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Aspergillus niger CJ-1 was applied to conduct fermentation process on tartary buckwheat leaves (TBL), and effect of fermentation time on antioxidant ability and chemical compositions was revealed. Antioxidant ability of TBL showed a three-stage change tendency during fermentation, which was relatively consistent with change in chemical compositions. Rutin and quercetin were identified as remarkable contributors among fermentation. Both total polyphenols, rutin and quercetin was positively being correlated with antioxidant activity of TBL. Results indicated that fermentation time influenced antioxidant activity by altering content of TBL phenolic compounds, which showed a three-stage change law: augmenting at the early stage of fermentation, declining at the medium stage of fermentation, and slightly changing during the last stage. This tendency suggested that fermentation was a potent means to promote antioxidant capacity of agro-food unless fermentation time was controlled. This study may provide a theoretical basis for producing fermented foods with high bioactivity.

Human breast cancer cells exhibit considerable diversity in the methylation status of genomic DNA CpGs that regulate metastatic transcriptome networks. In this study, we identified human Sipa1 promoter-proximal elements that contained a CpG island and demonstrated that the methylation status of the CpG island was inversely correlated with SIPA1 protein expression in cancer cells. 5-Aza-2’-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, promoted the expression of Sipa1 in the MCF7 breast cancer cells with a low level of SIPA1 expression. On the contrary, in MDA-MB-231 breast cancer cells with a high SIPA1-expression level, hypermethylation of the CpG island negatively regulated the transcription of Sipa1. In addition, the epithelial-mesenchymal transition (EMT) was reversed after knocking-down of Sipa1 in MDA-MB-231 cells. However, the EMT was promoted in MCF7 cells with over-expression of SIPA1 or treated with 5-Aza-CdR. Taken together, hypomethylation of the CpG island in Sipa1 promoter-proximal elements could enhance SIPA1 expression in breast cancer cells, which could facilitate EMT of cancer cells, possibly increasing a risk of cancer cell metastasis in patients treated with 5-Aza-CdR.

The phytochemical investigation of Abies chensiensis led to the isolation and identification of nine new compounds including eight triterpenoids (1-8) and a new abietane-type diterpene (9), along with three known compounds (10-12). The absolute configuration of 9 was assigned by X-ray diffraction analysis. Compounds 1-11 were evaluated for the anti-inflammatory activity. Among the tested compounds, 1, 2, 5 and 6 exhibited potent inhibitory activity with IC50 values of 15.97, 18.73, 20.18 and 10.97μM, respectively.

The S-adenosyl methionine synthetase gene FaSAMS1 is involved in the regulation of strawberry fruit ripening, however, the biochemical properties of FaSAMS1 protein remain unclear. Here, a coding cDNA sequence of FaSAMS1 was cloned by RT-PCR and inserted into a recombinant yeast expression vector pPICZA, then transformed into the yeast expression strain X-33. The fusion protein FaSAMS1 was induced, expressed, and purified. The enzymatic activity analysis of FaSAMS1 showed that the reaction system contains 0.09 mg of FaSAMS1, protein concentration reached at 0.454 mg·mL-1, the activity of the enzyme was 0.32 U, specific activity was 3.56 U·mg-1. This study has provided biochemical evidence for the involvement of ethylene in the regulation of strawberry fruit ripening.Â

International audience; Early detection of cancers through the analysis of ctDNA could have a significant impact on morbidity and mortality of cancer patients. However, using ctDNA for early cancer diagnosis is challenging partly due to the low amount of tumor DNA released in the circulation and its dilution within DNA originating from non-tumor cells. Development of new technologies such as droplet-based digital PCR (ddPCR) or optimized next generation sequencing (NGS) has greatly improved the sensitivity, specificity and precision for the detection of rare sequences. Areas covered: This paper will focus on the potential application of ddPCR and optimized NGS to detect ctDNA for detection of cancer recurrence and minimal residual disease as well as early diagnosis of cancer patients. Expert commentary: Compared to tumor tissue biopsies, blood-based ctDNA analyses are minimally invasive and accessible for regular follow-up of cancer patients. They are also described as a better picture of patients' pathology allowing to highlight both tumor heterogeneity and multiple tumor sites. After a brief introduction on the application of the follow-up of ctDNA using genetic or epigenetic biomarkers for prognosis and surveillance of cancer patients, potential perspectives of using ctDNA for early diagnosis of cancers will be presented.

Objective To investigate the expression of survivin gene among human normal endometrium, atypical hyperplasia of endometrium, and endometrial carcinoma. Methods Tissue samples of human normal endometrium during proliferative phase (NE, n=20), atypical hyperplasia of endometrium (AHE, n=11), and endometrial carcinoma (EC, n=9) were collected. Besides, Paraffin embedded sections of NE (n=20), AHE (n=20), and EC (n=20) were used. The expression of survivin gene was determined by immunohistochemistry and the real-time reverse transcription polymerase chain reaction. Results Survivin immunostaining appeared in the cytoplasm of endometrial epithelial cells. Both survivin staining and mRNA had higher levels in AHE or EC than those in NE (P 0.05). Conclusion High expression of survivin gene in human endometrium is associated with the risk of atypical hyperplasia progressing to endometrial carcinoma. The high level of survivin expression is useful as a predictive indicator for endometrial carcinoma.

To evaluate the effects of YC-1 on leukemia cell lines, PI incorporation was used to determine cell viability. YC-1 induced a dose- and time-dependent decrease in viability and apoptosis in YC-1-treated U937 cells. YC-1-induced apoptosis is a cyclic guanosine monophosphate (cGMP)-independent pathway. Proteomic analysis showed that the altered proteins include the significant regulation of HSP70, chaperonin, ATP synthase beta chains, and Chain F. Western blotting and immuno-cytochemistry stain showed that YC-1 treatment caused a time-dependent increase in cytosolic Cytochrome c, pro-caspase-9, Apaf-1, and the activation of caspase-9 and -3. Importantly, the in vivo antileukemia effects of YC-1 were evaluated in BALB/c mice inoculated with WEHI-3B orthotopic model. YC-1 enhanced survival rate and prevented the body weight loss in leukemia mice. The enlargement of spleen and lymph nodes were reduced in YC-1 treated than that in leukemia mice. H-E stain of spleen sections revealed that infiltration of immature myeloblastic cells into red pulp was reduced in YC-1-treated group. The apoptotic cells of splenocyte were significantly increased in YC-1 treated than that in leukemia mice by Tdt-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Taken together, we conclude that YC-1 acted against U937 cells in vitro via a mitochondrial-dependent apoptosis pathway, and in orthotopic leukemia model, YC-1 administered antileukemia activity.

Biosynthesis and secretion of two different types of antifungal compound [phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in Pseudomonas sp. M18] contribute to its suppression of soil-borne root pathogens. To better understand the correlation between two antifungal agents in secondary metabolism, a DNA fragment covering partial pltC and pltD coding sequences was obtained by screening the genomic library of Pseudomonas sp. M18. A mutant, M18T, was then constructed by insertion of the aacC1 gene cassette (encoding gentamycin resistance). With the same methods, one PCA biosynthetic gene cluster was insertionally inactivated and a mutant M18Z1 was created. The mutant strain M18T produces no Plt and the same amount of PCA in comparison with the wild-type strain M18. The mutant M18Z1, however, produces less PCA but more Plt than the wild-type strain M18. According to the documented data on strain M18, it is suggested that production of PCA is not influenced by Plt yield, but Plt biosynthesis is influenced by an alteration of PCA production.

The relationship between senescence process and photorespiration was investigated in Spirodela polyrrhiza P143. The senescence of half-fronds cultivated under long-day conditions was either delayed by elevating CO 2 or enhanced by elevating O 2 composition of the surrounding environment, which implicated the involvement of photorespiration in this process. Application of sucrose into the cultivation medium was not found to be able to postpone the senescence process. In testing the effect of upstream substances of photorespiratory pathway on senescence, it was observed that applied at 0.5 mM, l -glycine and l -serine, but not glycollate, glyoxylate or formate, did induce senescence of both half-fronds and intact plants. No similar effect was observed of other amino acids, such as Arg, Trp and Cys, etc. As the role of l -serine in inducing senescence has been reported earlier, the possible role of l -glycine in inducing senescence of half-fronds under long-day conditions was further investigated. It was found that the peak value of endogenous glycine accumulated during senescence occurred later than that of serine and that the application of MOA, a specific inhibitor of glycine decarboxylase, inhibited the accumulation of serine and resulted in the accumulation of glycine, but did not cause the irreversible process of senescence. Therefore, a causative role of l -glycine in the senescence of half-fronds under long-day conditions could be ruled out. Measurement of changes in the endogenous serine level demonstrated that the appearance of endogenous serine peak value was postponed when half-fronds were cultivated at elevated CO 2 , which provides further evidence that l -serine is a specific factor of inducing senescence in duckweed.

Half-fronds of Spirodela polyrrhiza accumulated high level of endogenous serine when they had been cultivated under long-day conditions and on a cytokinin-free medium for 4 days, after which the irreversible senescence process was triggered. When 1 μM 6-benzyl adenine (6-BA) was included in the medium, the accumulation of endogenous serine was significantly inhibited, and at the same time the biological activity of half-fronds was increased. Application of exogenous l -serine into medium at 1 mM initiated senescence of half-fronds, as well as intact plants, under the conditions of long-day and darkness. Application of 6-BA into the medium with l -serine postponed the appearance of serine level peak and senescence syndrome, although the senescence process was not prevented. It was also observed that half-fronds cultivated in the darkness on a medium with only inorganic nutrients had a very low level of endogenous serine and maintained their biological activity for up to 2 months. These data shows that high level of endogenous l -serine can initiate senescence in Spirodela polyrriza and cytokinin can inhibit the accumulation of endogenous l -serine. The possible reason for the accumulation of endogenous serine under long-day conditions was investigated. The activity of serine glyoxylate aminotransferase (SGAT), which catalyzes the conversion of l -serine to hydroxypyruvate, was found to decrease gradually in half-fronds during cultivation under long-day conditions, but to be maintained by 6-BA application. Northern blot analysis was used to detect the changes in the level of transcript encoding SAGT, and results showed that the expression of this gene was up-regulated by cytokinin but down-regulated during senescence.

Five new species of Spilopteron (Ichneumonidae: Acaenitinae), viz., S. crassifacialium, longitubus, flavescutatum, dualis, and bellulon, are described from China. The known species from China are listed, along with a key to the Chinese species.

Four new genera and eight new species of Ichneumonidae belonging to the subfamily Mesosteninae are described. These were collected from Great Yarluzangbo Canyon, P. R. China.

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Two new triterpenoids, 2 alpha-hydroxy-3 beta-methoxyurs-12-en-28-oic acid and 3 alpha-hydroxy-2 alpha-methoxyurs-12-en-28-oic acid as well as 25 known compounds were isolated from the whole plant of Salvia roborowskii Maxim. Their structures were elucidated by means of spectral data and chemical transformation. This is the first report on the chemical constituents of this plant. The presence of eugenyl beta-D-glucopyranoside 22 in a plant of the genus Salvia is also reported for the first time.

Background PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3β (GSK-3β) in chemosensitivity. Methods We examined PMS2 and phosphorylated GSK-3β(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3β after transfection with GSK-3β by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. Results We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3β (s9). Furthermore, we demonstrated GSK-3β transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. Conclusions Our results provide the evidence that stabilization of PMS2 production by GSK-3β was important to improve chemosensitization, indicating the significance of GSK-3β-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy.

SPA-1 (signal-induced proliferation associated gene-1) functions as a suppressor of myeloid leukemia by negatively regulating Rap1 signaling in hematopoietic progenitor cells (HPCs). Herein, we showed that transplantation of HPCs expressing farnesylated C3G (C3G-F), a Rap1 guanine nucleotide exchange factor, resulted in a marked expansion of thymocytes bearing unique phenotypes (CD4/CD8 double positive [DP] CD3(-) TCRbeta(-)) in irradiated recipients. SPA-1(-/-) HPCs expressing C3G-F caused a more extensive expansion of DP thymocytes, resulting in lethal T-cell acute lymphoblastic leukemia (T-ALL) with massive invasion of clonal T-cell blasts into vital organs. The C3G-F(+) blastic thymocytes exhibited constitutive Rap1 activation and markedly enhanced expression of Notch1, 3 as well as the target genes, Hes1, pTalpha, and c-Myc. All the T-ALL cell lines from C3G-F(+) SPA-1(-/-) HPC recipients expressed high levels of Notch1 with characteristic mutations resulting in the C-terminal truncation. This proliferation was inhibited completely in the presence of a gamma-secretase inhibitor. Transplantation of Rag2(-/-) SPA-1(-/-) HPCs expressing C3G-F also resulted in a marked expansion and transformation of DP thymocytes. The results suggested that deregulated constitutive Rap1 activation caused abnormal expansion of DP thymocytes, bypassing the pre-T-cell receptor and eventually leading to Notch1 mutations and Notch-dependent T-ALL.

Uterine leiomyomas were shown to be clonal lesions, but the relationship among different tumor nodules in multiple uterine leiomyomas remains unresolved. In this study, X-chromosomal inactivation patterns of these tumor nodules were shown by allelic polymorphism analysis through polymerase-chain reaction at the phosphoglycerate kinase and androgen receptor loci following pretreatment with the methylation-sensitive restriction enzyme HpaII or HhaI. A total number of 113 cases of uterine leiomyomas were examined. Monoclonality was demonstrated in all of the 315 nodules from 76 informative cases. The inter-nodular relationship was evaluated in 55 multiple cases with 294 tumor nodules. Different inactivation patterns were observed in 20 cases, demonstrating a multicentric origin, while an identical inactivated allele was found in all or most of the nodules in the rest of the cases, indicating a common clonal origin. The occurrence of the unicentric cases appeared to be associated with an elevated mitotic activity. Seven nodules from a multi-nodular case with a morphology indicative of mitotically active leiomyoma were shown to carry the identical inactivated allele, which demonstrates their unicellular origin and malignant nature. In addition, the same androgen receptor gene alteration was identified in two discrete leiomyoma nodules from a uterus. These results approve the monoclonality of uterine leiomyomas and demonstrate the presence of unicentric multiple leiomyomas.

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.

AIM:To establish an assay for detecting serum hepatitis B virus (HBV) transcripts including the full-length (fRNA) and truncated RNA molecules (trRNA) in chronic HBV carriers, and to investigate its clinical significance. METHODS:The serum viral nucleic acids were analyzed using PCR and RT-PCR targeted to the HBV x protein-encoding region. Reaction specificity was confirmed by Southern blotting. Representative amplification products were cloned and sequenced. RESULTS:Different viral transcripts including fRNA and trRNA were identified in sera of HBV carriers. Presence of fRNA was closely correlated with seropositivity of hepatitis B e antigen and viral DNA. However, trRNA did not depend on serum e antigen, its association with circulating viral DNA was weak. Similarly, no remarkable correlation was ob-served between the levels of trRNA and the degrees of liver damage. Different age-dependent representations for fRNA and trRNA were recognized, fRNA was progressively decreased to undetectable levels and trRNA increased to high levels in the second decade of life and then remained in high level in the rest of life.The trRNA and RNA without polyadenylation at either of the two addition sites were detected even in the absence of any other conventional HBV markers such as viral DNA. They were also observed in sera from patients with cryptogenic cirrhosis or chronic hepatitis C. CONCLUSION:The viral transcripts are potential serum markers for identifying replicative and non-replicative infection stages and diagnosis of occult HBV infection.

The complete loss of enzymic activity on reaction with alkylating agents, aromatic nitro compounds, or aliphatic mercaptans indicates the importance of sulfhydryl groups for catalysis. Analyses during the course of inactivation with any of these reagents revealed the loss of one of the two —SH groups in the rhodanese monomer when inactivation was complete. Amino acid analysis of iodoacetate-inactivated enzyme showed that half its cysteine residues had been carboxymethylated. Sedimentation experiments showed that dinitrobenzene-inactivated enzyme was an oxidized dimer, indicating the formation of an intermolecular disulfide bond between monomers. Reduction of mercaptoethanol-inactivated enzyme resulted in the appearance of two —SH-groups, suggesting the existence of a mixed disulfide of the reagent and the enzyme. In addition, competitive inhibition of rhodanese activity by aromatic ions, but not by the corresponding aliphatics, supported previous evidence suggesting the presence of a tryptophanyl residue at the active center. Further results suggested close proximity of the essential sulfhydryl and aromatic groups in the active site of rhodanese.

The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pH-induced structural transitions to inert forms that occur in the free enzyme species and other forms not covalently substituted.