Genome-Guided Transcriptomics, DNA-Protein Interactions, and Variant Calling

Nowadays, molecular biology has definitely become an interdisciplinary science. Toward the study of the functions and the interactions of the biological molecules, such as nucleic acids and proteins, computer science and engineering, along with chemistry and statistics, are routinely engaged. In molecular biology, techniques and methods are constantly developed, and new techniques emerge. Next-generation sequencing and bioinformatics have become the cornerstones of molecular biology. The developing technologies have led to a decrease of the cost per molecular unit analyzed, but at the cost of computer integration and intensification. Many research methods require a reference nucleic acid sequence. Considering the necessary integration of sequencing data and methodology, combining the “omics” approaches can help to elucidate more complex null hypotheses. Here, data processing basics, with an emphasis to commonly used techniques, are summarized. The knowledge gaps are discussed as well as further prospective for integrating next-generation sequencing data

Oral mucosal melanoma: a malignant trap

Oral mucosal melanomas are highly malignant tumors. The 'chameleonic' presentation of a mainly asymptomatic condition, the rarity of these lesions, the poor prognosis and the necessity of a highly specialized treatment are factors that should be seriously considered by the involved health provider. We present the case of a 75-year-old man who was referred to the Ear, Nose and Throat department. His symptoms were voice alteration and saliva drooling, progressively worsening during the last few weeks. The absence of pain was the reason for the delay of seeking medical care. The diagnosis was an oversized oral melanoma. This is an example of how the time of diagnosis and the evolution of a disease could be seriously influenced by patient's behavior. Melanomas arising from oral mucosa have poor prognosis unless they are discovered and treated early. The vigilance of the physicians is necessary to have success in this difficult task

Hox genes polymorphism depicts developmental disruption of common sole eggs

In sole aquaculture production, consistency in the quality of produced eggs throughout the year is unpredictable. Hox genes have a crucial role in controlling embryonic development and their genetic variation could alter the phenotype dramatically. In teleosts genome duplication led paralog hox genes to become diverged. Direct association of polymorphism in hoxa1a, hoxa2a & hoxa2b of Solea solea with egg viability indicates hoxa2b as a potential genetic marker. High Resolution Melt (HRM) analysis was carried out in 52 viable and 61 non-viable eggs collected at 54±6 hours post fertilization (hpf). Allelic and genotypic frequencies of polymorphism were analyzed and results illustrated a significantly increased risk for non-viability for minor alleles and their homozygous genotypes. Haplotype analysis demonstrated a significant recessive effect on the risk of non-viability, by increasing the odds of disrupting embryonic development up to three-fold. Phylogenetic analysis showed that the paralog genes hoxa2a and hoxa2b, are separated distinctly in two clades and presented a significant ω variation, revealing their diverged evolutionary rate.</p

Malpigmentation of common sole (Solea solea) during metamorphosis is associated with differential synaptic-related gene expression

In farmed flatfish, such as common sole, color disturbances are common. Dyschromia is a general term that includes the color defects on the blind and ocular sides of the fish. The purpose was to examine the difference in gene expression between normal pigmented and juveniles who present ambicoloration. The analysis was carried out with next-generation sequencing techniques and de novo assembly of the transcriptome. Transcripts that showed significant differences (FDR < 0.05) in the expression between the two groups, were related to those of zebrafish (Danio rerio), functionally identified, and classified into categories of the gene ontology. The results revealed that ambicolorated juveniles exhibit a divergent function, mainly of the central nervous system at the synaptic level, as well as the ionic channels. The close association of chromophore cells with the growth of nerve cells and the nervous system was recorded. The pathway, glutamate binding–activation of AMPA and NMDA receptors–long-term stimulation of postsynaptic potential–LTP (long term potentiation)– plasticity of synapses, appears to be affected. In addition, the development of synapses also seems to be affected by the interaction of the LGI (leucine-rich glioma inactivated) protein family with the ADAM (a disintegrin and metalloprotease) ones

Hox genes polymorphism depicts developmental disruption of common sole eggs

In sole aquaculture production, consistency in the quality of produced eggs throughout the year is unpredictable. Hox genes have a crucial role in controlling embryonic development and their genetic variation could alter the phenotype dramatically. In teleosts genome duplication led paralog hox genes to become diverged. Direct association of polymorphism in hoxa1a, hoxa2a & hoxa2b of Solea solea with egg viability indicates hoxa2b as a potential genetic marker. High Resolution Melt (HRM) analysis was carried out in 52 viable and 61 non-viable eggs collected at 54±6 hours post fertilization (hpf). Allelic and genotypic frequencies of polymorphism were analyzed and results illustrated a significantly increased risk for non-viability for minor alleles and their homozygous genotypes. Haplotype analysis demonstrated a significant recessive effect on the risk of non-viability, by increasing the odds of disrupting embryonic development up to three-fold. Phylogenetic analysis showed that the paralog genes hoxa2a and hoxa2b, are separated distinctly in two clades and presented a significant ω variation, revealing their diverged evolutionary rate

Hox gene expression profiles during embryonic development of common sole

Common sole (Solea solea) aquaculture production is based mostly on wild-caught breeders. Recently, the successful reproduction of first-generation fish that were reared in captivity was accomplished. A consistent good quality and quantity of produced eggs throughout the year, and of next-generationbroodstock, is important for reducing the overall cost of production. Hox genes play a pivotal role in normal embryonic development and alterations of their temporal expression level may be important for egg viability. Expression profile analysis of five hox genes (hoxa1a, hoxa2a, hoxa2b, hoxb1a and hoxb1b) involved in early embryonic development and of hoxa13a, which is involved in late stages, was carried out. Results revealed a premature and/or maternal expression of hoxa13a in sole embryos,and the detection of hoxa2a and hoxa2b genes as members of paralog group 2. Principal Component Analysis of hox gene expression in 54 ± 6 hours post fertilization embryos coming from wild-caught broodstock and a first-generation one reared in the hatchery, unveiled that these broodstocks are clearly distinct. In addition, their pairwise comparison revealed significant differences in the expression levels of hoxb1a and hoxb1b genes. Hox gene regulation during embryonic development could give valuable insight into rearing sole broodstocks with different origin in concert, and also into gaining a steady mass production of eggs, either in quality or quantity, all year round

High-Resolution Melting (HRM) Analysis for Rapid Molecular Identification of <i>Sparidae</i> Species in the Greek Fish Market

The red porgy (Pagrus pagrus) and the common dentex (Dentex dentex) are Sparidae species of high commercial value, traded in the Greek market. In some cases, fish species identification from Greek fisheries is difficult for the consumer due to the strong morphological similarities with their imported counterparts or closely related species such as Pagrus major, Pagrus caeroleustictus, Dentex gibbosus and Pagellus erythrinus, especially when specimens are frozen, filleted or cooked. Techniques based on DNA sequencing, such as COI barcoding, accurately identify species substitution incidents; however, they are time consuming and expensive. In this study, regions of mtDNA were analyzed with RFLPs, multiplex PCR and HRM in order to develop a rapid method for species identification within the Sparidae family. HRM analysis of a 113 bp region of cytb and/or a 156 bp region of 16s could discriminate raw or cooked samples of P. pagrus and D. dentex from the aforementioned closely related species and P. pagrus specimens sampled in the Mediterranean Sea when compared to those fished in the eastern Atlantic. HRM analysis exhibited high accuracy and repeatability, revealing incidents of mislabeling. Multiple samples can be analyzed within three hours, rendering this method a useful tool in fish fraud monitoring

Hox gene expression profiles during embryonic development of common sole: Supplementary material

Common sole (<i>Solea solea</i>) aquaculture production is based mostly on wild-caught breeders. Recently, the successful reproduction of first-generation fish that were reared in captivity was accomplished. A consistent good quality and quantity of produced eggs throughout the year, and of next-generation broodstock, is important for reducing the overall cost of production. <i>Hox</i> genes play a pivotal role in normal embryonic development and alterations of their temporal expression level may be important for egg viability. Expression profile analysis of six <i>hox </i>genes (<i>hoxa1a, hoxa2a, hoxa2b, hoxb1a </i>and <i>hoxb1b) </i>involved in early embryonic development and of <i>hoxa13a</i>, which is<i> </i>involved in late stages, was carried out. <a>Results revealed a premature and/or maternal expression of <i>hoxa13a </i>in sole embryos, and the detection of <i>hoxa2a </i>and <i>hoxa2b </i>genes as members of paralog group 2. Principal Component Analysis of <i>hox </i>gene expression in 54 ± 6 hours post fertilization embryos coming from wild-caught broodstock and a first-generation one reared in the hatchery, unveiled that these broodstocks are clearly distinct. In addition, their pairwise comparison revealed significant differences in the expression levels of <i>hoxb1a </i>and <i>hoxb1b</i> genes. </a>Hox gene regulation during embryonic development could give valuable insight into rearing sole broodstocks with different origin in concert, and also into gaining a steady mass production of eggs, either in quality or quantity, all year round

Early diagnosis of enteromyxosis in intensively reared sharpsnout seabream, Diplodus puntazzo

Myxozoans are metazoan parasites which are highly pathogenic in commercially important fish, particularly in aquaculture. Enteromyxosis is an economically significant disease caused by the myxosporean parasite Enteromyxum leei. Presently, early diagnosis of enteromyxosis in sharpsnout seabream was achieved by monitoring fish on a time-scale scheduled basis in combination with a validated molecular diagnostic technique. Two different successive fish batches of a commercial fish farm were monitored starting from the first month of on-growing in sea cages until fish reached commercial size. Fish weight and water temperature differentially ranged between them. E. leei detection was performed both by classical microscopic observation of fresh smears and by quantitative polymerase chain reaction (qPCR) while prevalence, mean intensity and mortality rate were calculated. In total, 520 fish were screened; 320 from the first batch and 200 from the second one. Parasitosis illustrated a seasonal occurrence pattern (spring and summer). When qPCR was used as a diagnostic method, enteromyxosis was diagnosed earlier with more increased prevalence compared to the microscopic observation. In this case, the parasite was detected at the lowest water temperature that has ever been recorded (14.7 degrees C), during the first month of on-growing in sea cages. The two-factor combination of water temperature and fish weight proved to be the most significant factor that could determine the emergence and development of parasitosis and smaller fish proved to be more susceptible. Mortality was influenced by both prevalence and intensity of parasitosis. An earlier diagnosis and identification of all developmental stages of the parasite throughout its life cycle was verified with qPCR, significantly before any outbreak occurred. The latter could become a key diagnostic tool for the control of enteromyxosis, allowing stakeholders and fish farmers to adopt all necessary precautionary measures minimizing economic loss in sharpsnout seabream intensive farming

Molecular cloning of four glutathione peroxidase (GPx) homologs and expression analysis during stress exposure of the marine teleost Sparus aurata

Glutathione peroxidase (GPx; EC 1.11.1.9) is an important family of enzymes that protects organisms from oxidative damage. Four full-length GPx cDNAs were cloned and characterized by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) from the liver of gilthead sea bream (Sparus aurata), an economically important species for Mediterranean aquaculture. Structural and functional annotations were performed for all paralogs, which suggested possible differences in function and subcellular localization. The phylogenetic analysis, based on the amino acid sequences, revealed four groups corresponding to teleostean GPx1a, GPx1b, GPx4a, and GPx4b and three groups for mammalian GPx1, GPx2 and GPx4. The tree topology indicated past duplication events for fish genes, unlike their mammalian homologs. Transcriptional analysis in ten tissues by reverse transcription quantitative polymerase chain reaction (RT-qPCR) evidenced a tissue-specific pattern for each GPx homolog. Fish experimental groups were exposed to stress factors such as fasting and confinement. Relative expression analysis in fish liver demonstrated that GPx1 genes were not regulated by dietary restriction; GPx4b was differentially expressed opposed to regularly fed fish. On the other hand, both GPx1 and GPx4 genes were up-regulated in fish post exposed to confinement, considered as a response to acute stress. The results underline the role of GPx genes as indicators of stress and welfare status in gilthead sea bream aquaculture. (C) 2013 Elsevier Inc. All rights reserved