Search Results (12)

Protein-rich microalgae have been increasingly recognized as viable alternatives to fish meal (FM) in aquaculture diets. In this study, we evaluated the effects of partial or total replacement of FM with the microalga Arthrospira platensis (Spirulina, SM) on the growth performance, reproductive parameters, and transcriptomic profile of zebrafish. Six isoproteic, isoenergetic experimental diets were formulated with increasing levels of SM (0, 10, 20, 30, 40, and 50 g kg⁻¹ feed) replacing FM. Fish were randomly assigned to six groups (five replicates each) and fed for 60 days. The diet containing 50 g kg⁻¹ SM resulted in the highest final body weight, weight gain, specific growth rate, and protein efficiency, as well as increased gonadosomatic index, eggs per female, fertilization rate, and hatching rate compared to the results for the control group (0 g kg⁻¹ SM). RNA-Seq transcriptomic analysis identified 2299 differentially expressed genes in the SM50 group, mainly associated with muscle development and energy metabolism. These findings offer new insights into the underlying molecular mechanisms and underscore the potential of Spirulina as a sustainable alternative for cultured fish nutrition. Full article

Three-dimensional (3D) scaffold systems have proven instrumental in advancing our understanding of polymicrobial biofilm dynamics and probiotic interactions within the oral environment. Among oral probiotics, Streptococcus salivarius K12 (Ssk12) has shown considerable promise in modulating microbial homeostasis; however, its long-term therapeutic benefits are contingent upon successful and sustained colonization of the oral mucosa. Despite its clinical relevance, the molecular mechanisms underlying the adhesion, persistence, and integration of Ssk12 into the native oral microbiome/biofilm remain inadequately characterized. In this pilot study, we explored the temporal colonization dynamics of Ssk12 and its impact on the structure and functional profiles of salivary-derived biofilms cultivated on melt-electrowritten poly(ε-caprolactone) (MEW-mPCL) scaffolds, which emulate the native oral niche. Colonization was monitored via fluorescence in situ hybridization (smFISH), confocal microscopy, and RT-qPCR, while shifts in community composition and function were assessed using 16S rRNA sequencing and meta-transcriptomics. A single administration of Ssk12 exhibited transient colonization lasting up to 7 days, with detectable presence diminishing by day 10. This was accompanied by short-term increases in Lactobacillus and Bifidobacterium populations. Functional analyses revealed increased transcriptional signatures linked to oxidative stress resistance and metabolic adaptation. These findings suggest that even short-term probiotic colonization induces significant functional changes, underscoring the need for strategies to enhance probiotic persistence. Full article

Background: Docosahexaenoic Acid (DHA) is an extensively used nutrition supplement in dairy food because of its beneficial effects on cognition. To find an effective DHA intervention for the synapses in the cortex during this period, this study aimed to use targeted lipidomics to evaluate the lipid composition of prefrontal-cortex (PFC) tissue in different DHA interference methods. Methods: Analyzed samples were taken from interfering feeding Bama pigs (BPs) (3 months) fed with soybean oil (Group B), blended oil (Group M), naturally DHA-supplemented milk with blended oil (Group OM), and DHA from fish oil (FO) with blended oil (Group Y). We also examined the protein expression levels of BDNF, GAP43, and MBP. Results: The lipidomics analysis identified 80 different related negative-ion lipid content and filtered the biomarker lipids in PFC tissue. We observed significant lipid composition changes between group Y and other groups, especially for content levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM). The same observations were made from mRNA and protein expressions related to lipid transportation, phosphatidylserine (PS) synthetase, and synaptic plasticity in PFC tissues between group Y and other groups, including the mRNA expression levels of CD36, BDNF, and PTDSS1. The analysis of protein expression levels showed that the metabolism mode of DHA intervention from FO benefited the PFC, PS metabolism, and PFC synaptic plasticity of infants. Conclusions: The results highlight further prospects for the DHA intervention mode, which provides new routes for other studies on polyunsaturated-fatty-acid (PUFA) interference for infants. Full article

Paraspeckles are nuclear condensates formed by NEAT1_2 lncRNA and different RNA-binding proteins. In general, these membraneless organelles function in the regulation of gene expression and translation and in miRNA processing, and in doing this, they regulate cellular homeostasis and mediate pro-survival in the cell. Despite evidence showing the importance of paraspeckles in the stress response, the dynamics of paraspeckles and their components under conditions of osmotic stress remain unknown. We exposed HEK293T cells to sorbitol and examined NEAT1_2 expression using real-time PCR. Localization and quantification of the main paraspeckle components, NEAT1_2, PSPC1, NONO, and SFPQ, in different cellular compartments was performed using smFISH and immunofluorescence. Our findings showed a significant decrease in total NEAT1_2 expression in cells after osmotic stress. Sorbitol shifted the subcellular localization of NEAT1_2, PSPC1, NONO, and SFPQ from the nucleus to the cytoplasm and decreased the number and size of NEAT1_2 foci in the nucleus. PSPC1 formed immunoreactive cytoplasmic fibrils under conditions of osmotic stress, which slowly disassembled under recovery. Our study deepens the paraspeckle dynamics in response to stress, suggesting a novel role for NEAT1_2 in the cytoplasm in osmotic stress and physiological conditions. Full article

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease. Full article

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19. Full article

The transient nature of RNA has rendered it one of the more difficult biological targets for imaging. This difficulty stems both from the physical properties of RNA as well as the temporal constraints associated therewith. These concerns are further complicated by the difficulty in imaging endogenous RNA within a cell that has been transfected with a target sequence. These concerns, combined with traditional concerns associated with super-resolution light microscopy has made the imaging of this critical target difficult. Recent advances have provided researchers the tools to image endogenous RNA in live cells at both the cellular and single-molecule level. Here, we review techniques used for labeling and imaging RNA with special emphases on various labeling methods and a virtual 3D super-resolution imaging technique. Full article

The Warburg effect, i.e., the utilization of glycolysis under aerobic conditions, is recognized as a survival advantage of cancer cells. However, how the glycolytic activity is affected during drug resistance acquisition has not been explored at single-cell resolution. Because the relative ratio of the splicing isoform of pyruvate kinase M (PKM), PKM2/PKM1, can be used to estimate glycolytic activity, we utilized a single-molecule fluorescence in situ hybridization (SM-FISH) method to simultaneously quantify the mRNA levels of PKM1 and PKM2. Treatment of HCT116 cells with gefitinib (GE) resulted in two distinct populations of cells. However, as cells developed GE resistance, the GE-sensitive population with reduced PKM2 expression disappeared, and GE-resistant cells (Res) demonstrated enhanced PKM1 expression and a tightly regulated PKM2/PKM1 ratio. Our data suggest that maintaining an appropriate PKM2 level is important for cell survival upon GE treatment, whereas increased PKM1 expression becomes crucial in GE Res. This approach demonstrates the importance of single-cell-based analysis for our understanding of cancer cell metabolic responses to drugs, which could aid in the design of treatment strategies for drug-resistant cancers. Full article

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating. Full article

Neurodevelopment is accompanied by a precise change in the expression of the translation elongation factor 1A variants from eEF1A1 to eEF1A2. These are paralogue genes that encode 92% identical proteins in mammals. The switch in the expression of eEF1A variants has been well studied in mouse motor neurons, which solely express eEF1A2 by four weeks of postnatal development. However, changes in the subcellular localization of eEF1A variants during neurodevelopment have not been studied in detail in other neuronal types because antibodies lack perfect specificity, and immunofluorescence has a low sensitivity. In hippocampal neurons, eEF1A is related to synaptic plasticity and memory consolidation, and decreased eEF1A expression is observed in the hippocampus of Alzheimer’s patients. However, the specific variant involved in these functions is unknown. To distinguish eEF1A1 from eEF1A2 expression, we have designed single-molecule fluorescence in-situ hybridization probes to detect either eEF1A1 or eEF1A2 mRNAs in cultured primary hippocampal neurons and brain tissues. We have developed a computational framework, ARLIN (analysis of RNA localization in neurons), to analyze and compare the subcellular distribution of eEF1A1 and eEF1A2 mRNAs at specific developmental stages and in mature neurons. We found that eEF1A1 and eEF1A2 mRNAs differ in expression and subcellular localization over neurodevelopment, and eEF1A1 mRNAs localize in dendrites and synapses during dendritogenesis and synaptogenesis. Interestingly, mature hippocampal neurons coexpress both variant mRNAs, and eEF1A1 remains the predominant variant in dendrites. Full article

Ewing’s sarcoma (ES) is caused by a chromosomal translocation leading to the formation of the fused EWSFLI1 gene, which codes for an aberrant transcription factor EWSFLI1. The transcriptional targets of EWSFLI1 have been viewed as promising and novel drug targets in the treatment of ES. One such target is six transmembrane epithelial antigen of the prostate 1 (STEAP1), a transmembrane protein that is upregulated by EWSFLI1 in ES. STEAP1 is a hallmark of tumor invasiveness and an indicator of tumor responsiveness to therapy. EWSFLI1 binds to the STEAP1 promoter region, but the mechanism of action by which it upregulates STEAP1 expression in ES is not entirely understood. Upon analysis of the STEAP1 promoter, we predicted two binding sites for NKX2.2, another crucial transcription factor involved in ES pathogenesis. We confirmed the interaction of NKX2.2 with the STEAP1 promoter using chromatin immunoprecipitation (ChIP) analysis. We used single-molecule RNA imaging, biochemical, and genetic studies to identify the novel role of NKX2.2 in regulating STEAP1 expression in ES. Our results show that NKX2.2 is a co-regulator of STEAP1 expression and functions by interacting with the STEAP1 promoter at sites proximal to the reported EWSFLI1 sites. The co-operative interaction of NKX2.2 with EWSFLI1 in regulating STEAP1 holds potential as a new target for therapeutic interventions for ES. Full article

Technological advances are continually improving our ability to obtain more accurate views about the inner workings of biological systems. One such rapidly evolving area is single cell biology, and in particular gene expression and its regulation by transcription factors in response to intrinsic and extrinsic factors. Regarding the study of transcription factors, we discuss some of the promises and pitfalls associated with investigating how individual cells regulate gene expression through modulation of transcription factor activities. Specifically, we discuss four leading experimental approaches, the data that can be obtained from each, and important considerations that investigators should be aware of when drawing conclusions from such data. Full article