Spatio-temporal metabolokinetics and therapeutic effect of CD106+ mesenchymal stem/stromal cells upon mice with acute lung injury.

Longitudinal investigations have revealed the unique attributes of mesenchymal stem/stromal cells (MSCs) in regenerative medicine. However, the spatio-temporal metabolokinetics and efficacy of MSCs with vascular cell adhesion molecule 1 (also known as CD106) expression in phenotypes and therapeutic effect upon acute lung injury (ALI) mice are largely obscure. For the purpose, we took advantage of the "3IL"-based strategy and Lentivirus-mediated green fluorescent protein (GFP) delivery for the generation of the CD106+ subset (denote as CD106+ -MSCs) from umbilical cord-derived MSCs (denote as NT-MSCs). Therewith, the cellular phenotypes of CD106+ -MSCs including immunophenotypes, multilineage differentiation potential towards adipocytes and osteoblasts were confirmed by flow cytometry and qRT-PCR assay. Meanwhile, multifaceted characteristics of transcriptomic features were analyzed by utilizing the RNA-SEQ and bioinformatics. Furthermore, to compare the therapeutic effects and spatio-temporal dynamics of CD106+ -MSCs, we conducted in vivo fluorescent tracer, hematoxylin and eosin staining, blood smear, blood routine and cytokine detection in mice. Herein, we generated CD106+ -MSCs with GFP expression and confirmed the conservative property of phenotypes. Compared to NT-MSCs with minimal CD106 expression, CD106+ -MSCs manifested consistent distribution and metabolokinetics in vivo but with preferable ameliorative effect upon the pathological appearance and proinflammatory cytokine secretion in ALI mice. Collectively, our data indicated the preferable therapeutic effects of CD106+ -MSCs upon ALI mice, which would benefit the further exploration of the CD106+ subset for pulmonary diseases and investigational new drug application purposes.

Natural Solar Irradiation Produces Fluorescent and Biodegradable Nanoplastics.

Nanoplastics (NPs) have raised global concern owing to their potential health effects. Herein, after simulated and natural solar irradiation, polyethylene, polypropylene, polystyrene, and poly(vinyl chloride) nanoplastics (PVC NPs) were observed to exhibit enhanced fluorescence, particularly PVC NPs. Furthermore, the role of photoaged NPs as a potential fluorescence indicator was evaluated by exposing a model aquatic organism Daphnia magna to these NPs. Our results revealed that photoaged NPs exhibited strong fluorescence owing to the generation of conjugated π bonds, which can achieve π-π* electron transition with low energy consumption. Photogenerated fluorescence also enabled the photoaged NPs to act as efficient fluorescent tracers, which can help track NP migration in various organisms. The results of two-photon laser confocal scanning microscopy revealed that the photoaged NPs could translocate across biological barriers and accumulate in extraintestinal tissues in addition to being ingested and excreted. Moreover, compared with pristine NPs, the photoaged NPs underwent biodegradation more easily, probably because of increased hydrophilicity due to photogenerated oxygen-containing moieties. Therefore, in addition to producing fluorescent NPs without the attachment of external fluorescent dyes, the natural photoaging process can promote the migration and degradation of photoaged NPs in food chains.

Synthesis of molecularly imprinted microspheres and development of a fluorescence method for detection of chloramphenicol in meat.

In this study, nitrobenzene was used as dummy template to synthesize a type of specific molecularly imprinted microspheres for chloramphenicol, and 4-nitroaniline was coupled with three fluorophores to synthesize three fluorescent tracers. Then a competitive fluorescence method was developed on a conventional microplate for detection of chloramphenicol in chicken and pork samples. This method contained only one sample-loading step, so one assay was finished within 30 min. The IC50 was 1.8 ng/ml, and the limit of detection was 0.06 ng/g. The recoveries from chloramphenicol-fortified blank meat samples were in the range 67.5-96.2%. Furthermore, this method could be recycled three times. The detection results for some real meat samples were identical to that of a LC-MS/MS method. Therefore, this method could be used as a practical tool for routine screening for the residue of chloramphenicol in large number of meat samples.

Quantitative analysis of droplet deposition produced by an electrostatic sprayer on a classroom table by using fluorescent tracer.

Due to the ongoing COVID-19 pandemic situation, measures to mitigate the risk of transmission of the SARS-CoV-2 virus in an indoor setting are urgently needed. Among the various types of disinfectant methods, electrostatic spraying is often applied to decontamination in public places. For quantitatively characterizing electrostatic spraying, we developed the novel evaluation method by using a fluorescent tracer. By applying this method, we performed three different experiment cases (static test on a table, static test on a cylinder, and dynamic test on a table) to figure out its unique characteristics (Coulombic fission and wraparound effect) and measure its performance in various aspects. To be specific, bimodal distribution with peak sizes of ~10 and ~100 µm was found due to Coulombic fission. Otherwise, a unimodal distribution with a peak size of ~100 µm occurred for the uncharged droplets. As a result, the effective contact area increased by 40-80 % due to small progeny droplets. The wraparound effect was examined on two different cylinders: copper (Cu) and polyvinyl chloride (PVC) pipe. When the target surface was not charged (Cu 0 kV and PVC 0 kV), the average normalized concentrations on the backside of the cylinder (θ = 180°) increased by around 67 % for charged droplets. Meanwhile, when the target surface was highly charged (PVC -19 kV), the average normalized concentrations at θ = 180° were increased more than two times for charged droplets.

Next tier in vitro and in vivo nonclinical studies further elucidating the safety and toxicity profile of MB-102, a novel fluorescent tracer agent for measurement of glomerular filtration rate.

MB-102 was designed for measurement of real-time glomerular filtration rate (GFR). Previously reported in vitro and in vivo nonclinical studies clearly demonstrated negligible toxicity, resulting in FDA clearance for First-in Human, proof of concept clinical studies. The next tier of safety and toxicity studies are reported herein. MB-102 did not demonstrate any phototoxic potential in a BALB/c 3T3 mouse fibroblast study. Co-administration of MB-102 and iohexol resulted in pharmacokinetic parameters virtually identical to the values observed upon individual administration in beagle dogs. A single dose of MB-102 administered either intravenously (18.6 mg/mL) or perivenously (0.25 mL) was well-tolerated in NZ white rabbits, with no adverse inflammation or irritation. MB-102 did not induce micronuclei in polychromatic erythrocytes for rat bone marrow cells treated up to 450 mg/kg/day, the maximum feasible dose. Two separate optical imaging studies demonstrated that MB-102 distributes rapidly and thoroughly throughout the test subjects, followed by rapid clearance from the body without any preferential localization in any particular tissue or organ, with the exception of the bladder, which is totally consistent with a known GFR agent. In addition, two-week repeat intravenous (once-daily) toxicity and toxicokinetic studies were conducted in rats and beagles, with no MB-102- related effects. Thus, for the studies reported herein, there were no toxicological effects of concern for MB-102.

Exploring non-curative endoscopic submucosal dissection: Current treatment optimization and future indication expansion.

The increasing popularity of endoscopic submucosal dissection (ESD) as a treatment for early gastric cancer has highlighted the importance of quality assessment in achieving curative resections. This article emphasizes the significance of evaluating ESD quality, not only for curative cases but also for non-curative ones. Postoperative assessment relies on the endoscopic curability (eCura) classification, but management strategies for eCuraC-1 tumour with a positive horizontal margin are unclear. Current research primarily focuses on comparing additional surgical procedures in high-risk patients, while studies specifically targeting eCuraC-1 patients are limited. Exploring management strategies and follow-up outcomes for such cases could provide valuable insights. Furthermore, the application of molecular imaging using near-infrared fluorescent tracers holds promise for precise tumour diagnosis and navigation, potentially impacting the management of early-stage gastric cancer patients. Advancing research in these areas is essential for improving the overall efficacy of endoscopic techniques and refining treatment indications.

A Simulation-based PPE orientation training curriculum for novice physicians.

Background: Personal protective equipment (PPE) is effective in preventing coronavirus disease (COVID-19) infection. Resident knowledge of proper use and effective training methods is unknown. We hypothesise that contamination decreases and knowledge increases after a formalised PPE educational session. Methods: Participants included first year interns during their residency orientation in June 2020. Before training, participants took a knowledge test, donned PPE, performed a simulated resuscitation, and doffed. A standardised simulation-based PPE training of the donning and doffing protocol was conducted, and the process repeated. Topical non-toxic highlighter tracing fluid was applied to manikins prior to each simulation. After doffing, areas of contamination, defined as discrete fluorescent areas on participants' body, was evaluated by ultraviolet light. Donning and doffing were video recorded and asynchronously rated by two emergency medicine (EM) physicians using a modified Centers for Disease Control and Prevention (CDC) protocol. The primary outcome was PPE training effectiveness defined by contamination and adherence to CDC sequence. Results: Forty-eight residents participated: 24 internal medicine, 12 general surgery, 6 EM, 3 neurology, and 3 psychiatry. Before training, 81% of residents were contaminated after doffing; 17% were contaminated after training (P<0.001). The most common contamination area was the wrist (50% pre-training vs. 10% post-training, P<0.001). Donning sequence adherence improved (52% vs. 98%, P<0.001), as did doffing (46% vs. 85%, P<0.001). Participant knowledge improved (62%-87%, P <0.001). Participant confidence (P<0.001) and preparedness (P<0.001) regarding using PPE increased with training. Conclusion: A simulation-based training improved resident knowledge and performance using PPE.

Effects and metabolism of fish collagen sponge in repairing acute wounds of rat skin.

Objective: Study the repair effect of tilapia collagen on acute wounds, and the effect on the expression level of related genes and its metabolic direction in the repair process. Materials and methods: After the full-thickness skin defect model was constructed in standard deviation rats, the wound healing effect was observed and evaluated by means of characterization, histology, and immunohistochemistry. RT-PCR, fluorescence tracer, frozen section and other techniques were used to observe the effect of fish collagen on the expression of related genes and its metabolic direction in the process of wound repair. Results: After implantation, there was no immune rejection reaction, fish collagen fused with new collagen fibers in the early stage of wound repair, and was gradually degraded and replaced by new collagen in the later stage. It has excellent performance in inducing vascular growth, promoting collagen deposition and maturation, and re-epithelialization. The results of fluorescent tracer showed that fish collagen was decomposed, and the decomposition products were involved in the wound repair process and remained at the wound site as a part of the new tissue. RT-PCR results showed that, without affecting collagen deposition, the expression level of collagen-related genes was down-regulated due to the implantation of fish collagen. Conclusion: Fish collagen has good biocompatibility and wound repair ability. It is decomposed and utilized in the process of wound repair to form new tissues.

In Vitro Methods for Measuring the Permeability of Cell Monolayers.

Cell monolayers, including endothelial and epithelial cells, play crucial roles in regulating the transport of biomolecules to underlying tissues and structures via intercellular junctions. Moreover, the monolayers form a semipermeable barrier across which leukocyte transmigration is tightly regulated. The inflammatory cytokines can disrupt the epithelial and endothelial permeability, thus the reduced barrier integrity is a hallmark of epithelial and endothelial dysfunction related with numerous pathological conditions, including cancer-related inflammation. Therefore, the assessment of barrier function is critical in in vitro models of barrier-forming tissues. This review summarizes the commercially available in vitro systems used to measure the permeability of cellular monolayers. The presented techniques are separated in two large groups: macromolecular tracer flux assays, and electrical impedance measurement-based permeability assays. The presented techniques are briefly described and compared.

Gain and retain - On the efficiency of modified agricultural drainage ponds for pesticide retention.

Drainage ponds have the potential to serve as long-term interface measures primarily for flood control, and mass retention. They are often considered as promising supplements for the mitigation of drainage pipe loads to improve the water quality in agricultural landscapes. In this study, a highly dynamic drainage pond system with non-steady inflows and groundwater interaction was modified and investigated regarding its potential for pesticide and transformation product (TP) retention. For this purpose, two 104-day monitoring campaigns were conducted before and after pond modification. Field experiments with fluorescent tracers, Uranine and Sulforhodamine-B, proved that structural modifications improved the hydraulic functionality of the ponds. The effective volume (Ɛ) increased from 20% to almost 100% in the modified pond and the mean hydraulic residence time (τ) was ten times longer. After a dry period, pesticide retention was high during slow refilling of the ponds, still TP loads posed a risk by infiltration into shallow groundwater due to the permeable ground. During wet periods, short nominal detention times together with high inflows led to rare high retention rates through peak attenuation. Moderate inflows resulted in extremely variable retention values, owing to the small pond storage capacity. Along with this, the total retention efficiency after modification reached up to 38% for mobile, 29% for sorptive pesticides, and 32% for mobile TPs. To achieve the best performances for ponds as natural landscape elements, they should be analysed for their hydrological functionality as a prerequisite and then modified for delayed pesticide and TP transport. Then, dynamic drainage ponds can utilize their full potential regarding mitigation of pesticide and TP loads in agricultural catchments.

Development of a molecularly imprinted microspheres-based microplate fluorescence method for detection of amantadine and rimantadine in chicken.

In this study, molecularly imprinted microspheres of a type capable of recognising amantadine and rimantadine were first synthesised, and three fluorescent tracers based on dansyl chloride, fluorescein isothiocyanate and 5-carboxytetramethylrhodamine were also synthesised. These reagents were used to develop and optimise a direct competitive fluorescence method on conventional 96-well microplate for detection of the two analytes. Results showed that this method achieved simple operation procedure, rapid assay process (30 min), high sensitivity (limits of detection 0.04-0.05 ng mL-1) and acceptable recycle performance (five times). After optimisation of several parameters, this method was used to detect amantadine and rimantadine in chicken muscle samples. Their recoveries from standards fortified blank samples were in the range of 62.3-93.7%. The analysis results for some real chicken samples were consistent with a confirmatory LC-MS/MS method. Therefore, this method could be used as a rapid, simple and accurate tool for routine screening the residues of amantadine and rimantadine in a large number of chicken muscle samples.

A novel, direct-push approach for detecting sulfidated nanoparticulate zero valent iron (S-nZVI) in sediments using reactive and non-reactive fluorophores.

Injection of microparticulate and nanoparticulate zero valent iron has become a regularly used method for groundwater remediation. Because of subsurface inhomogeneities, however, it is complicated to predict the ZVI transport in the subsurface, meaning that tools capable of determining its distribution after injection are highly useful. Here, we have developed a new direct-push based technique, which combines fluorescent and visible imaging, for detection of sulfidized nanoparticulate zero valent iron (S-nZVI) in the subsurface. Laboratory experiments show that the redox sensitive fluorophore riboflavin is rapidly reduced by S-nZVI within 200 s. Because the reduced riboflavin losses its green fluorescence, it can be used as S-nZVI sensitive indicator. Secondly, S-nZVI is black and tints light coloured sediment to a degree that allows detection in images. For quartz sand, 70 mg/kg of S-nZVI can be detected by visible imaging. Based on these results, a new direct-push probe (Dye-OIP) was designed based on Geoprobe's Optical Image Profiler (OIP), which was equipped with a fluorophore injection port below the OIP-unit. The injectant consisted of the redox active riboflavin mixed with the redox inactive fluorophore rhodamine WT, which fluoresces red and was used to verify that the mixture was indeed injected and detectable. Small scale experiments show that the fluorescence of this mixture in S-nZVI amended sand changes within 150 s from green with a hue of ~50 to red with a hue of ~30 when imaged with Dye-OIP. Tests of the Dye-OIP after a S-nZVI injection in a 1 m3 sized tank show that the tool could detect S-nZVI via fluorescence and visible imaging, when S-nZVI concentration was >0.2 mg per g dry sediment. Thus, these novel methods should be able to detect S-nZVI in the subsurface, without relying on infrastructure such as wells. Based on our results, the Dye-OIP could be further improved to make it suitable for regular use in the field.

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing detailed in situ hybridization analyses during zebrafish development, we discovered new expression domains for wt1a in the dorsal hindbrain, the caudal medulla and the spinal cord. Marker analysis identified wt1a expressing cells of the dorsal hindbrain as ependymal cells of the choroid plexus in the myelencephalic ventricle. The choroid plexus acts as a blood-cerebrospinal fluid barrier and thus is crucial for brain homeostasis. By employing wt1a mutant larvae and a dye accumulation assay with fluorescent tracers we demonstrate that Wt1a is required for proper choroid plexus formation and function. Thus, Wt1a contributes to the barrier properties of the choroid plexus in zebrafish, revealing an unexpected role for Wt1 in the zebrafish brain.

Field insights into leaching and transformation of pesticides and fluorescent tracers in agricultural soil.

The frequent detection of residues from pesticides in various natural water types has raised public awareness. This study investigated the pesticide transformation in soil and their loss to shallow groundwater in a small agricultural catchment in Northern Germany. The pesticide Flufenacet and its transformation product Flufenacet ESA were examined in Luvisol and Colluvic Gleyosol under field conditions during two consecutive years. In the second year, a fluorescent tracer experiment applying Uranine and Sulforhodamine - B was carried out to gain additional insights into leaching and formation of transformation products in soil during and after a drought. We found preferential flow in response to low precipitation as an important transport pathway for Flufenacet in dry soil, as a Flufenacet concentration (1.57 µg L-1) was detected in shallow groundwater within 10 days after application. Leaching of Flufenacet to shallow groundwater by preferential flow posed greater risks during the dry than during the wet period. In contrast, Flufenacet ESA was detected in all groundwater samples. During the dry period, we detected no formation of TP510 (tracer transformation product) in the immediate topsoil. A fraction of both tracers remained there, suggesting also long-term residues of pesticides in the topsoil caused by limited living conditions for microorganisms under dry conditions. Newly formed transformation products of Uranine and Flufenacet were mainly trapped in upper soil if capillary flow was marginal. Formation of TP510 could be related to a soil water optimum and a soil temperature threshold. The occurrence of increased TP510 amounts in soil after drought was concurrent with the main peak of Flufenacet ESA in shallow groundwater. This suggested similar retention and transformation processes of fluorescent tracers and organic pesticides inside the soil. This study contributed to an extended understanding of the leaching and transformation of organic pollutants in agricultural soil under real field conditions.

Reducing dermal exposure to agrochemical carcinogens using a fluorescent dye-based intervention among subsistence farmers in rural Honduras.

BACKGROUND: Occupational exposure to agrochemicals, some of which are known or suspected carcinogens, is a major health hazard for subsistence agricultural workers and their families. These impacts are more prevalent in low-and-middle income countries (LMIC) due to weak regulations, lack of awareness of the risks of contamination, predominant use of handheld backpack style spraying equipment, general lack of personal protective equipment (PPE), and low literacy about proper agrochemical application techniques. Reducing exposure to agrochemicals was identified as a paramount concern by rural Hondurans working with a community-engaged research initiative. Fluorescent tracer dyes have been described as a means of visualizing and quantifying dermal exposure to agricultural chemicals, and exposure models adapted for LMIC have been developed previously. Tracer dyes have also been used in educational simulations to promote pesticide safety. However, studies evaluating the effectiveness of these educational dye interventions in reducing future exposure have been lacking. AIM: To evaluate whether observing one's own chemical contamination after applying agrochemicals changed the amount of occupational dermal exposure during a subsequent chemical application. METHODS: We employed a multi-modal community intervention in a rural village in Honduras that incorporated chemical safety education and use of a fluorescent tracer dye during pesticide application on two consecutive occasions, and compared dermal exposure between the intervention group (previous dye experience and safety education, n = 6) and the control group (safety education only, n = 7). RESULTS: Mean total visual score (TVS) of the tracer dye, which accounts for both extent and intensity of whole-body contamination, was lower among those who had previously experienced the dye intervention (mean TVS = 41.3) than among participants who were dye-naïve (mean TVS = 78.4), with a difference between means of -37.10 (95% CI [-66.26, -7.95], p = 0.02). Stratifying by body part, contamination was significantly lower for the anterior left lower extremity and bilateral feet for the dye-experienced group vs. dye-naïve, with most other segments showing a trend toward decreased contamination as well. CONCLUSION: Participants who had previously experienced the dye intervention were significantly less contaminated than the dye-naïve control group during a subsequent spraying event. The findings of this small pilot study suggest that a multi-modal, community-based approach that utilizes fluorescence-augmented contamination for individualized learning (FACIL) may be effective in reducing dermal exposure to carcinogenic agrochemicals among subsistence farmers in Honduras and other LMIC.

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry.

Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity, and this will be useful in preclinical studies.

A hollow-structured nanohybrid: Intelligent and visible drug delivery and photothermal therapy for cancer.

It is of great valuable in drug delivery to fabricate multifunctional nanovehicle for cancer therapy. Herein, hollow-structured hCu2-xS@Au nanoshell/satellite composite with doxorubicin-carrying was designed and synthesized for intelligent and visible drug delivery and satisfactory photothermal therapy. By modification of disulfide linkage bridged Au nanoshell and multi-carboxylic graphene quantum dots (MC-GODs) on the surface of hCu2-xS@Au nanoparticles, both the high concentration of glutathione and low pH in cancer cell/tissue can induce responsive drug release. The satisfactory photothermal conversion efficiency (32%) of hCu2-xS@Au@MC-GODs under 808 nm near-infrared (NIR) laser irradiation ascribed to the reduced bandgap and more circuit paths for electron transitions for hCuS modified with Au nanoparticles depend on density functional theory, which antitumor therapy efficacy was greatly enhanced by combining chemo- and photothermal therapy. Moreover, the fluorescence of MC-GODs was quenched/"turn off" as linking to the surface of hCu2-xS@Au, and also restored/"turn on" as the MC-GODs detaching from the surface of hCu2-xS@Au. The fluorescent switch of MC-GODs can serve as both a controllable drug release "guard" and "eyes" for visualized monitoring. The multi-modality therapy with controllable drug delivery, visual monitoring and high photothermal conversion efficiency may be anticipated by this versatile strategy.

Validation studies of a fluorescent method to measure placental glucose transport in mice.

INTRODUCTION: Proper placental function is essential for optimal fetal growth in utero. Placental transfer of nutrients to the fetus can be measured using radiolabelled tracers, but non-radioactive methods have potential advantages. This study aimed to develop a fluorescence-based method to measure placental glucose transport in mice. METHODS: Time course and localisation of the IRDye 800CW 2-deoxyglucose were recorded (Lumina IVIS Live Imaging System) following tail vein injection into anaesthetised late pregnant mice. Fluorescent signals in placental and fetal tissues were assessed after injecting conscious dams with 10 nmol IRDye 800CW 2-deoxyglucose (3, 30, 60, 120 min) or vehicle. Specificity of dye uptake was determined by comparing uptake of IRDye 800CW conjugated to 2-deoxyglucose or carboxylate, at 2 and 24 h. Finally, we assessed relationships of fetal size and umbilical blood flow velocities with relative dye uptake. RESULTS: In late pregnant mice, uterine fluorescent signal localised rapidly over placentas and remained consistent for >1 h. Signal intensity in whole and homogenised tissues increased in fetuses and decreased in placentas after 3 min and stabilised by 30 min post-injection. Relative fetal dye uptake at 2 and 24 h was greater in littermates with the highest compared to lowest placental efficiency; signals were similar for 2-deoxyglucose- or carboxylate-conjugated dyes. Relative fetal dye uptake correlated positively with fetal weight and placental efficiency and negatively with umbilical artery resistance indices. CONCLUSIONS: Fetal uptake of IRDye 800CW correlates with markers of placental blood flow and fetal growth, but does not specifically measure placental glucose transport.

Quantification of Symplasmic Phloem Loading Capacity with Live-Cell Microscopy.

Sugars produced by photosynthesis in leaves get transported to other organs in the phloem vascular tissue. Three general mechanisms have been proposed for the loading of sugars into the phloem. These differ in the involvement of active transport across the phloem cell's membrane and their capacity for passive intercellular transport through plasmodesmata. This capacity for diffusion from the mesophyll into the phloem cells can be quantified by live-cell microscopy. Instead of sugar molecules, the movement of fluorescent tracers of similar size can be observed. In this chapter, a simple method is described that allows quantification of plasmodesmata-mediated intercellular diffusion across the mesophyll-bundle sheath interface and the bundle sheath-phloem cell interfaces. The fluorescent tracer carboxyfluorescein is loaded into intact leaves and its diffusion monitored with confocal microscopy after photobleaching of a bundle sheath cell.

In vivo distribution of Cornus cervi Colla and tracer kinetic analysis of its components that enter the blood and bone / 中国组织工程研究

BACKGROUND:Our previous studies found that the polypeptide of Cornus cervi Colla can promote bone growth,which has a good application prospect in the treatment of bone diseases.However,how Cornus cervi Colla works in the body and the principle are not clear. OBJECTIVE:To study the in vivo distribution and tracing of Cornus cervi Colla using fluorescence labeling and tracer technique. METHODS:Cornus cervi Colla was fluorescently labeled using fluorescein isothiocyanate,and the labeling results were detected by fluorescence imaging and UV spectral scanning.Successfully labeled Cornus cervi Colla was injected into mice by gavage,and the absorption of Cornus cervi Colla into blood was detected by laser confocal microscopy,and the distribution of Cornus cervi Colla in mice was detected by small animal in vivo imager.The distribution of Cornus cervi Colla in the mice was detected by laser confocal microscopy.Samples were taken from serum and bone at the time of the strongest fluorescence,and gel electrophoresis was carried out on serum and bone tissue protein solutions,and the components of Cornus cervi Colla absorbed into target organs were determined by secondary mass spectrometry. RESULTS AND CONCLUSION:The fluorescent markers were successfully separated by dextran gel chromatography,and the fluorescence imaging and ultraviolet spectrum scanning proved that the labeling was successful,and the fluorescence substitution degree of FITC-labeled Cornus cervi Colla was 0.953%.The fluorescence intensity of the components of Cornus cervi Colla in the blood showed that Cornus cervi Colla was most distributed in serum after oral administration for 2 hours.The fluorescence images of mice at different times were the same as those of bilateral femur and tibia,indicating that Cornus cervi Colla could play a role by entering the bone.Compared with UniProt database,secondary mass spectrometry showed that the peptide was a characteristic fragment of decorin.It is proved that decorin in Cornus cervi Colla can enter the bone to play a therapeutic role.