Confocal Fluorescence Imaging Research Articles

Targeted regulation of sterol biosynthesis genes according to perturbations in ergosterol biosynthesis in fungi.

The synthesis and regulation of ergosterol are vital for fungal growth and stress adaptation. While ergosterol-mediated feedback regulation is a recognized mechanism controlling sterol biosynthesis in fungi, prior research suggests the presence of additional regulatory mechanisms. However, the specifics of the alternative regulatory mechanisms have not been systematically investigated. We proposed that a regulatory network is likely to discern disturbances in sterol biosynthesis and trigger responses accordingly. This study aimed to validate the hypothesis and investigate the regulatory mechanisms. Quantitative Real-time PCR and HPLC-MS/MS were used to explore and compare the regulation of sterol biosynthesis in different fungi. Key transcription factors involved in the alternative regulatory mechanism in Neurospora crassa were identified by phenotypic profiling of a transcription factor mutant library. ChIP-qPCR, fluorescence confocal imaging, RNA sequencing, and gene set enrichment analysis (GSEA) reveal the mechanism of each transcription factor. Unlike the canonical ergosterol-mediated feedback regulation in fungi like C. neoformans, our study demonstrated that the inhibitions of ergosterol biosynthesis at specific steps triggered distinct transcriptional responses of erg genes in fungi, including N. crassa and Aspergillus fumigatus. In N. crassa, the responses were orchestrated by different transcription factors. Specifically, the inhibition of ERG24 and ERG2 activated transcription factors SAH-2 and AtrR, resulting in the upregulation of erg24, erg2, erg25, and erg3. Furthermore, the inhibition of ERG11/CYP51 activated transcription factor NcSR, leading to the upregulation of erg11 and erg6. Phenotypic profiles of mutants of various N. crassa erg genes and the aforementioned transcription factors implied that the targeted regulation of ergosterol biosynthesis could fortify fungal viability within complex habitats. Our study reveals a novel regulatory mechanism in fungi: targeted upregulation of specific sterol biosynthesis genes in response to given perturbations in ergosterol biosynthesis, exhibiting a higher degree of precision and sophistication in sterol biosynthesis regulation.

Confocal fluorescence imaging of levitated particles and microdroplets

Confocal fluorescence imaging of levitated particles and microdroplets

Scaffolds fabricated via two-photon polymerisation for regulating cell morphology and differentiation

ABSTRACT Three-dimensional (3D) cell culture scaffolds play a key role in guiding cell fate. The fine-tunability of these scaffolds is essential for accurately replicating in vivo conditions and revealing cellular behaviours. In this study, two-photon polymerisation (TPP) technology was employed to create 3D scaffolds with woodpile and honeycomb architectures. The influence of scaffold geometry and pore dimensions on the proliferation, morphology, orientation, and differentiation of human mesenchymal stem cells (hMSCs) was investigated through multi-staining fluorescence and 3D confocal imaging technique. It was revealed that hMSCs cultured within these 3D scaffolds demonstrated higher density up to 15.04 ± 1.46 cells/100 × 100 μm2 after a 6-day culture compared to their 2D counterparts (7.47 ± 1.42 cells/100 × 100 μm2). Furthermore, hMSCs grown on the woodpile scaffolds displayed elongated morphology, with approximately 50% aligning along the columns. In contrast, hMSCs cultivated on honeycomb structures exhibited triangular and crescent cellular shapes, with a random orientation. Notably, compared to the control group, the cells on the scaffolds exhibited smaller nucleus areas, lower circularity, and aspect ratios, but these values increased as pore size increased. Furthermore, ALP staining showed a greater tendency for osteogenic differentiation with larger pore sizes. These TPP-fabricated 3D scaffolds hold immense promise for advancing understanding of cellular behaviour.

Deep Learning-Assisted Fluorescence Single-Particle Detection of Fumonisin B1 Powered by Entropy-Driven Catalysis and Argonaute.

Timely and accurate detection of trace mycotoxins in agricultural products and food is significant for ensuring food safety and public health. Herein, a deep learning-assisted and entropy-driven catalysis (EDC)-Argonaute powered fluorescence single-particle aptasensing platform was developed for ultrasensitive detection of fumonisin B1 (FB1) using single-stranded DNA modified with biotin and red fluorescence-encoded microspheres as a signal probe and streptavidin-conjugated magnetic beads as separation carriers. The binding of aptamer with FB1 releases the trigger sequence to mediate EDC cycle to produce numerous 5'-phosphorylated output sequences, which can be used as the guide DNA to activate downstream Thermus thermophilus Argonaute (TtAgo) for cleaving the signal probe, resulting in increased number of fluorescence microspheres remaining in the final reaction supernatant after magnetic separation. Subsequently, through fast and accurate counting of red bright particles in the captured confocal fluorescence images from the supernatant via a YOLOv9 deep learning model, the sensitive and specific detection of FB1 could be realized. This approach has a limit of detection (LOD) of 0.89 pg/mL with a linear range from 1 pg/mL to 100 ng/mL, and satisfactory recovery (87.2-113.5%) in real food samples indicates its practicality. The integration of the aptamer and EDC with TtAgo broadens the target range of Argonaute and enhances sensitivity. Furthermore, incorporating deep learning significantly improves the analytical efficiency of single-particle detection. This work provides a promising analytical strategy in biosensing and promotes the application of fluorescence single-particle detection in food safety monitoring.

3D fluorescence staining and confocal imaging of low amount of intestinal organoids (enteroids): Protocol accessible to all.

The emerging field of 3D organ modeling encounters several imaging issues in particular related to antigen retrieval and sample loss during staining processes. Due to their compact shape, several antibodies fail to penetrate intact organoids or spheroids. Histology of organoids can be approached by paraffin inclusion and sectioning at 5 μm as performed for biopsies. However, to fully understand organoid behavior, including cellular organization, extracellular matrix structure, and their response to treatments, 3D imaging is essential. Here we propose an easy workflow allowing (1) immunostaining with a HIER step, (2) preservation of the intact shape of the organoids, (3) sample immobilization in a focal plane reachable for high resolution/short working distance lenses, and (4) minimizing the risk of loss of precious material.

Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics.

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semi-rigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically-relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semi-rigid linker provides enough molecular spacing to allow a single monomer to fully stack with base pairs, comparable to the monomeric parent ruthenium complex. The DNA base pairs in the equilibrium threading intercalated state are likely intact and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.

Endoplasmic Reticulum-Targeting Delayed Fluorescent Probe for Dual-mode Nitroreductase Sensing.

Organic thermally activated delayed fluorescence (TADF) materials, known for their long-lived emission properties, are highly sought after for background-free imaging of selective analytes in time-resolved modes. However, their practical application faces significant challenges, including the air sensitivity of triplet states, lack of organelle specificity, and the absence of precise analyte recognition centres. These limitations hinder their effectiveness in detecting key cancer biomarkers such as nitroreductase (NTR). Herein, we present the development of donor (triphenylamine)-acceptor (quinoxaline)-based probes, TPQS and TPNS, which are functionalized with a sulphonamide unit to offer endoplasmic reticulum specificity. TPQS exhibits delayed fluorescence, attributed to a minimal singlet-triplet energy gap, as confirmed by time-resolved fluorescence measurements. Additionally, a nonfluorescent probe, TPNS, is synthesized by introducing a nitro group into the sulphonamide unit of the TPQS backbone, serving as a recognition centre for NTR. Upon reacting with NTR, TPNS displays a "turn-on" luminescence and delayed fluorescence, enabling dual-mode detection of NTR through both confocal fluorescence imaging and time-resolved fluorescence imaging (TRFI) in cancer cells. These findings underscore the potential of delayed fluorescent emitters for the sensitive and specific detection of cancer biomarkers in complex biological environments.

Comparison between two artificial intelligence models to discriminate cancerous cell nuclei based on confocal fluorescence imaging in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) exhibits an exceptional intratumoral heterogeneity that might influence diagnosis and outcome. Advances in digital microscopy and artificial intelligence (AI) may improve the HCC identification of liver cancer cells. Two AI algorithms were designed to perform computer-assisted discrimination of tumour from non-tumour nuclei in HCC. Healthy livers and HCCs from commercially available tissue arrays were stained with an antibody against proliferating cell nuclear antigen and DRAQ5 dye with high affinity for double-stranded DNA, acquired by confocal microscopy imaging and then used to design machine learning (ML) and deep learning (DL) algorithms. Nuclei were segmented and then used to develop the Model 1 and Model 2 algorithms, using ML and DL respectively. Model 1 was trained with some texture nuclear features extracted using discrete wavelet transform and grey-level co-occurrence matrix. Model 2 was trained with the segmented images without any additional information. The comparative analysis of the models showed that DL was more effective than ML, achieving an average accuracy of 88 % in discriminating healthy from neoplastic nuclei in HCC samples. Our research shows that AI techniques and nuclear fluorescent staining could be useful tools for automatically detecting HCC cells in liver tissues.

Comprehensive property combination for biomedical application achieved in a Ti35Zr35Nb15Mo5Fe5Cr5 complex concentrated alloy

A novel Ti35Zr35Nb15Mo5Fe5Cr5 complex concentrated alloy (CCA) with potential for biomedical application was developed by vacuum arc melting and its microstructural evolution, mechanical properties, corrosion and wear behavior, and cytocompatibility were systematically evaluated. The alloy has an experimentally measured density of 6.37 g/cm³, suitable to fulfill the requirement of light weight. XRD analysis revealed that the as-cast and annealed specimens contain two BCC solid solution phases and a TiCr2 type Laves phase. The average microhardness (H) and elastic modulus (E) of the as-cast CCA are 618.39 ± 9.26 HV and 97.32 ± 3.58 GPa, respectively. Moreover, the yield strength (YS) of the as-cast alloy, estimated from elastoplastic analysis of the microindentation data, is 1203.94 ± 30.28 MPa. However, the CCA annealed at 1100˚C exhibits a microhardness of 833.08 ± 7.58 HV and a YS of 1669.65 ± 24.79 MPa, with the elastoplastic stress-strain response revealing no significant loss of plasticity due to the increased hard Laves phase. Corrosion and wear tests conducted in simulated body fluid confirmed the alloy's excellent corrosion and wear resistance. Cell culture experiments with MG-63 and HEK-293 cells demonstrated superior cell viability and proliferation compared to CP-Ti and 316 L SS. Moreover, confocal fluorescence images of MG-63 cells stained with AO/EtBr, Rh-123, and DCFH-DA revealed that the present CCA exhibits good biocompatibility. Thus, a comprehensive combination of low density, compatible elastic modulus, good strength with adequate plasticity, and sufficient corrosion resistance and biocompatibility were successfully achieved, unraveling significant potential of the alloy for biomedical applications.

Synthesis, characterisation, single crystal structure and evaluation of a redox innocent carbazate functionalized phenanthroline for antimycobacterial and anticancer activity

Development of bioactive candidates for cancer and bacterial infections are ever demanding challenges due to the resistance shown by these cancer and bacterial cells for already exposed drug molecules. Methyl carbazate derivatized phenanthroline compound 6-[2-(methoxycarbonyl)diazen-1-yl]-1,10-phenanthroline-5-one (MCDPO) is synthesized in a three step reaction from 1,10-phenanthroline. The spectroscopic features of MCDPO are studied by HRMS, IR, 1H NMR and 13C NMR, UV-visible spectroscopy. The three-dimensional molecular structure of the MCDPO is confirmed by single crystal XRD study. The MCDPO crystallized in a triclinic system P-1 space group with the following unit cell parameter: a = 5.0347(2) Å, b = 10.3509(4) Å, c = 11.8816(4) Å, α = 84.431(3)°, β = 84.172(3)°, γ = 81.380(4)°, and V = 606.92(4) Å3 at T = 133 K. The MCDPO is containing aromatic rings, hydrogen bonding donors as well as hydrogen bonding acceptor groups forming supramolecular molecular arrangements through C-H⋯O, CH⋯N, C-O⋯C, π-π and π⋯C non-covalent interactions. Electrochemical redox and electrochromic features of MCDPO are studied through cyclic voltammetry and UV-vis based spectroelectrochemistry. This compound shows good anticancer activity with IC50 values 1.438, 6.576 and 2.901 µM against 4T1, MCF-7 and PC-3 cancer cell lines respectively. The flow cytometry study on 4T1 cells suggests that the MCDPO promoted cancer cell death by inducing apoptosis, and cell cycle arrest in the G0/G1 phase. It also induces nuclear fragmentation and reactive oxygen species (ROS) generation, which was studied by DAPI, AO, and DCFH-DA based cellular staining studies by fluorescence confocal imaging. The MCDPO also studied for antibacterial activity against Escherichia Coil bacteria and Mycobacterium tuberculosis (Mtb). The MCDPO shows minimum inhibitory concentration (MIC) of 0.39 µg/mL against Mtb which is slightly better than the one of the clinically used drug candidates Ethambutol.

Betaine-Modified Green Carbon Dot for Cr(VI) Sensing, in Vivo Cr(VI) Imaging, and Growth Promotion in the Rice Plant.

Hexavalent chromium is a toxic environmental pollutant that damages plants due to disruption of nutrient uptake, photosynthesis metabolism, and oxidative stress, which suppresses the growth and development of the plant. In this work, we have developed a betaine-modified carbon dot (BT@CD) sensor for monitoring Cr(VI) in water and plants. Fluorescent carbon dots have been synthesized using jamun juice (Syzygium cumini) as the carbon source subjected to surface modification with betaine (BT@JCD). This BT@JCD exhibits strong blue fluorescence, which significantly decreases in the presence of Cr(VI) due to the inner filter effect in a range of 5-450 nM with a detection limit of 0.033 μM. Due to its easy translocation in the vascular bundles, these fluorescence nanosensors can be applied to detect Cr(VI) in rice plants Oryza sativa) through fluorescence confocal imaging. The treatment of rice plants with BT@JCDs in the concentration range of 0.2 to 1g/mL not only triggered photophysical parameters such as carbohydrates, chlorophyll, and carotenoids but also enhanced the antioxidant enzyme activity promoting plant growth.

Edwardsiella piscicida promotes mitophagy to escape autophagy-mediated antibacterial defense in teleost monocytes/macrophages

Edwardsiella piscicida is a Gram-negative intracellular pathogen. Currently, the immune evasion strategies of E. piscicida are not yet fully understood. In this study, we revealed that E. piscicida exploited mitophagy to enhance its intracellular survival in grass carp (Ctenopharyngodon idella) monocytes/macrophages. The results showed that in E. piscicida-infected cells, there was a significant loss of mitochondrial membrane potential and a marked increase in the production of mitochondrial reactive oxygen species (mtROS), indicating the damage to the mitochondria in the infected cells. Furthermore, fluorescence confocal images presented that E. piscicida infection elicited the colocalization of mitochondria with microtubule-associated protein 1 light chain 3 (LC3, a key marker molecule of autophagy) and lysosomes, suggesting the occurrence of mitophagy. This notion was supported by the findings that E. piscicida significantly stimulated LC3-II protein and damped Tom20 (a marker molecule of mitochondria) protein levels. Subsequently, a mitophagy inducer carbonyl cyanide m-chlorophenyl hydrazine (CCCP) and a mitophagy inhibitor mitochondrial division inhibitor 1 (Mdivi-1) were used to evaluate the role of mitophagy in modulating the intracellular survival of E. piscicida. The results revealed that CCCP significantly promoted intracellular survival of E. piscicida, whereas Mdivi-1 hindered the bacterial growth. Moreover, Mdivi-1 further increased the production of bacteria-induced mitochondrial ROS in an additive manner. In particular, CCCP suppressed and Mdivi-1 promoted the colocalization of LC3-II with E. piscicida, implying that E. piscicida-triggered mitophagy to facilitate its growth probably by subverting the autophagy targeting this bacterium, a previously known clearance pathway of E. piscicida. Overall, our findings suggest a new survival strategy employed by E. piscicida within fish monocytes/macrophages.

Supported Supramolecular Hydrogel Nanoarchitectonics for Tunable Biocatalytic Flow Activity.

Enzymatically-active polyelectrolyte multilayers containing n layers of phosphatase (APn-PEM) induce the formation of supported biocatalytic supramolecular hydrogels when brought in contact with the precursor tripeptide Fmoc-FFpY (Fmoc = N-fluorenylmethyloxycarbonyl; F = Phenylalanine; Y = Tyrosine; p = phosphate group). APn-PEM triggers the spatially-localized hydrogelation reaching 2, 17 and 350µm of thickness for n = 1, 2 and 3, respectively. As observed by cryo scanning electron microscopy, a dense nanofibrous network underpinning the hydrogel shows parallelly orientated Fmoc-FFY peptide-based fibrils, perpendicular to the substrate. For the gel generated by the AP3-PEM, fluorescence confocal microscopy images show that during the peptide self-assembly, some enzymes are distributed in the hydrogel, preferentially located in few dozens of micrometers above the substrate. In addition, a self-assembly growth rate of 5µmmin-1 is determined when the hydrogelation starts. Through transmission electron microscopy immuno-labelling experiments on self-assemblies generated in solution, we observe that AP are decorating the Fmoc-FFY nanofibers. It is observed both a long-term stability and a higher biocatalytic activity of the so AP-encapsulated hydrogel compared to the bare APn-PEM. This bioactivity can be tuned by the number n in batch and under continuous flow conditions. To illustrate the versatility of this enzyme-supported strategy, multi-catalytic transformations in continuous flow conditions have been successfully carried out using supported supramolecular hydrogel.

Mesoporous Silica Microparticle-Protein Complexes: Effects of Protein Size and Solvent Properties on Diffusion and Loading Efficiency.

Oral administration of protein-based therapeutics is highly desirable due to lower cost, enhanced patient compliance, and convenience. However, the harsh pH environment of the gastrointestinal tract poses significant challenges. Silica-based carriers have emerged as potential candidates for the delivery of protein molecules, owing to their tuneable surface area and pore volume. We explored the use of a commercial mesoporous silica carrier, SYLOID, for the delivery of octreotide and bovine serum albumin (BSA) using a solvent evaporation method in three different solvents. The loading of proteins into SYLOID was driven by diffusion, as described by the Stokes-Einstein equation. Various parameters were investigated, such as protein size, diffusion, and solubility. Additionally, 3D fluorescence confocal imaging was employed to identify fluorescence intensity and protein diffusion within the carrier. Our results indicated that the loading process was influenced by the molecular size of the protein as octreotide exhibited a higher recovery rate (71%) compared to BSA (32%). The methanol-based loading of octreotide showed uniform diffusion into the silica carrier, whereas water and ethanol loading resulted in the drug being concentrated on the surface, as shown by confocal imaging, and further confirmed by scanning electron microscopy (SEM). Pore volume assessment supported these findings, showing that octreotide loaded with methanol had a low pore volume (1.2cc/g). On the other hand, BSA loading was affected by its solubility in the three solvents, its tendency to aggregate, and its low solubility in ethanol and methanol, which resulted in dispersed particle sizes of 223 and 231μm, respectively. This reduced diffusion into the carrier, as confirmed by fluorescence intensity and diffusivity values. This study underscores the importance of protein size, solvent properties, and diffusion characteristics when using porous carriers for protein delivery. Understanding these factors allows for the development of more effective oral protein-based therapeutics by enhancing loading efficiency. This, in turn, will lead to advances in targeted drug delivery and improved patient outcomes.

Plant cell wall enzymatic deconstruction: Bridging the gap between micro and nano scales

Understanding lignocellulosic biomass resistance to enzymatic deconstruction is crucial for its sustainable conversion into bioproducts. Despite scientific advances, quantitative morphological analysis of plant deconstruction at cell and tissue scales remains under-explored. In this study, an original pipeline is devised, involving four-dimensional (space + time) fluorescence confocal imaging, and a novel computational tool, to track and quantify deconstruction at cell and tissue scales. By applying this pipeline to poplar wood, dynamics of cellular parameters was computed and cellulose conversion during enzymatic deconstruction was measured. Results showed that enzymatic deconstruction predominantly impacts cell wall volume rather than surface area. Additionally, a negative correlation was observed between pre-hydrolysis compactness measures and volumetric cell wall deconstruction rate, whose strength was modulated by enzymatic activity. Results also revealed a strong positive correlation between average volumetric cell wall deconstruction rate and cellulose conversion rate. These findings link key deconstruction parameters across nano and micro scales.

Novel High-Resolution Three-Dimensional Fluorescent Confocal Imaging Resolves Differences in Urinary Sediment

Novel High-Resolution Three-Dimensional Fluorescent Confocal Imaging Resolves Differences in Urinary Sediment

Sensitive Cancer Hypoxia Detection via a Dual-Locking Fluorescence Response System Using Two Hypoxia Indicators.

Hypoxia is intricately associated with various diseases, including ischemia, vascular disorders, and cancer. Particularly in cancer cells, hypoxia promotes tumor growth, cell proliferation, migration, and invasion and enhances treatment resistance, making its detection crucial for cancer diagnosis and therapy. However, methods for detecting hypoxia are limited, often relying on single-detection systems. In this study, we developed a dual-lock-based fluorescent probe that selectively exhibits strong green fluorescence under hypoxic conditions due to simultaneous activity of nitroreductases (NTRs) and hydrogen sulfide (H2S), with a high signal-to-background ratio. The biocompatibility and photophysical properties of the probes were thoroughly investigated through both extracellular and intracellular experimental analyses. Among the synthesized naphthalimide-based probes, the dual-detection probe DNNC demonstrated excellent selectivity and sensitivity to the simultaneous activity of NTR/H2S compared to other single-detection probes. The performance of DNNC was applied to various organ-derived cancer cells and tumor tissue models such as HeLa cell sparoids, enabling spatiotemporal confocal fluorescence imaging and quantitative analysis of hypoxic levels in cancer. Our development of DNNC is expected to significantly advance cancer diagnosis and treatment by molecularly detecting hypoxia associated with cancer aggressiveness, therapy resistance, and unfavorable prognosis.

Somatostatin modulation of initial fusion pores in Ca2+-triggered exocytosis from mouse chromaffin cells.

Somatostatin, a peptide hormone that activates G-protein-coupled receptors, inhibits the secretion of many hormones. This study investigated the mechanisms of this inhibition using amperometry recording of Ca2+-triggered catecholamine secretion from mouse chromaffin cells. Two distinct stimulation protocols, high-KCl depolarization and caffeine, were used to trigger exocytosis, and confocal fluorescence imaging was used to monitor the rise in intracellular free Ca2+. Analysis of single-vesicle fusion events (spikes) resolved the action of somatostatin on fusion pores at different stages. Somatostatin reduced spike frequency, and this reduction was accompanied by prolongation of pre-spike feet and slowing of spike rise times. This indicates that somatostatin stabilizes initial fusion pores and slows their expansion. This action on the initial fusion pore impacted the release mode to favour kiss-and-run over full-fusion. During a spike the permeability of a fusion pore peaks, declines and then settles into a plateau. Somatostatin had no effect on the plateau, suggesting no influence on late-stage fusion pores. These actions of somatostatin were indistinguishable between exocytosis triggered by high-KCl and caffeine, and fluorescence imaging showed that somatostatin had no effect on stimulus-induced rises in cytosolic Ca2+. Our findings thus demonstrate that the signalling cascades activated by somatostatin target the exocytotic machinery that controls the initial and expanding stages of fusion pores, while having no effect on late-stage fusion pores. As a result of its stronger inhibition of full-fusion compared to kiss-and-run, somatostatin will preferentially inhibit the secretion of large peptides over the secretion of small catecholamines. KEY POINTS: Somatostatin inhibits the secretion of various hormones by activating G-protein-coupled receptors. In this study, we used amperometry to investigate the mechanism by which somatostatin inhibits catecholamine release from mouse chromaffin cells. Somatostatin increased pre-spike foot lifetime and slowed fusion pore expansion. Somatostatin inhibited full-fusion more strongly than kiss-and-run. Our results suggest that the initial fusion pore is the target of somatostatin-mediated regulation of hormone release. The stronger inhibition of full-fusion by somatostatin will result in preferential inhibition of peptide release.

Characterizing thrombus adhesion strength on common cardiovascular device materials.

Thrombus formation in blood-contacting medical devices is a major concern in the medical device industry, limiting the clinical efficacy of these devices. Further, a locally formed clot within the device has the potential to detach from the surface, posing a risk of embolization. Clot embolization from blood-contacting cardiovascular devices can result in serious complications like acute ischemic stroke and myocardial infarction. Therefore, clot embolization associated with device-induced thrombosis can be life-threatening and requires an enhanced fundamental understanding of embolization characteristics to come up with advanced intervention strategies. Therefore, this work aims to investigate the adhesive characteristics of blood clots on common biocompatible materials used in various cardiovascular devices. This study focuses on characterizing the adhesion strength of blood clots on materials such as polytetrafluoroethylene (PTFE), polyurethane (PU), polyether ether ketone (PEEK), nitinol, and titanium, frequently used in medical devices. In addition, the effect of incubation time on clot adhesion is explored. Results from this work demonstrated strongest clot adhesion to titanium with 3h of incubation resulting in 1.06 ± 0.20kPa detachment stresses. The clot adhesion strength on titanium was 51.5% higher than PEEK, 35.9% higher than PTFE, 63.1% higher than PU, and 35.4% higher than nitinol. Further, adhesion strength increases with incubation time for all materials. The percentage increase in detachment stress over incubation time (ranging from 30min to 3h) for polymers ranged from at least 108.75% (PEEK), 140.74% (PU), to 151.61% (PTFE). Whereas, for metallic surfaces, the percentage rise ranged from 70.21% (nitinol) to 89.28% (titanium). Confocal fluorescence imaging of clot remnants on the material surfaces revealed a well-bounded platelet-fibrin network at the residual region, representing a comparatively higher adhesive region than the non-residual zone of the surface.

Dual-functional fluorescent probe for imaging ROS and hypoxia dynamic in cancer cells

Hypoxia is able to induce the overexpression of nitroreductase (NTR) in cancer cells. Moreover, there is still the controversy about reactive oxygen species (ROS) change under the hypoxic condition. Herein, we designed and synthesized a fluorescent probe WNO that could simultaneously visualize the fluctuation changes of NTR and ROS in hypoxic HeLa cells. WNO consists of 4-nitro-1,8-naphthalimide group and 2,3,3-trimethyl indolium salt. In buffer 4-nitro-1,8-naphthalimide group could selectively response to NTR in the presence of nicotinamide adenine dinucleotide phosphate (NADH), resulting in a strong green fluorescence. However, upon LED light irradiation 2,3,3-trimethyl indolium salt moiety could efficiently convert into Cy3 emitting a red fluorescence. Furthermore, the monitoring for NTR and photoconversion of WNO in hypoxic HeLa cells were investigated in detail by confocal fluorescence imaging through green and red channels, respectively. It was found that WNO could image the intracellular NTR or hypoxia level. Meanwhile, the WNO photoconversion was accelerated largely in hypoxic cells, which resulted from the production of excessive ROS. WNO and/or its photoconversion product mainly accumulated into the mitochondria. Moreover, MTT assay indicated that LED irradiation or hypoxia treatment made the cytotoxicity of WNO increase by 50 %. Additionally, based on ROS-stimulated the photoconversion of WNO, the intracellular ROS and NTR changes in mitochondrial dysfunction and oxidative stress were also investigated. It indicated that WNO was capable of sensitive and rapid imaging the intracellular ROS and NTR levels under both conditions. These findings would provide an excellent foundation for cancer diagnosis and therapy.