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Asone of the original plants of traditional Tibetan folk medicinal ‘Zangyinchen’, Swertia franchetiana (S. franchetiana) was used to treat a variety of ailments. Large amount of fruiting harvesting...

Gentianopsis paludosa (Mum.) Ma is an important species in Tibetan folk medicine, but its wild populations are shrinking roughly due to the increasing demand for it. Gentianopsis paludosa is presently at risk of over-exploitation, so it needs urgent conservation. Here, we report the complete sequence of the chloroplast genome of G. paludosa. The genome was 51,121 bp in length with 129 genes comprising 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall GC content of G. paludosa chloroplast genome was is 36.67%. The phylogenomic analysis suggested that G. paludosa forms a clade with species in Halenia and Swertia, indicating that the G. paludosa is more closely related to Halenia and Swertia than that of Gentiana.

Swertia tetraptera, native to the Qinghai-Tibetan Plateau, is an important traditional Chinese medicine. Although researchers have done a lot of work on it, the phylogenetic position of S. tetrapte...

Nitrogen (N) status has a great impact on methane (CH4) consumption by soils. Modeling studies predicting soil CH4 consumption assume a linear relationship between CH4 uptake and N addition rate. Here, we present evidence that a nonlinear relationship may better characterize changes in soil CH4 uptake with increasing N additions. By conducting a field experiment with eight N-input levels in a Tibetan alpine steppe, we observed a unimodal relationship; CH4 uptake increased at low to medium N levels but declined at high N levels. Environmental and microbial properties jointly determined this response pattern. The generality of the unimodal trend was further validated by two independent analyses: (i) we examined soil CH4 uptake across at least five N-input levels in upland ecosystems across China. A unimodal CH4 uptake-N addition rate relationship was observed in 3 out of 4 cases; and (ii) we performed a meta-analysis to explore the N-induced changes in soil CH4 uptake with increasing N additions across global upland ecosystems. Results showed that the changes in CH4 uptake exhibited a quadratic correlation with N addition rate. Overall, we suggest that the unimodal relationship should be considered in biogeochemistry models for accurately predicting soil CH4 consumption under global N enrichment.

Revegetation accelerates the recovery of degraded lands. Different microbial trophic groups underpin this acceleration from the aspects of soil structure stabilization, nutrient accumulation, and ecosystem functions. However, little is known about how revegetation influences the community and biodiversity of different soil microbial trophic groups. Here, six revegetation treatments with different plantings of plant species were established at an excavation pit in the Qinghai-Tibetan Plateau. Communities of plant, bacteria, and several key soil fungal groups were investigated after 12 years of revegetation. Plant and all microbial trophic group compositions were markedly influenced by revegetation treatments. Total fungal and pathogenic fungal compositions were not significantly predicted by any factor of plant and soil, but arbuscular mycorrhizal fungal composition could be mainly predicted by plant composition and plant P content. Bacterial composition was mainly determined by soil total N, organic carbon concentration, and moisture content; and saprotrophic fungal composition was mainly determined by soil organic carbon. Soil pH was the strongest factor to predict bacterial metabolic functions. Our findings highlight that even the differences of microbial compositions were because of different revegetation treatments, but each trophic microbial composition had different relations with plant and/or soil; especially, the bacterial community and metabolic functions and saprotrophic fungal community were more correlated with soil properties rather than plant community or characteristics per se.

Revegetation is widely used to enhance degraded topsoil recovery with the enhancements of soil nutrient accumulation and soil structure stabilization. Arbuscular mycorrhizal fungi (AMF) are important for the allocation of carbon into the soil and the formation of soil aggregates. Thus, we hypothesized that AMF could construct more niches for other microbes during revegetation, making AMF keystone taxa of soil. Soil fungal and bacterial communities were investigated under a revegetation experiment and correlation networks between soil fungi and bacteria were constructed. Simultaneously, the plant growth level, soil properties and structure, and soil microbial carbon decomposition abilities were measured. The results revealed that AMF were the most central fungi at the phylum (degree = 3), class (degree = 11), and family (degree = 15) levels. The reads number of AMF were positively correlated with both fungal (R2 = 0.431, P

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpesviruses (oHSV) has a major impact on cancer therapy marred by rare failures of healthy organs. Furthermore, tumors vary with respect to susceptibility to oncolytic effects of oHSV. Here we report the construction and properties of 3 families of oncolytic herpes simplex viruses expressing no immunomodulatory genes (T1 series), murine IL-12 (T2 series) or murine or human IL-12 and anti PD-1 antibody (T3 series). We report that insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). The T3 oHSV expressed IL-12, PD-1 Ab were restricted to the tumor bed whereas the induced IFN-γ accumulated to high levels both in tumor bed and in blood. Furthermore, the T3 oHSV was superior to systemic administration of IL-12 and antibody to PD-1. This report also shows that the oncolytic activity of T3 oHSV in a relatively resistant tumor was enhanced by concurrent intratumoral administrationof genetically engineered exosomes carrying miRNA targeting CTLA-4 checkpoint.

Tumor expression of immune co-inhibitory ligands, such as PD-L1 and Galectin-9, have potential prognostic value in Hepatocellular Carcinoma (HCC). Circulating levels of these molecules, however, have hardly been studied. This study aims to assess the prognostic significance of circulating PD-L1 and circulating Galectin-9 in patients with resected HCC, and to compare their prognostic significance to the intra-tumoral expression of these same molecules. Archived tissues and stored peripheral blood samples from 81 patients who underwent HCC resection or liver transplantation, with curative intent, were used. Immunohistochemistry was performed to determine intra-tumoral expression of PD-L1 and Galectin-9, while ELISA was used to quantify their respective circulating levels. High circulating PD-L1 (HR 0.12, 95%CI 0.16–0.86, p = 0.011) and high circulating Galectin-9 (HR 0.11, 95%CI 0.15–0.85, p = 0.010) levels were both associated with improved HCC-specific survival. Surprisingly, there was no correlation between circulating levels of PD-L1 and Galectin-9 and their intra-tumoral expression levels. In fact, circulating levels of PD-L1 and Galectin-9 were predictive of HCC-specific survival independently of intra-tumoral levels and baseline clinicopathologic characteristics. Combined analysis of circulating levels and intra-tumoral expression of PD-L1 (HR 0.33, 95%CI 0.16–0.68, p = 0.002) and Galectin-9 (HR 0.27, 95%CI 0.13–0.57, p = 0.001) resulted in more confident prediction of survival. In conclusion, circulating PD-L1 and Galectin-9 levels prognostically differentiate resected HCC patients, independently of their intra-tumoral expression. Combining circulating and intra-tumoral expression levels of PD-L1 or Galectin-9 further improves the prognostic values of these immune biomarkers.

Breeding new varieties of Michelia with Michelia maccluri as male parent and Michelia yunnanensis as female parent. The results showed that: ‘Yuxia’ has the characteristics of excellent ornamental characters, stable ornamental characters and wide adaptability. It is suitable for cultivation in southern China, especially in most areas of the Yangtze River, and has a good application prospect.

Magnolia ‘Wulin’ is a new cultivar of Magnolia officinalis. It belongs to small evergreen or semi-evergreen trees, with leathery leaves and slender petioles. It has fragrant flowers, and solitary branches top, petals are pink, filaments are deep red. The flowering period is from mid-April to mid-May. It is a kind of valuable plant with great potential value.Magnolia ‘Wulin’ is a new cultivar of Magnolia officinalis. It belongs to small evergreen or semi-evergreen trees, with leathery leaves and slender petioles. It has fragrant flowers, and solitary branches top, petals are pink, filaments are deep red. The flowering period is from mid-April to mid-May. It is a kind of valuable plant with great potential value.

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles

Oncolytic viruses are genetically engineered viruses designed for the treatment of solid tumors, and are often coupled with the antitumor immunity of the host. The challenge of using oncolytic herpes simplex virus (oHSV) as an efficacious oncolytic agent is the potential host tissue damage caused by the production of a range of cytokines following intratumoral oHSV injection. An HSV‑suppressor of cytokine signaling 4 (SOCS4) recombinant virus was created to investigate whether it inhibits cytokine storm. Recombinant HSV‑SOCS4 and HSV‑1(F) were used to infect mice, and levels of several representative cytokines, including monocyte chemoattractant protein‑1, interleukin (IL)‑1β, tumor necrosis factor‑α, IL‑6 and interferon γ, in serum and bronchoalveolar lavage fluid (BALF) of infected mice were determined, and immune cells in BALF and spleen were enumerated. Lung damage, virus titers in the lung, body weight and survival rates of infected mice were also determined and compared between the two groups. The cytokine concentration of HSV‑SOCS4‑infected mice was significantly decreased compared with that of HSV‑1(F)‑infected mice in BALF and serum, and a smaller number of cluster of differentiation (CD)11b+ cells of BALF, and CD8+CD62L+ T cells and CD4+CD62L+ T cells of the spleen were also identified in HSV‑SOCS4‑infected mice. HSV‑SOCS4‑infected mice exhibited slight lung damage, a decrease in body weight loss and a 100% survival rate. The results of the present study indicated that SOCS4 protein may be a useful regulator to inhibit cytokine overproduction, and that HSV‑SOCS4 may provide a possible solution to control cytokine storm and its consequences following induction by oncolytic virus treatment.

The stress response genes encoding GADD45γ, and to a lesser extent GADD45β, are activated early in infection with herpes simplex virus 1 (HSV-1). Cells that had been depleted of GADD45γ by transfection of short hairpin RNA (shRNA) or in which the gene had been knocked out (ΔGADD45γ) yielded significantly less virus than untreated infected cells. Consistent with lower virus yields, the ΔGADD45γ cells (either uninfected or infected with HSV-1) exhibited significantly higher levels of transcripts of a cluster of innate immunity genes, including those encoding IFI16, IFIT1, MDA5, and RIG-I. Members of this cluster of genes were reported by this laboratory to be activated concurrently with significantly reduced virus yields in cells depleted of LGP2 or HDAC4. We conclude that innate immunity to HSV-1 is normally repressed in unstressed cells and repression appears to be determined by two mechanisms. The first, illustrated here, is through activation by HSV-1 infection of the gene encoding GADD45γ. The second mechanism requires constitutively active expression of LGP2 and HDAC4. IMPORTANCE Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00111-6) contains supplementary material, which is available to authorized users.

Soil respiration (Rs) is an important source of atmospheric CO2 flux and is sensitive to changes in soil nutrient and water contents. Despite extensive studies on the effects of enhanced atmospheric nitrogen (N) deposition and changes in precipitation (P) on Rs, few studies have taken into account the effects of interactions between these factors on Rs of alpine grasslands. To address these questions, we investigated the effects of N addition (10 g N m−2 yr−1), changes in precipitation (±50% precipitation), and their interaction on soil respiration and its components, including heterotrophic respiration (Rh) and autotrophic respiration (Ra),in a Tibetan alpine steppe during three consecutive growing seasons. We found that Rs differed in its response to N addition and precipitation regimes. Specifically, decreased precipitation led to a significant reduction in Rs during the last two years, whereas N addition minimally impacted Rs. Another important finding was that soil respiration components differed in their response to N addition and precipitation regimes. Nitrogen addition significantly enhanced Ra, whereas Rh was not altered in response to N addition. By contrast, the precipitation regime led to marked changes in Rh, but exhibited marginally significant effects on Ra. Therefore, our findings highlighted that soil respiration differed in its response to N addition and precipitation regimes mainly due to the different responses of soil respiration components to these factors. Therefore, carbon dynamics should take soil respiration components into account under global change scenarios.

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

Anthraquinone glycosides, such as chrysophanol 1-O-β-d-glucoside, chrysophanol 8-O-β-d-glucoside and physion 8-O-β-d-glucoside, are the accepted important active components of Rheum tanguticum Maxim. due to their pharmacological properties: antifungal, anti-microbial, cytotoxic and antioxidant activities. However, an effective method for the separation of the above anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1-O-β-d-glucoside and chrysophanol 8-O-β-d-glucoside. This study demonstrated an efficient strategy based on preparative high-performance liquid chromatography and high-speed countercurrent chromatography for the separation of the above anthraquinone glycosides from Rheum tanguticum Maxim. This article is protected by copyright. All rights reserved

Background The inflammatory responses evoked by artificial organs and implantation of devices like biosensors and guide wires can lead to acute and chronic inflammation, largely limiting the functionality and longevity of the devices with negative effects on patients. Aims The present study aimed to reduce the inflammatory responses to biomaterials by covalent immobilization of glycosaminoglycans (GAGs) on amino-terminated surfaces used as model biomaterials here. Methods and Results Water contact angle (WCA) and zeta potential measurements showed a significant increase in wettability and negative charges on the GAG-modified surfaces, respectively, confirming the successful immobilization of GAGs on the amino-terminated surfaces. THP-1-derived macrophages were used as a model cell type to investigate the efficacy of GAG-modified surfaces in modulating inflammatory responses. It was found that macrophage adhesion, macrophage spreading morphology, foreign body giant cell (FBGC) formation, as well as β1 integrin expression and interleukin-1β (IL-1β) production were all significantly decreased on GAG-modified surfaces compared to the initial amino-terminated surface. Conclusions This study demonstrates the potential of covalent GAG immobilization to reduce the inflammatory potential of biomaterials in different clinical settings.

Soil moisture plays a vital role in regulating the direction and magnitude of methane (CH4) fluxes. However, it remains unclear whether the responses of CH4 fluxes to climate warming exhibit difference between dry and moist ecosystems. Based on standardized manipulative experiments (i.e., consistent experimental design and measurement protocols), here we explored warming effects on growing season CH4 fluxes in two alpine grasslands with contrasting water status on the Tibetan Plateau. We observed that experimental warming enhanced CH4 uptake in the relatively arid alpine steppe, but had no significant effects on CH4 emission in the moist swamp meadow. The distinct responses of CH4 fluxes were associated with the different warming effects on biotic and abiotic factors related to CH4 oxidation and production processes. Warming decreased soil water-filled pore space (WFPS) and increased the pmoA gene abundance and CH4 oxidation potential in the alpine steppe, which together led to a significant increase in CH4 uptake at this alpine steppe site. However, warming-induced enhancement in CH4 oxidation potential might be counteracted by the simultaneously increased CH4 production potential in the swamp meadow, which could then result in insignificant warming effects on CH4 emission at this swamp meadow site. Based on a meta-analysis of warming effects on CH4 fluxes across the entire Tibetan Plateau, we found that the entire alpine grasslands could absorb an extra 0.042 Tg CH4 (1 Tg = 1012 g) per growing season if soil temperature increased by 1 °C. These findings demonstrate that warming effects on CH4 fluxes differ between two alpine grasslands with contrasting moisture conditions and the entire alpine grasslands may not trigger a positive CH4 feedback to climate system with moderate warming.

Metabolic reprogramming universally occurs in cancer. Mitochondria act as the hubs of bioenergetics and metabolism. The morphodynamics of mitochondria, comprised of fusion and fission processes, are closely associated with mitochondrial functions and are often dysregulated in cancer. In this study, we aim to investigate the mitochondrial morphodynamics and its functional consequences in human liver cancer. We observed excessive activation of mitochondrial fusion in tumor tissues from hepatocellular carcinoma (HCC) patients and in vitro cultured tumor organoids from cholangiocarcinoma (CCA). The knockdown of the fusion regulator genes, OPA1 (Optic atrophy 1) or MFN1 (Mitofusin 1), inhibited the fusion process in HCC cell lines and CCA tumor organoids. This resulted in inhibition of cell growth in vitro and tumor formation in vivo, after tumor cell engraftment in mice. This inhibitory effect is associated with the induction of cell apoptosis, but not related to cell cycle arrest. Genome-wide transcriptomic profiling revealed that the inhibition of fusion predominately affected cellular metabolic pathways. This was further confirmed by the blocking of mitochondrial fusion which attenuated oxygen consumption and cellular ATP production of tumor cells. In conclusion, increased mitochondrial fusion in liver cancer alters metabolism and fuels tumor cell growth.

Background & Aims: Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. Methods: We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. Results: Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. Conclusions: Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. Lay summary: The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking “immune checkpoint” molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Host responses toward foreign implants that lead to chronic inflammation and fibrosis may result in failure of the biomedical device. To solve these problems, first a better understanding of the biomaterial-induced host reactions including protein adsorption, leukocyte activation, inflammatory and fibrotic responses to biomaterials is required; second an improved design of biomaterial surfaces is needed that results in an appropriate host response, causing less inflammatory response, and supporting tissue regeneration. Hence, this review provides a brief overview on the host response to implants, as well as in vitro models to study inflammatory and fibrotic responses to biomaterials to predict the clinical outcome of implantation. Moreover, the review highlights anti-inflammatory strategies to improve the biocompatibility of implants, which contain the modification of physicochemical surface properties of materials as well as the immobilization of anti-inflammatory reagents and bioactive molecules on biomaterials.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.

Background and Objectives Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8+ cytotoxic T‐cells and FoxP3+ regulatory T‐cells at the metastatic site of CRCLM patients. Methods TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin‐embedded tissue, from the same patients, was retrieved. CD8+ and FoxP3+ cells, both in the intra‐tumoral and the peri‐tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T‐cells (CD8+) and regulatory T‐cells (CD4+CD25+FoxP3+), within CD45+TILs, were measured by flow‐cytometry. Results By immunohistochemistry, individual densities of intra‐tumoral or peri‐tumoral CD8+ and FoxP3+ cells were not prognostic of survival. However, the intra‐tumoral, but not the peri‐tumoral, CD8+/FoxP3+ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19‐0.95, P = 0.032). By flow cytometry, the intra‐tumoral CD8+/regulatory T‐cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20‐0.99, P = 0.044). Conclusions The ratio of cytotoxic (CD8+) to regulatory (FoxP3+) T‐cells, in the intra‐tumoral compartment, but not in the peri‐tumoral compartment, can predict survival after resection of CRCLM.

Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC.

The separation of polar compounds by high-speed countercurrent chromatography is still regarded as a challenge. In this study, an efficient strategy for the separation of three polar compounds from Rheum tanguticum has been successfully conducted by using high-speed countercurrent chromatography. X-5 macroporous resin chromatography was used for the fast enrichment of the target compounds. Then, the target fraction was directly introduced into high-speed countercurrent chromatography for separation using ethyl acetate/glacial acetic acid/water (100:1:100, v/v/v) as the solvent system. Consequently, three polar compounds including gallic acid, catechin, and gallic acid 4-O-β-d-(6'-O-galloyl) glucoside were obtained with purities higher than 98%. The results showed glacial acetic acid could be such an appropriate regulator for the ethyl acetate/water system. This study provides a reference for the separation of polar compounds from natural products by high-speed countercurrent chromatography.

Chronic inflammatory responses after implantation of biomaterials can lead to fibrotic encapsulation and failure of implants. The present study was designed to reduce the inflammatory responses to biomaterials by assembling polyelectrolyte multilayers (PEMs) composed of glycosaminoglycans (GAGs) and chitosan (Chi) on glass as model surfaces through layer-by-layer (LBL) technique. Surface plasmon resonance (SPR) and water contact angle (WCA) investigations confirmed the multilayer build-up with alternating deposition of GAGs and Chi layers, while zeta potential measurements showed significant negative charges after multilayer deposition, which further proved the PEM formation. Macrophage adhesion, macrophage spreading morphology, foreign body giant cell (FBGC) formation, as well as β1 integrin expression and interleukin-1β (IL-1β) production were all significantly decreased by GAG-Chi multilayer deposition in comparison to the primary poly (ethylene imine) (PEI) layer. Thereby, the type of GAGs played a pivotal role in inhibiting the inflammatory responses to various extents. Especially heparin (Hep)-Chi multilayers hindered all inflammatory responses to a significantly higher extent in comparison to hyaluronic acid (HA)-Chi and chondroitin sulfate (CS)-Chi multilayer systems. Overall, the present study suggests a great potential of GAG-Chi multilayer coating on implants, particularly the Hep-Chi based systems, to reduce the inflammatory responses.

On entry into the body herpes simplex viruses (HSV) replicate in a series of steps that involves derepression of viral DNA activated by VP16, a virion protein, and sequential transcription of viral genes in a cascade fashion. HSV also enters into neurons in which viral DNA maintained as heterochromatin and with few exceptions viral gene expression is silenced. A third face of the interaction of HSV with its host cells takes place at the moment when the silenced viral genome in neurons is abruptly derepressed. The available data do no reveal evidence that HSV encodes different regulatory programs for each facet of its interaction with its host cells. Rather the data point to significant gaps in our knowledge of the mechanisms by which each facet is initiated and the roles of the infected cells at each facet of the interaction of viral gene products with the host cell.

Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4 + and CD8 + T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8 + and CD4 + T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8 + and CD4 + T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8 + TIL, compared with other CD8 + TIL. Compared with TIL that did not express these inhibitory receptors, CD8 + and CD4 + TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8 + and CD4 + TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

In plants with infrequent pollinator services, the benefits of reproductive assurance could be eroded by severe ovule discounting and inbreeding depression (ID). However, it remains unclear how selfing evolves under complete pollinator failure and strong ID. We examined the mating system and ID under netting and robbing conditions in Comastoma pulmonarium (Turcz.) Toyok. (Gentianaceae), an alpine annual experiencing a high ratio of nectar robbery on the Qinghai-Tibet Plateau. Comastoma pulmonarium produced seeds via selfing at the study site. No pollinator was observed and thus the nectar was consumed exclusively by robbers. Inbreeding depression occurred in the life stages of seed mass and germination, and the cumulative ID was much higher than 0.5 under netting and robbing conditions. Overall, in comparison with netting, the magnitude of ID under robbing conditions tended to decrease. Our results suggested that selfing could assure reproduction for plants under complete pollinator failure and strong ID, supporting the better than nothing role of selfing and providing one of the few cases of the evolution of selfing under strong ID.

Surface properties are believed to play important roles in initial inflammatory and subsequent wound healing/fibrotic responses after implantation of biomaterials. To investigate the surface property effect in mediating these host responses, we used an in vitro fibroblast/macrophage co-culture model established with a cell migration chamber, and a series of self-assembling monolayers (SAMs) bearing different terminal groups as model surfaces to study the effect of surface properties on macrophage fusion, fibroblast attachment, spreading morphology, proliferation, outgrowth, as well as pro-(interleukin-6) and anti-(interleukin-10) inflammatory cytokine production, expression of ED-A fibronectin (FN) and alpha-smooth muscle actin (α-SMA). The obtained results show that the hydrophobic CH3 surface caused high levels of inflammatory but low levels of wound healing/fibrotic responses, while the hydrophilic/anionic COOH surface resulted in both low levels of inflammatory and wound healing/fibrotic responses. Interestingly, the hydrophilic OH surface was found to possess a low potential of inducing inflammatory responses but high potential of inducing wound healing/fibrotic responses. These results reveal that the extent of inflammation and wound healing/fibrosis might not be always related in vitro. However, more important is the observation of the macrophage contributions in facilitating the wound healing and fibrotic responses by up-regulation of fibroblast outgrowth, cytokine production as well as ED-A FN and α-SMA expression. Overall, by linking the surface properties to cell activities with our established fibroblast/macrophage co-culture system, we could provide an useful model system for in vitro studies to design more biocompatible biomaterials for various biomedical and tissue engineering applications.

e14250 Background: Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Methods: We constructed 3 families of recombinant oHSVs expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). We compared the oncolytic effects of a single or multiple intratumoral injection(s) of T1, T2 and T3 series oHSVs. We also compared the effectiveness of intratumoral injection of T3 oHSV with systemic administration of anti PD-1 ab and IL12 alone or in combinations with T1. Results: Insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). In syngeneic mice the T3 series oHSV expressing murine IL-12 and PD-1 Ab was effective against a variety of murine tumors. Concurrent with enhanced cytolytic activity the T3 oHSV induced the production and significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ. Consistent with an earlier report we noted an inverse correlation between the volume of the tumor and the quantity of retained IFN-γ. The most effective oncolytic effect resulted from the administration of T3855 expressing both IL12 and anti PD-1 antibody or T2850 concurrently with intraperitoneal administration of anti PD-1 antibody alone. The least effective were single intraperitoneal administration of IL12 or PD-1 antibody. Intratumoral injection of T3 oHSV was most effective against murine tumors in comparison of either systemic administration of anti PD-1 ab and IL12 or intratumoral injection of T1 in combinations of systemic administration of anti PD-1 ab and IL12. Conclusions: We report the marriage in a single therapeutic agent of three distinct cancer therapies: the targeting of cancer cells by oncolytic viruses, the stimulation of immune system by IL12, and the production of immunotherapeutic antibodies against PD-1. The significant finding reported in this article is that the anti-tumor responses remain concentrated in the tumor environment.

High-latitude and high-altitude ecosystems store large amounts of carbon (C) and play a vital role in the global C cycle. Soil respiration (R S) in these ecosystems is believed to be extremely sensitive to climate warming and could potentially trigger positive C-climate feedback. However, this evidence is largely derived from wet ecosystems, with limited observations from dry ecosystems. Here, we explored the responses of R S, autotrophic (R A), and heterotrophic (R H) respiration under experimental warming in a dry ecosystem, an alpine steppe on the Tibetan Plateau. We assessed the effects of soil temperature and moisture dynamics on R S, R A, and R H and performed a meta-analysis to examine whether the warming effects observed were similar to those reported in wet ecosystems, including Tibetan alpine meadow and arctic ecosystem. Experimental warming did not alter R S, R A, and R H in this alpine steppe, likely because decreased soil moisture constrained positive warming effects. In contrast, the meta-analysis revealed that R S exhibited a significant increase under experimental warming in both the Tibetan alpine meadow and arctic wet tundra. These results demonstrate that R S exhibits different responses to climate warming between dry and wet ecosystems, suggesting potential more complex C-climate feedback in cold regions.

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Here we report the properties of 3 families of oHSV expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). Thus insertion of the genes encoding PD-1 Ab and IL-12 significantly augmented the oncolytic activity of oHSV bereft of immune-stimulatory genes (T1 series). In syngeneic mice the murine T3 oHSV induced significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ and was effective against a variety of murine tumors. Thus T3 oHSV concentrates the immunomodulatory products in the environment of the tumors thereby reducing the risk of toxicity associated with systemic administration. T3 oHSV injected into tumors was more effective than systemic administration of anti PD-1 Ab and IL12 alone or in combination with intratumoral injection of T1 oHSV. T3 induces local and systemic immune response characterized by cytokine signature. Citation Format: Grace Zhou, Dongyao Ni, Runbin Yan, Xiaoqing Chen, Xianjie Liu, Jie Ma, Yuxin Tang. New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-078.

Auxin and its homeostasis play key roles in many aspects of plant growth and development. Cadmium (Cd) is a phytotoxic heavy metal and its inhibitory effects on plant growth and development have been extensively studied. However, the underlying molecular mechanism of the effects of Cd stress on auxin homeostasis is still unclear. In the present study, we found that the root elongation, shoot weight, hypocotyl length and chlorophyll content in wild-type (WT) Arabidopsis seedlings were significantly reduced after exposure to Cd stress. However, the lateral root (LR) formation was markedly promoted by Cd stress. The level and distribution of auxin were both greatly altered in primary root tips and cotyledons of Cd-treated plants. The results also showed that after Cd treatment, the IAA content was significantly decreased, which was accompanied by increases in the activity of the IAA oxidase and alteration in the expression of several putative auxin biosynthetic and catabolic genes. Application of the auxin transport inhibitor, 1-naphthylphthalamic acid (NPA) and 1-naphthoxyacetic acid (1-NOA), reversed the effects of Cd on LR formation. Additionally, there was less promotion of LR formation by Cd treatment in aux1-7 and pin2 mutants than that in the WT. Meanwhile, Cd stress also altered the expression of PINs and AUX1 in Arabidopsis roots, implying that the auxin transport pathway is required for Cd-modulated LR development. Taken together, these findings suggest that Cd stress disturbs auxin homeostasis through affecting auxin level, distribution, metabolism, and transport in Arabidopsis seedling.

This study provides a vegetation and climate history of an alpine wetland in the centre of China’s Qinghai–Tibetan Plateau (QTP) over the last 14.8 ka. High resolution climate proxies include vegetation (pollen), geochemical (Ba, Rb, δ 13 C) and physical (particle size and magnetic susceptibility) data; optically stimulated luminescence (OSL) and AMS radiocarbon dating were used to reconstruct the environmental changes in the wetland ecosystem. Based on physical and geochemical proxies, the sucession divided into five stratigraphic units encompassing time intervals of 14.76–14.31, 14.31–13.17, 13.17–4.47, 4.47–0.51 and 0.51–0 ka. The average sedimentation rate of all these units was 0.098 mm/a. The study showed that this ecosystem was sensitive to climate change.

An effective high–speed counter–current chromatography (HSCCC) method was established for semi-preparative isolation and purification of bioactive chemical constituents from the Tibetan medicinal plant Pedicularis longiflora var. tubiformis (Klotzsch) P. C. Tsoong. With a two-phase solvent system composed of chloroform–methanol–water (8:4:5, v/v), 40 mg of extract was separated to yield luteolin (12.5 mg), apigenin (9.6 mg), and chrysoeriol (4.8 mg), with purities of 99.3, 98.2, and 98.6%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these three components were identified by 1H NMR and 13C NMR.

Unprecedented levels of nitrogen (N) have entered terrestrial ecosystems over the past century, which substantially influences the carbon (C) exchange between the atmosphere and biosphere. Temperature and moisture are generally regarded as the major controllers over the N effects on ecosystem C uptake and release. N-phosphorous (P) stoichiometry regulates the growth and metabolisms of plants and soil organisms, thereby affecting many ecosystem C processes. However, it remains unclear how the N-induced shift in the plant N:P ratio affects ecosystem production and C fluxes and its relative importance. We conducted a field manipulative experiment with eight N addition levels in a Tibetan alpine steppe and assessed the influences of N on aboveground net primary production (ANPP), gross ecosystem productivity (GEP), ecosystem respiration (ER) and net ecosystem exchange (NEE); we used linear mixed-effects models to further determine the relative contributions of various factors to the N-induced changes in these parameters. Our results showed that the ANPP, GEP, ER and NEE all exhibited nonlinear responses to increasing N additions. Further analysis demonstrated that the plant N:P ratio played a dominate role in shaping these C exchange processes. There was a positive relationship between the N-induced changes in ANPP (ΔANPP) and the plant N:P ratio (ΔN:P), while the ΔGEP, ΔER and ΔNEE exhibited quadratic correlations with the ΔN:P. By contrast, soil temperature and moisture were only secondary predictors for the changes in ecosystem production and C fluxes along the N addition gradient. These findings highlight the importance of plant N:P ratio in regulating ecosystem C exchange, which is crucial for improving our understanding of C cycles under the scenarios of global N enrichment. This article is protected by copyright. All rights reserved.

Anthraquinone glycosides, such as chrysophanol 1-O-β-d-glucoside, chrysophanol 8-O-β-d-glucoside, and physion 8-O-β-d-glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above-mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1-O-β-d-glucoside and chrysophanol 8-O-β-d-glucoside. This study demonstrated an efficient strategy based on preparative high-performance liquid chromatography and high-speed countercurrent chromatography for the separation of the above-mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.

High throughput sequencing technology is also called Next Generation Sequencing (NGS), which can sequence hundreds and thousands sequences in different samples at the same time. In the present study, the culture-independent high throughput sequencing technology was applied to sequence the fungi metagenomic DNA of the fungal internal transcribed spacer 1(ITS 1) in the root of Sinopodophyllum hexandrum. Sequencing data suggested that after the quality control, 22 565 reads were remained. Cluster similarity analysis was done based on 97% sequence similarity, which obtained 517 OTUs for the three samples (LD1, LD2 and LD3). All the fungi which identified from all the reads of OTUs based on 0.8 classification thresholds using the software of RDP classifier were classified as 13 classes, 35 orders, 44 family, 55 genera. Among these genera, the genus of Tetracladium was the dominant genera in all samples(35.49%, 68.55% and 12.96%).The Shannon's diversity indices and the Simpson indices of the endophytic fungi in the samples ranged from 1.75-2.92, 0.11-0.32, respectively.This is the first time for applying high through put sequencing technol-ogyto analyze the community composition and diversity of endophytic fungi in the medicinal plant, and the results showed that there were hyper diver sity and high community composition complexity of endophytic fungi in the root of S. hexandrum. It is also proved that the high through put sequencing technology has great advantage for analyzing ecommunity composition and diversity of endophtye in the plant.