Stonefish (Synanceia spp.) Ichthyocrinotoxins: An ecological review and prospectus for future research and biodiscovery.

Marine organisms possess a diverse array of unique substances, many with wide ranging potential for applications in medicine, industry, and other sectors. Stonefish (Synanceia spp.), a bottom-dwelling fish that inhabit shallow and intertidal waters throughout the Indo-Pacific, harbour two distinct substances, a venom, and an ichthyocrinotoxin. Stonefish are well-known for the potent venom associated with their dorsal spines as it poses a significant risk to public health. Consequently, much of the research on stonefish focusses on the venom, with the aim of improving outcomes in cases of envenomation. However, there has been a notable lack of research on stonefish ichthyocrinotoxins, a class of toxin that is synthesised within specialised epithelial cells (i.e., tubercles) and exuded onto the skin. This has resulted in a substantial knowledge gap in our understanding of these animals. This review aims to bridge this gap by consolidating literature on the ecological functions and biochemical attributes of ichthyocrinotoxins present in various fish species and juxtaposing it with the current state of knowledge of stonefish ecology. We highlight the roles of ichthyocrinotoxins in predator defence, bolstering innate immunity, and mitigating integumentary interactions with parasites and detrimental fouling organisms. The objective of this review is to identify promising research avenues that could shed light on the ecological functions of stonefish ichthyocrinotoxins and their potential practical applications as therapeutics and/or industrial products.

A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

Envenomations caused by fish in Brazil: an evolutionary, morphological, and clinical vision of a neglected problem.

Venomous fish are commonly found in Brazilian waters. The most important marine venomous fish species are stingrays (Dasyatidae, Gimnuridae, Myliobatidae, and Rhinopteridae families), catfish (Ariidae family), scorpionfish and lionfish (both Scorpaenidae family), and toadfish (Batrachoididae family). Meanwhile, Potamotrygonidae stingrays and Pimelodidae catfish are the most important venomous freshwater fish. The mechanisms of envenomation vary and involve various venomous apparatuses and glands. Despite not being highly developed, these venomous apparatuses in fish appear rudimentary, using structures such as fins and rays to inoculate toxins and rarely presenting with specialized structures. Toxins are produced by glandular tissue made up of proteinaceous cells, lacking true glands, and are positioned along the inoculation structures. However, systemic manifestations are rare. No antivenom serum has been developed for any species of American venomous fish. Brazilian venomous fish and their venoms have only recently attracted attention, leading to new studies not only addressing clinical issues in humans, but also exploring the discovery of new active substances with immense pharmacological potential.

Effective Pre-Clinical Treatment of Fish Envenoming with Polyclonal Antiserum.

Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of Thalassophryne nattereri, Scorpaena plumieri, Potamotrygon gr. Orbignyi, and Cathorops spixii and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of T. nattereri, P. gr orbygnyi, and C. spixii venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the S. plumieri venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.

Inflammasome Coordinates Senescent Chronic Wound Induced by Thalassophryne nattereri Venom.

Thalassophryne nattereri toadfish (niquim) envenomation, common in the hands and feet of bathers and fishermen in the north and northeast regions of Brazil, is characterized by local symptoms such as immediate edema and intense pain. These symptoms progress to necrosis that lasts for an extended period of time, with delayed healing. Wound healing is a complex process characterized by the interdependent role of keratinocytes, fibroblasts, and endothelial and innate cells such as neutrophils and macrophages. Macrophages and neutrophils are actively recruited to clear debris during the inflammatory phase of wound repair, promoting the production of pro-inflammatory mediators, and in the late stage, macrophages promote tissue repair. Our hypothesis is that injury caused by T. nattereri venom (VTn) leads to senescent wounds. In this study, we provide valuable information about the mechanism(s) behind the dysregulated inflammation in wound healing induced by VTn. We demonstrate in mouse paws injected with the venom the installation of γH2AX/p16Ink4a-dependent senescence with persistent neutrophilic inflammation in the proliferation and remodeling phases. VTn induced an imbalance of M1/M2 macrophages by maintaining a high number of TNF-α-producing M1 macrophages in the wound but without the ability to eliminate the persistent neutrophils. Chronic neutrophilic inflammation and senescence were mediated by cytokines such as IL-1α and IL-1ß in a caspase-1- and caspase-11-dependent manner. In addition, previous blocking with anti-IL-1α and anti-IL-ß neutralizing antibodies and caspase-1 (Ac YVAD-CMK) and caspase-11 (Wedelolactone) inhibitors was essential to control the pro-inflammatory activity of M1 macrophages induced by VTn injection, skewing towards an anti-inflammatory state, and was sufficient to block neutrophil recruitment and senescence.

How dangerous can a small fish be? A greater weever attack.

BACKGROUND: Thraconian fish (weever fish) are poisonous fish found mostly in the eastern Mediterranean region, but also in the eastern Atlantic Ocean, the North Sea and European coastal areas. Greater weever fish belong to the Trachinus draco family; these fish have spines on their dorsal fins and gill covers that secrete a high dose of dracotoxin venom. METHODS: This paper reports a 35-year-old female who presented with widespread body aches, respiratory distress and hoarseness following a greater weever attack. It discusses respiratory distress and other findings that occur after a greater weever attack in rare otolaryngological emergency cases. CONCLUSION: While greater weever fish are more likely to attack limbs such as arms and legs, patients stung on the neck who develop respiratory distress are considered an ENT emergency.

Inter-species variation in stonefish (Synanceia spp.) ichthyocrinotoxins; an ecological perspective.

Although stonefish (Synanceia spp.) are well-known to harbour a highly noxious defensive venom in their dorsal spines, very little is known about the composition and ecological function of the ichthyocrinotoxins that they secrete onto their epidermis. This study profiled reef (Synanceia verrucosa) and estuarine (Synanceia horrida) stonefish ichthyocrinotoxins via electrophoresis, liquid chromatography, and mass spectrometry to visualise and compare the composition of these toxins between the two species. Stonefish ichthyocrinotoxins were found to be multifarious concoctions that exhibited subtle differences between reef and estuarine species. We speculate that these variations and similarities are driven by the different and similar ecology of these fish species. Further research into the activity of the toxins components is now required to better understand their ecological role.

Preliminary report on the hemagglutinating activity of the Scorpaena plumieri fish venom.

The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 µg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 µg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.

Natterin-Induced Neutrophilia Is Dependent on cGAS/STING Activation via Type I IFN Signaling Pathway.

Natterin is a potent pro-inflammatory fish molecule, inducing local and systemic IL-1ß/IL-1R1-dependent neutrophilia mediated by non-canonical NLRP6 and NLRC4 inflammasome activation in mice, independent of NLRP3. In this work, we investigated whether Natterin activates mitochondrial damage, resulting in self-DNA leaks into the cytosol, and whether the DNA sensor cGAS and STING pathway participate in triggering the innate immune response. Employing a peritonitis mouse model, we found that the deficiency of the tlr2/tlr4, myd88 and trif results in decreased neutrophil influx to peritoneal cavities of mice, indicative that in addition to MyD88, TRIF contributes to neutrophilia triggered by TLR4 engagement by Natterin. Next, we demonstrated that gpcr91 deficiency in mice abolished the neutrophil recruitment after Natterin injection, but mice pre-treated with 2-deoxy-d-glucose that blocks glycolysis presented similar infiltration than WT Natterin-injected mice. In addition, we observed that, compared with the WT Natterin-injected mice, DPI and cyclosporin A treated mice had a lower number of neutrophils in the peritoneal exudate. The levels of dsDNA in the supernatant of the peritoneal exudate and processed IL-33 in the supernatant of the peritoneal exudate or cytoplasmic supernatant of the peritoneal cell lysate of WT Natterin-injected mice were several folds higher than those of the control mice. The recruitment of neutrophils to peritoneal cavity 2 h post-Natterin injection was intensely impaired in ifnar KO mice and partially in il-28r KO mice, but not in ifnγr KO mice. Finally, using cgas KO, sting KO, or irf3 KO mice we found that recruitment of neutrophils to peritoneal cavities was virtually abolished in response to Natterin. These findings reveal cytosolic DNA sensors as critical regulators for Natterin-induced neutrophilia.

Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles.

Tetrodotoxin (TTX)-bearing fish ingest TTX from their preys through the food chain and accumulate TTX in their bodies. Although a wide variety of TTX-bearing organisms have been reported, the missing link in the TTX supply chain has not been elucidated completely. Here, we investigated the composition of TTX and 5,6,11-trideoxyTTX in juveniles of the pufferfish, Chelonodon patoca, and toxic goby, Yongeichthys criniger, using LC-MS/MS, to resolve the missing link in the TTX supply chain. The TTX concentration varied among samples from different localities, sampling periods and fish species. In the samples from the same locality, the TTX concentration was significantly higher in the toxic goby juveniles than in the pufferfish juveniles. The concentration of TTX in all the pufferfish juveniles was significantly higher than that of 5,6,11-trideoxyTTX, whereas the compositional ratio of TTX and 5,6,11-trideoxyTTX in the goby was different among sampling localities. However, the TTX/5,6,11-trideoxyTTX ratio in the goby was not different among samples collected from the same locality at different periods. Based on a species-specific PCR, the detection rate of the toxic flatworm (Planocera multitentaculata)-specific sequence (cytochrome c oxidase subunit I) also varied between the intestinal contents of the pufferfish and toxic goby collected at different localities and periods. These results suggest that although the larvae of the toxic flatworm are likely to be responsible for the toxification of the pufferfish and toxic goby juveniles by TTX, these fish juveniles are also likely to feed on other TTX-bearing organisms depending on their habitat, and they also possess different accumulation mechanisms of TTX and 5,6,11-trideoxyTTX.

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development.

BACKGROUND: The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. RESULTS: Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. CONCLUSIONS: The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

Antiosteoporosis effects of a marine antimicrobial peptide pardaxin via regulation of the osteogenesis pathway.

Antimicrobial peptides (AMPs) are known to play an important role in natural immunity. Moreover, the diverse biological activities of AMPs showed great potency in treating many diseases. Thus, in this study, we used an AMP, that is, pardaxin, from a marine fish (Pardachirus marmoratus), which has been reported to possess antibacterial and antitumor activities. We first investigated the mechanisms of pardaxin in promoting osteogenic differentiation in vitro and in vivo. As per our data, it was determined that pardaxin could stimulate bone morphogenetic protein-2 (BMP-2) and downstream cascade. The activation of BMP-2 could further induce the phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Additionally, the activation of p-Akt and p-ERK could prompt the elevation and translocation of runt-related transcription factor 2 (runx-2), which is associated with osteoblast differentiation. The translocation of runx-2 initiated transcription and translation of osteogenesis-related markers, including alkaline phosphatase (ALP), osterix, and osteocalcin. Pardaxin significantly facilitated preosteoblast cells in mineralization and reversed dexamethasone- (DM-) induced zebrafish bone formation deficiency by activating the osteogenesis pathway. Therefore, we suggest that pardaxin could be a possible candidate for osteoporosis treatment and a promising therapeutic agent.

In Silico Protein-Protein Interaction of Pterois volitans Venom with Cancer Inducers of Helicobacter pylori.

Gastric cancer is a pathological condition induced by the bacteria Helicobacter pylori. Targeting the key virulence factors of H. pylori causing gastric cancer is a promising method for treating gastric cancer. Recently, research has been focused on analyzing the adrenergic, cholinergic, and anti-cancer properties of their venom proteins. Testing the anti-cancer activity of the lethal proteins in the venom of P. volitans provides a bioactive compound for cancer treatment. Still, it is also helpful to eliminate the ecological imbalance caused by these fish in the marine environment. This study focuses on an in silico approach using Z-dock to analyze the bioactive prospective of the venom proteins of P. volitans against the essential virulence proteins of H. pylori responsible for inducing cancer. Our in silico docking study using a computational model of the venom proteins and H. pylori proteins has displayed the possible interactions between these proteins. The results revealed that P. volitans hyaluronidase and PV toxin's venom proteins effectively interact with H. pylori proteins Cag A, Cag L, GGT, Cag D, and urease that may be promising proteins in cancer therapy.

Ciguatera fish poisoning in the age of discovery and the age of enlightenment.

BACKGROUND: Ciguatera fish poisoning (CFP) is the most common poisoning from seafood consumption with an estimated 50,000 cases per year worldwide. Attention to this malady in the English language literature has grown over the past five decades. Endemic areas include the South Pacific Ocean and the Caribbean Sea. It is likely that CFP has been present since ancient times, but records to substantiate this are scarce. OBJECTIVE: This historical review looks for clues in earlier writings about potential encounters with CFP as Europeans sailed farther from home into these endemic regions with little idea of what awaited them. We divide these records into the Age of Discovery and the Age of Enlightenment. METHODS: Review of available historical texts written by or about early European explorers with descriptions of illness attributed to eating fish. RESULTS: Fish poisonings appear in translated writings of early Spanish and Portuguese explorers in the 1500s, the writings of Captain James Cook's voyages to the South Pacific, and in Captain William Bligh's fateful voyage after the Mutiny on the Bounty. The most credible description of CFP comes from an early author in the Spanish colony of Cuba in the late 1700s. CONCLUSIONS: Although the quality of the observations varies, Parra in Cuba likely experienced CFP. Plausible CFP for Cook in the South Pacific and Locke in the Bahamas as both have elements of CFP. The descriptions from Quiros, Anghira, and Bligh lack sufficient detail to verify or to refute completely the possibility of CFP.

Fish Cytolysins in All Their Complexity.

The majority of the effects observed upon envenomation by scorpaenoid fish species can be reproduced by the cytolysins present in their venoms. Fish cytolysins are multifunctional proteins that elicit lethal, cytolytic, cardiovascular, inflammatory, nociceptive, and neuromuscular activities, representing a novel class of protein toxins. These large proteins (MW 150-320 kDa) are composed by two different subunits, termed α and ß, with about 700 amino acid residues each, being usually active in oligomeric form. There is a high degree of similarity between the primary sequences of cytolysins from different fish species. This suggests these molecules share similar mechanisms of action, which, at least regarding the cytolytic activity, has been proved to involve pore formation. Although the remaining components of fish venoms have interesting biological activities, fish cytolysins stand out because of their multifunctional nature and their ability to reproduce the main events of envenomation on their own. Considerable knowledge about fish cytolysins has been accumulated over the years, although there remains much to be unveiled. In this review, we compiled and compared the current information on the biochemical aspects and pharmacological activities of fish cytolysins, going over their structures, activities, mechanisms of action, and perspectives for the future.

Putative poison gland in the thorny catfish Acanthodoras spinosissimus (Siluriformes: Doradidae) / Suposta glândula de veneno no bagre rebeca Acanthodoras spinosissimus (Siluriformes: Doradidae)

Acanthodoras is the only genus of catfish known to secrete a conspicuous and abundant milky-looking substance through an axillary pore located just below the base of the posterior cleithral process. Despite this remarkable feature, there is no published information on the anatomical structures that produce the secretion and its possible biological/ecological functions. Dissection and histological analysis of preserved specimens of A. spinosissimus revealed the presence of a saccular axillary gland with large, binuclear secretory cells, similar to those found in other poisonous catfish. Secretory cells near the lumen appear to lose nuclei and become filled with secretory products, possibly with proteinaceous elements, as indicated by their eosinophilic appearance. As far as we know, the saccular morphology of the gland appears to constitute a unique characteristic of Acanthodoras among Doradidae catfishes. Further studies are necessary to determine the chemical composition of the secretion, as well as its possible uses by the catfish in its natural environment.

Acanthodoras é o único gênero de bagre conhecido por secretar uma substância de aparência leitosa conspícua e abundante através de um poro axilar localizado logo abaixo da base do processo cleitral posterior. Apesar dessa característica marcante, não há informações publicadas sobre as estruturas anatômicas que produzem a secreção, nem sobre suas possíveis funções biológicas/ecológicas. A dissecção e análise histológica de espécimes preservados de A. spinosissimus revelaram a presença de uma glândula axilar sacular com células secretoras binucleares, semelhantes às encontradas em outros bagres venenosos. As células secretoras próximas ao lúmen parecem perder os núcleos e são preenchidas com produtos secretores, possivelmente com elementos proteicos, conforme indicado por sua aparência eosinofílica. Até onde sabemos, a morfologia sacular da glândula parece constituir uma característica única de Acanthodoras entre os bagres Doradidae. Mais estudos são necessários para determinar a composição química da secreção, bem como seus possíveis usos pelo bagre em seu ambiente natural.

The Natterin Proteins Diversity: A Review on Phylogeny, Structure, and Immune Function.

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Anti-Glioma Effect of Pseudosynanceia melanostigma Venom on Isolated Mitochondria from Glioblastoma Cells

Background: Glioblastoma is the most common primary malignant tumor of the central nervous system that occurs in the spinal cord or brain. Pseudosynanceia melanostigma is a venomous stonefish in the Persian Gulf, which our knowledge about is little. This study's goal is to investigate the toxicity of stonefish crude venom on mitochondria isolated from U87 cells. Methods: In the first stage, we extracted venom stonefish and then isolated mitochondria have exposed to different concentrations of venom. Finally, mitochondrial toxicity parameters (Succinate dehydrogenase (SDH) activity, Reactive oxygen species (ROS), cytochrome c release, Mitochondrial Membrane Potential (MMP), and mitochondrial swelling) have evaluated. Results: To determine mitochondrial parameters, we used 115, 230, and 460 µg/ml concentrations. The results of our study show that the venom of stonefish selectively increases upstream parameters of apoptosis such as mitochondrial swelling, cytochrome c release, MMP collapse and ROS. Conclusion: This study suggests that Pseudosynanceia melanostigma crude venom has selectively caused toxicity by increasing active mitochondrial oxygen radicals. This venom could potentially be a candidate for the treatment of glioblastoma.

In Silico Target Prediction of Overexpressed microRNAs from LPS-Challenged Zebrafish (Danio rerio) Treated with the Novel Anti-Inflammatory Peptide TnP.

miRNAs regulate gene expression post-transcriptionally in various processes, e.g., immunity, development, and diseases. Since their experimental analysis is complex, in silico target prediction is important for directing investigations. TnP is a candidate peptide for anti-inflammatory therapy, first discovered in the venom of Thalassophryne nattereri, which led to miRNAs overexpression in LPS-inflamed zebrafish post-treatment. This work aimed to predict miR-21, miR-122, miR-731, and miR-26 targets using overlapped results of DIANA microT-CDS and TargetScanFish software. This study described 513 miRNAs targets using highly specific thresholds. Using Gene Ontology over-representation analysis, we identified their main roles in regulating gene expression, neurogenesis, DNA-binding, transcription regulation, immune system process, and inflammatory response. miRNAs act in post-transcriptional regulation, but we revealed that their targets are strongly related to expression regulation at the transcriptional level, e.g., transcription factors proteins. A few predicted genes participated concomitantly in many biological processes and molecular functions, such as foxo3a, rbpjb, rxrbb, tyrobp, hes6, zic5, smad1, e2f7, and npas4a. Others were particularly involved in innate immunity regulation: il17a/f2, pik3r3b, and nlrc6. Together, these findings not only provide new insights into the miRNAs mode of action but also raise hope for TnP therapy and may direct future experimental investigations.