Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging.

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

Young evolutionary origins of dioecy in the genus Asparagus.

PREMISE: Dioecy (separate sexes) has independently evolved numerous times across the angiosperm phylogeny and is recently derived in many lineages. However, our understanding is limited regarding the evolutionary mechanisms that drive the origins of dioecy in plants. The recent and repeated evolution of dioecy across angiosperms offers an opportunity to make strong inferences about the ecological, developmental, and molecular factors influencing the evolution of dioecy, and thus sex chromosomes. The genus Asparagus (Asparagaceae) is an emerging model taxon for studying dioecy and sex chromosome evolution, yet estimates for the age and origin of dioecy in the genus are lacking. METHODS: We use plastome sequences and fossil time calibrations in phylogenetic analyses to investigate the age and origin of dioecy in the genus Asparagus. We also review the diversity of sexual systems present across the genus to address contradicting reports in the literature. RESULTS: We estimate that dioecy evolved once or twice approximately 2.78-3.78 million years ago in Asparagus, of which roughly 27% of the species are dioecious and the remaining are hermaphroditic with monoclinous flowers. CONCLUSIONS: Our findings support previous work implicating a young age and the possibility of two origins of dioecy in Asparagus, which appear to be associated with rapid radiations and range expansion out of Africa. Lastly, we speculate that paleoclimatic oscillations throughout northern Africa may have helped set the stage for the origin(s) of dioecy in Asparagus approximately 2.78-3.78 million years ago.

Coupling of nanostraws with diverse physicochemical perforation strategies for intracellular DNA delivery.

Effective intracellular DNA transfection is imperative for cell-based therapy and gene therapy. Conventional gene transfection methods, including biochemical carriers, physical electroporation and microinjection, face challenges such as cell type dependency, low efficiency, safety concerns, and technical complexity. Nanoneedle arrays have emerged as a promising avenue for improving cellular nucleic acid delivery through direct penetration of the cell membrane, bypassing endocytosis and endosome escape processes. Nanostraws (NS), characterized by their hollow tubular structure, offer the advantage of flexible solution delivery compared to solid nanoneedles. However, NS struggle to stably self-penetrate the cell membrane, resulting in limited delivery efficiency. Coupling with extra physiochemical perforation strategies is a viable approach to improve their performance. This study systematically compared the efficiency of NS coupled with polyethylenimine (PEI) chemical modification, mechanical force, photothermal effect, and electric field on cell membrane perforation and DNA transfection. The results indicate that coupling NS with PEI modification, mechanical force, photothermal effects provide limited enhancement effects. In contrast, NS-electric field coupling significantly improves intracellular DNA transfection efficiency. This work demonstrates that NS serve as a versatile platform capable of integrating various physicochemical strategies, while electric field coupling stands out as a form worthy of primary consideration for efficient DNA transfection.

Disarm The Bacteria: What Temperate Phages Can Do.

In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.

Caspase-3-Responsive Fluorescent/Photoacoustic Imaging of Tumor Apoptosis.

Caspase-3 is an essential executor in apoptosis, and its activation has been regarded as a biomarker of cell apoptosis. The development of Caspase-3-responsive multimodal probes is a promising research prospect. Fluorescent/photoacoustic (FL/PA) imaging has attracted considerable attention due to the high sensitivity of FL as well as the high spatial resolution and penetration depth of PA. To our knowledge, there has been no tumor-targeted FL/PA probe to monitor the activity of Caspase-3 in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Bio-DEVD-HCy) for Caspase-3-responsive imaging of tumor apoptosis. Ac-DEVD-HCy without tumor-targeted biotin is used as a control probe. In vitro experiments indicated that Bio-DEVD-HCy is superior to Ac-DEVD-HCy because of the higher kinetic parameter of Bio-DEVD-HCy in comparison to Ac-DEVD-HCy. Cell and tumor imaging results suggested that Bio-DEVD-HCy could enter and accumulate in tumor cells with higher FL/PA signal with the help of tumor-targeted biotin. In detail, Bio-DEVD-HCy or Ac-DEVD-HCy could image apoptotic tumor cells with 4.3-fold or 3.5-fold FL enhancement and 3.4-fold or 1.5-fold PA enhancement. Bio-DEVD-HCy or Ac-DEVD-HCy could image tumor apoptosis with 2.5-fold or 1.6-fold FL enhancement and 4.1-fold or 1.9-fold PA enhancement. We envision that Bio-DEVD-HCy will be applied for FL/PA imaging of tumor apoptosis in clinical settings.

Synchronous Photoactivation-Imaging Fluorophores Break Limitations of Photobleaching and Phototoxicity in Live-cell Microscopy.

Fluorescence microscopy is one of the most important tools in the studies of cell biology and many other fields, but two fundamental issues, photobleaching and phototoxicity, associated with the fluorophores have still limited its use for long-term and strong-illumination imaging of live cells. Here, we report a new concept of fluorophore engineering chemistry, synchronous photoactivation-imaging (SPI) fluorophores, activating and exciting fluorophores by a single light source to thus avoid the repeated switches between activation and excitation lights. The chemically reconstructed, nonemissive fluorophores can be photolyzed to allow continuous replenishing of "bright-state" probes detectable by standard fluorescent microscopes in the imaging process so as to bypass the photobleaching barrier to greatly extend the imaging period. Equally importantly, SPI fluorophores substantially reduce photocytotoxicity due to the scavenging of reactive oxygen species (ROS) by a photoactivable group and the slow release of "bright-state" probes to minimize ROS generation. Using SPI fluorophores, the time-lapsed confocal (>16 h) and super-resolution (>3 h) imaging of subcellular organelles under intensive illumination (50 MW/cm2) were achieved in live cells.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Prosocial decision-making under time pressure: Behavioral and neural mechanisms.

The present study employed a novel paradigm and functional magnetic resonance imaging (fMRI) to uncover the specific regulatory mechanism of time pressure and empathy trait in prosocial decision-making, compared to self-decision making. Participants were instructed to decide whether to spend their own monetary interest to alleviate themselves (or another person) from unpleasant noise threats under high and low time pressures. On the behavioral level, results showed that high time pressure had a significant effect on reducing participants' willingness to spend money on relieving themselves from the noise, while there is a similar but not significant trend in prosocial decision-making. On the neural level, for self-concerned decision-making, low time pressure activated the bilateral insula more strongly than high time pressure. For prosocial decision-making, high time pressure suppressed activations in multiple brain regions related to empathy (temporal pole, middle temporal gyrus, and inferior frontal gyrus), valuation (medial orbitofrontal cortex), and emotion (putamen). The functional connectivity strength among these regions, especially the connectivity between the medial orbitofrontal cortex and putamen, significantly predicted the effect of time pressure on prosocial decision-making at the behavioral level. Additionally, we discovered the activation of the medial orbitofrontal cortex partially mediated the effect of empathy trait scores on prosocial decision-making. These findings suggest that (1) there are different neural underpinnings for the modulation of time pressure for self and prosocial decision-making, and (2) the empathy trait plays a crucial role in the latter.

Steroidal saponin profiles and their key genes for synthesis and regulation in Asparagus officinalis L. by joint analysis of metabolomics and transcriptomics.

BACKGROUND: Asparagus officinalis L. is a worldwide cultivated vegetable enrichened in both nutrient and steroidal saponins with multiple pharmacological activities. The upstream biosynthetic pathway of steroidal saponins (USSP) for cholesterol (CHOL) synthesis has been studied, while the downstream pathway of steroidal saponins (DSSP) starting from cholesterol and its regulation in asparagus remains unknown. RESULTS: Metabolomics, Illumina RNAseq, and PacBio IsoSeq strategies were applied to different organs of both cultivated green and purple asparagus to detect the steroidal metabolite profiles & contents and to screen their key genes for biosynthesis and regulation. The results showed that there is a total of 427 compounds, among which 18 steroids were detected with fluctuated concentrations in roots, spears and flowering twigs of two garden asparagus cultivars. The key genes of DSSP include; steroid-16-hydroxylase (S16H), steroid-22-hydroxylase (S22H) and steroid-22-oxidase-16-hydroxylase (S22O-16H), steroid-26-hydroxylase (S26H), steroid-3-ß-glycosyltransferase (S3ßGT) and furostanol glycoside 26-O-beta-glucosidases (F26GHs) which were correlated with the contents of major steroidal saponins were screened, and the transcriptional factors (TFs) co-expressing with the resulted from synthetic key genes, including zinc fingers (ZFs), MYBs and WRKYs family genes were also screened. CONCLUSIONS: Based on the detected steroidal chemical structures, profiles and contents which correlated to the expressions of screened synthetic and TFs genes, the full steroidal saponin synthetic pathway (SSP) of asparagus, including its key regulation networks was proposed for the first time.

Nanochannel Electro-Injection as a Versatile Platform for Efficient RNA/DNA Programming on Dendritic Cells.

The utilization of dendritic cell (DC) vaccines is a promising approach in cancer immunotherapy, and the modification of DCs for the expression of tumor-associated antigens is critical for successful cancer immunotherapy. A safe and efficient method for delivering DNA/RNA into DCs without inducing maturation is beneficial to achieve successful DC transformation for cell vaccine applications, yet remains challenging. This work presents a nanochannel electro-injection (NEI) system for the safe and efficient delivery of a variety of nucleic acid molecules into DCs. The device is based on track-etched nanochannel membrane as key components, where the nano-sized channels localize the electric field on the cell membrane, enabling lower voltage (<30 V) for cell electroporation. The pulse conditions of NEI are examined so that the transfection efficiency (>70%) and biosafety (viability >85%) on delivering fluorescent dyes, plasmid DNA, messenger RNA, and circular RNA (circRNA) into DC2.4 are optimized. Primary mouse bone marrow DC can also be transfected with circRNA with 68.3% efficiency, but without remarkably affecting cellular viability or inducing DC maturation. These results suggest that NEI can be a safe and efficient transfection platform for in vitro transformation of DCs and possesses a promising potential for developing DC vaccines against cancer.

Phage Engineering for Targeted Multidrug-Resistant Escherichia coli.

The lytic bacteriophages have potential application value in the treatment of bacterial infections. However, the narrow host spectrum of these phages limits their range of clinical application. Here, we demonstrate the use of scarless Cas9-assisted recombination (no-SCAR) gene-editing technology to regulate phage-host range. We used phage PHB20 as the scaffold to create agents targeting different multidrug-resistant Escherichia coli by replacing its phage tail fiber gene (ORF40). The engineered phages were polyvalent and capable of infecting both the original host bacteria and new targets. Phage-tail fiber genes can be amplified by PCR to construct a recombinant phage PHB20 library that can deal with multidrug-resistant bacteria in the future. Our results provide a better understanding of phage-host interactions, and we describe new anti-bacterial editing methods.

Recovery Mechanism of Endoplasmic Reticulum Revealed by Fluorescence Lifetime Imaging in Live Cells.

Endoplasmic reticulum (ER) is an important organelle of a membranous tubule network in cells for the synthesis, assembly, and modification of peptides, proteins, and enzymes. Autophagy and destruction of ER commonly occur during normal cellular activities. These processes have been studied extensively, but the spontaneous ER regeneration process is poorly understood because of the lack of molecular tools capable of distinguishing the intact, damaged, autophagic, and regenerative ER in live cells. Herein, we report a dual-localizing, environment-responsive, and lifetime-sensitive fluorescent probe for real-time monitoring ER autophagy and regeneration in live cells. Using this tool, the fluorescence lifetime imaging can quantitatively determine the degrees of ER destruction and spontaneous recovery. Significantly, we show that triglycerides supplied in lipid droplets can efficiently repair ER via the two critical pathways: (i) supplying materials for ER repair by converting triglycerides into fatty acids and diglycerides and (ii) partially inhibiting autophagy for stressed ER.

Fluorescence imaging of intracellular telomerase activity for tumor cell identification by oligonucleotide-functionalized gold nanoparticles.

As a specific biological marker for the occurrence and progression of tumor cells, detection of telomerase activity is of great importance for the physiological research of tumors. However, in situ measurement of telomerase activity in living cells still remains a challenge. Herein, we report a precisely designed oligonucleotide-functionalized gold nanoparticle probe that has realized high-efficiency detection of telomerase activity for cellular imaging toward the identification of tumors. Our method has achieved intracellular imaging of telomerase activity and shows good performance towards the distinction of tumor cells from normal ones. Moreover, the method reported here for tracking tumor cells in blood has wide applications in cancer diagnosis. This strategy offers an opportunity for cancer diagnosis, guiding therapy and evaluating prognosis.

Cathepsin B-Activated Fluorescent and Photoacoustic Imaging of Tumor.

Early diagnosis is crucial to the treatment of cancer. Cathepsin B (CTB) plays an important role in numerous cancers, which is a promising biomarker for early diagnosis of cancer. It is necessary to exploit new probes for visualization of CTB in vivo. Fluorescent/photoacoustic (FL/PA) imaging is a powerful tool for in vivo study which possesses both excellent sensitivity and spatial resolution. To our knowledge, there has been no FL/PA probe to image CTB in vitro or in vivo. Therefore, we developed two CTB-activated FL/PA probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly, which could successfully monitor CTB activity in vivo. Both two probes had excellent sensitivity and selectivity in vitro. Cell imaging showed that HCy-Cit-Val or HCy-Gly-Leu-Phe-Gly could image endogenous CTB in lysosome with 6.8-fold or 5.1-fold enhancement of the FL signal and 5.8-fold or 3.4-fold enhancement of the PA signal compared to their inhibitor contrast groups. Tumor imaging in vivo further confirmed the good applicability of these two probes to monitor CTB activity with high sensitivity and spatial resolution. Moreover, the property of HCy-Cit-Val is superior to HCy-Gly-Leu-Phe-Gly due to the higher catalytic efficiency of CTB toward HCy-Cit-Val than HCy-Gly-Leu-Phe-Gly. We envision that our FL/PA probe HCy-Cit-Val will be suitable for clinical early diagnosis of CTB-related cancer in the near future.

Revealing Sulfur Dioxide Regulation to Nucleophagy in Embryo Development by an Adaptive Coloration Probe.

Understanding signaling molecules in regulating organelles dynamics and programmed cell death is critical for embryo development but is also challenging because current imaging probes are incapable of simultaneously imaging the signaling molecules and the intracellular organelles they interact with. Here, we report a chemically and environmentally dual-responsive imaging probe that can react with gasotransmitters and label cell nuclei in distinctive fluorescent colors, similar to the adaptive coloration of chameleons. Using this intracellular chameleon-like probe in three-dimensional (3D) super-resolution dynamic imaging of live cells, we discovered SO2 as a critical upstream signaling molecule that activates nucleophagy in programmed cell death. An elevated level of SO2 prompts kiss fusion between the lysosomal and nuclear membranes and nucleus shrinkage and rupture. Significantly, we revealed that the gasotransmitter SO2 is majorly generated in the yolk, induces autophagy there at the initial stage of embryo development, and is highly related to the development of the auditory nervous system.

Mitochondria-Targeted Fluorescent and Photoacoustic Imaging of Hydrogen Peroxide in Inflammation.

Hydrogen peroxide (H2O2) is a prominent reactive oxygen species with relative stability, which makes it a potential diagnostic marker for pathological states. Excessive H2O2 in mitochondria leads to oxidative stress and inflammation. However, precisely monitoring the level of H2O2 at specific organelles (e.g., mitochondria) in vivo is still of urgent necessity. Therefore, we rationally designed a mitochondria-targeted near-infrared probe TPP-HCy-BOH for fluorescent/photoacoustic (FL/PA) dual-modal imaging of overproduced H2O2 in an inflamed mouse model. TPP-HCy-BOH had a low LOD (0.348 µM), which is comparable to those of recently reported probes for H2O2 detection. The high kinetic rate constant (kobs = 4.72 × 10-3 s-1) of TPP-HCy-BOH toward H2O2 is superior to recently reported H2O2 probes. Compared to control probe HCy-BOH without the mitochondrial targeting moiety, TPP-HCy-BOH successfully images exogenous or endogenous H2O2 in mitochondria with an additional 2.4-fold FL increase and 4.7-fold PA increase in HeLa cells or additional 2.1-fold FL increase and 3.3-fold PA increase in RAW 264.7 cells. In LPS-induced acute inflammation in vivo, TPP-HCy-BOH is more competent to image overproduced H2O2 with additional 1.6-fold higher sensitivity of FL in abdomen and 2.0-fold higher sensitivity of PA in liver and longer retention time of 0.5 h than HCy-BOH. We anticipate that TPP-HCy-BOH could be employed for the FL/PA dual-modal diagnosis of pathological inflammation in clinic in near future.

The molecular characters and antibiotic resistance of Clostridioides difficile from economic animals in China.

BACKGROUND: It has been performed worldwidely to explore the potential of animals that might be a reservoir for community associated human infections of Clostridioides difficile. Several genetically undistinguished PCR ribotypes of C. difficile from animals and human have been reported, illustrating potential transmission of C. difficile between them. Pig and calf were considered as the main origins of C. difficile with predominant RT078 and RT033, respectively. As more investigations involved, great diversity of molecular types from pig and calf were reported in Europe, North American and Australia. However, there were quite limited research on C. difficile isolates from meat animals in China, leading to non-comprehensive understanding of molecular epidemiology of C. difficile in China. RESULTS: A total of 55 C. difficile were isolated from 953 animal stool samples, within which 51 strains were from newborn dairy calf less than 7 days in Shandong Province. These isolates were divided into 3 STs and 6 RTs, of which ST11/RT126 was predominant type, and responsible for majority antibiotic resistance isolates. All the isolates were resistant to at least one tested antibiotics, however, only two multidrug resistant (MDR) isolates were identified. Furthermore, erythromycin (ERY) and clindamycin (CLI) were the two main resistant antibiotics. None of the isolates were resistant to vancomycin (VAN), metronidazole (MTZ), tetracycline (TET), and rifampin (RIF). CONCLUSIONS: In this study, we analyzed the prevalence, molecular characters and antibiotic resistance of C. difficile from calf, sheep, chicken, and pig in China. Some unique features were found here: first, RT126 not RT078 were the dominant type from baby calf, and none isolates were got from pig; second, on the whole, isolates from animals display relative lower resistant rate to these 11 tested antibiotics, compared with isolates from human in China in our previous report. Our study helps to deep understanding the situation of C. difficile from economic animals in China, and to further study the potential transmission of C. difficile between meat animals and human.

Silica-anchored cadmium sulfide nanocrystals for the optical detection of copper(II).

A fluorometric assay was developed for the determination of copper(II) ion based on its quenching effect on the green fluorescent probe of SiO2-anchored CdS nanocrystals (SiO2/CdS NCs). The fluorescent probe was prepared by a surface ion-directing strategy for homogeneous precipitation of CdS NCs onto the carboxyl-capped SiO2 core surfaces. In comparison to CdS NCs, the SiO2/CdS NCs has strong fluorescence emission and good photostability. Moreover, SiO2/CdS NCs show higher fluorescence selectivity for copper(II) ions than for other common metal ions because copper(II) ions have a strong fluorescence quenching effect on SiO2/CdS NCs. At excitation/emission wavelengths of 300/516 nm, the resulting fluorescent probe shows wide linear ranges from 0.01 to 2 µM with a detection limit of 6.3 nM for copper(II) ions. The method has been applied to the determination of trace copper(II) ions in tea infusions with satisfactory results. Graphical abstract.

Macrofungi: A review of cultivation strategies, bioactivity, and application of mushrooms.

The production of macrofungi (mushrooms) as well as their economic value have been steadily increasing globally. The use of functional foods, dietary supplements, and traditional medicines derived from macrofungi is increasing as they have numerous health benefits as well as abundant nutrients. Macrofungi are diverse with complex and highly varied growth conditions and bioactive constituents, most macrofungal resources have not yet been fully explored and applicated, leading to an urgent need for appropriate strategies to address the problem. Increasing attention has been paid to the macrofungal cultivation and application, in particular, potential prebiotics. Herein, the present review comprehensively summarizes recent progress in the cultivation, newly identified bioactive constituents, and their effects on gut microbiota as well as the potential ways in which they affect human health. Moreover, the macrofungal food development is discussed to improve food nutritional value and change the quality characteristics of food. Finally, the review addresses consumer safety concerns and the prospective genetic manipulation of macrofungi. We hope that this review can provide a comprehensive research reference for ensuring the safety and efficacy, along with maximizing the value and profitability of macrofungi production.

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging.

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H2 O2 ), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2 O2 level leads to disassociation of nucleic acid-protein complexes.