Alpinia oxyphylla is a common remedy in traditional Chinese medicine.Yakuchinone A is a major constituent of A. oxyphylla andexhibits anti-inflammatory, antitumor, antibacterial, and gastric protectiveactivities.Hymenolepis nana adults, and nematocidalactivities against Anisakis simplex larvae are investigated.Antioxidant and antitumor characteristics of yakuchinone A in skin cancer cells aswell as novel mechanisms for the inhibition of adipocyte differentiation,cestocidal activities against + free radicals and inhibition of lipid peroxidation.Yakuchinone A suppresses intracellular lipid accumulation during adipocytedifferentiation in 3 T3-L1 cells and the expressions of leptin andperoxisome proliferator-activated receptor γ(PPARγ). Yakuchinone A induces apoptosis and inhibits cellproliferation in skin cancer cells. The inhibition of cell growth by yakuchinone Ais more significant for non-melanoma skin cancer (NMSC) cells than for melanoma(A375 and B16) and noncancerous (HaCaT and BNLCL2) cells. Treatment BCC cells withyakuchinone A shows down-regulation of Bcl-2, up-regulation of Bax, and anincrease in cleavage poly (ADP-ribose) polymerase (PARP). This suggests thatyakuchinone A induces BCC cells apoptosis through the Bcl-2-mediated signalingpathway. The anthelmintic activities of yakuchinone A for A. simplex arebetter than for H. nana.Yakuchinone A presents the ability of the removal of DPPH·andABTSA.simplex and H. nana.In this work, yakuchinone A exhibits antioxidative properties, anti-adipocytedifferentiation, antitumor activity, and anthelmintic activities against  Cellular oxidative damagethat is caused primarily by ROS is a well-established general mechanism for cell as wellas tissue injury  assay . Aftercentrifugation , supernatants were collected.The cell lysates containing 40 μg of solubilized protein were subjected to12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andelectrophoretically transferred to nitrocellulose membranes. The membranes wereblocked in 5% skim milk. Blots were incubated with the antibodies against Bcl-2, Bax,PARP and β-actin . The membranes were incubated with theappropriate secondary antibody conjugated with horseradish peroxidase . Blotted antibodies were visualized by chemiluminescence method .Cells . They were then washed several times. The adult worms wereindividually observed under an inverted microscope, with subsequent discarding ofthose that exhibited internal or external damage. The adult worms were thenidentified by their morphological features, divided into groups and placed in 24-wellplates contained cultivated media RPMI-1640 plus 20% FBS, pH 7.4, in anatmosphere of 95% O2/5% CO2, 37°C. These cultureconditions have been shown to maximize the development and survival of H.nana.H. nana cultivated media were supplemented with L-glutamine(2 mM), penicillin (100 IU/ml), streptomycin (100 mg/ml) andamphotericin B (0.25 μg/ml), and then the effects of yakuchinone A atconcentrations of 10, 50 and 100 μM were tested. The survival and mobilityof the adult worm were assessed at 2, 4, 6, 12, 24, 48, and 72 h using astereomicroscope. They were observed for their spontaneous motility and evokedresponses at 2, 4, 6, 12, 24, 48, and 72 h using a stereomicroscope. Theoscillation and peristalsis states of adult worms were scored blindly by twoinvestigators. Cestode activity was scored by monitoring both oscillation andperistalsis. Oscillation was scored of movement at scolex and neck for each secondfor 30 seconds, and then the highest score was 30. Peristalsis was record thecontraction real times at scolex and neck. All data were compared with the initialtime before the test compounds had been added. Worms death and complete standstill asdetermined by none any oscillation and peristalsis changes for 30 seconds wereidentified. The mortality was recorded after ascertaining that the worms neithermoved when shaken vigorously nor when dipped in warm medium [The above m medium .Trichiuruslepturuss  that were purchased fromthe fish market of Kaohsiung, Taiwan. The AsL3 had an average length of20–22 mm, and were collected using a needle with a blunt tip, placed inPetri dishes with 0.9% NaCl and washed several times. The majority of the larvae wereencysted, but they quickly became excysted upon washing in NaCl solution. They wereindividually observed under an inverted microscope, with subsequent discarding ofthose that exhibited internal or external damage. The larvae were then identified bymorphological features, divided into groups and placed in 24-well plates containedcultivated media RPMI-1640 plus 20% FBS, pH 4.0, in an atmosphere of 95%O2/5% CO2, 37°C. These showed culture conditionsdemonstrated to provide for the maximum development and survival of A[.The AsL3 were obtained from the muscle and peritoneum of fresh ival of A,32.The above AsL3 cultivated media were supplemented with L-glutamine (2 mM),penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B(0.25 μg/ml), and tested of yakuchinone A for 10, 100, and200 μM. The survival and mobility of the larvae were assessed at 2, 4, 8,12, 24, 48 and 72 h using a stereomicroscope. Two investigators blindly scoredthe larvae as dead, with poor mobility or with normal mobility. The percentage lossesof spontaneous motion during 3 min periods immediately after incubation andcomplete standstill were determined by stimulation 4–5 h later (defined asdeath). The mortality was recorded after ascertaining that the worms neither movedwhen shaken vigorously nor when dipped in warm medium. The nematocidal activity wasmodified according to a scoring system that was developed by Kiuchi et al.  and Lin p value of 0.05was regarded as significant. The data were analyzed and the figures plotted usingsoftware .The results are expressed as mean ± standard deviation (SD).Statistical differences were estimated by one-way analysis of variance (ANOVA)followed by Dunnett’s test or the Tukey-Kramer test. A + radical has beenwidely used for assessment of radical scavenging because of the easy and convenientconsideration [50 values of yakuchinone A for thescavenging of DPPH · radicals were 33.5 (yakuchinone A) and17.9 μM (trolox). The generation of ABTS · +involves the direct production of the blue/green ABTS · +chromophore through the reaction of potassium persulfate and ABTS. The addition ofhydrogen-donating antioxidants to the preformed radical reduces it to ABTS [+. As increasing doses of 5,10, 20, 30, 40, 50, and 100 μM of yakuchinone A and trolox, the values ofABTS · + scavenging capacity were 5.7%, 11.7%, 22.5%,31.0%, 49.6%, 63.6%, and 70.6% for yakuchinone A and 21.9%, 27.3%, 47.6%, 49.6%,75.1%, 92.2%, and 98.0% for trolox, respectively. The EC50 values for thescavenging of ABTS · +-radicals were 40.2 (yakuchinone A)and 30.1 μM (trolox). The extent of decolorization as percentage inhibitionof the ABTS · + radical cation was proportional to theconcentration of antioxidants and calculated relative to the reactivity of trolox asa standard (TEAC). The TEAC value derived from the dose–response curve foryakuchinone A was 3.4 mM of trolox/g. These results suggest that yakuchinone Aexhibits an antioxidant capacity to scavenge DPPH · andABTS · + free radicals.The DPPH · and ABTS · deration . The sol to ABTS . Figure 50 values of the inhibition of lipid peroxidationefficiency by yakuchinone A, trolox and rutin were 10.3, 14.3 and 6.2 μM,respectively. Although the inhibition of lipid peroxidation activity by yakuchinone Awas weaker than by rutin, the inhibition efficiency of yakuchinone A exceeded trolox.The MDA lowering effect of yakuchinone A indicates a protective action against lipidperoxidation of unsaturated phospholipids.The antioxidant action is assessed by inhibiting the damage caused by free radicalsand the mechanisms involved in many human diseases such as hepatotoxicities,hepatocarcinogenesis, diabetes, malaria, acute myocardial infarction, and skin cancerto include lipid peroxidation as a main source of membrane damage . Lipid pleptin and PPARγ significantly asrevealed by RT-PCR. These results suggest that yakuchinone A inhibits andadipogenesis due in part to the inhibition of angiogenesis. These events may bemediated, in part, through antioxidative properties of yakuchinone A responsible forinhibition of angiogenesis.Numerous studies show that obesity may induce systemic oxidative stress, and theincrease in ROS in adipocytes contributes to deregulated expression of inflammatorycytokines such as tumor necrosis factor-α, which may be an early instigator ofthe obesity-associated diabetes and cardiovascular disease ,38. This50 values of yakuchinone A were 13.3, 11.3,18.7, 23.8, and 40.0 μM for A431, BCC, SCC25, A375, and B16 cells,respectively. Moreover, after 72 h treatment with yakuchinone A , the IC50 values of yakuchinone A againstnoncancerous cells  and mouse leukemic monocyte macrophage RAW 264.7 cells were 22.2,32.2, and 46.4 μM, respectively  for 48 and 72 h reduced theoscillation up to 21% and 31% or 47% and 73%, respectively. Yakuchinone A slowlyreduced oscillation from 2 to 72 h but did not cause death. Yakuchinone Areduced the oscillation activity of H. nana in a time- and dose-dependentmanner for 24 to 72 h  polymerase; PPARγ: Peroxisomeproliferator-activated receptor γ; ROS: Reactive oxygen species; TBARS:Thiobarbituric acid reactive substances; TNF-α: Tumor necrosis factor-α; TPA:12-The authors declare that they have no competing interests.RJL, LPC, HYD, CHL, acquisition of data; analysis and interpretation of data;statistical analysis; drafting of the manuscript; obtained funding; study supervision.All authors read and approved the final manuscript. CMY, FYC, administrative support;study supervision. THC, GHW: review of the manuscript. YPT, LW, TWH, HCW, acquisition ofdata.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/237/prepubYakuchinone A exhibits antioxidative properties, anti-adipocytedifferentiation, antitumor activity, and anthelmintic activities against A. simplex and H. nana.Click here for file


Bacillus subtilis DB1342 was used to improve its production, but the yield of CGA-N46 was still low. In the present study, response surface methodology (RSM) was used to optimize culture medium composition and growth conditions of the engineered strain B. subtilis DB1342(p-3N46) for the further increase in CGA-N46 yield. The results of two-level factorial experiments indicated that dextrin and tryptone were significant factors affecting CGA-N46 expression. Central composite design (CCD) was used to determine the ideal conditions of each significant factors. From the results of CCD, the optimal medium composition was predicted to be dextrin 16.6 g/L, tryptone 19.2 g/L, KH2PO4·H2O 6 g/L, pH 6.5. And the optimal culture process indicated inoculation of B. subtilis DB1342(p-3N46) seed culture into fresh culture medium at 5 % (v/v), followed by expression of CGA-N46 for 56 hours at 30 °C induced by 2 % (v/v) sucrose after one hour of shaking culture. To test optimal CGA-N46 peptide expression, the yeast growth inhibition assay was employed and it was found that under optimal culture conditions, CGA-N46 inhibited the growth of Candida albican by 42.17, 30.86 % more than that in the pre-optimization conditions. In summary, RSM can be used to optimize expression conditions of CGA-N46 in engineered strains B. subtilis DB1342(p-3N46).CGA-N46 is a small antifungal-derived peptide and consists of the 31st–76th amino acids of the N-terminus of human chromogranin A. Polycistronic expression of recombinant CGA-N46 in  Candida albican, a ubiquitously distributed opportunistic pathogen, is the leading cause of candidas and causes one of the highest numbers of deaths among patients with fungal infections in the world [C. albican. However, some Candida spp. are intrinsically resistant or have reduced susceptibility to antifungal agents [C. albican [he world , 2, 3. Al agents , 5, 6. Al agents , 8. CGA- albican . To meetcga-N46 gene be induced to express in one plasmid. Using this genetic strain, the expression of cga-N46 gene was increased by optimizing plasmid.There are numerous approaches to optimizing recombinant expression. They are mainly two classes, i.e., genetic engineering and culture conditions optimization. The approaches on high efficient expression optimization mainly were genetic engineering, such as fusion expression , 11, indProduction yield and cost of recombinant proteins are considerably influenced by bacterial culture conditions and medium composition. Optimization of culture conditions using biostatistic methods is another method to increase the expression of an engineered strain. Because of the difficulty of purification, one-factor-at-a-time was usually used . HoweverC. albican growth were tested.In this study, RSM was used to optimize the medium and culture conditions for maximal expression of CGA-N46. To quantify CGA-N46, its effectiveness on inhibiting B. subtilis DB1342(pSBPTQ) which bore plasmid pSBPTQ [B. subtilis DB1342(p-3N46) which bore plasmid p-3N46 [The engineered strains d pSBPTQ  and B. sd p-3N46  used in Escherichia coli and Bacillus subtilis shuttle plasmid, was a cloning plasmid with ampicillin resistance (Ampr) in E. coli and an expression plasmid with kanamycin resistance (Kanr) in B. subtilis. It had sacB promoter sequence (sacB ps) which can be induced by sucrose and promote the transcription of the exogenous gene downstream of sacB ps. It had multicloning sites between sacB ps and terminator sequence (T) which allowed inserting exogenous gene in pSBPTQ. The terminator could make an efficient stop of the transcription. Plasmid p-3N46 was a recombinant vector from pSBPTQ which bore three cistrons of cga-N46 gene between sacB ps and terminator in pSBPTQ. Each cga-N46 cistron had one ribosome binding site, one initiating codon, one copy of cga-N46 gene, one stop codon. Three cistrons of cga-N46 gene allowed three copies of CGA-N46 to be expressed in a single plasmid p-3N46. Their physical maps are shown in Fig. Plasmid pSBPTQ, an C. albican CSC314 was supplied by the Institute of Dermatology, Chinese Academy of Medical Sciences.Yeast strain B. subtilis DB1342 (pSBPTQ) and B. subtilis DB1342(p-3N46). The LB broth medium was prepared with the components as LB agar medium except agar as the growing culture for bacteria.Luria-Bertani (LB) agar medium  containing 10 μg/mL kanamycin was used to culture 2PO4 6 g in 1000 mL of distilled water, was used for expression culture optimization studies.2 × MSR medium , containC. albican CSC314.YPD medium  was used to culture B. subtilis DB1342(pSBPTQ) and B. subtilis DB1342(p-3N46) cells into 25 ml LB broth in 250 mL Erlenmeyer flask. The flask was incubated at 250 rpm at 37 °C for 16 h.The seed culture was prepared by transferring a loopful of fresh transformed strains g for 10 min at 4 °C. For antagonistic activity determination, 10 mL culture supernatant was filtered with 0.22 μm filter film.The production process was carried out, unless otherwise mentioned, in Erlenmeyer flasks (500 mL) containing 100 mL aliquots of the basal culture medium with 10 μg/mL kanamycin. After cultivated for 2 h at 37 °C 200 rpm, 5 mL 40 % sterile sucrose solution was added into culture medium to induce the expression of CGA-N46 for 48 h. During this period, CGA-N46 was expressed and secreted into culture supernatant. After expression, both of the culture were centrifuged at 12,000×C. albican (106 cells/mL) was inoculated into the mixture medium at 10 % (v/v) and then incubated at 26 °C, 200 rpm. The supernatant from B. subtilis DB1342(pSBPTQ) was used as control. After incubating for 24 h, the yeast cells were centrifuged at 10,000×g for 10 min at room temperature. The cell precipitates were dried in a constant temperature oven at 37 °C to remove all traces of water. The dried cell precipitates were weighed, and yeast growth inhibition caused by CGA-N46 was calculated according to Eq.  seed culture was inoculated into the carbon source modified B. subtilis DB1342(p-3N46) culture supernatant from each medium were measured.The carbon source in 3, ammonium sulfate, and urea, respectively. B. subtilis DB1342(p-3N46) seed culture was inoculated into nitrogen source modified B. subtilis DB1342(p-3N46) culture supernatant from each medium were measured.To study the effect of nitrogen source of the culture medium on the antagonistic activity of culture supernatant, the nitrogen source in The computer software, Design Expert 7.1 , was used for experimental design and regression analysis of the experimental data. For analysis of the fitted response nature and prediction of the maximum point, second-order equation was reduced to its canonical form.A), tryptone (B), initial medium pH (C), seed culture inoculation concentration (D), sucrose added time (E), induction period (F), and induction temperature (G). Design Expert 7.1 was used to design the experiments. Yeast growth inhibition rate (Y) was designed as response values. The significant factors affecting the expression of CGA-N46 would be determined according to the response values of different experimental conditions. Each experiment was conducted in three replicates.Two-level factorial experiments were used to find significant factors. Considering the culture conditions reported in previous studies, the main factors in this study were set as follow  culture supernatant from each carbon source modified Yeast growth inhibition rates of B. subtilis DB1342(p-3N46) culture supernatant from each nitrogen source modified Yeast growth inhibition rates of 2PO4·H2O 6 g/L. This culture medium was considered as pre-optimization medium. The antagonistic activity of B. subtilis DB1342(p-3N46) culture supernatant in pre-optimization conditions was calculated to be 11.31 %.From the results of one-factor-at-a-time method for screening carbon and nitrogen sources, the culture medium composition was obtained as follows: yeast extract 50 g/L, dextrin 10 g/L, tryptone 30 g/L, KHp < 0.05) that affected the yeast growth inhibition rate of B. subtilis DB1342(p-3N46) culture supernatant. There were 20 different experiments designed by software Design Expert 7.1 for two-level factorial experiment. The experiment results  are shown in Table A), tryptone (B), medium pH (C), seed culture inoculation concentration (D), sucrose added time (E), induction period (F), and induction temperature (G). From the coefficients of different variables in Eq.  for the independent variables was obtained by software analysis.A) and tryptone (B) in Eq. (B. subtilis DB1342(p-3N46) supernatant in run 6 (dextrin 15 g/L and tryptone 15 g/L) was 41.11 %, suggesting that the optimal concentrations of dextrin and tryptone for response value were near 15 g/L.The coefficients of significant factors dextrin  in Eq.  suggesteTo get the exact optimal concentrations of significant factors, central composite experiments were performed. 15 g/L dextrin and 15 g/L tryptone were set as the central value. The levels of dextrin and tryptone and central composite experiment designs are shown in Table The interaction effect of significant variables (dextrin and tryptone) on the response value (yeast growth inhibition rate) was studied by plotting three-dimensional surface curve. The three-dimensional curve of the calculated response (yeast growth inhibition rate) and contour plot from the interaction between the variables are shown in Fig. Bacillus subtilis DB1342(p-3N46) and DB1342(pSBPTQ) were cultured under the optimized conditions. The yeast growth inhibition rate of B. subtilis DB1342(p-3N46) supernatant was 42.17 %, which was very close to the theoretical result 43.98 %. The result suggested that the experimental model was quite accurate. The results also showed that the inhibition rate of DB1342(p-3N46) supernatant from optimal conditions was significantly increased compared to the original conditions.Compared with genetic engineering, the enhanced yield of CGA-N46 in culture conditions optimization was less, but it could be a valuable compensation to genetic engineering. Moreover, culture conditions optimization is a method to reduce the cost of expression for CGA-N46 industrial development.B. subtilis engineered strain DB1342(p-3N46) using mathematical and computational methods. The factors, such as nitrogen source and carbon source of medium components, initial medium pH, inoculum size, sucrose added time, and induction period, were considered. Although more factors were considered in this investigation than in other reports [This paper focused on investigation of significant factors that were responsible for peptide CGA-N46 expression in  reports , 26, somB. subtilis DB1342(p-3N46). The optimal medium composition was yeast 50 g/L, dextrin 16.6 g/L, tryptone 19.2 g/L, KH2PO4·H2O 6 g/L, pH6.5. The optimal culture conditions were inoculating seed culture at concentration of 5 % (v/v), adding sucrose up to 2 % one hour after the seed culture was inoculated, and allowing cultures to incubate at 30 °C for 56 h. Under optimal culture conditions, yeast growth inhibition rate of CGA-N46 was 31.53 % higher than that in original conditions. It was concluded that RSM was a method for efficiently improving the production of CGA-N46 in the engineered strain B. subtilis DB1342(p-3N46) and might provide an alternative approach to enhance the recombinant protein productivity in other engineered strains.Response surface methodology (RSM) experimental designs offered an efficient and feasible approach for culture conditions optimization of CGA-N46 expression in


Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, or the congenital absence of uterus and vagina, is the most severe anomaly of the female reproductive tract. It affects 1 in 5,000 females, and is the second most common cause of primary amenorrhea. The etiology remains unknown in most patients, although four single gene defects and some repetitive copy number variants (CNVs) have been identified. Translocations in MRKH patients are very rare, and reported only in three patients previously without breakpoint mapping. We have identified the fourth MRKH translocation patient and are the first to characterize the breakpoints mapped by molecular methods.TRIM71, CNOT10, ZNF200, OR1F1, ZNF205, and ZNF213—did not reveal any mutations. RT-qPCR of proband lymphoblast RNA for 20 genes near the breakpoints of 3p22.3 and 16p13.3 showed significantly altered expression levels for four genes in the proband compared to three white female controls, after correction for multiple comparisons. Reduced expression was seen for CMTM7 and CCR4 on 3p22.3, while increased expression was observed for IL32 and MEFV on 16p13.3.The proband is a 17- year old white female with agenesis of the uterus and vagina who had a peripheral blood karyotype revealing a de novo balanced translocation 46,XX,tdn. There were no known related anomalies present in the proband or her family. No CNVs were found by chromosomal microarray analysis, and no genes were directly disrupted by the translocation. DNA sequencing of six nearby candidate genes—We have mapped the breakpoints of our t translocation patient using molecular methods to within 13.6 kb at 3p22.3 and within 1.9 kb for 16p13.3 and have suggested 10 nearby genes that become plausible candidate genes for future study. Abnormal development of the uterus and vagina affects 7-10 % of women, comprising a significant cause of impaired reproductive function . OvariesMRKH is commonly classified with regard to the presence or absence of additional anomalies , 3. IsolAMH, AMHR, WT1, WNT7A, CFTR, GALT, HOXA7, HOXA13, PBX1, HOXA10, RARG, RXRA, CTNNB1, PAX2, LAMC1, DLGH1, and SHOX have been screened for mutations in small numbers of MRKH patients, but no mutations were found  . 2 μg of RNA were used in the 20 μl reaction with RT conditions of: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, 4 °C cooling. The cDNA product was diluted 1:5 for the qPCR reaction, which was run on a Roche LightCycler 96 using the FastStart Essential DNA Green Master Kit protocol  with SYBR green. In the 25 μl reaction volume 2 μl of cDNA was used and each primer concentration was 25 pmol. T values  and stanμg, microgram; μl, microliter; cDNA, complementary DNA; CGH, comparative genomic hybridization; CNVs, copy number variants; der(16), derivative chromosome 16; der(3), derivative chromosome 3; DNA, deoxyribonucleic acid; EBV, Epstein Barr virus; kb, kilobase; min, minute; MRKH, Mayer-Rokitansky-Kuster-Hauser syndrome; pmol, picomole; qPCR, quantitative polymerase chain reaction; RNA, ribonucleic acid; RT-qPCR, real time-quantitative polymerase chain reaction; t, translocation of chromosomes 3 and 16


Streptococcus pneumoniae isolates were submitted to the Canadian Bacterial Surveillance Network and underwent in vitro susceptibility testing. Fifteen percent of S. pneumoniae isolates were collected from pediatric patients (0–15 years old), 48.6 % of isolates were collected from adults between 16 and 64 years of age, and 36.1 % from adults aged ≥65 years; age data were not available for 11 patients. Forty-five percent of S. pneumoniae isolates were recovered from sterile specimens, and 55 % of isolates were from nonsterile specimens. Overall, 0.4 % of isolates were resistant to penicillin, 0.4 % to ceftriaxone, 3 % to amoxicillin, 25 % to erythromycin, and 13 % to trimethoprim/sulfamethoxazole; 6.6 % of isolates were multidrug resistant (MDR). Among MDR isolates, resistance rates exceeded 95 % for erythromycin, tetracycline, and trimethoprim/sulfamethoxazole. The MIC90 of cethromycin, ceftaroline, and ceftobiprole against MDR isolates were 0.12, 0.25, and 1 mg/L, respectively. Ceftaroline, the active form of the prodrug ceftaroline fosamil, exhibited potent in vitro activity against the tested S. pneumoniae including all 456 multidrug-resistant strains. No ceftaroline-resistant isolates were identified.Between 2008 and 2011, 6,895  Streptococcus pneumoniae is the most common bacterial pathogen associated with community-acquired bacterial pneumonia (CABP) [a (CABP) , 14. Thea (CABP) .S. pneumoniae and methicillin-resistant Staphylococcus aureus, and common gram-negative organisms [S. pneumoniae isolates collected across Canada between 2003 and 2008 [Ceftaroline, the active form of the prodrug ceftaroline fosamil, is a parenteral cephalosporin exhibiting broad spectrum in vitro bactericidal activity against gram-positive pathogens, including multidrug-resistant (MDR) rganisms , 12, 15.rganisms , 18. We and 2008 .S. pneumoniae isolates as part of a nationwide surveillance program since 1988. In recent years, there has been not only an increase in the prevalence of MDR S. pneumoniae, but also an increase in the degree of resistance to the β-lactam antibiotics. Surveillance studies in the United States also indicate an increase in nonsusceptibility of S. pneumoniae to common β-lactam antibiotics [S. pneumoniae isolates collected from 2008 to 2011.The Canadian Bacterial Surveillance Network (CBSN) has collected ibiotics , 7. The S. pneumoniae and serotyped using latex antisera  and Quellung reaction [The CBSN encompasses volunteer community and hospital-affiliated laboratories across Canada, which provide services to community and tertiary-care hospitals, community clinics, physician offices, and long-term care facilities. All ten Canadian provinces and two of three territories are represented. In total, 186 laboratories have participated in the CBSN, with 40 laboratories submitting annually since 1993. Only one isolate per patient episode is included; laboratories are asked to submit all sterile-site isolates and a defined number of consecutive nonsterile-site isolates annually, based on laboratory size. All isolates are submitted to a central laboratory where they are confirmed as reaction . IsolateS. pneumoniae isolates from 59 centers underwent antimicrobial susceptibility testing. There were 1,043 (15.1 %) isolates collected from pediatric patients (0–15 years old), 3,350 (48.6 %) isolates collected from adults between 16 and 64 years of age, and 2,491 (36.1 %) from adults aged ≥65 years; age data were not available for 11 patients. Of 6,895 isolates, 3,088 (45 %) were recovered from sterile specimens , and 3,796 (55 %) isolates were from nonsterile specimens .From 2008 to 2011, 6,895 Among sterile-site isolates, the most common serotypes were 19A (17 %), 7F (13 %), and 3 (8 %). Among nonsterile-site isolates, the most common serotypes isolated were 19A (11 %), 3 (9 %), and 11A (9 %). There were decreases in many common serotypes following the introduction of PCV10 in some provinces in 2009 and PCV13 in 2010. Serotypes included in PCV10  and PCV13  accounted for 53.9 % of isolates in 2008, but this decreased to 44.6 % of isolates in 2011.90 of cethromycin, ceftaroline, and ceftobiprole against MDR isolates were 0.12, 0.25, and 1 mg/L, respectively (Table The proportion of isolates resistant to more than two classes of antibiotics (MDR isolates) increased over time Fig. . Overall90 of ceftaroline was ≥8-fold lower, the MIC90 of ceftobiprole was ≥2-fold lower, and the MIC90 of cethromycin was ≥16-fold lower than the MIC90 of ceftriaxone across penicillin-, amoxicillin-, or erythromycin-resistant isolates and in MDR isolates (Table 90 of ceftriaxone increased over the study period from 0.25 mg/L in 2008 to 0.5 mg/L in 2011 (data not shown). Additionally, among all isolates, 13.2 % demonstrated high-level erythromycin resistance (MIC ≥16 mg/L) and 12.0 % demonstrated low-level erythromycin resistance (MIC ≥1 to <16 mg/L) , including PBPs 1a, 2b, and 2x [S.pneumoniae  [S. pneumoniae infection (P = 0.03) [Ceftaroline, ceftobiprole, and cethromycin exhibited more potent in vitro activity against MDR pneumococci than ceftriaxone. Potent in vitro activity of ceftaroline against pneumococci has also been reported from the Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) program . In vitr, and 2x , 11. In ctively) . An analS. pneumoniae isolates increased from approximately 5 % in 2008 to 8 % in 2011. Among the β-lactam antibiotics tested, ceftaroline demonstrated the most potent in vitro activity against MDR S. pneumoniae. The highest MIC observed for ceftaroline against any S. pneumoniae isolate was 0.5 mg/L. These data suggest that ceftaroline fosamil can play an important role in the treatment of infection caused by S. pneumoniae, including MDR strains. Based on the high clinical and microbiological response rates in clinical trials and the potent in vitro activity against S. pneumoniae in this analysis, ceftaroline fosamil is a useful option for management of CABP.In summary, the percentage of MDR


The chemical structure was elucidated by analysis of the spectroscopic data including one-dimensional (1D) and two-dimensional (2D) NMR spectra, and was further confirmed by X-ray crystallographic analysis. The compound was evaluated for its biological activities against a panel of cancer cell lines, dental bacterial biofilm formation, and HIV. It displayed anti-HIV potential with an IC50 value of 9.1 µM (SI = 12.2).Henrin A ( Ent-kaurane compounds are members of a class of diterpenes with a four-membered ring system, which are richly found in the Isodon genus (Lamiaceae) + ).Henrin A (www.ccdc.cam.ac.uk/data_request/cif). Crystal data of 1 (from MeOH): space group P212121, C20H34O3, M = 322.25, a = 6.462(2) Å, b = 12.320(4) Å, c = 22.311(7) Å, α = β = γ = 90.00°, V = 1776.3(9) Å3, T = 293(2) K, Z = 4. μ (Mo Kα) = 0.71073 mm−1. A crystal of dimensions of 0.26 mm × 0.25 mm × 0.24 mm was used for measurements on an APEX DUO diffractometer  with a graphite monochromator , Mo Kα radiation. The total number of independent reflections measured was 3113, of which 2250 were observed (|F|2 ≥ 2σ|F|2). The crystal structure was solved and refined by the direct method SHELXS-97 [2), s = 1.062. Flack parameter = −0.3(2).CCDC 1039988 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre (Available online: in situ with TCA to represent a no growth control at the time of drug addition (day 0). Then 10 μL 10% aqueous DMSO was used as control group. After 72 h incubation, the cells were fixed to plastic substratum by the addition of 50 μL cold 50% aqueous trichloroacetic acid and washed with water after incubation at 4 °C for 30 min. After staining cells with 100 μL of 0.4% sulforhodamine B in 1% aqueous AcOH for 30 min, unbound dye was removed by washing four times with 1% aqueous AcOH. Allowed the plates to dry at room temperature, then the bound dye was solubilized with 200 μL 10 mM unbuffered Tris base, pH 10. Shaken for 5 min or until the dye was completely solubilized and the optical density was measured at 515 nm using an ELISA plate reader . The average data were expressed as a percentage, relative to the control. Percentage growth inhibition was calculated as: (OD (cells + samples) − OD (day 0 only cells))/(OD (cells + 10% DMSO) − OD (day 0 only cells)) = % survival, Cytotoxicity = 1 − % survival.Cytotoxicity assays involving oral epidermoid , colon (HCT116), breast (MCF-7), and lung (A549) carcinoma cell lines  were performed using sulforhodamine B based on the slightly modified protocols used for individual cell lines . KB and Streptococcus mutans (ATCC35668) and S. sobrinus (ATCC33478) , and the antifungal activity was evaluated against Trichophyton rubrum (ATCC MYA-4438) . Biofilm formation was quantified according to a method previously described [wt/vol) was added to each sample well, and the mixture was incubated at room temperature for 15 min. Wells were washed four times with distilled water and were filled with 200 μL of 95% ethanol to solubilize crystal violet in the solvent. The eluent (150 μL) was transferred to a new microtiter well, and the absorbance was determined with a multimode microplate reader (Bio-Rad) at 570 nm.The anti-dental bacterial activity was evaluated against inus ATCC478 , supplemented with 2% glucose. The cell suspension was prepared in growth medium. Each test well received 190 µL of conidia at 2.0 × 104/mL, and 10 µL of the compound solution at 4 mg/mL, where the final concentrations in the well were 20 μg/mL. Positive (10 μL of miconazole with 190 μL of inoculum) and negative (200 μL of medium) controls were included in all experiments. The plates were incubated at 30 °C for 72 h. Then, 20 μL 5 mg/mL 3--2, 5-diphenyltetrazolium bromide (MTT) was added into the 96-well plate, then cultured 10 more hours. The pate was centrifuged at 2500 rpm for 10 min. Remove the supernatant and add 150 μL DMSO to dissolve the formazan by shaking for 30 min. The 96-well plate was centrifuged and the supernatant was transferred to a new 96-well plate, and OD reading was measured at 510 nm by using the microplate reader (Bio-Rad).A 96-flat-bottom-well microtiter plate  was used in experiments to evaluate the effectiveness of the compound in inhibiting ications ,36. The HIV/VSV-G were produced by co-transfecting 3 g of VSV-G envelope expression plasmid with 21 g of a replication-defective HIV vector (pNL4-3.Luc.R∙E) ,38 into 4 cells per well (96-well plate) in complete DMEM. Ten microliter compound for serial concentrations  and 190 µL of the pseudovirus were incubated with target cells. Forty-eight hours post-infection, cells were lysed and prepared for luciferase assay .Target A549 cells were seeded at 10ent-kaurane diterpene  was isolated from the leaves of P. henryi. The chemical structure was elucidated by analysis of the spectroscopic data and was further confirmed by the X-ray crystallographic analysis. The compound was evaluated for its biological activities against a panel of cancer cell lines, dental bacterial biofilm formation, and HIV. Our assay results showed that henrin A (1) had low cytotoxicity due to its lack of an α,β-unsaturated ketone group in the structure, but the compound has been determined as a potential anti-HIV agent in our antiviral assay study.A new


Clonostachys rosea is a promising mycoparasite. In this study, we sequenced the draft genome of the highly effective strain 67-1 using the Illumina HiSeq 2500 sequencing platform. The genome is 55.4 Mb with a G+C content of 49.2% and provides a powerful resource for future studies on the molecular mechanisms underlying Clonostachys rosea’s antagonism on fungal pathogens.  Clonostachys rosea (syn. Gliocladium roseum) is a promising biocontrol agent against various plant pathogenic fungi due to mycoparasitism, antibiosis, competition, and induced plant resistance  and incubated at 26°C for 7 days. Sufficient mycelia were collected and transferred onto a sieve with 25-µm pores using a sterile spatula and washed five times with sterile distilled water to remove fungal spores. Genomic DNA was extracted using a Biospin Fungus Genomic DNA extraction kit  according to the manufacturer’s instructions. Three paired-end libraries of 170,300 and 500 bp and one mate-pair library of 5 kb were constructed for high-throughput sequencing on the Illumina HiSeq 2500 platform. The raw data generated were filtered using FASTX-Toolkit software, and clean data were assembled using Velvet version 1.2.03 (n 1.2.03 .C. rosea 67-1 comprised 1,552 contigs, and further generated 475 scaffolds, with a longest size of 2,741,725 bp and an N50 value of 569,486 bp. In total, 20,747 genes were predicted by the Fgenesh software (The genome sequence coverage was at least 150×. The 5-kb mate-pair library included 8,626,470-bp clean reads, while 9,064,212-, 11,510,231-, and 8,769,057-bp clean reads were obtained from the 170-, 300-, and 500-bp paired-end libraries, respectively, for subsequent genome assembly. The draft genome of C. rosea is a mycoparasite in which cell wall-degrading enzymes such as chitinases and serine proteases play an important role in parasitizing on plant fungal pathogens (C. rosea.athogens , 14. A tathogens . Our futJYFM00000000. The version described in this paper is JYFM02000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Purpose. Cryoprotectants (CPA) for stem cells from umbilical cord blood (UCB) have been widely developed based on empirical evidence, but there is no consensus on a standard protocol of preservation of the UCB cells. Methods. In this study, UCB from 115 donors was collected. Each unit of UCB was divided into four equal parts and frozen in different kinds of cryoprotectant as follows: group A, 10% ethylene glycol and 2.0% dimethyl sulfoxide (DMSO) (v/v); group B, 10% DMSO and 2.0% dextran-40; group C, 2.5% DMSO (v/v) + 30 mmol/L trehalose; and group D, without CPA. Results. CD34+, cell viability, colony forming units (CFUs), and cell apoptosis of pre- and postcryopreservation using three cryoprotectants were analyzed. After thawing, significant differences in CD34+ count, CFUs, cell apoptosis, and cell viability were observed among the four groups (P < 0.05).  Conclusion. The low concentration of DMSO with the addition of trehalose might improve the cryopreservation outcome. In 1988, umbilical cord blood (UCB) was first successfully used as a source of stem cells for hematopoietic reconstitution in a 5-year-old boy with Fanconi anemia, an inherited bone-marrow-failure syndrome that could be cured only by allogeneic hematopoietic stem cell transplantation (HSCT) in Paris, France, by Dr. Eliane Gluckman and her colleagues. In recent decades, UCB stem cells have been used for the treatment of malignant diseases, such as hematological malignancies , Hurler UCB is cryopreserved in liquid nitrogen and is recovered when needed. During cryopreservation and thawing, the main cause of cell death is not long-term storage at low temperatures, but the processes of both cooling and warming through a range of temperatures, such as from −15 to −60°C. Ice crystal formation can also be reduced by the addition of sulfoxides or alcohols such as DMSO. DMSO freely permeates cell membranes due to its low hydrophilicity and molecular weight and is therefore thought to disrupt ice crystal nucleation and formation by forming hydrogen bonds with water . To miniDue to its stability upon freezing, disaccharides such as trehalose have been investigated as a CPA . Alterna+ cell count and cell viability were analyzed both before and after cryopreservation. The data showed that group C exhibited higher cell viability and CFUs and a lower apoptosis rate after thawing than either group A, B, or D.Assessment of stem cell content and viability after long-term storage is a critical step before successful stem cell transplantation. In this study, the three cryoprotectants were assessed. Most cord blood banks currently use 10% ethylene glycol (EG) and 2.0% DMSO (v/v) , 10% DMSUCB was obtained from healthy, full-term, naturally delivered newborns. Written informed consent was obtained from the mothers and their family members. The protocols were reviewed and approved by the Review Board and Ethics Committee of People's Hospital of Taizhou, Jiangsu, China. UCB units were processed within 4.0 h of collection. UCB weight was determined by weighing the collection bag and contents and then subtracting the weight of the empty collection bag and the citrate-phosphate-dextrose-adenine (CPDA) anticoagulant solution. A 10 mL UCB sample was gently mixed with 10 mL saline. Ten-milliliter lymphocyte separation medium (LSM1.077) was poured into a 50 mL tube. The 20 mL cell suspension on top of the separation medium was carefully added to the tube without disturbing the interphase. The tube was centrifuged at 440 ×g for 40 min. Most of the supernatant was then aspirated without disturbing the layer of mononuclear cells in the interphase. The mononuclear cells were then aspirated from the interphase, washed with saline, and centrifuged at 360 ×g for 10 min. The excess red blood cells and plasma were removed. Escherichia coli ATCC25922 for positive control.The plasma was detected using the BD BACTEC 9120 (BD Biosciences) Blood Culture System. The outer surface of the Standard/10 Aerobic/F and Lytic/10 Anaerobic/F  was cleaned with 75% alcohol. The plastic flip cap was removed and the exposed rubber septum was cleaned with an alcohol swab. Then, 3.0–10 mL plasma from UCB was collected for anaerobic bacteria and fungi cultures and another 3.0–10 mL for aerobic bacteria and fungi cultures. The inoculated culture vials were loaded into the instrument, and a temperature of (35 ± 1.5)°C in the racks and (30 ± 1.0)°C within the cabinet was maintained. The plasma was cultured for 7 d. The control assays were carried out by sterile saline for negative control andEach UCB unit was divided into four parts and each part was cryopreserved in one of the three cryoprotectants described above, which were added before the UCB units were frozen. A controlled-rate freezer (CRF) was used to slowly freeze the prepared stem cells to a temperature of −80°C. The following freezing protocol was used for cryopreservation of the stem cells subsequent to processing: wait at 4.0°C, stage 1: ramp 1.0°C/min until sample = −5.0°C; stage 2: ramp 21°C/min until chamber = −54.0°C; stage 3: ramp 17°C/min until chamber = −21.0°C; stage 4: ramp 2.0°C/min until sample = −40.0°C; and stage 5: ramp 10°C/min until sample = −80.0°C. After freezing, the units were immediately transferred from CRF to a liquid nitrogen vessel for storage.8 TNCs. TNC = white blood cell (WBC) + nucleated red blood cell (nRBC). The control assays were carried out for WBC  and nRBC . 103 qualified UCB units were analyzed at each of the data points.Six months after cryopreservation, the UCB units were retrieved from the liquid nitrogen and placed into a water bath at 37°C. To accelerate thawing, the units were carefully moved through the water and their contents were gently kneaded. As soon as the contents had thawed, the sample was removed from the water bath. Five minutes were allowed for equilibration. The tube was then centrifuged at 3000 revolutions per minute (rpm) for 5 min. After centrifugation, the supernatant was discarded by pipettor gently, except group D. A five classification hematology analyzer  was used for counting the total nucleated cells (TNCs) and the recovery of TNCs was calculated. Before freezing, the UCB unit was to have > 5.0 × 10μL CD45-FITC Ab, 10 μL CD34-PE Ab reagent, and 10 μL7-AAD reagent  were pipetted into a tube, and a 50 μL well-mixed sample was pipetted to the bottom of the tube. The tube was placed in a vortex, protected from light, and incubated at room temperature for 15 min. After incubation, 1.0 mL 1x lysing reagent was added to the tube. The tube was vortexed again and incubated at room temperature for 10 min. After incubation, the tubes were centrifuged at 300 ×g for 5.0 min. The supernatant was discarded and 1.0 mL PBS was added to each tube, the contents were mixed, and the tubes were centrifuged at 300 ×g for 5 min. The supernatant was again discarded, 350 μL PBS was added to each tube, and the contents were mixed. The UCB units were tested using BD FACSCanto II . The control assay was carried out for CD34+ count . The UCB units in which the CD34+ cells were >0.25% TNCs and the viability of TNCs was >85% before freezing were chosen. 103 qualified UCB units were analyzed at each of the data points.10 4Cl was added to the sample, and the solution was mixed and left to rest at room temperature for 10 min. The sample was then centrifuged at 300 ×g for 5 min. The supernatant was removed, 1.0 mL DMEM culture medium was added, and the contents were mixed and washed. The supernatant was again removed and DMEM was added to the suspended cells. To perform a cell count, 0.1 mL cell suspension was removed. The stem cells were cultured in 1.2 mL MethoCult GF H4434  culture medium. The final concentration was 1 × 105 cells/mL. Two wells of each sample were seeded in a 24-well plate with 0.5 mL cell suspension. The 24-well cell culture was placed in a humidified atmosphere with 5.0% CO2 at 37°C. After culturing for 14–16 d, the culture board was removed and an inverted microscope was used to count the colonies. The following standards were followed to count colonies: granulocytic, monocytic (GM) ≥ 30 cells/colony; granulocyte, erythrocyte, monocyte, and megakaryocyte (GEMM) ≥ 40 cells/colony; and burst-forming unit-erythroid (BFU-E) ≥ 50 cells/colony. 103 qualified UCB units were analyzed at each of the data points.One-tenth milliliter UCB was placed in a sterile EP tube, 1.0 mL NH4Cl was added to the sample. The sample was washed twice with PBS, and the cell concentration was adjusted to 2.0 × 106 cells/mL. 1.0 × 106 cells/mL were then centrifuged for 5.0 min at 300 ×g, the supernatant was discarded, and the cells were stained with 1.0 μg/mL annexin V and propidium iodide (PI). The cells were then incubated for 30 min at 4.0°C and fluorescence was measured by flow cytometry. 103 qualified UCB units were analyzed at each of the data points.0.1 mL sample of UCB was placed into an EP tube and 1.0 mL NHP < 0.05) was used as the level of statistical difference.Data were expressed as the mean ± SEM. Groups A, B, C, and D were compared using the analysis of variance (ANOVA). A 5.0% probability  was (87.35 ± 6.52)%, (82.43 ± 5.51)%, (91.18 ± 7.40)%, and (16.15 + 1.42)% after thawing, respectively. The viable TNC recovery of group C was higher than that of either group B (P < 0.05) or group D (P < 0.01). The recovery of group D was lower than that of either group A, B, or C (P < 0.01) (The gross weight of the UCB collection bags minus the weight of the collection bag itself and CPDA was the gross weight of UCB. UCB units > 100 mL were chosen for study. The average volume of UCB was (122.8 ± 17.8) mL. The mean TNC was (11.3 ± 3.4) × 10 < 0.01) .http://dx.doi.org/10.1155/2016/1396783). Because of the contaminated samples, the CFU assay could not be completed for these units and the positive samples were discarded.Five UCB units were contaminated with anaerobic bacteria. The BD BACTEC9120 system showed a typical S-shaped growth curve  × 105 and cell viability was (98.34 ± 1.23)% (fresh UCB). After thawing, the mean count of CD34+ was (27.13 ± 4.51) × 105 (group A), (24.57 ± 5.12) × 105 (group B), (30.34 ± 4.78) × 105 (group C), and (6.3 ± 0.51) × 105 (group D). A visible difference in the CD34+ count among the four groups (P < 0.05) was noted, with group C being the highest. The mean percentages of cell viability after thawing were (92.35 ± 5.26)% (group A), (89.43 ± 5.12)% (group B), (94.18 ± 3.97)% (group C), and (18.13 ± 0.98)% (group D). The cell viability of group C was higher than that of either groups A, B (P < 0.05) or group D (P < 0.01) (The mean count of CD34 < 0.01) .5 cells/mL, the average CFU was (36.14 ± 2.06) × 105 (fresh UCB). After thawing, the average CFU in the five different cryoprotectants was (27.78 ± 0.58) × 105 (group A), (22.25 ± 0.52) × 105 (group B), (31.86 ± 0.64) × 105 (group C), and (0.00 ± 0.00) × 105 (group D). There was almost no colony formation in group D. The CFU of group C was higher than that of either group A (P < 0.05) or groups B, D (P < 0.01) (When TNCs were plated at 1.0 × 10 < 0.01) .P < 0.05) or groups B, D (P < 0.01) % (fresh UCB), (9.24 ± 0.68)% (group A), (12.82 ± 0.83)% (group B), (7.01 ± 0.52)% (group C), and (19.01 ± 2.57)% (group D). The cell apoptosis of group C was lower than that of either group A ( < 0.01) .Current UCB cryoprotectants have been developed from empirical evidence; however, there is no consensus on a standard protocol of preservation. Different UCB banks have adopted the use of different cryoprotectants. In recent years, the cryoprotectants were 10% EG and 2.0% DMSO (v/v) , 10% DMS+ counts. The 103 qualified UCB units were divided into four groups and cryopreserved with one of the three different cryoprotectants. In CD34+ testing by FCM, the antibody could bind antigen, whether cells are dead or alive. But dead cells are more easily broken in potent hemolysin (containing sodium azide), so CD34+ cell count of group D was lower than that of group C (P < 0.01). Combined with cell viability and apoptosis assay, although some of the cells were alive, which had started apoptosis, CD34+ cells, cell viability, and CFUs were significantly higher in group C than in either of the other two groups. The results demonstrated that BFU-E were most likely to be affected during cryopreservation and recovery process. The outcomes were consistent with those reported by Motta et al. [In this study, UCB was collected from 115 donors; five were discarded because of bacterial contamination and seven were discarded because of low CD34a et al. –27.Several variables including the CPA concentration, composition, cooling rate, thawing rate, and hold temperatures all matter regarding the quality of UCB. The cryoprotectant in popular use is DMSO, and the use of a controlled rate freezing technique at 1 to 2°C/min and rapid thawing is considered standard. The standard temperatures currently in use are −196 to −80°C and the currently recommended optimal storage conditions are in the vapor nitrogen phase, at −156°C . −156°C Trehalose has also been adopted as a cryoprotective agent for mesenchymal stromal cells and porcine spermatogonial stem cells , 30. In The use of trehalose was shown to result in improved cell survival and differentiation capacity after thawing. A better understanding of the behavior and properties of UCB provides a valuable resource for understanding cryoprotective agents during cryopreservation. After thawing, group C, containing 2.5% DMSO (v/v) + 30 mmol/L trehalose, was shown to improve the survival of UCB stem cells compared to the other two groups prepared without trehalose.Legend: Five UCB units were contaminated by anaerobic bacteria. a) One of typical S-shaped growth curve was showed. b) The staphylococcus was discriminated by Gram staining in corresponding UCB


These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC50 5–19 μM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (RThe online version of this article (doi:10.1007/s00775-014-1187-5) contains supplementary material, which is available to authorized users. These complexes have gained our attention since they are not expected to have intercalative properties as confirmed by previous work 2+  while thous work , 15 and u(dip)2CHbpy-CH3]2ous work –18. The 3bpy-CH3) were purchased from Sigma-Aldrich. 4-carboxy-4′-methyl-2,2′-bipyridine(CH3bpy-COOH) and 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2′-bipyridine (bpy-NitroIm) were prepared according to the published procedures [2L]Cl2 where L denotes bpy, CH3bpy-CH3, CH3bpy-COOH or bpy-NitroIm were prepared following the literature procedures -urea)]Cl2 ([Ru(dip)2(CH3bpy-DCU)]Cl2) was synthesized as follows. To a stirred solution of [Ru(dip)2CH3bpy-COOH]Cl2  in dry CH2Cl2 (3 ml) with triethylamine , solution of N,N’-dicyclohexylcarbodiimide  in dry CH2Cl2 (2 ml) was added. The mixture was stirred at room temperature for 48 h and then the solvent was removed under reduced pressure. The resulting solid was chromatographed on aluminium oxide using chloroform/methanol (50:1) solution as the eluent to afford final compound  as an orange solid. 1H NMR : δ 0.95–1.17 , 1.32–1.70 , 1.80 , 2.14 , 2.63 , 3.33 , 4.31 , 7.42 , 7.49–7.62 , 7.65 , 7.71 , 7.75 , 7.78 , 7.88 , 7.95 , 8.07 , 8.09 , 8.16–8.25 , 8.30 , 8.48 , 8.77 , 9.00 , 9.30 , 9.49  ppm. 13C NMR : δ 21.42, 25.05, 25.16, 25.46, 26.01, 30.71, 31.04, 31.45, 31.64, 50.96, 55.35, 123.11, 125.75, 125.84, 126.21, 126.38, 126.40, 126.64, 126.91, 127.45, 127.84, 128.75, 128.88, 128.90, 129.06, 129.12, 129.23, 129.28, 129.39, 129.64, 129.70, 129.74, 129.78, 129.97, 130.03, 135.23, 135.31, 135.37, 135.44, 146.12, 147.62, 147.98, 148.54, 148.58, 149.01, 149.30, 149.34, 149.55, 150.91, 151.09, 151.15, 151.41, 151.92, 152.73, 153.71, 154.18, 155.97, 157.41, 164.70 ppm. HRMS: calcd. for C73H64N8O2Ru [M2+] 593.2058, found 593.2092 . IR-ATR: 3354w, 3194w, 3054w, 2928m, 2853w, 2157w, 1971w, 1689m, 1649s, 1621s, 1533m. HPLC: tR = 6.96 min, HILIC, acetonitrile/ammonium acetate (0.1 M), gradient from 95:5 to 1:1 .2+ with a small amount of DMSO (<0.008 % v/v) were used as standards . The emission spectra were recorded between 470 and 860 nm upon excitation at 463 nm. The average of three scans was subjected to smoothing. For determination of the quantum yield of luminescence . One-exponential fit was determined to be an optimal description of the obtained results for the ruthenium compounds.The luminescence lifetime measurements were performed with a single photon counting technique using Fluorolog-3, Horiba Jobin Yvon. The excitation wavelength was set at 464 nm (NanoLed Diodes) and the average lifetime of luminescence was monitored at 621 nm. Luminescence decays were collected with 1,000 counts in the peak. The instrument response functions were measured using a light scattering solution of Ludox . Experiments were conducted at room temperature. The DAS6 software (HORIBA Scientific) was used for deconvolution of the obtained decays and for calculation of the lifetime values. The quality of the fit was judged by the 4 cm−1 M−1 at 280 nm  of the MLCT absorption band at a given DNA concentration, fully bound and free ruthenium complex, respectively, Kb is binding constant.Calf thymus deoxyribonucleic acid was purchased from Sigma-Aldrich and its stock solution was prepared by dissolving of solid DNA in water. DNA concentrations per nucleotide were determined by absorption spectroscopy using the molar absorption coefficient of 6,600 Mf 260 nm . DNA-binf 260 nm .\documenThe emission titration studies were performed by using fixed concentration of ruthenium compound (3 µM). The DNA aliquots were added and after 5 min of incubation luminescence spectra upon excitation at 463 nm were measured. The average of three scans was subjected to smoothing and the luminescence intensities were corrected due to dilution effects.Po/w (n-octan-1-ol/water partition coefficient), was measured as following. Ruthenium complexes were dissolved in n-octan-1-ol (to mM concentration), then solutions were added to water and the mixtures were stirred sufficiently for partitioning at 25 °C for 24 h. After that the mixtures were left for equilibration for another 2 h. The concentration of the compounds in the water phase was measured spectrophotometrically and Po/w value was calculated according to the equation: cwater denotes final concentration in water 2+ (compare Table 2(CH3bpy-DCU)]2+ comparing to the parent complex. In contrast, for [Ru(dip)2(bpy-NitroIm)]2+ despite the decrease of the luminescence quantum yield, the luminescence lifetime increases probably due to interplay of the low-lying MLCT emitting levels and higher-lying nonemissive metal-centered levels [2(CH3bpy-DCU)]2+ oxygen does not influence strongly the quantum yield of luminescence, probably because of the increased complexes mobility and the vibration mode of relaxation caused by the expand ligand (bpy-DCU).The studied ruthenium complexes are found to be luminescent  and they all express a shift of the emission maxima toward a longer wavelengths as well as a significant decrease in the quantum yield of luminescence with the respect to the parent complex [Ru(dip)d levels . The lumd levels . Only in2(bpy-NitroIm)]2+ 2+ . SimilarroIm)]2+ . HSA is roIm)]2+ , it can 5 M−1 suggesting a moderate interaction between the investigated ruthenium complexes and albumin. The parent complex [Ru(dip)2(bpy)]2+ is characterized by the weakest interaction with albumin, while the attachment of an additional moiety for bpy ligand increases affinity of the complexes towards HSA, probably by increasing the lipophilicity or the hydrophobic surface of the complexes. The exception is complex with bpy-DCU ligand that is probably too bulky to achieve efficient binding.The association constants for formation of adducts between ruthenium complexes and HSA were determined using spectrofluorimetric method , 35 (det2(CH3bpy-DCU)]2+ exhibits the largest increase in luminescence parameters upon addition of HSA: in PBS in the absence of addition φ = 0.0076 while at [HSA]/[Ru] = 1, φ = 0.0472. This gives ca. ninefold increase in quantum yield of luminescence and makes HSA–[Ru(dip)2(CH3bpy-DCU)]2+ adduct the most luminescent species among the studied protein–ruthenium complex adducts. Correspondingly, the luminescence average lifetime of ruthenium complexes upon addition of HSA changes in a similar way, showing 2- to 4-fold increase at [HSA]/[Ru] = 1 ]2+ and DNA was thoroughly studied elsewhere [2(dppz)]2+), after addition of small excess of DNA luminescence of [Ru(dip)2(R1bpy-R2)]2+/+ complexes decreases as a result of diffusion quenching by DNA [2(CH3bpy-DCU)]2+ does not exhibit quenching of luminescence, but only small shift towards lower wavelengths . It is reasonable to assume, that its weak luminescence properties arising from an increased mobility of the complex caused by the attached expand ligand (bpy-DCU) making it no longer sensitive toward quenchers like DNA or O2 (Table The investigated ruthenium complexes have moderate DNA-binding constants (see Table lsewhere . Based og by DNA . InteresRu(dip)2Rbpy-R2]2+n-octan-1-ol/water partition coefficient (expressed in log Po/w) of the compounds, which was determined by a shaking method. It is well known that lipophilicity of the metal complexes is critical for their cellular selective uptake: cationic probes showing uptake in nuclei and lysosomes have −5 < log Po/w < 0, while dyes with 0 < log Po/w < 5 accumulate preferentially in mitochondria and endoplasmic reticulum [Po/w values of ruthenium(II) polypyridyl complexes are listed in Table 2(CH3bpy-DCU)]2+ is characterized by the highest log Po/w value arising from the expanded substituent in bpy ligand, while for [Ru(dip)2(CH3bpy-COO)]+the relatively high log Po/w is caused by monocationic character of the complex. The literature data have demonstrated that positively charged ruthenium complexes show higher uptake than neutral one 2+ and [Ru(dip)2(CH3bpy-CH3)]2+ were found to be the most toxic in the studied conditions. One of the possible explanations is the high lipophilicity of the former one and moderate liphophilicity and smaller size of the later one, which should facilitate their uptake. [Ru(dip)2(CH3bpy-COO)]+ is less cytotoxic despite the highest lipophilicity, this may be due to its lower accumulation as confirmed by uptake studies. The addition of serum containing as a major component bovine serum albumin to the incubation medium results in ca. twofold decrease in cytotoxicity In order to clarify how ruthenium complexes affected cell growth, after treatment with compound cells were examined by fluorescence microscope. Representative images are shown in Fig. 2(CH3bpy-DCU)]2+ that exhibits the highest accumulation. The actual values of the accumulated ruthenium concentration vary from 130 to 1,200 µM  and it correlates with previously reported ICP-MS measurement of ruthenium complexes uptake [2(CH3bpy-COO)]+ expresses much smaller accumulation than the rest of the studied complexes despite having the highest log Po/w value. The charge of the compound displays a stronger influence than lipophilicity on ruthenium accumulation. This can arise from a possible facilitated transport of ruthenium polypyridyl complexes into cells via passive diffusion due to membrane potential 2+ provided the highest luminescence of 4T1 cells. This complex is characterized by the smallest quantum yield of luminescence, but due to its interaction with protein the quantum yield greatly increases and its high lipophilicity intensifies its uptake. In general, for all the studied complexes the order of luminescence expressed by cells correlates with the cellular uptake determined by ICP-MS method as well as the cytotoxicity data. When the incubation of ruthenium complexes with cells was carried out in medium supplemented with serum, the cells expressed smaller luminescence intensity . It must be noted that the light emission by the cells was not proportional to the amount of ruthenium incorporated inside cells since the luminescence quantum yield for various ruthenium complexes is different. Furthermore, the luminescence of ruthenium complexes is substantially influenced by the interaction with proteins. The observed luminescence signal ]2+/+ that the higher luminescence parameters measured in aqueous solution do not directly correspond to the best cell staining properties. It is a combined effect of the luminescence expressed in cellular environment and the extent of its accumulation. For all studied complexes the interaction with human serum albumin results in pronounced increase of quantum yield and lifetime of luminescence. This phenomenon can be called “light switch” effect and for the studied complexes is observed only in the presence of albumin while DNA induces minor changes in their luminescence. As the intracellular protein content is high (ca. 50–400 mg/ml) we can expect that similar effect is taking place. Therefore, while designing cellular probes it is necessary to take into account not only the luminescence parameters of a single substance, but also the possible resulting adducts with biomacro molecules. The [Ru(dip)2(CH3bpy-DCU)]2+ regardless of its weakest luminescence parameters displays the best staining properties. Additionally, the lipophilicity and complex charge determine the level of its uptake which explains its cytotoxicity and imaging properties. The mechanism of action of these type of complexes remains still unknown, but our preliminary data point out that they can induce the apoptosis.We have shown for a series of complexes [Ru(dip)Supplementary material 1 (PDF 953 kb)Below is the link to the electronic supplementary material.


T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria. The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.A phylogenetically novel proteobacterium, strain Shr3 Culture-independent molecular approaches have revealed that microbial life is astoundingly diverse, and that most prokaryotic microbes have not yet been cultivated . While hIn scientific research and many medical and industrial applications, sterile filtration with 0.2-µm filters is a commonly used method of removing microorganisms. Several studies have reported, however, that previously uncultured bacteria can be isolated from 0.2-µm-filtered freshwater or seawater samples  on the eastern margin of the Sahara Desert in the Republic of Tunisia. Isolation of filterable bacteria was conducted as described previously . In our study, three filters were connected serially for secured filtration. The filtrate was then added aseptically to 5× R2A liquid medium  to obtain a final concentration of 1×. R2A medium is a relatively low-nutrient medium used for cultivation of environmental microorganisms   after 5 days at 25 °C. Cell morphology was examined by light microscopy  and transmission electron microscopy (TEM). Cells for light microscopic observation were grown on R2A for 5 days at 25 °C. Cells were filamentous (width 0.4–0.8 µm) or elongated spindle-shaped cells, some of which exhibited a thin and flexible spiral form similar to a spirillum , followed by post-fixation using osmium tetroxide. After dehydration in an ethanol series, the sample was embedded with resin and sectioned. Following staining with uranyl acetate and modified Sato’s lead staining solution , standard method agar (Nissui) and trypticase soy agar (Becton Dickinson). Even on R2A, the cells exhibited comparatively slow growth, with 3–5 days required before colonies were observable with the naked eye. Strain Shr3T did not require vitamins for growth. The isolate was able to grow in the presence of 0.5 % (w/v) NaCl, but not at 1 % or higher concentrations. The pH range for growth was 7.0–9.5, with an optimum range of pH 7.0–8.0. Strain Shr3T grew at 20–37 °C, with an optimum temperature of 25–30 °C; no growth was observed below 15 °C or above 40 °C. Tested carbohydrates were not utilized in either basal medium. Based on API 20NE results, strain Shr3T displayed a positive reaction for gelatin liquefaction, but was negative for the other tests. Sulfate reduction was not observed. Cells swollen by accumulated PHB were not observed when grown on PHB-containing medium. Particles stained by Sudan black B were observed inside the cells, however, when cells were grown on R2A or PHB-containing medium. Other detailed phenotypic features of the isolate are given in the species description.The isolate was Gram-negative, oxidase- and catalase-positive and non-spore-forming. Motility and flagellation of cells were not observed. Strain Shr3T were C16 : 1ω5c (65.7 %) and C16 : 0 (27.5 %); the major hydroxy acid was C12 : 0 3-OH (1.3 %). Other minor components are shown in Table S1 .To analyse the isoprenoid quinone system of the isolate, cells grown on R2A for 4 days at 30 °C were used. Quinone extraction and measurement was performed by HPLC (Waters) according to T was 54.0 mol%.Genomic DNA was extracted and purified as described by T (1458 bp) was compared with known sequences in the DDBJ nucleotide database (http://www.ddbj.nig.ac.jp/) using a blast search  clade nested deeply within the Proteobacteria .16 : 1ω5c and C16 : 0. The DNA G+C content of the type strain of the type species is 54.0 mol%. The type species is Oligoflexus tunisiensis.Gram-negative. Cells are non-motile and non-spore-forming. Aerobic. Cell shape is pleiomorphic, such as a filamentous, flexible, long fusiform shape or a helical shape similar to spirilla. Cells grow under low nutrient conditions. MK-7 is produced as a major quinone under aerobic cultivation conditions. The major cellular fatty acids are COligoflexus tunisiensis .D-glucose, l-arabinose, d-mannose, N-acetyl-d-glucosamine, d-maltose, lactose, myo-inositol, adonitol, d-sorbitol, d-mannitol, d-trehalose, glycerol, salicin, l-rhamnose, sucrose, d-cellobiose, citrate, fumarate and malate) are not utilized in two tested basal media. Based on the API 20NE test, positive for gelatin liquefaction, and negative for nitrate reduction, indole production, arginine dihydrolase, urease, aesculin hydrolysis, p-nitrophenyl β-d-galactopyranoside hydrolysis and utilization of d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-d-glucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Enzyme activities of esterase lipase (C8), leucine arylamidase, trypsin, naphthol-AS–BI-phosphohydrolase and α-mannosidase are positive. Alkaline phosphatase, esterase (C4), lipase (C14), valine arylamidase, cystine arylamidase, chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-fucosidase tests are negative (API ZYM). The major hydroxy fatty acid is C12 : 0 3-OH.This species has all the characteristics that define the genus, in addition to those described below. Catalase- and oxidase-positive. Non-pigmented. Does not require growth factors. Grows under low nutrient conditions, such as on R2A medium, but exhibits poor or no growth on nutrient agar, trypticase soy agar and standard method agar. Growth rate is slow. Colonies are circular to irregular, low-convex, undulate, pale creamy white and opaque, 1.5–2.0 mm in diameter after 3–5 days of incubation on R2A at 25 °C. Cells are 0.4–0.8 µm wide on R2A. Neisser staining is negative. Sudan black B-stained particles are observed. Non-halophilic and mesophilic. Grows at NaCl concentrations of <1.0 % (w/v). Growth occurs at 20–37 °C, with an optimum temperature of 25–30 °C, and at pH 7.0–9.5, with an optimum at pH 7.0–8.0. Sulfate reduction is not observed. Tested carbohydrates (T ( = JCM 16864T = NCIMB 14846T), was isolated from sand gravels collected from the Sahara Desert in the Republic of Tunisia. The DNA G+C content of the type strain is 54.0 mol%.The type strain, Shr3Oligoflexaceae .Oligoflexus. The type genus is Oligoflexus.The description is the same as for the genus Oligoflexales .Oligoflexus. The type genus is Oligoflexus.The description is the same as for the genus Oligoflexia .Oligoflexales.The class is defined on the basis of a phylogenetic analysis of 16S rRNA gene sequences of a single isolated strain and uncultured representatives from various environments. The type order is


Trypanosomiasis is a neglected tropical disease with complex clinical manifestations, tedious diagnosis, and difficult treatments. The drugs available for the treatment of this endemic disease are old, expensive, and associated with other problems including safety and drug resistant parasites. Therefore, there is an urgent need for the development of new, effective, cheap, and safe drugs for its treatment. Plants are potentially rich sources of leads for new drugs against trypanosomiasis.Vitex simplicifolia (Verbenaceae) is used traditionally for the treatment of tooth ache, edema, skin diseases, gout and trypanosomiasis in Nigeria. In a preliminary study, the methanol extract of Vitex simplicifolia was shown to exhibit a pronounced trypanocidal activity against T. b. rhodesiense.Vitex simplicifolia in the traditional treatment of trypanosomiasis in Nigeria and other African countries. Our investigations aim at assessing the plant as a new source of potential trypanocidal compounds.The present study was undertaken to investigate the active component responsible for the acclaimed activity of the leaves of The crude extracts were prepared from the dried leaves using methanol, successive extraction with hexane, dichloromethane, ethylacetate and butanol was also done. The ethylacetate fraction was further fractionated and compounds isolated using preparative chromatographic technique and their structures were elucidated by NMR, mass spectrometry and comparison with literature data. Trypanocidal activities and cytotoxicity, using rat skeletal myoblast (L6) cells were investigated and their selectivity indices were determined.2, 3, 6 and 7 exhibited promising trypanocidal activity with IC50 values ranging from 4.7-12.3 μg/ml and cytotoxicity in the range of 1.58- 46.20 μg/ml. Compound 6, however, showed the most selective trypanocidal activity with a selectivity index of 9.8. This is the first report of trypanocidal activity of flavonoids from this plant genus.The chromatographic separations of the methanol extracts gave rise to seven compounds. The isolated compounds Vitex simplicifolia exhibited noteworthy trypanocidal activities and hence may provide a source of new antitrypanosomal agents. These results also support the traditional use of Vitex simplicifolia in the treatment of trypanosomiasis. This is the first report of trypanocidal effect of flavonoids from this plant genus.The isolated compounds from  Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense in countries of sub-Saharan Africa [Human African trypanosomiasis also known as African sleeping sickness is caused by the protozoan parasites n Africa . World Hn Africa . There in Africa ,4. Drug n Africa -9.Vitex simplicifolia (Verbenaceae) is a sprawling shrub that can grow as tall as 1.5 m in height [V. simplicifolia are used in traditional medicine for the treatment of malaria, skin diseases, toothache, edema, gout and dermatitis [V. simplicifolia is used for the treatment of various pain disorders, such as stomach ache, hernia ache, dysmenorrhea, arthralgia, and piles [Vitex simplicifolia.n height . The lean height . The learmatitis . In soutnd piles . Over thnd piles ,15. Flavnd piles -18. Prevnd piles -23. So fH 7.26 (CDCl3) and 2.05 (acetone-d6) for 1H, and δC 77.0 (CDCl3) and 29.92 (acetone-d6) for 13C, respectively. Mass spectra were measured with a LCMS HP1100 Agilent Finnigan LCQ Deca XP Thermoquest and high-resolution electrospray ionization mass spectroscopy (HRESIMS) were recorded with an UHR-TOF maXis 4G  mass spectrometer. HPLC analysis was performed with a Dionex P580 system coupled to a photodiode array detector (UVD340S); routine detection was at 235, 254, 280, and 340 nm. The separation column (125 × 4 mm) was prefilled with Eurosphere-10 C18 , and the following gradient was used : 0–5 min (10% MeOH); 5–35 min (10-100% MeOH); 35–45 min (100% MeOH).Optical rotations were determined on a Perkin-Elmer-241 MC polarimeter. 1D and 2D NMR spectra were recorded on an Avance DMX 600 NMR spectrometer. Chemical shifts were referenced to the residual solvent peak at δSemi-preparative HPLC was performed using a Merck Hitachi HPLC System . Column chromatography was performed on Silica gel 60 M  and Sephadex LH-20 (Sigma). TLC was carried out on precoated silica gel plates  for monitoring of fractions. Detection was performed at 254 and 366 nm.Vitex simplicifolia were collected between March and April 2012 from Nsukka, Enugu State, Nigeria. The plant material was authenticated by Mr. Alfred Ozioko of the Centre for Ethnomedicine and Drugs Development, a subsidiary of Bioresources Development and Conservation Program (BCDP), Nsukka, Enugu State. The voucher specimens were deposited at the herbarium of the Department of Pharmacognosy, University of Nigeria, Nsukka. The leaves were cleaned, dried under room temperature and pulverized.The leaves of V. simplicifolia was extracted with 2.5 l of methanol for 24 hours with constant stirring using a magnetic stirrer. The methanol extract (45 g) was dispersed in water and successively extracted with hexane, dichloromethane (DCM), ethylacetate and butanol. The chromatographic separations of the DCM fraction (428.8 mg), using Vacuum Liquid Chromatography (VLC) 22 x 4 cm on silica gel (230–400 mesh) with gradient of n-hexane: ethylacetate led to 10 VLC fractions A-J. Fraction E (72.2 mg) eluted with 50% n-hexane: ethylacetate was subjected to semi-preparative HPLC  using a Eurosphere 100–10 C18 column  with the following gradient (MeOH:H2O): 0 mins, 50% MeOH; 0 mins, 50% MeOH; 4 mins, 60% MeOH; 5 mins, 65% MeOH; 12 mins, 85% MeOH; 14 mins 90% MeOH; 15 mins, 100% MeOH; 16 mins, 100% MeOH to obtain compounds 1 (8.0 mg), 2 (5.8 mg), 3 (1.8 mg), 4 (1.9 mg), 5 (3.0 mg), 6 (1.8 mg) and 7 (2.0 mg).About 600 g of the powdered leaves of 4 bloodstream forms of Trypanosoma brucei rhodesiense STIB 900 (a clone of a population isolated in 1982 from a patient in Tanzania) in 50 μl culture medium were added to each well and the plate incubated at 37°C under a 5% CO2 atmosphere for 72 h. Ten microlitres of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) were then added to each well and incubation continued for a further 2–4 h. The plate was then read in a Spectramax Gemini XS microplate fluorometer  using an excitation wavelength of 536 nm and emission wavelength of 588 nm [50 values. Melarsoprol was used as standard drug.Minimum essential medium (50 μl) supplemented according to a standard method , with 2-f 588 nm . Fluores3 2 g/l and 10% foetal bovine serum. Podophyllotoxin (Sigma-Aldrich) was used as the reference drug. The assay was performed following the antitrypanosomal assay protocol as described above. The IC50 values were calculated from the sigmoidal growth inhibition curves using Softmax Pro software (Molecular Devices Corp.). Tests were done in three independent experiments in duplicate.Cytotoxicity was determined using a rat skeletal myoblast cell line (L6 cells). The culture medium was RPMI 1640 supplemented with L-glutamine 2 mM, HEPES 5.95 g/l, NaHCOVitex simplicifolia was shown to exhibit a moderate trypanocidal activity against T. b. rhodesiense with an IC50 value of 14.2 μg/ml [1–7).In a previous preliminary study, the methanol extract of .2 μg/ml . The met1), 2-(5′-methoxyphenyl) 4′,5,7-trihydroxy-3-methoxychromen-4-one (2), Penduletin (3), 2-(4′-hydroxyphenyl)-5-hydroxy 3,7- dimethoxy chromen-4-one (4), 2-(4-hydroxyphenyl)-3,5,7-trihydroxy chromen-4-one (5), Artemetin (6) and 2--7-hydroxychromen-4-one (7). All the seven compounds in Figure Vitex simplicifolia for the first time. Compounds 3 and 6 have been isolated from a related Vitex species Vitex trifolia, while the others were isolated from the genus Vitex for the first time.The structures of the isolated flavonoid derivatives in Figure in vitro assays assessing the trypanocidal and cytotoxic activities. The results are shown in Table All isolated compounds were subjected to 2, 3, 6 and 7 exhibited moderate trypanocidal activities with IC50 values ranging from 4.7-13.8 μg/ml, however, selectivity versus mammalian L6 cells was completely missing for 2, 3, and 7. Compound 6 showed the most promising and selective trypanocidal activity (IC50 = 4.7 μg/ml) with a selectivity index of 9.8. A close look at the structures of the isolated compounds reveals some structural features that can be correlated with the activities. The trypanocidal activity appears to increase with increase in the methylation of the hydroxyl groups. A plausible explanation for this observation is the corresponding increase in lipophilicity, which increases the permeability of the compounds across the parasite’s membranes. This is in line with other investigations which stated that substitution at C-4” especially with methoxyl group seems relevant for antiplasmodial activity [Four of the isolated compounds activity . Similaractivity . Anotheractivity . It mighactivity .V. simplicifolia showed low to moderate trypanocidal activity in vitro with compound 6 as the most active of the isolated molecules with a promising trypanocidal activity and some selectivity. A similar study shows compounds isolated from plants with IC50s from 1.6 to 19.4 μM and selectivity indices between 0.5 and 6.5 [In conclusion, the flavonoids isolated from  and 6.5 . To the


Human milk (HM) is unique and the gold standard for feeding neonates. HM contains insufficient quantity of protein for preterm infants. Therefore, a new supplement (Aptamil Protein+®-Milupa/Danone) was developed.The aim was to learn about the capability of MIRIS Human Milk Analyzer to measure the composition, particular of the protein content, in HM after addition of Human Milk Fortifier (HMF) and a bovine protein powder (P+®).Nutrient composition of HM was measured baseline, after fortification with commercial HMF (Aptamil 4.3%FMS®-Milupa/Danone and BEBA 5%FM85®-Nestlé), and after addition of further protein (P+®) in 0.5g steps up to 4.0g/100ml. Different milk processing types and the influence on MIRIS measurements was evaluated.Preanalytic testing: MIRIS was capable to measure bovine protein dissolved in water accurately. Impreciseness started after adding 0.5g P+® to HM-fortifier mixtures and revealed up to 20% difference after addition of 4.0g P+®. Native HM  contained in mean 1.1±0.3g protein, 3.1±0.8g fat, 6.6±0.3g lactose, 11.7±0.8g of dry mass and 59±10kcal per 100ml HM. After fortification of HM with HMF and 4.0g P+® a deviation in protein content of 1.0g protein/100ml in FMS® group and in FM85® group was observed. MIRIS underestimated protein content of HM fortified with HMF and P+® mixtures (p<0.05). MIRIS underevaluated carbohydrates and energy content. Processing of HM had no effects on protein level. Lactationday correlated significantly with protein content .MIRIS is an adjuvant tool to provide the actual nutrient composition of unfortified HM. MIRIS has the ability to measure solely bovine protein (in water) and human protein in HM. A combination of both types of protein generated imprecise results of protein content altogether. Processing had no effect on protein content. Protein significantly decreased with lactation period in preterm HM.


Yisui Shengxue granules, which is a Chinese traditional medicine, can increase hemoglobin, red blood cells, and Ret of thalassemia patients with mild, moderate, and severe anemia and thus relieve clinical anemia symptoms. Studies on mechanism found that Yisui Shengxue granules can increase the proliferation ability of hematopoietic stem cells. Emodin promoted colony forming of hematopoietic stem cells. Yisui Shengxue granules can increase the activity of GSH-PX in bone marrow blood and decreased the severity of inclusion bodies on the cytomembrane of RBCs. YSSXG attenuated anemia symptoms in patients with thalassemia mostly by increasing the proliferation of hematopoietic stem cells and decreasing the hemolysis of RBCs. The most common types in China are alpha and beta thalassemia. Alpha thalassemia occurs with absence or mutation of α1 or α2 gene on chromosome 16, and beta thalassemia occurs with mutation of the β globin gene on chromosome 11 [Thalassemia is a type of hereditary hemolytic anemia, resulting from decreased expression of osome 11 . Hemogloosome 11 . The incosome 11  and 3–5%Majority of the patients with thalassemia require lifelong blood transfusions  and iron Cornus officinalis Sieb. et Zucc., Polygonum multiflorum Thunb., Rehmannia glutinosa Libosch., Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao., Codonopsis pilosula (Franch.) Nannf., Angelica sinensis (Oliv.) Diels, Psoralea corylifolia L., Equus asinus L., Spatholobus suberectus Dunn., Trionyx sinensis Wiegmann, and Amomum villosum Lour. A standard quality control of YSSXG has been previously described, including identification of five main herbal components of YSSXG by thin-layer chromatography and characterization of YSSXG by high-performance liquid chromatography [Yisui Shengxue granules (YSSXG) are a traditional Chinese medicine that has been used as an alternative therapy for almost 30 years (since 1989). It is the first and only traditional Chinese herbal medicine for the management of thalassemia. In 2006, a patent was awarded to YSSXG (number ZL200610078866X), which contains eleven components:tography –14.YSSXG can relieve some anemia-related clinical manifestations, including dizziness, palpitations, sallow complexion, tinnitus, and shortness of breath . PreviouEighty-seven thalassemia patients diagnosed according to “Diagnosis and Curative Effect Evaluation Standard of Hematopathy”  were recThe study identified 55 male and 32 female patients with an average age of 13.87 ± 8.00 (5~34). Of all the patients recruited, 21 had mild anemia, 56 had moderate anemia, and 10 had severe anemia . The disPatients were administered YSSXG (ratification number 20120516) for three months. Each packet of granules contained 10 g powder (1 g powder contains 2.368 g crude drug). The granules were dissolved in warm water and taken orally .Emodin used in the in vitro colony forming assay experiment was a purified component from YSSXG, which was dissolved in DMSO (37 mM) and stored at −20°C. Repeated freezing and thawing before use were avoided.An increase in Hb content of more than 5 g/L is considered to indicate effective outcome; Hb content >20 g/L is regarded as a significant curative effect. Clinical parameters: Hb; red blood cell (RBC); reticulocytes (Ret) of peripheral blood were recorded before and after one-, two-, and three-month treatment.Bone marrow blood was collected from the posterior superior iliac spine (ps.i), and CD34+ cells were separated using a magnetic separation method  from nucleated cells after density gradient centrifugation . All stages of the experiment were conducted in a sterile environment.μM 2-mercaptoethanol (AMRESCO 2617B036), 50 ng/mL stem cell factor , 50 ng/mL GM-CSF , 5 ng/mL interleukin-3 , 2 U/mL EPO , 100 UI/mL penicillin , and 100 mg/mL streptomycin .CD34+ cells were obtained from five patients before and after three months of treatment . CD34+ c5 cells/mL. A 96-well plate was prepared with 100 μL complete medium containing cells in each well, and 6 duplicated wells were prepared for each sample. Cells were incubated at 37°C and 5% CO2 in a humidified incubator without disturbance. CCK-8 reagent (10 μL) was added to each well received according to the instructions on the kit  on days two, five, six, and seven. After 2-hour incubation, OD value was detected at a wavelength of 450 nm using an enzyme standard instrument (BioTeSYNERGY2) to observe the proliferation ability of CD34+ cells.Cells were resuspended and adjusted to a concentration of 1 × 10μM, 4 μM, and 9 μM), and incubated in 24-well culture plates at 37°C and 5% CO2 in a humidified incubator without disturbance. The emodin concentration was referenced in the previous work [A hematopoietic colony forming assay was performed using methylcellulose semisolid medium . CD34+ cells were obtained from human donors, cultured in three groups with or without emodin . Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were detected with an enzyme-linked immunosorbent assay.μL of heparin-anticoagulated whole blood via centrifugation at 2500 r/min for 25 minute. The packed RBCs were washed twice with two volumes of phosphate buffered saline at pH 7.3 and fixed with 2.5% glutaraldehyde at 4°C for 2 hours. RBCs were subsequently dehydrated in a series of alcohol washes , and a 100% acetone wash for 10 min twice. Finally, the cells were embedded in epoxy resin (EPON812). Samples were sliced into 50 nm ultrathin sections using LKB-V-type ultramicrotome (Sweden), mounted on uncoated copper grids, and double stained with uranyl acetate and lead citrate. The inclusion bodies of the RBCs were observed under a JEM-1010 electron microscope (Japan) at ×8000 multiples [RBCs were separated from 100 ultiples .t-test if the difference value(d) followed a normal distribution. If the distribution was abnormal, they were analyzed with a nonparametric test . A Chi-Square test was used to analyze the clinical effectiveness rate. A probability value of less than 0.05 was considered statistically significant. Comparison of clinical effective rates between two groups was considered statistically significant at p value <0.0167.Data were entered in SPSS statistical software (v.11.5). Descriptive data were expressed as mean ± standard deviation. Comparison of data before and after treatment was analyzed by a paired-samples p = 0.007); further analysis showed that the effective rates in the moderate anemia group  and the severe anemia group  were higher than those in the mild anemia group (14.29%). There was no difference between moderate and severe anemia groups    . After t, >0.05) .Increase of hemoglobin after three months of treatment was presented in p = 0.010, <0.05) compared to that before treatment. In the moderate anemia group, content of Hb was 80.27 ± 10.29 g/L, 82.93 ± 8.95 g/L, and 83.55 ± 9.97 g/L after one, two, and three months of treatment, respectively; Hb content significantly increased compared with that before treatment, 75.93 ± 8.86 g/L . In the severe anemia group, Hb content was 51.40 ± 6.22 g/L, 60.10 ± 10.18 g/L, 61.30 ± 10.97 g/L, and 60.20 ± 13.04 g/L before treatment and after one, two and three months of treatment, respectively. There is a significant difference in Hb content compared to baseline after each month of treatment: first month , second month , and third month   .12/L), and there was no statistical change after treatment, with RBC levels of 4.82 ± 0.59 (×1012/L), 4.82 ± 0.59 (×1012/L), and 4.90 ± 0.58 (×1012/L), after one, two, and three months of treatment, respectively. The RBC count in the moderate anemia group was 4.26 ± 0.73 (×1012/L), 4.34 ± 0.67 (×1012/L), and 4.36 ± 0.76 (×1012/L), after one, two, and three months of treatment. The RBC count increased significantly compared to initial value 4.01 ± 0.57 (×1012/L), (p = 0.000 after each month). In the severe anemia group, the RBC count was 2.75 ± 0.52 (×1012/L) initially, 3.27 ± 0.71 (×1012/L) after the first month, 3.27 ± 0.90 (×1012/L) after the second month, and 3.32 ± 0.88 (×1012/L) after the third month. There was a significant difference in RBC count after one , two , and three months of treatment  compared to that before treatment  and third month (p = 0.010) compared to baseline. In the moderate anemia group, Ret count was 5.61 ± 2.87 , 6.11 ± 3.47 , and 6.71 ± 3.70  after one, two, and three months of treatment, respectively, with significant increase relative to initial value (4.01 ± 0.57). In the severe anemia group, Ret count was 4.01 ± 2.53 initially, 4.88 ± 3.16 after the first month, 5.60 ± 2.63 after the second month, and 5.78 ± 2.57 after the third month. There was a significant change in Ret count after two  and three months of treatment   .t-test was used to analyze the changes in the proliferation ability of CD34+ cells before and after YSSXG treatment. At seven days, OD value of CD34+ cells after three months of treatment was 0.62 ± 0.23 compared to that before treatment  (A paired-sample  < 0.05) .μM emodin medium, 25.00 ± 2.00 in 4 μM emodin medium, and 17.00 ± 2.65 in the control group. The number of colonies increased significantly in the 9 μM emodin group (p = 0.000) and the 4 μM emodin group (p = 0.005) compared with the control group. There was a significant difference in the number of colonies between 9 μM emodin (p = 0.003) and 4 μM emodin medium . Hemoglobinized cells stained blue with benzidine  , but CFU-GM began to increase rapidly and significantly in 9 μM emodin medium , relative to the control group (67.00 ± 23.12)  was significantly higher (p = 0.046) than in the control group (24.67 ± 5.51) and the 4 μM emodin group (23.00 ± 3.00)  colonies began forming after thirteen days, and the number of colonies in 9  Figures  and 8.p < 0.05) (There were no changes before and after treatment in SOD and MDA activity. Activity of GSH-PX in bone marrow blood significantly increased after treatment (183.11 ± 18.55) relative to baseline  .Dark grain from results of TEM images indicated that numerous inclusion bodies formed on RBC membrane because of unmatched denatured globin chains. After three months of YSSXG treatment, the dark grains numbers decreased .YSSXG is the only traditional Chinese medicine used in the treatment of thalassemia patients. It is an alternative to the lifelong blood transfusions and iron chelation therapies, which are the standard treatments. More important, using an alternative medicine can reduce the complications of blood transfusion in thalassemia patients, such as heart and liver iron accumulation. Therefore, it is important and necessary to evaluate the effect of YSSXG in the clinical setting and to explore the possible mechanisms of this effect.β thalassemia found that YSSXG could increase Hb content after treatment both in severe anemia and in moderate anemia [α or β thalassemia patients. And this Chinese medicine can relieve clinical anemia symptoms and prolong the interval between blood transfusion [Fang's study of 156 patients with e anemia . These rnsfusion , 21.Previous studies  of YSSXGRet is an important parameter to evaluate the proliferation ability of hematopoietic stem cells in bone marrow. Ret was 4.90 ± 2.67 in mild anemia group, 5.61 ± 2.87 in the moderate anemia group, and 4.01 ± 2.53 in the severe anemia group. The average Ret count in the three groups is 0.5–1.5% higher than the normal level, indicative of an increase in Ret count in thalassemia patients in compensation for hemolysis occurring within the body. Results showed that YSSXG can increase the percent of Ret and promote hemopoiesis.γ-globin, EpoR, Spi, and FKLF expression and the herbal medicine Radix polygoni multiflori can increase the Ckit expression.Proliferation of CD34+ cells cultured in vitro after three months of treatment had the same results seen in the mice study. A pharmacodynamics experiment showed that YSSXG can promote hematopoietic stem/progenitor cell proliferation in mice . Ma et aα or β globin genes lead to imbalance between α and β globin chains. And accumulation of relative surplus globin on the erythrocyte membrane makes the hemolysis more severe. Wang et al. [For thalassemia, abnormal g et al.  found thMechanism on YSSXG has been studied from different aspects in cellular level, and hematopoietic stem cells, K565 cells, and RBCs have been studied. Based on clinical trial and mechanism studies, we made a summary of the mechanism of YSSXG in reducing anemia in thalassemia patients, which focuses on the fact that YSSXG may promote hematopoietic ability and reduce hemolysis of red blood cells.We had some limitations in this study. Firstly, no previous studies indicated that colony forming stimulation with YSSXG or emodin could be applied in the clinical setting; future research will use transgenic mice to evaluate the function on hematopoiesis and explore the correlation between engraftment and resultant CFU assays. Secondly, the treatment duration is three months, which is quite short considering that Chinese medicine may need a longer time to take effect; therefore, further studies should focus on the clinical effect of long-term YSSXG therapy on thalassemia and characterize possible adverse effects.Eleven Chinese traditional medicine constituent Yisui Shengxue granules. A standard quality control of YSSXG has been described,including identification of five main herbal components of YSSXG by thin-layer chromatography and characterization of YSSXG by high-performance liquid chromatography.


Acute Respiratory Distress Syndrome(ARDS) is a common respiratory critical illness, usually develops in patients with predisposing conditions that induce systemic inflammatory response, such as sepsis. This study was undertaken to examine the effects of BMSCs injection on ARDS induced by sepsis.To assess the effect of mesenchymal stem cells (MSCs) on acute lung injury induced by sepsis.There were 25 rats in each group, including sepsis group, sepsis+antibiotic group, sepsis+antibiotic+MSCs group and sham group, twenty of which being sacrificed at 12 h, 18 h, 24 h and 48 h after surgery, respectively, five for survival analysis, and 5 normal controls. Survival rate, pulmonary physiological function, alveolar capillary barrier, lung inflammatory reaction and pathological injury were measured.P = 0.007), 35.77 ± 16.80 in the sepsis+antibiotic subgroup (P = 0.016) and 37.80 ± 22.96 in the normal group (P = 0.025). ②the protein concentration of bronchoalveolar lavage fluid in the sepsis+antibiotic+MSCs subgroup was less than that in the sepsis subgroup at 24h, .①at 18 h, total nucleated cell count of bronchoalveolar lavage fluid was 12.29 ± 7.03 in the sepsis+antibiotic+MSCs subgroup, as compared with 42.3 ± 8.18 in the sepsis subgroup