Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.

miR-1246-overexpressing exosomes improve UVB-induced photoaging by activating autophagy via suppressing GSK3ß.

Stem cell paracrine has shown potential application in skin wound repair and photoaging treatment. Our previous study demonstrated that miR-1246-overexpressing Exosomes (OE-EXs) isolated from adipose-derived stem cells (ADSCs) showed superior photo-protecting effects on UVB-induced photoaging than that of the vector, however, the underlying mechanism was unclear. The simultaneous bioinformatics analysis indicated that miR-1246 showed potential binding sites with GSK3ß which acted as a negative regulator for autophagy. This study was aimed to explore whether OE-EXs ameliorate skin photoaging by activating autophagy via targeting GSK3ß. The results demonstrated that OE-EXs significantly decreased GSK3ß expression, enhanced autophagy flux and autophagy-related proteins like LC3II, while suppressed p62 expression. Meanwhile, OE-EXs markedly reversed the levels of intracellular ROS, MMP-1, procollagen type I and DNA damage in human skin fibroblasts caused by UVB irradiation, but the ameliorating effects were significantly inhibited when 3-Methyladenine (3-MA) was introduced to block the autophagy pathway. Further, OE-EXs could reverse UVB-induced wrinkles, epidermal hyperplasia, and collagen fibers reduction in Kunming mice, nevertheless, the therapeutical effects of OE-EXs were attenuated when it was combinative treated with 3-MA. In conclusion, OE-EXs could cure UVB induced skin photoaging by activating autophagy via targeting GSK3ß.

Systemic metabolic engineering of Enterobacter aerogenes for efficient 2,3-butanediol production.

2,3-Butanediol (2,3-BDO) is an important gateway molecule for many chemical derivatives. Currently, microbial production is gradually being recognized as a green and sustainable alternative to petrochemical synthesis, but the titer, yield, and productivity of microbial 2,3-BDO remain suboptimal. Here, we used systemic metabolic engineering strategies to debottleneck the 2,3-BDO production in Enterobacter aerogenes. Firstly, the pyruvate metabolic network was reconstructed by deleting genes for by-product synthesis to improve the flux toward 2,3-BDO synthesis, which resulted in a 90% increase of the product titer. Secondly, the 2,3-BDO productivity of the IAM1183-LPCT/D was increased by 55% due to the heterologous expression of DR1558 which boosted cell resistance to abiotic stress. Thirdly, carbon sources were optimized to further improve the yield of target products. The IAM1183-LPCT/D showed the highest titer of 2,3-BDO from sucrose, 20% higher than that from glucose, and the yield of 2,3-BDO reached 0.49 g/g. Finally, the titer of 2,3-BDO of IAM1183-LPCT/D in a 5-L fermenter reached 22.93 g/L, 85% higher than the wild-type strain, and the titer of by-products except ethanol was very low. KEY POINTS: Deletion of five key genes in E. aerogenes improved 2,3-BDO production The titer of 2,3-BDO was increased by 90% by regulating metabolic flux Response regulator DR1558 was expressed to increase 2,3-BDO productivity.

Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

A united framework modeling of spatial-temporal characteristics for county-level agricultural carbon emission with an application to Hunan in China.

Effectively tackling extreme climate change requires sound knowledge about carbon emissions and their driving forces. Currently, agricultural carbon emission assessment often deals with its inventory, efficiency, determinants, and response independently, which will leave out the complex interactions among its various components, thus there is a lack of comprehensive, scalable, comparable explanations for agricultural carbon emissions. Herein, we introduce an integrated agricultural carbon emission assessment framework (IEDR): Inventory (I) × Efficiency (E) × Determinants (D) × Response (R), which was then applied to an illustration for the county-level agricultural carbon emissions in Hunan Province, China. Results show that: (1) Agricultural carbon emission inventory (ACEI) increased from 20.06 × 106 tC in 2006 to 21.99 × 106 tC in 2014 and decreased to 19.07 × 106 tC by 2020, depicting a fluctuating trend. Meanwhile, there was remarkable spatial heterogeneity, with higher ACEI in the North and South than in the East and West. (2) Agricultural carbon emission efficiency (ACEE) increased from 0.8520 in 2006 to 0.8992 in 2020, depicting a growing trend driven by technological progress. Spatially distributed in contrast to ACEI, regions with higher ACEE were located in the eastern and western areas. (3) ACEI was negatively correlated with ACEE (-0.657), indicating that increasing ACEE is a key strategy for reducing emissions. (4) The natural environment, rural development level, and policy support had critical impacts on ACEE and ACEI. In particular, the cultivated area and rural water affairs development were significant influences on ACEE and ACEI. Given the externalities of carbon emissions and its important public goods characteristics of the atmosphere, local carbon issues are also global concerns. Therefore, the case study of the IEDR model not only validates this theoretical paradigm and realizes regional responsibility for global carbon reduction but also supports and expands the theoretical and empirical corpus in the field of agricultural carbon emissions and efficiency, providing insights and references for other global regions facing similar challenges.

All-fiber orthogonal-polarized white-noise-modulated laser for short-coherence dynamic interferometry.

An all-fiber orthogonal-polarized white-noise-modulated laser (AOWL) for short-coherence dynamic interferometry is proposed. Short-coherence laser is achieved by current modulating of a laser diode with the band-limited white noise. A pair of orthogonal-polarized lights with adjustable delay for short-coherence dynamic interferometry are output by the all-fiber structure. In the non-common-path interferometry, the AOWL can significantly suppress the interference signal clutter with 73% side lobe suppression ratio, that improves the positioning accuracy of zero optical path difference. The wavefront aberrations of a parallel plate are measured with the AOWL in the common-path dynamic interferometers, avoiding the fringe crosstalk.

TfOH-Catalyzed Regioselective S-H Insertion of Cyclic Thioamide Derivatives with Diazo Compounds at Room Temperature.

We have developed a method for highly regioselective S-H bond insertion reactions of various diazo compounds and cyclic thioamide derivatives at room temperature. These reactions provide straightforward access to alkylated benzimidazoles, benzothiazoles, and benzoxazoles. This mild method uses readily available TfOH as a catalyst and features a broad substrate scope, good functional group tolerance, good to excellent yields, and high regioselectivities.

Machine learning-based models for predicting mortality and acute kidney injury in critical pulmonary embolism.

OBJECTIVES: We aimed to use machine learning (ML) algorithms to risk stratify the prognosis of critical pulmonary embolism (PE). MATERIAL AND METHODS: In total, 1229 patients were obtained from MIMIC-IV database. Main outcomes were set as all-cause mortality within 30 days. Logistic regression (LR) and simplified eXtreme gradient boosting (XGBoost) were applied for model constructions. We chose the final models based on their matching degree with data. To simplify the model and increase its usefulness, finally simplified models were built based on the most important 8 variables. Discrimination and calibration were exploited to evaluate the prediction ability. We stratified the risk groups based on risk estimate deciles. RESULTS: The simplified XGB model performed better in model discrimination, which AUC were 0.82 (95% CI: 0.78-0.87) in the validation cohort, compared with the AUC of simplified LR model (0.75 [95% CI: 0.69-0.80]). And XGB performed better than sPESI in the validation cohort. A new risk-classification based on XGB could accurately predict low-risk of mortality, and had high consistency with acknowledged risk scores. CONCLUSIONS: ML models can accurately predict the 30-day mortality of critical PE patients, which could further be used to reduce the burden of ICU stay, decrease the mortality and improve the quality of life for critical PE patients.

Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells.

Repetitive low-level blast (rLLB) exposure is a potential risk factor for the health of soldiers or workers who are exposed to it as an occupational characteristic. Alveolar macrophages (AMs) are susceptible to external blast waves and produce pro-inflammatory or anti-inflammatory effects. However, the effect of rLLB exposure on AMs is still unclear. Here, we generated rLLB waves through a miniature manual Reddy-tube and explored their effects on MH-S cell morphology, phenotype transformation, oxidative stress status, and apoptosis by immunofluorescence, real-time quantitative PCR (qPCR), western blotting (WB) and flow cytometry. Ipatasertib (GDC-0068) or PDTC was used to verify the role of the Akt/NF-κB signaling pathway in these processes. Results showed that rLLB treatment could cause morphological irregularities and cytoskeletal disorders in MH-S cells and promote their polarization to the M1 phenotype by increasing iNOS, CD86 and IL-6 expression. The molecular mechanism is through the Akt/NF-κB signaling pathway. Moreover, we found reactive oxygen species (ROS) burst, Ca2+ accumulation, mitochondrial membrane potential reduction, and early apoptosis of MH-S cells. Taken together, our findings suggest rLLB exposure may cause M1 polarization and early apoptosis of AMs. Fortunately, it is blocked by specific inhibitors GDC-0068 or PDTC. This study provides a new treatment strategy for preventing and alleviating health damage in the occupational population caused by rLLB exposure.

Oxygen-Free Csp3-H Oxidation of Pyridin-2-yl-methanes to Pyridin-2-yl-methanones with Water by Copper Catalysis.

Aromatic ketones are important pharmaceutical intermediates, especially the pyridin-2-yl-methanone motifs. Thus, synthetic methods for these compounds have gained extensive attention in the last few years. Transition metals catalyze the oxidation of Csp3-H for the synthesis of aromatic ketones, which is arresting. Here, we describe an efficient copper-catalyzed synthesis of pyridin-2-yl-methanones from pyridin-2-yl-methanes through a direct Csp3-H oxidation approach with water under mild conditions. Pyridin-2-yl-methanes with aromatic rings, such as substituted benzene, thiophene, thiazole, pyridine, and triazine, undergo the reaction well to obtain the corresponding products in moderate to good yields. Several controlled experiments are operated for the mechanism exploration, indicating that water participates in the oxidation process, and it is the single oxygen source in this transformation. The current work provides new insights for water-involving oxidation reactions.

Reversible Adsorption and Detachment of Saccharomyces cerevisiae on Thermoresponsive Poly(N-isopropylacrylamide)-Grafted Fibers for Continuous Immobilized Fermentation.

Biofilm-mediated continuous fermentation with cells immobilized has gained much attention in recent years. In this study, thermoresponsive poly(N-isopropylacrylamide)-grafted cotton fibers (PNIPAM-CF) were prepared via an improved surface-initiated atom transfer radical polymerization. The modification process imparted switchable wettability to the surface while maintaining the thermal stability and biocompatibility of the CF. During the ethanol transformation, the rapid, reversible cell adsorption and detachment of Saccharomyces cerevisiae were performed through the modulation of wettability, displaying the enhancement of immobilized biomass and immobilization efficiency from 2.20 g/L and 59.43% to 2.81 g/L and 93.32%, respectively. Moreover, the biofilm adsorption matched well with the Freundlich model, indicating that multilayer adhesion was the main mode of biofilm formation. Based on the accumulation of the biofilm, the fabrication and utilization of PNIPAM-CF improved the efficiency of continuous immobilized fermentation, making the ethanol production reach 26.34 g/L in the sixth batch of fermentation. Meanwhile, wettability regulation further enhanced the reusability of the carrier. Therefore, the findings of this study revealed that the application of smart materials in cell immobilization systems had broad prospects for achieving sustainable and continuous catalysis.

Transition-Metal-Free Synthesis of 3-Acyl Chromones by the Tandem Reaction of Ynones and Methyl Salicylates.

A facile and effective tandem reaction of ynones and methyl salicylates was developed to obtain a broad range of 3-acyl chromones in moderate-to-excellent yields. This protocol underwent a Michael addition and cyclization process, which exhibited easily accessible substrates, broad substrate scope, and high regioselectivity under mild and transition-metal-free conditions. Moreover, gram-scale reaction and further chemical transformation of the products were also further studied.

Microbiome-metabolome analysis reveals cervical lesion alterations.

Cervical cancer (CC) continues to be one of the most common cancers among females worldwide. It takes a few years or even decades for CC to arise in a minority of women with cervical precancers. An increasing corpus of studies today indicates that local microecology and carcinogenesis are intimately related. To investigate the changes in cericovaginal microecology with the development of cervical cancer, we performed 16S rDNA sequencing and metabolomic analysis in cericovaginal fluid from 10 LSIL patients, 10 HSIL patients, 10 CC patients and 10 healthy controls to reveal the differential flora and metabolites during cervical carcinogenesis. Carcinogenesis is associated with alterations in microbiome diversity, individual taxa, and functions with notable changes in Lactobacillus, Prevotella and Aquabacterium, as well as in cervicovaginal metabolites that correlate with cervicovaginal microbial patterns. Increased bacterial diversity and a decline in the relative abundance of Lactobacillus, the dominant species in the cericovaginal flora, are observed when cervical lesions advance. According to KEGG pathway enrichment analysis, lipids and organic acids change as cervical cancer progresses, and the phenylalanine, tyrosine, and tryptophan biosynthesis pathway is essential for the development of cervical cancer. Our results reveal that microbic and metabolomic profiling is capable of distinguishing CC from precancer and highlights potential biomarkers for the early detection of cervical dysplasia. These differential microorganisms and metabolites are expected to become a potential tool to assist in the diagnosis of cervical cancer.

[Expression and Role of PD-L1 in a Mouse Model of Necrotizing Enterocolitis].

Objective: To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC). Methods: A total of 20 wild-type C57 BL/6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L1 (+/+) mice and 14 PD-L1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors. Results: The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P<0.05). According to the results of the PD-L1 gene mouse experiment, the control groups of PD-L1 (+/+) mice and PD-L1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L1 (+/+) NEC mouse ( P<0.05). In addition, serum interleukin (IL)-10 in PD-L1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1ß all increased by more than 25% (all P<0.05). Conclusion: PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes.

Chitinase involved in immune regulation by mediated the toll pathway of crustacea Procambarus clarkii.

Chitinase can degrade chitin and play an essential role in animal immunity and plant defense. The immune functions of Chitinase in Procambarus clarkii (P. clarkii) remain to elucidate. Here, we identified PcChitinase 2 gene sequence from P. clarkii and studied its spatial and temporal expression profiles. The PcChitinase 2 transcribed unequally in different tissues; however, its expression was highest in those of stomach, gut, and hepatopancreas. The challenge with lipolysaccharide or peptidoglycan significantly up-regulated the expression of PcChitinase 2 in hepatopancreas. The knockdown of the PcChitinase 2 gene by double-stranded RNA suppressed most of the Toll-pathway-related immune genes (phospholipase, lectin, sptazle Cactus, serine proteikinase, anti-lipopolysaccharide factor, and Toll) production were significantly increased. Our results suggest PcChitinase 2 may be involved in the innate immune responses of P. clarkii by modulating the toll pathway.

Inhibitory effect of resveratrol on the pharmacokinetics of ticagrelor in vivo and in vitro.

This study was to evaluate the effect of resveratrol on the pharmacokinetics of ticagrelor in rats and the metabolism of ticagrelor in human cytochrome P450 (CYP) 3A4 (CYP3A4) and liver microsomes. Eighteen Sprague-Dawley rats were randomly divided into three groups: group A (control group), group B (50 mg/kg resveratrol), and group C (150 mg/kg resveratrol). After 30 min administration of resveratrol, a single dose of ticagrelor (18 mg/kg) was administered orally. The in vitro experiment was performed to examine the influence of resveratrol on ticagrelor metabolism in CYP3A4*1, human, and rat liver microsomes. Serial biological samples were assayed by validated ultra high-performance liquid chromatography - tandem mass spectrometer methods. For the in vivo study, the area under the concentration-time curve and mean peak plasma concentrations of ticagrelor in group B and C appeared to be significantly higher than the control group, while volume of distribution in terminal phase and apparent clearance of ticagrelor in group B and C were significantly decreased. For the in vitro study, resveratrol exhibited an inhibitory effect on CYP3A4*1, human and rat liver microsomes. The half-maximal inhibitory concentration values of resveratrol were 56.75 µM, 69.07 µM, and 14.22 µM, respectively. Our results indicated that resveratrol had an inhibitory effect on the metabolism of ticagrelor in vitro and in vivo. Further research should focus on the clinical combination of resveratrol with ticagrelor, and ticagrelor plasma concentration should be monitored to avoid the occurrence of adverse reaction.

Pronounced Dependence of All-Polymer Solar Cells Photovoltaic Performance on the Alkyl Substituent Patterns in Large Bandgap Polymer Donors.

For all-polymer solar cells which are composed of polymer donors and polymer acceptors, the effect of alkyl side chains on photovoltaic performance is a matter of some debate, and this effect remains difficult to forecast. In this concise contribution, we demonstrate that three alkyls namely branched alkyl 2-butyloctyl (2BO), long linear alkyl n-dodecyl (C12), and double-short linear alkyl n-hexyls (DC6) incorporated into the side chains of large bandgap polymer donor PBDT-TTz can induce considerable, of significance, and different electronic, optical, and morphological parameters. Systematic studies shed light on the critical role of the double-short linear alkyl n-hexyls (DC6) in (i) producing large ionization potential value, (ii) increasing propensity of the polymer to order along the π-stacking direction, (iii) generating polymer crystallites with more preferential "face-on" orientation, consequently, (iv) improvement of carriers transportation, (v) suppression of charge recombination, (vi) reduction of energy loss in all-polymer devices. In parallel, we unearth that the PBDT-TTz with double-short linear alkyl n-hexyls (DC6) represents the highest efficiency of 8.3 %, whereas, the other two PBDT-TTz analogues (2BO, C12) yield efficiencies of less than 3 % in optimized all-polymer solar cells. Though branched or long linear alkyl side chains (2BO, C12) have been applied to provide the solution processability of conjugated polymers, motifs bearing multiple short linear alkyl substituents (DC6) are proved critical to the development of high performing polymers.

6DoF Pose Estimation of Transparent Object from a Single RGB-D Image.

6DoF object pose estimation is a foundation for many important applications, such as robotic grasping, automatic driving, and so on. However, it is very challenging to estimate 6DoF pose of transparent object which is commonly seen in our daily life, because the optical characteristics of transparent material lead to significant depth error which results in false estimation. To solve this problem, a two-stage approach is proposed to estimate 6DoF pose of transparent object from a single RGB-D image. In the first stage, the influence of the depth error is eliminated by transparent segmentation, surface normal recovering, and RANSAC plane estimation. In the second stage, an extended point-cloud representation is presented to accurately and efficiently estimate object pose. As far as we know, it is the first deep learning based approach which focuses on 6DoF pose estimation of transparent objects from a single RGB-D image. Experimental results show that the proposed approach can effectively estimate 6DoF pose of transparent object, and it out-performs the state-of-the-art baselines by a large margin.

Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 - A key enzyme for heparin biosynthesis.

Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with the chaperone expression system (pGro7) was constructed and transferred to E. coli BL21 (DE3) to co-express recombinant strain E. coli BL21 (DE3)/pET-28a-3-OST-1 + pGro7. The soluble expression of 3-OST-1 was significantly improved in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/mL and having a specific activity of 0.83 U/mg. N-sulfo, N-acetylheparosan (NSNAH) was modified by the recombinant expressed 3-OST-1 and the product was confirmed by 1H NMR showing the sulfo group was successfully transferred to NSNAH.

[An Improved Acute Pancreatitis Apoptosis/Necrosis In vitro Model of Acinar Cells].

OBJECTIVE: To improve the apoptosis/necrosis In vitromodel of ascites-induced acute pancreatitic (AP) acinar cells by using the co-culture system and mixed ascites technology for the first time. Furthermore, we compared this improved model with cerulein and cerulein+LPS models, and observed the effects of three models on acinar apoptotic/necrotic-related indicators. METHODS: The In vitrocultured rat acinar cells AR42J were divided in four groups: control group (medium+PBS), cerulein group (medium+10 nmol/L cerulein), cerulein+LPS group (medium+10 nmol/L cerulein+10 mg/L LPS) and improved ascites group. In the improved ascites group, the ascites of sodium taurocholate-induced rat model was mixed and added into the co-culture system to stimulated In vitrocultured homogenous acinar cells. The co-culture system was set as follows: the chambers with the pore size of 1 µm were placed in the cultue plate, and the culture medium and mixed ascites were respectively added to the culture plate and the chamber at a ratio of 1:1. The acinar cells in each group were collected after 24 h stimulation. The apoptotic/necrotic rates, the expressions of apoptosis/necrosis related proteins [B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X protein (Bax) and receptor interacting protein 1 (RIP1)], and the ultra-structure of acinar cells were detected by flow cytometry, Western blot and transmission electron microscopy (TEM). RESULTS: The acinar cells in the improved ascites model were mainly characterized by necrosis; compared with the other 3 groups, the apoptosis rate and necrosis rate were both up-regulated, RIP1 and BAX protein expression levels were up-regulated, and Bcl-2 protein was down-regulated. TEM results showed the organelle structure of acinar cells was destroyed, and the cell membrane was degraded in the improved ascites model. Compared with the control group, apoptosis rate of acinar cells in the cerulein and cerulein+LPS models were increased and necrosis rate were not changed. The expression of pro-apoptotic protein Bax was increased, while the expression level of RIP1 was not significantly increased. TEM results showed that in cerulein group and cerulein+LPS group, the chromatin of the cells was condensed into a mass, the cytoplasm was degraded and the cell membrane was intact, showing typical apoptosis characteristics. CONCLUSION: Compared with cerulein and cerulein +LPS models, which mainly focus on apoptosis of acinar cells and applied to mild acute pancreatitis study, the improved ascites model mainly focuses on the necrosis of acinar cells and is a good model for studying acinar cell necrosis and severe acute pancreatitis.