A strategy to recover a poor-quality ligase product.

Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.

A new versatile peroxidase with extremophilic traits over-produced in MicroTom cell cultures.

Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H2O2. In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70-80 °C and the presence of NaCl (1.0-4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0-7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0-7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry.

Recombinant Expression of Archaeal Superoxide Dismutases in Plant Cell Cultures: A Sustainable Solution with Potential Application in the Food Industry.

Superoxide dismutase (SOD) is a fundamental antioxidant enzyme that neutralises superoxide ions, one of the main reactive oxygen species (ROS). Extremophile organisms possess enzymes that offer high stability and catalytic performances under a wide range of conditions, thus representing an exceptional source of biocatalysts useful for industrial processes. In this study, SODs from the thermo-halophilic Aeropyrum pernix (SODAp) and the thermo-acidophilic Saccharolobus solfataricus (SODSs) were heterologously expressed in transgenic tomato cell cultures. Cell extracts enriched with SODAp and SODSs showed a remarkable resistance to salt and low pHs, respectively, together with optimal activity at high temperatures. Moreover, the treatment of tuna fillets with SODAp-extracts induced an extension of the shelf-life of this product without resorting to the use of illicit substances. The results suggested that the recombinant plant extracts enriched with the extremozymes could find potential applications as dietary supplements in the nutrition sector or as additives in the food preservation area, representing a more natural and appealing alternative to chemical preservatives for the market.

Differential Distribution and Activity Profile of Acylpeptide Hydrolase in the Rat Seminiferous Epithelium.

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed.

In Vitro Assays for the Bifunctional Acylpeptide Hydrolase (APEH) Enzyme from Antarctic Fish.

The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme.

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.

The Bactericidal Activity of Protein Extracts from Loranthus europaeus Berries: A Natural Resource of Bioactive Compounds.

Loranthus europaeus is a well-known and important medicinal plant, with a long history of traditional medicine use. Several studies showed that it contains many bioactive compounds with a wide range of pharmacological effects. In light of these past researches, L. europaeus were chosen to consider its potential antimicrobial action. To this aim, different protocols were performed to selectively extract protein compounds, from L. europaeus yellow fruits, and evaluate the antimicrobial activity against four phytopathogenic fungi (Aspergillus niger, Alternaria spp., Penicillium spp., Botritis cinereus) and a number of foodborne bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus strains, Salmonella Typhimurium and Escherichia coli) by using serial dilutions and colony formation assays. Results evidenced no antifungal activity but a notable bactericidal efficiency of a crude protein extract against two foodborne pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 0.2 and 0.5 mg/mL, being S. aureus strains the most susceptible bacteria. Moreover, a strong bactericidal activity against S. aureus M7 was observed by two partially purified protein fractions of about 600 and 60 kDa molecular mass in native conditions. Therefore, these plant protein extracts could be used as natural alternative preventives to control food poisoning diseases and preserve foodstuff avoiding health hazards of chemically antimicrobial applications.

Dietary Supplementation with Fish Oil or Conjugated Linoleic Acid Relieves Depression Markers in Mice by Modulation of the Nrf2 Pathway.

Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and nuclear erythroid related factor 2 (Nrf2), a regulator of RedOx homeostasis and inflammation, is a promising target for depression prevention/treatment. As fish oil (FO) and conjugated linoleic acid (CLA) are known Nrf2 inducers, their protective ability is comparatively evaluated in a murine model of depression (MRL/MpJ-Faslpr ). Oxidative stress, fatty acids content, and critical factors reflecting brain functioning-namely brain-derived neurotrophic factor (BDNF), synaptic markers, and cholinergic signaling-are preliminarily evaluated in the frontal cortex of 8-week (Young) and in 22-week old animals (Old), which are used as model of depression. These markers are measured in Old mice at the end of a 5-week pretreatment with FO or CLA (728 or 650 mg kg-1 , respectively). Old mice exhibit disrupted Redox homeostasis, compensatory Nrf2 hyperactivation, lower docosaheaxaenoic acid (DHA), and lower BDNF and synaptic function proteins compared to Young mice. FO and CLA treatment relieves almost all the pathophysiological hallmarks at a level comparable to Young mice. Presented data provide the first evidence for the comparable efficacy of FO or CLA supplementation in preventing depression signs in Old MRL/lpr mice, likely through their ability of improving Nrf2-mediated antioxidant defenses.

The extraordinary resistance to UV radiations of a manganese superoxide dismutase of Deinococcus radiodurans offers promising potentialities in skin care applications.

An overproduction of free radicals or reactive oxygen species, often due to environmental factors, can alter the DNA structure and irreversibly modify proteins and lipids in the living cells. The superoxide anion (O2-) is one of the strongest oxidant molecules produced under oxidative stress conditions but it can be neutralized by the action of the enzymes SuperOxide Dismutases (SODs). In all the human tissues, SODs are essential for the prevention of serious diseases and the protection against oxidative stress damages. In the dermo-cosmetic sector, SODs have found promising applications, but their use is limited due to the loss of activity following the addition of the enzyme in the skin care formulas and the exposure of the skin to UV radiations and heat. Extremophile organisms, which proliferate in extreme physical and/or geochemical conditions, represent a potential source of stable SOD enzymes, able to function even in harsh conditions of high temperature, acid pH and long UV exposures. In the present study we investigated on a Mn-SOD deriving from the extremophilic bacterium Deinococcus radiodurans and, after its expression in E.coli, the Mn-SOD was characterized in terms of chemical and physical properties. Its extraordinary features in terms of UV resistance prompted us to investigate further about its potential applications in the dermo-cosmetic sector. It was expressed in Solanum lycopersicum (tomato) cell cultures with the main goal of developing a new ingredient, capable of keeping its ROS neutralizing activity once exposed to UV radiations and even when added to skin care formulas.

An alternative biocontrol agent of soil-borne phytopathogens: A new antifungal compound produced by a plant growth promoting bacterium isolated from North Algeria.

Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas protegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chromatographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Conjugated linoleic acid prevents age-dependent neurodegeneration in a mouse model of neuropsychiatric lupus via the activation of an adaptive response.

Oxidative stress is a key mediator of autoimmune/neurodegenerative disorders. The antioxidant/anti-inflammatory effect of a synthetic conjugated linoleic acid (CLA) mixture in MRL/MpJ-Fas lpr mice (MRL/lpr), an animal model of neuropsychiatric lupus, was previously associated with the improvement of nuclear factor-E2-related factor 2 (Nrf2) defenses in the spleen and liver. However, little is known about the neuroprotective ability of a CLA mixture. This study investigated the age-dependent progression of oxidative stress and the hyperactivation of redox-sensitive compensatory pathways (macroautophagy, Nrf2) in old/diseased MRL/lpr mice brains and examines the effect produced by dietary CLA supplementation. Disrupted redox homeostasis was evidenced in the blood, liver, and brain of 21- to 22-week-old MRL/lpr (Old) mice compared with 8- to 10-week-old MRL/lpr (Young) animals. This alteration was associated with significant hyperactivation of compensatory mechanisms (macroautophagy, Nrf2, and astrocyte activation) in the brains of Old mice. Five-week daily supplementation with CLA (650 mg/kg-1 body weight) of 16-week-old (CLA+Old) mice diminished all the pathological hallmarks at a level comparable to Young mice or healthy controls (BALB/c). Such data demonstrated that MRL/lpr mice can serve as a valuable model for the evaluation of the effectiveness of neuroprotective drugs. Notably, the preventive effect provided by CLA supplementation against age-associated neuronal damage and hyperactivation of compensatory mechanisms suggests that the activation of an adaptive response is at least in part accountable for its neuroprotective ability.

Low Erythrocyte Levels of Proteasome and Acyl-Peptide Hydrolase (APEH) Activities in Alzheimer's Disease: A Sign of Defective Proteostasis?

Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia. To date, there are no definitive diagnostic tests that can predict or assess onset and progression of the disease. Blood biomarkers for AD are being sought for many years but their identification remains a challenging task. In this study, we investigated the potential relationship between AD and levels of acyl-peptide hydrolase (APEH) and proteasome in erythrocyte samples of 52 participants (26 AD and 26 cognitively healthy controls). A statistically significant decrease in proteasome and exopeptidase/endopeptidase APEH activities was found in AD samples compared to those of healthy controls. Moreover, in contrast to what was observed for proteasome transcripts, APEH activities reduction in AD patients was unrelated to its gene expression levels, suggesting the occurrence of posttranslational modifications or the expression of endogenous inhibitors that might impair enzyme activity. These preliminary data further support a relationship between the APEH-proteasome system and AD molecular players, providing the first evidence of its potential use as a novel blood-based indicator for the routine detection of AD.

Unusual Antioxidant Properties of 26S Proteasome Isolated from Cold-Adapted Organisms.

The oxidative challenge represents an important factor affecting the adaptive strategies in Antarctic fish, but their impact on the protein degradation machinery still remains unclear. The previous analysis of the first 26S proteasome from the Antarctic red-blooded fish Trematomus bernacchii, evidenced improved antioxidant functions necessary to counteract the environmental pro-oxidant conditions. The purpose of this work was to carry out a study on 26S proteasomes from the temperate red-blooded Dicenthrarcus labrax and the icefish Chionodraco hamatus in comparison with the isoform already described from T. bernacchii, to better elucidate the cold-adapted physiological functions of this complex. Therefore, the 26S isoforms were isolated and the complementary DNAs (cDNAs) codifying the catalytic subunits were cloned. The biochemical characterization of Antarctic 26S proteasomes revealed their significantly higher structural stability and resistance to H2O2 with respect to that of the temperate counterpart, as also suggested by a comparative modeling analysis of the catalytic subunits. Moreover, in contrast to that observed in T. bernacchii, the 26S systems from C. hamatus and D. labrax were incapable to hydrolyze oxidized proteins in a ubiquitin-independent manner. Therefore, the 'uncommon' properties displayed by the Antarctic 26S proteasomes can mirror the impact exercised by evolutionary pressure in response to richly oxygenated environments.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Human semen as an early, sensitive biomarker of highly polluted living environment in healthy men: A pilot biomonitoring study on trace elements in blood and semen and their relationship with sperm quality and RedOx status.

The Campania region in Italy is facing an environmental crisis due to the illegal disposal of toxic waste. Herein, a pilot study (EcoFoodFertility initiative) was conducted to investigate the use of human semen as an early biomarker of pollution on 110 healthy males living in various areas of Campania with either high or low environmental impact. The semen from the "high impact" group showed higher zinc, copper, chromium and reduced iron levels, as well as reduced sperm motility and higher sperm DNA Fragmentation Index (DFI). Redox biomarkers (total antioxidant capacity, TAC, and glutathione, GSH) and the activity of antioxidant enzymes in semen were lower in the "high impact" group. The percentage of immotile spermatozoa showed a significant inverse correlation with TAC and GSH. Overall, several semen parameters (reduced sperm quality and antioxidant defenses, altered chemical element pattern), which were associated with residence in a high polluted environment, could be used in a further larger scale study, as early biomarkers of environmental pollution.

Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii.

Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its 'uncommon' ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery.

Adaptive response activated by dietary cis9, trans11 conjugated linoleic acid prevents distinct signs of gliadin-induced enteropathy in mice.

PURPOSE: The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor-2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). METHODS: Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg(-1)/bw) and indomethacin (15 mg L(-1)) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg(-1)/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. RESULTS: GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. CONCLUSIONS: The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice.

Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3' untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation.