Batrachochytrium dendrobatidis was identified from sick and dead frogs. Since then, several lines of evidence suggest that B. dendrobatidis may be involved in frog declines: the fungus has been found on frogs in afflicted areas; lab studies show that it's highly pathogenic to some frog species; and pathological evidence links it to host mortality. But with little information about the prevalence of this fungal infection in wild frogs, or how the disease impacts frogs in the wild, the causal role of this chytrid fungus remains unclear.Amphibian declines have reached crisis proportions in various parts of the world. In many areas, habitat loss is the likely culprit. But when mass die-offs suddenly occurred in relatively undisturbed habitats, the cause was far less obvious. Fourteen species suffered either extinctions or major declines in the pristine rainforests of Queensland, Australia, between 1979 and 1993. It was suggested in 1996 that some unknown disease had spread through the populations, but no pathogen was discovered until 1998, when the fungus B. dendrobatidis on frogs in their natural habitat, Richard Retallick et al. focused on six species living in the high-elevation rainforest streams of Eungella National Park in Queensland, Australia, where frog losses were “particularly catastrophic.” Two species vanished between 1985 and 1986: the Eungella Gastric-Brooding Frog (Rheobatrachus vitellinus), which is now thought extinct, and the Eungella Torrent Frog (Taudactylus eungellensis), which later reappeared in a few small populations. Other local frog species escaped this period relatively unscathed.To evaluate the effects of B. dendrobatidis had yet to be identified, but Retallick retained the toe tips, and the authors tested the toes for disease in 2002–2003. Fungal infections were found in two species—T. eungellensis and Litoria wilcoxii/jungguy ; the other four species were infection-free. L. wilcoxii/jungguy did not decline to any great extent during the 1985–1986 die-off.Retallick captured frogs from six sites from 1994 to 1998, clipped one or two toe tips from each frog to age and identify them, and then released the frogs back into the wild. At the time, T. eungellensis frogs was greatest at three particular sites, which showed peak infections during cooler months. Prevalence of infection was highest during winter and spring, but did not vary from year to year, suggesting that the infection is now endemic. Fungal infections were found in 27.7% of L. wilcoxii/jungguy frogs, with no evidence that prevalence differed among sites, seasons, or individuals . The probability of recapture was significantly lower for frogs that were already infected when first captured. While this might suggest a correlation between infection and death, it's impossible to distinguish death from simple failure to recapture the animal. On further analysis, McCallum and colleagues found no evidence that survival differed between infected and uninfected frogs, suggesting that this potentially devastating amphibian disease now coexists with the frogs, with little effect on their populations.The proportion of infected T. eungellensis, a rainforest frog listed as endangered, “now persist with stable infections of B. dendrobatidis.” While these findings do not exonerate the fungus as the agent of mass declines, they do rule out the possibility that the fungus caused the decline, then vanished from the area, allowing frog populations to recover. The authors allow that it's possible that B. dendrobatidis did not cause the initial T. eungellensis declines. Or alternately, the fungus could have emerged as a novel pathogen in the ecosystem, causing massive casualties before some form of evolutionary response took hold. Surviving frog populations may have evolved resistance to the pathogen, for example, or less virulent strains of the fungus may have evolved. If it turns out that frog populations can develop resistance to the chytrid fungus, the researchers point out, then a conservation program of captive breeding and selecting for resistance could potentially thwart the extinction of these, and other, critically endangered frogs. A critical next step, then, is to determine whether frogs and fungus do coevolve.These results, the authors conclude, “show unequivocally” that remaining populations of


Batrachochytrium dendrobatidis has been implicated in the decline and extinction of numerous frog species worldwide. In Queensland, Australia, it has been proposed as the cause of the decline or apparent extinction of at least 14 high-elevation rainforest frog species. One of these, Taudactylus eungellensis, disappeared from rainforest streams in Eungella National Park in 1985–1986, but a few remnant populations were subsequently discovered. Here, we report the analysis of B. dendrobatidis infections in toe tips of T. eungellensis and sympatric species collected in a mark-recapture study between 1994 and 1998. This longitudinal study of the fungus in individually marked frogs sheds new light on the effect of this threatening infectious process in field, as distinct from laboratory, conditions. We found a seasonal peak of infection in the cooler months, with no evidence of interannual variation. The overall prevalence of infection was 18% in T. eungellensis and 28% in Litoria wilcoxii/jungguy, a sympatric frog that appeared not to decline in 1985–1986. No infection was found in any of the other sympatric species. Most importantly, we found no consistent evidence of lower survival in T. eungellensis that were infected at the time of first capture, compared with uninfected individuals. These results refute the hypothesis that remnant populations of T. eungellensis recovered after a B. dendrobatidis epidemic because the pathogen had disappeared. They show that populations of T. eungellensis now persist with stable, endemic infections of B. dendrobatidis.The chytrid fungus  Recapture experiments provide evidence that some amphibian species can now persist with infections of the pathogenic chytrid fungus and suggests, for example, that frogs and fungus might be coevolving Lauranctil 1998  and was til 1998 . B. denddeclines , althougdeclines  had direB. dendrobatidis infection on toe tips collected between 1994 and 1998 from six species of frogs at Eungella National Park in east-central Queensland, Australia ((Rheobatrachus vitellinus) disappeared suddenly from relatively undisturbed rainforest streams, and it is now considered to be extinct ((Taudactylus eungellensis) also disappeared, but it was later found to have persisted in a few small populations  of the 474 toes assessed. Four species showed no sign of infection, while we detected infections on 58 (18.4%) of 316 toes of T. eungellensis and 13 (34%) of 47 toes of frogs identified at the time as Litoria lesueuri . The most parsimonious model  included each of these predictors but no interaction terms.Using a logistic model with season , species, and site as predictor variables, we found significant effects of each variable on infection, corrected for the effects of the other variables . A significantly smaller proportion was infected at Rawson Creek (10.5%) than at Dooloomai Falls (26.7%) or Tree Fern Creek (25.0%). There was a marginally significant difference overall in the infection levels among males, females, and subadults . However, when site was included in the model, any suggestion of a difference in infection level between males, females, or subadults disappeared. When we separated the main T. eungellensis populations, it was apparent that the difference in prevalence between those categories was influenced by the population at Tree Fern Creek, where 46% of males (n = 13) and no females or subadults were infected  differeinfected . When weT. eungellensis varied significantly among seasons , but not among years . Further, there was no evidence that the pattern of seasonal variation in infection changed among years . Infection was most prevalent (37.8%) during the winter months (1 June to 31 August) and least prevalent (11.3%) during the summer months (1 December to 28/29 February). Comparing the two sites with the largest sample sizes, Dooloomai Falls and Rawson Creek, there was no evidence that they had differing seasonal patterns of infection , although the level of infection overall was much higher at Dooloomai Falls . Infection levels were much higher in winter and spring combined than in summer and autumn , and there was no evidence of infection levels differing between winter and spring or between summer and autumn . The seasonal changes in infection at the two sites are shown in At these three sites, levels of infection among B. dendrobatidis was detected in 13 of the 47 L. wilcoxii/jungguy examined, giving an estimated prevalence of 27.7%, with a 95% confidence interval ranging from 15.6% to 42.6%. There was no evidence that prevalence of infection differed among males, females, and subadults ; among sites ; or among seasons  (each of the above Δdev terms is corrected for the other terms in the model).L. wilcoxii/jungguy and T. eungellensis was hampered by the fact that prevalence of infection on T. eungellensis differed between sites, and that the sampled populations of the two frog species had largely disjunct distributions. When the data were pooled over all sites, there was insufficient evidence to reject a null hypothesis of equal prevalence in the two species, whether the effects of season were allowed for  or not .Comparing the prevalence of infection between L. wilcoxii/jungguy and T. eungellensis recaptured at any stage later in the study, grouped by their infection status at first capture. A logistic model with recapture as the response and species and infection status as predictors produced no evidence that the effect of B. dendrobatidis infection on recapture probability differed between the species . In both species, the probability of recapture was significantly lower for infected frogs than for uninfected frogs , correcting for the substantially higher overall recapture rate of T. eungellensis relative to L. wilcoxii/jungguy.B. dendrobatidis may affect survival, but confounds the probability of recapturing a frog that is present at a site with the probability that a frog is still present at the site. The more sophisticated analysis that follows separates these two components, although it cannot distinguish between death and permanent emigration from the site. For brevity, we refer to continued presence at the site as “survival.”This simple analysis suggests that Investigating mark–recapture data for both Rawson Creek and Dooloomai Falls, we found that the best model (on the basis of minimizing the AIC) for which there were sufficient data to estimate almost all parameters had the probability of recapture varying with time but not group , and the survival probability differing between the groups but constant through time. However, further analysis in both cases showed that there was no evidence that survival differed between infected and uninfected frogs: At both sites, the group effect was due to survival being highest in females, intermediate in males, and lowest in subadults ; Table 4T. eungellensis, a rainforest frog that almost disappeared as a result of major die-offs in the Eungella area in the mid 1980s, now persist with stable infections of B. dendrobatidis. This does not imply that this pathogen cannot have been responsible for the decline. One hypothesis that can be discarded, however, is that B. dendrobatidis drove frog populations to low levels, consequently became locally extinct itself, and that frog populations subsequently recovered or stabilised in the absence of the pathogen. Two other hypotheses are consistent with our observed results.Our results show unequivocally that remnant populations of B. dendrobatidis was not responsible for the initial decline of T. eungellensis populations. These populations declined more than a decade before this chytrid was formally identified, and the declines occurred over a very short period , our results show that infection with B. dendrobatidis did not inevitably lead to rapid death in T. eungellensis.Our mark–recapture analysis did not find consistent evidence that captured . TogetheL. wilcoxii/jungguy appears not to have declined at the same time as T. eungellensis and R. vitellinus and, in our data, it has a high prevalence of infection. It therefore is a candidate reservoir host. Whether the prevalence of B. dendrobatidis differs between the sibling species L. wilcoxii and L. jungguy at sites where they are sympatric would be an interesting question, but cannot be answered from our study. The L. lesueuri complex has a widespread distribution throughout streams on the east coast of Australia  were histologically prepared in transverse sections stained with Ehrlich's haematoxylin for skeletochronological assessment . For both mark–recapture and analysis of prevalence, we grouped the data into 3-mo austral seasons . Mark–recapture modelling of survival requires that several assumptions be satisfied. Most critically, every marked individual present in the population at a given time has the same probability of recapture as all other members of its group, and the same probability of surviving to the next time interval. We used a parametric bootstrap (With the software MARK , we usedootstrap  to test ootstrap . Since t


SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a “master switch” in this differentiation. Heterozygous mutations in Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Our results provide a broad understanding of the strategies used by a “master” transcription factor of differentiation in control of the genetic program of chondrocytes. Sox9 cause Campomelic Dysplasia (CD), a generalized disease of cartilage characterized by hypoplasia of endochondral bones Sox9 gene at various times during mouse limb development also demonstrated that SOX9 is necessary for mesenchymal condensations, for the commitment to the chondrocyte fate at the time when the chondrocyte and osteoblast lineages segregate from a common progenitor, and for the overt differentiation of these cells into chondrocytes. SOX9 thus acts as a master regulator of chondrocyte differentiation Col2a1 revealed that this site appears to be depleted of nucleosomes.The transcription factor SOX9 plays a critical role in cell fate decisions of a discrete number of cell types Col2a1, Col11a1, Matrilin-1, Aggrecan, Syndecan-3, Cdrap, Fibromodulin and Prelp were also highly expressed in RCS cells. On the other hand, the Col1a1 gene was expressed at high level in Rat-2 cells but was not expressed in RCS cells. These results in addition to the previously reported data As chromatin source for ChIP-on-chip experiments, we used rat chondrosarcoma cells (RCS cells), because these cells display many chondrogenic characteristics including secretion of specific cartilage ECM proteins and high contents of SOX9, SOX5 and SOX6 AdamTS 5MMP9 and 13Cathepsin BSox9, Sox5 and Sox6, and in cartilage development such as PrxPGC-1αTCF4Lef1β-cateninStat1TIP60Runx2Osterix (Osx)MyoDMyogeninIntegrins α11BMP2 and 4NogginChordinTGFβ3WntsPatchedIhhShhSmoothenedVEGFCtgfEgf and EgfrIgf1 and Igf2rPTHrPGrb10TNF αIL1αPKC and p38Fgfr3PKANcamα-CateninCD44Because the sheared chromatin fragments had a size between 1 and 1.5 kb, the peaks of hybridization were relatively broad. To be counted as SOX9 interaction sites the peaks had to have similar heights at identical locations on both the forward and reverse DNA strand lanes. A so called smoothened lane was generated by subtracting the corresponding non-specific IgG hybridization signals from each SOX9 signal followed by averaging the two subtracted signals. Overall, except for a few genes discussed below, peaks, which on the “smoothened” lane had a cut off above the base 2 logarithm of 1.5 were further examined.Col2a1. The center of the peak located in intron 1 corresponds to a previously identified chondrocyte-specific enhancer in this gene Col2a1. Two SOX9 interaction sites were also identified in the Col11a2 gene. The centers of these hybridization peaks, one located in the promoter and the other in intron 1, correspond to previously characterized chondrocyte-specific enhancers bona fide specific SOX9 interaction sites. A total of 55 peaks with a cut off >log2 1.5 were identified in 30 genes. These genes and the locations of these peaks within these genes are listed in Col2a1 and Col11a2Col9a2Syndecan-3AggrecanEpiphycanChondroadherinBiglycan2 1.5 cut off, are, however, also likely to be direct targets of SOX9 and are likely to be part of the genetic program of chondrocytes controlled by SOX9. These genes are listed in a separate section of Col1a1 gene, which is not expressed in chondrocytes but is prominently expressed in mesenchymal precursors and in osteoblasts contains two SOX9 interaction peaks. Similarly the genes for Osx and Runx2, transcription factors required for osteoblast differentiation, contain distinct SOX9 interaction sites. One possible hypothesis is that at these sites in chondrocytes SOX9 might be part of repressor complexes. MyoD and Myogenin genes, which are master transcription factors for myoblast differentiation and are not expressed in chondrocytes. Other genes in this group include MMP13, those for several small leucine-rich proteoglycans, also the Wnt 3a, 5a, 7a, 9, Ihh and Shh genes. One surprise is that no clear SOX9-interaction site was found in the Sox6 gene, which together with Sox5, is required for overt chondrocyte differentiation and requires SOX9 for expression Col2a1, Col11a1, Col11a2 and Cdrap. The reason we chose these sites is that by analyzing the sites by EMSA, we could validate that the ChIP-on-chip peaks revealed true SOX9 binding sites. We chose the other sites based on potential SOX9 binding sequences within the peaks of hybridization. The potential SOX9 binding sites in most of these genes diverged from the consensus binding sites, WWCAAWG(N)nCWTTGWW  and SOX9 was indeed bound to each of these sequences. By comparing the mobility of SOX9-DNA complexes with that of a binding site in an enhancer in intron 1 of Col2a1, we concluded that SOX9 was mainly binding as a dimer to each of the peak sequences. We also asked whether the species conservation of sequences and the AT or GC content in the 610 bp centered on the hybridization peaks were different from 610 bp sequences surrounding random potential SOX9 binding sites outside the peaks. The conservation scores were higher in peaks than in non-peak DNA sequences  for 11 of these sites . These eequences . The AT equences .Col2a1, a peak was identified in intron 6  that expresses neither Col2a1 nor Sox9 , because these cells display many chondrogenic characteristics including secretion of specific cartilage ECM proteins and high contents of SOX9, SOX5 and SOX6 see  [19]. InSox5 and Sox6 in chondrocytes Sox5 contains several SOX9 interaction sites located in the promoter, introns 1 and 2. In contrast, we did not detect SOX9 interaction sites in the Sox6 gene. This suggests either that the Sox6 gene is not a direct target of SOX9 or that the SOX9 interaction sites are located outside the Sox6 DNA segments that were printed on the array. Our experiments detected interaction sites for SOX9 in the Sox9 gene located in introns 1 and 2. Although our previous results have indicated that chondrocyte-specific regulatory segments of Sox9 were still active even when the Sox9 gene was partially deleted, it is nevertheless possible that the SOX9 binding sites in introns 1 and 2 have an autoregulatory role in the transcriptional control of SOX9 in combination with other transcription factors. In Sertoli cells SOX9 binds to the promoter region of Sox9 but our results did not detect a SOX9 interaction sites in the chromatin of this segment in chondrocytes Sox9 is equally highly expressed. Through this receptor and via PKA, PTHrP stimulates the phosphorylation of SOX9 Bmp4 gene itself showed also a SOX9 interaction site. In addition to the genes specifically expressed in chondrocytes such as cartilage ECM genes, SOX9 interaction sites were also detected in ubiquitously expressed genes such as those for the transcription factors Tip60, Stat1 and Lef1, and for the ERK1, ERK2 and PKA signaling molecules. Thus in chondrocytes SOX9 also appeared to interact with a number of genes that are more broadly expressed. For example, we showed recently that Tip60 up-regulated expression of Col2a1, a direct target of SOX9 Tip60 promoter and up-regulated this promoter . Thus SOX9 interacts with the gene for a coactivator, which cooperates with SOX9 in activating a downstream target of SOX9. Previous experiments have shown that both ERK1 and ERK2 signaling in response to FGF increases SOX9 expression Col1a1 gene, which is not expressed in chondrocytes and in RCS cells, showed two clear interaction sites one in intron1, the other immediately 3′ to the gene. One possible hypothesis is that SOX9, which is known to be a transcriptional activator, might also be able to become part of a negative transcriptional complex and that in chondrocytes SOX9 may have a role in silencing genes that are active in Sox9-expressing osteochondroprogenitor cells as well as in osteoblasts. The SOX9 interaction sites in the genes for RUNX2 Our previous results have shown that in the absence of SOX9 there is no expression of Col2a1 gene is one that has been most intensively characterized so far in terms of response to SOX9. In this gene, SOX9 has been shown to bind to a sequence in a chondrocyte-specific enhancer in intron 1 and consequently to induce the activity of this enhancer. As shown here a ChIP-on-chip peak was clearly centered on this binding site in intron 1. In addition to this peak, another peak was detected in intron 6 of Col2a1. The binding of SOX9 at this site was validated by EMSA and ChIP-qPCR. Intron 6 increases the activity of a reporter containing the Col2a1 promoter and the enhancer of intron1, and the deletion of a short segment containing the SOX9 binding site in intron 6 from this reporter decreased its activity suggesting that this SOX9 binding site functions as a positive regulatory site in Col2a1. The 3′ sequence of a 48bp of intron 6 contained an inverted repeat sequence similar to the inverted repeat sequence in the 3′segment of the 48bp in intron 1 (Col2a1 gene in vivo still remains to be clarified.EMSA of a sample of SOX9 interaction sites confirmed that SOX9 was able to bind to specific sites found in the hybridization peaks of our ChIP-on-chip experiment. These sequences often diverged from the consensus dimeric binding sites WWCAAWGX(N)CWTTGWW (W = A or T) by several mismatches. Among the many genes having SOX9 binding sites, the intron 1 . The 3′ Saccharomyces cerevisiaeCol2a1 and Sox9 genes were not actively transcribed, did form nucleosome structures  were obtained from American Type Culture Collection (ATCC). Rat chondrosarcoma (RCS) cells were gifted from Dr. James H. Kimura, Henry Ford Hospital, Detroit, Michigan ChIP was performed according to the previously described method 32P-dCTP using Klenow fragment, and then EMSA was performed using recombinant human SOX9 proteins expressed in E.coli as described previously The probes used in EMSA shown in Suppl. Col2a1 intron 1 and intron 6 were cloned into pBluescript vector and then multimerized as previously described Renilla luciferase plasmid) using Fugene 6, The luciferase and β-galactosidase activities were obtained by use of a dual luciferase assay system (Promega Co. Ltd) and a Tropix Galacto Reaction kit (Applied Biosystems), respectively. Each value in the reporter assay was presented as the fold increase in Firefly luciferase activity units or β-galactosidase activity units per Renilla luciferase activity units from three independent experiments, each performed in triplicate.Oligonucleotides of 48 base pair fragments of both Table S1List of Primers for EMSA(0.05 MB DOC)Click here for additional data file.Table S2List of Primers for qPCR(0.08 MB DOC)Click here for additional data file.Table S3List of Primers for (0.05 MB DOC)Click here for additional data file.Figure S12y.mRNA expression levels in RCS cells compared to Rat-2 fibroblast cells. Total RNA was extracted from logarithmically growing RCS cells or Rat-2 cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was prepared from the RNA using AMV reverse transcriptase followed by qPCR with specific primer for each RNA  using SY(1.56 MB TIF)Click here for additional data file.Figure S2Sequence conservation of peaks. By use of the program, Multiz9way, obtained from UCSC genome browser, the evolutionary conservation was measured in nine vertebrates including rat, human, mouse, dog, cow, opossum, chicken, frog and zebrafish. In order to calculate the conservation scores, 72 regions out of 76 peaks that contain the consensus inverted repeat, WWCAAWG(N)nCWTTGWW  with a space (n) of 3 to 6. These regions also conserved a core inverted repeat sequence, AANG(N)nCNTT, and had a maximum of 2 mismatches in each half of the consensus repeat. 76 non-peak regions containing such repeat were also chosen. Note that such sequences are frequently found in both peak and non-peak regions of the genome. A two-sample t-Test showed that p-value was 0.01864. Readers interested in the detailed sequences that were used to compose this figure should contact the corresponding author.(1.56 MB TIF)Click here for additional data file.Figure S3−9.Box plot showing AT content of peak and non-peak regions. We compared AT or GC content in 610 bp sequences centered on the hybridization peaks were compared to 610 bp sequences surrounding random potential SOX9 binding sites outside the peaks. The bold horizontal lines show the mean of the data. Mean of AT content in peak regions was 46.7%, and mean AT content in non-peak regions was 53.8%. By Student's t-Test, p-value was measured at 3.425×10(1.56 MB TIF)Click here for additional data file.Figure S4Col2a1 on ChIP-on-chip microarray by ChIP-qPCR. The DNA obtained from ChIP of sheared chromatin of RCS cells with SOX9 antibodies was used as the template in real time qPCR to amplify a segment of intron 6 of Col2a1. A segment of intron 1 of Col2a1 previously identified as containing a functional SOX9 binding sites and another segment located 3′ to the Col2a1 gene served as positive and negative controls, respectively. Error bars represent standard deviations. The sequence of each probe is shown in Validation of SOX9 binding sites in (1.56 MB TIF)Click here for additional data file.Figure S5Col2a1 minimal promoter (89bp) followed by the firefly luciferase gene (Luc4) Renilla luciferase construct, to normalize for transfection efficiency.Functional analysis of the SOX9 binding site in intron 6. Five tandem repeats of the 48bp in intron1, the 48bp in intron6 or the segment conjugated each other were inserted in 5′ to the (1.56 MB TIF)Click here for additional data file.Figure S6Col2a1 gene of three different species are aligned. The inverted repeat of the rat SOX9 binding site is underlined. The bases in red are the bases that are not identical to the corresponding human sequence. The sequence of the binding sites between rat and mouse are completely conserved. The sequence of the binding site of the human is not identical to the rat sequence. B. An EMSA assay was performed to test whether SOX9 was binding to the human intron 6 sequence. The sequence of each probe is shown in Conservation of the SOX9 binding site in intron 6 among different species. The sequences of Sox9 binding sites in intron 6 of (1.56 MB TIF)Click here for additional data file.Figure S7CTGGGTTTCTGTAAAGAAGGCCTTCAGCTATCTGA Mutant; CGAAAAGGAAAACAGCTATCTGACTGGGTTTCTGT The ability of this mutated fragment to bind Sox9 was demonstrated as shown in Col2a1 intron 1), lane 2; SOX9 binding site of Col2a1 intron 6, lane 3; mutant SOX9 binding site of Col2a1 intron 6. B. Luciferase reporter assay of Chimera B and mutant Chimera B constructs. By use of this mutant fragment, the mutant Chimera B (Effect of mutation on the enhancer activity of Chimera B shown in himera B  construc(1.56 MB TIF)Click here for additional data file.Figure S8nd peak of Col2a1 gene in primary chondrocyte. Binding of H3 and Sox9 in Col2a1 gene of mouse rib chondrocyte primary culture and human lymphoblast, Reh cells, was demonstrated by ChIP-qPCR The mouse chondrocytes were cultured as shown previously st peak and 2nd peak correspond to the peaks in intron 1 and intron 6 of rat Col2a1 gene, respectively. Expression of Col2a1 and Sox9 was detected in primary chondrocyte cells but not in Reh cells by RT-qPCR method shown in Col2a1 gene is highly conserved in the human Col2a1 gene.Depletion of histone H3 in 2(1.56 MB TIF)Click here for additional data file.


Although the addition of bevacizumab significantly improves the efficacy of chemotherapy for advanced breast cancer, regulatory concerns still exist with regard to the magnitude of the benefits and the overall safety profile.A literature-based meta-analysis to quantify the magnitude of benefit and safety of adding bevacizumab to chemotherapy for advanced breast cancer patients was conducted. Meta-regression and sensitivity analyses were also performed to identify additional predictors of outcome and to assess the influence of trial design.st line , with a 1-yr absolute difference (AD) of 8.4% . A non-significant trend was found in overall survival (OS), and in PFS for 2nd line. Responses were improved with the addition of bevacizumab, without interaction between 1st line  and 2nd line . The most important toxicity was hypertension, accounting for a significant AD of 4.5% against bevacizumab . Other significant, although less clinically meaningful, adverse events were proteinuria, neurotoxicity, febrile neutropenia, and bleeding. At the meta-regression analysis for 1st-line, more than 3 metastatic sites (p = 0.032), no adjuvant chemotherapy (p = 0.00013), negative hormonal receptor status (p = 0.009), and prior anthracyclines-exposure (p = 0.019), did significantly affect PFS.Five trials  were gathered. A significant interaction according to treatment line was found for progression-free survival ; PFS was significantly improved for 1st-line chemotherapy significantly improves PFS, and overall activity. Hypertension should be weighted with the overall benefit on the individual basis.Although with heterogeneity, the addition of bevacizumab to 1 AboutDespite numerous advances in early diagnosis and treatment in local and systemic, metastatic breast cancer remains an incurable disease and the main objective of therapy is both the prolongation of survival and the improvement of associated symptoms , with particular reference to delay the onset of symptoms, improvement in progression-free survival , and improvement of quality of life .Metastatic breast cancer is a heterogeneous disease whose evolution is difficult to predict. Choosing the best treatment must necessarily be based to balance different aspects of patient characteristics, the disease characteristics and possible adjuvant treatment received . As a fuAngiogenesis represents an important step in the pathogenesis, invasion, progression and development of metastatic phenotype of breast cancer and is regulated by pro-angiogenic factors such as vascular endothelial growth factor (VEGF). High exAlthough bevacizumab showed modest benefits as single agent, numerous preclinical studies have demonstrated synergy between anti-angiogenic therapy and chemotherapy . The addIn order to explore the magnitude of the benefit of adding Bevacizumab to chemotherapy for metastatic breast cancer with particular attention to safety, we conducted a meta-analysis.The analysis was conducted following 4 steps: definition of the outcomes , definition of the trial selection criteria, definition of the search strategy, and a detailed description of the statistical methods used ,18.The combination of chemotherapy and Bevacizumab (Beva) was considered as the experimental arm and chemotherapy as the standard comparator. Analysis was conducted in order to find significant differences in primary and secondary outcomes. Primary outcomes for the magnitude of the benefit analysis were both the Progression Free Survival (PFS: time between randomization and progression or death from any cause) and the overall survival (OS: time between randomization and death for any cause). Secondary end-points were: overall response rate (ORR), and grade 3-4 toxicities.th, 2010. Updates of Randomized Clinical Trials (RCTs) were gathered through Medline (PubMed: http://www.ncbi.nlm.nih.gov/PubMed), ASCO , ESMO , FECS , and SABCS  website searches. Key-words used for searching were: advanced/metastatic breast cancer; chemotherapy; Bevacizumab; randomized; randomized; meta-analysis; meta-regression; pooled analysis; phase III; comprehensive review, systematic review. In addition to computer browsing, review and original papers were also scanned in the reference section to look for missing trials. Furthermore, lectures at major meetings  having 'advanced or metastatic breast cancer' as the topic were checked. No language restrictions were applied.Deadline for trial publication and/or presentation was June 30All prospective phase III RCTs published in peer-reviewed journals or presented at the ASCO, ECCO, ESMO and ASTRO meetings until June 2010, in which patients with advanced or metastatic breast cancer were prospectively randomized to chemotherapy with or without Bevacizumab were gathered, regardless of treatment lines.Hazard Ratios (HR) for PFS and OS and the number of events for secondary end-points were extracted; the last trial's available update was considered as the original source. All data were reviewed and separately computed by four investigators .HRs were extracted from each single trial for primary end-points ,20, and Five trials 3,841 patients) were identified , with an absolute benefit of 8.4%, corresponding to 12 patients to be treated for one to benefit, although with significant heterogeneity (p = 0.0001) , with specific regards to the lack of significant benefit in OS, and the toxicity profile. Moreover, the regulatory panel withheld the indication for breast cancer, and the final decision is still pending. The main question raised up by the regulatory committee refers to the eventual amount of benefit related to the addition of Bevacizumab. For this reason, a cumulative analysis specifically designed to weight that became mandatory.The addition of Bevacizumab to chemotherapy is considered one of the most viable treatment options in patients with HER-2 negative metastatic breast cancer, as distinct randomized studies so far presented and published consistently showed that this association resulted in significantly improved overall response rate and PFS. Notably, the therapeutic benefit was observed in all subgroup examined. Nevertheless, the issue of adding Bevacizumab to 1st line Bevacizumab is 32%, and 12 patients are needed to treat in order to see one patient who significantly benefit. This amount of benefit well compares with the benefits of other important therapeutic choices such as the addition of taxanes for the 1st line treatment of metastatic breast cancer, where the advantage in terms of relative risk is about 10%.The data presented herein show a statistically significant advantage in terms of either progression-free and responses, with an overall absolute benefit of 8% Table . The relFrom a global perspective, the hazard ratios for PFS obtained in the current analysis compare well with those obtained in other studies that have investigated the addition of another drug in the taxane-based chemotherapy. In the study of Albain et al , the addTaking into account the different approaches to treatment such as chemotherapy combination versus single agent therapy for first line treatment of metastatic patients with breast cancer, the HR for taxanes based combinations compared with control arm was 0.92 for PFS . Also wiWith regard to the concerns regarding the interpretation of those trials providing a significant  benefit in intermediate end-points (such as PFS) without any advantage in late-outcomes (such as OS), a recent original work has been published, trying to weight the impact of the post-progression survival  . To thisTwo meta-analysis analyzed the effect of the addition of Bevacizumab to chemotherapy in metastatic breast cancer ,33 in ovAt the last ESMO meeting, a meta-analysis of 530 elderly patients (older than 65 years) enrolled in the randomized trials ECOG 2100, AVADO and RIBBON-1, was presented . AlthougAnother relevant issue that emerges from our analysis is that the prior exposure to treatments containing taxanes does not affect the efficacy of bevacizumab Table . Indeed,With regard to serious adverse events, the main significant toxicity against the addition of bevacizumab was hypertension Table ; this reRecently, other anti-angiogenesis drugs have been studied in randomized trials for locally advanced or metastatic breast cancer -39. In tMore concerning data with regard to the overall safety profile of bevacizumab have been recently released ,42: in tOur data in context with the other exploring the safety-efficacy balance of the addition of bevacizumab to chemotherapy for advanced breast cancer do strengthen the need of a deep analysis of the correlation between adverse events and deaths on one side, and the maximization of the efficacy by restricting the drug to those patients who will really benefit. The latest approach is far to be understood, although positive hints with regard to polymorphisms analyses are encouraging. Bevacizumab, from a clinical practice standpoint, slightly increases the efficacy of chemotherapy in HER-2 negative advanced breast cancer, although a close follow-up monitoring for adverse events must be adopted.The authors declare that they have no competing interests.FCu, EB, VV, PC, MM and SG conceived the analysis, and supervised the calculations; FCu, EB, IS, and DG performed the calculations in a blinded fashion; VV, FB, AF, PC, MM, CN, MR, PP, and GF participated in the trials recruitment and selection process; FCu, EB, VV, FP, AF and MM drafted and revised the manuscript; EB, PC, MM, MA, DG and FC did coordinate the overall study process and did provide the funding. All authors read and approved the final manuscript.


We have shown that it is possible to tune, up to complete suppression, the photoluminescence superlinear dependence on the excitation density in quantum dot samples at high temperatures by annealing treatments. The effect has been attributed to the reduction of the defectivity of the material induced by annealing. Self-assembled semiconductor quantum dots (QDs) are zero-dimensional structures, being solid state systems with an atomic-like density of states. QDs are currently deeply investigated for their potentiality as building blocks for novel optoelectronic devices and for quantum information technologies. Several promising applications were recently developed in these fields, such as single photon sources for quantum cryptography, lasersSemiconductor QDs are known to efficiently capture the carriers generated by optical absorption. The electron hole pairs created in the barrier region rapidly relax to the QD ground state from where they radiatively recombine. The role of non-radiative channels on temperature dependence of the QD recombination efficiency has been analyzed in many details and with different experimental techniques-8. It isIL) on the excitation power density (Pexc) at high T that is sometimes observed in QD samples , which i0.3Ga0.7As barrier layer at 580°C by conventional MBE, the substrate temperature is lowered to 200°C and the As valve closed to deplete As pressure in the growth chamber. At this temperature and in the absence of As, only a flux of Ga atoms equivalent to 3.75 ML/s (where we indicate as 1 ML of Ga the amount of atoms necessary to obtain 1 monolayer of GaAs in the presence of As) is supplied at 0.5 ML/s rate. Due to the As-rich surface reconstruction, the initially deposited 1.75 ML/s are incorporated into the As terminated surface, resulting in the appearance of a Ga-stabilized surface, and the remaining 2 ML/s gives rise to the formation of tiny Ga droplets on the substrate. Following the deposition of the droplets, and maintaining the substrate temperature at 200°C, the As valve is opened to supply a molecular beam flux equivalent to 2 × 10−4 Torr beam on the surface. This growth step causes the complete arsenization of the Ga contained in the droplets, as demonstrated by the complete change of reflection high energy electron diffraction pattern from halo to spotty. A growth interruption of 10 min at 350°C under a constant As flux is then performed. This step did not introduce any change in the QD morphology[0.3Ga0.7As barrier layer of 50 nm is grown by migration-enhanced epitaxy (MEE)[λexc = 532 nm). The excitation power density is 10 W/cm2. The spectra are measured by a grating monochromator operating with a Peltier cooled CCD camera.A single GaAs/AlGaAs QD sample is grown by DE in a conventional molecular beam epitaxy (MBE) system provided with an As-valved cell. After the growth of the Alrphology. The AFMxy (MEE) at 350°CT = 15 K show a broad emission band located between the GaAs and the Al0.3Ga0.3As energy gaps at 1.72 eV  implies that the internal quantum efficiency increases with raising the excitation power. This could be understood easily if we consider that the value of the efficiency is limited to 1. In the case of efficiency equal to 1, all the absorbed photons are re-emitted, so the ratio between the incoming and emitted photons is constant and the dependence between IL and Pexc must be linear. On the other hand, in the case of samples where non-radiative recombination channels are present, the efficiency is reduced to values < 1. The increase of photogenerated carriers saturates the non-radiative recombination channels in the barrier, leading to a superlinear behavior and then α > 1. Therefore, the superlinearity of the QD emission intensity on the excitation power density could stem from the saturation of temperature-activated trap states, which affect the carrier diffusion in the barrier[IL on Pexc superlinerity. This can be appreciated in Figureα exponent at T = 190 K are reported. Clearly, α is in inverse relation with the PL yield, thus supporting the interpretation of the PL superlinarity as stemming from defect-related effects.The observed dependence of the  barrier. RTA treα exponent at high temperatures makes it possible to evaluate the quality of the QDs and of the surrounding barrier material by simple PL measurements. A linear dependence of IL on Pexc demonstrates the absence of temperature-activated non-radiative decay in the material.This has also an interesting applicative counterpart. The measurement of the In conclusion, we have shown that it is possible to tune, up to complete suppression, the PL superlinear dependence on the excitation density in QD samples at high temperatures by RTA treatments. The effect has been attributed to the reduction of the defectivity of the material induced by the annealing. This rules out possible explanation of the superlinear behavior as stemming from intrinsic carrier dynamics effects.Both authors declare that they have no competing interests.SS proposed and guided the overall project. SB performed material growth, AFM and PL measurements. Both authors read and approved the final manuscript.


An optimal life satisfaction (LS) is considered an important long-term outcome after a traumatic brain injury (TBI). It is, however, not clear to what extent a single instrument captures all aspects of LS, and different instruments may be needed to comprehensively describe LS. The aim of this study was to compare self-ratings of life satisfaction after a TBI with two commonly used instruments.Life Satisfaction Questionnaire (LiSat-11), comprising eleven items and Satisfaction With Life Scale (SWLS), comprising five items, were administered to 67 individuals (51 men and 16 women). Secondary analysis of data collected as part of a survey of individuals with TBI 6 to 15 years post TBI.p < 0.001). The total score in SWLS had the strongest correlation with items in LiSat-11. All items in LiSat-11, except ‘Family life’ and ‘Partner relationship’, were moderately to strongly correlated with items in SWLS. The item ‘Partner relationship’ in LiSat-11 did not correlate with any of the items in SWLS or the total score. The item ‘If I could live my life over, I would change nothing’ in SWLS had the weakest correlations with items in LiSat-11. Items ‘Vocation’ and ‘Leisure’ in LISat-11 were most strongly correlated with items in SWLS, whereas the item ‘ADL’ in LiSat-11 was more weakly correlated with items in SWLS.Item 1 in LiSat-11 (‘Life as a whole’) and the total SWLS score was strongly correlated (Spearman’s rho = 0.66; The strength of the relationships implies that the two instruments assess similar but not identical aspects of LS and therefore complement each other when it is rated. A high level of life satisfaction (LS) is considered an important outcome of rehabilitation and a long-term endpoint after a traumatic brain injury (TBI). LS is defined as an individual’s contentment with life. It is commonly referred to as the degree of an individual’s subjective appraisal if his or her aspirations or goals and achievements have been accomplished , 2.Two internationally validated instruments assessing LS after TBI are the Life Satisfaction Questionnaire (LiSat-11)  and the The aim of this study was to compare ratings of LS using the two instruments LiSat-11 and SWLS in individuals with TBI. This is a secondary analysis of data on LS, using LiSat-11  and SWLSParticipants were obtained from a sample of 332 individuals with a computed tomography (CT) verified TBI and brain injury symptoms who had been transferred to the only Neurosurgical Clinic in the region for neurosurgical care during the period 1 January 1992 to 31 December 2001 . From thThe LiSat-11 (11-item Life Satisfaction Questionnaire) , 5 assesThe SWLS  provides a global measure of satisfaction with life as an overall summation of a person’s LS . It consAll analyses were performed using IBM SPSS statistics 22.0 software .In Table p < 0.001 to p < 0.05); Spearman’s rho = 0.27 to 0.67) between 34 of the 55 items in LiSat-11. The item ‘Partner relationship’ was not significantly correlated with any of the other items in LiSat-11, and the item ‘Family life’ was significantly correlated with only 3 of the 11 items. All 5 items in SWLS were significantly correlated p < 0.01 to p < 0.001; Spearman’s rho = 0.35 to 0.81).There were significant correlations (p < 0.001). Overall, the total score in SWLS had the strongest correlation with items in LiSat-11. Generally, all items in LiSat-11, except ‘Family life’ and ‘Partner relationship’, were moderately to strongly correlated with items in SWLS. The item ‘Partner relationship’ in LiSat-11 did not correlate with any of the items in SWLS or the total score. Item ‘If I could live my life over, I would change nothing’ in SWLS had the weakest correlations with items in LiSat-11. Items ‘Vocation’ and ‘Leisure’ were most strongly correlated with the items in SWLS, whereas the item ‘ADL’ in LiSat-11 was more weakly correlated with items in SWLS.In Table Life satisfaction is often emphasized in modern rehabilitation and in outcome research of lifelong disabilities, such as TBI. Different instruments are available to assess LS, but as LS is multifaceted, it can be assumed that one instrument alone does not capture all aspects of LS. Consequently, there is a need for an in-depth knowledge of how different LS instruments are related. This study is, to the best of our knowledge, the first study that has pursued a face-to-face comparison of two commonly used instruments assessing LS in people with TBI. Overall, we found a moderate correlation between most items in the two instruments, which indicates that they measure similar but not the same aspects of LS.p < 0.001). They can both be considered to represent some form of global measure of satisfaction with life. One previous study has found a good convergent validity with other LS instruments in people with spinal cord injury [As expected, Item 1 in LiSat-11 (‘Life as a whole’) and the total SWLS score were strongly related  generally had the lowest correlation with items in LiSat-11. This was noted in the review of SWLS , where tThe items ‘Vocation’ and ‘Leisure’ in LiSat-11 were generally strongly correlated with items 1 to 4 in SWLS. In fact, satisfaction with ‘Vocation’ was the item most strongly correlated with a specific item in SWLS (Item 2). This supports previous studies that reported that people with TBI who are in gainful employment, regardless of the severity of their injury, are more satisfied with life than those who have a disability pension . SimilarThe results of this study have some general implications for clinical research and give rise to some aspects that need to be more carefully addressed. One is the content of the items in relation to the overarching research question. In situations where the purpose is to evaluate changes over time or effects of rehabilitation interventions, the global measure of SWLS can be considered together with item 1 in LiSat-11. On the other hand, if the purpose is to explore domains that are vital aspects of one’s LS and compare those, the LiSat-11 may be a better instrument.One limitation to this study is the relatively small and selected sample. Therefore, the results cannot be generalised to all people with TBI. It is known that cognitive impairments can affect the ability to respond to LS questionnaires. Thus, some of those with a severe TBI may have had difficulties to rate their life situation. Although none of the participants raised any concerns during the ratings, the inferences of this study should be treated with some caution.In conclusion, we found significant correlations between most items in the two LS instruments. The strength of the relationships implies that they assess similar but not identical aspects. The two instruments can be used simultaneously and complement each other and thereby cover life satisfaction in a broader sense.


Egretta eulophotes). Using a full length DNA and cDNA produced by PCR and RACE methods, these two MHC class I loci were characterized in the genome of the Chinese egret and were also found to be expressed in liver and blood. Both new genes showed the expected eight exons and were similar to two copies of the minimal essential MHC complex of chicken. In genetic diversity, 14 alleles (8 for UAA and 6 for UBA) in the MHC class I gene exon 3 were found in 60 individuals using locus-specific primers and showed little polymorphism. Only three potential amino acid residues were detected under positive selection in potential peptide-binding regions (PBRs) by Bayesian analysis. These new results provide the fundamental basis for further studies to elucidate the molecular mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids, finding that have not been previously reported.The genes of major histocompatibility complex (MHC) are important to vertebrate immune system. In this study, two new MHC class I genes, designated as Egeu-UAA and Egeu-UBA, were discovered in the vulnerable Chinese egret ( The major histocompatibility complex (MHC) is a gene complex that encodes for cell-surface proteins responsible for the recognition and presentation of foreign antigens to T-lymphocytes, and which triggers the adaptive branch of the immune system . The MHCThe MHC is known to be a complicated gene group in vertebrates, both in terms of allelic diversity and gene number . NumerouDespite continuing investigation of avian MHC class I genes, and an increasing interest in their use as markers of adaptive genetic variation in evolutionary ecology research –15, manyEgretta eulophotes) is listed as a vulnerable species with an estimated global population of 2600–3400 individuals [Egretta eulophotes), (2) design locus-specific primers to survey the variation in PBR-encoding exons for all loci and (3) gain insights into the selection pressures acting on PBR and non-PBR regions within the third exon of MHC class I loci in the Chinese egret.The Chinese egret . The scientific license for access to the study site was issued by the Administration Department of Xiamen Egret Natural Reserve (XMENR-1005). Blood samples (~0.5mL) were collected from 60 nestlings (aged around 15 days) of Blood samples of 60 nestlings were collected from 35 nests at Riyu Islet  in Fujian Province of China, where a total of 50 nests of the Chinese egret were found. The blood samples were frozen at −80°C until DNA extraction. Total genomic DNA was isolated using the Universal Genomic DNA Extraction Kit Ver. 3.0 (TaKaRa), and total RNA was extracted using Trizol  from the blood and liver of one individual Chinese egret that died at this Islet, respectively . One μg CGCTACAACCAGASCRRSG and MHCI-ex4R: GGGTAGAAGCCGTGAGCRC [2), 0.2 mM of each dNTP, 0.4 μM of each primer, 0.7 U of Taq polymerase (TaKaRa), 100 ng of genomic DNA. The conditions for PCR amplification were a denaturing step at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 10 min.A fragment from exon 2 to exon 4 of MHC class I genes in Chinese egret was first amplified with degenerate primers MHCI-ex2F: GTGAGCRC . PCR reaCGTGGTCCGAGTGTCG 13sp2: GACCCTGACCTTGTCCTGC) and 5’ RACE (15sp1: GGTACTTGTCCTTCTCCTCCG 15sp2: CCAGGTGTAGTAGGTGCCATC) respectively, using Oligo 6.0 (Molecular Biology Insights). PCR reactions followed the protocols and applications as specified in the kit. A 730bp and an 810bp segments were obtained from 3’ RACE and 5’ RACE, respectively. After removing their overlapping region, a 1380bp long intact gene fragment of MHC class I was obtained.The 3’ and 5’ rapid amplification strategy of cDNA ends (RACE)  was usedCAGAACTCTGCCCGGAGACGG and 13utrR AGCGTGCACAGGGAGCAGAAATCAG were designed to amplify the complete DNA sequence of the MHC class I gene. PCR reactions were carried out on a Biometra T gradient thermocycler in a final volume of 50 μL containing 1× LA PCR buffer II, 2.5 mM MgCl2, 0.4 mM dNTP, 0.5 μM of each primer, and 2 U TaKaRa, LA Taq. PCR conditions included an initial denaturation step at 95°C for 2 min, 35 cycles of denaturation at 98°C for 10 s, extension at 68°C for 15 min, and a final extension at 72°C for 10 min.According to the complete cDNA sequence, two primers, 15utrF Escherichia coli DH5α. The genes of over 3000 bp were sequenced by using Primer Walking method from both sides respectively. In this Primer Walking method, after a shorter stretch (about 800bp) had been sequenced using M13F or M13R, a new primer was generated from the end of what had been already sequenced and the process was continued until the sequence was overlapped. Ten positive colonies of each band were selected to sequence bidirectionally on an automatic sequencer  using universal M13 sequencing primers and BigDye version 3 (Applied Biosystems).All PCR products were purified using the Agarose Gel DNA Purification Kit Ver. 2.0 (TaKaRa), then ligated into pMD 18-T vector (TaKaRa) and transformed into CATCCCCGTGGCAGTCGAGA and UBAF: AATCCCATCTCCTTGTCCCATCTTCC). These were combined with the reverse primer 13exR: CGTAGCTCACGTATTTCCTCAGCCAC to amplify the two loci, respectively. PCR was carried out in a 20 μL reaction mixture containing 1 μL genomic DNA, 0.7 U of Taq polymerase (TaKaRa), 1.5 mM MgCl2, 200 μM of each dNTP and 0.4 μM of each primer, for 25 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1min. Second, to obtain suitable length fragments for SSCP genotyping and scanning of the variation in exon 3 of each locus, a second round PCR was carried out using the primer 13exF: ACTGAAGGCCCAGGGCTGC and 13exR was used to amplify the entire exon 3 in each locus. The reaction conditions for the second round PCR were identical with those described for the first round. The PCR products from the first round were diluted 100-fold and used as the template. The amplicons were separated by SSCP analyses as described by Fain [To detect polymorphism within the MHC class I genes in wild populations of the Chinese egret, 60 nestlings on the Riyu Islet were genotyped by semi-nested PCR combined with single strand conformation polymorphism (SSCP). First, two forward locus-specific primers in intron 2 were designed and designated as UAAF and UBAF  and non-synonymous (dN) substitutions were calculated for both the 18 possible PBRs as defined by Bjorkman and Saper [S) and nonsynonymous (dN) substitutions per site according to the Nei–Gojobori method with Jukes–Cantor correction for multiple substitutions [The complete DNA and cDNA sequences for MHC class I genes, derived from the Chinese egret, and were aligned using the DNAMAN software package . Exons and introns were distinguished using the GenScan Program . DNA frand Saper , 34, anditutions . Populatitutions . The proitutions  was usedGT/AG) and polyadenylation recognition sequences (AATAAA or ATTAAA) were present in all sequences.In this study, DNA sequences of two different MHC class I genes were isolated from one individual Chinese egret, and the sequences were highly similar over large stretches. They could be distinguished, however, by the divergent intron 2. The exon sizes were similar to those found in other birds , S2 Fig.Based on the Egeu-UAA and Egeu-UBA sequences, two forward locus-specific primers were designed on intron 2 for each locus, respectively. These two sequences were then combined with the universal primers to amplify the exon 3, as described above. Single locus genotyping of MHC class I was carried out on 60 nestlings. The SSCP results showed one or two alleles per individual at each locus , confirmIn this study, our sample size was appropriate for the analyses of allele frequency and Hardy-Weinberg Equilibrium because 60 samples were sufficient to represent the full genetic variation present in this islet population. According to the optimal sampling strategy of Marshall et al  and SjögN/dS) was analyzed in predicted peptide-binding region (PBR) sites, and non-PBR sites for each gene separately [N/dS ratio of 1.23 and 1.30 respectively. The ratios were not significantly different than 1, indicating that strong selection was not acting on these two loci in the Chinese egret  suggested that Egeu-UAA locus was under positive selection. These findings suggest that Egeu-UAA represents a major class I gene in the Chinese egret. In contrast to the greater sequence variation of Egeu-UAA, Egeu-UBA showed extremely limited variation and no evidence of positive selection on exon 3, suggesting that this locus may represent a minor MHC class I gene. Moreover, CODEML analyses revealed only 3 residues with posterior probabilities of positive selection (P≥0.95) in Egeu-UAA exon 3, which is a finding significantly difference with most previous research results [Tympanuchus cupido) [The “Major “class I genes with high sequence variation and high expression levels across tissue types have been described for some bird species such as the chicken, duck, red-billed gull and crested ibis , 48–50.  results , 51. Thi cupido)  and seve cupido) . A possiS1 FigTotal RNA is extracted from the liver and blood. The PCR products are amplified from liver and blood cDNA using primer MHCI-ex2F and MHCI-ex4R. M stands for marker DL2000(Takara).(TIF)Click here for additional data file.S2 FigThe sequences from the MHC class I loci in crested ibis (Nini) and red-billed gull (Lasc) are compared; an mRNA sequence is used to mark the location of the exons (Nini-UCA1M and Lasc-UAAM), respectively. Sequence alignment and the differences between loci in intron 2 from each species are depicted in the genomic plot. The red box is used to mark the position in full length DNA sequence. DNAMAN is used to align the sequences. Sequence sources are: crested Ibis (KP182409 KP182408) and red billed gull (HM008713 HM008714 HM008715 HM008716 HM015819).(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 TableThe typing results for the first 15 individuals of the UAA locus are shown in (XLS)Click here for additional data file.


In order to better assist medical professionals, this study aimed to develop and compare the performance of three models—a multivariate logistic regression (LR) model, an artificial neural network (ANN) model, and a decision tree (DT) model—to predict the prognosis of patients with advanced schistosomiasis residing in the Hubei province.Schistosomiasis surveillance data were collected from a previous study based on a Hubei population sample including 4136 advanced schistosomiasis cases. The predictive models use LR, ANN, and DT methods. From each of the three groups, 70% of the cases (2896 cases) were used as training data for the predictive models. The remaining 30% of the cases (1240 cases) were used as validation groups for performance comparisons between the three models. Prediction performance was evaluated using area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy. Univariate analysis indicated that 16 risk factors were significantly associated with a patient’s outcome of prognosis. In the training group, the mean AUC was 0.8276 for LR, 0.9267 for ANN, and 0.8229 for DT. In the validation group, the mean AUC was 0.8349 for LR, 0.8318 for ANN, and 0.8148 for DT. The three models yielded similar results in terms of accuracy, sensitivity, and specificity.Predictive models for advanced schistosomiasis prognosis, respectively using LR, ANN and DT models were proved to be effective approaches based on our dataset. The ANN model outperformed the LR and DT models in terms of AUC. Worldwide, approximately 240 million individuals are infected with schistosomiasis, a parasitic neglected tropical disease that continues to be a significant cause of morbidity and mortality, especially in China. Effective tools that can accurately predict the prognosis of patients with advanced schistosomiasis would aid in the treatment and management of the disease. To this end, we constructed and compared the performance of three predictive models—an artificial neural network (ANN) model, a logistic regression (LR) model and a decision tree (DT) model—in their ability to predict the prognosis of patients with advanced schistosomiasis. We found that while all three models proved effective, the ANN model outperformed the LR and DT models in terms of AUC and sensitivity. Yet, to achieve the highest level of prediction accuracy and to better assist medical professionals, we recommend comparing the performance of the three predictive models to select the optimal one, which will be better than select a model at random. The findings of this study not only provide valuable information on the construction of effective predictive models for the prognosis of advanced schistosomiasis, but also offer new methodology for clinically determining patient diagnosis and prognosis. Approximately 240 million individuals are infected worldwide by schistosomiasis, with an estimated 3.31 million disability-adjusted life years lost as a result of the disease . The thiOncomelania hupensis, which is the only intermediate host of Schistosoma japonicum. Moreover, Hubei has the highest rates of schistosomiasis transmission in China ,where X =  denotes the vector of independent variables. An ‘entry’ approach was used to construct the LR model using the 16 variables. The LR model was built using the training dataset and tested using the validation data.For the categorical dependent variables, a LR model was conducted to identify the risk factors of various diseases by using patient demographic characteristics and other disease parameters. The LR model formula calculates the probability of a given disease, y . If the subject suffers from the disease, the conditional probability is represented as p(y = 1∣X) = p(X), and the formula of the LR model is expressed as log [(p(x) ∣1− p(x)] = βThe model-based clinical data interpretation system C4.5 algorithm for the prognosis of advanced schistosomiasis is shown in The AUC was used to compare the prediction performance of the three data mining models. The classification accuracy referred to the fraction of cases classified correctly. Sensitivity referred to the proportion of positive cases that were classified as positive. Specificity referred to the proportion of negative cases that were classified as negative. The formulas are shown as follows, where TP, FP, TN, FN represent true positives, false positives, true negatives, and false negatives, respectively. The AUC value of ANN can be interpretedFor the training and validation group, the ROC curves for the ANN, LR, and DT models are shown in Figs Table 2. We evaluate the differences in order to see whether there was significance. AUC value could be shown as the normalized Mann–Whitney U statistics. Concerning the normalization denominator is universal for all models, we could thus show the superiority by the AUC value from nonparametric test perspective. Specifically, given the true label of each sample, the larger AUC value, the lager Mann–Whitney U statistics, the better classified capability of the model. We additionally conduct two pairwise tests for AUC values to substantiate the superiority.The performance comparison of the three models in the two groups is listed in For ANN and DT, the result shows the difference is significant. .For ANN and LR, we obtain the similar result as following.Advanced schistosomiasis, resulting from either repeated infection or acute infection without chemotherapy, is the most severe form of schistosomiasis and clinically presents with portal hypertension , periporData mining systems aim to extract implicit, previously unknown and potentially valuable relationships and patterns from large amounts of data to provide clear and useful information through advanced processes of selecting, exploring, and modeling , 28. RecOne of the most attractive features of ANN is the system’s ability to apply machine learning, also referred to as training. ANNs can continuously adjust parameters, such as connection weights, and store the sample set as a connection weight matrix under circumstance of external environment stimulation, such as the input of the sample set. When the ANN accepts the input again, the system can provide the appropriate output. In the present study, there were many neurons in the model and the sample size had rigorous requirements. Therefore, only the variables that were selected by single factor analysis and closely related to the prognosis of advanced schistosomiasis were used as input variables. A good predictive model can distinguish population at high risk from the one at low risk, which is so called discrimination. Discrimination is generally expressed as the area under the ROC curve, referred to as AUC. The higher the AUC value, the better the model can discriminate between high and low risk groups. Due to the serious adverse prognosis of advanced schistosomiasis patients, the sensitivity of the predictive model should be as high as possible in order to avoid false negatives on condition that the discrimination of the model is fine (e.g. AUC≥0.75).Mann–Whitney U test.Data from the designated training set was then used to evaluate the ANN model, and the prediction accuracy of the ANN model was 0.8660, which was better than the LR model  and the DT model (0.8194). The AUC of the ANN, LR, and DT models was 0.9267, 0.8276, and 0.8229, respectively, which indicates that the ANN model had the best prediction performance by In comparison to the LR and DT models, the ANN model had the best fitting effect for the relationship between advanced schistosomiasis and pathogenic factors. Schistosomiasis’ pathogenesis of disease is a complicated process influenced by multiple factors; thus, the use of traditional LR models to predict the development of disease is significantly limited by the inability to determine effects of multiple co-linearity between the independent variables. DT models can be easily applied to discrete values, but when there are more attribute values, the effect may be poor . While ADespite its limitations, the LR model has been widely adopted because it offers other advantages , 38. LR In comparison to LR models, DT models can not only detect statistically significant risk factors, the model can also intuitively compare the intensity of various risk factors on the prognosis of patients with advanced schistosomiasis , 43. TheANN simulates the function and structure of biological neural network to establish non-linear mathematical models with strong fault tolerance, adaptiveness, nonlinear comprehensive reasoning ability, and the powerful ability to solve co-linearity and interactions between variables , 48. AltThe advantages and disadvantages between these models on the implementation of them in the medical practice are noteworthy. A study that used ANN models and generalized additive models (GAM) to estimate glomerular filtration rate (GFR) in patients with chronic kidney disease found that the advantage of ANN is obvious only when multiple variables added to the model, especially the multicollinearity existed . ANN is The present study constructed three predictive models—the ANN model, the LR model, and the DT model—to predict advanced schistosomiasis prognosis. While each of the predictive models proved effective and had their own advantages, the ANN model outperformed the LR and DT models in terms of AUC and sensitivity. However, to achieve the highest level of prediction accuracy and better assist medical professionals, the three predictive models should be applied after model comparison.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.


In 2012, 12 medical schools were opened in Ethiopia to tackle the significant shortage of doctors. This included Aksum School of Medicine situated in Aksum, a rural town in Northern Ethiopia. The new Innovative Medical Curriculum (NIMC) is a four-year programme designed by the Ethiopian Federal Ministries of Health and Education. The curriculum is designed to train biomedical science graduates to become doctors in 4 years, with a focus on the healthcare needs of rural people living in poverty.This research was conducted at Aksum School of Medicine and included two hospitals . This study focused on medical students during their clinical years across multiple specialities (61 Clerkship 1 students and 13 Clerkship 2 students). We used primarily qualitative research methods supplemented with quantitative measures. There were 3 stages of data collection over a 1 month period, this included qualitative group interviews, direct observation of students in a clinical setting and direct observation of skills sessions followed by a questionnaire on the sessions. We analysed the data by reconstructing the student experience and comparing it with the NIMC.The proposed typical week set out in the NIMC tended to differ from the real clinical experience of these students. Through qualitative group interview and direct observation of teaching, the main theme that was consistent throughout was the lack of doctors with specialist postgraduate training. Clinical need often took priority over education. However, students enjoyed taking early responsibility and gaining practical experience. Through direct observation of skills sessions and short questionnaires, these sessions were highly valuable to the students and they felt confident in carrying out the taught procedures in the future.The combination of poorly resourced hospitals and lack of specialist doctors provides a challenging environment for medical students to learn. However, it is a unique clinical experience that is rarely seen in developed countries and facilitates the acquirement of skills from an early stage. Supervision and specialist input is fundamental in enabling students to learn and this is a key area that was lacking in the students’ clinical experience. Ethiopia is a country with a severe shortage of doctors especially in the rural areas . AccordiAksum is a town in Tigray, Northern Ethiopia with an estimated population of 66,800 people . Aksum UThe four-year curriculum is split into two pre-clinical years and two clinical or clerkship years, known as Clerkship 1 (C1) and Clerkship 2 (C2) . The research included 61 C1 students and 13 C2 students.Qualitative group interviewsDirect observation of students in a clinical settingDirect observation of skills sessions followed by a student questionnaire on the sessionsThere were 3 stages of data collection, which we completed over a 1 month period:We completed Stages 1 and 2 over a 2-week period. Stage 1 consisted of eight structured group interviews, involving 2–4 participants per interview. We conducted the interviews using a convenience sample of available students in the clinical area. We used this method as it allowed the students to decide if they wished to participate, any students who wanted to participate were included in the study. It was not possible to pre-arrange interviews with the students due to network and power outages which sometimes lasted multiple days making communication and organisation of meetings very difficult.We used an interview guide that covered the students’ experience in Ophthalmology, Dermatology, Obstetrics and Gynaecology and Surgery. CM and MT developed the guide after discussion with senior faculty members from Aksum University and review of the NIMC. We asked the students to describe their current clinical experience, including details of their attachment and their average day. We also asked what they felt worked well on their attachment and areas that could be improved. The lead researcher (CM) attended both hospitals  alternating sites over a two-week period to carry out the structured group interviews alongside a General Practitioner from Aksum (MT). CM and MT conducted these interviews together in the morning after locating the medical students in the relevant departments within the hospital. The morning was the time when the highest number of students were present. The lead researcher (CM) collected the data through note taking. The Aksum GP was known to the students which enabled a strong rapport and trust to be built during the interview.We obtained verbal consent prior to interviewing the students and all data was anonymised to maintain confidentiality. We retained a written register of the students who verbally consented. We offered the students the choice of participating and we explained that it would not affect their training in any way if they chose not to participate. We also obtained verbal consent from the consultant in charge of each department prior to direct observation and interviews. All interviews and teaching sessions were conducted in English, which is the same language the students are taught in. In Stage 2, CM observed a selection of students, based on convenience sampling, in the clinical environment. This involved both hospital wards and outpatient clinics. CM made observational notes to compare with the described student experience in the NIMC. We obtained ethical approval from both Aksum University and Queen Mary University of London. All researchers had received training in collecting qualitative data. How useful did you find the teaching? Did you feel the teaching was right for your level? How confident would you be in using the skills you learnt in the session? Was the session well organised? What other aspects of this topic would you like to see included?Two medical students (RB & KS) carried out Stage 3 of data collection. This involved direct observation of skills sessions using a structured observation teaching form, followed by an evaluation of the students’ experience using a cross-sectional survey. We anonymised the survey to maintain the students’ confidentiality. We (RB & KS) collected data by observational note keeping. A debrief was undertaken after each session. The researchers (RB & KS) then compared the notes, collaborated findings and any differences were discussed. Both medical students (RB & KS) were present at all skill sessions. The survey consisted of the following 5 questions :The lead researcher (CM) collated the observational notes from the three researchers who had taken notes  into a written document. This data included qualitative group interviews, direct observation of teaching and direct observation of skills sessions. The lead researcher (CM) developed deductive codes which were derived from the interview guide, these included ‘positive experience’, ‘areas for improvement’ and ‘structure’. CM then developed inductive codes from further reading and analysis of the data. These included ‘supervision’, ‘teaching’, ‘level of responsibility’ and ‘workplace dynamics’. Coding of the data allowed common themes to be identified. The lead researcher (CM) then placed the data into different categories, consisting of headings and subheadings which involved the year and speciality of the medical students. CM then mapped the data to the NIMC. We categorised the data in this way as it allowed conclusions to be drawn and recommendations to be made to the faculty leadership and individual departments. Using the Likert-type scale, researchers RB and KS collated the results from the cross-sectional survey into a simple bar chart. An experienced qualitative researcher (TCB) checked the data analysis and coding framework. This helped to ensure reliability in the data analysis and credibility of the coding. TCB identified any discrepancies in the coding and revisions were made.The main theme that was consistent throughout was the lack of doctors with specialist postgraduate training. Students enjoyed being taught by specialists when available, however, there were different levels of engagement across the different specialities. This was often due to clinical need taking priority over education and specialists having to leave to attend emergencies, particularly in the case of Surgery and Obstetrics. There was often poor access to basic teaching materials such as ophthalmoscopes and specialist textbooks. However, students enjoyed taking early responsibility and gaining practical experience, for example delivering babies and performing minor surgical procedures. The proposed typical week set out in the NIMC for C1 and C2 students  and Ophthalmology (31) at Aksum Referral Hospital. The students were split into two groups and then into further subgroups of approximately 8, each student spent 3 weeks on Ophthalmology and 3 weeks on Dermatology. Aksum Referral Hospital had just recently opened and all clinical exposure was outpatient based. There was one specialist Ophthalmologist and one specialist Dermatologist. An average day would involve teaching by the specialist in the morning, via a PowerPoint presentation that was then followed by a clinic. Groups had to have at least 1 day per week away from the clinical environment due to there being only one specialist in each department and 61 students.From direct observation, the ophthalmology clinic consisted of the ophthalmologist, specialist nurse and medical students. There was one ophthalmoscope in the department used by the specialist and a slit lamp; the students used a torch and goggles to examine the eyes. Jointly the students would take a very brief history from the patient with the nurse. There appeared to be a good relationship between the specialist nurse and the students, allowing the students time to examine the patients. The students would then observe the ophthalmologist who at times gave teaching. The relationship between the students and ophthalmologist appeared strained, primarily due to the clinical work load. From observation, it was clear that the students were being exposed to a wide variety of ocular pathology with advanced clinical signs. During the interviews the students spoke about the lack of hard copy Ophthalmology textbooks in the library and their reliance on a limited Internet connection for resources. They also spoke about their desire to learn how to use equipment such as an ophthalmoscope.The dermatology clinic comprised of the dermatologist and the medical students. From direct observation, approximately 8 students stood around the patient and the students were asked to take a history from the patient, formulate differential diagnoses and consider appropriate investigations. Possible management plans were then discussed with the students. From observation, the dermatologist appeared highly motivated with a passion for teaching. The dermatologist ensured all students were involved in the teaching and reading materials were given prior to the clinic. Much like Ophthalmology, when interviewing the students, they spoke about limited access to textbooks and difficulty accessing online resources. However, they enjoyed seeing a wide variety of dermatological conditions and were very positive in regards to the dermatologist.Thirteen students were on the Emergency Surgery and Lifesaving skills module at St Mary’s District Hospital Aksum. This is a 12-week module made up of 6 weeks of Surgery (6 students) and 6 weeks of Obstetrics and Gynaecology (7 students) and is the final module before they sit their final qualifying exam. The students were split into two groups and rotated after 6 weeks.There was one general surgeon covering the entire hospital and the different surgical subspecialties. The students’ surgical rotation was split into 2 weeks of theatre and 4 weeks of ward work. Theatre involved assisting in major surgery, involving all sub surgical sub-specialities, which included plastics, vascular, orthopaedics and paediatric surgery. They also undertook minor procedures unsupervised, such as draining abscesses. From direct observation on the wards the students would clerk patients, prescribe medications, consent patients for surgery and manage uncomplicated patients. From group interview the students sometimes felt overwhelmed by the responsibility given to them and the number of unwell patients. They also found the working conditions hard, these included no running water in the hospital and lack of basic medical supplies. However, as they were in their final year, they felt this responsibility and experience was important in preparing them for their internship year.The responsibility given to these final year medical students was significant. There was one specialist across the entire department. The students were running their own clinics and admitting, managing, prescribing medications and discharging patients (see Table The skills sessions consisted of 60-min-long tutorials, delivered by two senior clinicians from the obstetrics department, to groups of 15 students. The sessions were conducted in a well-equipped skills lab. The content of the session would be oriented to lecture based teaching that had been delivered by residents in the same week. For example, on a week discussing termination of pregnancy, the skills session was to familiarise students with manual vacuum aspiration. These topics would also be selected based on feedback from previous students who had struggled with this area in their assessments. The initial skills session would act more as an interactive tutorial, establishing the students’ prior knowledge and introducing them to the equipment generally, before being followed by a supervised practice session.These skills sessions were well received by the students who mostly had little or no prior experience of the procedure being taught Fig. . The intMedical student comments were also broadly positive about the session stating that it was “necessary” and should be “continued” while also seeking further opportunity to practice, either in the skills lab or on patients.The combination of poorly resourced hospitals and lack of specialist doctors provides a difficult environment for medical students to learn. A shortage of specialist doctors is a huge problem across Ethiopia, especially in rural areas such as Aksum. Supervision is fundamental in enabling students to learn and progress into safe clinicians  and thisFurthermore, Fig. A common theme that should be discussed is the different levels of engagement from the specialist doctors. Each department had only one specialist which placed enormous strain on these doctors. St Mary’s, a 150 bed district general hospital in Aksum is estimated to serve 1.5 million people. The burden of disease in Ethiopia measured in terms of premature death is estimated at 350 disability adjusted life years lost per 1000 population, which is the highest in Sub-Saharan Africa . The comHigh levels of responsibility and a huge disease burden can be advantageous to a medical student’s education. Exposure to tropical diseases such as Leishmaniasis and patients consistently presenting at extremely advanced stages of disease is a unique experience that is rarely seen in developed countries. This allows Aksum medical students to continue to develop their history and examination skills and formulate diagnoses without becoming dependent on investigations. The Aksum students enjoyed gaining practical experience which involved delivering babies and performing minor surgical operations. A study carried out in Tanzania found that clinicians felt clerkship students should be performing procedures such as a lumbar puncture under minimal supervision . AllowinIt does appear that the curriculum undergoes some degree of tailoring to knowledge gaps identified in assessment, as evidenced by the skills sessions. The fact that these sessions were engaging, interactive and resulted in the students having a high level of confidence in their ability to perform the taught practical skills, are all examples of good educational practice. The development of confidence in practical procedures is hugely important for when these skills are transferred into a clinical setting, especially if the students are performing skills with limited supervision. Students’ positive comments in feedback further illustrates the value of this practice. Further collection of feedback and tailoring of the curriculum to the students’ needs should be encouraged .A limitation of this study is the fact that convenience sampling was used, which may not be representative of the whole cohort of students. Only 13 C2 students were involved in this study due to the remaining C2 students being placed at hospitals outside of Aksum; these hospitals were not accessible by public transport. To ensure the data is fully representative further research could be conducted in these hospitals. Further research could also explore different specialties that were not covered in this study. Another limitation to the data collection is that the interviews were not directly recorded and transcribed due to the lack of available equipment in Ethiopia.Access to basic teaching materials for specialities such as Ophthalmology and Dermatology are very important in the students’ learning and their absence was a common theme highlighted by the students. This was the first time that Aksum Referral Hospital had Dermatology and Ophthalmology teaching clinics for the students as the hospital had recently opened and these departments were the first to become operational. The medical school recruited the patients for the clinic by organising a campaign in the week prior to the clinic. This involved an individual with a ‘megaphone’ driving up and down the main road in Aksum inviting anyone who had any eye or skin problems to come to the hospital. This again demonstrates the creativity of the medical school in organising and delivering the curriculum. As more departments open this will provide invaluable learning opportunities across different specialities for the medical students. It is important that Ophthalmology and Dermatology resources including textbooks are available for the students to help consolidate their learning. Investing in equipment such as ophthalmoscopes and teaching the students how to use them will provide them with important skills that they can apply in hospitals throughout Ethiopia .A recent study looking at how medical students contribute to healthcare in Sub-Saharan Africa showed students are valuable resources in the health facilities . They haClinical medical students at Aksum University have high levels of experience, expertise and responsibility. However, there is a need for more senior supervision across the field to maximise the students’ clinical experience and to optimise patient care. The implementation of the NIMC is an important area that should continue to be reviewed regularly, not only in Aksum but across all medical schools throughout Ethiopia.