The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation. An emnd ASD-2 , the Droster HOW  and KEP1ster HOW , the Xenpus Xqua , QUAKINGpus Xqua ,9, Sam68pus Xqua , Sam likpus Xqua ,12 and Spus Xqua ,14. ThesN terminus of KH (NK or Qua1) and C terminus of KH (Qua2) [C-terminal tail [This family of proteins owes its name and its dual role to the presence of a structural domain for the binding of RNA, the GRP33/SAM68/GLD-1 (GSG) domain of 200 amino acids, flanked by regulatory regions containing motifs for protein–protein interactions and residues that are modified post-translationally . The GSGH (Qua2) . Two proH (Qua2) –18 and tH (Qua2) . Moreovenal tail .2.Sam68 (Src-associated in mitosis 68 kDa), also known as KHDRBS1  is the prototypic member of the STAR family of RNA-binding proteins, which regulate RNA metabolism in response to signaling cascades. Sam68 was the first STAR member to be characterized and was initially described as a cell cycle regulated phosphorylation target of c-Src and cdc2 kinases –21. Morein vitro [in vivo, including beta-actin mRNA and hnRNP A2/B1 mRNA among others [The KH domain of Sam68 allows the binding of certain RNA sequences with high affinity and specificity. Thus, Sam68 was first shown to bind nonspecifically to poly(U) and poly(A) RNA, and specifically to the high-affinity binding sequences UAAA or UUAA in vitro ,23. 3′-Ug others . Other mg others , and it g others . Regardig others .C-term which can be phosphorylated and mediate SH2-dependent interplay with numerous cell signaling components in response to different stimuli [N-methyltransferases is required for RNA export inducing localization to the nuclear compartment [For its role as a docking protein, Sam68 contains 5 proline-rich motifs which represent binding sites for SH3 and WW domain–containing proteins and a tyrosine rich  stimuli ,23. In a stimuli . Besides stimuli ,30. Othe stimuli , and metpartment ,33. Alsopartment .3.C-terminus [Despite its predominantly nuclear localization determined by a nuclear localization signal (NLS) in the terminus , Sam68 wterminus . In thisSam68 has been found to bind the SH3 domains of p85 phosphatidylinositol 3-kinase (PI3K) , phosphoThus, Sam68 acts as a scaffold protein in response to activation of membrane-bound receptors and extracellular signaling pathways, such as the T-cell receptor ,48,50–52C-terminal domain at Tyr440 by Breast cancer kinase (Brk) has been reported to direct Sam68 to nuclear localization and cell cycle progression [Regarding the biological function of Sam68, it has been considered both as a tumor suppressor and a proto-oncogene regulating cell cycle progression and apoptosis through both RNA dependent and RNA-independent mechanisms ,34,64–66gression . Moreovegression ,59,67, cgression ,68–73.Sam68−/− mice have shown normal life span and development [Further insights of Sam68 function and physiologic role have been provided by studying Sam68 haploinsufficiency. Even though most elopment , some inelopment ,76, delaelopment , basal melopment , maintenelopment  or proteelopment . These i4.In addition to the docking role of Sam68 in cytosolic cell signaling, Sam68 has been described to take part in some ribonucleoprotein complexes. Various other RNPs and proteins were found to be directly associated to Sam68 in the nucleus. For example, the binding to hnRNP K was described to cause antagonistic effect to their respective transcriptional activities . Sam68 aIn cancer cells, apart from Sam68 protein showing a general nucleoplasmic distribution, Sam68 has been found to be also concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signaling components and nucleic acids . These nSome cellular stresses, such as UV light, chemical insults or hyper-osmotic condition, as well as heat shock stress can induce accumulation of several RBPs in nuclear stress bodies . This isA number of different cellular structures have been demonstrated to include Sam68 in cytoplasmic localization. For instance, some other stress granules (SG), such as cytoplasmic foci, appear in cells exposed to various environmental stress agents. They are mainly composed of non-functional translation pre-initiation complexes that aggregate after phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) in response to oxidative stress, hypoxia, UV exposure or heat shock . Particu5.Alternative splicing (AS) is a major cause of mRNA variability and protein diversity in eukaryotes. Some sequences of RNA in the splice site or its vicinity interact with proteins that can be subdivided into SR proteins and hnRNPs , recruitDue to the binding of the splicing factor YT521-B to SAF-B to regulate the selection of alternative splice sites, and the evidence of Sam68 binding to YT521-B, Sam68 was suggested, for the first time, to be part of a signal transduction pathway that can influence splice site selection . There iThe molecular mechanism through which Sam68 regulates AS decisions in response to signaling cascades is poorly understood. All Sam68 and Sam68-like mammalian proteins have been implicated in alternative splicing of target pre-mRNAs ,80,100. in vivo, which affects the splice site occupancy of its binding partner U2AF65 as a step to control spliceosome assembly at regulated splice sites [Signaling pathways attributed to growth factors receptor activation, such as ERK1/2 pathway, seem to modulate alternative splicing through Sam68 phosphorylation, promoting some tumoral effects. CD44 encodes a cell surface molecule involved in cell adhesion, proliferation, and migration of cancer cells . Ras-patce sites .In addition to Sam68 participation in CD44 alternative splicing correlating with enhanced malignancy and invasiveness of some tumors, this protein has been related to some other effects promoting tumorigenesis and proliferation through AS regulation. Thus, in response to extracellular stimuli, Sam68 was found to regulate the alternative splicing of the SR protein splicing factor and proto-oncogene SF2/ASF through the nonsense-mediated mRNA decay pathway (AS-NMD). Through SF2/ASF AS regulation, Sam68 has also been shown to play a role in the epithelial-to mesenchymal transition during tumor metastasis . Other rMoreover, Sam68 has also been shown to play a role in the regulation of Bcl-x alternative splicing in cooperation with hnRNP A1 in HEK293 cells . Even thThere is in addition, mounting evidence that indicates a role of Sam68 in the regulation of alternative splicing during cellular differentiation in different tissues, namely in spermatogenesis, neurogenesis and adipogenesis.Sgce [In spermatogenesis, it has been suggested that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells. Sam68 interacts with the phosphorylated form of the RNA polymerase II (RNAPII) and binds to transcriptionally active chromatin in pachytene spermatocytes, where it seems to modulate alternative splicing of mRNA targets in male germ cells, such as murine Sgce ,114.The participation of Sam68 in alternative splicing has been demonstrated also in neurogenesis, where Sam68 seems to regulate the splicing of specific pre-mRNAs, which are important for neural development ,115. MorMore recently, Sam68 has been reported to regulate alternative splicing of the PI3K downstream effector mTOR in adipogenesis. Sam68 deficiency resulted in mTOR intron 5-retention through the introduction of a premature termination codon and the subsequent reduction of protein levels of mTOR . Thus, S6.prostate-specific antigen (PSA) gene, where Sam68 seems to have some effect on AR-regulated transcriptional activity independently of its ARN binding capacity and splicing regulatory properties in LNcaP cells [Sam68 could also have a role in transcriptional regulation and may behave as a competitive inhibitor of positive regulators of transcription, as has been shown by repressing various mammalian and viral promoter constructs. Thus, binding of Sam68 to hnRNP K inhibits the function of hnRNP K in transcriptional activation of a reporter driven by the CT promoter element of the proto-oncogene c-myc . Most ofaP cells . Sam68 maP cells . MoreoveaP cells .In addition to transcriptional and posttranscriptional functions of Sam68, increasing evidence has shown that Sam68 could also play a role in translation . In male−/− germ cells. These miRNAs included miR-29b, which belongs to a family of miRNAs that are often down-regulated in human cancers [Recent studies have also proposed a role for Sam68 in the miRNA pathway during its translocation and accumulation in chromatoid bodies in spermatogenesis . Based o cancers . Thus, i cancers ,125.Most of the evidence regarding the role of Sam68 in RNA export has been based on studies of retroviral mRNA processing . The hum7.Sam68 has been suggested to participate in a variety of biological processes regarding its apparent multifunctionality as a prototypic member of STAR family of proteins. Sam68 structure justifies its docking function in signaling, where SH3 and SH2 interactions, in addition to Sam68 phosphorylation, mediate signal transduction of extracellular stimuli.Sam68 posttranscriptional modifications, its association to other proteins and RNA targets seem to account for both Sam68 function and its intracellular dynamics. Thus, Sam68 shows different biological functions depending on the signaling status of the cell, which may mediate a role of Sam68 in transcriptional processes, posttranscriptional events, such as alternative splicing, regulation of miRNA pathway, translation, or RNA transport. Nevertheless, further studies are needed to understand the specific role of Sam68 integrating the broad variety and complexity of external and internal signals in relation to its participation in multi-molecular complexes and the modulation of key steps of RNA processing. More precisely, a better knowledge of the contextual interaction of many proteins and RNA molecules that may associate with Sam68 is needed. Moreover, further studies investigating the biological effects of Sam68 downregulation or knockout may be useful to clarify the role of Sam68 in the processing of specific RNA targets and how it may affect biological processes. Finally, regarding Sam68 biological functions and its implication in some pathophysiological conditions see , further


Comparison of expression in wildtype, ache and fixesop mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein  Understanding of chemical-induced biological responses and their underlying pathways is indispensable to evaluate the impact of chemicals on organisms. Toxicogenomic analysis is an effective approach to better understand these pathways and their related adverse effects ache) or nAChR (fixesop) activity are available to support functional analyses.In the present study, we examined the toxicogenomic response to azinphos-methyl (APM), an AChE-inhibitor, using zebrafish embryos as a model. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) and is important for the cholinergic neurotransmission. Furthermore, non-cholinergic functions of AChE have been described in neuronal development 50 is 1340fold below the predicted acute toxicity hspb11 in wildtype, ache and fixesop mutant embryos in more detail and study its role during development. For the first time, we demonstrate that developmental hspb11 expression is muscle-activity dependent and is indirectly regulated by intracellular calcium levels. Furthermore, Hspb11 is required for slow muscle myofibril organization in the embryo. These results show that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.First, we compared changes in gene expression patterns provoked by APM with those of two non-AChE inhibiting compounds, 1,4-dimethoxybenzene (DMB), a narcotic (baseline toxic) compound and 2,4-dinitrophenol (DNP), an oxidative decoupling agent. The test compounds were selected to allow the identification of genes specifically responding to compounds inhibiting acetylcholine esterase. Neurotoxicity is a common MoA among compounds exhibiting an excess toxicity (i.e. ratio of predicted baseline toxicity versus measured toxicity). The organophosphate insecticide and known acetylcholine esterase inhibitor APM 10  as exposure concentrations for the microarray study  we selected the modeled LCay study . Exposuray study . It is kFor the microarray analysis we exposed zebrafish embryos for 24 h (26–50 hpf) and 48 h (2–50 hpf). The different exposure durations were chosen in order to identify potential differences with respect to primary or secondary responses. By hierarchical cluster analysis we identified an APM specific cluster of significantly induced genes after the 24 h as well as the 48 h exposure . The obshspb11 , pdlim3b (PDZ and LIM domain 3b), and socs3a  for concentration-dependent quantitative expression analysis. The selection of these genes was based on an initial RT-PCR verification experiments (not shown) and the genes covered different potential gene functions, e.g. stress response (hspb11), immuno response (socs3a) and cytoskelatal assembly (pdlim3b). The expression of these genes was gradually increased with elevated APM concentrations. By means of qPCR an about two-fold (pdlim3b), 8-fold (hspb11) and 6-fold (socs3a) maximum induction after 24 h of exposure was observed  APM exposures whereas the induction of hspb11 (approx. 20-fold) exceeded the levels of expression after 24 h of exposure  did not result in further induction of hspb11 expression, whereas fixesop siblings exposed to APM showed a 9-fold up-regulation  hspb11 gene expression. Thapsigargin induced hspb11 mRNA expression 11-fold and caffeine 23-fold  by calcium channel blockers  23-fold .hspb11, we first analyzed the spatiotemporal hspb11 expression. At 18 hpf hspb11 is expressed in the adaxial cells  resulted in a significant hspb11 repression (hspb11 expression pattern was lost (hspb11 gene expression is muscle activity dependent.In order to determine the function of pioneers . To elucwas lost  indicatiotochord . Interesotochord . To test fashion . Further pattern . To exclt 18 hpf . These apression  and the was lost . This suhspb11 and MO(5′UTR)-hspb11, and a mismatch control morpholino (mmMO-hspb11) that harbors five mismatches compared to the MO(ATG)-hspb11 were used. To avoid interference with nonspecifically induced apoptosis a morpholino directed against p53 was co-injected hspb11 morpholinos are specific to hspb11 mRNA we performed additional control experiments. First, we injected a gfp mRNAs that contains either the morpholino binding site for MO(ATG)-hspb11 or MO(5′UTR)-hspb11 upstream of the GFP coding sequence and we detected a bright GFP fluorescence (gfp mRNA with either MO(ATG)-hspb11 or MO(5′UTR)-hspb11 strongly reduced the GFP fluorescence -rescence . Coinjecrescence . Furtherrescence .hspb11 and MO(5′UTR)-hspb11 morphants showed a ventral body curvature. In MO(ATG)-hspb11 and MO(5′UTR)-hspb11 injected embryos, 82%  and 73%  showed a morphant phenotype, respectively. The MO(5′UTR)-hspb11 phenotype was much stronger and mmMO-hspb11 control injected embryos showed no effects (hspb11 mRNA that is not targeted by MO(ATG)-hspb11 and MO(5′UTR)-hspb11. Coinjection of the hspb11 mRNA with either MO(ATG)-hspb11 or MO(5′UTR)-hspb11 reduced the ventral body curvature phenotypes to 53%  and 51% , respectively.Both MO(ATG)- effects . Additiohspb11, MO(5′UTR)-hspb11 and mmMO-hspb11 injected embryos, we detected a wildtype eng2a expression pattern in muscle pioneers at 18 hpf -t 18 hpf . Furthert 18 hpf . However embryos . There ahspb11, pdlim3b and socs3a) were differently expressed in response to various AChE inhibitors. Pdlim3b, also known as Alp-like, is a structural protein and a member of the PDZ/LIM protein family which are characterized by the presence of both, a PDZ and a LIM domain pdlim7pdlim3b  is expressed in myotomes pdlim3b expression in response to AChE inhibitors might be related to early disturbances of muscle structure and differentiation. The comparison of the responses to different AChE inhibitors and 4-nitrophenol indicated that socs3a was not induced specifically by AChE inhibitors. Socs3a belongs to the suppressor of cytokine signaling (Socs) protein family. It negatively regulates cytokine signaling in order to prevent excessive cytokine signaling that may disturb the normal homeostasis and cellular functions hspb11 was the only significantly induced member of sHSPs in zebrafish embryos exposed to APM. Commonly, the chaperone function, i.e. the support of correct protein folding, is considered as major physiological role of sHSPs and is regarded as the reason for their induction by a variety of stress factors hspb11 is strongly induced after heat shock and transcripts are found throughout the somites and the hearts Hspb11 orthologs seem to exist in nearly all vertebrates except mammals.By a comparative toxicogenomic approach we demonstrated that the AChE inhibitor APM induced specific responses on the gene expression levels in zebrafish embryos. Other non-AChE inhibiting compounds (DMB and DNP) did not affect the same cluster of genes. Gene set enrichment analyses (GSEA) revealed APM affected genes as enriched in a couple of gene sets involved in neuronal/muscle structure and differentiation. The enrichment of genes within these gene sets particularly after 48 h of exposure might indicate subtle morphological changes caused by APM. This is supported by the birefringence analysis which revealed structural alterations in muscular tissue. It is known that the lack of AChE activity in zebrafish results in an increase of muscular activity and can cause a progressive myopathy hspb11 could be mediated via the ACh/nAChR/calcium pathway. In this case, transcriptional up-regulation would primarily be observed in skeletal muscle tissues. An inactivation of nAChR would result in a loss of hspb11 expression and ache mutants should exhibit elevated hspb11 levels as well. Furthermore, it would be likely that compounds, which do not activate nAChR but increase cytosolic calcium levels, induce the expression of hspb11. Indeed, the experimental observations clearly support the anticipated role of nAChR and calcium, i.e. the ache −/− mutant exhibited an elevated hspb11 expression in muscle tissue similar as in embryos exposed to APM. In fixe−/−sop mutants, which do not express a functional nAChR, the hspb11 developmental expression pattern was completely lost and an induction of hspb11 with APM was not possible. This data and the repressed transcription of hspb11 by MS-222 provided evidences that developmental hspb11 expression is muscle activity dependent.ACh is a neurotransmitter which is released by excitation of motoneurons at the neuromuscular junction. Binding of acetylcholine to nAChR depolarizes membranes of adjacent muscle cells and induces an intracellular calcium increase. Calcium acts as a messenger linking excitation events at the membrane with downstream effects, like contraction, ATP production and transcription hspb11 induction. Calcium dependent signal transduction cascades play important roles in the control of skeletal muscle gene expression in mammals and increased calcium levels result in, for instance, suppression of gene expression of myogenin and nAChR ache −/− mutant zebrafish embryos the sub-cellular localization of nAChRs is disturbed ache, fixesop or acc, showed disarrayed muscle fibers hspb11 expression is modulated in ache and fixesop mutants. A continuous elevation of calcium levels and subsequently enhanced calcium-activated protease activity has been shown to cause myofibre degeneration hspb11 gene expression and myofibril organization and Hspb11 may compensate for calcium-induced protein degradation. We demonstrate that down-regulation of Hspb11 has no effect on muscle pioneer development, which is supported by the eng2a expression. Furthermore, morpholino knockdown of Hspb11 alters the distribution of slow muscle myosin. Hence, Hspb11 is important for myofibril organization and/or maintenance. Hspb11 may play a critical role in folding and assembly of various sarcomeric proteins during myofibrillogenesis and it remains to identify Hspb11 client proteins.Modulation of intracellular calcium levels with thapsigargin or caffeine, which most likely affect the whole zebrafish embryo and is not restricted to muscles, resulted in hspb11 can be mediated via specific transcription factors, e.g. Creb, Nfat, Nf-κB, which are important components of intracellular signaling pathways and are regulated by calcium hspb11 transcriptional start site, several putative binding sites of these transcription factors are present . However, the precise mechanism of the hspb11 transcriptional regulation remains to be further investigated.The transcriptional regulation of hspb11 as a possible marker for interference with neuromuscular signaling and/or calcium signaling. Further research need to address how elevated calcium levels and hspb11 expression are linked.In summary, the combination of comparative toxicogenomics and functional analysis has led to the identification of Guidelines on the protection of experimental animals by the Council of Europe, Directive 2010/63/EU, which allows zebrafish embryos to be used up to the moment of independent feeding (approximately 5 days after fertilization). Because embryos used here were no more than 4 days old, no license is required by Council of Europe (1986), Directive 2010/63/EU or the local authority.All zebrafish husbandry and experimental procedures were performed in accordance with the German animal protection standards and were approved by the Government of Saxony, Landesdirektion Leipzig, Germany (Aktenzeichen 75-9185.64). Based on the achesb55, fixesop and smub577 have been described previously and were kept at the Institute of Toxicology and Genetics, Karlsruhe A wildtype zebrafish strain (WiK) was obtained from the Max Planck Institute of Developmental Biology  and cultured at 26°C according to standard procedures 2*2H2O, 123.3 mg/L MgSO4*7H2O, 64.7 mg/L NaHCO3, 5.7 mg/L KCl; according to ISO guideline 15088 or DMSO (thapsigargin)). Exposure concentrations were prepared by dilution of stock solutions. In case of thapsigargin DMSO concentration in exposure and control media was adjusted to 0.01%. Exposure concentrations of APM, DMB and DNP were analyzed by GC-MS by the TZW . After 48 h of exposure 90% , 75%  or 74% , respectively, of the nominal concentrations could be detected.Embryos were exposed from 2–50 hpf, 26–50 hpf, 48–50 hpf and/or 12 hpf (5-somite stage) to 72 hpf in 100 ml crystallization dishes covered with watchmaker glasses with the chemicals. The following chemicals were used: APM , DMB , 2,4-DNP , GAL , propoxur , disulfoton , 4-nitrophenol , thapsigargin , caffeine  and MS-222 . Stock solutions of the chemicals were prepared either in exposure medium . We used the 4×44 K Agilent D. rerio oligo microarrays . The synthesis/labeling of cRNA with Cy3 (Agilent Low RNA Input Linear Amplification Kit) and hybridizations with the microarray slides were performed according to the manufacturer's instructions. Slides were scanned with an Agilent DNA Microarray Scanner.RNA was extracted from 50 embryos for each treatment . Log2-transformed fluorescent intensities were quantile normalized and used to calculate the ratio for each treatment versus the mean fluorescent value of the respective control. Analysis of statistical significance with SAM www.broadinstitute.org/gsea/index.jsp] http://www.ensembl.org/biomart/index.html]. GSEA was performed using the complete dataset whereas 5961 genes, i.e. those for which a human ortholog was identified, could be associated with gene sets from the database. The gene sets of the MSigDB (v3.0) databases C2  and C5 (Gene sets of Gene Ontology terms) were used for the analysis.For the identification of biological functions and pathways associated with the changes in gene expression caused by the APM treatment, Gene Set Enrichment Analyses (GSEA) [Zebrafish embryos were exposed with different APM concentrations for 24 h (26–50 hpf). 30 embryos were used for AChE activity assay and were performed as previously described ™ SYBR with ROX as passive reference (Bioline). qPCR experiments were performed with three independent biological replicates and each single run was performed in three replicate samples. Statistical analysis of qPCR experiments were performed with one-way ANOVA followed by Dunnett's post test using GraphPad Prism 5.0 software . Primer sequences are given in Total RNA was extracted from 50 control, mutant or exposed zebrafish embryos at 50 hpf. 2 µg of total RNA was reversely transcribed with RevertAid™ H Minus Reverse Transcriptase (Fermentas) according to the manufacturer instructions. qPCR was carried out using Step-One-Plus PCR System (Applied Biosystems) and we used the SensiMixhspb11 was amplified with ORF primers (in situ hybridization probes. GFP constructs were amplified with F1-MOATG-gfp/R1-GFP primers for atg-gfp and F2-MOUTR-gfp/R1-GFP primers for utr-gfp (hspb11-F/hspb11-ORF_Rev primers (hspb11-ORF-pCRII vector as template. Products were cloned into pCRII blunt vector (Invitrogen) and subcloned into pCS2P+. We performed whole-mount in situ hybridization (WISH) and immunohistochemistry as described previously The open reading frame (ORF) of  primers , cloned  utr-gfp  using pE primers  and hspbhspb11 (5′- TCGGGCAAAGCATCTTCAGTGGATT- 3′); 0,25 mM–1 mM mmMO-hspb11 (5′-TCcGGgAAAGgATCTTCAcTGcATT-3′) was a 5 base mismatch control for MO(ATG)-hspb11; 0,25 mM–0,5 mM MO(5′UTR)-hspb11 (5′- TTTGCTGTTGAGCTGTTTGGCTTCT- 3′); 0,25 mM–1 mM MO-p53 (5′-GACCTCCTCTCCACTAAACTACGAT-3′). 5′ capped mRNAs were transcribed from linearized vectors using the mMESSAGE mMACHINE SP6 Kit . Injection needles were pulled from borosilicate glass capillary tubes with filament  using a Narishige micropipette puller . Embryos were injected with the Eppendorf FemtoJet  through the chorion into the yolk compartment at the one-cell stage.Morpholinos  were dissolved in water and injected at the following concentrations: 0,25 mM – 1 mM MO(ATG)-Figure S1Acute toxicity of AMP, DMB and DNP in zebrafish embryos. Dose response curves were determined by recording mortality in 2–50 hpf exposed embryos. Non linear regression modeling was performed with SigmaPlot version 11  using the Hill 4 parameter equation (f = y0+a*x∧b/(c∧b+x∧b)).(TIF)Click here for additional data file.Figure S2AChE enzymatic activity in response to various APM concentrations. AChE enzyme activities are expressed as fold change of controls (based on specific activity). .(TIF)Click here for additional data file.Figure S3Heat map of a hierarchical cluster analysis of genes significantly differentially expressed in zebrafish embryos. Embryos were exposed for 24 h (26–50 hpf) or 48 h (2–50 hpf) to azinphos-methyl (APM), 1,4-dimethoxybenzene (DMB) or 1,2-dinitrophenol (DNP). Genes with significantly altered expression were identified using SAM (TM4 software suite). Treatments were performed by two separate series of experiments, one for the 24 h exposure and one for the 48 h exposure. C24 and C48 are control samples that refer to these separate experiments. Data represent the log2-ratio of each treatment or control to the average of control levels (either C24 or C48). Numbers 1–4 indicate different independent biological replicates. The cluster of genes, which are specifically regulated by APM, is marked by orange lines.(TIF)Click here for additional data file.Figure S4qPCR analysis of hspb11, pdlim3b and socs3a expression in zebrafish embryos. Embryos were exposed from 26–50 hpf to different AChE inhibitors. (A) Propoxur, (B) disulfoton, and (C) galantamine. (D) 4-nitrophenol served as unspecific (non-acetylcholinesterase inhibiting) control. Concentrations are given in µM if not differently labeled. Bars represent the relative gene expression as fold change of the respective untreated control as mean ± standard deviation of three replicate exposures. Control  =  ctrl. * P<0.05.(TIF)Click here for additional data file.Figure S5Muscle pioneer specific expression analysis of hspb11 and eng2a expression. (A) Transcripts of hspb11 mRNA are localized in muscle pioneers of wildtype or smu heterozygous embryos (+/?). (B) smu−/− embryos lack muscle pioneers and hspb11 expression is absent. (C) Homozygous fixe−/−sop mutants show wildtype eng2a expression in muscle pioneers and at the midbrain-hindbrain boundary (mhb). (D and E) Muscle pioneer development in MO(UTR)-hspb11 and mmMO-hspb11 injected embryos was not effected, confirmed by eng2a expression.(TIF)Click here for additional data file.Figure S6MO(ATG)-hspb11 and MO(5′UTR)-hspb11 morpholinos specifically target their hspb11 binding sites. Embryos injected with gfp mRNA that contains the MO binding site expresses GFP. Co-injection of MO(ATG)-hspb11 or MO(5′UTR)-hspb11 with the gfp mRNA abolishes GFP expression, whereas with the mmMO-hspb11 morpholino does not reduce GFP expression. atg-gfp mRNA contains MO(ATG)-hspb11 binding site. utr-gfp mRNA contains MO(UTR)-hspb11 binding site.(TIF)Click here for additional data file.Table S1Gene sets exhibiting greatest overlap to differentially expressed genes in APM treated zebrafish embryos. The analysis was performed by the software GSEA  for all genes of which a human ortholog could be identified. Gene expression patterns of APM-treatments were compared to all other treatments . Only gene sets with a p-value<0.01 are shown.(XLS)Click here for additional data file.Table S2List of primers used in this study.(XLS)Click here for additional data file.


Differences between urban and rural settings can be seen as a very important example of gaps between groups in a population. The aim of this paper is to compare an urban and a rural area regarding child growth during the first two years of life as related to mother’s use of antenatal care (ANC), breastfeeding and reported symptoms of illness.The studies were conducted in two Health and Demographic Surveillance Sites, one rural and one urban in Hanoi, Vietnam.We found that children in the urban area grow faster than those in the rural area. There were statistical associations between growth and the education of the mother as well as household resources. There were positive correlations between the number of ANC visits and child growth. We also saw a positive association between growth and early initiation (first hour of life) of breastfeeding but the reported duration of exclusive breastfeeding was not statistically significantly related to growth. Reporting symptoms of illness was negatively correlated to growth, i.e. morbidity is hampering growth.All predictors of growth discussed in this article, ANC, breastfeeding and illness, are associated with social and economic conditions. To improve and maintain good conditions for child growth it is important to strengthen education of mothers and household resources particularly in the rural areas. Globalization and urbanization means obvious risks for increasing gaps not least between urban and rural areas. Improvement of the quality of programs for antenatal care, breastfeeding and integrated management of childhood illness are also needed in Vietnam. Birth weight and child growth are important predictors for the future health of a person and for the public health of a population. Abnormal growth in utero and during infancy can have immediate negative effects but may also lead to adverse health effects later in life e.g. as stated by the Barker hypothesis . SuboptiThe conceptual framework for this paper Figure  is basedbasic factors possibly indirectly influencing child growth are the general social, cultural, economic and political contexts. These are fundamental for establishing human, social, financial, physical and natural capital, all determinants for living conditions and distinctively different between urban and rural contexts.The most important Underlying factors are primarily characteristics of persons and households. For persons, the traditional demographic factors like the age of the mother and, to some extent, of the father are of interest. Education and occupation of parents [ parents ,9 partic parents . At the  parents . SatisfaImmediate factors are directly influencing child growth at the individual level. The birth weight of a child is the result of intrauterine growth as well as the nutritional conditions and gestational age at birth. It is also reflecting the mother’s health, nutritional status and behavior during pregnancy including e.g. use of antenatal care and smoking. Birth weight is the starting point for the infant’s further growth. After birth, nutritional practices, primarily breastfeeding, and child illness are likely to influence growth.This article will study three immediate factors, antenatal care (ANC), breastfeeding and child illness. Education about nutrition and counseling provided in ANC during pregnancy can help to reduce the risk of anemia, increase gestational weight gain and improve birth weight . The couWeight and length at birth have been reported to be important determinants of infant growth and future nutritional status. Low Birth Weight (LBW) infants have shown difficulties to achieve the standard weight or length at 12 months . Later iExclusive breastfeeding of infants has been shown to give faster growth, regarding both weight and height, during the first 6 months of life compared to weaned and partially breastfed infants . BreastfThe main aim for this study is to describe the weight and length growth during a two-year follow-up of children in one urban and one rural cohort in Hanoi, Vietnam and the importance for growth of the three above mentioned immediate factors. The article is also an extension of a previously published article describing growth during the first year of life , where dAll studies were conducted in one rural and one urban area of Hanoi, in northern Vietnam. Dongda is an old, central district of Hanoi. The population is about 352,000 persons. The socio-economic characteristics are typical for the urban areas of big Vietnamese cities. Bavi is a rural district of Hanoi with 250,000 persons, about 60 km from the city center with farming as the main occupation. Two Health and Demographic Surveillance Sites (HDSS) were established to provide information for planning and policy making. The urban HDSS, DodaLab, was started 2007 in three communes with 11,000 households and 38,000 inhabitants . The roust March, 2009 to 30th June, 2010, in DodaLab and FilaBavi, were invited to enroll their children in the study. These mothers had taken part in a previous study of antenatal and delivery care [All mothers with children born alive from 1ery care . About 9Birth weight information was provided in the first interview when the mothers reported the measurement made at the hospital or commune health centers immediately after birth. For less than 1% of the children birth weight information was not obtained. These children were still used in the postnatal growth analysis.Totally 1,466 children, 540 in DodaLab and 926 in FilaBavi, were scheduled for monthly measurements of weight and length each month during the first year and every three months during the second year of life. At the same time, mothers were interviewed about breastfeeding and symptoms of illness. The total number of interviews with weight and length measurements was 17,148, 73% of the total 23,456 scheduled. Data about antenatal care and delivery were obtained from the antenatal care study conducted earlier in the two sites [Data about economy and education of mothers were obtained from the household surveys conducted 2009 at the two sites. To describe the household economical level we used the reported yearly household income and the household assets available (according to a specified list) as indicators of economic resources. For the mothers social position we included age, education and occupation. Information about fathers was incomplete since it was not routinely registered .. We could not use birth weight information from birth certificate of hospitals or commune health centers. These are kept by the commune administrations in different offices and it was not possible for the field workers to examine all birth certificates. However, we compared the birth weight information from the mothers with the birth certificates in a sample of more than 10% of the infants. The results were found to match very well. The means from the two sources differed about 10 grams. In another sample, information from the hospital records and health centers was used to evaluate the quality of the mothers birth weight information. Again there was no large difference between the different sources of birth weight information.Mothers reported the information they received in hospitals or community health centers immediately after deliveryThe date of the last menstruation as reported by the mothers was intended to be used for the estimation of the gestational age at birth. This information must however be considered as fairly imprecise since Vietnamese women in general do not remember this date well. It was used nevertheless as a crude proxy for gestational age.These are the absolute measurements for a child at any specific child age.Stunting, underweight and wasting in this article are defined as low length-for-age (below mean minus two standard deviations), weight-for-age (below mean minus two standard deviations) and weight-for-length (below mean minus two standard deviations) according to WHO standards [tandards .Standardized equipment for measuring the child recommended from Hanoi Medical University was used. A number of commune health center staff members in DodaLab were trained specifically to measure children. In FilaBavi, a number of the permanent interviewers were trained to measure children. The principle of measurement was that the same field worker should assess a child at each visit using the same equipment. Weight was measured to the nearest 10 gram with the child in light clothes using a Vietnamese mechanical infant scale.Length was measured to the nearest centimeter in horizontal position using a length board. Two person worked together in order to have valid and reliable measurements .Age is defined as the date of interview and measurement minus the date of birth.The education of the mother was used as one indicator of the socio-economic situation. Three levels were used: primary school or less, secondary school and higher than secondary school.To describe the household economy we investigated different forms of wealth and assets indices and the reported household income. For this study we actually used the number of assets in the following list: bicycle, motorbike, car, telephone, radio, television, video player, sewing machine computer, refrigerator and buffalo.Three variables aimed to describe the use of ANC: (i) The number of antenatal care visits during pregnancy, (ii) ANC reported to contain counseling and advice and (iii) First antenatal visit during first trimester. These variables were found to have the strongest simple correlations to child growth.Three variables were used to describe breastfeeding during infancy:Early initiation of breastfeeding defined as breastfeeding starting during the first hour after birth[ter birth.“Exclusive breastfeeding: The infant receives breast milk, from the breast of the mother or a wet nurse or expressed, with the only additional oral intake of oral rehydration solutions (ORS) or medication including vitamins or minerals”[.minerals”.“Any breastfeeding: The infant receives breast milk, from the breast of the mother or a wet nurse or expressed, with or without additional oral foods. This category includes the WHO definitions of exclusive breastfeeding as well as non-exclusive breastfeeding, that is predominant breastfeeding and complementary feeding according to the WHO definitions”[.initions”.The indicator reported illness symptoms  at the child level was defined as the number of interviews with one or more reported symptoms divided by number of interviews.Standard simple and multiple regression models where used for the analysis of birth weight and associations with time and other factors. Mean growth curves were fitted using fractional polynomial (FP) linear regression models .For the analysis of association between attained weight and various factors we used the relative residuals from the predicted curve. The relative residuals were defined as the deviations, positive or negative, of measurements from the FP predicted curve divided by the predicted value.Two approaches were used to study associations. This main analysis used the means of residuals for individuals in a collapsed dataset (one record per child). The results were compared to those from repeated measurement analyses using linear mixed regression models applied to all residuals. Results were given as correlations, crude or partial (adjusted). The latter correspond to partial regression coefficients in multiple linear regression but are standardized to have values between minus one and plus one.All statistical analysis used commands available in the software STATA version 11.Approval for the project was given by the Scientific and Ethical Committee of Hanoi Medical University, Hanoi Health Bureau and Dong Da district authorities. The data collection in the two sites was approved by the Ministry of Health. The participants were informed about the purpose of the studies and their right to decline participation or withdraw. Consent was obtained from all the study participants. Data was analyzed and presented anonymously. All results have been duly disseminated to communities and authorities.Table Figures Table The strongest positive correlations between weight and length and the ANC variables were with the number of ANC visits and a positive answer to the question if the mother was given advice and counseling which was reported by 44% of all women Table . The parEarly initiation of breastfeeding and the duration of exclusive breastfeeding were positively correlated to weight growth. For length the latter correlation was small and insignificant Table . After aIncreasing numbers of interviews with reported symptoms was statistically significantly associated with reduced weight growth Table . IllnessOne finding of this paper is that the differences in growth between children in the studied Vietnamese urban and rural areas previously reported during the first year of life , remain Much evidence supports that breastfeeding provides good nutrition for children as it reduces the severity of e.g. respiratory and gastrointestinal infections in children -34. ChilPoor nutrition has been seen as the most important risk for poor growth  and diffAccording to WHO and UNICEF, the prevalence of stunting among children under five in Asia was 27% in 2011 . In VietThe Vietnamese government has noted stunting as a public health problem. A plan to reduce the incidence of stunting to 23% by 2020 and underweight to 12.5% by the same year in children under five was launched in 2012 . A contrWe observed some statistically significant simple (unadjusted) correlations between growth and ANC use. In, regression models where both underlying and immediate variables are included though, the education and assets variables turn out to be more important than the ANC indicators. The partial correlations between growth and ANC use, adjusting for education level of the mother and the household resources, are small and not statistically significant. The simple explanation can be that the ANC variables are themselves associated with education and economy. Socially and economically resourceful mothers possibly use, and benefit, more from ANC than others.Child illness stands out among the studied immediate factors. A fairly strong association between growth of children and reported illness symptoms was found, particularly in the rural area.Symptoms of illness were most commonly reported in the rural area. The risk for illness reporting at a particular visit was 0.40 compared to about 0.20 in the urban. The high incidence of illness could be important to explain the slow growth of rural infants. The possible negative influence on weight growth was also stronger in the rural area.The most common causes of illness in children under five and especially during infancy are diarrhea and acute respiratory infection. This has been observed in several studies -44. DiarA possible intervention may be that the Vietnamese Ministry of Health work to enhance the quality of the Integrated Management of Childhood Illness program aimed to help lay community health workers assess and treat sick children. Improvements of health staff skills as well as the health system itself seem also to be needed particularly in the rural area.Early initiation of breastfeeding, within the first hour of life, can be a positive factor for growth and has been claimed to protect newborn from acquiring infections . The maiExclusively breastfed infants have been seen to grow faster during the first 6 months of life compared to groups of weaned and partially breastfed children ,47. ExclDecisions taken by mothers about use of ANC, breastfeeding, nutrition and child health care utilization are related to the educational level of mothers and the household resources. Likewise, the risks for illness are associated with education and economy. In the present study about 19% of the variation in weight growth and 12% for length growth are explained by the variation in education of the mother and household wealth. Adding the ANC indicators, early initiation of breastfeeding and illness symptoms as independent variables in the regression model increased the determination coefficient by about two percent units. However, more than 80% of the total growth variation is left unexplained.The associations between growth and the immediate factors in the conceptual framework for this study are to large extents reflections of the associations between growth and the underlying socio-economic factors. Thus these turn out to be the most important to explain growth variation. Interventions aimed at improved provision and use of antenatal care, promoting good breastfeeding practices and preventing child morbidity will have their effects. These risk to be limited as long as the underlying social and economic conditions are not strong and equitable. In the end the basic factors with its political, social, economic, cultural and other contexts will determine the conditions for child growth.A child with a complete weight and length measurement set has been measured 12 times during the first year of life and four times during the second. As can be seen in Table The dropout rates were clearly different between the urban and the rural area and to some extent between boys and girls in the rural area. The first mentioned difference could be expected since many mothers in the urban area work outside the household and visits could be more difficult to arrange.The dropouts cannot be expected to be random but systematic and possibly creating bias. To investigate we compared the growth curves fitted using all available observations with other curves using only the data from children with complete sets. The former curves came out systematically lower than the latter but the differences were small, 30 to 50 gram after two years of age, largest in DodaLab. Another approach used to investigate possible bias was to correlate the means of relative residuals for the first half-year to the number of visits. Very weak positive correlations were found. Both approaches thus indicate that the risk to dropout is higher for children with slower growth. Correlations between birth weight and number of measurements however, did not support that conclusion.The study is to a large extent dependent on information reported by the mothers, e.g. birth weight. . Possible sources of errors are both how the measured birth weight was reported to the mother and the recall of mothers. It could be suspected that the hospital or health center staff tends to report a higher weight to please the mother. The proportion of low birth weight newborn  is lower than expected. On the other hand there is no heaping e.g. at 2,500 gram in the birth weight distribution. The precision of birth weight reporting is 100 gram. Systematically and incorrectly rounding upwards would create a bias of that size. Recall biases are likely to be small as it is considered important for a mother to remember the birth weight of a child in the Vietnamese tradition. Another important piece of information provided by the mothers was the date of the last menstruation before pregnancy. This is needed to calculate the gestational age at birth. The adequacy of this information turned out to be somewhat problematic. The information is missing for quite many women and it seems that by using the available information we underestimate the gestational age. Too many women appear to be classified as giving birth prematurely. However, using or not using the gestational age as an explanatory variable for growth does influence the results only marginally.Household assets have been used as an indicator of household economical resources. The available alternatives could be the reported household incomes or reported expenditures. A third alternative could be the “Wealth index” which is a wider combination of housing characteristics and assets ,50. All Globalization and urbanization means obvious risks for increasing social and economic gaps between urban and rural areas. The predictors of growth discussed studied in this article, antenatal care, breastfeeding and reported illness, are associated to social and economic conditions as underlying factors. In order to improve and maintain good conditions for child growth it is important to strengthen the education of mothers and the household resources, particularly in the rural areas. The high prevalence of stunting observed underscores this. In addition, improvement of the quality of programs for antenatal care, postnatal care, breastfeeding and integrated management of childhood illness are needed in Vietnam.The authors declare that our findings have not been influenced by our personal or financial relationship with other person or other organization.HNT led and supervised the fieldwork and data management. She also drafted and completed this paper. BE assisted in the research design as well as in the statistical analyses, interpretation of results and revision of the manuscript. HA, LNT, MP, TTK and GB were involved in the design of the study, supervised the study and revised the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/13/149/prepub


We report a baby with an unusual true umbilical cord cyst detected at 12 weeks gestation which as the pregnancy progressed became increasingly difficult to distinguish from a pseudocyst of the umbilical cord. Concern of the possibility of cord compression/cord accident led to an elective caesarean section being performed at 35+ week’s gestation with delivery of a healthy female infant weighing 2170g. At birth the cyst ruptured and the resultant thickened elongated cord was clamped accordingly. After the cord clamp fell off at 5 days post delivery an elongated umbilical stump was left behind from which a stream of urine surprisingly jetted out from the umbilicus each time the baby cried. A patent urachus was confirmed on ultrasound and the umbilical jet of urine resolved at 4 weeks post delivery after treatment of an Escherichia coli urinary tract infection. At 11 weeks post delivery a laparoscopic excision of the urachus was successfully performed. The baby, now 18 months of age, continues to thrive without incident. Just over 100 cases in the neonatal period have been documented after the first report in the 16th Century2.A persistent urachus is a result of failure of involution at 10–12 weeks gestation of the allantois which communicates from the dome of the bladder to the umbilicus. Embryologically, the allantois is an endodermal diverticulum which becomes the urogenital sinus with the cranial portion developing as the bladder. Persistence of the urachus may be partial resulting in an urachal cyst, diverticulum or sinus, or it may be completely patent allowing communication with the bladderA 36 year old Caucasian woman with a 4 year history of infertility presented at 9 weeks gestation for antenatal care following a successful single embryo transfer after intra-cytoplasmic sperm injection (ICSI). First Trimester screening at 12 weeks gestation demonstrated a trisomy 21 risk of 1:2760 and a trisomy 18 risk of 1:4060. However, detailed ultrasonic examination of the fetus revealed a hypoechoic area on the anterior abdominal wall which was thought possibly to be fluid distending the urethra or an extra-abdominal mass such as an omphalocoele. At 16 weeks gestation a repeat ultrasound examination identified an umbilical cyst, of dimensions 1.7 cm x 1.6 cm x 1.8 cm with vessels coursing around it with no gut present within and a normal anterior abdominal and bladder wall. The uncertainty of the type of cyst and possible reduction in the fetal dimensions required trisomy 18 to be excluded and an amniocentesis revealed a normal XX karyotype. The umbilical cyst increased progressively with advancing gestation, increasing in dimension, to 4.9 x 4.5 x 4.7 cm at 35 weeks gestation See.On the ultrasound, the cord developed homogeneous echogenicity, which was thought to be due to increased Wharton’s jelly, seen more commonly with pseudocysts of the cord; or due to oedema which was a more sinister sign of possible cord compression/cord accident. In view of the latter, after a course of preoperative maternal cortico-steroids to enhance fetal lung maturity, the patient underwent an elective caesarean section under a combined epidural/spinal anaesthetic block, with delivery of a healthy female infant. Unfortunately, during the process of the delivery the cord cyst ruptured making accurate diagnosis impossible. However the cord was thickened with Wharton’s jelly and was elongated with an umbilical stump with overlying skin up to 2 cm in length. The cord was divided in the standard fashion and a normal placenta was removed.th day post-delivery, the cord clamp fell off and each time the baby cried a stream of urine jetted out from the umbilicus 4. True cysts are less common, ranging in size between 4 and 60 mm, are located towards the anterior abdominal wall of the fetus, split the umbilical vessels, contain fluid and if present in the second and third trimesters, are associated with an omphalocoele or patent urachus5.The prevalence of umbilical cord cysts detected by ultrasound in the first trimester is of the order of 0.4–3.4%, with a patent urachus being a rare anomaly with an incidence of 1–2.5 per 100,0005. Post-delivery the differential diagnosis of an umbilical cord cyst is between an umbilical pseudo-cyst and an allantoic cyst with a patent urachus which can be distinguished by ultrasound. Ultrasound shows the channel communicating from the umbilicus to the bladder. Bladder outlet obstruction can be excluded by a micturating cysto-urethrogram preoperatively or by an intra-operative cystoscopy. The contemporary treatment of a patent urachus is by laparoscopic surgical excision6 whilst other urinary tract abnormalities  are investigated and treated appropriately7. Post-operatively these babies are given antibiotic prophylaxis until a normal ultrasonogram of the urinary tract is demonstrated.Pseudo cysts are more common, are usually smaller, and may be located anywhere along the length of the umbilical cord. The umbilical vessels are pushed to one side as a result of local oedema and liquefaction of Wharton’s jelly and may be associated with omphalocoele, hydrops and aneuploidy (trisomy 18)Antenatal ultrasound detection of an umbilical cyst, particularly if located close to the anterior abdominal wall of the fetus should stimulate a search for a patent urachus post-natally. Laparoscopic surgical correction is the treatment of choice for a baby with a patent urachus. Reduced post operative morbidity and length of hospital stay are important advantages in comparison to the open surgical approach with both methods being equally successful in closing a patent urachus.Written informed consent for publication of clinical details and clinical images was obtained from the mother of the neonate. This is an excellent case report with appropriate referencing and balanced discussion. I would have liked to see in the conclusion a comment about the lack of evidence on antenatal scanning of the patent urachus. For the reader, this would further highlight the need for the postnatal assessment of a baby with the antenatal diagnosis of cord/umbilical cyst(s), even if the antenatal scan suggested normal bladder anatomy.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I think this case presentation is well written and appropriately referenced. I have one question for the authors:In retrospect, do the authors believe that early delivery at 35+ weeks gestation by LSCS would have been justifiable if they had have made the diagnosis antenatally? And further to this, is there an increase in perinatal mortality if these babies with a patent urachus are allowed to delivery at term? I think the Conclusion should also be changed to read: 'Antenatal ultrasound detection of an umbilical cyst, particularly if located close to the anterior abdominal wall of the fetus should alert the clinician to the possibility of an underlying patent urachus. Once diagnosed post delivery, the laparoscopic approach is the preferred surgical route.'I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


HWP1 gene in non-Candida albicans Candida species and the differential expression of HWP1 following treatment with Piper betle and Brucea javanica aqueous extracts. All candidal suspensions were standardized to 1 × 106 cells/mL. The suspension was incubated overnight at 37 °C . Candidal cells were treated with each respective extract at 1, 3, and 6 mg/mL for 24 h. The total RNA was extracted and reverse transcription-polymerase chain reaction was carried out with a specific primer of HWP1. HWP1 mRNAs were only detected in C. albicans, C. parapsilosis, and C. tropicalis. Exposing the cells to the aqueous extracts has affected the expression of HWP1 transcripts. C. albicans, C. parapsilosis, and C. tropicalis have demonstrated different intensity of mRNA. Compared to P. betle, B. javanica demonstrated a higher suppression on the transcript levels of HWP1 in all samples. HWP1 was not detected in C. albicans following the treatment of B. javanica at 1 mg/mL. In contrast, C. parapsilosis and C. tropicalis were shown to have HWP1 regulation. However, the expression levels were reduced upon the addition of higher concentration of B. javanica extract. P. betle and B. javanica have potential to be developed as oral health product.The study aimed to identify the  Candida is a genus of yeast-like fungi that are commonly part of the normal flora of the mouth, skin, intestinal tract, and vagina. Areas of recovery of Candida species in the oral cavity include the dentition, tongue, cheeks, and palatal mucosa, as well as from restorative materials and prostheses [Candida cell is about 4–6 μm. Approximately, around 196–200 species of Candida have been identified so far [Candida species, Candida albicans is most frequently isolated from patients with candidiasis. C. albicans has been reported to have the ability to grow as yeast, hyphae, and pseudohyphae [-Candida albicans Candida (NCAC) species such as Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis being associated with oral candidiasis [ostheses . Morpholdohyphae . Howeverdidiasis .Candida species allows the transition from harmless to a pathogenic microorganism and is responsible for a wide range of systemic and superficial infections in the immunocompromised hosts [The opportunistic characteristic of ed hosts . A numbeHWP1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo [HWP1 functions as adhesins in the cell wall, promoting the attachment of candidal cells to host mucosal surface. Although it has been described as hypha-specific adhesins [HWP1 transcript may arise from pseudohyphal growth forms [Candidal cell wall is a dynamic structure and represents the prime site to interact with oral epithelial cells.  in vivo . The HWPadhesins , recent th forms . Brucea javanica (L.) Merr is a member of the family Simaroubaceae. The seeds of this plant have been used in traditional medicine of Indonesia and China [Piper betle L. (Piperaceae). The P. betle leaves have long been used in the Indian local system of medicine for its antioxidant and antimicrobial properties [P. betle [Malaysia is well known for its plant biodiversity. Some of them are utilized for medicinal purposes. nd China , 11. It nd China  and usednd China . Anotheroperties , 15. It operties , 17. DueP. betle . HWP1 in the NCAC species. HWP1 of C. albicans has been extensively studied, reflecting the growing concern over its role in various stages of infections. Information on the expression levels of HWP1 transcripts that encode adhesins amongst the NCAC species, however, is scarce. The hyphal formation between Candida species has the propensity to express distinctive pattern of HWP1. Therefore, this study aimed to identify the expression of HWP1 mRNA transcript in NCAC species and to examine the regulation of HWP1 following treatment with B. javanica and P. betle aqueous extracts.In the present study, we focused on the association of Piper betle and the seeds of Brucea javanica were collected from local areas in Brickfields and Sekinchan, Malaysia. Crude aqueous extracts of the plants were prepared [Fresh leaves of prepared . Each spCandida albicans (ATCC 14053), Candida dubliniensis (ATCC MYA-2975), Candida glabrata (ATCC 90030), Candida krusei (ATCC 14243), Candida lusitaniae (ATCC 64125), Candida parapsilosis (ATCC 22019),d and Candida tropicalis (ATCC 13803), were purchased from the American Type Culture Collection (ATCC), USA. The candidal cells were revived in 10 mL Yeast Peptone Dextrose (YPD) broth and the turbidity of the suspension was adjusted spectrophotometrically to an optical density (OD550 nm) of 0.144 which is equivalent to 1 × 106 cells/mL. This standard suspension was used throughout the experiment.Seven candidal strains, 6 cells/mL) of each strain was incubated overnight at 37°C  to allow the propagation of cells. The cultures were acclimatized for 1 h, followed by the addition of sub-MICs of 1, 3, and 6 mg/mL. The MICs of Piper betle and B. javanica were determined between 25 to 12.5 mg/mL [The standardised suspension  to amplify the sequences that encode for HWP1 in Candida species. ACT1 primer was used as a positive control to detect Candida species carrying HWP1 gene. A pair of primer specifically designed for Annealing temperature is a key factor in performing PCR amplification for reaction specificity. The optimization was carried out with the Mastercycler gradient. A gradient of 60°C ± 5°C was programmed and the temperature distribution in the individual columns of the block was set at 55°C, 57°C, 59°C, 61°C, 63°C, and 65°C.Freshly prepared candidal suspensions were inoculated into 10 mL YPD broth and incubated overnight at 37°C in a rotary shaking incubator. 3 mL of the respective cell suspension was centrifuged at 2,000 ×g for 10 min in a 4°C refrigerated centrifuge and the supernatant was discarded. The pellet formed was washed with sterile PBS, pH 7.2. Following that, total RNA was extracted using an easy-RED BYF Total RNA Extraction kit (Intron Biotechnology Inc.) according the manufacturer's instruction. μL of prelysis buffer was added and re-suspended thoroughly. A 750 μL of easy-RED solution was added, vigorously mixed for 15 s, and left at room temperature for 5 min. Following the addition of 200 μL of chloroform, the samples were vigorously mixed for 15 s and left at room temperature for 5 min. Next, 250 The samples were centrifuged at 8,000 ×g (4°C)  for 15 min, and the colourless aqueous phase formed was transferred to a new microcentrifuge tube. An equal volume of isopropanol (2-propanol) was added, mixed by inverting the tube for 6-7 times, and left at room temperature for 10 min. The suspension was recentrifuged at 8,000 ×g (4°C) for 10 min, and the supernatant was discarded without disturbing the pellet. μL of RNase-free water and stored at −80°C.One millilitre of 70% ethanol was added and mixed by inverting the tube several times. The mixture was centrifuged at 8,000 ×g (4°C) for 5 min. The supernatant was discarded and the RNA pellet was left to dry. The pellet was dissolved in 25 The RNA samples were analyzed for its integrity using the Agilent 2100 Bioanalyzer and all samples were considered to have high quality RNA by referring to the RIN number . μL of Prime RT-PCR Premix 2X (GENET BIO) which contained HS Prime Taq DNA polymerase, Prime MMLV reverse transcriptase, reaction buffer, 0.1 mM dNTPs mixture, RNase inhibitor, protein stabilizer, and enhancers for cDNA synthesis. RNase-free water was added up to a total reaction volume of 20 μL.Fifty ng/mL of the RNA template and 1 mL of each primer were mixed in 10 HS Prime Taq DNA polymerase. The samples were subjected to 30 cycles of denaturation (94°C), annealing (56°C to 64°C), and extension (72°C), each for 30 s. Lastly, the final extension was at 72°C for 5 min. Reverse transcription was carried out at 42°C for 30 min to synthesis the cDNA, followed by denaturation at 94°C for 10 min to deactivate the reverse transcriptase and activate the μL was separated by electrophoresis in 1.5% (w/v) of agarose gel and stained with ethidium bromide. A Tris-borate-EDTA (TBE) was used as a running buffer and 100 bp DNA ladder (BIO-RAD) was used as a molecular weight marker. The expression of the gene was visualized by ultraviolet (UV) illumination . The amplicon of 6 HWP1 transcript was investigated in seven Candida species cultured in YPD broth for 24 h. mRNAs from all candidal spp. were loaded accordingly and results of gel electrophoresis after RT-PCR are shown in ACT1 was used as the positive control, it was observed that HWP1 mRNAs were only expressed in C. albicans, C. parapsilosis, and C. tropicalis after three different independent experiments. No HWP1 transcripts were detected in C. dubliniensis, C. glabrata, C. krusei, and C. lusitaniae  [C. albicans but there is lack of information of its presence and expression in NCAC species [HWP1 forms covalent attachments to proteins on human buccal epithelial cells in host tissue [HWP1 is also required as first cell surface protein in vivo for biofilm formation [Piper betle and Brucea javanica—specifically on the regulation of HWP1. These two plant extracts have been reported to exhibit antifungal effect on seven oral Candida species [ Most microorganisms including otein 1) . It has  species . Unlike t tissue . HWP1 isormation . Further species . HWP1 mRNA transcripts were positively regulated in C. albicans, C. parapsilosis, and C. tropicalis. The expression of HWP1 in these three Candida species indicates the ability of the cells to produce adhesins that are covalently linked to the cell wall glucan through a remnant of its glycosylphosphatidylinositol (GPI) anchor, leading to cell adhesion and biofilms formation [HWP1 can be detected by the primers  and exists as a multilayer structure of the yeast form [C. glabrata does not rely on the production of HWP1 to establish an infection.ast form . TherefoHWP1 transcripts was drastically reduced following treatment with the extracts. The addition of extracts probably has created environmental stress and demonstrated fungistatic effect on the cells. The uptake of ions and nutrients which depend on the integrity of the cell wall may be restricted. Several regulatory elements as well as the transcription of respective genes may be deactivated while waiting for the environment to be adequate for growth. This could lessen the chance for the cells to propagate and thus inhibits the hyphal formation. This could possibly explain that the transcription of HWP1 was suppressed. The low expression of HWP1 indicates that the level of adhesins was affected by the extracts. The lack of HWP1 being produced will distort the virulence trait of the candidal cells.  Findings showed that the expression of HWP1 transcript may indicate the production of adhesins in C. albicans, C. parapsilosis, and C. tropicalis. The transcript levels were affected following treatments with P. betle and B. javanica extracts, suggesting that the level of adhesins being produced by the candidal cells may be lacking. Subsequently, the integrity of the cell wall will be compromised and the adhesion progress will be interrupted. Findings concluded that P. betel and B. javanica aqueous extracts have a great potential to be developed as oral health care product. The expression of


The influence of multiple maternal and pregnancy characteristics on offspring cardiometabolic traits at birth is not well understood and was evaluated in this study.The Family Atherosclerosis Monitoring In earLY life (FAMILY) Study prospectively evaluated 11 cardiometabolic traits in 901 babies born to 857 mothers. The influence of maternal age, health , health behaviors  and pregnancy characteristics  were examined. Greater gestational age influenced multiple newborn cardiometabolic traits including cord blood lipids, glucose and insulin, body fat and blood pressure. In a subset of 442 singleton mother/infant pairs, principal component analysis grouped 11 newborn cardiometabolic traits into 5 components , accounting for 74% of the variance of the 11 outcome variables. Determinants of these components, corrected for sex and gestational age, were examined. Baby anthropometry/insulin was independently predicted by higher maternal pre-pregnancy weight (standardized estimate 0.30) and gestational weight gain  and was inversely related to smoking during pregnancy  and maternal polyunsaturated to saturated fat intake . Component 2 (HDL-C/Apo Apolipoprotein1) was inversely associated with maternal age. Component 3 (blood pressure) was not clustered with any other newborn cardiometabolic trait and no associations with maternal pregnancy characteristics were identified. Component 4 (triglycerides) was positively associated with maternal hypertension and triglycerides, and inversely associated with maternal HDL and age. Component 5 (glycemia) was inversely associated with placental/fetal ratio . LDL-C was a bridging variable between the lipid factors and glycemia.Maternal health, health behaviours and placenta to fetal weight ratio are associated with newborn cardiometabolic traits over and above gestational age. Future investigations are needed to determine if these factors remain important determinants of cardiometabolic health throughout childhood. Cardiovascular (CV) disease is the leading cause of morbidity and mortality worldwide among adults and 9 modifiable risk factors account for the majority of this risk We conducted a prospective investigation designed to understand determinants of cardiometabolic traits early in life in the Family Atherosclerosis Monitoring In earLY life (FAMILY) study, a longitudinal cohort study in which mothers were enrolled during pregnancy The study was approved by the Research Ethics Boards at the participating hospitals .The study rationale and design have been previously published Maternal demographic, pregnancy history, laboratory and physical measurements were obtained from the mother at the baseline visit which occurred between 21 and 39 (median 28.6) weeks of gestation and chart review after delivery. Maternal height, weight, fasting lipid profile and glycemic status were measured. Maternal glycemic status was assessed using self-reported history of diabetes that preceded the pregnancy and results of a 75 g oral glucose tolerance test (OGTT) carried out on the non-diabetic pregnant mothers including fasting, 1 hour and 2 hour plasma glucose levels. Dysglycemia included pre-existing diabetes, impaired glucose tolerance (IGT) of pregnancy or gestational diabetes. Gestational diabetes was present when 2 or more plasma glucose values were equal to or exceeded the thresholds of: fasting 5.3 mmol/L, 1 hour 10.6 mmol/L, 2 hour 8.9 mmol/L set out by the Canadian Diabetes Association, and participants were classified with IGT of pregnancy when one threshold was exceeded Socioeconomic status was reported as annual household income. Family history of CV disease and diabetes and maternal health behaviours /birth weight (g). Newborn outcomes of interest included anthropometric characteristics , blood pressure, glycemia (cord blood glucose), cord blood insulin and lipid profile . NewbornStatistical analysis was conducted using SAS version 9.1 for unix, SAS Institute Inc., Cary, NC, USA. The distribution of all continuous variables was examined. Insulin and triglyceride were not normally distributed and were log transformed for further analysis. The impact of gestational age on the key newborn cardiometabolic traits  was assessed using linear regression. Principal components analysis was used in a subset of mother/infant pairs to group the following potentially correlated newborn variables: birthweight, length, body fat, cord blood glucose and insulin, LDL-C, HDL-C, triglyceride, apoA1 and newborn systolic and diastolic blood pressure at the birth visit, into a smaller set of uncorrelated variables. Only data from singletons were included in the principal component analysis because multiple births influence birthweight, gestation at delivery, LDL-C, triglyceride and cord blood glucose levels and these multiple births shared common maternal environments. Prior to inclusion in the principal component analysis, each variable was adjusted for gestational age and sex, as these were strongly associated with birthweight and length, lipids, glucose, insulin and ApoA1 and blood pressure levels . Blood pPrincipal component analysis utilizes the linear relationship among variables to reduce the number of variables to summary factors or components while retaining as much of the total variance in the original data as possible Using the resulting 5 newborn principal components as dependent variables, we then examined associations with each maternal characteristic using simple linear regression. We considered maternal age, pre-pregnancy weight, gestational weight gain, glucose intolerance, mid-pregnancy lipid profile , smoking during the pregnancy, elevated blood pressure prior to or during pregnancy, placental-fetal weight ratio, major macronutrient intake , physical activity during pregnancy, maternal education, and annual household income as independent variables. These variables were selected based on prior reports of association in the medical literature and our beliefs regarding possible pathophysiological mechanisms linking maternal characteristics to newborn cardiometabolic traits. All factors which were associated with a newborn component with p<0.10 were entered into a multivariate linear regression model which included maternal age.We enrolled 857 families between 2002 and 2009, with 857 mothers and 901 babies (816 singleton and 85 twins or triplets). Most mothers (92.3%) were married, of European ethnicity (84.6%) and had attended college or university (83.9%). Approximately 11% reported a family income<$30,000 CDN annually (the Canadian poverty cut-off level) and 35% had a household income that exceeded $100,000 CDN annually. While 37.8% of mothers were current or former smokers, 14.1% reported smoking for some time during the pregnancy. Hypertension predating the pregnancy was reported in 4.1% and a very small percentage (0.7%) developed pre-clampsia during pregnancy. Abnormal glycemic status (dysglycemia) was present in 111 (13.0%) mothers during the pregnancy; 35 (4.1%) had pre-existing diabetes, 35 (4.1%) were diagnosed with gestational diabetes and 41 (4.8%) had impaired glucose tolerance of pregnancy. A family history of CV disease  was reported by 19.7% of mothers. Dietary records were available for 765 mothers. Maternal energy intake averaged 2156±749 kcal/day and reported carbohydrate and protein intakes met current guidelines, whereas the consumption of saturated fat was high relative to polyunsaturated fat intake (P:S ratio = 0.36). .There were 901 live births and no stillbirths; two infants died at 1 and 2 days of age; 454 (50.4%) were male and 377(41.8%) were first babies for the mothers. The mean gestational age at birth was 38.9 (2.2) weeks; 98 (10.9%) were born prior to 37 weeks gestation (3). Cord blood was successfully collected in 683 (75.8%) of the births and lipid profile, glucose and insulin levels were measured in these samples. In 218 births, cord blood was not available for one or more of several reasons including birth outside of hospital, family participation in cord blood banking, precipitous or complicated delivery or failure of the labour and delivery unit staff to note that the family was participating in the study. Weight was taken at birth and birth length, skinfolds and blood pressure were measured at the birth visits on average 2.6 (3.5) days after birth. Blood pressure measurements could not be made in 110 (12.2%) babies because they were restless or crying or in 25 (2.9%) who had no birth visit due to early discharge.Newborn levels of LDL-C, HDL-C (but not ApoA1) and insulin were lower with more advanced gestational age, whereas glucose and triglycerides were higher . Similarly, percent body fat (p = 0.0002) and systolic BP (p = 0.03), but not diastolic BP, were higher with increasing gestational age .Both sysPrincipal component analysis was conducted in a subset of 442 singletons for whom all 11 outcome variables of interest were available. There were no differences in maternal characteristics between those who were included and those who were not, except for the newborn characteristics that were related to exclusion of twins and triplets . .As shown in A bridging variable is identified when a newborn variable is correlated with more than one component. Newborn LDL-C bridged the two newborn lipid components (components 2 and 4) and newborn glycemia (component 5) and in each component had the minor role compared to the other newborn cardiometabolic traits. Maternal characteristics which were associated with each newborn component with p<0.10 and which were included in multivariable models are shown in In this prospective birth cohort of primarily white Caucasian women from Ontario Canada, we identified 5 clusters of newborn cardiometabolic factors including anthropometrics, two lipid components, blood pressure and a glycemia component which collectively account for 74% of the variance of the newborn cardiometabolic traits. We then found that key maternal characteristics including maternal age, pre-pregnancy weight, pregnancy weight gain, the relative intake of polyunsaturated to saturated fat, hypertension, tobacco use, second trimester HDL-C, triglyceride and the placental to fetal weight ratio are significantly associated with these characteristics . MaternaGestational age is strongly associated with lower cord blood LDL-C, HDL-C and insulin and higher newborn triglycerides, glucose, body fat and systolic blood pressure. The influence of gestational age on newborn cardiometabolic factors has been previously documented but was not always considered in previous studies examining maternal influences on cardiometabolic outcomes at birth. Premature  newborns have increased cardiovascular risk factors as adults We show that newborn cardiometabolic traits group into 5 clusters. Of these, the newborn anthropometry/insulin cluster alone explains 23% of the variance in newborn cardiometabolic traits. Maternal pre-pregnancy weight and weight gain throughout pregnancy are strongly positively associated, whereas maternal smoking and relative consumption of polyunsaturated to saturated fat are negatively associated with newborn anthropometry/insulin. Maternal glucose status was strongly correlated to the anthropometric/insulin factor in univariate analysis , but only weakly related in multivariate analysis  when the influence of maternal weight and pregnancy weight gain were included. While maternal weight and maternal smoking behaviours have been previously related to newborn body size, some findings are new, including the influence of maternal diet (P:S fat intake ratio) on offspring body size. Saturated fats in this study were mostly derived from consumption of dairy products, red meat and chocolate whereas polyunsaturated fats were mostly derived from peanut butter and chicken. The average P/S ratio in our study of 0.36 reflects a diet predominantly comprised of saturated fats, and is significantly lower than the Canadian average (0.55) Cord blood LDL-C levels bridged three components including 2 lipid components and the glycemia component. Together, the HDL-C/ApoA cluster and the newborn triglyceride cluster explain 28% of the variance in newborn cardiometabolic traits. We have demonstrated lower newborn HDL-C and LDL-C with advancing gestational age as previously reported, but no difference in ApoA1 was identified Component 5, newborn glycemia, explained 9% of the variance in newborn cardiometabolic traits. The influence of placental function is demonstrated by the strong inverse association between placental-fetal weight ratio and newborn glycemia that is independent of maternal glucose intolerance. As the organ for nutrient transport and gas exchange between mother and fetus, the placenta is implicated in disturbances of fetal growth. Some studies suggest that the relative size of the placenta to birth weight is associated with antenatal factors including cigarette smoking, hypertension, and maternal weight gain The mean systolic blood pressure (69.3±9.6 mmHg) was within the normal range expected for sleeping newborns of a similar age and systolic blood pressure was directly related to gestational age at birth and time since delivery. This is consistent with previous studies Our study is the first to explore the clustering of cardiometabolic traits at birth using principal component analysis and to simultaneously examine the association between the derived components and maternal determinants in a large number of babies and their mothers. Previous studies typically reported multiple single factor associations without performing a unifying analysis Maternal health , health behaviours  and placental to fetal weight ratio are important determinants of newborn cardiometabolic health over and above gestational age. Future investigations are required to determine if these variables remain important determinants of cardiometabolic health as the offspring grow.Table S1Maternal and Newborn Demographic Characteristics and Biochemical Measures for those included and not included in the Principal Component Analysis.(DOCX)Click here for additional data file.


Ficus deltoidea Jack (Ficus: Moraceae) from the Malay Peninsula of Malaysia. Several molecular-based classifications on the genus Ficus had been proposed, but neither had discussed the relationship between seven varieties of F. deltoidea to its allies nor within the varieties. The relationship between seven varieties of F. deltoidea is still debated due to the extreme morphological variabilities and ambiguous boundaries between taxa. Thus, the correct identification of these varieties is important as several morphological characters are variety-specific. To test the monophyly and further resolved the relationship in F. deltoidea, a morphological phylogenetic analysis was conducted based on herbarium specimens representing the seven varieties of F. deltoidea that were collected from the Malay Peninsula of Malaysia, by using related species of the genus Ficus; F. grossularioides, F. ischnopoda and F. oleifolia as the outgroups. Parsimony and neighbour-joining analyses indicated that F. deltoidea is monophyletic, in that the seven varieties of F. deltoidea nested into two clades; clade subspecies deltoidea  and clade subspecies motleyana (var. intermedia and var. motleyana).This study is the first report to suggest a morphological phylogenetic framework for the seven varieties of  Ficus deltoidea Jack (Ficus: Moraceae) is a diverse species of subgenus Ficus, section Ficus and subsection FrutescentiaeFicus deltoidea is a native and widely distributed throughout Malaysia, Thailand, Sumatra, Java, Kalimantan, Sulawesi and Moluccas F. deltoidea as ‘Mas Cotek’ due to the presence of golden dots on the upper surface of its lamina F. deltoidea; namely var. deltoidea, var. bilobata Corner, var. angustifolia (Miq.) Corner, var. intermedia Corner, var. kunstleri (King) Corner, var. motleyana (Miq.) Corner and var. trengganuensis Corner that were found in the Malay Peninsula were described by Kochummen Ficus deltoidea is commonly cultivated as a houseplant for decorative purposes and traditional medicinal uses by the local people bilobata, var. angustifolia, var. intermedia, var. kunstleri, var. motleyana and var. trengganuensis are commonly used in the Malay traditional medicine F. deltoidea has been exploited in many different ways, the taxonomy of this species is still controversial at the varietal level. Historically, several botanical names of F. deltoidea have been reported; namely F. diversifolia Blume, F. motleyana Miq. and F. oleifolia King F. deltoidea varieties. The leaf characters are probably the most variable and showed heterophylly in the species F. deltoidea and to differentiate intra-specific variation based on information contributed by overall morphological characters.Although Morphological data were scored from 108 herbarium specimens, with prior permissions from four different herbaria; the Herbarium of the Universiti Kebangsaan Malaysia (UKMB), Herbarium of the Forest Research Institute of Malaysia (FRIM), Herbarium of Sarawak (SAN) and the National Herbarium of Singapore (SING). All measurements and observations were taken from the herbarium specimens except data for flowers, which were gathered from the literatures Ficus grossularioides Burm. f. (Selangor: Anuar 115670 UKMB); Ficus ischnopoda Miq. (Kelantan: Zainudin 5726 UKMB); Ficus oleifolia King (Sarawak: Jamre 70886 SAN); Ficus deltoidea var. angustifolia (Miq.) Corner ; F. deltoidea var. bilobata Corner ; F. deltoidea var. deltoidea Corner ; F. deltoidea var. trengganuensis Corner . F. deltoidea var. intermedia Corner . F. deltoidea var. kunstleri (King) Corner ; F. deltoidea var. motleyana (Miq.) C. C. Berg .The characters were surveyed throughout the ingroup and outgroup taxa using criteria of putative homology or hypothetical homology The phylogenetic analyses were performed using the maximum parsimony (MP) and Wagner approaches and trees were generated using the PAUP* version 4.0b10 software motleyana and var. intermedia, which was supported and characterized by tepals longer than ovary (#29) and a synapomorphy of smooth and slightly angular ovary (#31). There was no character found to support this group, thus collapsed into polytomy in the other tree. Other character transformation series within the whole tree were found to be almost identical in both trees. The results were then compared with the neighbour-joining (NJ) tree (The resultant data matrix is shown in NJ) tree . Noted tFicus deltoidea formed a weakly-moderately supported clade with bootstrap support of 70% and 82% in the MP and NJ trees, respectively. The group was characterized by the important characters of leaf base acute (#6), stipule length equal or less than 0.5 cm (#16), periderm not persistent (#17), ostiole diameter less than 1.5 mm (#27) and color of ripening fig that is rose red to dark purple (#28). The species was divided into two clades; clade subspecies deltoidea (BS  = 75%/79%) and clade subspecies motleyana (BS  = 51%/61%), of which the first clade comprised of five varieties namely, var. angustifolia, var. bilobata, var. deltoidea, var. kunsleri and var. trengganuensis, whereas the second clade contained two varieties namely, var. intermedia and var. motleyana.deltoidea was moderately supported with 75% and 79% bootstrap values in the MP and NJ analyses, respectively. It was defined by the following characters; leaf length equal or less than 5 cm (#1), midrib forked near the middle of the lamina (#3), leaf apex rounded to truncate and minutely emarginate to form bilobed (#5), leaf spatulate (#7), waxy gland beneath the lamina is equal or more than three (#11), gland present at the forked midrib (#12) and commonly seen at the subsequent dichotomies of the midrib (#13). Within subspecies deltoidea, var. kunsleri and var. trengganuensis formed a strongly supported clade, 90% and 91% in the MP and NJ trees, respectively. They were described by having obtuse leaf base (#6), peduncle length more than 1 cm (#18), fig borne in pairs (#19), fig length more than 1 cm (#22), fig cuneate at the base (#24), ostiole diameter between 1.5–2.5 mm (#27) and flowers with yellowish-red tepals (#30). However, var. kunstleri was discriminated from its ally by having leaf length and width that is more than 5 cm each (#1), (#2), veins are deeply impressed (#9), petiole length more than 1.5 cm (#14), glabrous at the tip (#15) and glabrous fig (#20), whereas var. trengganuensis was identified by having midrib forked less than 45 degrees (#4), stipule length more than 0.5 cm (#16) and tepals longer than ovary (#29). The positions of var. deltoidea, var. bilobata and var. angustifolia were defined by extremely low confidence in the MP analysis and therefore, relationship between these varieties were not certain. However, the NJ analysis support their relationship by 70% bootstrap support and they share the peduncle length that is equal or less than 1 cm (#18), fig solitary (#19), flowers with red and dark purple tepals (#30) and rugose-angular ovary (#31). Within this group, var. angustifolia was identified by having fig cuneate at the base (#24), ripening fig yellow to orange to brownish red (#28) and ovary with fleshy exocarp (#32), whilst var. bilobata showed a unique autapomorphy of tepals length that is as long as ovary (#29).The placement of clade subspecies motleyana received weak bootstrap support of 51% in the MP analysis, but was moderately supported, 61% in the NJ analysis. It was distant to subspecies deltoidea by having midrib not forked to forked near the apex (#3), tepals longer than ovary (#29) and smooth or slightly angular ovary (#31). Within subspecies motleyana, var. intermedia was distinguished by having stipule length more than 0.5 cm (#16), fig cuneate at the base (#24), protracted at the apex (#25), ostiole diameter between 1.5–2.5 mm (#27) and ovary with fleshy exocarp (#32). On the other hand, var. motleyana showed an autapomorphy of fig oblong in shape (#21).The clade subspecies F. deltoidea into twelve varieties and four forma, namely, var. deltoidea, var. angustifolia f. angustissima Corner, var. arenaria Corner, var. bilobata, var. borneensis Corner f. subhirsuta Corner, var. intermedia, var. kunstleri, var. lutescens (Desf.) Corner f. longipedunculata Corner, f. subsessilis (Miq.) Corner, var. motleyana, var. oligoneura (Miq.) Corner, var. peltata Corner and var. trengganuensis. Later, he introduced a new variety, var. kinabaluensis Stapf which probably is a synonym of var. intermedia of Borneo with larger peduncle and leaves F. deltoidea into seven varieties namely, var. deltoidea, var. bilobata, var. angustifolia, var. intermedia, var. kunstleri, var. motleyana and var. trengganuensis, which were available in the Malay Peninsula of Malaysia or formerly known as Malaya, and described them. Later on, two endemic varieties of Borneo, namely var. subhirsuta Kochummen and var. recurvata Kochummen were added deltoidea and subspecies motleyana) which seems to be more practical and satisfactory in handling the variation. They further mentioned that as strong phytogeographical and ecological support is lacking, the rank of variety proposed by others might be more correct, but the chosen rank allows recognition of varieties for regional use. Therefore, the sampling of this study focused on the earlier classification scheme of Kochummen Most of the previous classifications  were basFicus deltoidea formed one major clade. Nevertheless, character variation in the dataset was insufficient to resolve all the phylogenetic relationships, especially among the internal branching of subspecies deltoidea. The divergence of var. deltoidea, var. angustifolia and var. bilobata received extremely low bootstrap support, thus the branches collapsed in the strict consensus tree. The placement of these varieties, as revealed by a previously published RAPD analysis, were also uncertain due to the variable banding patterns and hence showed that similar varieties were grouped into different clusters In this study, F. deltoidea of Malesian region can be placed into two subspecies: subspecies deltoidea consisted of var. deltoidea, var. bilobata, var. angustifolia, var. kunstleri and var. trengganuensis. Within the subspecies deltoidea, var. kunstleri was shown to be more closely related to var. trengganuensis than to the other varieties. The positions of var. deltoidea, var. bilobata and var. angustifolia received extremely low support in the MP tree, suggesting that the analysis did not assure the relationships between these varieties. However, they were genetically closely related to each other and were fairly supported in the NJ analysis. This should, however, be confirmed by a detailed molecular studies. The second, subspecies motleyana comprised var. intermedia and var. motleyana. Our results showed an agreement with Berg intermedia was excluded in their classifications and then was transferred to F. oleifolia subspecies intermedia, because of mixture in characters of forked and non-forked midrib, and leaf shapes ranging from spatulate to obovate to oblanceolate intermedia and var. motleyana was constantly supported and defined in both the MP and NJ analyses.From the results obtained, the seven varieties of F. deltoidea, F. ischnopoda Miq., F. oleifolia King and F. grossularioides Burm.f., F. oleifolia was found to be the closest ally to F. deltoidea, whilst F. ischnopoda and F. grossularioides were placed at the base of trees and did not get any bootstrap support Click here for additional data file.