Critical period of weed control is the foundation of integrated weed management and, hence, can be considered the first step to design weed control strategy. To determine critical period of weed control of aerobic rice, field trials were conducted during 2010/2011 at Universiti Putra Malaysia. A quantitative series of treatments comprising two components, (a) increasing duration of weed interference and (b) increasing length of weed-free period, were imposed. Critical period was determined through Logistic and Gompertz equations. Critical period varied between seasons; in main season, it started earlier and lasted longer, as compared to off-season. The onset of the critical period was found relatively stable between seasons, while the end was more variable. Critical period was determined as 7–49 days after seeding in off-season and 7–53 days in main season to achieve 95% of weed-free yield, and 23–40 days in off-season and 21–43 days in main season to achieve 90% of weed-free yield. Since 5% yield loss level is not practical from economic view point, a 10% yield loss may be considered excellent from economic view point. Therefore, aerobic rice should be kept weed-free during 21–43 days for better yield and higher economic return. Critical period of weed control (CPWC) is an integral part of integrated weed management (IWM) and can be considered the first step to design weed control strategy . The CPWCritical period of weed control has got a beginning and an end as well. The beginning of CPWC is determined by estimating critical time for weed removal (CTWR) after which weed control must be initiated to ensure potential yield , 10. TheThe CPWC is likely to be unique for every crop species because of their morphophysiological makeup , but it Aerobic rice, growing rice in nonpuddled and nonsaturated soil like an upland crop, is gaining popularity day by day as a waterwise technology , 21. But The trial was carried out during off-season 2010 (May–July) and main season 2010/2011 (November–January) under field condition at the experimental farm, Universiti Putra Malaysia, Malaysia . The local climate was hot humid tropic with high humidity and abundant rainfall. During the off-season, monthly average maximum and minimum temperature and relative humidity ranged from 33.5 to 35.0°C, 23.6 to 24.6°C, and 93.3 to 93.9%, respectively, while rainfall, evaporation, and sunshine hours ranged from 4.2 to 10.8 mm/day, 3.65 to 4.90 mm/day, and 5.90 to 7.37 hrs/day, respectively. During the main season, monthly average maximum and minimum temperature and relative humidity ranged from 31.7 to 33.3°C, 22.9 to 23.8°C, and 94.1 to 94.6%, respectively, while rainfall, evaporation, and sunshine hours ranged from 6.1 to 9.9 mm/day, 2.64 to 4.66 mm/day, and 3.95 to 6.34 hrs/day, respectively. The experimental soil was sandy clay loam in texture  and acidic in reaction (pH 5.8) with 1.85% organic carbon, 1.43 g/cc bulk density, and 17.36 me/100 g soil CEC. Soil nutrient status was 0.33% total N, 21.2 ppm available P, 143 ppm available K, 794 ppm Ca, and 163 ppm Mg. At field capacity, soil water retention was 22.64% (wet basis) and 29.27% (dry basis).An aerobic rice line AERON 1, sourced from International Rice Research Institute (IRRI), was used as the plant material in this study. The rice line AERON 1 was selected as the plant material because it performed well under aerobic soil conditions in previous study . The experimental design was a randomized complete block with three replications. To determine critical period of weed control, a quantitative series of treatments comprising two components, (a) increasing duration of weed interference and (b) increasing length of weed-free period, were imposed. Timing of weed removal was based on the number of weeks after rice seeding. To determine the beginning of the CPWC, the first component, increasing duration of weed interference, was established by allowing the weeds to compete with the crops for 2, 4, 6, and 8 weeks after seeding (WAS) (referred to as weedy plots), after which plots were maintained weed-free until harvest. To evaluate the end of the critical period of the CPWC, the second component, increasing length of weed-free period, was established by maintaining weed-free condition for 2, 4, 6, and 8 WAS (referred to as weed-free plots) before allowing subsequent emerging weeds to compete for the remainder of the growing season. In addition, season long weedy check and weed-free check were included as control. No herbicide was used as weed control was accomplished by hand weeding. The experiment was conducted under naturally occurring population of mixed weed species.The experimental field was dry-ploughed and harrowed but not puddled during land preparation. Each plot was 5.0 m long and 3.0 m wide and accommodated 12 rows with 25 cm interrow spacing. Rice seeds were directly dry-seeded in rows with 15 cm intrarow spacing at the rate of eight seeds/hill. Each plot was fertilized with triple superphosphate (TSP) and muriate of potash (MP) at 100 kg P/ha and 100 kg K/ha, respectively, as basal application; urea was top-dressed thrice each at 50 kg N/ha at 2, 4, and 6 WAS. Field was maintained under nonsaturated aerobic conditions throughout the growing season. In both the seasons, the trial was primarily rain-fed, but supplemental sprinkler irrigation was applied when needed. Overflow canals were maintained to facilitate drainage whenever heavy rainfall resulted in ponding. Plant protection measures were taken, as needed, to avoid confounding effect of competition with insect and/or disease injury. Different intercultural operations and plant protection measures were conducted following standard practices.2 and g/m2, respectively. Dominant weed species were identified using the summed dominance ratio (SDR) computed as follows [ At each weed removal time, a 25 cm × 25 cm quadrate was randomly placed lengthwise at four spots in each plot for recording weed data. Weeds were clipped to ground level, identified and counted by species, and separately oven dried at 70°C for 72 hours. Weed density (WD) and weed dry weight (WDW) were expressed as no./m follows :2 and grains/panicle were recorded from ten randomly selected hills. Central 3 m2 area of each plot was hand harvested to record grain yield and thousand-seed weight. Grain yield and thousand-seed weight were adjusted to 14% moisture content. Growing degree day (GDD) was accumulated from the date of seeding considering base temperature as 10°C [Four central rows, excluding the harvesting area, were used for data recording. Aboveground crop biomass was recorded at panicle initiation, heading and harvesting stages from ten randomly selected hills of each plot by oven drying at 70°C for 72 hours. At maturity, yield components like panicle/m as 10°C . The timTmax and Tmin are daily maximum and minimum air temperature (°C), and Tb is the base or threshold temperature below which physiological activities are inhibited.Statistical Analysis System (SAS 9.1) software was used to analyze the data, including analysis of variance (ANOVA) and comparison of means based on a protected LSD procedure at 5% level of probability . Data foPhysalis heterophylla Nees, Scoparia dulcis L., Cleome rutidosperma DC, and Cyperus rotundus L. were the most common species dominating the community in both the seasons. Physalis heterophylla Nees appeared as the most abundant species in both the seasons with about 58 and 71 plants/m2 in off and main seasons, respectively. Cyperus sphacelatus Rottb., Cyperus aromaticus, Axonopus compressus (Sw.) Beauv, Amaranthus viridis L., and Eclipta prostrata (L.) L. were present in off-season but not found in main season. In the experiment, the naturally occurring weed community had a wide weed spectrum including broadlaf, sedges, and grasses. Weed population was mostly dominated by broadleaf weeds with very little contribution of grasses. The weed community composed of 23 species in off-season 2010 and 18 species in main season 2010/2011 representing 12 different families . PhysaliCyperus rotundus L., representing sedge group, was the most dominant species at early growth stage of rice, But with the advancement of time, sedges were gradually replaced by broadleaf weeds. Among the grasses, only Axonopus compressus (Sw.) Beauv was recorded as one of the five most dominant species at early growth stages in both seasons and thereafter disappeared, whereas Eleusine indica (L.) Gaertn. and Leptochloa chinensis (L.) Nees were amongst the most dominant species in off-season and main season, respectively, in later growth stages. Physalis heterophylla Nees, Scoparia dulcis L., and Cleome rutidosperma DC were the broadleaf weeds started dominating the community from midgrowth stage of rice till maturity in both the seasons. Weed species dominance pattern was not found similar throughout the growing period . In both2 and 520–540 g/m2, resp.) in both the seasons   was determined by using relative rice yield (% of season long weed-free yield) and growing degree days (GDDs) as quantitative variables in the regression analysis. Rice seeding date was used as the reference point for accumulation of GDD for accounting the possibility of weeds emerging before the rice. The CPWC was determined based on arbitrarily chosen yield loss levels (AYLs) of 5% and 10%, which are judged to be acceptable considering the present economics of weed control. Predicted and observed relative rice yield as affected by weed interference and weed-free periods in off and main seasons are shown in ng (DAS) . In contOver the past few decades, weed management has been mostly herbicide dependent resulting raised public concern about the residual toxicity of herbicides, which necessitates the development of a less herbicide-dependent weed management system . Study oPhysalis heterophylla > Scoparia dulcis > Cleome rutidosperma > Cyperus rotundus > Fimbristylis miliacea > Eleusine indica > Leptochloa chinensis. Similarity in weed composition between seasons might be due to proximity of the experimental sites and similarity of cropping pattern and weed management practices. The weed community was mostly dominated by broadleaf weeds followed by sedges and grasses, which is rather different from that of a typical aerobic rice field. In Karnataka, India, Gowda et al. [The experiment was accomplished under naturally occurring weed population comprising 23 species in off-season and 18 species in main season. Based on summed dominance ratio (SDR), averaged over seasons, the most dominant weed species could be arranged in the order of a et al.  reporteda et al.  also acca et al. . Weed spa et al. . Conflica et al. , who rec2 in between seasons, whereas, in this study, weed dry matter ranged from 390 to 431 g/m2, a 14–37% lower weed dry matter. The dissimilarity could be due to the contrasting rice variety, weed flora, soil moisture regimes, and agroclimatic conditions between experimental sites.Weed density and dry matter were recorded very high in both the seasons. The weed pressure was higher in main season than in off-season because of more favorable conditions in terms of soil moisture status. Differences in weed dry matter between rice seasons has also been reported by Johnson et al.  and ChauBiomass accumulation by AERON 1 was adversely affected by the increasing length of weed interference period and, conversely, favorably influenced by the increasing span of weed-free period up to 6 or 8 WAS. Maintaining weed-free conditions after 6 or 8 WAS failed to improve biomass accumulation of rice. Rice grown in a competitive advantage in comparison with weed will grow better than its counterpart and vice versa. At early crop growth stage, weeds may be better competitor than the crop, which is likely due to competitive advantages for the weeds in terms of preemption of resources. But with the advancement of times, crop start dominating over weeds, and after a certain stage, weed is no more a threat for crop growth, controlling weeds after that resulting no significant enhancement in crop growth. Similar findings have been reported by many researchers , 18, 35,2, grains/panicle, and thousand-seed weight which ultimately translated into lower grain yield. Increased biomass accumulation by weeds with the increasing span of weed interference period might also be a plausible cause of yield reduction in rice. As Woolley et al. [Rice grain yield decreased with prolonged delays in weed removal; conversely, grain yield increased with the increasing length of weed-free period in both the seasons. Weed competition throughout reduced crop yield by approximate 55% in both seasons as compared with season long weed-free period. These values are very close to those reported in a previous study, where season long weed competition reduced yield by approximately 50% . Juraimiy et al.  stated, Based on 5% AYL, our results imply that under similar experimental conditions rice can tolerate weed interference until 137 GDD or 7 DAS in off-season and 131 GDD or 7 DAS in main season, suggesting that control measures should start at that stage; the crop should be kept weed-free until 987 GDD corresponding to 49 DAS in off-season and 1044 GDD corresponding to 53 DAS in main season in order to prevent appreciable economic yield loss. Therefore, weed control measures must begin as soon as possible after rice emergence to prevent yield loss more than 5% in both seasons. At 10% AYL, the beginning of the CPWC was determined 456 GDD for off-season and 412 GDD in main season; to prevent more than acceptable yield loss, aerobic rice field should be maintained weed-free until 832 GDDs have been accumulated in off-season, while in main season, fields should continue to be scouted until 847 GDDs have been accumulated. This equates to controlling weeds from 23 to 40 DAS and 21 to 43 DAS in off and main seasons, respectively. This implies that keeping rice weed-free for that stipulated period is equivalent to keeping rice weed-free season long; presence of weeds before or after that period will not pose a threat [As Zimdahl  stated, The onset of the critical period was found relatively stable between seasons, while the end was more variable. This phenomenon is supported by many researchers , 38, 39,The study portrays the significance of CPWC determination for sustainable weed management in aerobic rice. The practical implication of this study is that under the similar experimental conditions aerobic rice field should be kept weed-free during 7–49 DAS in off-season and 7–53 DAS in main season to achieve 95% of weed-free yield, and 23–40 DAS in off-season and 21–43 DAS in main season to achieve 90% weed-free yield. Since 5% yield loss level would not be practical from economic view point, a 10% yield loss may be considered excellent in terms of economic return, and this level can be achieved by early postemergence application of herbicide or weeding between 10 and 15 DAS followed by a post emergence application or weeding between 30 and 35 DAS. Since weeds emerge after this period are supposed to cause no substantial yield looses, the need for applying additional herbicides or weeding more than 2 times as practiced by most farmers would not be warranted and could lead to significant cost savings. Nevertheless, weed management can be extended beyond that period if the objective is not only to have higher yield but also to avoid weed seed rain to prevent buildup of the weed seed bank, which is of major concern for long-term sustainability of weed management.


Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species.Chinese bayberry  were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species. Chinese bayberry is an important commercial horticultural crop. It has been cultivated for more than 7,000 years in southern China, but is little known elsewhere. The production area is currently 340,000 ha, with an annual production of 1.1 million tons. The plant is diploid 2n = 16), generally dioecious, with the female plants cultivated for fruit[n = 16, gMyrica in China, namely Myrica rubra Sieb. & Zucc., M. esculenta Buch.-Ham., M. nana Cheval., and M. adenophora Hance. M. rubra is widely distributed, with many local cultivars in the Zhejiang, Jiangsu, Fujian and Guangdong provinces and a few from Guizhou, Yunnan and Hunan provinces. The best known cultivars are Biqi and Dongkui, both from the Zhejiang province. Although there are abundant germplasm resources, studies on genetics and breeding of the species are still in their infancy. Molecular marker technology is a popular tool for breeding and genetic research, and with the construction of a genomic library, 13 polymorphic microsatellite loci have been developed in M. rubra[6 as the primer for enriching microsatellite sequences. Reports on the genetic diversity in Chinese bayberry using SSR markers have also recently been published[There are four species within the genus  M. rubra. Recentl M. rubra with GSGublished, but theThe reproducibility, multiallelicism, co-dominance, relative abundance and good genome coverage of SSR markers have made them one of the most useful tools for genetic diversity and linkage mapping. Genomic SSRs and EST-SSRs, considered complementary to plant genome mapping, have been reported in many fruit crops, such as walnut, cherryWith genetic maps serving as the basis for future positional gene cloning, making map-based cloning and marker-assisted selection possible, the development of more SSRs is essential. As sequencing technologies advance, whole-genome shotgun (WGS) sequences are becoming increasingly available. These DNA sequences are valuable resources for SSR development in many plant species, such as rice and papa’ end, acts as the forward primer in a ‘one-tube’ reaction. As this method allows for high-throughput genetic analyses, with a high number of microsatellite markers widely used, we considered the possibility of using this approach for multiplex PCR, to improve the efficiency and save costs.As a way of reducing the cost of genotyping research, Schuelke proposedMyrica species. These markers are useful for genotyping and genetic diversity analysis and linkage mapping of Myrica rubra and other Myrica species.In this study, we mined and validated 158 SSR markers and describe their application for understanding the genetic relationship among 29 Chinese bayberry accessions and other WGS generated 273,161 (>100 bp) high quality sequence reads from two DNA libraries (250 bp and 500 bp) of the androphyte individual ‘C2010-55’. We used 9.01 G raw data for K-mer analysis and heterozygous simulation. For the 17-mer frequency distribution  retrieved from the genome survey sequence contained 28,602 SSRs , and 581 (96.8%) of these were amplified successfully in Biqi and Dongkui, and 400 (66.7%) in M. cerifera. The SSR loci  were identified as heterozygous loci either in Biqi or in Dongkui. Subsequently, they were used to screen 32 accessions, and detected an average of 8.25 alleles and from 3 to 15 alleles per locus , and the remainder trinucleotide. Initially, the effectiveness of these primer pairs was detected in two cultivars (Biqi and Dongkui) and M. adenophora (91.14%) and M. nana (89.87%) and low rates for M. cerifera (46.84%), indicating that these markers are suitable for genetic diversity analyses in other Myrica species.The PIC at each locus ranged from 0.256 to 0.883 with an average of 0.67 loci. The PCR product size ranged from 110 to 274 bp. All the primers produced amplicons in agreement with the expected sizes. Most of the SSR primers (139 primer pairs) showed significant deviation from HW equilibrium (P < 0.05). Partial correlation analysis showed that significant positive correlations existed between the repeat unit length and PIC . This showed that these SSRs had high rates of transferability for 1 populations between Biqi and Dongkui.One of the objectives of this study was to develop potential suitable SSR markers for genetic mapping using Biqi and Dongkui as crossing parents. We selected 99 heterozygous loci in Biqi and 105 in Dongkui Table: 135 priM. nana and M. cerifera into two distinct groups. The genetic similarity between M. cerifera and M. rubra was 0.54, lower than the 0.74 previously reported by Xie[The 32 accessions were divided into three groups  and Hangzhou (3), where the popular and dominant cultivar is Biqi. This demonstrates that these natural elite seedling selections are truly distinct from the local cultivars. For their genetic relationships Figure, the rarM. adenophora’ grouped together, and are possibly in the same population. Six androphyte accessions, distributed in group A, are close to cultivars of the same geographic origin.Subgroup A-2 includes 14 accessions, with four from Wenzhou, two from Taizhou, and one each from the cities of Hangzhou and Ningbo, and the Hunan, Guangxi, Guizhou and Jiangsu provinces. This group includes the popular cultivar Dongkui. The four accessions from Wenzhou distributed in this cluster have genetic similarity. The accession ‘Tongzimei’ from the Hunan province is on an independent branch, showing that it is genetically distinct. ‘Xiaolejiangchonghei’ and ‘Myrica nana’ from Yunnan and ‘Myrica cerifera’ from the USA were independently classified as the ‘B’ and ‘C’ group, indicating a distant relationship with cultivated Myrica rubra.The accessions ‘Myrica rubra and understand the genetic diversity and relationship among representative cultivars, androphyate and related species. This research paves the way for constructing an SSR-based linkage map in Myrica.Our major aims were to find a large set of SSR markers for http://www.rosaceae.org/peach/genome), but close to our estimate of 250 Mb from flow cytometry using rice as the reference (date not shown). Although the highly homozygous material was selected on a limited number of SSR loci assays, the actual heterozygous rate, as revealed by 185 new SSR markers, was very high (63%). To overcome the key issue of heterozygosity and allow us to generate a high-quality genome sequence, we can use a unique homozygous form such as monoploid, derived using tissue culture or from nature and worth further study.Shotgun sequencing is suitable for simple genomes, with no or few repeat sequences, such as Chinese bayberry. For such genomes, the genome can largely be assembled simply by merging together reads containing overlapping sequence. We repon as the most common repeat unit[SSRs have been widely used for high-throughput genotyping and map construction as they have the advantage of high abundance, random distribution within the genome, high polymorphism information content and stable co-dominance-20. Theyeat unit.1 populations, although they are useful for mapping in F2 or backcross populations[1 populations  were annotated, which provides an excellent platform for future EST-SSR development and functional genomic research.Normally, a universal M13 primer is labelled with one of a number of fluorescent dyes. The tailed primer provides a complementary sequence to the fluorescent labelled M13 primer, leading to the amplification of fluorescent PCR products, and then the PCR products of different sizes and/or labelled with different fluorescent dyes are mixed and tested. In thisM. nana and M. cerifera were clearly genetically distant to M. rubra. M. nana, also known as the dwarf or Yunnan arbutus, is indigenous to the Yunnan and Guizhou provinces, and has a plant height of < 2 m. The juvenile period of fruit tree can be shortened for breeding purposes. Studies on embryo culture in vitro of the F1 seeds of crosses between M. rubra and M. nana,[M. rubra and M. nana, resulting in 70.5% normal seeds with intact embryo. M. adenophora and M. nana grow as wild trees, with the fruit of M. adenophora only suitable for medical purposes and not edible.In this study, both cultivated species and wild species were analysed and their genetic diversity could easily be differentiated. M. nana,, has shoM. adenophora and M. rubra, which are considered a progenitor-derivative species pair, are consistent with a previously published figure of 0.897[Our findings on the genetic similarity between of 0.897. An earlof 0.897. The marMyrica genus is small, about 320 Mb. A large set of SSRs were developed from a genome survey of Myrica rubra. The results suggest that they have high rates of transferability, making them suitable for use in other Myrica species.In summary, the genome size of We selected an androphyte ‘C2010-55’ for the genome survey because it was the most homozygous (10 out of 14 SSRs) individual among 230 accessions. Two DNA libraries of 250 and 500 bp insert size were constructed and sequenced by Illumina Hi-Seq 2000.M. rubra) and 3 related species , collected from different provinces in China  to identify microsatellite repeats in our sequence database. The SSR loci containing perfect repeat units of 2-6 nucleotides only were considered. The minimum SSR length criteria were defined as six reiterations for dinucleotide, and five reiterations for other repeat units. Mononucleotide repeats and complex SSR types were excluded from the study.We used MISA scripting language (http://pgrc.ipk-gatersleben.de/misa/primer3.html). We selected 600 primers that met the following parameters: 110–230 final product length, primer size from 18 to 22 bp with an optimum size of 20 bp, and the annealing temperature was set at 60°C. The repeat units over eight were used.The SSR primers were designed using BatchPrimer3 interface modules (M. cerifera for 600 SSR loci by PAGE (polyacrylamide denaturing gel) to confirm their suitability. Tail-1, Tail-2, Tail-3 and Tail-4 labelled with one of the following dyes: NED, PET, FAM, and HEX, respectively.Tail-1, Tail-2(CGAGTCAGTATAGGGCAC), Tail-3(ATCACGAAGCAGATGCAA) and Tail-4(GAGCATCTCGTACCAGTC) were added to the 5’ end of each 150 forward primers of pairs respectively. Tail-2, Tail-3 and Tail-4 were designed so that the primer size was 18 bp and the annealing temperature was 53°C. Primers were synthesised by Invitrogen Trading (Shanghai) Co., Ltd. We primarily tested two cultivars (Biqi and Dongkui) and Each 20 μl reaction mixture contained 10 × PCR buffer (plus Mg2+), 0.2 mM of each dNTP, 5 pmol of each reverse, 4 pmol of the tail primer, 1 pmol of the forward primer, 0.5 units of rTaq polymerase  and 40 ng genomic DNA template. Each primer pair had an interval of 20 bp according to the expected size of amplicons.DNA amplification was in an Eppendorf Mastercycler (Germany) programmed at 94°C for 5 min for initial denaturation, then 32 cycles at 94°C (30 s)/58°C (30 s)/72°C (30 s), followed by 8 cycles of 94°C (30 s)/53°C (30 s)/72°C (30 s). The final extension step was 10 min at 72°C. Each PCR product was run on 1% agarose gel at 110 V for a quality check.Subsequently, PCR products were electrophoresed on 8% denaturing PAGE, according to Myers et al., at 60 W2+), 0.8 mM of each dNTP, 1 unit of rTaq polymerase, 40 ng genomic DNA template and a total of four primer pairs with 5 pmol of each reverse primer, 4 pmol of each tail primer, and 1 pmol of each forward primer. The PCR products were diluted, mixed with the internal size standard LIZ500 (Applied Biosystems) and loaded on an ABI 3130 Genetic Analyzer. Alleles were scored using GeneMapper version 4.0 software .Multiplex PCR was designed and tested with products of different sizes and labelled with different fluorescent dyes. Each 20 μl reaction mixture contained 10 × PCR buffer (plus Mghttp://soap.genomics.org.cn/soapdenovo.html) denovo software to contig, scaffold and fill in gaps. In addition, we used SSPACE software to build the scaffold. K-mer analysis was to help estimate the genome size and characters, such as heterozygosis and repeats.The raw genome sequence data was first filtered to obtain high quality reads, then assembled using SOAP . Chi-square test for Hardy-Weinberg equilibrium allele frequencies and polymorphism information content (PIC) was calculated using PowerMarker version 3.25[http://statgen.ncsu.edu/powermarker/downloads.htm). Microsoft office Excel 2007 was used to analyse the correlation. Genetic similarity among all the accessions was calculated according to Dice’s coefficients using Nei's coefficient index[http://www.natur.cuni.cz/~flegr/programs/freetree.htm) software, and the dendrogram constructed using the unweighted pair-group method with arithmetic averaging (UPGMA) option. The confidence of branch support was then evaluated by bootstrap analysis with 1,000 replicates. The dendrogram was printed using MEGA version 5.05 software[http://www.megasoftware.net/mega.php).The number of alleles (A), observed heterozygosity (Ho) and expected heterozygosity (He) were calculated using POPGENE version 1.32 (ion 3.25 (http:/nt index with thent index (http:/software (http:/ZSG: HJJ: EVW and MLC designed the experiments. YJ: CYC: GYW collected plant materials. YJ: HMJ and XWL performed the SSR experiments and analysed the data. The whole genome shotgun and sequencing assembly was performed by ZC. YJ: ZSG and EVW drafted this manuscript.Occurrence of different SSRs in the genome survey of Chinese bayberry.Click here for file


Libidibia ferrea has been used in folk medicine throughout Brazil, and this study evaluated the biological activities of crude extract (CE) as well as a partially purified fraction (F80) obtained from its pods. Results from the MTT assay revealed that only F80 inhibited NCI-H292 cell growth; however, neither CE nor F80 reduced HEp-2 cell growth or sarcoma 180 tumor weight with the in vivo assay. Acute oral toxicity of the extract and fraction was evaluated following the steps of Guideline 423, using female mice; LD50 for both preparations was determined as 2,500 mg/kg body weight. CE and F80 promoted a reduction of the leukocyte number and nitrite level in inflammatory exudates when the anti-inflammatory assay (carrageenan-induced peritonitis) was performed. CE and F80 inhibited writhing regarding antinociceptive activity (acetic acid-induced writhing response in mice). In conclusion, CE and F80 have no significant cytotoxic or antitumor activities in cell lines showing low toxicity and no action against tumors in vivo. Both preparations revealed anti-inflammatory and antinociceptive activities, corroborating the pharmacological basis of L. ferrea for ethnomedical use. Anticancer, pain relief, and anti-inflammatory drugs with weak adverse effects are some of the most important goals in modern research; natural products, especially plant-derivative substances, play a role in this search for an ideal treatment.Libidibia ferrea var. parvifolia (Mart. ex Tul.) L. P. Queiroz (Leguminosae), whose basionym is Caesalpinia ferrea Mart. ex Tul.  -2,5-biphenyl tetrazolium bromide (MTT) in PBS  was added, and the plates were maintained at 37°C for 2 h. Culture medium with MTT was suctioned, and dimethylsulphoxide (DMSO) was added to dissolve formazan crystals  × 100. Animal experiments were performed according to the NCI protocol [CE, F80, and methotrexate (MTX) were calculated according to animal body weight index . Malignant tumor cells (sarcoma 180) from donor animals with 8 days of implanting were used. All animals were previously hygienized in an experimental surgery room. Donor mice were anaesthetized for tumor suctioning, and the ascitic form of the tumor was introduced under the right axilla of the receptor animals. Treatment, by i.p. route, began 24 h after tumor implantation for 7 days. The negative control group received only saline, and the standard group (positive) received MTX as referential antitumor drug. The animals were sacrificed on the eighth day, by cervical dislocation; solid tumors were excised and weighed. Tumor inhibition was expressed as the mean of tumor weight for the treated animal group (T) in comparison to the untreated control group (C). The tumor inhibition was then calculated according to percentage tumor inhibition = [; the standard drugs: dexamethasone, piroxicam, and indomethacin ; CE; F80 (100 mg/kg for both) were administered by oral route to the correspondent groups . After one hour, 0.25 mL carrageenan (1% in 0.9% NaCl), intraperitoneally injected, was used as a phlogistic agent. Four hours later, animals were sacrificed by cervical dislocation, and immediately the abdomen was opened . The per2−) in the peritoneal exudates was measured as an indicator of NO production according to a colorimetric assay based on Griess reaction [μL) were subjected to reaction with 100 μL Griess reagent (6 mg/mL) at room temperature for 10 min, and then NO2− concentration was determined by measuring absorbance at 540 nm. A standard curve was constructed using known concentrations of sodium nitrite (NaNO2).Accumulated nitrite . Natural products, known as secondary metabolites of plants or animals, continue to be an important segment of research into new drugs. In fact, many compounds frequently used in chemotherapy, such as vincristine, taxol, and camptothecins, were isolated from plants or derived from natural prototypes –20.Additionally, the search for new drugs that effectively interfere with the inflammatory process and pain is currently of great relevance; plants traditionally used as well as their derivative substances have historically been valued as a source of anti-inflammatory agents and pain killers , 22.μg/mL, F80 inhibited NCI-H292 cell growth by 25.6%, 14.3%, and 7.8%, respectively. A concentration of 6.25 μg/mL and HEp-2 cell line induced no growth inhibition. CE did not show significant cytotoxic activity for either cell line. IC50 up to 50.0 μg/mL used cell lines did not allow calculation of IC50. Therefore, both extract and fraction presented low cytotoxicity.The assays revealed that, at 50.0, 25.0, and 12.5 in vitro or in vivo. Nakamura et al. [L. ferrea pod extracts using the in vitro Epstein-Barr virus early-antigen activation assay and found that the ethyl acetate extract exhibited the strongest inhibitory activity. Nozaki et al. [L. ferrea, showed that pauferrol, a compound obtained from the stem, possessed a cell proliferation inhibitory activity through the induction of apoptosis in human leukemia HL60 cells.Only F80 showed some cytotoxic activity; CE did not present cytotoxicity against the cell lines used. Several previous studies have demonstrated that plant extracts and derivative compounds have an anticancer potential a et al.  tested di et al. , also wo50 cutoff of CE and F80 was determined as 2,500 mg/kg body weight; both concentrations used were considered as safe (category 5) [The doses of 300 mg/kg and 2,000 mg/kg did not induce mice weight loss or death from either extract or fraction. LDegory 5) .L. ferrea pods is practically nontoxic via oral route in mice using an acute single dose, indicating that the popular use of this kind of extract can be considered secure.Despite widespread use , few scientific studies have been undertaken to ascertain the safety and efficacy of traditional remedies . The preL. ferrea pod and tested them against skin carcinogenesis in mice, finding a reduction in the average number of papillomas per mouse. Studying another Caesalpinia species, C. bonducella, Gupta et al. [Caesalpinia spp. may vary according to extract (and/or isolated constituents) and tumor types used. F80 showed a tumor growth inhibition of 8.68%, but this value is not significant when compared with the control group. CE did not reveal antitumor activity ; CE and a et al.  evaluateCE and F80 exhibited anti-inflammatory activity, reducing cell migration. CE and F80 showed significant reduction  in the number of PMNL in the inflammatory exudate, similar to piroxicam (46.7%) .β-glucan, etc.), carrageenan is perhaps the most commonly used and well studied [Among the phlogistic agents available  synthesis by inducible nitric oxide synthase (iNOS) is increased in inflammation and leads to cellular injury. This assay is an indirect method to quantify NO, which rapidly reduces to nitrate and nitrite.Nitric oxide derived from iNOS is involved in various pathological conditions such as inflammation and autoimmune diseases leading to tissue damage . Thus, sCE and F80 promoted a high reduction in the content of nitrite, especially CE, which decreased it to a level smaller than those presented by the standard drugs, as shown in Gastrodia elata rhizomes, discovered that the extract and its n-butanol fraction decreased the nitrite content in the exudates obtained from the carrageenan-induced air-pouch model in mice. Koo et al. [Gardenia jasminoides fruit in rats.Plant extracts showing immunomodulatory activities have been extensively described, especially for their inhibitory effect upon the NO production by macrophages , 34. Ahno et al.  also ver There are different routes to evaluate nociception; however, the assay using intraperitoneally acetic acid was the chosen approach. The animals were pretreated one hour before administration of phlogistic agent, then apparently, there was no influence of the administration effect. Other authors have also used parenteral route in their researches , 37. It is well known that the intraperitoneal administration of agents that irritate serous membranes, such as acetic acid, causes a stereotypical behavior in mice characterized by abdominal contractions, movements of the body as a whole, twisting of dorsoabdominal muscles, and a decrease in motor activity and coordination . Using tAcetic acid causes algesia by releasing endogenous substances, which then excite the pain nerve endings; the abdominal constriction is related to sensitization of nociceptive receptors to prostaglandins . AlthougDipyrone (metamizol) is a widely used nonsteroidal anti-inflammatory drug (NSAID). In addition to the well-known peripheral effects of NSAIDs—especially prostaglandin synthesis inhibition—and the fact that dipyrone is able to induce a significant antinociceptive effect in the absence of an anti-inflammatory response, it has been proposed that it produces antinociception at least partially by acting upon central nervous system structures , 44.Since CE and F80 were confirmed to have anti-inflammatory activity in carrageenan-induced peritonitis , their aCarvalho et al.  also evaL. ferrea. Further investigations are necessary to elucidate the precise mechanisms of action and the compounds responsible for these effects.CE and F80 have neither significant cytotoxic nor antitumor activities over NCI-H292 and HEp-2 cell lines or the solid tumor (sarcoma 180) assayed. However, further studies are necessary to evaluate these activities using other cell lines and tumors to elucidate whether these preparations are effective against any of them, as this plant is used popularly for cancer prevention. On the other hand, their low toxicity allows the use of CE and F80 with a degree of safety in other situations. Furthermore, the antinociceptive and anti-inflammatory effects demonstrated in the present study contributed to the ethnomedical uses of


M.tuberculosis (M.tb) is associated with enhanced T helper cell type 1 (Th1) immune responses while helminth infection is associated with T helper cell type 2 (Th2) immune responses. Our aim was to investigate whether helminth infection could influence the ability to generate an appropriate Th1 immune response that is characterized by a positive tuberculin skin test (TST), in M.tb exposed children.M.tb exposure. We documented demographics, clinical characteristics, HIV status, M.tb exposure (using a standard contact score) and M.tb infection status (TST > = 10 mm). Ascaris lumbricoides-specific IgE was used as proxy for Ascaris infection/exposure.We completed a community-based, cross sectional household contact tracing study, using matched enrolment of HIV negative children with and without documented household Ascaris IgE. There were 168 (62%) children with a documented household tuberculosis contact and 107 (40%) were (TST) positive overall.Of 271 children (median age 4 years (range: 4 months to 15 years)) enrolled, 65 participants (24%) were serum positive for M.tb contact score , previous tuberculosis treatment  and previous isoniazid preventive treatment . A visible bacillus Calmette-Guérin (BCG) scar was associated with reduced odds of being TST positive .A positive TST was associated with increasing age (Odds Ratio (OR) =1.17, p < 0.001), increasing Ascaris IgE was not associated with TST status in univariate analysis , but multivariable logistic regression analysis suggested an inverse association between Ascaris IgE status and a positive TST , when adjusted for age, and M.tb contact score. The addition of an age interaction term to the model suggested that the age effect was stronger among Ascaris IgE positive children; the effect of being Ascaris IgE positive significantly reduced the odds of being TST positive amongst younger children while this effect weakened with increasing age.Ascaris exposure/infection and M.tb infection in children in an urban setting. Helminth exposure/infection may reduce the immune response following M.tb exposure when controlling for epidemiological and clinical covariates. These findings might be relevant to the interpretation of immunological tests of M.tb infection in children.Our preliminary findings highlight a high prevalence of both  Mycobacterium tuberculosis (M.tb) are at high risk for disease progression in the absence of effective chemoprophylaxis [Infectious diseases are important causes of morbidity and mortality in children living in the developing world. Tuberculosis is a major global threat with an estimated 9.4 million incident cases reported globally in 2009 , with Sophylaxis .Poor socio-economic circumstances, prevalent in many settings with a high burden of tuberculosis, also predispose to poor living conditions, overcrowding and increased risk of helminth exposure and infection ,5. HelmiM.tb exposure involves complex immune processes and leads to a spectrum of infection and clinical disease outcomes in children, including M.tb-exposed but uninfected, infected non-diseased and diseased [M.tb requires an effective adaptive cellular immune response characterized by robust T helper cell type 1 (Th1) T-cell immunity and relative weaker T helper cell type 2 (Th2) T-cell immune responses [M.tb infection, is a marker of a Th1-type delayed type hypersensitivity reaction. Previously M.tb-sensitized circulating lymphocytes are attracted to the intra-dermal tuberculin injection site, followed by monocyte, macrophage, CD4 + − and CD8+ T-lymphocyte accumulation and release of inflammatory mediators that causes clinical erythema and oedema [diseased ,12. In aesponses -17. The Ascaris lumbricoides [Trichiuris trichiura infection are inconsistent [M.tb and to routine vaccinations [M.tb [Th2 cells produce cytokines which play a role in protection against extracellular organisms and are increased in the presence of helminth infection -21, specricoides  and hookricoides ,21. Datansistent ,22. Th2 inations . Helmintns [M.tb .Ascaris-specific IgE positive status on a commonly used marker of M.tb infection (TST), in children in a setting with a high burden of tuberculosis. We hypothesized primarily that helminth infection in children, while controlling for M.tb exposure and other relevant covariates, would decrease the ability to generate an appropriate Th1 immune response characterized by a positive TST in children.The current limited evidence supports a potential immune modulating effect of helminths on mycobacterial immune responses in humans ,21,23, aThis was a prospective cross-sectional community-based study. Study participants were recruited from three high burden tuberculosis communities: Ravensmead, Uitsig (R/U) and Site C, Khayelitsha (Site C), in Cape Town, Western Cape Province, South Africa. The communities are demographically heterogeneous, and respectively represent ethnic groups that are predominantly of South African Mixed (R/U) and Xhosa African ancestry (Site C).The tuberculosis notification rate in the province was 994 per 100 000 in adults and 671 per 100 000 in children aged 0 – 14 years in 2008 . There is near universal infant bacille Calmette-Guérin (BCG) vaccination.Ascaris lumbricoides and 51% for Trichuris trichiura [Prior surveys from school-going children in R/U found that 25% were stool positive for richiura . RoutineParticipants were recruited following identification of consenting adults (≥ 18 years of age), who routinely started tuberculosis treatment in the preceding 3 months (“source case”) at the local clinic. Participants were eligible for enrolment if they were a household child contact between 3 months and 15 years of age. Households were identified through TB source cases (“tuberculosis households”), and matched neighbouring households without known tuberculosis exposure (“neighbouring households”). The ratio between tuberculosis and neighbouring households was approximately 1:1.M.tb exposure and demographic characteristics. In addition, all adult household members were screened for symptoms of tuberculosis followed by sputum collection, if they were symptomatic.We identified tuberculosis households within 2–4 weeks following the adult source case identification, then completed a home visit and collected data on Following written informed consent from the parents/caregivers, children aged 3 months – 15 years who were not currently on tuberculosis therapy, were enrolled in the study. Children were deferred for enrolment if there was a) prior TST administration in the preceding 3 months, b) live measles or polio vaccine within the past 6 weeks or c) recent acute illness requiring hospitalization (preceding 6 weeks). Children on isoniazid preventive therapy (IPT) were not excluded, whereas HIV-infected children were.M.tb infection and disease using standard protocols [All children were investigated for rotocols ,28, inclThe TST  was administered intra-dermally on the volar aspect of the left forearm by trained standardized study nurses, and read within 48–72 hours using the ball-point and ruler method and callipers. The TST was regarded as positive if measuring ≥10 mm in HIV-negative children for clinical care. Unless already known to be HIV-infected, participants were tested for HIV (Abbott Determine Rapid HIV test), followed by a confirmatory laboratory based ELISA/PCR test if positive or indeterminate.M.tb exposure (M.tb contact score) [M.tb infection [Data were collected to quantify the degree of t score) . In the nfection -31 [Tablnfection ,30.We also recorded information on BCG vaccination history and scar presence, previous and current isoniazid preventive therapy, previous tuberculosis treatment, nutritional status (z-scores), household composition, measures of socio-economic status (household assets), and reported history of deworming during the preceding 6 months. Z-scores were calculated with reference to the 2006 World Health Organization Child Growth Standards for children up to five years of age and the 2000 Centres for Disease Control and Prevention growth charts reference population for children that were older than 5 years . ReporteAscaris lumbricoides was determined by the ImmunoCAP specific-IgE testing which measures circulating Ascaris-specific IgE concentration in serum. This method was developed for clinical diagnosis and laboratory use where reagins belonging to the IgE antibody class, which develop shortly after exposure, are measured by a fluoroenzymeimmunoassay [Sensitization to unoassay . ResultsM.tb infection, measured by a positive TST (≥10 mm). The main exposure of interest, Ascaris IgE, was categorised according to RAST categories and dichotomized (positive if ≥0.35kU/L). This measure, consistent with previous work (25) was used as a measure of Ascaris infection or sensitization.Data analysis was performed using STATA/SE version 12.0 . An alpha level of less than or equal to 0.05 was regarded as statistically significant. The main outcome of interest was M.tb infection in univariate analysis, with the primary determinant being Ascaris IgE status in 271 enrolled participants. Child baseline characteristics were described overall, and stratified according to Ascaris IgE status for baseline characteristics. Continuous data were described using mean and standard deviations and median and inter-quartile ranges, dependent on data distribution. Categorical variables were described using frequencies and percentages.We assessed the effect of predictors of Ascaris IgE status, age and the M.tb contact score. These measures were significant during univariate analysis or were clinically or epidemiologically relevant as covariates. Additionally, the developed model was examined for interactions between the explanatory and environmental variables in order to establish if possible effect modification is present. Results of the regression model are presented as adjusted odds ratios (aORs) and corresponding 95 percent confidence intervals (95% CIs).We developed logistic regression models that included We complied with the Helsinki Declaration and the study was approved by the Health Research Ethics Committee of the Stellenbosch University (reference number N05/07/129 and N08/08/207).Of 337 total child participants enrolled, 271 HIV-negative children were included in the analysis . The median age was 4 years (range: 4 months to 15 years).Few children (2%) were receiving IPT at the time of enrolment despite the fact that 67% of children were household contacts of a tuberculosis index case currently on therapy. Of children with a documented contact (n = 181), 121 were below 5 years of age and were referred for IPT. Previous IPT use was reported in 9% and 3% had previously received tuberculosis treatment.Housing and socio-economic indicators indicate the poor living conditions prevalent in these communities: almost 20% of participants did not have access to a fridge or radio at home while almost 5% relied occasionally on selling household assets. Only 68% of children lived in a house with a flushing toilet and 83% had access to piped water in the household. The majority of children resided in formal brick housing (77%) [Table Ascaris IgE results are described in Table Ascaris IgE positive. Of these, 37% were also TST positive. Most Ascaris IgE positive participants were within RAST range 2  followed by ranges 1  and 3  [Table M.tb contact score , had received previous TB treatment  or previous IPT . A visible BCG scar  was associated with reduced odds of infection [Table Ascaris IgE and positive TST status .Overall, 40% of children were TST positive at enrolment. Children were more likely to have a positive TST if they were older , had a higher M.tb contact score and binary Ascaris IgE status in a regression model that demonstrated an increased risk for a positive TST in older children  and those with a higher M.tb contact score . This model also showed an inverse association between TST and Ascaris IgE positivity .Based on the univariate analysis, we included age, M.tb contact score, age, Ascaris IgE and an age interaction term showed that the effect of age was stronger among those children with a positive Ascaris IgE status. Being Ascaris IgE positive significantly reduced the odds of being TST positive amongst younger children, while this effect weakened as age increased .Older children  were more likely to be Few children were on IPT at enrolment which could reflect the active contact tracing strategy used by the study; however previous studies from these communities indicate poor IPT uptake and adherence in young children under routine programmatic conditions -43.M.tb exposure and included children from neighbouring “control” households as a measure of background M.tb transmission. Finally, the proxy used for Ascaris in our study does not apply to all potential helminth species, and might underestimate the general and differential effect of helminth infection. Deworming was only reported on a questionnaire basis and its impact on mycobacterial and other immune responses could therefore not be rigorously evaluated.This study is limited by its cross-sectional nature, the limited sample size and the exclusion of HIV-infected children, due to small numbers. The enrolment strategy was purposeful to ensure a high proportion of children with recent In light of these limitations, these results need to be verified in paediatric cohort studies, preferably with additional measures of helminth infection including stool samples and the standard evaluation of the effect of deworming on mycobacterial immune responses.M.tb infection, especially in high prevalence tuberculosis and helminth infection settings.The results support our hypothesis that helminth infection may change immune responses in children. This may play a clinically measureable role in widely used immune diagnostic tests that measures M.tb:Mycobacterium tuberculosis; OR Odds ratio; R/U: Ravensmead and Uitsig; Site C: Site C, Khayelitsha; TB: Tuberculosis; Th1: T helper cell type 1; Th2: T helper cell type 2; TST: Tuberculin skin test; WHO: World Health Organisation.95% CI: 95 percent confidence interval; aOR: Adjusted odds ratio; BCG: Bacillus Calmette-Guérin; IMCI: Integrated Management of Childhood Illness; IPT: Isoniazid preventive therapy; All authors declare that they have no competing interests.NVS was involved in study design, data analysis, interpretation and manuscript preparation and primarily responsible for manuscript writing. AMM participated in design, implementation, analysis and writing. HLK participated in analysis, interpretation and writing. GW participated in design, writing and interpretation. HMSG was involved in study design and interpretation. MJ participated in writing and interpretation. ACH participated as academic supervisor in all phases of the study and manuscript preparation including design, implementation, analysis and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/211/prepub


Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) often receive intravenous liposomal amphotericin B (L-AmB) as antifungal prophylaxis. There are no guidelines for antifungal prophylaxis in children in this situation. Caspofungin (CAS), a broad-spectrum echinocandin, could be an effective alternative with lower nephrotoxicity than L-AmB.2/day) as antifungal monoprophylaxis starting on day one after HSCT. The median ages of patients receiving L-AmB and CAS were 7.5 years and 9.5 years, respectively.We retrospectively analyzed the safety, feasibility, and efficacy of CAS in our center, and compared the results with L-AmB as antifungal monoprophylaxis in pediatric patients undergoing HSCT. 60 pediatric patients received L-AmB (1 or 3 mg/kg bw/day) and another 60 patients received CAS (50 mg/mNo proven breakthrough fungal infection occurred in either group during the median treatment period of 23 days in the L-AmB group and 24 days in the CAS group. One patient receiving CAS developed probable invasive aspergillosis. During L-AmB treatment, potassium levels significantly decreased below normal values. Patients treated with L-AmB had more drug-related side effects and an increased need for oral supplementation with potassium, sodium bicarbonate and calcium upon discharge as compared with the CAS group. CAS was well-tolerated and safe in this cohort of immunocompromised pediatric patients, who underwent high-dose chemotherapy and HSCT.Prophylactic CAS and L-AmB showed similar efficacy in this biggest cohort of pediatric patients after allogeneic HSCT reported, so far. A prospective randomized trial in children is warranted to allow for standardized guidelines. Candida and Aspergillus species are the most common fungal pathogens in this setting, with Aspergillus infections being associated with higher mortality [Pediatric patients undergoing hematopoietic stem cell transplantation (HSCT) are at high risk of acquiring severe invasive opportunistic fungal infections. Risk factors include extensive immunosuppression, cytopenia, T-cell depletion of the graft, graft-versus-host disease (GvHD), underlying disease and viral or bacterial infections ,2. Candiortality -6. Studiortality -10.Pediatric patients < 18 years) most frequently received antifungal prophylaxis with liposomal amphotericin B (L-AmB) after allogeneic HSCT -12. Howe years moCaspofungin (CAS) was shown to be effective in treatment of invasive candidiasis and aspergillosis with low toxicities in adults -21. In cThis retrospective survey was conducted in accordance with the declaration of Helsinki and performed with approval by the University Children’s Hospital Tübingen’s Institutional Review Board. Data were collected retrospectively, entered in a standardized case report form, and anonymized.The study is a single centre, retrospective survey on antimycotic prophylaxis in pediatric patients under eighteen years of age who underwent allogeneic HSCT between January 2006 and June 2010 at the University Children’s Hospital Tübingen, Germany. The allogeneic stem cell transplantation occurred in high-efficiency particle-arrestance filtered environment. All pediatric patients in our clinic received L-AmB exclusively as antifungal prophylaxis during conditioning and after HSCT until August 2008. As we observed a high incidence of nephrotoxicity, we changed our antifungal prophylaxis beginning from day 1 after allogeneic HSCT, from L-AmB to CAS in September 2008. Other supportive standards remained unchanged during this short period of 4.5 years.The primary objective of this study was to evaluate safety of CAS as antifungal prophylaxis in pediatric patients following allogeneic HSCT as compared to L-AmB. The secondary objective was the incidence of aspergillosis, candidiasis, and other mycoses.The observation period in this trial was defined as the time from start of intravenous antimycotic monoprophylaxis at the beginning of conditioning until three weeks after switching to oral antimycotic prophylaxis 3–4 days before inpatient discharge.All 120 pediatric patients included in the survey received antimycotic prophylaxis with L-AmB (1 mg/kg/day) from the beginning of conditioning until day 0   would have been possible [The second group of patients, who had undergone stem cell transplantation between September 2008 and June 2010, received the same dosage as the pediatric patients in group 1 until day 0, that is, they received L-AmB at a dosage of 1 mg/kg/day, and CAS at a dosage of 1 × 50 mg/mpossible ,27. A lo120 pediatric patients under eighteen years of age with hemato-oncological malignancies, non-malignant hematological diseases, and inborn errors of metabolism undergoing allogeneic HSCT were included in this retrospective analysis. Exclusion criteria were the presence of proven or probable invasive fungal infections before the start of conditioning according to the definitions of invasive fungal disease by the European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) .Aspergillus enzyme-linked immunosorbent assay . In instances of positive galactomannan antigens, measurements were repeated again with the same blood specimen. After the confirmation of positive results, daily measurements of the galactomannan antigen took place, until a negative result was obtained.Patients were examined daily for signs of invasive fungal breakthrough infection on the ward and twice per week for three weeks after switching to oral antimycotic prophylaxis. At least once per week for the entirety of the observation period galactomannan antigens were measured by PLATELIA™ Successful intravenous antifungal monoprophylaxis was defined as the absence of proven or probable fungal infection from the post-transplant period through the date of discharge, as well as three weeks after the end of intravenous antifungal monoprophylaxis.This endpoint was comprised of clinical and laboratory parameters. Clinical side effects were recorded according to current Common Toxicity Criteria by the US National Cancer Institute . HepaticAll 120 pediatric patients who received monoprophylaxis with L-AmB or CAS were included in the assessments of safety and efficacy. The statistical comparison of the difference between the results and normal values was done with the one-sample t-test. The Wilcoxon matched pairs signed rank test was applied for a statistical comparison of these parameters: between the baseline before conditioning and day 0, the baseline and maximum/minimum during intravenous antifungal prophylaxis, the baseline and end of intravenous antifungal prophylaxis, day 0 and maximum/minimum, day 0 and end, and maximum and end. The hepatic and renal function data are presented as mean values ± standard deviation. P values of ≤ 0.05 (*), P ≤ 0.01 (**) and P ≤ 0.001 (***) were defined as statistically significant. The Two Independent Proportion Z-Test was used to compare percentage of patients with hypokalemia during intravenous antimycotic prophylaxis as well as the percentage of electrolytes substitution between two groups. Graphs were created with GraphPad Prism 4 for Windows, version 4.03 . The statistical analysis was carried out with the statistical program XLStat2010 .2/day beginning on day one after allogeneic HSCT, but no more than 50 mg/day, between September 2008 and June 2010. Fever greater than or equal to 38.5°C came about in 27 (43%) of the 60 pediatric patients treated with CAS.In total, 120 pediatric patients under the age of eighteen years, divided into two groups, were evaluated in this analysis. All pediatric patients included in the survey, a total of 60 patients in the first group and an additional 60 patients in the second group, received antimycotic prophylaxis with L-AmB (1 mg/kg/day) from the beginning of conditioning until day 0. The 60 pediatric patients in group 1 underwent allogeneic HSCT between January 2006 and August 2008, and received L-AmB at a dosage of 1 mg/kg/day as antifungal prophylaxis from day one after allogeneic HSCT. In 34 (56.7%) of the 60 pediatric patients in the L-AmB group, the antimycotic prophylaxis with L-AmB was adjusted after HSCT to 3 mg/kg/day from the initial dosage of 1 mg/kg/day due to fever greater than or equal to 38.5°C. The second group also consisted of 60 pediatric patients, all of whom were treated intravenously with CAS at a dosage of 1 × 50 mg/mThe median age in the L-AmB group was 7.5 years (range 4 months to 17.6 years), while the CAS group had a median age of 9.5 years (range 8 months to 17.5 years) (Table Leukocytopenia was observed for a median duration of 12 days in both groups. 56.7% (n = 34) of the pediatric patients in the L-AmB group and 21.7% (n = 13) in the CAS group received methylprednisolone during conditioning. 61.7% (n = 37) of patients in the CAS group were treated with prednisolone after transplantation in comparison to 38.3% (n = 23) in the L-AmB group. The median duration of intravenous antifungal monoprophylaxis was approximately the same for each group, 23.0 days (range 9–72 days) in the L-AmB group and 24.0 days (range 14–49 days) in the CAS group after allogeneic HSCT.Prophylaxis was effective with L-AmB as well as with CAS. There was no incidence of proven invasive aspergillosis or another invasive fungal infection in either group. This also remained true during the conditioning phase, when both groups received liposomal amphothericin B, and also during the post-transplant period, in which group one received L-AmB and group two CAS. Furthermore, no proven fungal breakthrough infections were observable in either group three weeks after the conclusion of intravenous antifungal prophylaxis.In the CAS group, one severely immunocompromised pediatric patient showed clinical and serological indicators of a probable invasive fungal infection, i. e. increase of galactomannan antigen in more than two consecutive blood samples and signs of pulmonary aspergillosis in computed tomography thirteen days after start of monoprophylaxis with CAS. However, bronchopulmonary lavage failed to detect fungi by culture or PCR. The patient died on day 94 after allogeneic HSCT of veno-occlusive disease (VOD), the parents did not consent with an obduction. There was no incidence of probable invasive fungal infections in the L-AmB group.None of the patients in both cohorts died from an invasive fungal infection during the observation period. In addition to the single pediatric patient from the CAS group who succumbed to VOD, one patient from the L-AmB group died from a relapse of AML three weeks after the end of the intravenous antifungal prophylaxis.Clinical side effects directly related to intravenous treatment with L-AmB were observed in 5 (8.3%) and directly related to CAS in 2 (3.3%) pediatric patients  as well as to day 0 (P < 0.001) Figure . An incrA total of 25% (n = 15) of pediatric patients in the L-AmB group required oral potassium supplementation and spironolactone upon discharge. This compares to only 11.7% (n = 7) in the CAS group. Sodium bicarbonate substitution was required in 5 (8.33%) and calcium in 3 (5%) cases upon discharge in the L-AmB group. In the CAS group, calcium was given in 2 (3.3%) cases and sodium bicarbonate in one (1.7%) case. In the L-AmB group, there was a trend towards higher percentage of patients requiring potassium (P = 0.0591) and bicarbonate substitution (P = 0.094) after end of intravenous antifungal prophylaxis. All pediatric patients in the L-AmB group who required oral supplementation with potassium and bicarbonate had received L-AmB after HSCT at a dosage of 3 mg/kg/day.Clinical practice guidelines have been published and adapted over the years for primary antifungal prophylaxis in adult patients undergoing allogeneic HSCT . Yet priIn our BMT unit, children receive a comparably low dose of 1 mg/kg/day L-AmB during conditioning in order to reduce drug interactions. When using L-AmB at a therapeutic dosage (3 mg/kg/day) in pediatric patients after allogeneic HSCT, we often observed an increased need for supplementation of potassium, bicarbonate, and calcium during and within the first two weeks following intravenous antifungal prophylaxis. In a randomized, double-blind, multinational trial, CAS was better tolerated than L-AmB in 556 adults with persistent fever and neutropenia . CAS in 2/day up to a maximum of 50 mg/day, the case of probable aspergillosis might argue to increase the dose of caspofungin to 70 mg/m²/d  in children due to pharmacological studies and its low toxicity [Although caspofungin showed good efficacy at a dosage of 50 mg/mtoxicity ,27. The toxicity . In our toxicity ,24,34. Ctoxicity . In the This is the first retrospective observational trial of antifungal prophylaxis after allogeneic HSCT that has been carried out with a large cohort of pediatric patients under eighteen. In this retrospective survey, the efficacy of antifungal prophylaxis was good in both groups, since no proven invasive fungal infection occurred in either group. During the early transplant period, patients received several antibiotics and virostatics, which did not affect the tolerability of CAS. The combination of L-AmB with certain virostatics such as ganciclovir, foscarnet or cidofovir may have potentiated nephrotoxicity requiring prolonged therapeutic intervention more often here than in the CAS group. The present study is limited in that it was not randomized, and it retrospectively analyzed two periods of time, where minimal changes in daily management cannot be precluded. Prospective studies with larger cohorts due to the low incidence of invasive aspergillosis in this setting must be undertaken in order to derive clinical guidelines on antifungal prophylaxis in children undergoing stem cell transplantation.The authors declare that they have no competing interest.No substantial overlapping with previous papers.MD carried out the clinical analysis, performed the statistical analysis and drafted the manuscript. UH performed a portion of the clinical analysis. AE assisted in both the clinical and statistical analyses. PL helped to draft the manuscript. RH helped to draft the manuscript. IM conceived of the study, and participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/151/prepub


The aim of this study was to evaluate the protective effects of Fufang Xueshuantong (FXT) on diabetic retinopathy in rats induced by streptozotocin (STZ). Diabetes was induced in Sprague-Dawley rats by a single injection of 60 mg/kg STZ. One week after STZ, FXT 0.525 g/kg or 1.05 g/kg was administrated to the rats by intragastric gavage (ig) once daily consecutively for 24 weeks. The control rats and untreated STZ rats received vehicle the same way. At the end of the experiment, the erythrocyte aggregation and blood viscosity were assayed. The retina vessel morphology was observed in retinal digestive preparations. Expression of occludin and intercellular adhesion molecule-1 (ICAM-1) in retina was measured by western blotting. Expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in retina was detected by immunohistochemistry. The activity of aldose reductase in retina was investigated with a NADPH oxidation method. The results showed that, in STZ rats, there were distinct lesions in retinal vessel, including decrease of pericytes and increase of acellular capillaries, together with dilatation of retinal veins. The expression of VEGF and ICAM-1 increased, while the expression of PEDF and occludin decreased. The activity of aldose reductase elevated, and the whole blood viscosity, plasma viscosity, and erythrocyte aggregation also increased after STZ stimulation. FXT 0.525 g/kg and 1.05 g/kg demonstrated significant protective effects against STZ induced microvessel lesion in the retina with increased pericytes and reduced acellular capillaries. FXT also reduced the expression of VEGF and ICAM-1 and enhanced the expression of PEDF and occludin in STZ insulted rats. The activity of aldose reductase, the whole blood viscosity, plasma viscosity, and erythrocyte aggregation also decreased after FXT treatment. The results demonstrated that FXT has protective effect on STZ induced diabetic retinopathy in rats. Diabetic retinopathy (DR) is a leading cause of adult vision loss in developed countries and the prevalence of retinopathy is directly related to the duration of diabetes , 2. The α-lipoic acid and L-carnosine, significantly reduced retinal levels of TNF-α, VEGF, and plasma TBARS in diabetic rats [The blood-retinal barrier (BRB) plays a critical role in partitioning the neural elements of the retina from the circulation and protecting them from circulating inflammatory cells and their cytotoxic products . Long-tetic rats . All thePanax notoginseng, Salvia miltiorrhiza, Astragalus membranaceus, and Scrophularia ningpoensis. It was found that FXT could dilate the blood vessels and promote blood circulation [FXT is a widely used Chinese herbal formula in ophthalmologic clinical application, which consists of culation , 18. A lMale Sprague-Dawley (SD) rats were purchased from Vital River (China) at a weight of 230 to 250 g (SCXK (Jing) 2007-0001). Experiments performed in this study adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the “Use of Animals in Ophthalmic and Vision Research.” Animal experimental protocol was approved by the BUCM Institutional Animal Care and Use Committee. Rats were acclimated for one week with a 12-hour light/dark cycle, constant temperature and humidity, and abundant access to food and water. STZ was purchased from Sigma (USA). Antibodies were purchased from Abcam (UK) or Santa Cruz (USA). All other reagents were procured from standard commercial suppliers unless otherwise noted. 1, ginsenoside Rg1 and Rb1, cryptotanshinone, tanshinone I, harpagoside, and astragaloside IV. The content of these constituents in FXT was determined to be 3.27 mg/g, 15.7 mg/g, 15.5 mg/g, 26.6 μg/g, 31.3 μg/g, 69.0 μg/g, and 0.23 mg/g, respectively, by HPLC.FXT, a Chinese patent drug provided by Zhongsheng Pharmaceutical Co, Ltd, Guangdong, China, was produced as described in the Chinese Pharmacopoeia 2010. The main peaks in HPLC profile of FXT were identified to be notoginsenoside RAfter 16 hours of fasting, rats received a single dose of 60 mg/kg STZ  by intraperitoneal injection. Control rats received citrate buffers alone. After one week, STZ treated rats with blood glucose levels greater than 16.7 mmol/L were considered to be diabetic and randomly divided into three groups: (1) STZ group, 70 rats; (2) FXT 1.05 g/kg group, 60 rats; (3) FXT 0.525 g/kg group, 60 rats. And then FXT-diabetic rats received FXT 1.05 g/kg or 0.525 g/kg by intragastric gavage once daily for consecutively 24 weeks. FXT was dissolved in 0.5% CMC and the daily dosage of drug was 1 mL/100 g weight. Control (30 rats) and untreated STZ rats received the isometric 0.5% CMC the same way.Fasting body weight and fasting blood glucose were recorded every four weeks throughout the study. The general physical signs, including coat color, skin, bite and sup, urine and stool, and crystalline lens, were observed and recorded carefully. Blood samples were collected at the end of experiments for hemorheology test.Trypsin digestion of the retina was performed according to the method of Cogan et al.  with somAs indicated in −1, respectively, were determined via a testing program. Erythrocyte aggregability was measured by RBC Deformation and Aggregation Test Instrument. .Fresh blood samples were collected from rats by arteriopuncture into liquaemin (10 mg/mL) test tubes. Plasma was separated from blood by centrifugation at 3000 r/min for 15 min. Whole blood viscosity and plasma viscosities were measured using a blood viscometer . The serial blood viscosities at different shear rates, 1, 60, and 200 sβ-actin. Rabbit anti-occludin antibody  and mouse anti-intercellular adhesion molecule (ICAM)-1 antibody , goat anti-rabbit lgG (1 : 500), and goat anti-mouse lgG (1 : 2000) were used in the study. The proteins were visualized with ECL, kit . The Image J Analysis Software  was used to analyze the gray intensity of target band.Western blot analysis was performed as described by Park et al.  and ChenImmunohistochemical analysis was performed as described by Ogata et al.  and Juann = 8). The retina was dissected by a posterior approach and homogenized in 10 volumes of 100 mM potassium phosphate buffer (pH 6.2). The homogenate was centrifuged at 15,000 r/min for 30 min and the supernatant was kept in −20°C refrigerator. The protein concentration of the supernatant was determined using Bradford Protein Assay (Biomiga). AR activity was assayed according to the method described by Akileshwari et al. [Crude aldose reductase (AR) was prepared from rats retinas of each group , indicating significant retinal venular dilatation, which was also supported by arteriolar-venular ratio as listed in Quantitative analysis demonstrated that the retinal venular caliber in STZ rats significantly increased, with 88.90 ± 2.07 The whole blood viscosity at three different shear rates and the plasma viscosity increased significantly after STZ treatment. The erythrocytes aggregability also elevated in STZ animals. FXT 1.05 g/kg and FXT 0.525 g/kg significantly inhibited the alterations in erythrocytes aggregation and blood viscosity induced by STZ treatment as shown in Western blot analysis demonstrated that the retina ICAM-1 significantly increased and occludin decreased after STZ treatment, indicating an obvious inflammation in retina and a possible blood-retinal barrier lesion as shown in Immunohistochemistry studies have shown that in control rats VEGF was localized to the pigment epithelium layer (PEL), the outer nuclear layer (ONL), the outer plexiform layer (OPL), inner plexiform layer (IPL), the ganglion cell layer (GCL), and also the nerve fiber layer (NFL), while PEDE was mainly localized to the PEL and the inlayers of retina. STZ treatment caused a strong upregulation of VEGF in all layers of retina, particularly in PEL, the inner nuclear layer (INL), and GCL; however, STZ induced an overall decrease in PEDE expression. As shown in P < 0.05 versus control). FXT 0.525 g/kg and FXT 1.05 g/kg effectively attenuated the enzyme activity increase induced by STZ though still higher than that in controls rats.Increased aldose reductase activity is one of the important initiating factors in diabetic retinopathy. As shown in The aim of the study was to investigate the effects of FXT on microvessel lesion on early stage of diabetic retinopathy. The results suggest that FXT 0.525 g/kg and 1.05 g/kg have distinct protective effect on STZ induced microvessel injury.When the blood glucose persistently increases, the activity of aldose reductase in pericytes of retinal capillaries increases too, resulting in enhanced level of metabolites (sorbitol and fructose) in intracellular pericytes leading to an elevation of intracellular osmotic pressure,cell swelling and metabolic disorders, eventually bringing about the loss of pericytes and damage of their primary function  , 12. WitElevated glycosylated hemoglobin in red blood cell (RBC) triggered by increased blood glucose results in cellular stiffening, decreasing their deformation capacity which leads to an increase in blood viscosity and a constant, elevated sheer stress at the endothelium of retinal vessel. The integrity of blood vessel could be damaged by increased sheer stress together with pericytes loss , 26. TheVEGF plays a key role in the onset and development of diabetic retinopathy , 29. AccAngiogenesis in retina is a hallmark of diabetic retinopathy turning into proliferative phase, which can be controlled by inhibitors. Admittedly, VEGF is the most important mediator in triggering angiogenesis and there are large numbers of endogenic angiogenic inhibitors. As the most effective and natural angiogenic inhibitor, PEDF has better control effect on angiogenesis than angiotensin, thrombospondin-1, and other angiogenic inhibitors . The balDiabetic cataract is another diabetic eye diseases, whose harm ranks only second to diabetic retinopathy –36. DiabDiabetic retinopathy, a microcirculation disturbance triggered by a persistent increase in blood glucose levels, is a multifactorial, complicated illness expressed in abnormality of vessel and blood in retina. In this study we found that FXT has protective effect on STZ induced diabetic retinopathy in rats with possible mechanisms of inhibiting AR activity and VEGF expression. However, there are lots of questions need to be answered before we elucidate the mechanisms of FXT protecting the diabetic retina.


Vitex rotundifolia has shown antitumor, anti-inflammatory, and immunoregulatory properties. This work is designed to evaluate the antiangiogenic effects of VIT and address the underlying action mechanism of VIT by a network pharmacology approach. The results validated that VIT can act as a novel angiogenesis inhibitor. Firstly, VIT can exert good antiangiogenic effects by inhibiting vascular-endothelial-growth-factor- (VEGF-) induced endothelial cell proliferation, migration, and capillary-like tube formation on matrigel in a dose-dependent manner. Secondly, VIT was also shown to have an antiangiogenic mechanism through inhibition of cell cycle progression and induction of apoptosis. Thirdly, VIT inhibited chorioallantoic membrane angiogenesis as well as tumor angiogenesis in an allograft mouse tumor model. We further addressed VIT's molecular mechanism of antiangiogenic actions using one of our network pharmacology methods named drugCIPHER. Then, we tested some key molecules in the VEGF pathway targeted by VIT and verified the inhibition effects of VIT on AKT and SRC phosphorylation. Taken together, this work not only identifies VIT as a novel potent angiogenesis inhibitor, but also demonstrates that network pharmacology methods can be an effective and promising approach to make discovery and understand the action mechanism of herbal ingredients.Vitexicarpin (VIT) isolated from the fruits of  Panax ginseng can inhibit tumor angiogenesis and metastasis by inhibiting the release of VEGF from tumour cells , and then stained with 0.25% Coomassie Blue R250.Gelatin zymography was used to examine the activity of MMP2. All media were collected and subjected to SDS-PAGE using 0.01% w/v gelatin containing 10% polyacrylamide gel. After electrophoresis, the gels were equilibrated in 50 mM Tris-HCl (pH 7.5) with 2.5% Triton X-100 for 30 min at room temperature. Gels were then incubated at 37°C for 20 h in an incubation buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 10 mM CaClX, Ydenote the drugCIPHER-CS score of vitexicarpin and FDA-approved drugs, respectively, and N is the total number of proteins in the target profiles. The target-related proteins of vitexicarpin were predicted using the drugCIPHER-CS step in our drugCIPHER method, whose good performance on target prediction was demonstrated in our previous work . BrieflyP value less than 0.05 after Bonferroni's correction.For functional analysis, we use DAVID web server to analyze the enriched KEGG pathways for the top 10% target-related proteins of vitexicarpin with a false discovery rate less than 0.05 by the Fisher Exact test . We onlyThe high-resolution protein structures of SRC and AKT1 were obtained from the refined X-ray crystal structure of 2H8H.pdb and 3MVH.pdb, which is available from the Protein Data Bank. The Autodock 4.0 suite of programs uses an automated docking approach that allows ligand flexibility as described to a full extent elsewhere . Defaultt-test. P < 0.05 was considered statistically significant.The data are presented as mean ± SD, and statistical comparisons between groups were performed using Student's μM. Vitexicarpin significantly inhibited VEGF-induced lateral migration and decreased wound healing potential at 5 μM  were incubated for 24 h in 96-well microplates with various final concentrations of vitexicarpin . As shown in μM, with an IC50 of approximately 2.5 μM. As a solvent control, we also examined the effect of 0.1% DMSO on cell viability. Upon supplementation of the culture medium with 0.1% DMSO, our data indicated that 0.1% DMSO has no effects on HUVEC proliferation and viability. The cytotoxic activity of vitexicarpin against HUVECs was assessed without the supplementation of proangiogenic factors in 5% FBS-containing endothelial cell medium (condition did not support proliferation). Vitexicarpin treatment at 5 μM for 24 h only decreased the percentage of metabolically viable HUVECs by approximately 25% -treated HUVECs confirmed DNA fragmentation  polymerase (PARP). These results demonstrated that vitexicarpin inhibited proliferation and induced early and late apoptosis in VEGF-induced HUVECs.To verify the proapoptotic effects of vitexicarpin on VEGF-stimulated HUVECs, we evaluated the apoptotic cell death effect of vitexicarpin by annexin V-PI staining and flow cytometry, in which only annexin V-stained cells were considered as early apoptotic cells . We alsoentation . To gainentation . The potentation . A clearin vivo, blood vessel growth was stimulated on the chorioallantoic membranes (CAMs) with VEGF in the presence or absence of different concentrations of vitexicarpin (0.1–5 μM). This assay measures developmental angiogenesis, and it is routinely used to obtain the first indication of angiogenic activity in vivo. VEGF induced a robust angiogenic response, whereas vitexicarpin disrupted VEGF-mediated angiogenesis (P < 0.001). The development of vasculature at day 10 treated with 0.1 μM, 1 μM, 2.5 μM, and 5 μM vitexicarpin caused a 24, 36, 45, and 84% reduction in the infiltration of blood vessels, respectively. These results suggested that vitexicarpin is a potent antiangiogenic molecule ex vivo. To assess the effect of vitexicarpin on angiogenesis in vivo. H&E staining showed that treatment of tumors with vitexicarpin appeared to increase the extent of necrosis within tumor  were dissected, fixed, and imbedded. Tumors from the mice with vitexicarpin treatment were smaller than that from NS group, suggesting that vitexicarpin inhibits tumor growth in tumor . Furtherin tumor . Based oTo identify the target-related proteins of vitexicarpin, we used drugCIPHER-CS  to calcuP = 3.63E − 05). By using top 10% targets of vitexicarpin, we also found significantly enriched KEGG pathways vitexicarpin may affect cancer-related pathways , T cell receptor signaling pathway, and VEGF signaling pathway. Interestingly, it has been previously reported that vitexicarpin exhibits broad cytotoxicity against human cancer cell lines and exerts an inhibitory effect on T-lymphocyte proliferation. The comparison results with literature suggest that target profiles predicted by drugCIPHER can reveal the polypharmacological activities of vitexicarpin.Enrichment analysis helps to better understand the biological functions of target proteins and pathways involved in the activities of vitexicarpin. By establishing a set of the known angiogenesis-related drug targets , we found that top 10% target proteins are significantly enriched in the set of known angiogenesis-related drug targets . These results indicate the potential of SRC and AKT1 as direct targets of vitexicarpin, which deserve further experimental verifications.To evaluate the possibility of SRC and AKT1 as the direct targets of vitexicarpin, we performed docking analysis to examine the mode of vitexicarpin-binding SRC and AKT1. The available X-ray crystal structures of kinases from the PDB database were used for validation of target prediction results. We found that the binding energy of vitexicarpin with SRC and AKT1 is −9.23 kcal/mol and −9.21 kcal/mol, respectively. Vitexicarpin was docked into the ligand-binding sites and occupied the hydrophobic pocket of SRC and AKT1 . CompareV. rotundifolia, which can inhibit tumor growth and inflammation. However, little information is known about its functions in angiogenesis. In this study, we demonstrate that vitexicarpin (0.1–5 μM) exhibited Antiangiogenic activities in vitro, as shown in the results of endothelial cells migration, proliferation, and Matrigel tube formation assays  inhibited ECs proliferation via cell cycle arrest and induction of apoptosis (in vitro results, 5 μM vitexicarpin inhibited the angiogenesis sprouting from CAM (In recent years, substantial effort has been dedicated to identifying Antiangiogenic agents from TCM herbs , 41. Vit Figures  and 3. Fpoptosis . Consistfrom CAM . In addifrom CAM . It shouAt present, one of the major challenges in the use of herbal compounds in drug discovery is the unexpected interactions with on-target and off-target proteins due to the less specific binding characteristics of small molecules , 42. In The top 10% target-related proteins of vitexicarpin are significantly enriched in the 38 KEGG pathways. 14 cancer-related pathways and T cell receptor signaling pathway exactly account for the known biological functions of vitexicarpin. Interestingly, VEGF signaling pathway can partly elaborate the Antiangiogenic mechanisms of vitexicarpin, consistent with our experimental results. Of the remaining pathways, Neurotrophin signaling pathway, Insulin signaling pathway, and Type II diabetes mellitus are enriched by target-related proteins of vitexicarpin, suggesting that it may be potentially used for the diabetes therapy. Thus, vitexicarpin's result provides an example of how “target profiles” predicted by drugCIPHER can comprehensively shed light on the mechanisms of action of herbal compounds. The available target profile makes it possible to infer the mechanism of a herbal compound of interest by comparing profiles with existing FDA-approved drugs. As shown in in vitro and in vivo. We successfully applied drugCIPHER to predict and experimentally validate key proteins SRC and AKT in the target network of vitexicarpin, revealing a previously unreported anti-angiogenesis molecular basis of vitexicarpin on vascular endothelial cell migration, apoptosis, and proliferation. This work also highlights the considerable potential for future herb medicine researches in terms of TCM network pharmacology.In summary, this work suggests that vitexicarpin is an angiogenesis inhibitor