Aridity threshold for alpine soil nitrogen isotope signature and ecosystem nitrogen cycling.

Determination of tipping points in nitrogen (N) isotope (δ15N) natural abundance, especially soil δ15N, with increasing aridity, is critical for estimating N-cycling dynamics and N limitation in terrestrial ecosystems. However, whether there are linear or nonlinear responses of soil δ15N to increases in aridity and if these responses correspond well with soil N cycling remains largely unknown. In this study, we investigated soil δ15N and soil N-cycling characteristics in both topsoil and subsoil layers along a drought gradient across a 3000-km transect of drylands on the Qinghai-Tibetan Plateau. We found that the effect of increasing aridity on soil δ15N values shifted from negative to positive with thresholds at aridity index (AI) = 0.27 and 0.29 for the topsoil and subsoil, respectively, although soil N pools and N transformation rates linearly decreased with increasing aridity in both soil layers. Furthermore, we identified markedly different correlations between soil δ15N and soil N-cycling traits above and below the AI thresholds (0.27 and 0.29 for topsoil and subsoil, respectively). Specifically, in wetter regions, soil δ15N positively correlated with most soil N-cycling traits, suggesting that high soil δ15N may result from the "openness" of soil N cycling. Conversely, in drier regions, soil δ15N showed insignificant relationships with soil N-cycling traits and correlated well with factors, such as soil-available phosphorus and foliage δ15N, demonstrating that pathways other than typical soil N cycling may dominate soil δ15N under drier conditions. Overall, these results highlight that different ecosystem N-cycling processes may drive soil δ15N along the aridity gradient, broadening our understanding of N cycling as indicated by soil δ15N under changing drought regimes. The aridity threshold of soil δ15N should be considered in terrestrial N-cycling models when incorporating 15N isotope signals to predict N cycling and availability under climatic dryness.

Responses of root-associated fungal community structure of mycorrhizal plants to nitrogen and/or phosphorus addition in a subtropical forest.

Root-associated fungi play a vital role in maintaining nutrient absorption and health of host plants. To compare the responses of root-associated fungal community structures to nitrogen (N) and/or phosphorus (P) additions across differential mycorrhizal types, we collected roots of nine plant species belonging to three mycorrhizal types (arbuscular mycorrhiza, ectomycorrhiza, and ericoid mycorrhiza) under control and N and/or P addition treatments from a subtropical forest, and detected the diversity and community composition of fungi inhabiting roots through the high-throughput sequencing technique. The results showed that root-associated fungal communities of all nine plant species were mainly composed of Basidiomycota and Ascomycota. The relative abundance of Ascomycota and Basidiomycota was significantly lower and higher under the P addition than that under control, respectively. The relative abundance of Ascomycota of ericoid mycorrhizal trees was significantly higher than those of arbuscular mycorrhizal and ectomycorrhizal trees, while the relative abundance of Basidiomycota was significantly lower than the other two mycorrhizal types. Compared with the control, P addition significantly reduced the α-diversity and changed community composition of root-associated fungi across different mycorrhizal plant types, while no effect of N addition or mycorrhizal type was observed. Compared with the control and N addition treatments, NP addition caused root-associated fungal communities of all plants becoming integrally divergent. In addition, the fungal communities of ectomycorrhizal mycorrhizal trees became apparently convergent in comparison with those of arbuscular and ericoid mycorrhizal trees under the NP addition. Collectively, our results highlighted that P was a critical factor influencing community structures of tree root-associated fungi in subtropical forest soils. This study would enhance our understanding of the responses and maintenance mechanisms of plant root-associated fungal diversity under global environmental changes in the subtropical region.

Bacterial communities in the phyllosphere are distinct from those in root and soil, and sensitive to plant species changes in subtropical tree plantations.

Deciphering the local diversity and community composition of plant-associated microorganisms is crucial to predict their ecological functions in forest ecosystems. The differences in microbial diversity and community composition between the aboveground and belowground tree compartments remain largely unknown. Here, we examined bacterial communities in the leaf surface (phyllosphere) and root-associated (root and rhizospheric soil) habitats of 13 tree species. Bacterial richness substantially differed across the three compartments, with the highest value observed in rhizospheric soil. Tree species exerted a significant effect on α-diversity of leaf- and soil- but not root-inhabiting bacteria. Bacterial communities were distinct across habitats and were significantly more divergent in leaf- than in root-associated habitats. Leaf nutrients and soil pH and NH4+-N were the main factors regulating leaf- and root-related community composition, respectively. This study highlights that host selection effects on bacterial community structure were more prominent in aboveground than in belowground habitats. Our findings contribute to a better understanding of the effect of compartments and subtropical tree species on microbial diversity, with crucial implications for sustainable forest plantation management.

Rapid Quantification of Infectious Cucumber green mottle mosaic virus in Watermelon Tissues by PMA Coupled with RT-qPCR.

Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 102 copies·µL-1. PMA at 120 µmol·L-1 was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 102~105 copies·g-1. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources.

[Responses of soil microbial biomass and carbon source utilization to simulated nitrogen deposition and drought in a Cunninghamia lanceolata plantation]. / 杉木人工林土壤微生物生物量和碳源利用能力对模拟氮沉降和干旱的响应.

Chinese fir (Cunninghamia lanceolata) plantation is a dominant forest type and carbon sink in the subtropical region in China. An experiment with simulated nitrogen deposition (addition of 40 kg N·hm-2·a-1) and drought (50% of precipitation exclusion, PE) was established in Chinese fir plantation in 2018. Soil samples (0-15 cm) were collected in summer (July 2020) and winter (January 2021). Soil microbial biomass, colony forming units (CFUs) and carbon source utilization were determined through phospholipid fatty acids (PLFAs), plate count, and Biolog methods, respectively. The results showed significant seasonal variations of PLFAs-related microbial biomass and composition. Soil bacterial and fungal CFUs tended to be decreased by nitrogen addition or precipitation exclusion treatment, and bacterial CFUs were more sensitive to the two treatments than fungal CFUs. Soil microbial function (i.e. carbon source utilization) was not affected by nitrogen addition, but significantly decreased by precipitation exclusion. There was a significant positive correlation between bacterial CFUs and microbial function, indicating the crucial roles of culturable bacteria in microbial carbon transformation. Our results highlight the critical effects of nitrogen deposition and 50% reduced precipitation on microbes in topsoil of fir plantation, with implications for unraveling soil microbial ecological function of subtropical forest ecosystem under global changes in future.

Compartment and Plant Identity Shape Tree Mycobiome in a Subtropical Forest.

Deciphering the relationships between microbes and their host plants is critical for a better understanding of microbial diversity maintenance and community stability. Here, we investigated fungal diversity and community assembly in the phyllosphere and rhizosphere of 13 tree species in a subtropical common-garden experiment. The results showed that fungal community structures significantly differed across compartments (leaf, root, and soil) and different tree species. Higher α-diversity was observed in the phyllosphere than in the roots and rhizospheric soil. Fungal community composition (ß-diversity) was significantly affected by both compartment and species identity. The fungal community compositions were significantly correlated with soil pH in the roots and the soils as well as with soil nitrate and leaf total phosphorus in the leaves. We found that fungal community assemblies were mainly driven by deterministic processes, regardless of compartments. Moreover, host preference analyses indicated that stronger plant/fungus preferences occurred in leaves than in roots and soils. Our results highlight the differences in tree mycobiome between aboveground and belowground compartments and have important implications for the promotion of biodiversity conservation and management sustainability for the subtropical forest. IMPORTANCE Subtropical mountain forests are widely distributed in Southern China and are characterized by high biodiversity. The interactions between plants and fungi play pivotal roles in biodiversity maintenance and community stability. Nevertheless, knowledge of fungal diversity and of the community assembly patterns of woody plants is scarce. Here, we investigated fungal diversity and community assembly in the phyllosphere and rhizosphere of 13 tree species in a common-garden experiment. We found that both compartment and plant identity influenced fungal diversity, community, and guild compositions, while deterministic processes mainly governed the fungal community assembly, especially in the rhizospheric fungal communities. Our results demonstrate that tree leaves represent stronger host/fungi preferences than do roots and soils. Together, our findings enhance the understanding of the roles of compartment and plant identity in structuring fungal communities as well as promote fungal diversity maintenance in subtropical mountain forest ecosystems.

[Optimization for the determination of phenol oxidase activity in subtropical forest soils developed on sandstone]. / äºšçƒ­å¸¦ç ‚å²©å‘è‚²æ£®æž—åœŸå£¤é šæ°§åŒ–é ¶æ´»æ€§æµ‹å®šæ–¹æ³•ä¼˜åŒ–.

Phenol oxidase plays an important role in the degradation of soil organic matter. There was no standard method to determine soil phenol oxidase activity. To fill such knowledge gap, we investigated the effects of substrate type, pH, soil storage conditions, storage time, substrate concentration, water-soil ratio, incubation time and incubation temperature on soil phenol oxidase activity in three different subtropical forest soils developed on sandstone. The pH of extraction buffers significantly affected the phenol oxidase activity. Using 2,2'-azinobis-(-3-ethylbenzo-thiazoline-6-sulfononic acid)-diammonium salt (ABTS) as substrate acquired higher oxidase activity and was applicable to wider pH range than using 3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) as substrate, indicating that ABTS was more suitable as a substrate for measuring phenol oxidase activity in acidic soils of subtropical forests. The storage condition significantly affected phenol oxidase activity. The phenol oxidase activity declined with time in all the three types of soil. The decreasing rate was air-dried > 4 ℃ refrigerated > -20 ℃ frozen > -80 ℃ frozen, suggesting that the frozen storage method was better than others in maintaining soil phenol oxidase activity if the determination of phenol oxidase activity in fresh soil samples cannot be immediately done. Substrate concentration, water-soil ratio, and incubation time and temperature all affected the activity of soil phenol oxidase. The condition of soil: buffer ratio of 1:100, 2 mmol·L-1 concentration of ABTS with an incubation time of 4 h at 25-30 ℃ was optimal for measuring phenol oxidase activity in acidic soils of subtropical forests, with high repeatability and sensitivity.

[Responses of arbuscular mycorrhizal fungi to elevated atmospheric CO2 concentration and warming: A review]. / ä¸›æžèŒæ ¹çœŸèŒå¯¹å¤§æ°”CO2æµ“åº¦å‡é«˜å’Œå¢žæ¸©å“åº”ç ”ç©¶è¿›å±•.

Global changes have profound impacts on biodiversity and ecological functioning of terrestrial ecosystems. Arbuscular mycorrhizal (AM) fungi can form symbiotic associations with most terrestrial plant species and play an important role in nutrient acquisition of host plants, promotion of plant growth, and maintenance of plant diversity. In this review, we primarily focused on the responses and feedbacks of AM fungal community and functioning to elevated atmospheric CO2(eCO2) and warming in forest and grassland ecosystems. eCO2 influenced AM fungi mainly through indirectly impacting host plants and soil carbon inputs. A majority of previous studies reported that eCO2 could enhance the abundance and activity of AM fungi, and influence their diversity and community composition. Warming could have direct and indirect (via plant and/or soil pathways) impacts on AM fungi. Warming significantly altered the community compositions of AM fungi in forest soils. But the results from grassland were not consistent. We identified some outstanding problems in current studies and proposed future research topics which deserve more attentions. Our aim was to elucidate the AM fungal responses and adaptation to eCO2 and warming and to improve our understanding of AM fungal functioning in soil ecological processes. This review could provide insights into the implications of AM fungi to mitigate global change and improve the resilience of soil functions, as well as climate change adaptation of ecosystems.

Percolation thresholds of randomly rotating patchy particles on Archimedean lattices.

We study the percolation of randomly rotating patchy particles on 11 Archimedean lattices in two dimensions. Each vertex of the lattice is occupied by a particle, and in each model the patch size and number are monodisperse. When there are more than one patches on the surface of a particle, they are symmetrically decorated. As the proportion χ of the particle surface covered by the patches increases, the clusters connected by the patches grow and the system percolates at the threshold χ_{c}. We combine Monte Carlo simulations and the critical polynomial method to give precise estimates of χ_{c} for disks with one to six patches and spheres with one to two patches on the 11 lattices. For one-patch particles, we find that the order of χ_{c} values for particles on different lattices is the same as that of threshold values p_{c} for site percolation on these lattices, which implies that χ_{c} for one-patch particles mainly depends on the geometry of lattices. For particles with more patches, symmetry becomes very important in determining χ_{c}. With the estimates of χ_{c} for disks with one to six patches, using analyses related to symmetry, we are able to give precise values of χ_{c} for disks with an arbitrary number of patches on all 11 lattices. The following rules are found for patchy disks on each of these lattices: (1) as the number of patches n increases, values of χ_{c} repeat in a periodic way, with the period n_{0} determined by the symmetry of the lattice; (2) when mod(n,n_{0})=0, the minimum threshold value χ_{min} appears, and the model is equivalent to site percolation with χ_{min}=p_{c}; and (3) disks with mod(n,n_{0})=m and n_{0}-m (m

Prognostic value of preoperative inflammatory markers in patients with hepatocellular carcinoma who underwent curative resection.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is not optimistic. Our study focused on present inflammatory markers, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), gamma-glutamyl transpeptidase-to-platelet ratio (GPR), aspartate aminotransferase-to-lymphocyte ratio (ALR) and fibrinogen-to-albumin ratio (FAR), and explored their optimal combination for the prognosis of HCC after resection. METHODS: A total of 347 HCC patients who underwent curative resection were enrolled. The optimal cutoff values of the inflammatory markers were calculated using receiver operating characteristic (ROC) curve analysis, and used to divide patients into two groups whose differences were compared by Kaplan-Meier analysis. Cox univariate and multivariate analyses were used to analyze the independent prognostic inflammatory markers. The χ2 test was chosen to determine the relationship between independent prognostic inflammatory markers and clinicopathological features. We created combined scoring models and evaluated them by Cox univariate and multivariate methods. The concordance index (C-index), Akaike information criterion (AIC) and likelihood ratio were calculated to compare the models. The selected optimal inflammatory markers and their combinations were tested in different stages of HCC by Kaplan-Meier analysis. RESULTS: The ALR and GPR were independent prognostic factors for disease-free survival (DFS); the ALR, PLR, and GPR were independent prognostic factors for overall survival (OS). The proposed GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively. CONCLUSION: The preoperative GPR and ALR-GPR-PLR score models were independent predictors for DFS and OS, respectively, and performed well in stratifying patients with HCC. The higher the score in the model was, the worse the prognosis.

[Long-term effects of imbalanced fertilization with lime and gypsum additions on denitrifying functional genes of an Ultisol at Yingtan, Jiangxi, China]. / é•¿æœŸç¼ºç´ æ–½è‚¥åŠçŸ³ç°çŸ³è†æ–½ç”¨å¯¹æ±Ÿè¥¿é¹°æ½­çº¢å£¤åç¡åŒ–å¾®ç”Ÿç‰©åŠŸèƒ½åŸºå› ä¸°åº¦çš„å½±å“.

The abundance of denitrifying functional genes plays a key role in driving the soil nitrous oxide (N2O) emission potential. Nitrite reductase genes (nirK and nirS) and nitrous oxide reductase genes (nosZ I and nosZ II) are the dominant denitrifying funtional genes. In this study, real-time quantitative PCR was conducted to evaluate the effects of 32-year imbalanced fertilization and lime and gypsum additions on the abundances of nirK, nirS, nosZ I and nosZ II genes in an Ultisol at Yingtan, Jiangxi Province. We further explored the underlying driving factors. The results showed that, compared with the balanced fertilization treatment, fertilization without phosphorus (P) signifi-cantly decreased the abundances of nirK, nirS, nosZ I and nosZ II genes. Fertilization without nitrogen (N) significantly reduced the abundances of nirK, nosZ I and nosZ II, but did not affect the abundance of nirS. Fertilization without potassium (K) did not affect the abundances of all denitri-fying functional genes. Results of stepwise regression analysis and random forest analysis showed that soil pH was a key environmental factor affecting the abundances of nosZ I and nosZ II. The application of lime or lime + gypsum significantly increased soil pH, which subsequently increased the abundances of nosZ II and nosZ II/nosZ I by 150%-231% and 127%-155%, respectively. Our results suggested that application of lime or lime + gypsum favored nosZ II more than nosZ I in upland Ultisols, which might enhance the relative importance of nosZ II in N2O reduction. Overall, fertilization without P would reduce denitrifying gene abundances, while the application of lime or lime + gypsum enriched nosZ II and increased ratio of nosZ II/nosZ I, which might be beneficial for reducing N2O emission potential in the Ultisols.

Oncogenic Activation of YAP Signaling Sensitizes Ferroptosis of Hepatocellular Carcinoma via ALOXE3-Mediated Lipid Peroxidation Accumulation.

Ferroptosis, a form of programmed cell death process driven by iron-dependent lipid peroxidation, plays an important role in tumor suppression. Although previous study showed that intracellular Merlin-Hippo signaling suppresses ferroptosis of epithelial tumor cells through the inactivation of YAP signaling, it remains elusive if the proto-oncogenic transcriptional co-activator YAP could serve as a potential biomarker to predict cancer cell response to ferroptosis-inducing therapies. In this study, we show that both total YAP staining and nuclear YAP staining were more prevalent in HCC tissues than in nontumorous regions. Compared to low-density HCC cells, high-density cells showed decreased nuclear localization of YAP and conferred significant resistance to ferroptosis. Oncogenic activation of YAP signaling by overexpression of YAP(S127A) mutant sensitized ferroptosis of HCC cells cultured in confluent density or in the 3D tumor spheroid model. Furthermore, we validated the lipoxygenase ALOXE3 as a YAP-TEAD target gene that contributed to YAP-promoted ferroptosis. Overexpression of ALOXE3 effectively increased the vulnerability of HCC cells to ferroptotic cell death. In an orthotopic mouse model of HCC, genetic activation of YAP rendered HCC cells more susceptible to ferroptosis. Finally, an overall survival assay further revealed that both a high expression of YAP and a low expression of GPX4 were correlated with increased survival of HCC patients with sorafenib treatment, which had been proven to be an inducer for ferroptosis by inhibition of the xc-amino acid antiporter. Together, this study unveils the critical role of intracellular YAP signaling in dictating ferroptotic cell death; it also suggests that pathogenic alterations of YAP signaling can serve as biomarkers to predict cancer cell responsiveness to future ferroptosis-inducing therapies.

Identification of a cartilage specific novel miRNA which directly targets PRMT3 in rats.

Through experiments to testify a candidate novel miRNA previously discovered by us is a real miRNA and involved in cartilage development. DESIGN: The miR-novel and the newly hairpin miRNA transcribed sequence (pre-miR-novel) was verified as a genuinely existing miRNA by northern blotting. The predicted secondary structure, sequence alignment and targets of pre-miR-novel were performed by "RNAstructure 5.3" program, LASTN2.8.0+/miRbase22 program and RNA hybird program, respective. GO/KEGG pathway analysis also were performed. The miR-novel expression in cartilage tissue during development was detected by RT-qPCR and dot blotting. The chondrocyte differentiation model was established to examine whether miR-novel is involved in cartilage development. The regulation of PRMT3 expression by novel miRNA was determined with the luciferase reporter gene assay and Western blotting after novel miRNA mimic or inhibitor transfection. RESULTS: It's potential role in specifically regulating rodent cartilage development and associated cellular processes. Furthermore, the expression of protein arginine N-methyltransferase 3 (PRMT3), as a predicted target of the novel miRNA, was found consistently downregulated at rat cartilage during developmental stages and RCJ3.1C5.18 (C5.18) cells during the proliferating and hypertrophic phases of the cartilage development, where the miR-novel expression was significantly up-regulated. Both the dual-luciferase reporter gene assay and the up- or down-regulation of miR-novel suggest that the later can specifically bind with the Prmt3 3'-UTR. CONCLUSION: Overall, this study provides the first comprehensive evidence that a genuine cartilage-specific novel miRNA directly targets PRMT3 and may regulate multitudinous cellular processes and signal transduction during cartilage development.

The resurgent landscape of xenotransplantation of pig organs in nonhuman primates.

Organ shortage is a major bottleneck in allotransplantation and causes many wait-listed patients to die or become too sick for transplantation. Genetically engineered pigs have been discussed as a potential alternative to allogeneic donor organs. Although xenotransplantation of pig-derived organs in nonhuman primates (NHPs) has shown sequential advances in recent years, there are still underlying problems that need to be completely addressed before clinical applications, including (i) acute humoral xenograft rejection; (ii) acute cellular rejection; (iii) dysregulation of coagulation and inflammation; (iv) physiological incompatibility; and (v) cross-species infection. Moreover, various genetic modifications to the pig donor need to be fully characterized, with the aim of identifying the ideal transgene combination for upcoming clinical trials. In addition, suitable pretransplant screening methods need to be confirmed for optimal donor-recipient matching, ensuring a good outcome from xenotransplantation. Herein, we summarize the understanding of organ xenotransplantation in pigs-to-NHPs and highlight the current status and recent progress in extending the survival time of pig xenografts and recipients. We also discuss practical strategies for overcoming the obstacles to xenotransplantation mentioned above to further advance transplantation of pig organs in the clinic.

[Responses of denitrifying functional gene abundance to long-term fertilization regimes in an upland Ultisol]. / æ—±åœ°çº¢å£¤åç¡åŒ–åŠŸèƒ½åŸºå› ä¸°åº¦å¯¹é•¿æœŸæ–½è‚¥çš„å“åº”.

Fertilization affects soil nitrogen cycling and nitrous oxide (N2O) emissions, which are mainly driven by microbes. A 32-year field experiment was conducted to investigate the effects of chemical fertilizers and their combination with organic materials on the abundance of denitrifying functional genes (nirS, nirK, nosZ I and nosZ II) in Ultisol. The treatments comprised no fertilizer (CK), chemical fertilizer, chemical fertilizer+peanut straw, chemical fertilizer+rice straw, chemical fertilizer+radish and chemical fertilizer+pig manure. Compared with the single chemical fertilizer treatment, soil pH and organic carbon content increased in the chemical fertilizer plus organic material treatments, with chemical fertilizer+pig manure having the strongest effect. Long-term fertilization did not affect the abundance of nirK gene, but significantly altered the nirS gene abundance. Compared to CK, long-term chemical fertilizer application increased the abundance of nirS gene by 426%. However, partial replacement of chemical fertilizer by organic materials decreased the abundance of nirS gene. The abundance of nosZ I gene was one order of magnitude higher than that of nosZ II, indicating the domination of nosZ I in the acidic Ultisol. Long-term fertilization did not affect the abundance of nosZ II, whereas chemical fertilizer+pig manure increased the abundance of nosZ I by 138%. Results of stepwise regression analysis showed that available phosphorus content was the primary factor regulating the abundance of nosZ I gene, whereas the abundance of the nosZ II gene was mainly regulated by nitrate content. Moreover, the lowest (nirS+nirK)/(nosZ I+nosZ II) value in the chemical fertilizer+pig manure treatment indicated that long-term manure application might reduce N2O emission potential in Ultisols.

Adoptive transfer of polarized M2c macrophages ameliorates acute rejection in rat liver transplantation.

Hepatic macrophages play pivotal roles in tolerance induction after liver transplantation (LT). However, macrophages possess functional heterogeneities, and the protective role of M2c macrophages, a macrophage subtype characterized by the surface marker CD163 that secretes interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1), in acute rejection following LT, has not been addressed. The aim of this study was to determine whether polarized macrophages of the M2c subtype could improve outcomes after LT for rats, including survival rate, liver function, and inflammatory infiltration. In our study, the numbers of CD163-positive cells were found to be increased in tolerant liver grafts. Immediately following the surgery, M2c macrophages induced from rat bone marrow-derived cells were infused into recipients; this significantly improved survival rate and liver function. The expression levels of IL-10 and TGF-ß1 were markedly increased in these rats compared to those in the control group. Furthermore, CD8+ T-cell infiltration was reduced, whereas the numbers of apoptotic cells increased, in rats treated with M2c. To explore the mechanisms of the protective role of M2c, the numbers of major histocompatibility complex (MHC) class II positive cells were found to be decreased and the expression of N-acetylglucosaminyltransferase V (MGAT5) was up-regulated in M2c infusion groups. Together, these findings demonstrate that polarization of macrophages towards the M2c phenotype ameliorated acute rejection in a rat LT model and may provide a novel and effective therapeutic approach for AR after transplantation.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

BACKGROUND: In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. METHODS: Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. RESULTS: GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. CONCLUSIONS: Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

A review of pig liver xenotransplantation: Current problems and recent progress.

Pig liver xenotransplantation appears to be more perplexing when compared to heart or kidney xenotransplantation, even though great progress has been achieved. The relevant molecular mechanisms involved in xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia, are complex, and need to be systematically investigated. The deletion of expression of Gal antigens in the liver graft highlights the injurious impact of nonGal antigens, which continue to induce humoral rejection. Innate immunity, particularly mediated by macrophages and natural killer cells, interplays with inflammation and coagulation disorders. Kupffer cells and liver sinusoidal endothelial cells (LSECs) together mediate leukocyte, erythrocyte, and platelet sequestration and phagocytosis, which can be exacerbated by increased cytokine production, cell desialylation, and interspecies incompatibilities. The coagulation cascade is activated by release of tissue factor which can be dependent or independent of the xenoreactive immune response. Depletion of endothelial anticoagulants and anti-platelet capacity amplify coagulation activation, and interspecies incompatibilities of coagulation-regulatory proteins facilitate dysregulation. LSECs involved in platelet phagocytosis and transcytosis, coupled with hepatocyte-mediated degradation, are responsible for thrombocytopenia. Adaptive immunity could also be problematic in long-term liver graft survival. Currently, relevant evidence and study results of various genetic modifications to the pig donor need to be fully determined, with the aim of identifying the ideal transgene combination for pig liver xenotransplantation. We believe that clinical trials of pig liver xenotransplantation should initially be considered as a bridge to allotransplantation.

Berberine Inhibits Growth of Liver Cancer Cells by Suppressing Glutamine Uptake.

INTRODUCTION: Glutamine metabolism is essential for the proliferation of cancer cells. Transported by SLC1A5, a Na+ dependent transporter, glutamine is absorbed for further use. Recent studies have revealed the anti-tumor effect of berberine. The present study aimed to evaluate the effect of berberine on cancer cell glutamine metabolism. MATERIALS AND METHODS: The inhibitory effect of berberine on liver cancer cells was analyzed by CCK-8 and EdU assay. The glutamine concentrations were detected by ELISA and UHPLC-MRM-MS analysis. Glutamine metabolism-related proteins were determined by Western blot, immunofluorescent analysis and immunohistochemistry. RESULTS: Berberine inhibited the proliferation of Hep3B and BEL-7404 cell in vitro. Berberine suppressed the glutamine uptake by inhibiting SLC1A5. The upregulation of SLC1A5 led to an increased glutamine uptake and improved tolerance to berberine. Berberine suppresses SLC1A5 expression by inhibiting c-Myc. Furthermore, berberine suppresses the growth of tumor xenografts, and the expression of SLC1A5 and c-Myc in vivo. The high expression of SLC1A5 in hepatocellular carcinoma (HCC) tissues is associated with poor prognosis. CONCLUSION: Berberine can suppress the proliferation of liver cancer cells by reducing SLC1A5 expression.

Circular RNA circRHOT1 is upregulated and promotes cell proliferation and invasion in pancreatic cancer.

AIM: We aimed to identify the roles of circRHOT1 in pancreatic cancer. MATERIALS & METHODS: The circRHOT1 was acquired from our previous study followed by quantitative real-time PCR and fluorescence in situ hybridization validation in pancreatic cancer. We used siRNA and shRNA to explore the function of circRHOT1 in pancreatic cancer cells. Bioinformatic analyses were applied to study the potential mechanism of circRHOT1. RESULTS: The circRHOT1 was upregulated in pancreatic cancer and predominantly located in the cytoplasm. Reducing the circRHOT1 expression may inhibit the pancreatic cancer cell proliferation, invasion and migration. The circRHOT1 may play a role in pancreatic cancer through binding miR-26b, miR-125a, miR-330 and miR-382 to regulate multiple tumor-associated pathways. CONCLUSION: This study demonstrated that circRHOT1 may serve as an oncogenic circRNA that promotes tumor progression.