Insight into effects of terbium on cell growth, sporulation and spore properties of Bacillus subtilis.

Recovery of rare earth elements (REEs) from wastewater with Bacillus subtilis (B. subtilis) during culture is promising due to its environmental benefits. However, the effects of REEs in the culture media on B. subtilis are poorly understood. This study aims to investigate the effects of the terbium (Tb(III)), a typical rare earth element, on the cell growth, sporulation, and spore properties of B. subtilis. Tb(III) can suppress bacterial growth while enhancing spore tolerance to wet heat. Spore germination and content of dipicolinic acid (DPA) were promoted at low concentrations of Tb(III) while inhibited at a high level, but an inverse effect on initial sporulation appeared. Scanning electron microscope and energy dispersive spectrometer detection indicated that Tb(III) complexed cells or spores and certain media components simultaneously. The germination results of the spores after elution revealed that Tb(III) attached to the spore surface was a key effector of spore germination. In conclusion, Tb(III) directly or indirectly regulated both the nutrient status of the media and certain metabolic events, which in turn affected most of the properties of B. subtilis. Compared to the coat-deficient strain, the wild-type strain grew faster and was more tolerant to Tb(III), DPA, and wet heat, which in turn implied that it was more suitable for the recovery of REEs during cultivation. These findings provide fundamental insights for the recovery of rare earths during the culture process using microorganisms.

Synthesis of folic acid functionalized terbium-doped dendritic fibrous nano-silica and Interaction with HEK 293 normal, MDA breast cancer and HT 29 colon cancer cells.

A novel folic acid functionalized terbium-doped dendritic fibrous nanoparticle (Tb@KCC-1-NH2 -FA) with high surface area was synthesized using a novel hydrothermal protocol. In the present work, we report the fluorescent Tb-doted nanomaterial with emission wavelength at 497 nm which confirms the formation of Tb@KCC-1-NH2 -FA. Synthesized nanoparticles were investigated through transmission electron microscope, field emission scanning electron Microscopy, Fourier transform infrared spectra, Brunauer-Emmett-Teller, energy dispersive X-ray, Zeta potential and particle size distribution values and AFM (Atomic force microscopy) techniques. Specially, our desired nanomaterial which has FA moieties on the surface of Tb@KCC-1-NH2-FA where interact with folate receptor (FR) which there is on the surface of the various cancer cells. For this purpose, fluorescence microscopy images were used to prove the uptake of FA based nanomaterial with FR-positive MDA breast cancer and HT 29 colon cancer cells. Also HEK 293 normal cells as FR-negative cells verified the specificity of our desired nanomaterial toward the FR-positive cells. The cytotoxicity survey of Tb@KCC-1-NH2 -FA was examined by MTT assays against MDA breast cancer, HT 29 colon cancer and HEK 293 Normal cell lines which confirmed their biocompatible nature with any significant cytotoxic effects even for concentration higher than 900 µg/mL which could be used as a non-toxic catalyst or carrier in biological ambient. Hence, Tb@KCC-1-NH2 -FA were synthesized using green and hydrothermal method; the process was simple with good productivity and desired nanocomposite was non-toxic.

Luminescent Coordination Polymer for Picric Acid Detection and Treatment on Spinal Cord Injury Model Via Upregulating the trka Expression.

A luminescent coordination polymer based on Tb(III) has been synthesized with the tripodal carboxylic acid ligand containing N,O codonors (H2PBA = 5-[4-pyridin-3-yl-benzoylamino]-isophthalic acid) as ligand under solvothermal conditions. The chemical formula of this polymer is {[Tb2(PBA)3(H2O)3]·DMF·3H2O}n (1). Complex 1 has good sensitivity and selectivity to picric acid (PA). At 0-30 µmol/L, 1's quenching constant is 4.5 × 104 L/mol. In the biological function study, the motor function of spinal cord-injured animals after different treatments was evaluated using the blood-brain barrier (BBB) method. The trka expression level on the neural stem cells after treatment was measured to reveal the underlying mechanism.

Fluorescent Tb(III)-MOF for Fe(III) Ion Detection and Treatment Effect in Aortic Dissection by Reducing ß-receptor Expression and D-Dimer Production.

Iron is a class of essential elements involved in the metabolic process in all living organisms. However, excessive or deficient iron levels from normal ranges can lead to severe diseases. In this study, a fluorescent Tb(III)-based metal-organic framework with the chemical formula of [Tb(cptpy)3]n (1, Hcptpy = 4'-(4-carboxyphenyl)-2,2':6',2''-terpyridine) has been prepared under the hydrothermal condition successfully and its properties were determined by X-ray single-crystal diffraction, IR spectra, powder X-ray diffraction (PXRD) thermogravimetric analyses (TGA) and elemental analyses. Luminescent and sensing properties of complex 1 were examined closely, and it is shown that the Tb-MOF has the distinct ability to efficiently and selectively detect the Fe3+ ion and acetone. Furthermore, the RT-PCR was employed to determine the effect of compound on the ß-receptor and mmp-9 genes expression in cardiomyocytes. And the ELISA assay was used for the measurement of D-Dimer in the serum after compound treatment.

Bactericidal effects and accelerated wound healing using Tb4O7 nanoparticles with intrinsic oxidase-like activity.

BACKGROUND: Nanomaterials that exhibit intrinsic enzyme-like characteristics have shown great promise as potential antibacterial agents. However, many of them exhibit inefficient antibacterial activity and biosafety problems that limit their usefulness. The development of new nanomaterials with good biocompatibility and rapid bactericidal effects is therefore highly desirable. Here, we show a new type of terbium oxide nanoparticles (Tb4O7 NPs) with intrinsic oxidase-like activity for in vitro and in vivo antibacterial application. RESULTS: We find that Tb4O7 NPs can quickly oxidize a series of organic substrates in the absence of hydrogen peroxide. The oxidase-like capacity of Tb4O7 NPs allows these NPs to consume antioxidant biomolecules and generate reactive oxygen species to disable bacteria in vitro. Moreover, the in vivo experiments showed that Tb4O7 NPs are efficacious in wound-healing and are protective of normal tissues. CONCLUSIONS: Our results reveal that Tb4O7 NPs have intrinsic oxidase-like activity and show effective antibacterial ability both in vitro and in vivo. These findings demonstrate that Tb4O7 NPs are effective antibacterial agents and may have a potential application in wound healing.

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.

This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

Terbium promotes adhesion and osteogenic differentiation of mesenchymal stem cells via activation of the Smad-dependent TGF-ß/BMP signaling pathway.

With its special physical and chemical properties, terbium has been widely used, which has inevitably increased the chance of human exposure to terbium-based compounds. It was reported that terbium mainly deposited in bone after introduction into the human body. Although some studies revealed the effects of terbium on bone cell lines, there have been few reports about the potential effect of terbium on adhesion and differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the effects of terbium on the adhesion and osteogenic and adipogenic differentiation of MSCs and the associated molecular mechanisms. Our data reveal that terbium promoted the osteogenic differentiation in a time-dependent manner and conversely inhibited the adipogenic differentiation of MSCs. Meanwhile, the cell-cell or cell-matrix interaction was enhanced by activating adherent-related key factors, which were evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Real-time RT-PCR and Western blot analysis were also performed to further detect osteogenic and adipogenic biomarkers of MSCs. The regulation of terbium on differentiation of MSCs led to the interaction between the transforming growth factor ß/bone morphogenetic protein and peroxisome-proliferator-activated receptor γ (PPARγ) signaling pathways, resulting in upregulation of the osteogenic master transcription factors, such as Runt-related transcription factor 2, bone morphogenetic protein 2, collagen I, alkaline phosphatase, and osteocalcin, and downregulation of the adipogenic master transcription factors, such as PPARγ2. The results provide novel evidence to elucidate the mechanisms of bone metabolism by terbium and may be helpful for more rational application of terbium-based compounds in the future.

Effect of lanthanide complex structure on cell viability and association.

A systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. The terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (DOTA) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. Three sensitizing antennas were investigated in terms of their function in vitro: 2-methoxyisophthalamide (IAM(OMe)), 2-hydroxyisophthalamide (IAM), and 6-methylphenanthridine (Phen). Of these complexes, Tb-DOTA-IAM exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related Tb-DOTA-IAM(OMe) platform renders it more attractive. Further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ED50 > 300 µM) regardless of the lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of 100 µM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM(OMe)) disfavors the cellular association of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species.

How predicted temperature and salinity changes will modulate the impacts induced by terbium in bivalves?

The threat of climate change, which includes shifts in salinity and temperature, has generated a global concern for marine organisms. These changes directly impact them and may alter their susceptibility to contaminants, such as terbium (Tb), found in electronic waste. This study assessed how decreased and increased salinity, as well as increased temperature, modulates Tb effects in Mytilus galloprovincialis mussels. After an exposure period of 28 days, Tb bioaccumulation and biochemical changes were evaluated. Results indicated no significant modulation of salinity and temperature on Tb accumulation, suggesting detoxification mechanisms and adaptations. Further analysis showed that Tb exposure alone caused antioxidant inhibition and neurotoxicity. When exposed to decreased salinity, these Tb-exposed organisms activated defense mechanisms, a response indicative of osmotic stress. Moreover, increased salinity also led to increased oxidative stress and metabolic activity in Tb-exposed organisms. Additionally, Tb-exposed organisms responded to elevated temperature with altered biochemical activities indicative of damage and stress response. Such responses suggested that Tb effects were masked by osmotic and heat stress. This study provides valuable insights into the interactions between temperature, salinity, and contaminants such as Tb, impacting marine organisms. Understanding these relationships is crucial for mitigating climate change and electronic waste effects on marine ecosystems.

Folate-based radiotracers for PET imaging--update and perspectives.

The folate receptor (FR) is expressed in many tumor types, among those ovarian and lung cancer. Due to the high FR affinity of folic acid, it has been used for targeting of FR-positive tumors, allowing specific delivery of attached probes to the malignant tissue. Therefore, nuclear imaging of FR-positive cancer is of clinical interest for selecting patients who could benefit from innovative therapy concepts based on FR-targeting. Positron emission computed tomography (PET) has become an established technique in clinical routine because it provides an increased spatial resolution and higher sensitivity compared to single photon emission computed tomography (SPECT). Therefore, it is of critical importance to develop folate radiotracers suitable for PET imaging. This review article updates on the design, preparation and pre-clinical investigation of folate derivatives for radiolabeling with radioisotopes for PET. Among those the most relevant radionuclides so far are fluorine-18 (t(1/2): 110 min, E(av) ß⁺: 250 keV) and gallium-68 (t(1/2): 68 min, E(av) ß⁺: 830 keV). Recent results obtained with new PET isotopes such as terbium-152 (t(1/2): 17.5 h, Eß⁺: 470 keV) or scandium-44 (t(1/2): 3.97 h, (Eav) ß⁺: 632 keV) are also presented and discussed. Current endeavors for clinical implementation of PET agents open new perspectives for identification of FR-positive malignancies in patients.

Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq.

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.

Assessing the impact of terbium on Mytilus galloprovincialis: Metabolic and oxidative stress responses.

The increasing demand for electric and electronic equipment has led to a rise in potentially hazardous electronic waste, including rare-earth elements (REEs), such as terbium (Tb), which have been already detected in aquatic systems. This study investigated the biochemical effects of anthropogenic Tb on mussels over a 28-day period. The mussels were exposed to different concentrations of Tb (0, 5, 10, 20, 40 µg/L), and biomarkers related to metabolism, oxidative stress, cellular damage, and neurotoxicity were evaluated. Bioaccumulation of Tb in the mussels' tissue increased with exposure concentrations, but the bioconcentration factor remained similar between treatments. Exposure to Tb enhanced glycogen consumption and decreased metabolic capacity which could be seen as a physiological adaptation to limit Tb accumulation. Antioxidant defenses and glutathione S-transferases showed a more complex dose-response, with enzymatic responses increasing until 10 µg/L but then returning to control levels at 20 µg/L. At 40 µg/L, enzymatic responses were also enhanced but to a lower extent than at 10 µg/L. The presence of Tb had clearly an inhibitory effect on biotransformation enzymes such as carboxylesterases in a dose-dependent manner. Likely, thanks to biochemical and physiological adaptations, no cellular damage or neurotoxicity was observed in any treatments, confirming the mussels' ability to tolerate Tb exposure. Nevertheless, prolonged exposure to these concentrations could lead to harmful consequences when facing other environmental stressors, such as misallocating energy resources for growth, reproduction, and defense mechanisms.

Hormetic activation of nano-sized rare earth element terbium on growth, PSII photochemistry, antioxidant status and phytohormone regulation in Lemnaminor.

Soils contaminated with rare earth elements (REEs) can damage agriculture by causing physiological disorders in plants which are evaluated as the main connection of the human food chain. A biphasic dose response with excitatory responses to low concentrations and inhibitory/harmful responses to high concentrations has been defined as hormesis. However, not much is clear about the ecological effects and potential risks of REEs to plants. For this purpose, here we showed the impacts of different concentrations of nano terbium (Tb) applications (5-10-25-50-100-250-500 mg L-1) on the accumulation of endogeneous certain ions and hormones, chlorophyll fluoresence, photochemical reaction capacity and antioxidant activity in duckweed (Lemna minor). Tb concentrations less than 100 mg L-1 increased the contents of nitrogen (N), phosphate (P), potassium (K+), calcium (Ca2+), magnesium (Mg2+), manganese (Mn2+) and iron (Fe2+). Chlorophyll fluorescence (Fv/Fm and Fv/Fo) was suppressed under 250-500 mg L-1 Tb. In addition, Tb toxicity affected the trapped energy adversely by the active reaction center of photosystem II (PSII) and led to accumulation of inactive reaction centers, thus lowering the detected level of electron transport from photosystem II (PSII) to photosystem I (PSI). On the other hand, 5-100 mg L-1 Tb enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), NADPH oxidase (NOX), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione S-transferase (GST). Tb (5-50 mg L-1) supported the maintenance of cellular redox status by promoting antioxidant pathways involved in the ascorbate-glutathione (AsA-GSH) cycle. In addition to the antioxidant system, the contents of some hormones such as indole-3-acetic acid (IAA), gibberellic acid (GA), cytokinin (CK) and salicylic acid (SA) were also induced in the presence of 5-100 mg L-1 Tb. In addition, the levels of hydrogen peroxide (H2O2) and lipid peroxidation (TBARS) were controlled through ascorbate (AsA) regeneration and effective hormonal modulation in L. minor. However, this induction in the antioxidant system and phytohormone contents could not be resumed after applications higher than 250 mg L-1 Tb. TBARS and H2O2, which indicate the level of lipid peroxidation, increased. The results in this study showed that Tb at appropriate concentrations has great potential to confer tolerance of duckweed by supporting the antioxidant system, protecting the biochemical reactions of photosystems and improving hormonal regulation.

Terbium-Rose Bengal Coordination Nanocrystals-Induced ROS Production under Low-Dose X-rays in Cultured Cancer Cells for Photodynamic Therapy.

X-ray-triggered scintillators (Sc) and photosensitizers (Ps) have been developed for X-ray-induced photodynamic therapy (X-PDT) to selectively destruct deep tissue tumors with a low X-ray dose. This study designed terbium (Tb)-rose bengal (RB) coordination nanocrystals (T-RBNs) by a solvothermal treatment, aiming to reduce photon energy dissipation between Tb3+ and RB and thus increase the reactive oxygen species (ROS) production efficiency. T-RBNs synthesized at a molar ratio of [RB]/[Tb] = 3 exhibited a size of 6.8 ± 1.2 nm with a crystalline property. Fourier transform infrared analyses of T-RBNs indicated successful coordination between RB and Tb3+. T-RBNs generated singlet oxygen (1O2) and hydroxyl radicals (•OH) under low-dose X-ray irradiation (0.5 Gy) via scintillating and radiosensitizing pathways. T-RBNs produced ∼8-fold higher ROS amounts than bare RB and ∼3.6-fold higher ROS amounts than inorganic nanoparticle-based controls. T-RBNs did not exhibit severe cytotoxicity up to 2 mg/mL concentration in cultured luciferase-expressing murine epithelial breast cancer (4T1-luc) cells. Furthermore, T-RBNs were efficiently internalized into cultured 4T1-luc cells and induced DNA double strand damage, as evidenced by an immunofluorescence staining assay with phosphorylated γ-H2AX. Ultimately, under 0.5 Gy X-ray irradiation, T-RBNs induced >70% 4T1-luc cell death via simultaneous apoptosis/necrosis pathways. Overall, T-RBNs provided a promising Sc/Ps platform under low-dose X-PDT for advanced cancer therapy.

Therapeutic potentials of terbium hydroxide nanorods for amelioration of hypoxia-reperfusion injury in cardiomyocytes.

Myocardial hypoxia reperfusion (H/R) injury is the paradoxical exacerbation of myocardial damage, caused by the sudden restoration of blood flow to hypoxia affected myocardium. It is a critical contributor of acute myocardial infarction, which can lead to cardiac failure. Despite the current pharmacological advancements, clinical translation of cardioprotective therapies have proven challenging. As a result, researchers are looking for alternative approaches to counter the disease. In this regard, nanotechnology, with its versatile applications in biology and medicine, can confer broad prospects for treatment of myocardial H/R injury. Herein, we attempted to explore whether a well-established pro-angiogenic nanoparticle, terbium hydroxide nanorods (THNR) can ameliorate myocardial H/R injury. For this study, in vitro H/R-injury model was established in rat cardiomyocytes (H9c2 cells). Our investigations demonstrated that THNR enhance cardiomyocyte survival against H/R-induced cell death. This pro-survival effect of THNR is associated with reduction of oxidative stress, lipid peroxidation, calcium overload, restoration of cytoskeletal integrity and mitochondrial membrane potential as well as augmentation of cellular anti-oxidant enzymes such as glutathione-s-transferase (GST) and superoxide dismutase (SOD) to counter H/R injury. Molecular analysis revealed that the above observations are traceable to the predominant activation of PI3K-AKT-mTOR and ERK-MEK signalling pathways by THNR. Concurrently, THNR also exhibit apoptosis inhibitory effects mainly by suppression of pro-apoptotic proteins like Cytochrome C, Caspase 3, Bax and p53 with simultaneous restoration of anti-apoptotic protein, Bcl-2 and Survivin. Thus, considering the above attributes, we firmly believe that THNR have the potential to be developed as an alternative approach for amelioration of H/R injury in cardiomyocytes.

Three-dimensional analysis and computer modeling of the capillary endothelial vesicular system with electron tomography.

OBJECTIVE: We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. MATERIALS AND METHODS: Mouse abdominal muscle capillaries were perfused with terbium to label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to -60° at 1° increments, were acquired in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular associations not evident in thin sections. RESULTS: Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. CONCLUSIONS: Uncertainties associated with other three-dimensional methods to study the capillary wall are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries.

Activation of the SK potassium channel-calmodulin complex by nanomolar concentrations of terbium.

Small conductance Ca(2+)-activated K(+) (SK) channels sense intracellular Ca(2+) concentrations via the associated Ca(2+)-binding protein calmodulin. Structural and functional studies have revealed essential properties of the interaction between calmodulin and SK channels. However, it is not fully understood how the binding of Ca(2+) to calmodulin leads to channel opening. Drawing on previous biochemical studies of free calmodulin using lanthanide ions as Ca(2+) substitutes, we have used the lanthanide ion, Tb(3+), as an alternative ligand to study the activation properties of SK channels. We found that SK channels can be fully activated by nanomolar concentrations of Tb(3+), indicating an apparent affinity >100-fold higher than Ca(2+). Competition experiments show that Tb(3+) binds to the same sites as Ca(2+) to activate the channels. Additionally, SK channels activated by Tb(3+) demonstrate a remarkably slow deactivation process. Comparison of our results with previous biochemical studies suggests that in the intact SK channel complex, the N-lobe of calmodulin provides ligand-binding sites for channel gating, and that its ligand-binding properties are comparable to those of the N-lobe in isolated calmodulin.

Efficient inhibition of germination of coat-deficient bacterial spores by multivalent metal cations, including terbium (Tb³+).

Release of dipicolinic acid (DPA) and its fluorescence with terbium (Tb(3+)) allow rapid measurement of the germination and viability of spores of Bacillus and Clostridium species. However, germination of coat-deficient Bacillus spores was strongly inhibited by Tb(3+) and some other multivalent cations. Tb(3+) also inhibited germination of coat-deficient Clostridium perfringens spores.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458 nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.

Redistribution of terbium ions across acetylcholine receptor-enriched membranes induced by agonist desensitization.

Using small-angle x-ray diffraction from centrifugally oriented acetylcholine receptor (AChR) enriched membranes coupled with anomalous scattering from terbium ions (Tb3+) titrated into presumed Ca2+ binding sites, we have mapped the distribution of Tb3+ perpendicular to the membrane plane using a heavy atom refinement algorithm. We have compared the distribution of Tb3+ in the closed resting state with that in the carbamylcholine-desensitized state. In the closed resting state we find 45 Tb3+ ions distributed in 10 narrow peaks perpendicular to the membrane plane. Applying the same refinement procedure to the data from carbamylcholine desensitized AChR we find 18 fewer Tb3+ ions in eight peaks, and slight rearrangements of Tb3+ density in the peaks near the ends of the AChR ion channel pore. These agonist dependent changes in the Tb3+ stoichiometry and distribution suggest a likely role for multivalent cations in stabilizing the different functional states of the AChR, and the changes in the Tb3+ distribution at the two ends of the pore suggest a potential role for multivalent cations in the gating of the ion channel.