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In vitro cell culture systems serve as instrumental platforms for probing biological phenomena and elucidating intricate cellular mechanisms. These systems afford researchers the opportunity to scrutinize cellular responses within a regulated environment, thereby circumventing the ethical and logistical challenges associated with in vivo experimentation. Three-dimensional (3D) cell cultures have emerged as a viable alternative to mimic in vivo environments. Within this context, spheroids are recognized as one of the most straightforward and efficacious models, presenting a promising substitute for conventional monolayer cultures. The application of 3D cultures of fish cells remains limited, focusing mainly on physiological and morphological characterization studies. However, given the capacity of spheroids to emulate in vivo conditions, researchers are exploring diverse applications of these 3D cultures. These include eco-toxicology, immunology, drug screening, endocrinology, and metabolism studies, employing a variety of cell types such as fibroblasts, hepatocytes, embryonic cells, gonadal cells, gastrointestinal cells, and pituitary cells. This review provides a succinct overview, concentrating on the most frequently employed methods for generating fish cell spheroids and their applications to date. The aim is to compile and highlight the significant contributions of these methods to the field and their potential for future research. [ABSTRACT FROM AUTHOR]

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-Y, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naive monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated FcY receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches. [ABSTRACT FROM AUTHOR]

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually fed with diets supplemented with Omega-3 Long Chain Polyunsaturated fatty acids (LC-PUFAs), the lack of knowledge about the optimal requirements for fatty acids or about their impact on mechanisms that regulate skeletal development has impeded the design of diets that could improve bone formation during larval stages when the majority of skeletal anomalies appear. In this study, Argyrosomus regius larvae were fed different levels of Omega-3s (2.6% and 3.6% DW on diet) compared to a commercial diet. At 28 days after hatching (DAH), their transcriptomes were analyzed to study the modulation exerted in gene expression dynamics during larval development and identify impacted genes that can contribute to skeletal formation. Mainly, both levels of supplementation modulated bone-cell proliferation, the synthesis of bone components such as the extracellular matrix, and molecules involved in the interaction and signaling between bone components or in important cellular processes. The 2.6% level impacted several genes related to cartilage development, denoting a special impact on endochondral ossification, delaying this process. However, the 3.6% level seemed to accelerate this process by enhancing skeletal development. These results offered important insights into the impact of dietary Omega-3 LC-PUFAs on genes involved in the main molecular mechanism and cellular processes involved in skeletal development. [ABSTRACT FROM AUTHOR]

Simple Summary: Probiotic supplementation plays a vital role in European sea bass wellbeing. Accordingly, it is important to increase our knowledge of and experience on their mechanisms of action and host effects. Although information on these aspects is available, further studies are needed to achieve optimal European sea bass aquaculture. European sea bass production has increased in recent decades. This increase is associated with an annually rising demand for sea bass, which encourages the aquaculture industries to increase their production to meet that demand. However, this intensification has repercussions on the animals, causing stress that is usually accompanied by dysbiosis, low feed-conversion rates, and immunodepression, among other factors. Therefore, the appearance of pathogenic diseases is common in these industries after immunodepression. Seeking to enhance animal welfare, researchers have focused on alternative approaches such as probiotic application. The use of probiotics in European sea bass production is presented as an ecological, safe, and viable alternative in addition to enhancing different host parameters such as growth performance, feed utilization, immunity, disease resistance, and fish survival against different pathogens through inclusion in fish diets through vectors and/or in water columns. Accordingly, the aim of this review is to present recent research findings on the application of probiotics in European sea bass aquaculture and their effect on growth performance, microbial diversity, enzyme production, immunity, disease resistance, and survival in order to help future research. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) infection is the major risk factor associated with the development of gastric cancer. The transition from normal mucosa to non-atrophic gastritis, triggered primarily by H. pylori infection, initiates precancerous lesions which may then progress to atrophic gastritis and intestinal metaplasia. Further progression to dysplasia and gastric cancer is generally believed to be attributable to processes that no longer require the presence of H. pylori. The responses that develop upon H. pylori infection are directly mediated through the action of bacterial virulence factors, which drive the initial events associated with transformation of infected gastric cells. Besides genetic and to date poorly defined environmental factors, alterations in gastric cell stress-adaptivemechanisms due to H. pylori appear to be crucial during chronic infection and gastric disease progression. Firstly, H. pylori infection promotes gastric cell death and reduced epithelial cell turnover in the majority of infected cells, resulting in primary tissue lesions associated with an initial inflammatory response. However, in the remaining gastric cell population, adaptive responses are induced that increase cell survival and proliferation, resulting in the acquisition of potentially malignant characteristics that may lead to precancerous gastric lesions. Thus, deregulation of these intrinsic survival-related responses to H. pylori infection emerge as potential culprits in promoting disease progression. This review will highlight the most relevant cellular adaptive mechanisms triggered upon H. pylori infection, including endoplasmic reticulum stress and the unfolded protein response, autophagy, oxidative stress, and inflammation, together with a subsequent discussion on how these factors may participate in the progression of a precancerous lesion. Finally, this review will shed light on how these mechanisms may be exploited as pharmacological targets, in the perspective of opening up new therapeutic alternatives for non-invasive risk control in gastric cancer. [ABSTRACT FROM AUTHOR]

Flow cytometry (FC) is a straightforward, highly specific and sensitive procedure, which provides objective and quantitative recording of fluorescent signals from individual cells. FC has been used widely in the area of aquaculture research due mainly to the development of monoclonal antibodies (mabs). Phagocyte activity of head–kidney macrophages of several fish species, antibody production against specific antigens or detection of viruses inside immune cells are some of the several possible analysis approached by this assay. The aim of this work is to analyse and to compare kinetics of IgM-positive cells in the gill and spleen of sea bream juveniles after bath immunization against Photobacterium damselae subsp. piscicida (Phdp) using a direct staining method. Fish were immunized with three different inactivated vaccines prepared with 94/99 antigen and a commercial vaccine was used as a positive control. The effect on IgM-positive cells post-vaccination was compared among groups. Results show that the direct staining method with mabs is an effective and repeatable method for staining sea bream IgM-positive cells. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1a knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori-induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection. [ABSTRACT FROM AUTHOR]

Hafnia alveiis a Gram negative bacterium that belongs to the familyEnterobacteriaceae. It is part of the intestinal flora of humans and animals, moreover it has been described from natural environments and foodstuffs. In human medicine is considered an opportunistic bacterium that causes different infections associated to underlying illnesses or predisposing factors as immunocompromised patients. The knowledge and literature about this bacterium in veterinary medicine is scarce, highlighting its importance in gastropods and bees, and it has also been described in poultry, mares, fish and ruminants producing various clinicopathologic signs. [ABSTRACT FROM PUBLISHER]

In this work, we evaluated the effect of the probioticEnterococcus gallinarumL-1 on the cellular immune system of four different fish species of great interest in aquaculture such as gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax), meagre (Argyrosomus regius) and red porgy (Pagrus pagrus). Phagocytic activity, respiratory burst and peroxidase content of leucocytes were observed 30 minutes after incubation with the probioticE. gallinarumstrain L-1, alive or inactivated with heat shock or ultraviolet (UV) light at different concentrations of 107, 108and 109cfu mL−1(final concentration 106, 107and 108cfu mL−1).E. gallinarumproduced dose-dependent increments in respiratory burst in red porgy, sea bream and sea bass leucocytes. About 106and 107cfu mL−1of live and inactivated bacteria with no stimulation of the respiratory burst activity of sea bream and red porgy head kidney leucocytes was shown. The highest values of peroxidase content were observed in red porgy cells with stimulation indexes higher than 1 in each treatment. Statistical analysis revealed that differences were only significant in sea bream where UV light-inactivated bacteria denote statistically significant differences (P< 0.05) with respect to other treatments. Highest values of phagocytic activity were obtained in sea bream leucocytes incubated with live bacteria (26% ± 1.88), where significant differences (P< 0.05) with other species were detected. Our results suggest that thein vitroassays may be useful in optimising their effective dose and viability for the immunomodulatory effects of probiotic bacteria, althoughin vivostudies are necessary to confirm the immunomodulatory effect of this strain. [ABSTRACT FROM PUBLISHER]

Coffee consumption decreases the risk of oxidative stress-related diseases. The by-product obtained after brewing process (spent coffee) also has antioxidant capacity. Spent coffee and coffee brews (filter and espresso) extracts were obtained from Arabica and Robusta coffees, respectively. Spent coffee showed slightly high amounts in chlorogenic acids, but caffeine content was similar to their respective coffee brew. All samples exhibited strong protection activity against indirect acting mutagen 2-AF (≤92%), whereas the protection against NPD (direct mutagen) was 12–35% (Ames Test). The growth inhibition of common food-borne pathogen and food spoilage microorganisms by coffee extracts was also studied. Spent coffee showed antimicrobial activity, mainly against Gram-positive bacteria ( Staphylococcus aureus, Listeria monocytogenes ) and yeast ( Candida albicans ). The role of phenolic acids, caffeine and melanoidins in the antimutagenic and antimicrobial activities is discussed. Thus, spent coffee extracts could be a potential source of bioactive compounds, thereby becoming a promising new functional food ingredient. [ABSTRACT FROM AUTHOR]

Simple Summary: Dietary hydroxy-selenomethionine (OH-SeMet) reduces oxidative stress and modulates immune response against bacterial infection in fish. However, the effect of OH-SeMet on essential fatty acids with a high oxidation risk or on the response against viral infection has not been sufficiently studied. This study aimed to assess the impact of dietary OH-SeMet supplementation on whole-body fatty acid profiles and response against viral infection. Gilthead seabream (Sparus aurata) juveniles were fed for 91 days with three experimental diets, a control diet without Se supplementation (0.29 mg Se kg diet−1) and two diets supplemented with OH-SeMet (0.52 and 0.79 mg Se kg diet−1). Afterwards, a crowding stress challenge and an anti-viral response challenge were conducted. Selenium (Se), proximate and fatty acid composition of diets and body tissues were analyzed, as well as plasma cortisol and the antiviral response protein Mx gene expression. Elevation in dietary Se (from 0.29 to 0.79 mg kg−1) proportionally raised Se contents in body tissues (from 0.79 to 1.35 mg kg−1), increased lipid contents in whole body (from 9.46 to 10.83%), and promoted the retention and synthesis of n-3 polyunsaturated fatty acids (from 44.59 to 72.91%), reducing monounsaturated (from 44.07 to 42.00 %) and saturated fatty acids (29.77 to 26.92 %) contents in whole-body lipids. Additionally, it increased 2 h post-stress plasma cortisol levels and after poly I:C injection up-regulated Mx and other immune response related genes, showing, for the first time in gilthead seabream, the importance of dietary Se levels on antiviral defense. The supplementation of fish diets with OH-SeMet reduces oxidative stress and modulates immune response against bacterial infection. However, despite the importance of essential polyunsaturated fatty acids in fish nutrition and their high risk of oxidation, the potential protective effect of OH-SeMet on these essential fatty acids has not been studied in detail. Moreover, while viral infection is very relevant in seabream production, no studies have focused the Se effects against viral infection. The aim of the present study was to assess the impact of dietary supplementation with OH-SeMet on gilthead seabream fatty acid profiles, growth performance and response against viral infection. Gilthead seabream juveniles (21.73 ± 0.27 g) were fed for 91 days with three experimental diets, a control diet without supplementation of Se (0.29 mg Se kg diet−1) and two diets supplemented with OH-SeMet (0.52 and 0.79 mg Se kg diet−1). A crowding stress test was performed at week 7 and an anti-viral response challenge were conducted at the end of the feeding trial. Selenium, proximate and fatty acid composition of diets and body tissues were analyzed. Although fish growth was not affected, elevation in dietary Se proportionally raised Se content in body tissues, increased lipid content in the whole body and promoted retention and synthesis of n-3 polyunsaturated fatty acids. Specifically, a net production of DHA was observed in those fish fed diets with a higher Se content. Additionally, both monounsaturated and saturated fatty acids were significantly reduced by the increase in dietary Se. Despite the elevation of dietary Se to 0.79 mg kg−1 not affecting basal cortisol levels, 2 h post-stress plasma cortisol levels were markedly increased. Finally, at 24 h post-stimulation, dietary OH-SeMet supplementation significantly increased the expression of the antiviral response myxovirus protein gene, showing, for the first time in gilthead seabream, the importance of dietary Se levels on antiviral defense. [ABSTRACT FROM AUTHOR]

The risk of developing gastric cancer is strongly linked to Helicobacter pylori (H. pylori) infection. Alternatively, autophagy is a conserved response that is important in cellular homeostasis and provides protection against bacterial infections. Although H. pylori is typically considered an extracellular bacterium, several reports indicate that it internalizes, possibly to avoid exposure to antibiotics. Mechanisms by which H. pylori manipulates host cell autophagic processes remain unclear and, importantly, none of the available studies consider a role for the secreted H. pylori virulence factor gamma-glutamyltranspeptidase (HpGGT) in this context. Here, we identify HpGGT as a novel autophagy inhibitor in gastric cells. Our experiments revealed that deletion of HpGGT increased autophagic flux following H. pylori infection of AGS and GES-1 gastric cells. In AGS cells, HpGGT disrupted the late stages of autophagy by preventing degradation in lysosomes without affecting lysosomal acidification. Specifically, HpGGT impaired autophagic flux by disrupting lysosomal membrane integrity, which leads to a decrease in lysosomal cathepsin B activity. Moreover, HpGGT was necessary for efficient internalization of the bacteria into gastric cells. This important role of HpGGT in internalization together with the ability to inhibit autophagy posits HpGGT as a key virulence factor in the development of gastric cancer. [ABSTRACT FROM AUTHOR]