AbstractAncistrus species were analyzed from streams and rivers of three hydrographic Brazilian basins. All populations showed different diploid numbers (2n), fundamental numbers (FNs), and karyotypes. Some representatives of Loricariidae have 2n = 54 chromosomes, which is very likely an ancestral cytotaxonomic characteristic, but many other representatives show extensive karyotype diversification. In the Ancistrus species studied, extensive karyotypic differentiation, which is generally associated with chromosome number reduction and rearrangement of the ribosomal RNA gene (rDNA) sites, was verified. Chromosomal locations of 18S and 5S rDNA were jointly detected using fluorescence in situ hybridization (FISH). In all the Ancistrus species analyzed, 18S rDNA sites were detected only on one chromosome pair, though this differed among species. 5S rDNA was located on 1–3 chromosome pairs either separately or in synteny with 18S rDNA in four of the seven species/populations. Hence the karyotype differentiation in Ancistrus species could be associated with a morphological speciation process, suggesting that chromosome fusions, inversions, deletions, duplications, and heterochromatination could contribute to the karyotype evolution of these neotropical armored catfishes.Populations of seven  Silver  regions .Pimelodidade and Pseudopimelodidae, where non-syntenic 5S and 18S ribosomal regions were observed  and chromosomal features, although some evolutionary trends can be defined among the distinct subfamilies  of Mato Grosso state, Brazil: (a) Paraguay basin, Coxipó river, 15°21'59"S, 55°57'11"W, Ancistrus claro Knaack, 1999 (11 M and 10 F);Sepotuba river, 14°41'35"S, 57°48'14"W, Ancistrus sp. 04 (12 M and 15 F); Currupira river, 15°07'59"S, 56°49'47"W, Ancistrus sp. 08 (7 M and 8 F); Flechas stream, 15°58'7"S, 57°19'7"W, Fundo stream, 16°14'17"S, 56°37'31"W, and Pari stream, 15°36'6"S, 56°12'19"W, Ancistrus cf. dubius Eigenmann and Eigenmann, 1889 ; Arrombado bay, 16°21'21"S, 56°27'55"W, Ancistrus cuiabae Knaack, 1999(15 M and 15 F); (b) Araguaia–Tocantins basin, Salgadinho stream, 14°40'14"S, 52°21'50"W, Ancistrus sp. 13 (11 M and 6 F); and (c) Amazon basin, Matrixã river, 10°3'7"S, 57°36'27"W, Ancistrus sp. 06 (9 M and 5 F).One hundred and thirty six specimens [male (M) and female (F)] of seven Ancistrus sp. 04, 06, 08, and 13 present discriminative morphological characteristics that have not yet been described.Specimens were morphologically identified and deposited in the Museu de Ciências da Pontíficia Universidade Católica do Rio Grande do Sul  and Núcleo de Pesquisas Limnológicas da Universidade Estadual de Maringá, Paraná . in vivo treatment with colchicine  for the 18S rDNA and anti-digoxigenin–rhodamine (Roche Applied Science) for 5S rDNA at 1:1000 and 1:200 dilutions, respectively, in non-fat dry milk buffer (5% non-fat dry milk in 4×SSC). The chromosomes were counterstained with DAPI and analyzed under an epifluorescence microscope (Olympus BX41) coupled to an image capturing system (Olympus DP71). Approximately 30 metaphases were analyzed to determine the 2n, karyotypic formulae, and the presence or absence of rDNA sites on the chromosomes.FISH was performed according to iz, 1829  and a 5Ses, 1850 . The 18SAncistrus claro to 34 chromosomes in Ancistrus cuiabae. The fundamental number (FN) varied from 68 to 86 chromosome arms , metacentric (m), submetacentric (sm), subtelocentric (st), acrocentric (a), fundamental number (FN), sex chromosome system (SC).Ancistrus sp. 06  carrying 5S rDNA sites  in the Ancistrus species studied could possibly involve several chromosomal rearrangements and gene flow restriction in different hydrographic basins or rivers . In addition to Ancistrus claro, species from the Amazon basin (Ancistrus sp. 06) have 2n = 50 chromosomes, considered to have been derived from the family. However, this species retains a primitive single NOR pair with syntenic 5S rDNA site and no additional site. Thus, the chromosome number reduction in Ancistrus sp. 06 is attributable to chromosome fusion. Ancistrus sp. 08 and Ancistrus dubius from the Paraguay basin have chromosome number reduction  by fusion and independent pathways to differentiated sex chromosome systems (Ancistrus sp. 08 shows a ZZ/ZW system relative to pair 20 (Ancistrus dubius has a XX/XY heteromorphic sex system in pair 19 (The other lineage consists of species that retain the syntenic rDNA sites ( systems . Ancistr pair 20 , whereas pair 19 .Ancistrus sp. explain the high degree of karyotypic diversification in this taxon. Also, the 18S rDNA marker, which is mostly considered to be conserved in a single chromosome pair in the interstitial position, showed different site locations in different types of chromosomes.Nevertheless, there is large chromosome plasticity among the species from the Paraguay basin, and the diversity of chromosome types with 5S and 18S ribosomal cistrons in Ancistrus sp. could be attributed to structural and numeric chromosome rearrangements. The karyotypic data presented here are important tools for taxonomy of Ancistrus species. The karyotype differentiation in Ancistrini could be associated with a morphological speciation process, suggesting that chromosome fusions, inversions, deletions, duplications, and heterochromatination could contribute to the chromosomal differentiation of Ancistrini.The variation observed in the 2n, FN, and rDNA sites of the


All of these compounds enhanced neurite outgrowth in NB cells. Dose-dependent responses to compounds 1–3 were observed within the concentration range of 10–100 nM, and dose-dependent responses to glycosides 4–6 were observed at concentrations of 1–50 nM. All the tested substances exhibited notable synergistic effects with trace amounts of nerve growth factor  or brain-derived neurotrophic factor . Using NB cells and OHSCs, it was shown for the first time that starfish steroids 1–6 act as neuroprotectors against oxygen-glucose deprivation (OGD) by increasing the number of surviving cells. Altogether, these results suggest that neurotrophin-like neuritogenic and neuroprotective activities are most likely common properties of starfish polyhydroxysteroids and the related glycosides, although the magnitude of the effect depended on the particular compound structure.The neuritogenic and neuroprotective activities of six starfish polar steroids, asterosaponin Р Neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), are essential for development, differentiation, survival and functional maintenance of neurons in the central and peripheral nervous systems ,2. AlthoLinckia laevigata was revealed for the first time by Qi, Han and co-authors [1, (25S)-5α-cholestane-3β,4β,6α,7α,8,15α,16β,26-octaol, and (25S)-5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, designated as PP1, PP2, and PP3 (1–3), from Patiria (=Asterina) pectinifera [1–D3, designated as D1, D2, and D3 (4–6), from Distolasterias nipon [1–6 at low concentrations, we analyzed their neuroprotective ability during oxygen-glucose deprivation using the mouse neuroblastoma C-1300 and organotypic rat hippocampal slice cultures. The starfishes P. pectinifera and D. nipon are common shallow waters species in the Northwestern Pacific. Compounds 1–3 are the most abundant polar steroid constituents in P. pectinifera [4–6 are the major polar steroid constituents in D. nipon [. Asterosaponin Р1 represents the example of monoside with the 3-O-methyl-5-O-sulfonato-α-l-arabinofuranose unit attached to C-24 in a side chain while the both 2 and 3 are free nonsulfated polyhydroxylated compounds. Distolasterosides D1–D3 (4–6) are the biosides, containing β-d-xylopyranose unit at C-3 and other monosaccharide residue at C-24 . Moreover, compound 5 have a double bond in the side chain at the position 22(23).Polyhydroxylated steroids and related steroid glycosides are the predominant secondary metabolites in starfish  ,7,8. Pol-authors ,9,10 on -authors ,12,13. Ttinifera , and distinifera , whereasNeuroblastoma (NB) cell cultures are widely used to study neuritogenic and neuroprotective properties of bioactive substances ,16. Usua1–3, manifested significant and nearly identical neuritogenic effects at doses of 2–20 μM, but these effects were slight or even absent at higher concentrations of approximately 40 μM [4–6 from D. nipon were effective at lower concentrations (0.1–2 μM) but demonstrated neurotoxic properties at higher concentrations (20–40 μM) [The neuritogenic activity of starfish polar steroids in living NB cells can be detected beginning at the second day of cultivation, as we have described previously ,12,13. Ply 40 μM . Polar s1–6 at much lower concentrations within 10–100 nM for 1–3 and 1–50 nM for 4–6 in NB cells using silver-impregnated preparations. Our experiments demonstrated that compounds 1 and 3 increased the number of differentiated neurons (percentage of cells bearing neurites longer than two cell diameters or bearing more than two processes) after four days of incubation at concentrations of 50 nM and higher. Compound 2 had the same effect at doses of 10 nM and above, as did compounds 4–6 at doses of 5 nM and above. Dose-dependent responses to all of the compounds were also observed after four days of incubation  were 91.56 ± 5.5 (РР1), 136 ± 7.8 (РР2), and 95.31 ± 4.6 (РР3) μm compared to 105.4 ± 4.3 μm  and 79.61 ± 4.7 μm in the controls. In the experiments using polar steroids 4–6 from D. nipon (at a concentration of 50 nM), the process lengths ± SEM were 132.37 ± 8.6 (D1), 119.77 ± 7.19 (D2), and 113.18 ± 7.1 (D3) μm compared to 115.11 ± 2.5 μm  and 103.94 ± 5.7 μm in the controls. Thus, the neuritogenic effects of the tested compounds in NB cell cultures were comparable to those of NGF. Compound 3  significantly increased the mean number of processes per cell (2.4 ± 0.2 times) compared to the control (1.7 ± 0.2 times) (p ≤ 0.05); a similar effect was not observed in NB cells cultivated with the other tested steroids or NGF.Culturing NB cells in the presence of the starfish steroid compounds also resulted in rapid primary neurite elongation. The mean process lengths ± SEM in the NB cells cultivated with polar steroids 4–6 from D. nipon compared to other starfish steroids. Generally, the number of such neurons in self-differentiated NB C-1300 culture is insignificant  concentrations were determined to be 1 ng/mL of NGF and 0.1 ng/mL BDNF. The simultaneous treatment of NB cultures with low concentrations of ntiation .Henricia leviucula that action of this compound on mouse skin JB6 Cl 41 cells is rather complicated and includes the inhibition of ERKs, NF-κB and AP-1 activities [It is known that some endogenous steroids in mammals stimulate the synthesis of neurotrophins ,22. The tivities . We suggThe neuroprotective effects of starfish polar steroids were assessed using NB C-1300 and organotypic hippocampal slice cultures. Both models are used in studies of the protective abilities of different drugs during oxygen-glucose deprivation (OGD) ,24,25,261–3 from P. pectinifera and 4–6 from D. nipon were observed. Although protective effects were not observed for steroids 1–3 from P. pectinifera at the concentration of 25 nM, reliable neuroprotective properties were detected for the same compounds at a concentration of 50 nM (D. nipon were more toxic and reduced the number of surviving cells in OGD-treated NB cells at concentrations of 0.5 or 2 μM (data not shown). However, at concentration of 50 nM, these substances showed significant neuroprotective effects  was identified as a neuroprotector in studies on OGD in NB cell lines . Interesin vitro to study the protective effects of various substances because they maintain cell architecture, neuronal connections and electric activity, allowing for extended survival studies [in vivo ischemia-induced injuries [1), D1 (3) and D3 (6), because these compounds were some more effective in neuritogenic and neuroprotective assays on NB culture and were available for further studies. OHSC survival in the presence of 1, 3, and 6 (1 μM) was estimated visually during cultivation. After 7–8 days of cultivation, the OHSCs were fixed, stained with cresyl violet or impregnated with silver and studied under a light microscope. None of the tested compounds affected the development of hippocampal slices in culture or their adhesion to collagen in the absence of OGD. Slices maintained the normal arrangement of cellular layers and developed a growth area containing migrated fibroblasts and extended processes similar to control cultures. DMSO added to the culture medium in control OHSCs (0.025%) did not affect the development of slices.Organotypic hippocampal slice cultures (OHSCs) demonstrate advantages compared with other organotypic systems used  studies . In terminjuries ,26. To s1), D1 (4) and D3 (6) at a concentration of 1 μM were tested in OHSCs under OGD conditions. These starfish steroids reduced the number of propidium-labeled dead cells  pectinifera, and compounds 4–6 were isolated from the starfish Distolasterias nipon, as described previously, and were pure according to NMR, MS, TLC, and HPLC data [Dulbecco’s modified Eagle’s medium (DMEM), Iscove’s modified DMEM (IMEM), RPMI 1640 medium, Hank’s balanced solution, penicillin, streptomycin, NGF, BDNF, dimethylsulfoxide (DMSO) and the antimitotic drugs were obtained from Sigma-Aldrich; heat-inactivated fetal bovine serum was obtained from BioloT . The mouse neuroblastoma C-1300 cell line was a kind gift from Professor A.S. Holansky . Compounds PLC data . Starfis2. The cells were passaged every 2–3 days.NB C-1300 cells were grown in culture medium containing DMEM:IMEM (3:1), 5% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin at 36 °C in a 90% humidified atmosphere containing 5% CO4 cells per well) with glass coverslips. The cells were cultivated overnight, and the medium was replaced with that containing 2% fetal bovine serum and different concentrations of the tested compounds. DMSO in equal concentration was added to culture medium in control. At day 4 of incubation, the cultures on coverslips were fixed with bromformol for 24 h at room temperature and impregnated with silver according to routine protocols. The silver-impregnated preparations were assayed under a Biolar D light microscope . Digital images were used for analysis of the neuritogenic effects using the Image Tool program (Union D) according to 3 parameters: % of cells bearing processes at least 2× the cell body diameter or bearing more than 2 processes, the number of neurites per cell, and the length of the primary neurites. In the latter case, the measured segments were summated and converted to μm using an appropriative conversion factor. A total of >100 cells were examined in 10 randomly chosen fields in 4 slides for each concentration of a compound, and 3 independent sets of experiments were conducted for each compound. Data are presented as the means ± SEM. Comparisons between the control and steroid-treated groups were performed using Student’s t-tests. A p-value less than 0.05 was considered statistically significant.The assays for neuritogenic activity in live cells were performed according to previously described methods ,12,13. F4 cells per well in 3 mL) in serum-free RPMI 1640 medium without glucose that was previously aerated with a 95% nitrogen–5% CO2 gas mixture for 1–1.5 h [2 at 36 °C. The steroid compounds were added to the culture medium twice for the OGD period and subsequent normoxic culture period. DMSO in equal concentration was added to culture medium in control. Cell survival was assessed by adding 0.08% trypan blue dye to the culture wells for 5 min at 25 °C, replacing with dye-free buffer, and counting the dye-containing (injured) and dye-excluding (viable) cells in five 40× microscope fields per well; in most cases, approximately 200 cells per well were counted. Three independent sets of experiments were conducted for each concentration of a compound. The data are reported as the means ± SEM. Comparisons between the control and steroid-treated groups were performed using Student’s t-tests. A p-value of less than 0.05 was considered statistically significant.To induce OGD, NB cells in the logarithmic phase of growth were seeded in 24-well plates  were added to the culture medium to final concentrations of 1−0.1 μM for 24 h. After the removal of the antimitotics, the OHSCs were cultivated in standard culture medium (control group) or were treated with the steroid compounds at a final concentration of 1 μM. DMSO in equal concentration (0.025%) was added to culture medium in control group. The medium was changed twice a week; the state of the OHSCs was controlled visually. To determine the effects of tested compounds on OHSC development after 7–10 days of cultivation, the cultures were fixed with 4% paraformaldehyde and stained with cresyl violet or impregnated with silver according to routine methods. All experimental procedures involving rats were approved by the Institutional Animal Care and Use Committee and performed according to the Directive 2010/63/EU.Organotypic hippocampal slice cultures were prepared according to previously described protocols . Briefly2 gas mixture and treated with the same medium for 35 min in a chamber containing 95% nitrogen–5% CO2. Then, the coverslips with OHSCs were rinsed twice with standard medium, placed into Petri dishes, and 100 μL of culture medium containing the examined steroid compounds was added. The cultures were returned to the CO2-incubator. In control OHSCs, the standard culture medium was replaced with RPMI 1640 containing glucose for 35 min and then again replaced with standard medium. After 24 h, the OHSCs were stained with propidium iodide (PI) , rinsed with Hank’s balanced solution, fixed with 4% paraformaldehyde (60 min) and embedded in glycerol. Finally, the OHSCs were analyzed with a LSM510 confocal laser scanning microscope . Propidium iodide was excited with monochromatic light at 543 nm, and images were generated using a diachronic beam splitter (FT 488/543) and a BP 585–605 nm emission bandpass filter. In order to quantify cell death, the optical density of PI fluorescence was recorded. The representative images were captured at the same exposure and digital gain settings to eliminate confounds of differential background intensity or false-positive fluorescent signal across sections. PI fluorescence was quantified using NIH ImageJ software (version 1.46). Cell death area of the 35 min OGD-exposed OHSCs was considered 100%. Compared with this 100% value, cell death in starfish steroids-treated hippocampal slices was calculated. Statistical comparison between OGD and the starfish steroid treatments was done by oneway ANOVA. Five independent experiments were carried out. The data was expressed as a mean ± SEM. Differences with a p-value ≤ 0.05 were considered statistically significant.To analyze protective effects, the steroid compounds at the final concentration of 1 μM were added to the culture medium on day 8 of cultivation. After 24 h, the cultures were rinsed twice with RPMI 1640 medium without glucose that had been aerated previously with a 95% nitrogen–5% COPolar starfish steroids, analyzed in the present study, manifest neuritogenic abilities in NB cell cultures, and these effects were synergistic with the actions of NGF and BDNF. They also acted as neuroprotectors in oxygen-glucose deprivation conditions in NB cells and hippocampal slice cultures. Altogether, these results suggest that neurotrophin-like neuritogenic and neuroprotective activities are most likely common properties of starfish polyhydroxysteroids and the related glycosides, although the magnitude of these effects depends on the specific compound structures. The molecular mechanisms of these effects remain to be determined, but polar starfish steroids appear to be promising candidates for further investigation as potential neurotrophic and neuroprotective therapeutics.


Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 μg·mL−1 (0.17 μM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 μg·mL−1 (13.3 μM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 40 μg·mL−1 (66.7 μM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained. Discovering new drugs that are more active, more selective, and less toxic, limiting deleterious side effects and tumor multidrug resistance, will obviously be a challenge for the 21st century. The isolation of potent anticancer molecules from the marine environment has generated interest in many groups to purify original compounds, understand their biological activity, and also identify the pharmacological targets of molecules previously known for their ecological function. Extensive screening of marine microalgae has led to the isolation and chemical determination of over 15,000 compounds, including fatty acids, sterols, phenolic compounds, terpenes, enzymes, polysaccharides, alkaloids, toxins and pigments ,4. Marinpoptosis –15. The Dunaliella tertiolecta. Violaxanthin exerted a potent antiproliferative activity on MCF-7 breast cancer cells, and induced biochemical changes typical of early apoptosis.As part of our ongoing activity dedicated to the research and pharmacomodulation of natural anticancer compounds, we screened extracts from various microalgae species, in order to purify and identify antiproliferative molecules. We report here the bioassay-guided isolation of violaxanthin as the major antiproliferative pigment in the dichloromethane extract of the Chlorophyceae 2.2.1.50) was determined . The DT DCM extract also inhibited LNCaP growth, with a GI50 close to the value determined on MCF-7 (GI50 = 60.9 μg·mL−1). No extract inhibited MDA-MB-231 growth. The DT DCM extract, active both on MCF-7 and LNCaP cells, was selected to purify antiproliferative molecules by fractionation.DCM and EtOH 2.2.DT DCM extract chromatogram obtained at 435 nm, with the definition of the fractions and sub-fractions tested on MCF-7.DT DCM fractions and the four F1 sub-fractions on MCF-7.Identification of the 11 major peaks present on the chromatogram was not performed at this step as we hypothesized that some fractions may not be active on cancer cells. The antiproliferative activity of each fraction was studied on the MCF-7 cell line as it was the most sensitive to the starting DCM extract, and grew faster than LNCaP. 1) was identified as the only active fraction in the DT DCM extract, with a GI50 = 14.3 μg·mL−1. Decrease of the GI50 value compared to the DCM extract confirmed that this fraction was concentrated in active molecules  measured on MCF-7 with the starting DT DCM extract, the F1 fraction and the F1.4 subfraction.Fraction 1  of the freeze-dried microalgae content, considering the extraction yield to be 100%. The absorption spectrum of M was characteristic of a carotenoid pigment and presented maximal absorption peaks at 417.2, 441.5 and 471.9 nm, with a band III:II ratio of 96%  (66.4 μM), but no DNA fragmentation was observed even after 72 h incubation  belongs to the Chlorophyceae class . The genus Dunaliella exhibit a small ovoid unfrustulated cell (<20 μm), covered of cellulose, xylans, mannans and/or glycoproteins, with two equal flagella inserted at the apexes of the cell. Chlorophyll a, chlorophyll b, β,β-carotene, neoxanthin, lutein and violaxanthin are the main pigments described in DT [DT was selected as it does not contain fucoxanthin, which has already been extensively studied for its antiproliferative activity [ed in DT . DT was activity –15.3.2.DT strain CCMP364  was cultivated in 10 L flasks under continuous illumination at an average light intensity of 180 μmol·m−2s−1. Growth was performed at 20 °C, in pH unregulated batch culture, in Walne (Conway) medium diluted in 0.22 μm sterile-filtered natural seawater. The DT cell suspension was harvested at the end of the exponential growth phase, and cells were separated from culture medium by soft centrifugation . Cells were frozen at −20 °C, sent to laboratory LIENSs, La Rochelle and freeze-dried at −55 °C and P < 1 hPa, on a freeze-dryer equipped with a HetoLyoPro 3000 condenser and Heto cooling trap .Microalgae were grown at IFREMER PBA, Nantes, France. 3.3.In order to extract most microalgal organic molecules, on a wide range of polarity, successive extractions were performed in dichloromethane (DCM), ethanol (EtOH) and ultrapure water. A 1 g sample of freeze-dried microalgae powder was first extracted for 2 h in 100 mL DCM (1% w/v), at room temperature, under continuous shaking and in the dark. The mixture was filtered through a PVDF 0.22 μm membrane, and the DCM extract was evaporated to dryness in dark vials . The insoluble residue was collected, dried and successively extracted in ethanol and ultrapure water, in the same conditions, except that the water extract was collected by centrifugation  and filtered through a nitrocellulose membrane. The EtOH extract was evaporated to dryness in dark vials  and the aqueous extract was freeze-dried.3.4.−1 and elution was monitored at 435 nm. Fractionation of the active extract was performed in semi-preparative conditions, using a semi-preparative Phenomenex Luna C18 (2) column  with a flow rate fixed at 5 mL·min−1.The RP-HPLC system was composed of a binary pump , an autosampler , a thermostated (20 °C) column compartment, and a photodiode-array detector . Analytical RP-HPLC of the DCM extract was performed on a 20 μL sample injected in a Phenomenex Luna C18 (2) analytical column , the mobile phase consisting of a ternary gradient of solvent A (Methanol/water (80/20)); solvent B (Acetonitrile/water (90/10)) and solvent C (ethyl acetate). The gradient flow program was adapted from Jeffrey and collaborators [3.5.2–95% air humidified atmosphere, in DMEM  supplemented with 10% heat-inactivated  FCS  to which penicillin 100 U·mL−1 and streptomycin 100 μg·mL−1 were added. LNCaP human prostatic carcinoma was grown as monolayer in RPMI (devoid of phenol red) supplemented with 10% heat-inactivated  FCS, penicillin 100 U·mL−1 and streptomycin 100 μg·mL−1.MCF-7 and MDA-MB-231 human mammary carcinoma cell lines, as well as A549 human lung adenocarcinoma cell line  were grown as monolayers, at 37 °C in a 5% CO3.6.The algal extracts and related fractions were evaporated to dryness and stock solutions were prepared in DMSO before being diluted in the cell culture medium. The final DMSO concentration was lower than 1% and tested as a negative control. Cell viability was studied using the MTT assay.3.7.3.7.1.Ten thousand cells were grown for 24 h on epifluorescence live cell array slides  and treated for 72 h with control culture medium or microalgae extracts diluted in cell culture medium. Phosphatidylserines translocation onto the outer side of the plasma membrane of early apoptotic cells was detected using Annexin-V-Alexa 568 fluorochrome . Since necrotic cells also expose phosphatidylserines because of the loss of membrane integrity, necrotic cells were distinguished from apoptotic cells by BOBO-1 labeling. BOBO-1 is a DNA-binding fluorochrome, excluded from living and apoptotic cells. Cells were incubated for 15 min at 20 °C with the labeling mix solution, and observed using a Leica epifluorescence microscope, equipped with an I3 epifluorescence filter block (blue excitation 450–490 nm) and a numeric camera. Early necrotic cells, having lost their cytoplasmic membrane integrity but with a round shaped nucleus, were labeled by BOBO-1 only and appeared as round green spots. Early apopotic cells, having exposed phosphatidylserines but with no cytoplasmic membrane damages, appeared as red spots. Late necrotic cells exhibited a red and green co-staining and a shrunk nucleus.3.7.2.6 MCF-7 cells were lysed at 70 °C for 30 min in a lysis buffer . Then, DNA was purified using 360 μL of BB buffer (“Circulating DNA from Plasma” kit), which allows the purification of high and low molecular weight DNA fragments. DNA samples (5 μg) from control cells, cells treated with violaxanthin or cell treated with a control apoptosis inducer, DIM , were separated by electrophoresis on a 1.5% agarose/Tris–borate–EDTA (TBE) gel, stained with ethidium bromide and compared to standards ranging from 100 to 3000 bp (Fermentas) using a UV transilluminator.DNA was extracted and purified from cancer cells using a combination of two kits (Macherey-Nagel). In a first step, 103.8.DT extracts was determined by HRMS at the “Centre Régional de Mesures Physiques de l’Ouest”, University of Rennes 1, France. The mass spectrometer was a Bruker MicrO-Tof-Q 2, equipped with an ESI source, and samples were dissolved in CH2Cl2:CH3OH (90:10).Accurate molecular weight of the bioactive molecules contained in 3.9.All solvents used in this study were HPLC grade. Standard pigments were obtained from Chromadex, France. Ultra-pure water was obtained using a Milli-Q system .4.in vitro and suggests that violaxanthin and derivatives obtained by pharmacomodulation should be studied as possible new drugs to cure breast cancer. Violaxanthin was already identified as possibly involved in the strong antiproliferative and pro-apoptotic activity of Chlorella ellipsoidea extracts on HCT116 human colon cancer cells [DT content, with a theoretical extraction yield of 100%. Thus, it seems unrealistic to consider that violaxanthin could be purified from DT for therapeutic applications, even if physiological studies in Dunaliella or Chlorella may be carried out to increase production yields. Moreover, there is no clear evidence that epoxycarotenoids, despite their abundance in dietary fruits and vegetables, are absorbed per os by humans [per os in mice [in vivo.Breast cancer is a major cause of mortality worldwide, and the development of new drugs is necessary to reduce mortality and limit tumor resistance to chemotherapy. This study unequivocally demonstrates the strong antiproliferative activity of violaxanthin on MCF-7 human mammary cancer cells grown er cells . Furthery humans . Recent  in mice , suggest in mice . Additio


Despite the variability in the triacylglycerols and volatile compounds composition, the quality indices  all of the virgin olive oils samples studied met the commercial standards. Decanal was the major constituent, accounting for about 30% of the whole volatiles. Moreover, the chemical composition of the volatile fraction of the oil from fruits of trees grown at the planting density of 156, 100, and 51 trees ha−1 was also characterised by the preeminence of 1-hexanol, while oils from fruits of trees grown at the planting density of 69 trees ha−1 had higher content of (E)-2-hexenal (20.3%). Our results confirm that planting density is a crucial parameter that may influence the quality of olive oils.The present study focused on the comparison the chemical composition of virgin olive oil samples obtained from fruits of the main Tunisian olive cultivar  grown in four planting densities (156, 100, 69, and 51 trees ha Virgin olive oil composition determines its intrinsic quality and could be influenced by several factors. Cultivar, environment, and agronomic practices affect the fruit physiology, whereas processing and storage conditions alter oil composition. Virgin olive oil is composed of triacylglycerols around 97-98%), minor variable amounts of free fatty acids, and minor glyceridic compounds—partial glycerides, phospholipids, and oxidized triacylglycerols—and around 1% of unsaponifiable constituents of varied structure and polarity . Further-98%, minOlea europaea L.) is present practically in every region of the country, up to the border of the southern desert.Tunisia is a very important country in the olive oil producing world, the largest African exporter and fourth worldwide after Spain, Italy, and Greece . The oli−1.Historically, Tunisian olives were produced under dry-land conditions where trees were spaced widely to take full advantage of the stored soil water from winter rains for spring and summer growth. Many studies have been carried out to categorise virgin olive oils from different cultivars and different geographical origins according to their different chemical properties. However, there is no study on the effect of the planting density on the volatile and triacylglycerols composition of olive oil. The present research aimed to study the relationships between the aroma profile and triacylglycerols composition of virgin olive and the planting density. For these purposes, the volatile compounds and triacylglycerols composition of virgin olive oils were compared in samples obtained from fruits of olive trees cultivated in four planting densities ranging from 51 to 156 tree ha−1, respectively, and were subjected to the same fertilization and common olive cultivation practices. Tree heights were about 3 m. Higher trees productions were about 1300 kg ha−1. The same laboratory mill was used to prepare the olive oil samples. Only healthy fruits, without any kind of infection or physical damage, were processed. After harvesting, fresh olives (1.5–2.0 kg) were washed and deleafed, crushed with a hammer crusher, and the paste mixed at 25°C for 30 min, centrifuged without addition of warm water (oil produced from each extraction was 200–250 mL/kg) and then transferred into dark glass bottles, and stored (one week) in the dark at 4°C until analysis.Olive oil samples were obtained from fruits of the main Tunisian olive cultivar, Chemlali, which were picked by hand at the same stage of maturity from three trees during the crop season 2009/2010 (October) in a 4 ha olive orchard located in Souassi center of Tunisia . The climate of the study area is Mediterranean with an average annual rainfall of 250 mm, mostly distributed outside a 4-month summer drought period. Olive samples were collected from trees planted in 1988, at four-tree spacing: 8 × 8 m, 10 × 10 m, 12 × 12 m, and 14 × 14 m corresponding to 156, 100, 69, and 51 trees ha2/kg); and UV absorption characteristics (K232 and K270) were determined according to the analytical methods described in the European Union Commission Regulations EEC/2568/91 and EEC/1429/92. Free acidity, expressed as percent of oleic acid (%18 : 1); peroxide value, given as milliequivalents of active oxygen per kilogram of oil  was used at room temperature. All solvents were filtered through a 0.45 μm nylon filter disk  prior to use. The injection volume was 10 μL. The eluent used was a gradient of 2-propanol as mobile phase A, and acetonitrile as mobile phase B of the solvent system. Elution was performed at a solvent flow rate of 0.7 mL min−1 with a linear gradient as follows: from 0 to 2 min 52% B, up to 4 min 58% B, 25 min 58% B, 30 min 10% B, and 37 min 52% B. Peak assignment was carried out by comparison with chromatograms reported in the literature [−1. A 0.3 g oil sample was dissolved in 10 mL of 2-propanol/acetonitrile/n-hexane  and homogenized by stirring. High-performance liquid chromatography analyses were performed using an HP 1100 Series instrument  equipped with a binary pump delivery system, degasser, and autosampler and an evaporative light scattering detector (ELSD) PL-ELS model 1000 Series . Luna C18  column, 5 −1; carrier gas, helium at 1 mL min−1; and splitless injection. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and homemade library mass spectra built from pure substances and components of known oils and MS literature data [Solid-phase microextraction devices coated with polydimethylsiloxane were used to sampling the headspace of 2 mL of olive oil inserted into a 5 mL glass septum vial and allowed to equilibrate for 30 min. After the equilibration time, the fibre was exposed to the headspace for 50 min at 25°C room temperature. Extraction temperature and times were optimised in previous studies. Once sampling was finished, the fibre was withdrawn into the needle and transferred to the injection port on the GC-FID and GC-MS system. GC-EIMS separations were performed with a Varian CP 3800 gas chromatograph equipped with a DB-5 Capillary column  and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were as follows: injector and transfer line temperature at 250 and 240°C, respectively; oven temperature programmed from 60 to 240°C at 3°C minure data –14. MoreP < 0.01). The statistical analysis was performed using SPSS 13.0 for Windows . Significant differences between means were determined by an analysis of variance, which applied a Duncan's test. Differences were considered statistically significant when the probability was greater than 99%  and OOL (17.50%). As for fatty acids [Results for triacylglycerol contents, expressed in percentage of total triacylglycerols of oil samples, are shown in  and OOL .50%. As E)-2-hexenal,ortho-guaiacol, (E)-β-ocimene, phenyl ethyl alcohol, and -α-farnesene. As shown in Aroma is an important criterion for virgin olive oils. Volatile components of olive oil are of great interest since they are related to its quality and are used to detect adulteration . All theE,E)-α-farnesene (1.2–15.3%) was the main one. The hydrocarbons of olive oil have been studied by different authors as possible markers to distinguish virgin olive oil from different olive varieties or geographical origins [−1 was characterised by the preeminence of two compounds: 1-hexanol (15.1%) and decanal (37.4%). The other main compounds detected were 1-hexyl acetate (11.8%), (E)-2-hexenal (7.4%), and phenyl ethyl alcohol (7.7%) (−1 was characterised by the preeminence 1-hexanol (32%), Decanal (39%), (E)-2-hexenal (10.9%), and (E)-β-ocimene (4.1%) -α-farnesene, (E)-2-nonenal, and benzyl alcohol. The major constituents of the volatile fraction obtained from of the oil from fruits of trees grown at the planting density of 69 trees ha−1 were identified as decanal (28.6%), (E)-2-hexenal (20.3%), and 1-heptadecene (15.4%). Other important volatiles were 1-hexanol (7.3%), Hexanal (4.4%), and pentadecane (2.8%) , 1-hexanol (19.3%), -α-farnesene (15.3%), and ortho-guaiacol (10.1%). Other minor volatile compounds were observed in the virgin olive oils (E)-β-ocimene (4.7%), 2,3-butanediol (3.3%) 2-ethylbenzaldehyde (2.2%), and (Z)-3-hexen-1-ol (3%). Therefore, the differences between the four planting densities were mainly quantitative because most of the compounds were detected in all the olive oils. The variation in levels of C6 aldehydes and alcohols for oil samples from different regions implies that planting density may influence the activity of Alcohol Dehydrogenase (ADH). Differences in the levels of esters in olive oil were also observed between olive oil samples, suggesting a dependence of Alcohol Acetyl Transferase (AAT) activity on planting density. Several terpene hydrocarbons (mono- and sesquiterpenes) were often detected, and they totally accounted for 0–15.3% of the whole volatiles . -α origins –24. The l (7.7%) . Therefoe (4.1%) . Other me (2.8%) . Also, tive oils : (E)-β-o−1  that may influence the quality of olive oils.


Retinoblastoma is a malignant tumor of the retina usually occurring in young children. To date, the conventional treatments for retinoblastoma have been enucleation, cryotherapy, external beam radiotherapy, or chemotherapy. Most of these treatments, however, have possible side effects, including blindness, infections, fever, gastrointestinal toxicity, and neurotoxicity. More effective treatments are therefore imperative. Gossypol has been reported as a potential inhibitor of cell proliferation in various types of cancers, such as prostate cancer, breast cancer, leukemia, and lung cancer. This study investigates the possible antiproliferative effect of gossypol on retinoblastoma.Human retinoblastoma cells were cultured with various concentrations of gossypol and checked for cell viability with a 3--2,5-diphenyltetrazolium bromide (MTT) assay. Nuclear condensation caused by cell apoptosis was detected by staining retinoblastoma cells with 4',6-diamidino-2-phenylindole (DAPI), counting those with condensed nuclei, and determining the percentage of apoptotic cells. In addition, the stages of apoptosis and phases in cell cycles were examined with flow cytometry. The possible signal transduction pathways involved were examined with a protein array assay and western blot analysis.After incubation, the cell survival rate was significantly lower after treatment with 5, 10, and 20 µM of gossypol. The maximum antisurvival effect of gossypol was observed at 20 µM, and the number of apoptotic cells was higher in the preparations cultured with 10 and 20 µM of gossypol. The results in flow cytometry indicated that at concentrations of 10 and 20 µM, gossypol increased the proportion of early- and late-apoptotic retinoblastoma cells and induced cell arrest of retinoblastoma cells at the same concentrations. This antiproliferative effect was later confirmed by upregulating the expression of death receptor 5 (DR5), caspase 8, caspase 9, caspase 3, cytochrome C, tumor protein 53 (p53), and second mitochondria-derived activator of caspases (Smac) in the signal transduction pathways.We concluded that gossypol has an antiproliferative effect on retinoblastoma cells. RB1) mutations, which account for 60% of all retinoblastoma cases [RB1 have a bilateral retinoblastoma tumor [Retinoblastoma is the most common intraocular malignant tumor in infants and children, where it might occur unilaterally or bilaterally. Most cases of unilateral retinoblastoma involve somatic nonhereditary retinoblastoma 1 gene –mediated apoptosis [Gossypol is a polyphenolic extract of cottonseeds. The biologic effects of gossypol are attributable to 6 hydroxyl and 2 aldehyde groups, which also make the molecule soluble in organic solvents. To date, gossypol has had a wide range of uses. In agricultural applications, it reduces larval weight and the metabolic rate in invertebrate pest species by inhibiting mitochondria ATPase activity . Gossypopoptosis ,14. In spoptosis ,16, and poptosis . Studiespoptosis ,19. We tGossypol  was dissolved in dimethyl sulfoxide (DMSO) to make a 25 mM stock solution. The stock solution was diluted to concentrations of 0.5, 1, 3, 5, 10, and 20 mM in DMSO before the experiments were started, and then added  to the cell culture medium.2, and the culture medium was changed every 3 days.The human retinoblastoma Y79 (HTB-18) and the human retinal pigmented epithelium (ARPE) cell lines were purchased from Bioresource Collection and Research Center . Cells were cultured in RPMI 1640 medium  containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were incubated at 37 °C with 5% CO4 cells/well) were cultured in 24-well plates with 1 ml RPMI 1640 medium and 0.5, 1, 3, 5, 10, or 20 μM of gossypol for 6 to 48 h. The preparations for the control group were cultured without gossypol but with the same amount of DMSO as the experimental groups. After incubation, 100 μl of MTT was added in each well, and incubated at 37 °C for another 2 h. Triton  solution was then added into the preparations to dissolve the purple formazan from each cell. The amount of soluble formazan was measured with an enzyme-linked immunosorbent assay (ELISA) plate reader  at 550 nm absorption.Cell viability was measured with the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Y79 cells (5×104 per well) with 1 ml RPMI 1640 medium, and treated with 0, 1, 10, or 20 μM of gossypol for 24 h. After incubation, cells were collected and centrifuged at 92.5 ×g for 5 min at room temperature, washed twice in phosphate-buffered saline (PBS), fixed with 4% formaldehyde at room temperature for 30 min, and washed with PBS twice again after fixation. These fixed cells were further incubated with 4,6-diamidino-2-phenylindole  for 30 min at room temperature, avoiding light exposure. After DAPI staining, the cells were washed with PBS twice to remove the redundant fluorescent dye, the remaining liquid was removed, and the preparation was completely resuspended in the antifading mounting medium . The preparation was then transferred to glass slides for observation under a fluorescence microscope , and images were taken and counted using a cooled charge-coupled device (CCD) camera .Y79 cells were cultured in each well of a 24-well culture plate  and propidium iodide (PI), instead of DAPI. Cells were washed twice with PBS and then resuspended in 500 μl of binding buffer; 5 μl of annexin V-FITC and 5 μl of PI , and then incubated at room temperature for at least 10 min in the dark. After staining, the preparations were washed and transferred to glass slides for observation under a fluorescence microscope  with a cool CCD camera .6) were cultured in six-well plates with 2 ml of RPMI 1640 medium (Gibco) and 0, 1, 10, and 20 μM of gossypol for 24 h. After incubation, these cells were collected and washed in PBS, and then stained using an annexin V-FITC and PI kit . The preparations were transferred into the sample tube and diluted with PBS . For cell cycle study, the cells were stained with PI only. Data were collected from 4×104 cells in the gated region of each preparation.Y79 cells  with 0.1% EDTA and then stored at −20 °C. Total cellular protein extract was resolved using 10 or 12% sodium dodecyl sulfate–PAGE and electrophoretically transferred to Hybond-P polyvinylidene fluoride membrane . The blot was blocked with 5% non-fat dry milk in TBST  containing 0.1% Tween-20) for 1 h at room temperature, and then incubated with primary antibodies  in blocking solution at 4 °C overnight. After incubation, the blot was washed in TBST, incubated for 1 h at room temperature, and then incubated with horseradish peroxidase (HRP)-linked secondary antibody. The binding antibodies were detected using an electrochemiluminescence (ECL) assay.t-test and one-way ANOVA with the Bonferroni multiple comparisons were used to evaluate statistical significance.Experimental data were analyzed by using SPSS version 18 . The Student 50 calculated as 8.4 μm  -22. GossThe upregulation of mitochondria-related apoptotic proteins observed in the present study suggests that gossypol molecules induce mitochondrial stress, thereby increasing mitochondrial membrane permeability and causing Smac and cytochrome C to be released through the membrane to the cytoplasm. This might lead to procaspase 3 cleavage and subsequent cell apoptosis. Some researchers have suggested that this protein release mechanism shares the same pathway as Bak- and Bax-dependent cytochrome C release . BecauseIn our preliminary protein array screening assay, the expression of insulin-like growth factor-binding protein 6 (IGFBP-6) increased more than twofold in the gossypol-treated preparations. Other studies have indicated that Y79 occasionally uses autocrine signaling through insulin-like growth factor (IGF) types I and II to stimulate cell growth . IGFBP-6Gossypol has been shown to have antiangiogenesic ,28 and a


Purpose. The immunological mechanisms of peri-implant crestal bone loss have, hitherto, not been elucidated. We hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri-implant cells, which results in osteoclast formation and, ultimately, in bone resorption. Materials and Methods. We used RT-PCR and ELISA to assay mediators of osteoclastogenesis in rat and human macrophages (r-and hMO); bone marrow derived stromal cells (r-and hBMCs); and human gingival fibroblasts (hGF)—with or without stimulation by LPS. TRAP positive multinucleate cells were assessed for their resorptive ability. Results. We show that IL-1α, IL-1β, and IL-6 were expressed by all examined cell types, and TNF-α was upregulated in hGF. Secretion of IL-1α and IL-1β proteins was stimulated in hMO by LPS, and IL-6 protein secretion was highly stimulated in hBMCs and hGF. Both LPS and RANKL stimulated macrophages to form osteoclast-like TRAP positive cells, which resorbed calcium phosphate substrates. Conclusion. Taken together, the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri-implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts. Previous evidence has shown that Gram-positive, and Gram-negative bacteria found in the oral cavity can colonize the inner surfaces of implants and the microgap area [Dental implants facilitate the rehabilitation of edentulous patients, and markedly improve both function and esthetics. However, crestal bone loss on the order of 1.5 mm-1.6 mm has been observed radiographically around some dental implants within the first year of loading . The congap area –5; and cThe existence of an inflammatory cell infiltrate at the level of the implant-abutment junction—even around implants placed in areas of meticulous plaque control and clinically healthy, soft tissues—has been described in a series of animal studies –10. HoweBroggini et al. demonstrated that in dogs, the major cell type constituting the inflammatory infiltrate around the microgap of submerged and nonsubmerged two-piece implants was the neutrophil . In theiThe pathophysiological consequences of the implant-abutment interface position have clinically important implications, since esthetic demands encourage the placement of implants in a more apical position . Such plκB activation independent of host factors such as PGE2, RANKL, IL-1, and TNF-α [β and TNF-α while enhancing the expression of RANKL [A. actinomycetemcomitans and P. gingivalis [E. coli bacterial culture solution to demonstrate that implant design affects invasion of oral microorganisms into the fixture—abutment interface microgap under dynamic-loading conditions.It is widely known that bone matrix is resorbed by osteoclasts, which are multinucleated giant cells that originate from hematopoietic progenitors of the monocyte/macrophage lineage . It has nd TNF-α . Osteoclof RANKL . LPS derngivalis , Koutouzngivalis  employedin vitro. Thus, it has been shown that inflammatory cells, microbial cells and/or fluid , 14, 30 Porphyromonas gingivalis , Prevotella intermedia (P. intermedia), and commercially available, Escherichia coli (E. coli), LPS on human gingival fibroblasts, since concern has recently been raised over the use of “home made” LPs preparations [We hypothesized that bacterial products originating from the microgap cause the upregulation of cytokines, in healthy peri-implant macrophages, mesenchymal stromal cells and gingival fibroblasts that result in the recruitment of resorptively active osteoclasts and, ultimately, bone resorption. The notion that healthy fibroblasts in the periodontium could play an important role in providing sustainable inflammatory mediators in the transition from gingivitis to periodontitis has also recently been discussed , althougarations .α-MEM supplemented with Penicillin G (167 units/mL), Gentamicin (50 μg/mL), and 10% FBS, following which it was cut into small pieces and digested with a mixture of 3 mg/mL collagenase type 1 (Sigma-Aldrich) and 4 mg/mL Trypsin in SM for 1 hour at 37 degrees. The digested tissue was centrifuged for 5 minutes at 1150 rpm, and the supernatant was discarded. Then, 10 mL of SM was added to the tube containing the pellet, and the cell suspension was transferred to a T-75 culture flask. When cells reached 80–90% confluence, they were passaged to new T-75 flasks and plated at a concentration of 1.0 × 106 cells/cm2 in 6-well plates at Passage 3. This procedure was repeated five times.Samples of gingival tissues were obtained, with informed consent, from 5 patients who showed no clinical signs of periodontal disease. The tissues were collected from different sites: (1) the palate, used for free connective tissue gingival grafting; (2) the marginal gingivae, following extraction of third molars; (3) the gingiva around dental implants during 2nd stage surgery. The collected tissue was washed 3 times with 2 in a final volume of 2 mL. The cells were allowed to adhere to plastic dishes for 90 minutes at 37 degrees, 5% CO2, in RPMI medium. The cells were vigorously washed with PBS three times, and the nonadherent cells (mainly lymphocytes) were removed. Cell viability was 91% before cells were seeded and characterized by immunofluorescence analysis with Human anti-CD14, Human MHC II, and FITC-conjugated Mouse IgG .Two healthy volunteers, a 35-year-old female and a 50-year-old male, donated blood for this experiment, after providing informed consent. Venous blood (30 mL) was collected from a brachial vein into heparinized tubes and immediately processed. Peripheral blood mononuclear cells (PBMC) were isolated using gradient centrifugation in Histopaque solution (Sigma-Aldrich). Isolated PBMCs were incubated in RPMI  medium-1640 (Gibco) with 10% FBS in 6 well-plates at a density of approximately 5 × 10^6 cells/cm2 flasks in 10 mL of SM. Cultures were incubated at 37 degrees with 5% CO2. After 24 hours, non-adherent cells were removed. When cells achieved 70–80% confluency, they were trypsinized (0.25% trypsin in PBS at 37 degrees for 5 minutes), harvested, and expanded in new 75 cm2 flasks. Cells were expanded to achieve 80–85% confluency at Passage 1 through Passage 4. At this stage, no hemopoietic lineage cells were detectable. The hBMCs were suspended in SM and seeded at a concentration of 1.0 × 106 cells/cm2 in 6-well plates at Passage 4. We collected filters from six different donor aspirates and repeated the hBMCs culture procedures six times.hBMCs were obtained, following local Research Ethics Board (REB) approval, from the collection kit filters  normally discarded following harvesting of bone marrow, from healthy donors, for transplantation. The cells were washed with PBS and then plated in 75 cmμg/mL Gentamicin), and 0.3 μg/mL Fungizone . Subsequently, the cells were plated in 75 cm2 (T-75) polystyrene tissue culture flasks , and incubated at 37 degrees with 5% CO2. The medium was replaced every 2 or 3 days. The cells grew to 80% confluency, as observed by inverted phase microscopy, after which they were washed with PBS  and removed from the culture flask after the addition of 2.5 mL 0.25% trypsin (Gibco) in PBS for 5 minutes at 37 degrees. To inactivate the trypsin, 5 mL SM was added, and the cell suspension was then centrifuged at 1150 rpm for 5 minutes. The total viable cell count was obtained from a ViCell-XR Automated Cell Counter . This cell culture protocol was followed for cell expansion from passage 0 to passage 2. On passage 3, RAW 264.7 cells were plated at a concentration of 2.0 × 105 cells/well in 6-well plates. These cell culture procedures were repeated 6 times.The mouse macrophage cell line, RAW 264.7 , was initially suspended in fresh supplemented medium (SM) composed of 10% fetal bovine serum (FBS) , 80% DMEM (ATCC) and 10% antibiotics  supplemented with 10% antibiotics (as above). Both epiphyses were cut off and the bone marrow was gently flushed out from both sides with a 10 mL syringe (fitted with a 20-gauge needle) filled with fully supplemented medium . The volume of bone marrow suspension was adjusted to 30 mL with FSM and divided in two aliquots of 15 mL each, which were distributed in two T-75 flasks. The first medium change was done at 24 hours and then three times per week subsequently. Cells were grown for 5 to 6 days to 80–85% confluence. At each passage, and up to passage 8, 1.0 × 10^6 cells were collected and fixed with formalin in a 1.5 mL microcentrifuge tube for flow cytometry  analysis of the CD45 positive cell population , for investigation of the presence of hematopoietic cells. Aliquots of 2.0 × 10^6 cells/cm2 of the rSBMCs at passage 7 were seeded into 6-well plates. The passage 7 was chosen in this assay because passage 7 included only 0.36% of CD45+ cells. Stromal cells were isolated from bone marrow of two rats for quantification of CD45+ cells.rSBMCs were harvested from the femora of 100–120 g Wistar rats. The harvested femora were soaked and washed for 10 minutes three times with P. gingivalis, P. intermedia, and E. coli LPS . Both LPS of P. gingivals and P. intermedia were isolated from colonies of these bacterial cultures. The strains used in this study were P. intermedia ATCC 25611, P. gingivalis ATCC 33277. All anaerobes were maintained on Brucella HK agar  supplemented with 5% laked sheep blood in an atmosphere of 80% N2, 10% CO2, and 10% H2O in the aerobic chamber for 48 hours 37 degrees. LPS was extracted by using an LPS extraction kit , dissolved in 10 mM Tris-HCl buffer, and freeze-dried.hGFs were stimulated with different types of LPS: E. coli LPS (0 μg/mL or 0.1 μg/mL or 1 μg/mL) for 24, 48, or 96 hours, and they were stimulated with P. gingivalis LPS or P. intermedia LPS (1 μg/mL) for 2 or 24 hours .In separate experiments, hBMCs and hGFs were stimulated with E. coli LPS  (0 μg/mL or 1 μg/mL) for 12, 24, 36, 48, and 60 hours; hMO were stimulated with LPS (0 μg/mL or 1 μg/mL) for 24 hours; rSBMCs were stimulated with E. coli LPS (0 μg/mL or 0.1 μg/mL or 1 μg/mL) for 6, 24, 48 and 96 hours; hBMCs and hGFs were stimulated with E. coli LPS (0 μg/mL or 0.1 μg/mL or 1 μg/mL) for 24, 48, and 96 hours. In each case, LPS was added to the culture medium when cells reached 75–80% confluence, which was approximately at day 3 for mMO and day 7 for rSBMCs, hGFs, and hBMCs. LPS was added into the culture medium every 24 hours. Cell culture supernatants of hBMCs and hGFs were harvested and stored at −80 degrees prior to enzyme-linked immunosorbent assay . RNA was isolated from both LPS stimulated cells and cell, lysate after the culture medium was removed (see below).RAW 264.7 cells were stimulated with 2 culture dish area of TRI Reagent . cDNA templates of these cells were produced using a reverse transcriptase kit . GAPDH, a house-keeping gene, was used for all species of human, rat, and mouse. Specific primers of IL-1α, IL-1β, TNF-α, IL-6, RANK, RANKL, TRL-2, TRL-4, CD14 and MD-2 were designed for human or rat, and mouse individually using an electronic gene database . The specific primers  and the protein lysates were stored in the same manner. ELISA kits were used to quantify IL-1α, IL-1β, and TNF-α concentration in cell lysates according to the manufacturer's protocols. The absorbance at 450 nm was read with wavelength correction set at 550 nm. The sensitivities of the commercial ELISA kits were <2 pg/mL for IL-1α, <1 pg/mL for IL-1β, <2 pg/mL for TNF-α, and <1 pg/mL for IL-6. The number of samples of cell-free culture supernatant samples, the cell lysate samples, and the times of repeated analysis of each sample are shown in α, IL-1β, IL-6, and TNF-α was analyzed in cell-free culture supernatants and the cell lysate for hBMCs and hGFs. In hMO, we analyzed only the concentration of IL-1α and IL-1β. Culture supernatants from each cell type were removed and stored at −80 degrees prior to ELISA analysis. ELISA kits were used to quantify IL-1mMO (RAW 264.7) were washed with PBS and fixed with 10% formalin at room temperature for 5 minutes. Then, they were washed with distilled water and incubated in michaelis veronal acetate buffer (pH 5.0) containing naphtol AS-BI phosphate as substrate, pararosanilin-HCL as coupler, and tartaric acid for detection of TRAP activity, for 40 minutes at 37 degrees. After removal of the TRAP incubation buffer, cells were washed with distilled water. They were analyzed and counted in micrographs taken with a digital camera attached to the light microscope. μL/well of the assay solution: 1 ml of 0.1 M pNPP (disodium p-Nitrophenylphophate hexahydrate), 1 ml of ×10 buffer (1 M sodium acetate and 0.1 M sodium tartrate), 1 ml of 0.1% Triton-X, and fill up to 10 ml with distilled water. Incubate the plate for 10 minutes at room temperature and add 50 μL/well of 0.2 M NaOH as a stop solution. Then, measure the absorbance at 405 nm.mMO cells in a 96-well plate were washed with PBS and add 100 μg/mL), or RANKL (receptor activator of nuclear factor κB ligand) (200 ng/mL) was added to the cell culture medium, and cells were stimulated with either LPS or RANKL for 7 days. Un-stimulated cells were used as controls. After 7 days, the cell culture medium was removed and cells were fixed with 10% formalin. Micrographs of the OSTEOLOGIC plates were taken with SEM (Scanning electron microscopy). This cell culture procedure was repeated three times. The osteoclastic bone resorption assay was performed using the commercially available OSTEOLOGIC culture system , in which the substrate is coated with a thin layer of osteoclast resorbable calcium phosphate ceramic . RAW 264We investigated the effects of different types of LPS on pro-inflammatory and osteoclastogenic cytokine gene expression following exposure to LPS for 2 hours in hGF. E.coli, P. gingivalis, P. intermedia.) showed similar upregulation of IL-1β, IL-6, and RANKL gene expression  cycles; the expression of IL-1α, IL-1β, and TNF-α was observed at 35 cycles. Interestingly, all gene expression was stronger at 0.1 μg/mL than at 1 g/mL LPS  in cultures of hMOs, hBMCs, and hGFs. ELISA was used to assess levels of protein secretion  were cultured in medium with 300 ng/mL GST-RANKL for 3 days demonstrated the highest TRAP positive reaction compared to negative controls, without RANKL, and LPS stimulated mMO . The varμg/mL LPS for 5 to 7 days and observed under phase contrast for the presence of multinucleated cells (MNCs) (mMO were cultured in medium containing 1 s (MNCs) . The MNCs (MNCs) .Osteoclast formation was examined by TRAP staining, which is a selectable staining to identify osteoclasts and activated macrophages. TRAP-positive mononuclear cells and MNCs were observed in mMO cultures in the presence of LPS  and in tPit forming activity was analyzed using the commercially available BioCoat Osteologic Bone Cell Culture System (Osteologic), which consists of quartz plates with a thin film surface coating of calcium phosphate (CaP). Pit formation on the CaP substrates was observed in mouse macrophage cultures in the presence of LPS  and RANKE. Coli LPS, as a model bacterial endotoxin, can selectively stimulate the upregulation of pro-inflammatory cytokines in healthy candidate resident cells, which can cause the genesis of osteoclasts that are resorptively active. Although gingival and periodontal pathologies are known to cause the upregulation and secretion of pro-inflammatory cytokines, –36 our sin vitro studies [Despite differences in implant/abutment designs, implant/abutment connections will invariably form a microgap, that under function (cyclic opening/closing), will allow passage of organic fluids containing bacteria and endotoxin from and to the implant cavities . Further studies –5. In fa studies . The int studies ); the am studies , 13. Ind studies . Interes studies , 40, whi studies , have shα and IL-6 gene expression in all the examined cells types; while IL-β, and TNF-α were only upregulated in cultures of hBMCs, and hGFs. On the contrary, analysis of protein secretion showed very low or undetectable levels IL-1α, IL-1β and TNF-α in cultures of hM, hBMCs and hGFs. However, there was a clear increase in IL-6 secretion, with LPS treatment, for both hBMCs and hGFs, a finding that corresponds to previous reports [β release [Our results show that LPS upregulated IL-1 reports , 42. Ind reports  and oste reports , an effe reports . Further release . Thus, I release –45. β, IL-10, IL-12, IL-8, IL-6, and TNF-α have all been found to be present in peri-implant crevicular fluid, recorded levels tended to be higher than those observed around teeth, [α and TNF-α in hGFs [IL-6 has also been implicated to have a role on the pathogenesis of periodontitis. Gingival tissues obtained from diseased periodontal sites contain higher levels of IL-6 as compared to healthy sites  and crevd teeth,  and IL-6 in hGFs . Osteobl in hGFs . Thus, i in hGFs  destruct in hGFs , 51. Sin in hGFs  this may in hGFs , 54 contα, IL-1β, and TNF-α were also produced and secreted. The IL-1α and IL-1β have been shown to enhance RANKL expression in both osteoblasts and stromal cells [α promotes the survival of osteoclasts through NF-kB activation which results in induction of osteoclast activation [α induces differentiation and activation of multinucleate osteoclasts [α stimulates the differentiation and survival of osteoclasts, but not the function of osteoclasts [α, IL-1β, and TNF-α. Our results show that LPS activated macrophages can form resorptively active osteoclast-like TRAP positive cells and suggest that LPS may have a direct action on resident macrophages in addition to its known ability to act indirectly on preosteoclasts [In comparison to the specific expression and abundant secretion of IL-6 by hBMCs and hGFs, it is noteworthy that low amounts of IL-1al cells , 45, whitivation . These feoclasts . TNF-α seoclasts . Thus, teoclasts . Taken together, our results support the hypothesis that bacterial endotoxins may upregulate pro-inflammatory genes in a number of resident cells found in the healthy peri-implant compartment, and, that the local synergistic action of cytokines secreted by such cells result in the genesis of resorptively active osteoclasts.


Volleyball is a physically demanding sport and success is based on aspects speed, power, agility, endurance, rapid processing and focus. Nutrition plays a significant role in maximizing performance and volleyball athletes need to be well-informed. Meanwhile, players can be self-conscious of body size and appearance especially in lieu of body contour revealing uniforms. At this time research-based information of this athletic population with regard to body composition, nutrition intake, habits and perceptions is limited and was studied.Twelve Division I women volleyball players aged 18.33±2.9 with 8.8±1.9 years of competitive volleyball experience participated in a study to assess body weight, composition and self-image as well as nutrition knowledge, perceptions, information resources and intake. Body composition was assessed using BOD POD  and a 50-question survey was administered including questions addressing nutrition habits, perceptions and knowledge as well as self-image. Nutrition knowledge questions were validated and multiple choice while answers regarding nutrition and health perceptions and self-image were designed on a Likert scale and ranged from “Strongly Disagree” to “Strongly Agree”. Meanwhile a 3-day Food Journal was completed including two weekdays and one weekend day.1, B2, B3, B6 B12 and zinc were above DRI recommendations while folate, calcium, iron and magnesium were below. Meanwhile 75% ofplayers alleged using one or more nutrition supplements ≥ 2 days/week. Only two of the players had taken a college nutrition course while seven indicated that they dedicated personal time to nutrition study and all ranked their coaches, friends and the internet as the primary sources of nutrition information. However, the players scored 38%±12% of the answers correct on a nutrition questionnaire while ranking water (hydration), protein and then carbohydrate in order of importance to maximizing sport performance. Related to health, 67% and 33% alleged never having their blood glucose and blood pressure and lipids checked. Furthermore, 75% either agreed or strongly agreed that they would like to change the way their body looks and worry about becoming fat while all players disagreed that skipping meals was a good way to control weight.Results of anthropometric measures included height (176.2±7.4 cm), weight (73.3±6.8 kg), BMI (23.57±2.4), FM% (22.1±5.7%) and FFM% (77.9±5.7%). The average energy and protein intake was 1577±451 kcal/day and 1.04±0.23 g/kg with 52%, 28%, 20% of energy derived from carbohydrate protein and fat. The average intake of Vitamin C, BIn conclusion, the volleyball players assessed were lean on average and most were concerned about body weight and are calorie conscious and have a strong sense of self-image. Meanwhile, average energy intake was below estimated needs while energy distribution suggests emphasis on carbohydrate and protein food choices.


Yunnan has the longest endured Human Immunodeficiency Virus-1 (HIV-1) epidemic in China, and the genetic diversity of HIV-1 constitutes an essential characteristic of molecular epidemiology in this region. To obtain a more comprehensive picture of the dynamic changes in Yunnan’s HIV-1 epidemic, a cross-sectional molecular epidemiological investigation was carried out among recently infected individuals.gag (HXB2∶781–1861) and env (HXB2∶7002–7541) genes from 308 plasma samples of recently infected patients. With phylogenetic analysis, 130 specimens generated interpretable genotyping data. We found that the circulating genotypes included: CRF08_BC (40.8%), unique recombinant forms , CRF01_AE (18.5%), CRF07_BC (9.2%), subtype B (2.3%) and C (1.5%). CRF08_BC was the most common genotype, and was predominant in both intravenous drug users (IDUs) and heterosexually transmitted populations. CRF08_BC and CRF07_BC still predominated in eastern Yunnan, but CRF08_BC showed increasing prevalence in western Yunnan. Strikingly, the URFs raised dramatically in most regions of Yunnan. Seven different types of URFs were detected from 12 prefectures, suggesting that complicated and frequent recombination is a salient feature of Yunnan’s HIV-1 epidemic. Among URFs, two BC clusters with distinctive recombination patterns might be potential new CRF_BCs. CRF01_AE was no longer confined to the prefectures bordering Myanmar, and had spread to the eastern part of Yunnan, especially the capital city of Kunming, with a large number of infections in the transient population. The ratios of the main genotypes showed no statistical differences between infected IDUs and heterosexually transmitted infections.We sequenced partial The changing patterns of the dominant HIV-1 genotypes in Yunnan indicate the complex evolving dynamic nature of the epidemic. Understanding new trends in molecular epidemiology of HIV-1 infection is critical for adjusting current prevention strategies and vaccine development in Yunnan. Yunnan is located in southwestern China and has 16 prefectures and 129 counties. Geographically, Yunnan borders Myanmar, Laos and Vietnam and is situated along the drug trafficking routes that channel heroin into China from southeast Asia’s opium-producing “Golden Triangle” region. In 1989, the first Human Immumodeficiency Virus (HIV) epidemic in China was identified among intravenous drug users (IDUs) in Ruili County of Dehong Prefecture In the late 1980s, China’s first HIV-1 epidemic among IDUs was initiated by both subtype B (circulating in United States and Europe) and subtype B’  strains imported from Thailand to Yunnan by drug trafficking through Myanmar Though CRF07_BC and CRF08_BC were first detected from IDUs in Xinjiang Province and Guangxi Province in 1997, respectively Subsequently,studies carried out from 2001 to 2006 showed that there were three major groups circulating in Yunnan: C/CRF07_BC/CRF08_BC, CRF01_AE and B HIV-1 molecular epidemiological surveillance in new infections is of prime importance for understanding the real-time dynamics of the HIV-1 epidemic in a region with complicated HIV-1 subtype prevalence, such as Yunnan. The “gold standard” method for measuring recent infections is a prospective cohort study. However, cohort studies are difficult and expensive to implement and prone to biases that could reduce the general applicability of the results. As an alternative method, the BED-capture enzyme immunoassay (CEIA) has been widely used to measure the proportion of HIV-1-specific IgGs among total IgGs in blood samples for the purpose of identifying infections that were acquired recently A total of 3034 HIV-1-positive plasma samples were identified between January 2009 and March 2009 from 461,661 individuals at local voluntary counseling and testing sites (VCT), medical institutions and sentinel surveillance sites in Yunnan Province. HIV-1 infection status was determined by an enzyme immunoassay and confirmed by Western blot assay . A total of 308 plasma samples identified as recent infections among 3034 HIV-1-positives were used for genotype analysis. Plasma was separated from whole blood and used to obtain HIV-1 RNA for subsequent analysis. All HIV tests are informed and voluntary. Written consents for HIV testing were obtained, in which the subjects agreed that if they have HIV, their samples can be used in the researches for the purpose of controlling and preventing HIV. Because no human experimentation and no investigation of host genetics were conducted, the authors were exempted from approval by the local ethical review committee at the Yunnan Centers for Disease Control and Prevention.BED-CEIA was performed according to the manufacturer’s instructions  gag gene (HXB2∶781–1861) and the env gene (HXB2∶7002–7541). The gag fragment was amplified using One Step reverse transcription PCR  with primers GAG-L (5′- TCGACGCAGGACTCGGCTTGC -3′) and GAG-E2 (5′- TCCAACAGCCCTTTTTCCTAGG -3′) in 25 µl reaction volume. Cycling conditions were as follows: 50°C for 30 min;94°C for 5 min, 55°C for 1 min,72°C for 2 min;94°C for 30 s,55°C for 45 s,72°C for 1 min 30 s,30 cycles;72°C for 10 min. The nested gag PCR was performed using 2×Taq PCR MasterMix  with primers GUX (5′-AGGAGAGAGATGGGTGCGAGAGCGTC-3′) and GDX (5′- GGCTAGTTCCTCCTACTCCCTGACAT-3′) in 50 µl reaction volume. Cycling conditions: 94°C for 2 min, 55°C for 1 min,72°C for 1 min 30 s;94°C for 30 s,55°C for 45 s,72°C for 1 min 30 s,30 cycles;72°C for 10 min. The env fragment was amplified with primers 44F (5′-ACAGTRCARTGYACACATGG-3′) and 35R (5′-CACTTCTCCAATTGTCCITCA-3′) with cycling conditions: 50°C for 30 min;94°C for 2 min, 50°C for 1 min,72°C for 4 min;94°C for 30 s,55°C for 30 s,72°C for 2 min,30 cycles;72°C for 10 min. The nested env PCR was performed with primers 33F (5′- CTGTTIAATGGCAGICTAGC -3′) and 48R (5′- RATGGGAGGRGYATACAT -3′) in 50 µl reaction volume; Cycling conditions: 95°C for 2 min; 95°C for 15 s,55°C for 30 s,72°C 1 min 15 s, 5 cycles;95°C for 15 s,60°C for 30 s,72°C for 1 min,25 cycles; 72°C for 10 min. Each step was carried out with appropriate negative controls to detect possible contamination during the experiments. The generated products were analyzed using 1% agarose gel electrophoresis. Positive samples were sent to Biomed Co.  for sequencing by using an ABI 3730XL automated DNA sequencer  with sequencing primers: GUX/GDX (for gag) and 33F/48R (for env).Three hundred and eight plasma samples identified as recent infections were used for genotype analysis. Viral RNA was extracted from 140 µl of plasma by using the QIAamp Viral RNA Mini kit  according to the manufacturer’s instructions, and was then subjected to nested polymerase chain reaction (PCR) to generate the fragments of the http://hiv-web.lanl.gov/content/index), covering the major HIV-1 subtypes/CRFs. Some reference sequences which were previously characterized in China and countries surrounding Yunnan were included. Phylogenetic tree analyses were performed using the neighbor-joining method based on Kimura 2-parameter model with 1000 bootstrap replicates, using MEGA version 4.0 http://hiv-web.lanl.gov) using the appropriate parameters. Similarity plot analyses  were further performed using reference strains of subtype A1, subtype A2, subtype B, subtype C, subtype D, subtype F1, subtype J, CRF01_AE and CRF08_BC. The conditions are further descried in the figure legends.The contig assembly of sequences was performed using DNA sequence analysis software Sequencher 4.9 . The ClustalW Multiple alignment and manual editing were performed using Bio-Edit 7.0 software. The reference sequences were obtained from the NIH/NIAID-funded HIV Databases . A Dot Density Map was used to display the distribution density of each HIV-1 genotype within Yunnan Province. For each genotype, the number of patients with the genotype in each prefecture was divided by 130  to obtain the percentage of each genotype in each prefecture. When using PHGIS to map the data, one dot was defined as 0.025% of the population.All the sequences obtained in this study were submitted to GenBank under accession numbers JX263434 to JX263661.2. All tests were two-tailed and a p value <0.05 was considered statistically significant.Statistical analyses for this report were conducted using the SPSS 17.0 statistical analysis software package . Categorical variables were compared using χgag and env genes were amplified and sequenced. In total, we obtained 120 gag sequences and 108 env sequences. Combining the phylogenetic tree analysis of gag and env, we successfully genotyped 130 samples with a rate of 42.2% (130/308). The failure of amplification was most commonly due to poor storage and transportation conditions of samples. The constituent ratios of the 130 subjects genotyped and the total of 308 recently HIV-infected individuals showed no statistical differences by geographical area, gender, age, ethnicity, marital status and infection routes , and env fragments  were performed , 12 (10.0%), 3 (2.5%), 5 (4.2%) and 24 (20.0%) sequences were identified as CRF08_BC, CRF07_BC, subtype C, subtype B and CRF01_AE, respectively  CRF01_AE, 3 (2.8%) subtype B, 71 (65.7%) subtype C, 2 (1.9%) CRF01_AE/C and 1 (0.9%) A1/B  A1/B .gag and env, a total of 130 samples generated interpretable sequence data, revealing 2 subtypes, 3 CRFs and 7 discrete URFs. Among the recent infections, CRF08_BC was the most common genotype , followed by the URFs , CRF01_AE , CRF07_BC , subtype B  and subtype C   .gag or env region , the other 13 prefectures have more than one viral genotype present, and Dehong, Honghe and Kunming showed more complex genetic diversity .gag and env, we described the distribution characteristic of HIV-1 subtypes, CRFs and URFs in this area. We found that CRF08_BC was the most common CRF, which predominated in both east and west Yunnan among IDUs and the sexually transmitted population. Exceeding the frequency of CRF01_AE, URFs became the second most common HIV-1 strain which suggests continuous risk behavior still exists among certain groups. Importantly, the distribution of these genotypes was not closely associated with transmission route.In the present work, we conducted an HIV-1 molecular epidemiological study to disclose new trends in recently-infected individuals of Yunnan Province, the province most severely affected by HIV/AIDS in China. By analyzing the HIV-1 genes By the end of 2011, the cumulative number of reported HIV/AIDS in Yunnan was 95296, accounting for 21.0% of the total national figure. Yunnan has shown more diverse HIV-1 genetics than any other HIV-1 high-prevalent province in China, including Guangxi, Sichuan, Xijiang, Guangdong and Hennan In the early 2000s, CRF08_BC and CRF01_AE were dominant in IDUs and those with sexually transmitted infections, respectively env region only in earlier studies env region alone would have been difficult. It has been reported that the nearly full-length genome analyses of the env-based subtype C samples revealed that they were actually BC recombinants gag region, which is distinct from those of CRF07_BC and CRF08_BC. All of this suggests that new CRFs_BC might have been established in this area. To prove this hypothesis, the complete sequencing of full-length HIV-1 genomes is required in future research.Recombination between HIV-1 genotypes is an important mechanism that contributes substantially to the genetic complexity of HIV-1 and may result in establishing epidemiologically important founder strains. The coexistence of multiple genotypes in the same area always causes the formation of CRFs. Typically, CRF07_BC and CRF08_BC have been thought to originate among IDUs in Yunnan Besides CRFs, another striking result is that diverse URFs arise continually. URFs reported before mainly included new BC recombinants, CRF07_BC/CRF08_BC recombinants, and C/CRF01_AE recombinants Furthermore, the HIV-1 strains showed different temporal and spatial dynamics in the process of spreading. We found that CRF07_BC and CRF08_BC were both highly prevalent in east Yunnan, but CRF08_BC was distributed more widely than CRF07_BC, covering almost the entire province. Presently, we do not know whether the transmission capacities of these two CRFs differ. CRF01_AE spread to east Yunnan, particularly in Kunming where the proportion of CRF01_AE remained at a low level in the early 2000s Since the first acknowledged HIV-1 epidemic in China began in Yunnan, this region has become a critical area bridging HIV-1 epidemics in southeast Asia with the subsequent inland epidemic. Through durg trafficking, some genotypes originally found in Yunnan had spread not just to the neighboring provinces of Guangxi (CRF08_BC) gag: 39.0%, 120/308; env: 35.1%, 108/308), which also occurrd in our previous work One limitation of this work was the relatively low PCR amplification rate (In summary, we found that six HIV-1 genotypes circulated in the recently infected individuals of Yunnan, and the distribution of the main genotypes was not closely associated with transmission route. HIV-1 genetics became more diverse because of frequent intersubtypes/CRFs recombinations. These findings showed new trends of HIV-1 molecular epidemiology with dynamic changes in the HIV-1 epidemic and will contribute to a better understanding of the distribution and evolution of HIV-1 in Yunnan as well as help to establish and modify public health efforts to prevent new infections.


P = 0.03, P = 0.007) 140 min after last sprint, but not in placebo. Activity and protein expression of hVps34 were not significantly changed from rest in EAA + Carb 140 min after the last sprint. However, hVps34 activity and protein expression tended to increase in placebo (both P = 0.08). In conclusion, on the contrary to the hypothesis, no increase in activation of hVps34 was found following sprint exercise in EAA + Carb condition. In spite of this, the results support an activation of mTOR during this condition. However, this does not exclude the permissive role of hVps34 in mediating the amino acid-induced activation of mTOR and muscle protein synthesis.Nutrient provision after sprint exercise enhances mammalian target of rapamycin (mTOR) signaling. One suggested that nutrient sensor is the class III phosphatidylinositol 3-kinase, vacuolar protein sorting 34 (Vps34), not previously studied in human skeletal muscle. It is hypothesized that oral ingestion of essential amino acids (EAA) and carbohydrates (Carb) increases Vps34 activity and mTOR signaling in human skeletal (hVps34) muscle after sprint exercise. Nine subjects were performed 3 × 30-sec all-out sprints with or without ingestion of EAA + Carb or placebo drinks in a randomized order with a month interval. Muscle biopsies were performed at rest and 140 min after last sprint and analyzed for p-mTOR, p-p70S6k, p-eEF2 and for hVps34 activity and hVps34 protein content. Venous blood samples were collected and analyzed for amino acids, glucose, lactate, and insulin. During the sprint exercise session, EAA, glucose, and insulin in blood increased significantly more in EAA + Carb than in placebo. P-mTOR and p-p70S6k were significantly increased above rest in EAA + Carb ( In sprint exercise, with great power development, there is a need for both large muscle mass and fast contracting muscles. Therefore, it is important to study the effect of sprint exercise on factors that regulate muscle protein metabolism and the possible additional effects of nutritional supplements. The few studies, that have been performed on this topic, show that there is low or no activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6k after sprint exercise, at least in males  overexpression, in vitro has been shown to activate p70S6k, whereas an hVps34 knockdown was shown to block amino acid-induced activation of p70S6k  was determined using a standardized incremental cycle test to exhaustion on a cycle ergometer . Oxygen uptake was measured continuously utilizing a gas analyzer Vmax Encore System . The subjects' physical characteristics are summarized in Table Nine healthy physically active subjects  were recruited from the university area and sports/health clubs for the study. The inclusion criteria include: in good health, participation in leisure-time sports, but not at an elite level and age 20–35 years. The exclusion criteria were chronic disease, acute infection, severe asthma, or use of products containing nicotine. Health was checked by a general health questionnaire, followed by a 12-lead electrocardiogram registration. A questionnaire was used to estimate the physical activity level during leisure time. The subjects answered nine different questions from which an activity index  was calculated  and maltodextrin, 1 g × kg−1 body weight . Both EAA + Carb and placebo drinks were “Wild Berrie” flavored and contained salts and artificial sweetener, all to conceal the taste of the amino acids . The subjects performed the sprint exercise on a mechanically braked cycle ergometer  according to the protocol in −1 body weight was applied to the flywheel, while the subjects continues to pedal “all out” for 30 sec. The subjects were performed in total three 30-sec sprint exercise bouts with 20-min rest between the sprints. A sensor-microprocessor assembly counted flywheel revolutions and the flywheel progression per pedal revolution was 6 m. Average power output was automatically printed every 5th second. Peak power  and mean power (the average power during the 30-sec sprint exercise) output was expressed in absolute values (W), relative to body mass (W × kg−1 body mass) and relative to FFM (W × kg−1 FFM), see Table All subjects repeated the sprint exercise protocol with or without nutrient ingestion with a month interval between occasions. The order of the experiments was randomized. On each of the two occasions, the subjects reported to the laboratory in the morning after an overnight fast. The subjects were instructed to ingest either an essential amino acid and maltodextrin drink (EAA + Carb) containing eight EAA, 300 mg × kgFor the collection of blood samples, a Teflon catheter was inserted into an antecubital vein. Sampling occurred at 10 min before the first sprint, 9 and 18 min after each sprint and finally 80 and 140 min after the last sprint Fig.  from lefg (4°C) for 10 min. One-milliliter aliquots of the plasma were frozen in liquid nitrogen and stored at −80°C. The other portion of blood was transferred to a serum tube stored at room temperature for 20 min and then treated the same way as the heparinized blood. Plasma lactate concentration was analyzed using a Radiometer ABL 800 Flex blood gas analyzer . An electrochemiluminescence immunoassay was used to analyze serum insulin . Plasma glucose was analyzed enzymatically by a Beckman-Coulter (LX-20) instrument according to kit instructions. Briefly, amino acids were analyzed using high performance liquid chromatography as previously described by Vesali et al. (Each blood sample (10 mL) was divided into two equal portions. One of these was transferred into sodium-heparinized tubes, and immediately centrifuged at 2000i et al.  using pr−1 wet weight) containing 20 mmol L−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 1 mmol L−1 ethylene diamine tetraacetic acid (EDTA) (pH 7.4), 1 mmol L−1 Na3VO4, 5 mmol L−1 ethylene glycol tetraacetic acid (EGTA) (pH 7.4), 10 mmol L−1 MgCl2, 50 mmol L−1 β-glycerophosphate, 2 mmol L−1 dithiothreitol, 1% Triton X-100, and one tablet (per 10 mL) of Complete mini protease inhibitor tablets  diluted in Milli Q® water . Homogenates were rotated  for 60 min at 4°C and centrifuged at 15,000g for 10 min at 4°C to remove cell debris. The supernatants were collected and stored at −80°C. The protein concentration was determined using the Bio-Rad Bradford protein assay using a spectrophotometer . The samples were diluted with homogenization buffer and Laemmli buffer , 40% glycerol, 5% β-mercaptoethanol, and 0.002% bromophenol blue) to a final protein concentration of 2 μg μL−1 containing 25% Laemmli buffer. Following dilution, the samples were heated at 95°C for 5 min to denature proteins and stored at −20°C until further analysis.Muscle samples (∼30 mg) were homogenized on ice using glass homogenizers in ice-cold buffer  used were the following: phospho-mTOR , phospho-p70S6k , phospho-eEF2 , hVps34 , and monoclonal α-tubulin . The secondary antibodies used were all from Cell Signaling Technology: anti-rabbit , anti-mouse , and anti-sheep  IgG antibodies conjugated with horseradish peroxidase.Due to the lack of biopsy material, two of the nine subjects in the study were not included in the Western blot analyses.−1 Tris-base pH 7.5, 1 mmol L−1 EGTA, 1 mmol L−1 EDTA, 1 mmol L−1 sodium orthovanadate, 10 mmol L−1 β-glycerophosphate, 50 mmol L−1 sodium fluoride, 5 mmol L−1 sodium pyrophosphate, 0.27 mol L−1 saccharose, 0.1% (v/v) β-mercaptoethanol and 0.3% (w/v) 3[(3-Cholamidopropyl)dimethylammonio]-propanesulfonic acid, and one tablet (per 10 mL) of Complete protease inhibitor  to a final protein extract concentration of 2–5 mg muscle powder mL−1. The protein extract was agitated  at 1000 rpm, for 10 min at 4°C and centrifuged at 4°C for 15 min at 16,000g, to remove insoluble material. The protein concentration was determined using the DC protein assay (Bio-Rad) utilizing a spectrophotometer .To produce protein extracts, muscle samples (∼30 mg) were powdered under liquid nitrogen using a mortar and pestle. Powdered frozen extracts were lysed using analysis buffer containing 50 mmol L−1 Tris-base, pH 7.5, 150 mmol L−1 NaCl, 1 mmol L−1 EDTA, 0.1 mmol L−1 Na3VO4), resuspended in TNE+ , and incubated with 20 μg hVps34 antigen peptide. Substrates for the assay were prepared by adding 10 μL of 100 mmol L−1 MnCl2 and 10 μL of 2 mg mL−1 phosphoinositol (PtdIns)  to each sample. PtdIns was sonicated for 5 min prior to addition to the assay to generate micelles. Reactions were performed at 30°C with shaking throughout the assay and initiated with the addition of ATP mix . After 10 min, reactions were terminated by the addition of 20 μL of 8 mol L−1 HCl and phase separated with 160 μL 1:1 chloroform: methanol and centrifuged for 1 min at 16,000g. The lower organic phase was spotted on an aluminum-backed silica TLC gel 60 F254 plate  and analyzed in a TLC chamber solvent system . After the plates had developed sufficiently, they were air dried, wrapped in protective film, and exposed to a phosphorimager screen for 24 h before imaging with a phosphorimager . Incorporation of [γ-32P] ATP was quantified using Multi-Gauge software (Fujifilm).Details of the hVps34 activity assay procedures have been previously published , a coefficient of variation (CV) for the hVps34 activity of around 30% was found. However, this does not represents the imprecision of the method because a biological variation, over the 1 month separating the experiments, adds to the true and lower value of imprecision. The CV for the comparison of postexercise values was around 60%, indicating a greater variation after exercise, that is, in a situation where changes in hVps34 activity were expected to occur. This together with our control experiments in cell lines (see below) and the finding of the significant interaction between the two regression lines  of repeated measures design was used to study the effect of condition (placebo or EAA + Carb) and time . In the case of a significant interaction between condition and time, a one-way ANOVA of repeated measures design or Student's t-test for paired observations was used to evaluate changes by time separately for each condition. Student's t-test for group mean was used to evaluate difference between conditions at a few points of time.Values in the text are means ± SD unless stated otherwise. The The relationship between hvps34 activity and plasma glucose  was studied using linear regression model for each condition. To study possible interactions between condition and these regressions (differences in slope between conditions) a mixed model analysis with one within-group factor was performed with the independent variable plasma glucose as covariate.The peak and mean power was not significantly changed on comparing sprint 1, 2, or 3 nor in placebo neither EAA condition (Table P = 0.007) 140 min after sprint exercise in EAA + Carb condition, but not in placebo condition . A similar pattern was shown for the phosphorylation of mTOR at Ser2448, which also was increased above rest  140 min after sprint exercise in the EAA + Carb condition but not in placebo . Phosphorylation of eEF2 at Thr56 was decreased below rest by ∼56%; (P = 0.03) 140 min after sprint exercise independent of condition . A similar trend was seen for hVps34 protein expression .Concentrations of plasma leucine, sum of EAA and of nonessential amino acids (NEAA), tryptophan, serum insulin, plasma glucose, and plasma lactate did not differ between conditions at rest . After the last sprint, plasma glucose decreased in both conditions by approximately 25% . An accentuated increase was seen for serum insulin in EAA + Carb conditions from 18 min to 80 min after the last sprint (P = 0.08), while a decrease was seen in the placebo condition during this period (P = 0.01), leading to sevenfold higher concentration of serum insulin after EAA + Carb than placebo at 80 min after last sprint (P = 0.02). At 140 min after the last sprint, serum insulin was close to basal resting levels in both conditions, . However, the peak level at 9 min after the last sprint 3 was 5% lower in EAA + Carb compared to placebo (condition: P = 0.03). Plasma lactate decreased similarly in both condition from 18 min to 140 min after last sprint, . These two regression lines had significantly different slopes (P = 0.01). This indicates that in placebo condition, the increase in hVps34 activity was greater the lower plasma glucose concentration, while in EAA + Carb the increase in hVps34 activity was greater the higher plasma glucose concentration. The five subjects with the lowest plasma glucose levels (AUC) during placebo condition showed a significant increase in Vps34 activity after the sprint exercise (P = 0.013). Plasma leucine was higher in EAA + Carb condition as compared to placebo, which may add to explain this difference between conditions regarding the relationship between hVps34 and plasma glucose. A similar pattern was seen if AUC was calculated for extended time up to 18 min after the last sprint, but not if the whole area from rest to 80 or 140 min after the last sprint was included . In spite of this, there was a marked increase both in plasma EAA and p70S6k Thr389 phosphorylation in skeletal during the current experimental setup. This is, to the best of our knowledge, the first report of hVps34 activity measurements in human skeletal muscle.The hypothesis in this study was based on in vitro experiments, showing that Vps34 is activated by amino acids or glucose  after sprint exercise in the nutrient poor (fasted) condition in this study, that is, the placebo condition. The novel interaction reported in this study, between increased hVps34 activity and plasma glucose levels in the EAA + Carb and placebo conditions, emphasizes the complexity of hVps34 activation and regulation  as earlier demonstrated after skeletal muscle contractions in rat (MacKenzie et al.