Cypripedium lichiangense (Orchidaceae) mimics a humus-rich oviposition site to attract its female pollinator, Ferdinandea cuprea (Syrphidae).

Most species in the genus Cypripedium (Cypripedioideae) produce trap flowers, making it a model lineage to study deceptive pollination. Floral attractants in most species studied appear to target bee species of different sizes. However, more recent publications report fly pollination in some subalpine species, suggesting novel suites of adaptive floral traits. Cypripedium lichiangense (section Trigonopedia) is an endangered subalpine species endemic to the Hengduan Mountains, China. We observed and analysed its floral traits, pollinators and breeding systems over 2 years in situ and in the lab. Cypripedium lichiangense was visited by females of Ferdinandea cuprea (Syrphidae). The pollinia were carried dorsally on the fly thoraces. The eggs of this fly were frequently found in the saccate labellum and on other floral organs, suggesting brood-site mimesis. The orchid is self-compatible, but cross-pollination produces more viable embryos. We propose a new mode of floral mimesis, humus-rich oviposition site mimicry, for C. lichiangense. Compared with the mimesis of aphid colonies attracting syrphid pollinators (subfamily Syrphinae), whose larvae are entomophagic, as reported in some Paphiopedilum species (Cypripedioideae), pollination by deceit in C. lichiangense represents a distinct and separate mode of exploitation of another saprophagic (or phytophagic) larvae syrphid lineage in the subfamily Eristalinae and appears to indicate diversity of pollination strategies in Section Trigonopedia of Cypripedium. However, this new brood-site mimesis seems to be less attractive to pollinators. As a possible adaptation to the weak attracted pollination strategy, this plant species has a long flowering period and extended lifespan of individual flowers to ensure reproductive success.

[The effect and mechanism of transient receptor potential M(2) in antigen-induced arthritis mice].

The purpose of this study was to explore the role and mechanism of transient receptor potential M(2) (TRPM(2)) in antigen-induced arthritis (AIA) mice. Twelve C57BL/6 mice and 12 TRPM(2) knockout mice were divided into 4 groups, includingwild type control group, wild type AIA group, TRPM(2) knockout control group and TRPM(2) knockout AIA group, with 6 mice in each group. Methylated bovine serum albumin (mBSA) was used to establish AIA mouse model. The degree of joint swelling and inflammatory cell infiltration were recorded, as well as synovial hyperplasia of the knee joints. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of interleukin (IL)-6, IL-8, chemokine ligand 6 (CXCL-6) and tumor necrosis factor alpha (TNFα) mRNA in synovial cells of knee joints. The results showed that compared with the wild-type AIA group, the TRPM(2) knockout AIA group had more significant synovial proliferation and inflammatory cell infiltration in the synovial tissue.The neutrophil and macrophage counts rather than monocytes in the knee joints of TRPM(2) knockout AIA group were higher than those in wild-type AIA mice. The expression of IL-6, IL-8 and CXCL-6 mRNA were significantly increased in the knock out mice. In summary, TRPM(2) may inhibit inflammatory cytokines such as IL-6 and IL-8 in knee joints of AIA mice by reducing the infiltration of neutrophils and macrophages, the refore alleviates the manifestations of knee arthritis.

Combined mTOR/MEK inhibition prevents proliferation and induces apoptosis in NF2-mutant tumors.

OBJECTIVE: Merlin is encoded by Neurofibromatosis type 2 gene (NF-2), a tumor suppressor gene, which causes some multiple tumors forming disease of the nervous system in case of function loss. Bioinformatics analysis suggested that patients with NF-2 mutation had a worse prognosis, while it was associated with PI3K/mTOR activation, implying abnormal apoptosis in NF-2 mutation related tumors. Hence, we supposed that the inhibitors of PI3K/mTOR pathway might play a role in suppressing the tumor proliferation. MATERIALS AND METHODS: Two representative NF-2 mutation tumor model of NCI-H2452 and HEI193 cell lines were adopted, while two PI3K/mTOR pathway inhibitors Trametinib and Vistusertib were chosen to study the proliferation and apoptosis of the tumor cells. RESULTS: CCK8 cell counting experiment showed that both Trametinib and Vistusertib could inhibit the proliferation of NCI-H2452 cell in vitro, while the combination of Trametinib and Vistusertib was more significant. Flow cytometry results showed that both Trametinib and Vistusertib could enhance apoptosis of NCI-H2452 cell in vitro, while the combination of Trametinib and Vistusertib was more significant. Similar results were also achieved for HEI193 cell lines. In vivo tumorigenicity experiments demonstrated that the tumor volume and weight were significantly decreased by both Trametinib and Vistusertib, while their combination had the most significant effect. Western blot results demonstrated that both Trametinib and Vistusertib could inhibit PI3K/mTOR /MEK pathway and enhance the expression of merlin. CONCLUSIONS: We found that PI3K/mTOR inhibitor could decrease the proliferation of NF-2 mutation tumor cell lines by enhancing apoptosis, while the combination of two drugs might have a better effect.

[Clinical research progression of molecular-targeted drugs and PD-1 inhibitors for advanced hepatocellular carcinoma].

Since sorafenib has been first-line molecular-targeted drug for advanced hepatocellular carcinoma (HCC), clinical studies in the last 10 years failed to confirm that a new molecular-targeted drug or immune checkpoint inhibitor was superior or non-inferior to sorafenib, or approved second-line treatment for patients with the failure of sorafenib. However, many clinical studies published in 2017 have changed people's previous understanding. REFLECT trial showed that as the first-line treatment of advanced HCC, lenvatinib was non-inferior than sorafenib. In addition, RESORCE trial and CheckMate-040 trial confirmed respectively that regorafenib and PD-1 inhibitor nivolumab were options of second-line treatment for patients with advanced HCC after sorafenib treatment. The development of these drugs will bring a new prospect for advanced HCC patients.

Clinical efficacy and mechanism of lymphoplasma exchange in the treatment of Guillain-Barre syndrome.

Guillain-Barre syndrome (GBS) is an autoimmune disease of the nervous system and is the most common acute polyneuropathy. Both cellular and humoral immunity are believed to be involved in the pathogenesis of GBS, and various types of activated CD4+ T cells are thought to orchestrate the onset and progression of GBS. Lymphoplasma exchange (LPE) filtering out activated lymphocytes while exchanging plasma has been used for GBS treatment for years. However the treatment is still not yet optimal. In order to assess the efficacy of this treatment, we evaluate the effect of LPE and determine the appropriate frequency of LPE treatments for GBS patients through comparing the neurological deficit scores and the changes in related immunology indicators of GBS patients before and after LPE treatment. Twenty-four patients with GBS who received LPE were evaluated for immunologic indicants before treatment, on the second day, and the fourth day after the treatment. The immunoglobulin complement and CD4+ T lymphocyte subsets were tested by flow cytometry. The patients' Medical Research Council sum scores were increased from 25.7±10.4 up to. 36.7±10.4 (P=0.019) and their Hughes scores decreased from 3.7±0.76 to 3.1±0.73 (P=0.027) at 7 days after LPE. In the peripheral blood from patients received LPE treatment, the levels of immunoglobulin, complement, monocytes and fibrinogen were significantly reduced. The percentages of Th1 and Th17 cells in the CD4+ T lymphocyte subsets were significantly decreased, whereas the Th2 and Treg cells were increased in patients after treatment. The changes in CD4+T lymphocyte subsets were correlated with patient MRC score changes. Our data indicate that LPE is effective in treating GBS patients by directly removing immunoglobulin, complement, monocytes, and fibrinogen as well as regulating lymphocyte subsets in the peripheral blood.

Effects of DNA damage and short-term spindle disruption on oocyte meiotic maturation.

DNA damage has recently been shown to inhibit or delay germinal vesicle breakdown (GVBD) in mouse oocytes, but once meiosis resumes, DNA-damaged oocytes are able to extrude the first polar body. In this study, using porcine oocytes, we showed that DNA damage did not affect GVBD, but inhibited the final stages of maturation, as indicated by failure of polar body emission. Unlike mitotic cells in which chromosome mis-segregation causes DNA double-strand breaks, meiotic mouse oocytes did not show increased DNA damage after disruption of chromosome attachment to spindle microtubules. Nocodazole-treated oocytes did not display increased DNA damage signals that were marked by γH2A.X signal strength, but reformed spindles and underwent maturation, although aneuploidy increased after extended nocodazole treatment. By using the mouse for parthenogenetic activation studies, we showed that early cleavage stage embryos derived from parthenogenetic activation of nocodazole-treated oocytes displayed normal activation rate and normal γH2A.X signal strength, indicating that no additional DNA damage occured. Our results suggest that DNA damage inhibits porcine oocyte maturation, while nocodazole-induced dissociation between chromosomes and microtubules does not lead to increased DNA damage either in mouse meiotic oocytes or in porcine oocytes.

Purification, characterization, and milk coagulating properties of ginger proteases.

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31 kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60 °C and maximum milk clotting activity at 70 °C. They were capable of hydrolyzing isolated α(S1)-, ß-, and κ-casein, of which α(S1)-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than α(S1)- and ß-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods.

The association of sporadic inclusion body myositis and Sjögren's syndrome in carriers of HLA-DR3 and the 8.1 MHC ancestral haplotype.

Sporadic inclusion body myositis (sIBM) usually occurs as an isolated condition, but it may occur in association with another autoimmune disorder such as Sjögren's syndrome. We reviewed sIBM cases with Sjögren's syndrome (sIBM/SS) from the Perth Inflammatory Myopathies Database to determine whether they are distinguishable from other sIBM cases. Six such cases were identified, representing 12% of all sIBM cases. Muscle biopsies confirmed the presence of an inflammatory myopathy with rimmed vacuoles and the characteristic muscle fibre inclusions of sIBM. Five of the six were females, contrasting with a 2:1 male preponderance in the rest of the sIBM cohort. The mean age-at-onset and the pattern of muscle weakness were similar in the two groups. Four out of five sIBM/SS patients treated with immune therapies had improvement in muscle strength lasting for 6-24 months, whereas only 27% of other sIBM patients improved. All 6 patients with sIBM/SS carried the HLA-DRB1*0301 allele, or its equivalent HLA-DR3 serological specificity, compared with 83% of other sIBM cases and all carried some or all of the major markers of the 8.1 MHC ancestral haplotype which is also associated with Sjögren's syndrome. Patients with sIBM/SS represent a subgroup of sIBM cases who are more likely to be female and carriers of HLA-DR3 and the 8.1 MHC ancestral haplotype, and are more likely to respond to treatment. The association of sIBM and Sjögren's syndrome is likely to be due to a common genetic predisposition linked to the MHC and supports the notion that sIBM has an autoimmune basis.

Structural characterization of the exopolysaccharide produced by Streptococcus thermophilus 05-34 and its in situ application in yogurt.

An exopolysaccharide (EPS) producing strain was isolated from Tibetan kefir grains in China, which was identified by 16S rDNA tests and designated as Streptococcus thermophilus 05-34. The high-performance liquid chromatography analysis showed that it was composed of galactose and glucose in a molar ratio of 1.0:0.8 with a molecular mass of 2.5 × 10(4) Da. EPS was further revealed to have α-d-glucose, α-d-galactose, ß-d-glucose, and ß-d-galactose by Fourier transform infrared spectroscopy combined with 1D (1) H nuclear magnetic resonance spectroscopy. The length of EPS ranged from 10 to 100 nm and the maximal height of lumps was 2.5 nm through atomic force micrograph analysis. Furthermore, yogurt fermented with EPS-producing S. thermophilus 05-34 exhibited lower susceptibility to whey separation, higher viscosity, and sensory scores than those made with non-EPS-producing strain in yogurt production. These results suggested that EPS-producing Streptococcus thermophilus 05-34 provided a potential application in the fermented dairy industry.

Reduced recombination in gynodioecious populations of a facultative apomictic orchid.

Many plants combine sexual reproduction with some form of asexual reproduction to different degrees, and lower genetic diversity is expected with asexuality. Moreover, the ratios of sexual morphs in species with gender dimorphism are expected to vary in proportion to the reproductive success of the sexual process. Hence, sex ratios can directly influence the genetic structure and diversity of a population. We investigated genotypic diversity in 23 populations of a facultative, apomictic gynodioecious orchid, Satyrium ciliatum, to examine the effect on genotypic diversity of variation in the frequency of females and in the amount of sexual reproduction. The study involved one pure female, seven gynodioecious (both females and hermaphrodites present) and 15 hermaphroditic populations. Pollinia receipt was higher in hermaphroditic than in gynodioecious populations. Analyses of variation in ISSRs demonstrated that genotypic diversity was high in all populations and was not significantly different between hermaphroditic and gynodioecious populations. We used character compatibility analysis to determine the extent to which recombination by sexual reproduction contributed to genotypic diversity. The results indicate that the contribution of recombination to genotypic diversity is higher in hermaphroditic than in gynodioecious populations, consistent with the finding that hermaphroditic populations received higher amounts of pollinia. Our finding of reduced recombination in gynodioecious populations suggests that maintenance of sex in hermaphrodites plays an important role in generating genotypic diversity in this apomictic orchid.

Pollination by deceit in Paphiopedilum barbigerum (Orchidaceae): a staminode exploits the innate colour preferences of hoverflies (Syrphidae).

Paphiopedilum barbigerum T. Tang et F. T. Wang, a slipper orchid native to southwest China and northern Vietnam, produces deceptive flowers that are self-compatible but incapable of mechanical self-pollination (autogamy). The flowers are visited by females of Allograpta javana and Episyrphus balteatus (Syrphidae) that disperse the orchid's massulate pollen onto the receptive stigmas. Measurements of insect bodies and floral architecture show that the physical dimensions of these two fly species correlate with the relative positions of the receptive stigma and dehiscent anthers of P. barbigerum. These hoverflies land on the slippery centralised wart located on the shiny yellow staminode and then fall backwards through the labellum entrance. They are temporarily trapped in the inflated chamber composed of the interconnected labellum and column. The attractive staminode of P. barbigerum strongly reflects the colour yellow (500-560 nm), a colour preferred innately by most pollen-eating members of the Syrphidae. No scent molecules were detected using GC mass spectrometry analysis, showing that the primary attractant in this system is visual, not olfactory. Pollination-by-deceit in P. barbigerum is contrasted with its congener, P. dianthum, a brood site mimic that is pollinated by ovipositing females of E. balteatus. As the natural rate of fruit set in P. barbigerum (mean 26.3% pooled over three seasons) is lower than that of P. dianthum (mean 58.5% over two seasons), the evolution of false brood sites in some Paphiopedilum spp. should be selectively advantageous as they may provide an increase in the attention and return rates of dependable pollinators to flowers that always lack a reward.

Comparison of grain from corn rootworm resistant transgenic DAS-59122-7 maize with non-transgenic maize grain in a 90-day feeding study in Sprague-Dawley rats.

DAS-59122-7 (59122) is a transgenic maize (Zea mays L.) that contains genes encoding Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis Berliner strain 149B1 and phosphinothricin acetyltransferase (PAT) protein from Streptomyces viridochromogenes. Expression of these proteins in planta confers resistance to corn rootworms and other Coleopteran parasites and tolerance to herbicides containing glufosinate ammonium, respectively. In the current study, processed flours from 59122 maize grain or its near isogenic control line (091) were used at two concentrations (50% and 70% wt/wt) to produce diets that were fed to rats for 90 days in accordance with Chinese toxicology guidelines (GB15193.13-2003). A commercial AIN93G diet was used as an additional negative control. No significant differences in body weight and feed utilization were observed between rats consuming diets formulated with 59122 and 091 Control corn. Statistical differences (p<0.05) were observed in certain hematology and serum chemistry response variables between rats consuming diets formulated with 59122 or 091 Control flour compared to AIN93G diet. However, the mean value of these response variables in the 59122 groups were not statistically different from those observed in diets formulated with corresponding high and low concentrations of the flour from the 091 Control maize grain. Therefore, the statistical differences were considered to be related to consumption of diets containing high concentrations of maize flour (compared to AIN93G diets) regardless of source rather than to consumption of flour from 59122 maize grain. The results from this study demonstrated that 59122 maize grain is as safe as non-transgenic maize grain.