Roles and occurrences of microbiota in the osmoregulatory organs, gills and gut, in marine medaka upon hypotonic stress.

Gills and gut are the two primary osmoregulatory organs in fish. Recently, studies have expanded beyond the osmoregulatory mechanisms of these organs to explore the microbiota communities inhabiting them. It is now known that microbial communities in both organs shift in response to osmotic stress. However, there are limited studies identifying the major contributors and co-occurrence among these microbiota in both organs under seawater and freshwater transfer conditions. The current data mining report performed a bioinformatics analysis on two previous published datasets from our group, aiming to provide insights into host-bacteria relationships under osmotic stress. We divided the samples into four groups: control seawater gills (LSW); control seawater gut (TSW); freshwater transfer gills (LFW); and freshwater transfer gut (TFW). Our results showed that LSW had higher diversities, richness, and evenness compared to TSW. However, both the LFW and LSW did not show any significant differences after the freshwater transfer experiment. We further applied co-occurrence network analysis and, for the first time, reported on the interactions of taxa shaping the community structure in these two organs. Moreover, we identified enriched ectoine biosynthesis in seawater samples, suggesting its potential role in seawater environments. Increased mRNA expression levels of Na+/K+-atpase, and cftr, were observed in gills after 6 h of ectoine treatment. These findings provide a foundation for future studies on host-bacteria interactions under osmotic stress.

Screening for oncogenic AF1q expression predicts disease recurrence in gastric cancer patients.

AF1q associates with tumor progression and metastases upon WNT signaling. The downstream WNT target CD44 has demonstrated prognostic significance in gastric cancer (GC). This study evaluates the impact of AF1q on tumor stage and survival in GC patients. Immunohistochemical marker expression was analyzed and data were processed to correlation and survival analysis. Out of 182 GC samples, 178 (97.8%) showed moderate to high AF1q expression (p < 0.001), these samples correlated with positive lymph node stage (p = 0.036). In a subgroup analysis of patients with nodal-positive GC (n = 129, 70.9%), enhanced tumoral AF1q expression resulted in impaired recurrence-free survival (RFS, p = 0.030). Enhanced tumoral CD44 expression resulted in impaired disease-specific survival (DSS) in the subgroup of patients with nodal-positive GC (p = 0.031) as well as in the overall GC group (p = 0.005). AF1q demonstrated as an independent prognostic marker for RFS (p = 0.035) and CD44 for DSS (p = 0.036). AF1q has shown potential for prognostication of RFS in GC patients and is predominantly expressed in nodal-positive GC. Testing AF1q provides a possibility of identifying patients with locoregional (and advanced) disease, particularly at risk for disease recurrence. Implementing AF1q into the diagnostic process may facilitate screening, prognosis estimation as well as consideration of preoperative multimodal treatment in patients qualifying for elective upfront surgery.

Microplastic exposure disturbs sleep structure, reduces lifespan, and decreases ovary size in Drosophila melanogaster.

The organ-specific toxicity resulting from microplastic (MP) exposure has been extensively explored, particularly concerning the gut, liver, testis, and lung. However, under natural conditions, these effects are not restricted to specific organs or tissues. Investigating whether MP exposure presents a systemic threat to an entire organism, impacting factors such as lifespan, sleep, and fecundity, is essential. In this study, we investigated the effects of dietary exposure to two different doses of MPs (1-5 µm) using the terrestrial model organism Drosophila melanogaster. Results indicated that the particles caused gut damage and remained within the digestive system. Continuous MP exposure significantly shortened the lifespan of adult flies. Even short-term exposure disrupted sleep patterns, increasing the length of daytime sleep episodes. Additionally, one week of MP exposure reduced ovary size, with a trend towards decreased egg-laying in mated females. Although MPs did not penetrate the brain or ovaries, transcriptome analysis revealed altered gene expression in these tissues. In the ovary, Gene Ontology (GO) analysis indicated genotoxic effects impacting inflammation, circadian regulation, and metabolic processes, with significant impacts on extracellular structure-related pathways. In the brain, GO analysis identified changes in pathways associated with proteolysis and carbohydrate metabolism. Overall, this study provides compelling evidence of the systemic negative effects of MP exposure, highlighting the urgent need to address and mitigate environmental MP pollution.

Microplastics from face mask impairs sperm motility.

The COVID-19 pandemic has resulted in unprecedented plastic pollution from single-used personal protective equipment (PPE), especially face masks, in coastal and marine environments. The secondary pollutants, microplastics from face masks (mask MP), rise concern about their detrimental effects on marine organisms, terrestrial organisms and even human. Using a mouse model, oral exposure to mask MP at two doses, 0.1 and 1 mg MP/day for 21 days, caused no change in animal locomotion, total weight, or sperm counts, but caused damage to sperm motility with increased curvilinear velocity (VCL). The high-dose mask MP exposure caused a significant decrease in linearity (LIN) of sperm motility. Further testicular transcriptomic analysis revealed perturbed pathways related to spermatogenesis, oxidative stress, inflammation, metabolism and energy production. Collectively, our findings substantiate that microplastics from face masks yield adverse effects on mammalian reproductive capacity, highlighting the need for improved plastic waste management and development of environmentally friendly materials.

Size-dependent deleterious effects of nano- and microplastics on sperm motility.

INTRODUCTION: Growing concerns regarding the reproductive toxicity associated with daily life exposure to micro-/nano-plastics (abbreviated as MNPs) have become increasingly prevalent. In reality, MNPs exposure involves a heterogeneous mixture of MNPs of different sizes rather than a single size. METHODS: In this study, an oral exposure mouse model was used to evaluate the effects of MNPs of four size ranges: 25-30 nm, 1-5 µm, 20-27 µm, and 125-150 µm. Adult male C57BL/6 J mice were administered environmentally relevant concentrations of 0.1 mg MNPs/day for 21 days. After that, open field test and computer assisted sperm assessment (CASA) were conducted. Immunohistochemical analyses of organ and cell type localization of MNPs were evaluated. Testicular transcriptome analysis was carried out to understand the molecular mechanisms. RESULTS: Our result showed that MNPs of different size ranges all impaired sperm motility, with a decrease in progressive sperm motility, linearity and straight-line velocity of sperm movement. Alterations did not manifest in animal locomotion, body weight, or sperm count. Noteworthy effects were most pronounced in the smaller MNPs size ranges (25-30 nm and 1-5 µm). Linear regression analysis substantiated a negative correlation between the size of MNPs and sperm curvilinear activity. Immunohistochemical analysis unveiled the intrusions of 1-5 µm MNPs, but not 20-27 µm and 125-150 µm MNPs, into Leydig cells and testicular macrophages. Further testicular transcriptomic analysis revealed perturbations in pathways related to spermatogenesis, oxidative stress, and inflammation. Particularly within the 1-5 µm MNPs group, a heightened perturbation in pathways linked to spermatogenesis and oxidative stress was observed. CONCLUSIONS: Our data support the size-dependent impairment of MNPs on sperm functionality, underscoring the pressing need for apprehensions about and interventions against the escalation of environmental micro-/nano-plastics contamination. This urgency is especially pertinent to small-sized MNPs.

p53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model.

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

Transcriptomic analysis reveals mitochondrial dysfunction in the pathogenesis of Nager syndrome in sf3b4-depleted zebrafish.

Nager syndrome (NS) is a rare acrofacial dysostosis caused by heterozygous loss-of-function variants in the splicing factor 3B subunit 4 (SF3B4). The main clinical features of patients with NS are characterized by facial-mandibular and preaxial limb malformations. The migration and specification of neural crest cells are crucial for craniofacial development, and mitochondrial fitness appears to play a role in such processes. Here, by analyzing our previously published transcriptome dataset, we aim to investigate the potential involvement of mitochondrial components in the pathogenesis of craniofacial malformations, especially in sf3b4 mutant zebrafish. We identified that oxidative phosphorylation (OXPHOS) defects and overproduction of reactive oxygen species (ROS) due to decreased antioxidants defense activity, which leads to oxidative damage and mitochondrial dysfunction. Furthermore, our results highlight that fish lacking sf3b4 gene, primarily display defects in mitochondrial complex I. Altogether, our findings suggest that mitochondrial dysfunction may contribute to the development of the craniofacial anomalies observed in sf3b4-depleted zebrafish.

Transcriptome alterations in sf3b4-depleted zebrafish: Insights into cataract formation in retinitis pigmentosa model.

Posterior subcapsular cataract (PSC) frequently develops as a complication in patients with retinitis pigmentosa (RP). Despite numerous scientific investigations, the intricate pathomechanisms underlying cataract formation in individuals affected by RP remain elusive. Therefore, our study aims to elucidate the potential pathogenesis of cataracts in an RP model using splicing factor subunit 3b (sf3b4) mutant zebrafish. By analyzing our previously published transcriptome dataset, we identified that, in addition to RP, cataract was listed as the second condition in our transcriptomic analysis. Furthermore, we confirmed the presence of nucleus retention in the lens fiber cells, along with abnormal cytoskeleton expression in both the lens fiber cells and lens epithelial cells in sf3b4-depleted fish. Upon closer examination, we identified 20 differentially expressed genes (DEGs) that played a role in cataract formation, with 95 % of them related to the downregulation of structural lens proteins. Additionally, we also identified that among all the DEGs, 13 % were associated with fibrotic processes. It seems that the significant upregulation of inflammatory mediators, in conjunction with TGF-ß signaling, plays a central role in the cellular biology of PSC and posterior capsular opacification (PCO) in sf3b4 mutant fish. In summary, our study provides valuable insights into cataract formation in the RP model of sf3b4 mutants, highlighting its complexity driven by changes in structural lens proteins and increased cytokines/growth factors.

PFHxS Exposure and the Risk of Non-Alcoholic Fatty Liver Disease.

Perfluorohexanesulfonic acid (PFHxS) is a highly prevalent environmental pollutant, often considered to be less toxic than other poly- and perfluoroalkyl substances (PFASs). Despite its relatively lower environmental impact compared to other PFASs, several studies have suggested that exposure to PFHxS may be associated with disruptions of liver function in humans. Nevertheless, the precise pathomechanisms underlying PFHxS-induced non-alcoholic fatty liver disease (NAFLD) remain relatively unclear. Therefore, this study applied our previously published transcriptome dataset to explore the effects of PFHxS exposure on the susceptibility to NAFLD and to identify potential mechanisms responsible for PFHxS-induced NAFLD through transcriptomic analysis conducted on zebrafish embryos. Results showed that exposure to PFHxS markedly aggravated hepatic symptoms resembling NAFLD and other metabolic syndromes (MetS) in fish. Transcriptomic analysis unveiled 17 genes consistently observed in both NAFLD and insulin resistance (IR), along with an additional 28 genes identified in both the adipocytokine signaling pathway and IR. These shared genes were also found within the NAFLD dataset, suggesting that hepatic IR may play a prominent role in the development of PFHxS-induced NAFLD. In conclusion, our study suggests that environmental exposure to PFHxS could be a potential risk factor for the development of NAFLD, challenging the earlier notion of PFHxS being safer as previously claimed.

Association between glaucoma susceptibility with combined defects in mitochondrial oxidative phosphorylation and fatty acid beta oxidation.

Glaucoma is one of the leading causes of visual impairment and blindness worldwide, and is characterized by the progressive damage of retinal ganglion cells (RGCs) and the atrophy of the optic nerve head (ONH). The exact cause of RGC loss and optic nerve damage in glaucoma is not fully understood. The high energy demands of these cells imply a higher sensitivity to mitochondrial defects. Moreover, it has been postulated that the optic nerve is vulnerable towards damage from oxidative stress and mitochondrial dysfunction. To investigate this further, we conducted a pooled analysis of mitochondrial variants related to energy production, specifically focusing on oxidative phosphorylation (OXPHOS) and fatty acid ß-oxidation (FAO). Our findings revealed that patients carrying non-synonymous (NS) mitochondrial DNA (mtDNA) variants within the OXPHOS complexes had an almost two-fold increased risk of developing glaucoma. Regarding FAO, our results demonstrated that longer-chain acylcarnitines (AC) tended to decrease, while shorter-chain AC tended to increase in patients with glaucoma. Furthermore, we observed that the knocking down cpt1a (a key rate-limiting enzyme involved in FAO) in zebrafish induced a degenerative process in the optic nerve and RGC, which resembled the characteristics observed in glaucoma. In conclusion, our study provides evidence that genes encoding mitochondrial proteins involved in energy metabolisms, such as OXPHOS and FAO, are associated with glaucoma. These findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and may offer potential targets for therapeutic interventions in the future.

Insights from metabolomics and transcriptomics studies on Perfluorohexanesulfonic acid (PFHxS) exposed zebrafish embryos.

Perfluorohexanesulfonic acid (PFHxS) is a short-chain perfluoroalkyl substance widely used to replace the banned perfluorooctanesulfonic acid (PFOS) in various industrial and household products. It can be found in the environment and human bodies; however, its potential toxicities are not well studied. Zebrafish have been extensively used as a model for studying toxicants, and currently, two studies have reported on the toxicity of PFHxS in zebrafish from different approaches. Ulhaq and Tse (J Hazard Mater. 2023; 457: 131722) conducted general biological experiments and applied transcriptomics to demonstrate that PFHxS at a concentration of 5 µM could affect glucose and fatty acid metabolism, leading to oxidative stress, developmental defects, and cell cycle arrest. Xu et al. (Sci Total Environ. 2023; 887: 163770) employed metabolomics and showed that concentrations of various metabolites changed after exposure to 3 and 10 µM PFHxS. As we observed a match between the metabolomics data and our biochemistry experimental findings, we integrated the two studies, which enabled us to unfold the possible mechanism of the deregulated metabolites. We identified 22 differential expressed genes (DEGs) in the tricarboxylic acid (TCA) cycle, 17 DEGs in glcyolytic process, including the critical glucokinase under the carbon metabolism. Besides, genes likes aldehyde dehydrogenases, and histone-lysine N-methyltransferases that participate in lipid peroxidation and amino metabolism respectively were spotted. Lastly, we further strengthen our discoveries by undergoing the gene set enrichment analysis. This article could provide insights into the toxicity of PFHxS, as well as prospects for environmental studies.

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies.

Background: Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations - demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency - enabling unlimited production of modified "off-the-shelf" hPSC-NKs. Methods: hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results: HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions: Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research - improving patient survival and quality of life.

Real-world evidence of incidence and outcomes of aplastic anaemia following administration of immune checkpoint inhibitors.

Aplastic anaemia (AA) is a rare immune-related adverse events (irAEs) after immune checkpoint inhibitors (ICIs) administration with poorly understood incidence and outcomes. We analysed an electronic health record database of 52 303 ICI-treated patients and found 77 (0.15%) cases of AA, with a median onset of 126 days (interquartile range, 58-363 days). The most used treatment for AA was systemic glucocorticoids 60 (77.9%) and 32 (41.6%) patients were able to resume ICI within 1 year. Patients diagnosed with AA had a steep decline in overall survival (OS) within the first 120 days; when compared to propensity score-matched patients without AA, they had a significantly worse OS (hazard ratio 1.72, 95% confidence interval 1.19-2.50; p = 0.003).

Perfluorohexanesulfonic acid (PFHxS) induces oxidative stress and causes developmental toxicities in zebrafish embryos.

Perfluorohexanesulfonic acid (PFHxS) is a short-chain perfluoroalkyl substance widely used to replace the banned perfluorooctanesulfonic acid (PFOS) in different industrial and household products. It has currently been identified in the environment and human bodies; nonetheless, the possible toxicities are not well-known. Zebrafish have been used as a toxicant screening model due to their fast and transparent developmental processes. In this study, zebrafish embryos were exposed to PFHxS for five days, and various experiments were performed to monitor the developmental and cellular processes. Liquid chromatography-mass spectrometry (LC/MS) analysis confirmed that PFHxS was absorbed and accumulated in the zebrafish embryos. We reported that 2.5 µM or higher PFHxS exposure induced phenotypic abnormalities, marked by developmental delay in the mid-hind brain boundary and yolk sac edema. Additionally, larvae exposed to PFHxS displayed facial malformation due to the reduction of neural crest cell expression. RNA sequencing analysis further identified 4643 differentiated expressed transcripts in 5 µM PFHxS-exposed 5-days post fertilization (5-dpf) larvae. Bioinformatics analysis revealed that glucose metabolism, lipid metabolism, as well as oxidative stress were enriched in the PFHxS-exposed larvae. To validate these findings, a series of biological experiments were conducted. PFHxS exposure led to a nearly 4-fold increase in reactive oxygen species, possibly due to hyperglycemia and impaired glutathione balance. The Oil Red O' staining and qPCR analysis strengthens the notions that lipid metabolism was disrupted, leading to lipid accumulation, lipid peroxidation, and malondialdehyde formation. All these alterations ultimately affected cell cycle events, resulting in S and G2/M cell cycle arrest. In conclusion, our study demonstrated that PFHxS could accumulate and induce various developmental toxicities in aquatic life, and such data might assist the government to accelerate the regulatory policy on PFHxS usage.

Dysregulation of Spliceosomes Complex Induces Retinitis Pigmentosa-Like Characteristics in sf3b4-Depleted Zebrafish.

The SF3B4 gene encodes a highly conserved protein that plays a critical role in mRNA splicing. Mutations in this gene are known to cause Nager syndrome, a rare craniofacial disorder. Although SF3B4 expression is detected in the optic vesicle before it is detected in the limb and somite, the role of SF3B4 in the eye is not well understood. This study investigated the function of sf3b4 in the retina by performing transcriptome profiles, immunostaining, and behavioral analysis of sf3b4-/- mutant zebrafish. Results from this study suggest that dysregulation of the spliceosome complex affects not only craniofacial development but also retinogenesis. Zebrafish lacking functional sf3b4 displayed characteristics similar to retinitis pigmentosa (RP), marked by severe retinal pigment epithelium defects and rod degeneration. Pathway analysis revealed altered retinol metabolism and retinoic acid signaling in the sf3b4-/- mutants. Supplementation of retinoic acid rescued key cellular phenotypes observed in the sf3b4-/- mutants, offering potential therapeutic strategies for RP in the future. In conclusion, this study sheds light on the previously unknown role of SF3B4 in retinogenesis and provides insights into the underlying mechanisms of RP.

Integrated network findings reveal ubiquitin-specific protease 44 overexpression suppresses tumorigenicity of liver cancer.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third leading cause of cancer-related deaths worldwide. HCC is a multistep disease marked by various signaling alterations. A better understanding of the new molecular drivers of HCC could therefore provide an opportunity to develop effective diagnostic and therapeutic targets. Ubiquitin-specific protease 44 (USP44), a member of the cysteine protease family, has been reported to play a role in many cancer types. However, its contribution to HCC development remains unknown. In the present study, we observed suppression of USP44 expression in HCC tissue. Clinicopathologic analysis further showed that low USP44 expression correlated with poorer survival and a later tumor stage in HCC, suggesting that USP44 could be a predictor of poor prognosis in patients with HCC. Gain-of-function analysis in vitro demonstrated the importance of USP44 in HCC cell growth and G0/G1 cell cycle arrest. To investigate the downstream targets of USP44 and the molecular mechanisms underlying its regulation of cell proliferation in HCC, we conducted a comparative transcriptomic analysis and identified a cluster of proliferation-related genes, including CCND2, CCNG2, and SMC3. Ingenuity Pathway Analysis further delineated the gene networks controlled by USP44 through the regulation of membrane proteins and receptors, enzymes, transcriptional factors, and cyclins involved in the control of cell proliferation, metastasis, and apoptosis in HCC. To summarize, our results highlight, for the first time, the tumor-suppression role of USP44 in HCC and suggest a new prognostic biomarker in this disease.

Deciphering the pathogenesis of retinopathy associated with carnitine palmitoyltransferase I deficiency in zebrafish model.

Fatty acid oxidation disorders (FAODs) are a group of rare genetic metabolic disorders caused by mutations in genes responsible for transporting and metabolizing fatty acids in the mitochondria. One crucial enzyme involved in this process is carnitine palmitoyltransferase I (CPT1), which transports long-chain fatty acids to the mitochondrial matrix for beta-oxidation. Defects in beta-oxidation enzymes often lead to pigmentary retinopathy; however, the underlying mechanisms are not entirely understood. To investigate FAOD and its impact on the retina, we employed zebrafish as a model organism. Specifically, we used antisense-mediated knockdown strategies to target the cpt1a gene and examined the resulting retinal phenotypes. We demonstrated that the cpt1a MO-injected fish significantly reduced the length of connecting cilia and severely affected photoreceptor cell development. Moreover, our findings highlight that the loss of functional cpt1a disrupted energy homeostasis in the retina, leading to lipid droplet deposition and promoting ferroptosis, which is likely attributed to the photoreceptor degeneration and visual impairments observed in the cpt1a morphants.

FGF8 rescues motor deficits in zebrafish model of limb-girdle muscular dystrophy R18.

Variants in the gene encoding trafficking protein particle complex 11 (TRAPPC11) cause limb-girdle muscular dystrophy R18 (LGMD R18). Although recently several genes related to myopathies have been identified, correlations between genetic causes and signaling events that lead from mutation to the disease phenotype are still mostly unclear. Here, we utilized zebrafish to model LGMD R18 by specifically inactivating trappc11 using antisense-mediated knockdown strategies and evaluated the resulting muscular phenotypes. Targeted ablation of trappc11 showed compromised skeletal muscle function due to muscle disorganization and myofibrosis. Our findings pinpoint that fish lacking functional trappc11 suppressed FGF8, which resulted in the aberrant activation of Notch signaling and eventually stimulated epithelial-mesenchymal transition (EMT) and fibrotic changes in the skeletal muscle. In summary, our study provides the role of FGF8 in the pathogenesis and its therapeutic potential of LGMD R18.