frequencies of these SNPs were similar to infer- tile individuals without protamine deficiency (37). Tanaka et al. recently reported a C248T SNP in Protamine2 gene (25). They identified presence of this SNP among of 266 azoospermic individuals. This SNP results in appearance of a stop codon and premature termination of the protamine2 mRNA leading to azoospermia. Iguchi et al. also reported occurrence of G197T SNP in patients with a fairly normal sperm count but with a markedly altered morphology based upon Kruger’s strict criteria with a frequency rate of 3 to 30 (24). The latter SNP converts the highly conserved arginine to a serine residue, which can serve as a potential phos- phorylation site for the enzyme serine/arginine- rich protein specific kinase1 (SRPK1). Improper phosphorylation can substantially alter both DNA binding and protamine-to-protamine interaction ability of protamine1 in the sperm nucleus. The aim of this study was to evaluate the occurrence rate of these two SNPs in our study population. The results of SNP analysisin 273 infertile and 35 fertile individuals revealed absence of the two mentioned SNPs in our studied population. The 273 individuals included 96 oligozoospermic and 177 azoospermic individuals. Although the above two SNPs were reported in smaller population than the studied population, it is not uncommon to ob- served absence of previously reported SNPs. In- deed a more recent study, evaluating two SNPs in PRM1 in 1195 individuals, one of which was the same as the SNP assessed in this study and they concluded that this SNP has no significant effect in male infertility (38). Even though considerable number of SNPs has been detected in PRM1 and 2, but the overall conclusion derived from these study suggest that except the SNPs that lead to ter- mination codons, these SNPs do not result in the aberrant expression of P1 or P2. Therefore, recent attention has been directed on the promoters of the PRM1 and PRM 2. Indeed, Gazquez et al., recently, reported an SNP in promoter of PRM1 which lead to aberrant ratio of P1/P2 and results in abnormal morphology (39,40). The frequency of the reported SNP was significantly different compared to nor- mal fertile males, which suggest that such SNP may serve as a good molecular marker for genetic diagnosis of male infertility.
In summary, this is the first report investigating miRSNPs involved in the miRNA network in liver cancer. A polymorphismin the binding site for diverse miRNA clusters in XPO5 was associated with a significantly OS in hepatocellular carcinoma patients. MiRSNPs emerged as new promising markers for disease progression in cancer. Although miR-SNP studies for miRNA processing machinery genes are still preliminary, our results are encouraging, as they indicate that miR-SNPs could be used as cancer prognosis marker. However, the results from this study require validation in another larger HCC cohort study and in laboratory-based functional studies. MicroRNAs have been emphasised as a key factor in patients’ susceptibility to therapeutic response in many complex diseases, including cancer . The analysisof genetic polymorphisms in miRNA processing genes may help to identify patient subgroups with poor prognoses and may, accordingly, help to refine therapeutic decisions regarding HCC patients.
In this study, we present the analysisof natural polymorphism with respect to three major bacterial blight resistance genes: Xa21, Xa26 and xa5, at the sequence level in a set of rice germ- plasm. Xa21 is a broad spectrum bacterial blight resistance gene, originally derived from a wild rice O. longistaminata and introgressed into cultivated rice . Song et al.  reported the cloning of this gene which encodes a LRR threonine rich receptor like kinase domain contain- ing protein. Xa26 is a dominant R gene, which provides resistance against Chinese, Japanese and Korean Xoo strains. Both Xa21 and Xa26 genes had been mapped on the long arm of Chromosome 11 of rice. The presence of Xa26 in rice was first reported in Chinese O. sativa cultivar Minghui 63. Plants with this gene exhibited broad resistance both at seedling and adult stages. Xa26 also encodes a LRR receptor kinase-type protein . Both Xa26 and Xa3 have been shown to be the same gene  and is an important gene in the breeding of japonica culti- vars with BB resistance in China . xa5 is a recessive gene, which confers race specific blight resistance against Philippines Xoo race 1 (PXO86). The xa5 gene is a mutant form of rice tran- scription factor OsTFIIAγ5 (Xa5) where there is a substitution variant of a single amino acid: V39E . xa5 codes for 106 amino acids and both the dominant and recessive genes are con- stitutively expressed in different tissues of the plant . The predicted 3-D structure of xa5 protein after its superimposition with Xa5 and TFIIA shows that due to the substitution of V39E, a minor variation occurs in the third helix domain of xa5 protein .
Bax gene is a well-studied tumor suppressor gene in various cell types. Bax is encoded by six exons and expresses a complex pattern of alternative RNA splicing that forms a β1kd membrane (α) and two forms of cytosolic protein ( and ). It belongs to a proapoptotic member of the Bcl-β family and has been implicated in the induction of apoptosis . It remains inactive in cytosol of non-apoptotic cells during homeostatic conditions. Upon a cell death signal, Bax undergoes a conformational change that leads to their insertion, oligomerization and formation of large pores through the outer mitochondrial membrane . Many apoptogenic factors like cytochrome c [10,11], Smack/Diablo [1β], Omi/HtrAβ [1γ], endonuclease G  and apoptosis inducing factor  are released through this pore and recruit various molecules of apoptotic pathways. Numerous studies have demonstrated that alternation of Bax expression plays an important role in pathogenesis of cancer [16–β1]. Genetic change of the Bax gene may have distinguished significance in cancer initiation and progression since it has a series of target genes including many oncogenes and tumor suppressor genes [ββ–β6]. Studies on the association between SNPs in Bax and human cancer have provided new insights into the molecular mechanisms of cancer development. To date, at least 111 Bax SNPs have been reported in Enterzdatabase. In case of gastrointestinal cancer missense mutations of the Bax gene in codon 169 (Thr > Ala or Thr > Met) causes inhibition of the proapoptotic activity of the protein and enhance the development of cancer [β7]. A guanine substituting adenosine at position 1β5 (G1β5A) in the Bax promoter is associated with higher stages of chronic lymphocytic leukemia (CLL) and failure to treatment response [β8]. A silent point mutation in Bax codon 184
Background. Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. Principal Findings. We found that single-nucleotidepolymorphism (SNP) analysisof DNA repair, recombination and replication (3R) genesin a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets ofgenes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. Conclusions/ Significance. This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis . They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.
where different on all populations, what nowadays we know that each population show different frequencies not only on the polymorphism ABO but practically in all other genetic polymorphic marker studied [Cavalli-Sforza, 1996; Cavalli-Sforza et al., 2003]. Subsequent analysisof proteins [Pauling et al., 1949; Lewis et al., 1958] enabled the analysisof a large number ofgenes, where the first results show a higher diversity than expected in the human population, with a large genetic variation within populations (≈85%) and only a small part (≈15%) attributable to differences between populations [Lewontin, 1967, 1972, 1974]. However, protein based genetic was limited due to the low discrimination power of the system [Budowle et al., 2008]. From this moment on, genetic markers become a valuable tool for anthropology, as long as they meet a number of characteristics: to be monophyletic, neutral, permit to establish a direct relationship between antecessor and offspring without the distortion of environmental adaptations and allowing comparisons of interpopulation variability [Susanne et al., 2007]. Finally, the direct analysisof DNA became possible, thanks to advances in techniques such as polymerase chain reaction [Saiki et al., 1985]. Since then, the knowledge on genetic markers have progressed and nowadays the most important markers are based on polymorphisms at the DNA, which have fewer limitations against, for example, the use of proteins [Budowle et al., 2008]. The most common forms ofpolymorphism used are: restriction length fragment polymorphism (RFLP), minisatellites (Variable Number of Tandem Repeats – VNTR), microsatellites (Short Tandem Repeats - STRs) and singlenucleotidepolymorphism (SNP) [Budowle et al., 2008; Griffiths et al., 1993].
Molecular phylogenetic analysis is essential to eluci- date the ecological, evolutionary, and taxonomic character- istics of organisms. To date, most phylogenetic methods have used only a tiny portion of the nuclear and chloroplast genomes, generally the phylogeny ofgenes or intergenic spacers. With next generation sequencing technologies, whole genome sequence data have become faster and chea- per to obtain, and phylogenetic reconstruction has dramati- cally accelerated, for instance, high-throughput sequencing has facilitated phylogenomic studies based on multilocus phylogeny (Comer et al., 2015), repetitive DNA (satellites and mobile elements) (Macas et al., 2015), chloroplast genomic DNA (Barrett et al., 2016), and nuclear ribosomal DNA (nrDNA) (Weitemier et al., 2015). Although nrDNA is not suitable for phylogenetic studies because the copies are assembled as tandem repeats and homologous recombi- nation occurs frequently, nrDNA alleles may become spe- cies-specific, thereby allowing hybrid detection. Singlenucleotidepolymorphism (SNP) analysis has been utilized in studies of genomic characterization and diversity, but heretofore the SNP approach has not been used for genomic analysisof hybrids. The aim of the present study was to de- termine the hybrid origin of S. bahiensis using high- throughput sequencing to analyze SNP variants, nrDNA al- leles, and the nuclear and chloroplast phylogenomics. DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2017-0256
important roles in cell differentiation and proliferation, animal growth, and metabolism. IGF-1 function is par- ticularly important for embryonic development (10). The porcine IGF-1 gene is also important for regulating body growth, development, and metabolism. Due to the wide and diverse geographic features of China, many minipig breeds are naturally distributed around the country. Over the last two decades, some of these minipig breeds have been adopted and used as laboratory animals, including Tibetan minipigs (TBs), Guizhou xiang minipigs, Guangxi Bama minipigs (BMs), Wuzhishan minipigs, and Banna minipigs (11). The Laboratory Animal Center of Southern Medical University (China) first imported TBs from Tibet to Guangzhou for laboratory animal research in 2004. The acclimatization and experimental animalization of these minipigs have been completed (12). TB is a unique breed that lives in high altitude environments (13). These pigs, which grow slowly and have thin skin, high meat cutability, and extra-fine muscle fibers, exhibit strong adaptability and resistance to harsh environments (14).
Abstract —cancer represents one of the greatest medical causes of mortality. The majority of Hepatocellular carcinoma arises from the accumulation of genetic abnormalities, and possibly induced by exterior etiological factors especially HCV and HBV infections. There is a need for new tools to analysis the large sum of data to present relevant genetic changes that may be critical for both understanding how cancers develop and determining how they could ultimately be treated. Gene expression profiling may lead to new biomarkers that may help develop diagnostic accuracy for detecting Hepatocellular carcinoma. In this work, statistical technique (discrete stationary wavelet transform) for detection of copy number alternations to analysis high-density single-nucleotidepolymorphism array of 30 cell lines on specific chromosomes, which are frequently detected in Hepatocellular carcinoma have been proposed. The results demonstrate the feasibility of whole-genome fine mapping of copy number alternations via high-density single-nucleotidepolymorphism genotyping, Results revealed that a novel altered chromosomal region is discovered; region amplification (4q22.1) have been detected in 22 out of 30-Hepatocellular carcinoma cell lines (73%). This region strike, AFF1 and DSPP, tumor suppressor genes. This finding has not previously reported to be involved in liver carcinogenesis; it can be used to discover a new HCC biomarker, which helps in a better understanding of hepatocellular carcinoma.
The analyses were performed using the R packages qtl (Broman et al., 2003), eqtl (Khalili and Loudet, 2014), and qtlbim (Yandell et al., 2007) (see the codes in Appendix). For the maximum likelihood and least squares methods we defined a threshold of three for the LOD score. For the Bayesian analysis, we used the tool qb.mainmodes to determine the number of QTLs per chromosome and to estimate peaks and valleys (cutoff = 25). The tools qb.mcmc , qb.varcomp , and qb.hpdone were used to compute the additive and dominance variances, and the highest probability density (HPD) region across genome. We defined the prior expected number of QTLs as 4, 8, and 12. The number of iterations was 60,600, with a burn-inof 600 and a thin of 20 (3000 iterations saved). For all approaches, pseudo-markers were defined every 0.5 cM and the additive-dominance model was fitted (no epistasis and gene x environment interaction).
as well as, association with cardiovascular disease, association to additional risk of neural tube defect and association with preeclampsia 13,21 . Nowadays, a few papers have described the genetic risk of oral mucositis development and relating to the variable response to the toxicity of chemotherapeutic drugs 22-24 . Singlenucleotide polymorphisms (SNPs) affecting enzymes needed for metabolism of chemotherapeutic agents, were identified as important prognostic determinants of response to chemotherapy 25 and individuals who express different phenotypes can have increased risk of drugs toxicities 3 . Two common SNPs of metilenetetrahidrofolate reductase (MTHFR - C677T and A1298C) have been described as having opposite effects on cancer patients, reducing cancer susceptibility and increasing drug-related toxicities, when methotrexate are used 22,25 . C677T is characterized by cytosine to valine change in codon 222 causing an alanine to valine substitution in DNA coding sequence 24 . The clinical relevance of this SNP on HSCT patients is related to a decrease of enzyme activity, reported as 30% in homozygous mutant genotype (TT) and 60% in heterozygous genotype (CT) 24,26 . A1298C polymorphism clearly reduces enzyme activity, albeit to a lesser extent than the C677T 27 .
Postpartum depression (PPD) is a frequent condition with major consequences for both mother and child. 1 A genetic determinant for PPD has been suggested by several reports. The first genome-wide study of PPD was recently published, and showed that the hemicentin-1 (HMNC1) gene had the strongest association with postpartum mood symptoms. 2 This gene encodes an extracellular protein that contains four estrogen receptor- binding sites and is involved mainly in cell migration, protein anchorage, and the formation of hemidesmo- somes in the epidermis. 3
SNP analysis and phylogenomics. The genome sequence of M. avium subspecies paratuberculosis K10 (22) (GenBank accession number NC_002944.2) was used as the reference genome for the phylogenetic analyses. Raw genome sequence data for the M. avium subspecies paratu- berculosis isolates are available from the European Nucleotide Archive under accession number PRJEB2204. The assembly of the reads into con- tigs was performed using the Velvet assembler program (freely available at https://www.ebi.ac.uk/⬃zerbino/velvet/), and the alignment of the sequences and positioning of SNPs were executed using the MUMmer package (freely available at http://mummer.sourceforge.net/) with M. avium subspecies paratuberculosis K10 as the reference sequence. SNPs were then extracted and concatenated, and a phylogenetic tree was calculated using phyML (freely available at http://atgc.lirmm.fr/phyml). This phylogenetic tree was then imported into R using the ade4 package to explore the paths which exist between the genomes. These paths are a description of the strains which group on the same branch as the structure of the phylogenetic tree descends (i.e., all strains are included on the root branch, which then bifurcates to include a subset of strains on one branch and the remaining strains on the other, and so on). Using these paths to assign groups of genomes, SNPs which were shared by all strains on each branch were compiled. These data then allowed comparison of the SNPs present in reducing sized groups of genome sequences. (As the tree de- scends through specific branches, the number of genomes remaining in
Genomic DNA was isolated from peripheral blood of patients and controls using the commercial Qiamp DNA Blood Mini Kit (Qiagen, Valencia, CA). Amplification of the target DNA was performed by polymerase chain reaction (PCR). The primers used in this study are presented in Table 4. A 15 m l reaction mixture, which consisted of 7.5 m l GoTaq Green Mater Mix (Promega, Madison, WI), 50 pmoles primers, and 0.2 m g of genomic DNA, was amplified by PCR. PCR conditions were as follows: initial denaturation at 95 uC for 3 min followed by 35 cycles of denaturation at 94 uC for 30 s, annealing at different temperatures (63uC for rs2227981, rs10204525 and rs1970000, and 58uC for rs7854303) for 30 s, extension at 72 uC for 30 s, and a final extension at 72 uC for 3 min. The SNPs were genotyped by PCR- restriction fragment length polymorphism (RFLP) analysis. The PCR products of four SNPs were respectively digested with 2 U of Pvu II (Promega, Madison, WI), Hsp92 II (Promega, Madison, WI), Ban II (Promega, Madison, WI) and Mnl I (Fermentas, MBI) restriction enzymes (Table 4) in a 10 m l reaction volume overnight. Digestion products were visualized on 3% agarose gels and stained with GoldView TM (SBS Genetech, Beijing, China). To confirm the
The Taihang Mountain range of north-central China, the Southern region area of Fujian province, and the Chaoshan plain of Guangdong province are 3 major regions in China well known for their high incidence of esophageal cancer (EC). These areas also exhibit high incidences of gastric cardia cancer (GCC). The ancestors of the Chaoshanese, now the major inhabitants in the Chaoshan plain, were from north-central China. We hypothesized that EC and GCC patients in Chaoshan areas share a common ancestry with Taihang Mountain patients. We analyzed 16 East Asian-specific Y-chromosome biallelic markers (singlenucleotide polymorphisms; Y-SNPs) and 6 Y-chromosome short tandem repeat (Y-STR) loci in 72 EC and 48 GCC patients from Chaoshan and 49 EC and 63 GCC patients from the Taihang Mountain range. We also compared data for 32 Chaoshan Hakka people and 24 members of the aboriginal She minority who live near the Chaoshan area. Analysis was by frequency distribution and principal component, correlation and hierarchical cluster analysisof Y-SNP. Chaoshan patients were closely related to Taihang Mountain patients, even though they are geographically distant. Y-STR analysis revealed that the 4 patient groups were more closely related with each other than with other groups. Network analysisof the haplogroup O3a3c1-M117 showed a high degree of patient-specific substructure. We suggest that EC and GCC patients from these 2 areas share a similar patrilineal genetic background, which may play an important role in the genetic factor of EC and GCC in these populations.
Inhibition of H6PDH activity, and thus depletion of the ER luminal store of NADPH, sensitizes cells to oxidative stress and thus promotes apoptosis, which is a key process in the vessel wall in the initiation and progression of atherosclerosis . Although PolyPhen analysis and sequence alignments suggested the Pro554Leu variant to be ‘‘probably damaging’’, in vitro functional assays did not reveal any significant, large scale effects of this mutation on NADPH generation compared to WT H6PDH. There are however, certain caveats to the functional assay used in this study. Firstly, the method would not be able to detect subtle differences (decreases or increases) in the kinetic parameters that might arise due to this mutation, since the assay uses only crude cell extracts and measures only initial rates, with saturating substrate concentrations. Secondly, H6PDH is a bifunctional enzyme with both glucose-6-phosphate dehydrogenase activity (which is responsible for the NADPH generation) and 6- phosphogluconolactonase activity . The P554L mutation is situated at the junction between the enzyme domains responsible for these two functions. The assay carried out in this study only directly measured NADPH generation (glucose-6-phosphate dehydrogenase activity) and not 6-phosphogluconolactonase activity, which is much more difficult to assay. 6-phosphogluco- nolactonase activity accelerates the hydrolysis of the reactive/toxic intermediate 6-phosphogluconolactone . Any interference with the functionality of the second domain could also affect the generation of NADPH by the first domain. In order to carry out further analyses of the effect of the mutation on 6-phosphogluco- nolactonase activity, further experiments using highly purified protein will be required. Finally, H6PDH and 11b-HSD1 have
The authors express their most sincere gratitude to Drs. David Lipman, Graham Cameron, Joakim Lundeberg, and Francis Collins for their support, the Research Association for Biotechnology of Japan, the International Human Genome Sequencing Consortium, and the Chromosome 22 Group at the Sanger Institute for providing sequence and annotation data. We are grateful to T. Hasui, T. Habara, K. Yamaguchi, H. Kawashima, F. Todokoro, N. Yamamoto, Y. Makita, R. Aono, Y. Tanada, H. Kubooka, H. Maekawa, Y. Sasayama, T. Yamamoto, S. Okiyama, K. Nakamura, A. Matsuya, Y. Mimiura, R. Matsumoto, K. Takabayashi, Y. Hayakawa, H. Zhang, S. Nurimoto, T. Sugisaki, T. Kawamura, O. Nakano, S. Hosoda, N. Yoshimura, and T. Endo for their technical support. This research is ﬁnancially supported by the Ministry of Economy, Trade, and Industry of Japan (METI), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), the Japan Biological Informatics Consortium (JBIC), the New Energy and Industrial Technology Development Organization (NEDO), the United States Department of Energy, the National Institutes of Health of the United States, the Bundesministerium fu¨r Bildung und Forschung (BMBF) of Germany, the European Union through the EURO-IMAGE Consortium (grant BMH4-CT97-2284 coordinated by Charles Auffray), the 863 and 973 Program of the Ministry of Science and Technology of China, and CNRS of France. The work on Module 3D-keynote is supported by Grants-in-Aid for Scientiﬁc Research on Priority Areas (C) ‘‘Genome Information Science’’ to Mitiko Go and Kei Yura, and for Scientiﬁc Research (B) to MG, from MEXT. KY is also supported by a Grant-in- Aid for Encouragement of Young Scientists from MEXT. The work on subcellular localization is supported by a Grant-in-Aid for Scientiﬁc Research on Priority Areas (C) ‘‘Genome Information Science’’ from MEXT and the National Project on Protein Structural and Functional Analyses from the same Ministry.
Major players in this role are Google and Yahoo. This model is also based on the pay- per click strategy discussed above. The Ad-publishers are supposed to give permission to Search Engine to place advertisements in their website and for every click a user makes on the advertisement the website owner gets paid by the Search Engine. People with creative ideas can get rich relatively quickly by permitting advertisers to piggyback on any web site that attracts a lot of viewers. It has been analyzed that Google pays Adsense publishers 78.5 cents on the dollar as revenue share. Google never officially disclosed details on revenue sharing but it has been observed that, as per rule Google shares 50 cents of every dollar to partners and can range up to the full dollar, depending on the size of the relationship. The study on revenue generated by Website owner’s shows that most of the revenue is generated by Search Engine Advertising Industry e.g. Google Adsense program. The revenue generated by some websites using Adsense has been summarized in Table . Many Indian companies are surviving on Adsense business and many Indian are earning month’s salary just through such pay per click programs. Many companies like ACT Media, Web Techies are specialized for successful Adsense campaign integration to the client’s website. The revenue generated by the program highly depends on the traffic of the website. Amit Aggarwaal (2006), owner of Digital Inspiration has revealed that his blog gets around 1.25 million hits per month with a majority originating from Google followed by direct traffic (like bookmarks, rss feeds, etc). The major traffic comes from four countries - US, India, UK and Australia (in the same order). The maximum site traffic comes from four countries - US, India, UK and Australia (in the same order) and the majority of revenue earned is from Adsense. Priya Shah, an Editor, Editor Http://EbizWhizPublishing.com in her article states that “Google's TOA prevents me from making any disclosures here, but I can safely say that I now make in a day what I used to earn in a month(over a year ago)”.
The other three listed genes, BMPR1A, MSGN1 and FLT1, belong to less interrelated pathways. BMPR1A has been found to regulate the recruitment of prospective paraxial mesoderm cells in the mouse epiblast. Mutant embryos have a shorter primitive streak, a more proximal node and a defective extension of the primitive streak . MSGN1 is a direct target of Wnt and TBX6 during the specification, maturation and segmentation of the paraxial mesoderm in the mouse . FLT-1 encodes VEGFR-1, a vascular endothelial growth factor receptor expressed in the endothelium of blood vessels . Vasculogenesis at the chorionic villi tree is evident by post conceptional day 21, during the four- somite embryonic stage (reviewed in ). A FLT-1 mutation resulted in endothelial disorganization and abnormal vessel formation during early embryo development . During the fourth week of life the human embryo switches from the exchange of nutrients and wastes by simple diffusion to establishment of utero-placental circulation. It is conceivable that failure to properly transition through these two stages could impair critical processes as mesodermal proliferation or neurulation.