulation with GPC3 antigen, GPC3CAR-T cells could be effectively activated, proliferate strongly and secrete high levels of cytokines. It also was demonstrated that GPC3CAR-T cells displayed potent cytotoxicity against GPC3-positive HCC cell lines in vitro by using real-time cell analyser (RTCA) system and the JuLI™ Stage Cell History Recorder. More importantly, in a Huh-7 xenograft mouse model, GPC3CAR-T cells significantly reduced the tumour burden companied with the secretion of high levels of IFN-γ. Moreover, T cells in mice treated with GPC3CAR-T cells could infiltrate into tumour tissues and persist as effector memory T cells (TEM). Overall, our study suggests that the use of PB system-based GPC3CAR-T cell therapy could be a promising clinical strategy for patients with HCC.

Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor.

Mesothelin (MSLN) is an attractive antigen for chimeric antigen receptor (CAR) T therapy and the epitope selection within MSLN is essential. In this study, we constructed two types of CARs targeting either region I of MSLN (meso1 CAR, also known as a membrane-distal region) or region III of MSLN (meso3 CAR, also known as a membrane-proximal region) using a modified piggyBac transposon system. We reported that, compared with meso1 CAR T cells, meso3 CAR T cells express higher levels of CD107α upon activation and produce increased levels of interleukin-2, TNF-α, and IFN-γ against multiple MSLN-expressing cancer cells in vitro. In a real-time cell analyzer system and a three-dimensional spheroid cancer cell model, we also demonstrated that meso3 CAR T cells display an enhanced killing effect compared with that of meso1 CAR T cells. More importantly, in a gastric cancer NSG mice model, meso3 CAR T cells mediated stronger antitumor responses than meso1 CAR T cells did. We further identified that meso3 CAR T cells can effectively inhibit the growth of large ovarian tumors in vivo. Collectively, our study provides evidences that meso3 CAR T-cell therapy performs as a better immunotherapy than meso1 CAR T-cell therapy in treating MSLN-positive solid tumors.

Preventive impacts of PAS-Na on the slow growth and activated inflammatory responses in Mn-exposed rats.

BACKGROUND: Sodium para-aminosalicylic acid (PAS-Na), an anti-tuberculosis drug, has been demonstrated its function in facilitating the Mn elimination in manganism patients and Mn-exposed models in vivo and improving the symptoms of Mn poisoning. But whether it can improve the growth retardation and inflammatory responses induced by Mn have not been reported. OBJECTIVES: This study was designed to investigate the preventive effects of PAS-Na on the development of retardation and inflammatory responses in Mn-exposed rats. METHODS: Male Sprague Dawley (SD) rats (8 weeks old, weighing 180 ± 20 g) were randomly divided into normal control group and Mn-exposed group in the 4 weeks experiment observation and normal control group, Mn-exposed group, PAS-Na preventive group and PAS-Na control group in the 8 weeks experiment observation. The Mn-exposed group received an intraperitoneal injection (i.p.) of 15 mg/kg MnCl2 and the normal control group i.p. physiological Saline in the same volume once a day for 4 or 8 weeks, 5 days per week. The PAS-Na preventive group i.p. 15 mg/kg MnCl2 along with back subcutaneous (s.c.) injection of 240 mg/kg PAS-Na once a day for 8 weeks, 5 days per week. PAS-Na control group received s.c. injection of 240 mg/kg PAS-Na along with i.p. injection of saline once daily. The body weight was determined once a week until the end of the experiment. The manganese contents in the blood were detected by graphite furnace atomic absorption spectrometry. The inflammatory factor levels (TNF-α, IL-1ß, IL-6, and PGE2) in the blood were detected by using enzyme-linked immunosorbent assay (Elisa) and each organ taking from rats were weighed and recorded. RESULTS: Mn exposure significantly suppressed the growth in rats and increased heart, liver, spleen and kidney coefficients as compared with the control group. The whole blood Mn level and serum levels of IL-1ß, IL-6, PGE2, and TNF-α in sub-chronic Mn-exposure group were markedly higher than those in the control group. However, preventive treatment with PAS-Na obviously reduced the whole blood Mn level, the spleen and liver coefficients of the Mn-exposed rats. And serum levels of IL-1ß and TNF-α were significantly reduced by 33.9% and 14.7% respectively in PAS-Na prevention group. CONCLUSIONS: PAS-Na could improve the growth retardation and alleviate inflammatory responses in Mn-exposed rats.

Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer.

Patients with pancreatic cancer have a poor prognosis largely due to the poor efficacy of the available treatment modalities. In this study, we engineered mesothelin-targeting chimeric antigen receptor T cells (mesoCAR T) using the piggyBac transposon based plasmid electroporation technique for specific targeting of pancreatic cancer cells expressing mesothelin. In vitro, mesoCAR T cells exhibited rapid and robust killing effect against ASPC1 cells with high expression levels of mesothelin with high production of IFN-γ; the cytotoxic effect on PANC1 cells with low expressions of mesothelin was relatively attenuated. In the ASPC1 xenograft mice model, mesoCAR T cells significantly suppressed the tumor growth accompanied with higher-level IFN-γ secretion as compared to control T cells. Besides, more mesoCAR T cells differentiated into memory T cells after tumor remission, whilst causing minimal lesions in major organs. Our study suggests promising efficacy of piggyBac transposon-based mesoCAR T cell therapy for pancreatic cancer, which is a potential candidate for clinical translation.

Sodium P-aminosalicylic acid inhibits sub-chronic manganese-induced neuroinflammation in rats by modulating MAPK and COX-2.

Excessive manganese (Mn) accumulation in the brain may induce an extrapyramidal disorder known as manganism. Inflammatory processes play a critical role in neurodegenerative diseases. Therapeutically, non-steroidal anti-inflammatory drugs or analogous anti-inflammatory therapies have neuroprotective effects. As a non-steroidal anti-inflammatory drug, p-aminosalicylic acid (PAS) has anti-inflammatory effects, which are mediated by decreased prostaglandins E2 (PGE2) levels. The aim of the current study was to investigate whether PAS-Na treatment prevents Mn-induced behavioral changes and neuroinflammation in vivo. Male Sprague-Dawley rats were intraperitoneally (i.p.) injected with MnCl2·4H2O (15mg/kg) for 12 weeks, followed by 6 weeks PAS-Na treatment. Sub-chronic Mn exposure increased Mn levels in the whole blood, cortex, hippocampus and thalamus, and induced learning and memory deficits, concomitant with astrocytes activation in the cortex, hippocampus and thalamus. Moreover inflammatory cytokine levels in serum and brain of Mn-treated group were increased, including IL-1ß, IL-6, TNF-αand PGE2, especially in the hippocampus and thalamus. Furthermore, sub-chronic Mn exposure also increased inflammatory cytokines and COX-2 in transcription levels concomitant with increased MAPK signaling and COX-2 in the same selected brain regions. PAS-Na treatment at the highest doses also decreased Mn levels in the whole blood and selected brain tissues, and reversed the Mn-induced learning and memory deficits. PAS-Na inhibited astrocyte activation as well as the Mn-induced increase in inflammatory cytokine levels, reducing p38, ERK MAPK pathway and COX-2 activity. In contrast PAS-Na had no effects on the JNK MAPK pathway. These data establish the efficacy of PAS-Na not only as a chelating agent to mobilize whole blood Mn, but also as an anti-inflammatory agent.

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein.

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61ß, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.

[Determination of Arsenic in Food Package Aluminum by Ultrasound Assisted Solid Phase Extraction/ICP-AES].

Determination of arsenic in pure aluminum by inductively coupled plasma atomic emission spectrometry was interfered by aluminum matrix. The experiment showed that when the mass concentration of Al was greater than or equal to 5 000 times the As in the test solution, the measurement error was greater than 5%. In order to eliminate the interference, strong acid cation exchange fiber (SACEF) was used as solid phase extraction agent to adsorb Al(3+). The extraction conditions included amount of SACEF, extraction time, temperature and pH were investigated. The optimal extraction conditions were that 0.9000 g SACEF was used to extract the aluminum from the sample solution of pH 2.0 at 55 °C for 5 min with the ultrasonic assist, and in this case, the arsenic in the form of arsenic acid was not extracted and left in the solution for the determination. The results showed that after treating 10. 00 mL test solution containing 1.00 µg arsenic and 20.0 mg aluminum, arsenic did not lose. The mass concentration of residual aluminum in the raffinate was about 2,000 times the As, which had not interfered the determination of arsenic. The detection limit (3 s) was 0.027 µg · mL(-1) and quantification limit (10 s) was 0.0091 µg · mL(-1). The proposed method was successfully applied to the separation and determination of arsenic in the synthetic samples, the aluminum cans and the barbecue aluminum foil. Recovery was in the range of 98.3%-105% and RSD (n = 3) was in the range of 0.1%-4.3%. The results showed that the content of arsenic in the aluminum cans and the aluminum barbecue foil was below the limited value of national standard (GB/T 3190-2008).

Effect of conventional interferon-α in patients with HBeAg-positive chronic hepatitis B: a systematic review and meta-analysis.

BACKGROUND: Although a few studies have tested the effect of interferon-α on chronic hepatitis B, its treatment effect remains uncertain, and the association of treatment effect with intervention characteristics has not been thoroughly explored. This study examined the effect of IFN-α in patients with HBeAg-positive chronic hepatitis B, and investigated the characteristics associated with treatment effect. METHODS: We searched MEDLINE, Scientific Citation Index, Current Content Connect, Cochrane Controlled Trial Register, and Chinese Biomedical Database, all up to 15 September 2009. We included randomized trials comparing IFN-α to placebo, no treatment, or standard care (SC) in patients with HBeAg-positive chronic hepatitis B. Two reviewers assessed the risk of bias and extracted data, independently and in duplicate. We conducted meta-analyses of the included studies, and subgroup analyses to examine the association of pre-specified characteristics (eg, dose, treatment duration) with treatment effect. RESULTS: A total of 31 randomized controlled trials, involving 2164 patients, were included. The risk of bias varied across studies. Compared with placebo, no treatment, or SC, IFN-α improved loss of HBeAg (OR 2.36, 95% CI 1.83 to 3.04), HBV DNA undetectability (OR 2.04, 95% CI 1.28 to 3.32), HBeAg seroconversion (OR 1.82, 95% CI 1.26 to 2.62), ALT normalization (OR 1.24, 95% CI 1.01 to 1.56), and loss of HBsAg (OR 2.45, 95% CI 1.22 to 4.91). Treatment effects differed in high versus low dose, and long versus short duration of IFN-α. The effect of high dose IFN-α (OR 3.28, 95% CI 2.31 to 4.66) is statistically larger than that of low dose IFN-α (OR 1.58, 95% CI 1.10 to 2.28) on loss of HBeAg (interaction P = 0.017), and longer IFN-α treatment durations produce greater effects (OR 3.28, 95% CI 2.16 to 5.00) than do shorter durations (OR 1.94, 95% CI 0.42 to 2.66, interaction P = 0.038). High dose IFN-α had a significant effect on HBV DNA undetectability (OR 2.80, 95% CI 2.03 to 3.86), while low dose IFN-α did not (OR 0.93, 95% CI 0.61 to 1.41, interaction P = 0.01); longer treatments significantly improved HBV DNA undetectability (OR 2.58, 95% CI 1.62 to 4.12), but shorter durations did not (OR 1.28, 95% CI 0.83 to 1.97, interaction P = 0.024). CONCLUSIONS: IFN-α can improve serological, biomedical, and virological response. Higher doses and prolonged treatments appear to have larger treatment benefits than lower doses and shorter treatments. However, the increased adverse reactions and costs associated with higher doses and prolonged treatment warrant caution in applying these results.