OBJECTIVE: This study aimed to elucidate whether the administration of parathyroid hormone (PTH) results in remodeling- or modeling-based bone formation in different regions of the murine femora, and whether the PTH-driven bone formation would facilitate osteoblastic differentiation into osteocytes.
METHODS: Six-week-old male C57BL/6J mice were employed to examine the distribution of alkaline phosphatase (ALP), PHOSPHO1, podoplanin, and calcein labeling in two distinct long bone regions: the metaphyseal trabeculae close to the chondro-osseous junction (COJ) and those distant from the COJ in three mouse groups, a control group receiving a vehicle (sham group) and groups receiving hPTH (1-34) twice a day (PTH BID group) or four times a day (PTH QID group) for two weeks.
RESULTS: The sham group showed PHOSPHO1-reactive mature osteoblasts localized primarily at the COJ, whereas the PTH BID/QID groups exhibited extended lines of PHOSPHO1-reactive osteoblasts even in regions distant from the COJ. The PTH QID group displayed fragmented calcein labeling in trabeculae close to the COJ, whereas continuous labeling was observed in trabeculae distant from the COJ. Osteoblasts tended to express podoplanin and PHOSPHO1 independently in the close and distant regions of the sham group, while osteoblasts in the PTH-administered groups showed immunoreactivity of podoplanin and PHOSPHO1 together in the close and distant regions.
CONCLUSIONS: Administration of PTH may accelerate remodeling-based bone formation in regions close to the COJ while predominantly inducing modeling-based bone formation in distant regions. PTH appeared to simultaneously facilitate osteoblastic bone mineralization and differentiation into osteocytes in both remodeling- and modeling-based bone formation.

The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.

Responsiveness of liposomes to external stimuli, such as light, should allow a precise spatial and temporal control of release of therapeutic agents or ion transmembrane transport. Here, some aryl-azo derivatives of thymol are synthesized and embedded into liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to obtain light-sensitive membranes whose photo-responsiveness, release behaviour, and permeability towards Cl- ions are investigated. The hybrid systems are in-depth characterized by dynamic light scattering, atomic force microscopy and Raman spectroscopy. In liposomal bilayer the selected guests undergo reversible photoinduced isomerization upon irradiation with UV and visible light, alternately. Non-irradiated hybrid liposomes retain entrapped 5(6)-carboxyfluorescein (CF), slowing its spontaneous leakage, whereas UV-irradiation promotes CF release, due to guest trans-to-cis isomerization. Photoisomerization also influences membrane permeability towards Cl- ions. Data processing, according to first-order kinetics, demonstrates that Cl- transmembrane transport is enhanced by switching the guest from trans to cis but restored by back-switching the guest from cis to trans upon illumination with blue light. Finally, the passage of Cl- ions across the bilayer can be fine-tuned by irradiation with light of longer λ and different light-exposure times. Fine-tuning the photo-induced structural response of the liposomal membrane upon isomerization is a promising step towards effective photo-dynamic therapy.

In advanced drug delivery, versatile liposomal formulations are commonly employed for safer and more accurate therapies. Here we report a method that allows a straightforward production of synthetic monodisperse (~ 100 μm) giant unilamellar vesicles (GUVs) using a microfluidic system. The stability analysis based on the microscopy imaging showed that at ambient conditions the produced GUVs had a half-life of 61 ± 2 h. However, it was observed that ~ 90% of the calcein dye that was loaded into GUVs was transported into a surrounding medium in 24 h, thus indicating that the GUVs may release these small dye molecules without distinguishable membrane disruption. We further demonstrated the feasibility of our method by loading GUVs with larger and very different cargo objects; small soluble fluorescent proteins and larger magnetic microparticles in a suspension. Compared to previously reported microfluidics-based production techniques, the obtained results indicate that our simplified method could be equally harnessed in creating GUVs with less cost, effort and time, which could further benefit studying closed membrane systems.

Controlling the morphology of molecular assemblies formed by surfactants by photoirradiation enables the controlled release of incorporated substances, which can be applied to delivery systems for drugs and active ingredients. On the other hand, conventional photoresponsive surfactants and molecular assemblies have a slow response speed, making it difficult to control their functions at the desired time. In this review, I discuss our recent progress in the accelerated control of functions of photoresponsive molecular assemblies by using lophine dimer as a photochromic compound. The lophine dimer derivative dissociates into a pair of lophyl radicals that upon ultraviolet (UV) light irradiation, and these radical species thermally recombine although the recombination reaction is extremely slow due to the diffusion of lophyl radicals. By using the confined inner space of micelles formed by surfactants, the recombination reaction was extremely accelerated. With UV light irradiation, rapid morphological changes in micelles, formed by amphiphilic lophine dimers were observed by using in situ small-angle neutron scattering (in situ SANS) system. Moreover, the rapid controlled release of calcein as a model drug was achieved by UV light irradiation using the photoresponsive micelles. This rapid system can realize the controlled release of drugs truly at the desired time, developing an efficient and precise drug delivery system (DDS). Furthermore, it can be applied in a wide range of fields such as release control of active ingredients, efficient heat exchange control, and actuating systems.

Over the course of the last twenty years, there has been a growing recognition of the pig\'s potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs\' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays.

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it\'s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.