Effects of rpl1001 Gene Deletion on Cell Division of Fission Yeast and Its Molecular Mechanism.

The rpl1001 gene encodes 60S ribosomal protein L10, which is involved in intracellular protein synthesis and cell growth. However, it is not yet known whether it is involved in the regulation of cell mitosis dynamics. This study focuses on the growth, spore production, cell morphology, the dynamics of microtubules, chromosomes, actin, myosin, and mitochondria of fission yeast (Schizosaccharomyces pombe) to investigate the impact of rpl1001 deletion on cell mitosis. RNA-Seq and bioinformatics analyses were also used to reveal key genes, such as hsp16, mfm1 and isp3, and proteasome pathways. The results showed that rpl1001 deletion resulted in slow cell growth, abnormal spore production, altered cell morphology, and abnormal microtubule number and length during interphase. The cell dynamics of the rpl1001Δ strain showed that the formation of a monopolar spindle leads to abnormal chromosome segregation with increased rate of spindle elongation in anaphase of mitosis, decreased total time of division, prolonged formation time of actin and myosin loops, and increased expression of mitochondrial proteins. Analysis of the RNA-Seq sequencing results showed that the proteasome pathway, up-regulation of isp3, and down-regulation of mfm1 and mfm2 in the rpl1001Δ strain were the main factors underpinning the increased number of spore production. Also, in the rpl1001Δ strain, down-regulation of dis1 caused the abnormal microtubule and chromosome dynamics, and down-regulation of hsp16 and pgk1 were the key genes affecting the delay of actin ring and myosin ring formation. This study reveals the effect and molecular mechanism of rpl1001 gene deletion on cell division, which provides the scientific basis for further clarifying the function of the Rpl1001 protein in cell division.

Role of SARS­CoV­2 nucleocapsid protein in affecting immune cells and insights on its molecular mechanisms.

The present study aimed to explore the immune regulatory function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein and related mechanisms. In a series of protein activity experiments, SARS-CoV-2 N protein promoted proliferation of three immune cell lines: mouse Raw264.7, human Jurkat and human Raji in a dose-dependent manner. A total of 10 µg/ml N protein could significantly change cell cycle progression of the aforementioned three immune cell lines and could promote quick entry of Raw264.7 cells into G2/M phase from S phase to achieve rapid growth. Additionally, the N protein could also stimulate Raw264.7 cells to secrete a number of proinflammatory factors such as TNF-α, IL-6 and IL-10. RNA sequencing analysis indicated that the N protein changed the expression of certain genes involved in immune-related functions and four important signaling pathways, including JAK-STAT, TNF, NF-κB and MAPK signaling pathways, which suggested that the N protein may not only regulate the expression of genes involved in the process of resisting viral infection in macrophages of the immune system, but also change cellular signal processing.

Comparative Studies on the Structure and Biological Activities of Two New Polysaccharides from Tricholoma sinoportentosum (TS-P) and Termitomyces albuminosus (TA-P).

Polysaccharides are important active ingredients of living organisms. In this study, two new polysaccharides, Tricholoma sinoportentosum polysaccharide (TS-P) and Termitomyces albuminosus (TA-P), were extracted and purified using anion exchange column chromatography. The structure of each polysaccharide was identified by HPGPC, FT-IR, HPLC, GC-MS and NMR, and the biological activities were also investigated. The results of the structure identification showed that TS-P was composed of arabinose, mannose, glucose and galactose at a ratio of 1:1:3:2 and its main chain was composed of (1→4)-Arap residues, (1→4,6)-D-Manp residues and two (1→6)-Galp residues. The TA-P was composed of arabinose, glucose and galactose at a ratio of 2:4:8. Its main chain was composed of two (1→4)-ß-L-Arap residues, one (1→4)-Glcp residues, three (1→2,6)-Galp residues and five (1→6)-Galp residues. The immunoassay showed that TS-P and TA-P could significantly promote the proliferation of T cells, B cells and RAW264.7 cells. The cell cycle results showed that for B cells and macrophages, TS-P and TA-P mainly affected the G0/G1 phases of the cell cycle; for T cells, TS-P affected G2/M phase, while TA-P mainly affected the G0/G1 phases. TS-P could significantly promote B cells to secrete IgA, IgG and IgD (p < 0.01), while TA-P could significantly promote the secretion of IgA and IgG (p < 0.01). The chemical structure and biological activity of TS-P and TA-P were first studied and compared to lay a theoretical foundation for the application of fungal polysaccharide.

The Multiple Influences of Natural Farming Environment on the Cultured Population Behavior of Kuruma Prawn, Penaeus japonicus.

Recent years have witnessed a tremendous development in shrimp farming around the world, which, however, has raised a variety of issues, possibly due to a lack of knowledge of shrimp behavior in farms. This study focused on the relationship between shrimp behavior and the various factors of natural farming environment through situ surveys, as distinguished from the majority of laboratory studies on shrimp behavior. In the survey, the behaviors of kuruma prawn (Penaeus japonicus) were investigated in the groups of swimming in the water, crawling on the sand, resting on the sand, and hiding in the sand, followed by the quantification of the sex ratio, water quality, density, and light intensity. The results showed the average proportions of resting, hiding, crawling, and swimming activities of 69.87%, 20.85%, 8.24%, and 1.04%, respectively, of P. japonicus. The behavior of hiding, resting, and crawling is significantly affected by the sex ratio of the shrimp (p < 0.05). The proportions of hiding behavior exhibited a negative connection with density and a positive connection with light intensity, while the proportions of resting behavior showed the opposite according to both Pearson correlation analysis and multiple linear regression analysis. The light intensity was the only factor that significantly influenced the swimming behavior, in which the probability of the swimming behavior was reduced from 48% to 5% when light intensity varied from 0 to 10 lx, as determined by the generalized linear model. It could be speculated that P. japonicus prefers a tranquil environment. Female shrimp might exhibit less aggression and more adventure compared to male shrimp. The findings suggested light intensity, followed by density, as the most crucial element influencing the behavior of P. japonicus in the culture environment. These findings will contribute to the comprehension of the behavior of P. japonicus and provide a novel perspective for the formulation of its culture management strategy.

Loss of FZO1 gene results in changes of cell dynamics in fission yeast.

Mitochondrial fission and fusion dynamics are critical cellular processes, and abnormalities in these processes are associated with severe human disorders, such as Beckwith­Wiedemann syndrome, neurodegenerative diseases, Charcot­Marie­Tooth disease type 6, multiple symmetric lipomatosis and microcephaly. Fuzzy onions protein 1 (Fzo1p) regulates mitochondrial outer membrane fusion. In the present study, Schizosaccharomyces pombe (S. pombe) was used to explore the effect of FZO1 gene deletion on cell dynamics in mitosis. The mitochondrial morphology results showed that the mitochondria appeared to be fragmented and tubular in wild­type cells; however, they were observed to accumulate in fzo1Δ cells. The FZO1 gene deletion was demonstrated to result in slow proliferation, sporogenesis defects, increased microtubule (MT) number and actin contraction defects in S. pombe. The FZO1 gene deletion also affected the rate of spindle elongation and phase time at the metaphase and anaphase, as well as spindle MT organization. Live­cell imaging was performed on mutant strains to observe three distinct kinetochore behaviors (normal, lagging and mis­segregation), as well as abnormal spindle breakage. The FZO1 gene deletion resulted in coenzyme and intermediate metabolite abnormalities as determined via metabolomics analysis. It was concluded that the loss of FZO1 gene resulted in deficiencies in mitochondrial dynamics, which may result in deficiencies in spindle maintenance, chromosome segregation, spindle breakage, actin contraction, and coenzyme and intermediate metabolite levels.

Hepatopancreas Transcriptome Profiling Analysis Reveals Physiological Responses to Acute Hypoxia and Reoxygenation in Juvenile Qingtian Paddy Field Carp Cyprinus carpio var qingtianensis.

The Qingtian paddy field carp (Cyprinus carpio var qingtianensis) is a local carp cultivated in the rice field of Qingtian county, Zhejiang province, China. Its high tolerance to hypoxia makes it an ideal organism for studying the molecular regulation mechanism during hypoxia process as well as reoxygenation following hypoxia in fish. In this study, we counted the differentially expressed genes (DEGs) altered during hypoxic exposure and reoxygenation process. The results indicated that 2236 genes (1506 up-regulated genes and 730 down-regulated genes) were differentially expressed between the control and hypoxic groups. The results from Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that 1152 of 2236 genes were enriched, and those genes participated in energy metabolism, reactive oxygen species (ROS) elimination, acceleration of cell apoptosis, inhibition of growth, and other processes. We found activation of the pentose phosphate pathway in hypoxia treatment, suggesting that carbohydrates not only provide energy for metabolism but also provide NADPH for protecting the body from oxidative damage and ribosomes for promoting RNA synthesis. During reoxygenation, 4509 genes (1865 up-regulated genes and 2644 down-regulated genes) were differentially expressed. The results of KEGG enrichment analysis indicated that 2392 of 4509 genes were enriched, and participated in pyruvate and lactic acid metabolism, synthesis of amino acids and lipids, inhibition of cell apoptosis, regulation of cell growth and differentiation, and other processes. These differentially expressed genes effectively alleviate the body acidosis and promote the normal growth and development of the body. Through the analysis of KEGG pathway enrichment, we observed that the physiological regulation of Qingtian paddy field carp during the processes of hypoxia and reoxygenation is not a simple and reversible process. This work first reported the adaptive mechanism of hypoxia and the recovery mechanism of reoxygenation after hypoxia in common carp, and also provided new insights for the physiological regulation of fish under hypoxia treatment.

Structure identification, antitumor activity and mechanisms of a novel polysaccharide from Ramaria flaccida (Fr.) Quél.

It is an important aspect of current cancer research to search for effective and low-toxicity anticancer drugs and adjuvants. Polysaccharides, as immunomodulators, can improve the immune function of the body, kill tumor cells directly and prevent tumor development by increasing the resistance of the body to carcinogenic factors. The aim of the present study was to identify natural polysaccharide compounds with novel structure and antitumor activity via the separation and analysis of polysaccharide components from Ramaria flaccida (Fr.) Quél. (RF-1). In the present study, high-performance gel permeation chromatography, gas chromatography-mass spectrometry and nuclear magnetic resonance were used to identify the structure of polysaccharides from RF-1. Subsequently, the antitumor activity and mechanism of RF-1 were studied by establishing an in vivo S180 tumor model, and by using Illumina sequencing technology and enzyme-linked immunosorbent assay (ELISA). The present results revealed that the average molecular weight of RF-1 was 17,093 Da and that RF-1 was composed of the monosaccharides glucose and galactose, with a 2:1 ratio. The main chain of RF-1 consisted of (1→6, 2)-α-D-galactopyranose and (1→6, 4)- α-D-glucopyranose. One of the branched chains was linked to 4-O of the main glucose chain by (1→6)-α-D-glucopyranose and next linked by one (→4)-ß-D-glucopyranose. The other two branched chains were both linked to 2-O of the main glucose chain by one (→4)-ß-D-glucopyranose. In addition, RF-1 inhibited the growth of S180 tumors in vivo. When the concentration of RF-1 was 20 mg/kg, the inhibition rate of S180 tumors in mice was 48.4%. Compared with the blank control group, 1,971 differentially expressed genes were identified, of which 818 were upregulated and 1,153 were downregulated in the RF-1 group. A Gene Ontology enrichment analysis generated 47,091 assignments to biological processes, 5,250 assignments to cellular components, and 6,466 assignments to molecular functions. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the Wnt and MAPK signaling pathways were significantly enriched. The number of differentially annotated genes in these two pathways was 19 and 33, respectively. Additionally, ELISA results revealed that the protein levels of interleukin (IL)-1ß, IL-6, vascular endothelial growth factor (VEGF) and VEGF receptor in the RF-1 group were significantly downregulated compared with the S180 blank control group (P<0.01).

Comparative analysis of transcriptomes revealed the molecular mechanism of development of Tricholoma matsutake at different stages of fruiting bodies.

The purpose of the study is to investigate the molecular mechanisms of development of Tricholoma matsutake fruiting body at the primordial stage (TM-1), the intermediate stage (TM-2) and the mature stage (TM-3) using RNA-Seq sequencing technology. The analysis of gene expression level revealed that the Spn2 and Eef1a1 gene were the key genes in the primordial stage of T. matsutake by regulating cytokinesis, protein synthesis, and cell growth. And the Ubc, Atp6, Cytb, and Pth2 gene were the key genes in the mature stage of T. matsutake by regulating energy metabolism and protein synthesis. Differential expression genes (DEGs) analysis results showed that Cdc28, Rad53, Dun1, Pho85 and Pho81 were the key DEGs regulating cell cycle genes of T. matsutake from primordial stage to intermediate stage. And APC, Cyr1, Cdc45, Spo11 and Rec8 genes were the key DEGs for the meiosis and sporogenesis of T. matsutake from the intermediate stage to the mature stage.

Overexpression of PTEN may increase the effect of pemetrexed on A549 cells via inhibition of the PI3K/AKT/mTOR pathway and carbohydrate metabolism.

Lung cancer is one of the leading causes of tumor­associated mortality, and >75% of patients with lung cancer have non­small cell lung cancer (NSCLC). Pemetrexed, a folate antagonist, is a first­line chemotherapy drug for NSCLC that is administered alone or in combination with cisplatin. The present study established in vitro cell models of PTEN inhibition and overexpression, and the effects of the treatment with pemetrexed were investigated in these cell models. Result from the present study demonstrated that treatment with pemetrexed suppressed lung cancer cell proliferation, inhibited mRNA and protein expression levels of anti­apoptotic Bcl2, and increased the mRNA and the protein expression levels of pro­apoptotic p53 and apoptosis regulator BAX. The present study suggested that pemetrexed regulated apoptosis via the inhibition of the mTOR/PI3K/AKT signaling pathway. Additionally, cellular processes associated with the aerobic oxidation of carbohydrates were identified to be significantly inhibited. The present findings suggested that treatment with pemetrexed may exhibit synergistic effects with PTEN on lung cancer cells via the inhibition of the PI3K/AKT/mTOR signaling pathway and through carbohydrate metabolism, and treatment with pemetrexed combined with PTEN overexpression may represent a novel therapeutic strategy for the treatment of NSCLC.

Structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray.

The present study investigated the structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray. Chemical methods, high performance gel permeation chromatography, fourier transform infrared spectroscopy, nuclear magnetic resonance spectrum and gas chromatography­mass spectrometry were used to characterize the polysaccharide structure. The immunomodulatory abilities of the Maerkang L. deliciosus Gray polysaccharide (LDG­M) were also investigated. LDG­M was primarily composed of ß­D­glucose and α­D­lyxose with the ratio of 2:1. The possible structure of LDG­M had a backbone of 1,6­linked­ß­D­glucose and 1,4,6­linked­ß­D­glucose, with branches primarily composed of one (1→4)­linked­α­D­lyxose residue. The immunoregulatory activity results demonstrated that LDG­M promoted the proliferation and phagocytosis of macrophages, and induced cytokine release. LDG­M also promoted the proliferation of B cells by affecting the G0/G1, S and G2/M phases. The present study introduced LDG­M as a valuable source with unique immunoregulatory properties.

Protein chip analysis of cytokines reveals a key mechanism of the antitumor and immunostimulatory activities of Tricholoma matsutake polysaccharide.

Polysaccharide has been widely used in medical and health field because of its function of immune regulation. The aim of present study was to use protein chip to test the 200 cytokines secreted by macrophages which were induced by the polysaccharides of Tricholoma matsutake (TMP-A) to study the role of TMP-A acting on macrophages and its mechanism, further understanding the mechanism of the TMP-A effect on immune activity. The results of the analysis indicated that among all of these cytokines, including IL-1ß, IL-10, IL-23, TNF-α, CD40L, G-CSF, etc. there are 73 up-regulated and 43 down-regulated cytokines. The KEGG analysis indicated that T. matsutake polysaccharide can influence the immune response of macrophages through a series of signaling pathways, and the three major signaling pathways are Jak-STAT signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Those three signaling pathway are closely related to the pathogenesis of many diseases. The results showed that TMP-A can activate immune cells to regulate the immunity.

Effect on macrophage proliferation of a novel polysaccharide from Lactarius deliciosus (L. ex Fr.) Gray.

The fundamental mechanisms underlying the preventional and therapeutic effects of polysaccharides from fungi, including the immunostimulatory, antiviral and antitumor effects, are considered to occur through the modulation and stimulation of the macrophage and complement system. LDG-A, a novel polysaccharide from Lactarius deliciosus (L. ex Fr.) Gray exhibits marked antitumor activities in vivo. However, the underlying molecular mechanism of the antitumor activities of LDG-A remains unclear. In the present study, cell cycle analysis was performed in macrophages and B cells, and the transcriptomes of macrophages in the control group and LDG-A group were sequenced using Illumina sequencing technology to analyze the differentially expressed genes (DEGs), and elucidate the molecular mechanisms underlying the immunomodulatory and antitumor activities of LDG-A. The cell cycle analysis results indicated that LDG-A was able to promote the proliferation of B cells by promoting cell cycle progression in S phase and G2/M phase and eliminating cell cycle arrest in G0/G1, and promote the proliferation of macrophages by promoting cell cycle progression in G0/G1 phase and eliminating cell cycle arrest in G2/M phase. Of the total number of genes (8,140), ~77.00% were expressed [reads per kilobase per million reads (RPKM) ≥1] and 1,352 genes were highly expressed (RPKM >60) in the LDG-A group. Of 775 unigenes which were identified as DEGs, 469 were downregulated and 306 genes were upregulated. A protein chip method was also used to determine the cytokines secreted by macrophages. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and GO enrichment analysis indicated that the Janus kinase/signal transducer and activator of transcription, mitogen-activated protein kinase, chemokine, vascular endothelial growth factor and transforming growth factor ß signaling pathways are markedly enriched for DEGs.

Structure elucidation and bioactivities of a new polysaccharide from Xiaojin Boletus speciosus Frost.

A new water-soluble polysaccharide named BSF-X was extracted and purified from the fruiting bodies of Boletus speciosus Frost which had a molecular weight of 141,309 Da. The structure identification results showed that BSF-X was mainly composed of ß-d-glucose and α-D-galactose. BSF-X had a backbone of 1, 4-linked ß-d-glucose of which branches were mainly composed of two 1, 6-linked α-D-galactose residue and a 4-linked ß-d-glucose at the end of the branches. Antitumor activities results showed that BSF-X could inhibit the proliferation of L929 cells in vitro and S180 tumor cells in vivo. Immunoregulatory activities results showed that BSF-X could promote the proliferation of T cells, B cells and macrophages by promoting the cells into S phase from G0/G1 phase. Polysaccharide BSF-X can also enhance the phagocytes is and cytokine secretion of macrophages. This study introduced the new polysaccharide BSF-X as a valuable source which exhibit unique antitumor and immunoregulatory properties.

Structural characterization, immune regulation and antioxidant activity of a new heteropolysaccharide from Cantharellus cibarius Fr.

A new heteropolysaccharide was extracted and purified from the fruiting bodies of Cantharellus cibarius Fr. The Cantharellus cibarius Fr. polysaccharide (CC-1) had a molecular weight of 61,056 kDa and was mainly formed of the glucose and xylose at ratio of 5:1. Structure identification of CC-1 was analysed by a combined application of total hydrolysis, high performance liquid chromatography (HPLC), methylation analysis, gas chromatography-mass spectrometry (GC-MS), infrared (IR) spectra and nuclear magnetic resonance (NMR) spectroscopy. The experimental results showed that CC-1 had a backbone of 1,4-linked-ß-D-glucose which branched at O-6 and the branches were mainly composed of 6→1)-α-D-xylopyranose residue. CC-1 exhibited significant in vitro antioxidant effect and proliferation effect of immune cells. The activity study showed CC-1 has ability to clear the ABTS+ free radical and DPPH- free radical in a certain range of concentration. The proliferation activity of the immune cells showed that the proliferation effect on B cells was very significant (P<0.001) in the concentration of 0.625-80 mg/ml; and the effect of T cell proliferation was also very significant (P<0.001) in the concentration of 5-20 mg/ml. The result of this study introduced Cantharellus cibarius Fr. as a possible valuable source in exhibiting unique immunoregulatory and antioxidant properties.

Comparative Analysis of Transcriptomes of Macrophage Revealing the Mechanism of the Immunoregulatory Activities of a Novel Polysaccharide Isolated from Boletus speciosus Frost.

BACKGROUND: The mechanism of the immunoregulatory activities of polysaccharide is still not clear. MATERIALS AND METHODS: Here, we performed the B-cell, T-cell, and macrophage cell proliferation, the cell cycle analysis of macrophage cells, sequenced the transcriptomes of control group macrophages, and Boletus speciosus Frost polysaccharide (BSF-1) group macrophages using Illumina sequencing technology to identify differentially expressed genes (DEGs) to determine the molecular mechanisms of immunomodulatory activity of BSF-1 in macrophages. RESULTS: These results suggested that BSF-1 could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell division. A total of 12,498,414 and 11,840,624 bp paired-end reads were obtained for the control group and BSF-1 group, respectively, and they corresponded to a total size of 12.5 G bp and 11.8 G bp, respectively, after the low-quality reads and adapter sequences were removed. Approximately 81.83% of the total number of genes (8,257) were expressed reads per kilobase per million mapped reads (RPKM ≥1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 group. A gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functions. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathways are significantly enriched for DEGs between the two cell groups. CONCLUSION: An analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of BSF-1. Based on the experimental data, we believe that the significant antitumor activities of BSF-1 in vivo mainly involve the MAPK signaling pathways. SUMMARY: Boletus speciosus Frost-1 (BSF-1) could promote the proliferation of B-cell, T-cell, and macrophages, promote the proliferation of macrophage cells by abolishing cell cycle arrests in the G0/G1 phases, and promote cell cycle progression in S-phase and G2/M phase, which might induce cell divisionApproximately 81.83% of the total number of genes (8257) were expressed (reads per kilobase per million mapped reads [RPKM] =1) and more than 1366 genes were highly expressed (RPKM >60) in the BSF-1 groupA gene ontology-enrichment analysis generated 13,042 assignments to cellular components, 13,094 assignments to biological processes, and 13,135 assignments to molecular functionsA Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the mitogen-activated protein kinase signaling pathways are significantly enriched for DEGs between the two cell groups. Abbreviations used: BSF-1: Boletus speciosus Frost polysaccharide.

Structure elucidation and antitumor activity of a new polysaccharide from Maerkang Tricholoma matsutake.

A new heteropolysaccharide was isolated from the fruiting bodies of Tricholoma matsutake which had a molecular weight of 12078 Da. The results of structural features analysis showed that T. matsutake polysaccharide, here named TMP-B, was mainly composed of α - D - glucose and α - D - galactose which ratios were 7:2 and had a backbone of 1, 4 - linked α - D - glucose which branches were mainly composed of two 6 - linked α - D - galactose residue, and the α - D - galactose was 1, 6 - linked. Antitumor activity results showed that heteropolysaccharide TMP-B could inhibit the growth of S180 tumor in vivo and promote the apoptosis of L929 cells in vitro. Immunoregulatory activity results showed that TMP-B could promote the proliferation of macrophages by affecting G0/G1 phase, S phases and G2/M phases and promote cytokines release and gene expression. The result of this study introduced Maerkang T. matsutake as a possible valuable source which helped to exhibit unique antitumor and immunoregulatory properties.

[Tumor Associated Fibroblasts Promote PD-L1 Expression in Lung Cancer Cells].

BACKGROUND: Tumor-associated fibroblasts (TAF) is an important part of TME, which inhibits the function of immune cells. CD8+ T cells play a significant role in tumor immunity. T-cell membrane possesses a distinct type of molecule with a negative regulatory function. Upon interaction with its corresponding ligand [programmed death factor ligand 1 (PD-L1)], programmed death factor 1 (PD-1) is activated and thus inhibits the kinase activity of T cells. This study aims to explore the possible effects of TAF on PD-L1 expression in lung cancer cells. METHODS: Lung cancer cell lines H1975 and H520 were co-cultured with (experiment) or without TAF (control) via Transwell assay for through 48 hours under the same culture condition. H1975 and H520 cells were counted using a microscope. The protein and mRNA expression levels of PD-L1 were detected by FCM assay and PCR analysis, respectively. RESULTS: The numbers of lung cancer cells in 100 µm2 for H1975 and H520 cells are (46±21) and (38±10) in the experiment group, respectively, and (16±5) and (12±5) in the control group, respectively (P<0.05). The expression levels of the PD-L1 protein in H1975 and H520 cells are (20.93%±3.54%) and (19.26%±3.04%) in the experiment group, respectively, and (12.58%±2.52%) and (11.60%±2.65%) in the control group, respectively (P<0.05). The mRNA expression levels in H1975 and H520 cells are (16.45±1.25) and (15.38±2.02) pg/mL in the experiment group, respectively, and (7.78±1.27) and (7.20±1.58) pg/mL (P<0.05) in the control group, respectively (P<0.05). CONCLUSIONS: TAF promotes the growth and increases the expression of PD-L1 in H1975 and H520 cells.
.

[Altered Treg and IL-1A Expression in the Immune Microenvironment 
of Lung Squamous-cell Cancer after EGFR Blockade].

BACKGROUND: Targeting the mutations and amplifications in the epidermal growth factor receptor (EGFR) gene has curative effects on cancers of the lung, oral cavity, and gastrointestinal system. However, a systemic immune inflammation is an adverse effect of this therapeutic strategy. In this study, we aimed to identify the possible changes in the tumor microenvironment that contribute to the anti-cancer activity of EGFR inhibition. METHODS: Squamous-cell cancers were induced by the syngeneic transplantation of either EGFR-null or wild-type mouse primary keratinocytes that had been transduced with an oncogenic H-ras retrovirus. The mice were treated with gefinitib. Then, flow cytometric was used to detect the ratio of T cells and the expression of programmed cell death receptor 1 (PD-1). RT-PCR was used to detect the expression of cytokines and chemokines. RESULTS: Tumors that formed from EGFR-null keratinocytes were smaller, had fewer infiltrating FoxP3+ Treg cells, lower Foxp3 RNA, and lower percentage of PD-1 positive CD4 cells than those formed from wild-type keratinocytes. These results indicated that tumor cells can autonomously regulate the tumor microenvironment. Hosts with wild-type cancers and that were treated with gefitinib for 1 week tended to have smaller tumors. The treated mice in the short-term pharmacological model tended to have reduced FoxP3+ cells and FoxP3 RNA in the tumor microenvironment, as well as a substantially increased ratio of IL-1A/IL-1RA transcripts. These results suggested that the brief systemic inhibition of EGFR signaling alters the immune environment of the targeted cancer. CONCLUSIONS: The autonomous (genetic) or systemic (pharmacologic) inhibition of EGFR signaling in tumor cells reduces tumor growth and Treg infiltration in the tumor microenvironment. An EGFR-dependent Treg function supports the growth of squamous cancers. Therefore, Treg is a target in the therapeutic strategy of EGFR inhibition.

Structure and antioxidant activity of a novel polysaccharide derived from Amanita caesarea.

A heteropolysaccharide was isolated from the fruiting bodies of Amanita caesarea using a diethylaminoethyl-cellulose column, Sephacryl S­300 gel column and Sephadex G­200 column. The Amanita caesarea polysaccharide was predominantly composed of α-D-glucose and α-D-lyxose at a ratio of 2:1, and it had a molecular weight of 19,329 Da. The structural features of the Amanita caesarea polysaccharide were investigated by a combination of total hydrolysis, methylation analysis, gas chromatography-mass spectrometry, and infrared spectra and nuclear magnetic resonance spectroscopy. The results showed that Amanita caesarea polysaccharide (termed AC­1) had a backbone of 1,4­linked α­D­glucose and 1,3,6­linked α­D­glucose, with branches of one 1­linked α­D­lyxose residue. The antioxidant activity of AC­1 was evaluated by two biochemical methods, 2,2-azino-bis diammonium (ABTS+) radical scavenging activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH-) radical scavenging activity. The uncontrolled production of free radicals is involved in various diseases, including cancer, atherosclerosis and degenerative aging processes. The results indicated that the Amanita caesarea polysaccharide exhibits strong antioxidant activity, thus, it may be a useful natural product antioxidant.

Structure elucidation, proliferation effect on macrophage and its mechanism of a new heteropolysaccharide from Lactarius deliciosus Gray.

A new heteropolysaccharide was isolated from the fruiting bodies of Lactarius deliciosus Gray which had a molecular weight of 16kDa and was mainly composed of the galactose and glucose. Structural elucidation results indicated that Lactarius deliciosus Gray polysaccharide (LDG-B) had a backbone of (1,6)-linked d-galactose and (1, 2, 6)-linked d-galactose which branches were mainly composed of 4-linked d-glucose and 6-linked d-galactose residue. Cell cycle test results showed that LDG-B could promote the proliferation of B cells and macrophage cells by affecting G0/G1 phase, S phases and G2/M phases. The analysis of transcriptomes sequence of macrophages showed a total of 1839 genes were identified as DEGs, and approximately 708 genes were up-regulated, whereas 1131 genes were down-regulated in LDG-B group. KEGG pathway enrichment analysis showed that the MAPK, JAK-STAT and NF-κB signaling pathways are significantly enriched for DEGs in LDG-B group. Analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of LDG-B.