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N 6 -Adenosine methylation (m 6 A) is a prevalent post-transcriptional modification of mRNA, with YTHDC1 being the reader protein responsible for recognizing this modification in the cell nucleus. Here, we present a protein structure-based medicinal chemistry campaign that resulted in the YTHDC1 inhibitor 40 , which shows an equilibrium dissociation constant ( K d ) of 49 nM. The crystal structure of the complex (1.6 Å resolution) validated the design. Compound 40 is selective against the cytoplasmic m 6 A-RNA readers YTHDF1-3 and YTHDC2 and shows antiproliferative activity against the acute myeloid leukemia (AML) cell lines THP-1, MOLM-13, and NOMO-1. For the series of compounds that culminated into ligand 40 , the good correlation between the affinity in the biochemical assay and antiproliferative activity in the THP-1 cell line provides evidence of YTHDC1 target engagement in the cell. The binding to YTHDC1 in the cell is further supported by the cellular thermal shift assay. Thus, ligand 40 is a tool compound for studying the role of YTHDC1 in AML.

Methylation of adenine N6 (m 6 A) is the most frequent RNA modification. On mRNA, it is catalyzed by the METTL3-14 heterodimer complex, which plays a key role in acute myeloid leukemia (AML) and other types of blood cancers and solid tumors. Here, we disclose the first proteolysis targeting chimeras (PROTACs) for an epitranscriptomics protein. For designing the PROTACs, we made use of the crystal structure of the complex of METTL3-14 with a potent and selective small-molecule inhibitor (called UZH2). The optimization of the linker started from a desfluoro precursor of UZH2 whose synthesis is more efficient than that of UZH2. The first nine PROTAC molecules featured PEG- or alkyl-based linkers, but only the latter showed cell penetration. With this information in hand, we synthesized 26 PROTACs based on UZH2 and alkyl linkers of different lengths and rigidity. The formation of the ternary complex was validated by a FRET-based biochemical assay and an in vitro ubiquitination assay. The PROTACs 14 , 20 , 22 , 24 , and 30 , featuring different linker types and lengths, showed 50% or higher degradation of METTL3 and/or METTL14 measured by Western blot in MOLM-13 cells. They also showed substantial degradation on three other AML cell lines and prostate cancer cell line PC3., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The skin represents an indicator of an animal's health status. Causes of cutaneous diseases in fish most often trace back to biological agents. However, fish skin diseases can also arise from a complex interaction of infectious and non-infectious causes, making it more difficult to identify a specific aetiology. In the period between April and September of the years 2019-2022, four koi carp (Cyprinus carpio koi) from two European countries presented with multifocal, irregularly round, few mm to 1 cm, variably raised cutaneous reddened areas. The fish displayed good general condition. Cutaneous samples, investigated by microbiological and molecular methods and microscopy, did not indicate a primary pathogenic agent. Gross and histological findings of the cutaneous biopsies were consistent with a multifocal/reactive process centred on dermal vessels. The histological features were reminiscent of angiomatosis, a benign proliferative condition affecting the dermal vessels of mammals, including human patients. The clinical-pathological presentation and the dermatologic condition that affected the koi carp are discussed and compared with the veterinary and human literature., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Aquaculture is the fastest-growing food-producing sector, with a global production of 122.6 million tonnes in 2020. Nonetheless, aquatic animal production can be hampered by the occurrence of viral diseases. Furthermore, intensive farming conditions and an increasing number of reared fish species have boosted the number of aquatic animals' pathogens that researchers have to deal with, requiring the quick development of new detection and study methods for novel unknown pathogens. In this respect, the molecular tools have significantly contributed to investigating thoroughly the structural constituents of fish viruses and providing efficient detection methods. For instance, next-generation sequencing has been crucial in reassignment to the correct taxonomic family, the sturgeon nucleo-cytoplasmic large DNA viruses, a group of viruses historically known, but mistakenly considered as iridoviruses. Further methods such as in situ hybridisation allowed objectifying the role played by the pathogen in the determinism of disease, as the cyprinid herpesvirus 2, ostreid herpesvirus 1 and betanodaviruses. Often, a combination of molecular techniques is crucial to understanding the viral role, especially when the virus is detected in a new aquatic animal species. With this paper, the authors would critically revise the scientific literature, dealing with the molecular techniques employed hitherto to study the most relevant finfish and shellfish viral pathogens.

Aquaculture is a constantly growing sector. The intensification of fish production and the movement of aquatic animals could cause the spread of infectious diseases. Remarkably, the diffusion of viral agents represents the major bottleneck for finfish production, and viral encephalopathy and retinopathy (VER) is considered the most impacting disease for Mediterranean aquaculture. No effective therapies are available to contrast VER, and vaccination can be applied only in grow-out facilities. Hence, programs to minimize the sanitary risks in farms are paramount to implementing hygienic standards and biosecurity. This study aimed to evaluate the in vitro virucidal activity of a peroxy-acid disinfectant (Virkon ® S, DuPont, Sudbury, UK) towards the two NNV strains most widespread in the Mediterranean Sea. Remarkably, two protocols were applied to assess the virucidal activity under different conditions of use: the suspension test and the net test. The latter has been applied to evaluate the efficacy of the biocide on instruments, simulating the in-field application. The obtained results demonstrated the suitability of the tested biocide for NNV inactivation, being effective under some of the tested conditions. However, the presence of organic matter, the concentration of the product, and the application conditions can significantly affect the result of the disinfection procedure.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method's detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains., Competing Interests: The authors have declared that no competing interests exist.

Marine gastropods represent a major food source for higher trophic levels and an important source of animal protein for humans. Like bivalve molluscs, gastropods can accumulate several types of contaminants; however, the bioaccumulation of microorganisms, particularly viruses, has been poorly investigated in these animals. This study focused on gastropods ( Tritia mutabilis , Bolinus brandaris and Rapana venosa ) collected during the fishing season from 2017 to 2021 in the north-western Adriatic Sea, and on clams ( Ruditapes philippinarum ) harvested in the same geographical area, in order to evaluate the presence of human and fish viruses in their tissues. A virological investigation was carried out on the digestive gland using molecular methods. The presence of hepatitis A virus was detected in one sample, whereas noroviruses were not present in the investigated specimens. Regarding fish viruses, it was possible to detect the presence of nervous necrosis virus (NNV) in 26.5% of the analyzed gastropods; however, the histological examination did not show any pathological changes in the nervous tissue in both NNV-positive and -negative batches. As a whole, the investigated gastropods showed the ability to bioaccumulate viruses; however, lower contamination by human viruses compared to bivalve molluscs was pointed out, posing a minor concern to human health.

Cichlids include hundreds of species with a high economic value for aquaculture. These fish are subjected to intensive trade and farming that expose them to the risk of infectious diseases. This work focuses on ornamental cichlids held in an aquarium commercial facility presenting emaciation, in order to evaluate the presence of lesions in fish skin and organs. The fish were sampled during routine management activities and subjected to pathological and molecular investigations. The presence of lymphocystis disease virus, typically associated with cutaneous nodular disease, was ruled out. Histologically, they presented granulomas in the spleen, sometimes extending to the other visceral organs. Bacterial heat-shock protein 65 PCR products were detected in tissues associated, in the majority of cases, with granulomas; molecular investigation identified Mycobacterium spp. in two cases and Cutibacterium acnes in seven cases. Immunoreactivity to anti-Mycobacterium and anti-C. acnes antibodies was detected within granulomas. The presence of C. acnes within granuloma is elucidated for the first time in fish; however, similarly to what is found in humans, this bacterium could be harmless in normal conditions, whereas other contributing factors would be required to trigger a granulomatogenous response. Further confirmation by bacterial culture, as well as using large-scale studies in more controlled situations, is needed.