Spatial heterogeneity of the effects of river network patterns on water quality in highly urbanized city.

River water quality deterioration is a serious problem in urban water environments. River network patterns affect water quality by influencing the flow, mixing, and other processes of water bodies. However, the effects of urban river network patterns on water quality remain poorly understood, thereby hindering the urban planning and management decision-making process. In this study, the geographically weighted regression (GWR) model was used to explore the spatial heterogeneity of the relationship between river network pattern and water quality. The results showed that the river network has a complex structure, high connectivity, and relatively even distribution and morphology. Important river structure indicators affecting water quality included the water surface ratio (Wp) and multifractal features (∆α, ∆f) while important river connectivity indicators included circuitry (α) and network connectivity (γ). River structure has a more complex effect on water quality than connectivity. This study recommends that the Wp should be increased in agricultural areas and appropriately reduced in urban built-up areas, and the number of river segments and nodes should be controlled within a rational configuration. Our study provides key insights for evaluating and optimizing the river network patterns to improve water quality of urban rivers. In the future, the land use intensity, hydrological processes, and human activities should be coupled with the river network pattern to deepen our understanding of urban river environment.

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.

OBJECTIVE: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins. RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures. CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.

Exploration of the interaction mechanism of lignocellulosic hybrid systems based on deep eutectic solvents.

The interactions of three deep eutectic solvents (DES) choline chloride-glycerol (ChCl-GLY), ChCl-lactic acid (ChCl-LA) and ChCl-urea (ChCl-U) with cellulose-hemicellulose and cellulose-lignin hybrid systems were investigated using the simulated computational approach. Aiming to simulate DES pretreatment of real lignocellulosic biomass in nature. DES pretreatment could disrupt the original hydrogen bonding network structure among the lignocellulosic components and reconstruct the new DES-lignocellulosic hydrogen bonding network structure. ChCl-U had the highest intensity of action on the hybrid systems, removing 78.3% of the hydrogen bonds between cellulose-4-O-methyl Gluconic acid xylan (cellulose-Gxyl) and 68.4% of the hydrogen bonds between cellulose-Veratrylglycerol-b-guaiacyl ether (cellulose-VG), respectively. The increase of urea content facilitated the interaction between DES and lignocellulosic blend system. Finally, the addition of appropriate water (DES:H2O = 1:5) and DES formed the new DES-water hydrogen bonding network structure more favorable for the interaction of DES with lignocellulose.

Theoretical study of lactic acid-based deep eutectic solvents dissociation of hemicellulose with different hydrogen bonding acceptors.

This paper explored the mechanism of dissociation of hemicellulose using lactic acid (LA)-based deep eutectic solvents (DESs) synthesized with different hydrogen bond acceptors (HBAs) via simulations. Density functional theory (DFT) calculations and molecular dynamics (MD) simulations revealed that DESs synthesized with guanidine hydrochloride (GuHCl) as hydrogen bond acceptor (HBA) demonstrated better hemicellulose solubilization compared to the conventional DESs synthesized using choline chloride (ChCl) as HBA. The best interaction with hemicellulose was achieved at GuHCl:LA = 1:1. The results showed that CL- played a dominant role in the dissolution of hemicellulose by DESs. Unlike ChCl, the guanidine group in GuHCl had the delocalized π bond, which made CL- have stronger coordination ability and promoted dissolution of hemicellulose by DESs. Moreover, multivariable analysis was employed to establish the correlation between the effects of different DESs on hemicellulose and the molecular simulation results. Additionally, the influence of different HBAs functional groups and carbon chain length on the solubilization of hemicellulose by DESs were analyzed.

Multi-Electron Visible Light Photoaccumulation on a Dipyridylamine Copper(II)-Polyoxometalate Conjugate Applied to Photocatalytic Generation of CF3 Radicals.

This article describes the synthesis and characterization of an organic-inorganic hybrid polyoxometalate functionalized by a short link with a tripodal N-based ligand and its copper complex. Upon visible light irradiation, the latter is able to store up to three reducing equivalents. The locus of the reduction is discussed based on physicochemical measurements and DFT calculations. In the presence of Togni's reagent, this complex allows for the photocatalytic generation of CF3 radicals, opening the road to valuable synthetic applications.

Mechanism study of ternary deep eutectic solvents with protonic acid for lignin fractionation.

This paper investigated the fractionation of lignin by ternary DES of different polyols using simulation calculation. ChCl-EG-PTSA showed the highest degradability of lignin with the absolute value of total interaction energy of -8023.80 kJ/mol, and the total number of hydrogen bonds was 91.4. The highest degradability was observed for ChCl:EG: PTSA = 2:4:1. The results show that CL- plays a dominant role in lignin fractionation and readily forms hydrogen bonds with γ-OH. The difference is that the polyol preferred to form hydrogen bonds with α-OH in lignin. The addition of PTSA provided protons to the original system. It formed a new π-π stacking interaction with the lignin benzene ring, which destroyed the π-π stacking interaction between the original lignin. And increased the interaction of DES on lignin from -39.73 kcal/mol to -58.15 kcal/mol based on DFT.

Macromolecular protein crystallisation with biotemplate of live cells.

Macromolecular protein crystallisation was one of the potential tools to accelerate the biomanufacturing of biopharmaceuticals. In this work, it was the first time to investigate the roles of biotemplates, Saccharomyces cerevisiae live cells, in the crystallisation processes of lysozyme, with different concentrations from 20 to 2.5 mg/mL lysozyme and different concentrations from 0 to 5.0 × 107 (cfu/mL) Saccharomyces cerevisiae cells, during a period of 96 h. During the crystallisation period, the nucleation possibility in droplets, crystal numbers, and cell growth and cell density were observed and analysed. The results indicated the strong interaction between the lysozyme molecules and the cell wall of the S. cerevisiae, proved by the crystallization of lysozyme with fluorescent labels. The biotemplates demonstrated positive influence or negative influence on the nucleation, i.e. shorter or longer induction time, dependent on the concentrations of the lysozyme and the S. cerevisiae cells, and ratios between them. In the biomanufacturing process, target proteins were various cells were commonly mixed with various cells, and this work provides novel insights of new design and application of live cells as biotemplates for purification of macromolecules.

A modified method of gene disruption in Komagataella phaffii with Cre/loxP system.

To increase protein production, technologies of gene manipulation for engineering the yeast Komagataella phaffii are extensively exploited. In this study, we developed a convenient gene disruption method in the yeast via Cre/loxP system. First, the simple gene disruption cassette [upstream homologous region (UP)-lox71-Sh ble-lox66-downstream homologous region (DW)] was constructed and transformed into the yeast to replace target gene. Second, the Sh ble gene of the cassette integrated in the chromosome was inserted with the auxiliary plasmid pPICZαA/cre/his4, resulting in an expanded cassette of UP-lox71-Sh ble-pPICZαA/cre/his4-lox66-DW. The auxiliary plasmid was generated via sequential insertion of cre and his4 genes into pPICZαA, and linearized with SmaI before its transformation. Finally, for deletion of the sequence between lox71 and lox66 sites in the expanded cassette, CRE protein responsible for Cre/loxP-mediated recombination was produced by methanol induction. Consequently, the corresponding sequence was eliminated permanently, only leaving a scar of lox72 site in the disrupted genes. This strategy was verified by disrupting two genes in the yeast. As the markers were recycled, it was also suitable for multiple gene disruption.

Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

BACKGROUND: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR). OBJECTIVE: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. METHOD: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. RESULTS: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. CONCLUSION: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.

Establishing quality evaluation system of nursing management in fever clinics: A Delphi method.

AIM: The aim of this study is to construct a quality evaluation system for fever clinic nursing management. BACKGROUND: Fever clinic is the first line of defence against the epidemic during COVID-19 in China. METHODS: Our study combines the Delphi method and the analytic hierarchy process. Delphi method was used to carry out two rounds of consultation for 18 experts, to select and revise indicators at all levels. Analytic hierarchy process was used to calculate the weight of indicators at all levels. RESULTS: A quality evaluation system of nursing management for fever clinics is built using Delphi method. It includes five first-level indexes, 14 second-level indexes and 82 third-level indexes. A two-round expert consultation is used to build the indicators. The recovery rates of expert questionnaires in the two rounds were, respectively, 100% and 94%, and expert authority coefficients were 0.925. The Kendall coefficients in the two rounds were, respectively, 0.205 and 0.162 (P < .001). The weight analysis shows that health management of nursing staff (0.2803) and disinfection isolation and treatment of medical waste (0.2803) are most important, followed by nursing post management and personnel training (0.1889), configuration and management of equipment (0.1427) and patient consultation management and nursing (0.1078). CONCLUSION: The quality evaluation system of nursing management in the constructed fever clinic is used to put forward a specific, objective and quantifiable evaluation criteria of nursing quality for fever clinic management, which can better meet the needs of epidemic prevention and control, and has a certain application and promotion value. IMPLICATIONS FOR NURSING MANAGEMENT: The establishment and improvement of a quality system for fever clinic care management will help to respond to outbreaks such as COVID-19.

Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the "immunological scar" left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19.

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.

BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

The speciation, transformation kinetics and fate of spiked Pu (IV) in highly saline groundwater.

The mobility of plutonium (Pu) in groundwater is dependent of its speciation distribution and transformation. The speciation and transformation kinetics of Pu(IV) and its colloids in highly saline groundwater have, however, been rarely studied. In the present study, groundwater (Ionic strength 1 M) from Dunhuang region, NW China, was collected for investigating the speciation, transformation kinetics and fate of spiked Pu (IV) with aging time. The results showed that ~99% of the spiked Pu (IV) (over initial concentration c0 range 2.5 × 10-10-7.8 × 10-7 mol·L-1) was easily associated with the natural colloids and transformed into relatively unstable Pu pseudo-colloids in 1 day, which then gradually deposited and/or adsorbed on the container walls with aging. The suspended Pu pseudo-colloids decreased in similar exponential models, with rate equations r(t) = -3.1 × 10-10e- t/4 and -1.3 × 10-8e-/3 for c0 = 1.25 × 10-9 mol·L-1and 4.17 × 10-8 mol·L-1, respectively. The chemical speciation of the suspended colloidal Pu was dominated by "Fe/Mn Oxides" at the early time, while "Carbonates" with slower depositing rate (r(t) = -6.9 × 10-12e- 0.149t) dominated it (~82%) at equilibrium state. Whatever the c0 was, the concentration of dissolved Pu (i.e., the apparent solubility of Pu) kept at 0.7 × 10-11 mol·L-1 over aging. The valence of dissolved Pu was dominated by Pu(IV) at early time, while Pu(V + VI) would become dominant (~95%) at equilibrium state with transformation rate of r(t) = -92.9e- t/16.6 + 96.9. The equilibrium times of Pu deposition (and/or adsorption), speciation transformation of the suspended colloidal Pu, and valence change of the dissolved Pu were 30 d, 80 d and 120 d, respectively. The kinetic process for each Pu species could be well fitted with exponential model. These results suggest that the majority of released Pu(IV) into highly saline groundwater will be easily associated with natural aquatic colloids and then become immobile in short time due to deposition (and/or adsorption) onto the environmental medium, but potential migration risk caused by stable suspended Pu colloids cannot be ignored.

Disperse magnetic solid phase microextraction and surface enhanced Raman scattering (Dis-MSPME-SERS) for the rapid detection of trace illegally chemicals.

The technique of solid phase micro-extraction (SPME) is an important method for sample pretreatment in analytical chemistry, especially for the analysis in micro-systems. Surface enhanced Raman scattering (SERS) is an ultra-sensitive and fast detection technique. Both are particularly important in qualitative analysis of trace amount of substance. In this study, combining the magnetic nanoparticles with magnetic SPME device, we develop a high efficient new pretreatment method named as disperse magnetic solid phase micro-extraction (Dis-MSPME). In comparison to the traditional SPME, the proposed Dis-MSPME realizes solid phase micro-extraction from dispersive system, which improved the extraction efficiency largely. Conjunction the advantages both of Dis-MSPME and SERS is proposed as Dis-MSPME-SERS as a new detection method, which realize enrichment, magnetic separation and detection all-in-one. Making it a simpler, more efficient and sensitive approach in identifying the illegal additives. Sildenafil citrate (SC) in 500µL health wine as an example of illegal additive was successfully detected in a LOD of 1.0 × 10-8M. Moreover, comparative study on the extract efficient of Dis-MSPME-SERS with SPME-SERS shows it takes only 10min to detect sildenafil citrate in the health wine, from enrichment to detection by Dis-MSPME-SERS.

Effects of humic acid concentration on the microbially-mediated reductive solubilization of Pu(IV) polymers.

The role of humic acid concentration in the microbially-mediated reductive solubilization of Pu(IV) polymers remains unclear until now. The effects of humic concentration (0-150.5mg/L) on the rate and extent of reduction of polymeric Pu(IV) were studied under anaerobic and pH 7.2 conditions. The results show that Shewanella putrefaciens, secreting flavins as endogenous electron shuttles, cannot notably stimulate the reduction of polymeric Pu(IV). In the presence of humic acids, the reduction rate of polymeric Pu(IV) increased with increasing humic concentrations (0-15.0mg/L): e.g., a 102-fold increase from 4.1×10-15 (HA=0) to 4.2×10-13mol Pu(III)aq/h (HA=15.0mg/L). The bioreduced humic acids by S. putrefaciens facilitated the extracellular electron transfer to Pu(IV) polymers and thus the reduction of polymeric Pu(IV) to Pu(III)aq became thermodynamically favorable. However, the reduction rate did not increase but decrease with increasing humic concentrations from 15.0 to 150.5mg/L. Humic coatings formed on the polymer surfaces at relatively high humic concentrations limited the electron transfer to the polymers and thus decreased the reduction rate. The finding of the dynamic role of humic acids in the bioreductive solubilization may be helpful in evaluating Pu mobility in the geosphere.

Regulation on the aggregation-induced emission (AIE) of DNA-templated silver nanoclusters by BSA and its hydrolysates.

The aggregation-induced emission (AIE) phenomenon of the metal nanoclusters (NCs) has been discovered quite recently, which is considered to be creative to improve the optical properties of NCs upon ligand aggregation. In the present study, we report an AIE system for double-stranded DNA-templated silver nanoclusters (AgNCs-dsDNA) by using bovine serum albumin (BSA) in solution, which induce a moderate emission enhancement of AgNCs (5-fold) and a blue-shift through a loosen interaction. In addition, significant luminescence enhancement (30-fold) is further stimulated by the addition of digestive enzyme to the system by altering the surface structure of the aggregated particles. The processes are observed directly through transmission electron microscopy (TEM), where the dispersed AgNCs spheres are aggregated in a large incompact sheet-like film by BSA; while further trypsin addition lead them into larger particles (>50nm) with higher density. And the intrinsic mechanism is explained well by UV-vis absorption and time-resolved luminescence spectra. The observed luminescence enhancement in step-II is attributed to the AIE through stronger interaction between the hydrolysates of BSA and the ligand dsDNA. Therefore, the optical properties of AgNCs-dsDNA are regulated well by the addition of BSA and trypsin in different amounts, which relates directly to the degree of aggregation and can be extended to other metal nanoclusters by employing the pairs of protein and related enzyme.

Specific and sensitive detection of Plasmodium falciparum lactate dehydrogenase by DNA-scaffolded silver nanoclusters combined with an aptamer.

Innovative nanomaterials offer significant potential for diagnosis of severe diseases of the developing world such as malaria. Small sized silver nanoclusters have shown promise for diagnostics due to their intense fluorescence emission and photo-stabilities. Here, double-stranded DNA-scaffolded silver nanoclusters (AgNCs-dsDNA) were prepared to detect the established malaria biomarker, Plasmodium falciparum lactate dehydrogenase (PfLDH). Significant luminescence enhancement over a wide concentration range of PfLDH was demonstrated. In addition, a low limit of detection at 0.20 nM (7.4 pg µL-1) was achieved for PfLDH in buffer solution, sensitive enough for practical use correlating with the clinical level of PfLDH in plasma from malaria-infected patients. Unique specificity was observed towards Plasmodium falciparum over Plasmodium vivax and human lactate dehydrogenase, as well as other non-specific proteins, by combining the use of AgNCs-dsDNA with a DNA aptamer against PfLDH. Moreover, the intrinsic mechanism was revealed in detail for the two-step luminescence response. The combination of DNA-scaffolded silver nanoclusters coupled to a selective single-stranded DNA aptamer allows for a highly specific and sensitive detection of PfLDH with significant promise for malaria diagnosis in future.

Thermally prepared ultrabright adenosine monophosphate capped gold nanoclusters and the intrinsic mechanism.

Gold nanoclusters (AuNCs) have been widely applied in fluorescence sensing, bioimaging and phototherapy. Although great progress has been made, the relatively low quantum yield (QY, <10%) in most currently reported AuNCs limits their application greatly. In the present study, adenosine monophosphate (AMP) capped gold nanoclusters (AuNC@AMP) are prepared by using a newly developed heating method in a short time (within 60 min), and are found to show a strong and stable luminescence emission in a relative high QY (14.52%). In addition, an in-depth investigation by employing infrared, 1H and 31P NMR spectroscopy has attributed the origin of such a high luminescence to both the binding of the purine ring and/or the phosphate moiety of AMP as well as their orientations at the gold core surface. In addition, electron-rich atoms such as nitrogen and oxygen, or group moieties such as -NH2 in the ligands can largely promote the luminescence emission of AuNCs. The present study reveals the intriguing generation of ultrabright luminescence from metal nanoclusters, and it will stimulate more research both on the fabrication and practical applications of luminescent metal NCs. With regard to the high QY of these AuNCs, they have great potential for biological applications as adenine is crucially important in life sciences.

Pentoxifylline inhibits liver fibrosis via hedgehog signaling pathway.

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-ß gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

Enhanced expression of recombinant elastase in Pichia pastoris through the substitution of Thr for Ser in Asn-Xaa-Ser sequons.

N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.