HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29’s ability to modulate actions of HS-binding growth factors (HBGFs) in angiogenesis. Between 1–2.5 μg/ml (ca. 60–150 nM), HIP/RPL29 inhibited HBGF-stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 μg/ml). At this concentration, HIP/RPL29 had no effect on HBGF- stimulated MAPK phosphorylation or VEGF-stimulated receptor-2 phosphorylation at site Y-996. Partial inhibition occured at VEGF receptor-2 site Y951, associated with cell migration. HBGF displacement from HS-bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 μg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE-dependent release of HS-bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 μg/ml and by 77% at 40 μg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 μg/ml (ca 2.5 mM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors.
HIP/RPL29; heparan sulfate; VEGF; FGF2; heparanase
The effect of prostaglandin E2 (PGE2) on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regulates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteoblasts in the presence of fluid shear stress (FSS), which could better uncover the anabolic effect of PGE2
in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A (PKA) pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 min significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells' calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the ‘reset’ process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone formation as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.
prostaglandin E2; intracellular calcium; cytoskeleton; PKA pathway; fluid shear stress (FSS)
The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.
super-resolution microscopy; single-particle tracking; mRNA; nuclear export; nuclear pore complex
The aim of the present case study was to investigate the advantages of intraoperative magnetic resonance imaging (iMRI) on the real-time guidance and monitoring of a stereotactic biopsy. The study describes a patient with intracranial lesions, which were examined by conventional MRI and diffusion tensor imaging using a 1.5T intraoperative MRI system. The digital and pre-operative positron emission/computed tomography image data were transferred to a BrainLAB planning workstation, and a variety of images were automatically fused. The BrainLAB software was then used to reconstruct the corticospinal tract (CST) and create a three-dimensional display of the anatomical association between the CST and the brain lesions. A Leksell surgical planning workstation was used to identify the ideal target site and a reasonable needle track for the biopsy. The 1.5T iMRI was used to effectively monitor the intracranial condition during the brain biopsy procedure. Post-operatively, the original symptoms of the patient were not aggravated and no further neurological deficits were apparent. The histopathological diagnosis of non-Hodgkin’s B-cell lymphoma was made. Using high-field iMRI, the multi-image fusion-guided stereotactic brain biopsy allows for a higher positive rate of biopsy and a lower incidence of complications. The approach of combining multi-image fusion images with the frame-based stereotactic biopsy may be clinically useful for intracranial lesions of deep functional areas.
intraoperative magnetic resonance imaging; multi-image fusion; stereotactic; biopsy
The nuclear envelope (NE) of eukaryotic cells provides a physical barrier for messenger RNA (mRNA) and the associated proteins (mRNPs) traveling from sites of transcription in the nucleus to locations of translation processing in the cytoplasm. Nuclear pore complexes (NPCs) embedded in the NE serve as a dominant gateway for nuclear export of mRNA. However, the fundamental characterization of export dynamics of mRNPs through the NPC has been hindered by several technical limits. First, the size of NPC that is barely below the diffraction limit of conventional light microscopy requires a super-resolution microscopy imaging approach. Next, the fast transit of mRNPs through the NPC further demands a high temporal resolution by the imaging approach. Finally, the inherent three-dimensional (3D) movements of mRNPs through the NPC demand the method to provide a 3D mapping of both transport kinetics and transport pathways of mRNPs. This review will highlight the recently developed super-resolution imaging techniques advanced from 1D to 3D for nuclear export of mRNPs and summarize the new features in the dynamic nuclear export process of mRNPs revealed from these technical advances.
mRNA; nuclear export; nuclear pore complex; super-resolution microscopy; single-molecule tracking
Brain metastases generally present in the parenchyma of the brain. In the current report, a very rare case of brain metastasis, which simultaneously invaded the subgaleal region, the skull, and the dural and cavernous sinuses is presented. The patient, a 54-year-old female, complained of a progressive headache and exhibited the symptoms of intracranial hypertension. Coronal contrast-enhanced T1-weighted magnetic resonance imaging (MRI) showed high intensity signals in the subgaleal tissue of the left frontoparietal area, as well as in the dural and the cavernous sinuses. The patient was initially diagnosed with an intracranial infection, however, the administered treatment was ineffective. The patient subsequently underwent a biopsy and the pathological diagnosis was determined as a metastatic adenocarcinoma; a primary tumor was not identified during the examinations. Surgical removal of certain metastases and a decompressive craniectomy were performed to relieve the intracranial hypertension. However, the prognosis was unsatisfactory. The patient’s neurological condition progressively worsened and an axial computed tomography scan with a bone window demonstrated a bulging growth in the brain tissue. The patient succumbed after one month due to the widespread metastasis. Thus, this case presents the unusual clinical development of this type of metastatic adenocarcinoma. In addition, due to the intracranial hypertension, the unusual sites of the high intensity signals in the MRI and the lack of a primary tumor, the patient was misdiagnosed with an intracranial infection. Furthermore, this case highlights the necessity for conducting a biopsy as soon as possible and demonstrates the poor prognosis associated with this type of patient.
brain metastases; metastatic adenocarcinoma; surgery; brain tumor
A peptide containing Asn-Gly-Arg(NGR) sequence was synthesized and directly labeled with 99mTc. Its radiochemical characteristics, biodistribution, and SPECT imaging were evaluated in nude mice bearing human HepG2 hepatoma. Nude mice bearing HepG2 were randomly divided into 5 groups with 5 mice in each group and injected with ~7.4 MBq 99mTc-NGR. The SPECT images were acquired in 1, 4, 8, and 12 h postinjection via caudal vein. The metabolism of tracers was determined in major organs at different time points, which demonstrated rapid, significant tumor uptake and slow tumor washout. The control group mice were blocked by coinjecting unlabelled NGR (20 mg/kg). Tumor uptake was (2.52 ± 0.83%) ID/g at 1 h, with the highest uptake of (3.26 ± 0.63%) ID/g at 8 h. In comparison, the uptake of the blocked control group was (1.65 ± 0.61%) ID/g at 1 h after injection. The SPECT static images and the tumor/muscle (T/NT) value were obtained. The highest T/NT value was 7.58 ± 1.92 at 8 h. The xenografted tumor became visible at 1 h and the clearest image of the tumor was observed at 8 h. In conclusion, 99mTc-NGR can be efficiently prepared and it exhibited good properties for the potential SPECT imaging agent of tumor.
The nuclear pore complex (NPC), which provides the permeable and selective transport path between the nucleus and cytoplasm of eukaryotic cells, allows both the passive diffusion of small molecules in a signal-independent manner and the transport receptor-facilitated translocation of cargo molecules in a signal-dependent manner. However, the spatial and functional relationships between these two transport pathways, which represent critical information for unraveling the fundamental nucleocytoplasmic transport mechanism, remain in dispute. The direct experimental examination of passive and facilitated transport with a high spatiotemporal resolution under real-time trafficking conditions in native NPCs is still difficult. To address this issue and further define these transport mechanisms, we recently developed single-point edge-excitation sub-diffraction (SPEED) microscopy and a deconvolution algorithm to directly map both passive and facilitated transport routes in three dimensions (3D) in native NPCs. Our findings revealed that passive and facilitated transport occur through spatially distinct transport routes. Signal-independent small molecules exhibit a high probability of passively diffusing through an axial central viscous channel, while transport receptors and their cargo complexes preferentially travel through the periphery, around this central channel, after interacting with phenylalanine-glycine (FG) filaments. Strikingly, these two distinct transport zones are not completely separate either spatially or functionally. Instead, their conformations are closely correlated and simultaneously regulated. In this review, we will specifically highlight a detailed procedure for 3D mapping of passive and facilitated transport routes, demonstrate the correlation between these two distinct pathways, and finally, speculate regarding the regulation of the transport pathways driven by the conformational changes of FG filaments in NPCs.
nucleocytoplasmic transport; deconvolution; single-particle tracking; three-dimensional; probability density map; super-resolution microscopy; single-molecule fluorescence
The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.
The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.
Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.
Recently, many scientists including bacteriologists have begun to focus on social aspects of antibiotic management especially the knowledge, attitude and practice (KAP) among the general population regarding antibiotic use. However, relatively few works have published on the relationship between KAP and medical education. In this study, we analyze the present status of Chinese medical (MS)- and non-medical (NS) students’ KAP on the use of antibiotics, and examine the influence of Chinese medical curriculum on the appropriate usage of antibiotics among medical students.
In this study, 2500 students from 3 universities (including one medical university) in Northeastern China participate in the questionnaire survey on students’ knowledge, attitude and practice toward antibiotic usage. Wilcoxon rank sum test and Chi square test were used to analyze questionnaire-related discrete and categorical variables respectively, in order to assess the impact of the medical curriculum on students’ KAP towards antibiotics.
2088 (83.5%) respondents (MS-1236 and NS-852) were considered valid for analysis. The level of knowledge of MS on the proper use of antibiotics was significantly higher than that of NS (p < 0.0001). However, based on their responses on actual practice, MS were found to rely on antibiotics more than NS (p < 0.0001). Moreover, the knowledge and attitude of MS towards antibiotic use improved with the increase in grade with discriminate use of antibiotics concurrently escalating during the same period.
This study indicates that Chinese medical curriculum significantly improves students’ knowledge on antibiotics and raises their attention on antibiotic resistance that may result from indiscriminate use of antibiotics. The study also shows an excessive use of antibiotics especially among the more senior medical students, signifying a deficiency of antibiotics usage instruction in their curriculum. This might explain why there are frequent abuses of antibiotics in both hospital and community settings from a certain angle.
Anbiotics usage; KAP; Questionnaire survey; Medical students; Statistical analysis
Cerenkov luminescence imaging (CLI) has been successfully utilized in various fields of preclinical studies; however, CLI is challenging due to its weak luminescent intensity and insufficient penetration capability. Here, we report the design and synthesis of a type of rare-earth microparticles (REMPs), which can be dually excited by Cerenkov luminescence (CL) resulting from the decay of radionuclides to enhance CLI in terms of intensity and penetration. Methods: Yb3+- and Er3+- codoped hexagonal NaYF4 hollow microtubes were synthesized via a hydrothermal route. The phase, morphology, and emission spectrum were confirmed for these REMPs by power X-ray diffraction (XRD), scanning electron microscopy (SEM), and spectrophotometry, respectively. A commercial CCD camera equipped with a series of optical filters was employed to quantify the intensity and spectrum of CLI from radionuclides. The enhancement of penetration was investigated by imaging studies of nylon phantoms and nude mouse pseudotumor models. Results: the REMPs could be dually excited by CL at the wavelengths of 520 and 980 nm, and the emission peaks overlaid at 660 nm. This strategy approximately doubled the overall detectable intensity of CLI and extended its maximum penetration in nylon phantoms from 5 to 15 mm. The penetration study in living animals yielded similar results. Conclusions: this study demonstrated that CL can dually excite REMPs and that the overlaid emissions in the range of 660 nm could significantly enhance the penetration and intensity of CL. The proposed enhanced CLI strategy may have promising applications in the future.
Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.
Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by 18F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.
IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).
The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.
MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of 131I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo
99mTc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.
The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2 free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans, and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2 treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.
Perlecan; Hyaluronic Acid; Heparan Sulfate; Osteoarthritis; Cartilage Repair; Bone Morphogenetic Protein
Radioiodine therapy has proven to be a safe and effective approach in the treatment of differentiated thyroid cancer. Similar treatment strategies have been exploited in nonthyroidal malignancies by transfecting hNIS gene into tumor cells or xenografts. However, rapid radioiodine efflux is often observed after radioiodine uptake, limiting the overall antitumor effects. In this study, we aimed at constructing multicistronic co-expression of hNIS and hTPO genes in tumor cells to enhance the radioiodine uptake and prolong the radioiodine retention. Driven by the cytomegalovirus promoter, hNIS and hTPO were simultaneously inserted into the expression cassette of adenoviral vector. An Ad5 viral vector (Ad-CMV-hTPO-T2A-hNIS) was assembled as a gene therapy vehicle by Gateway technology and 2A method. The co-expression of hNIS and hTPO genes was confirmed by a double-label immunofluorescence assay. The radioiodine (125I) uptake and efflux effects induced by co-expression of hNIS and hTPO genes were determined in transfected and non-transfected PC-3 cells. Significantly higher uptake (6.58 ± 0.56 fold, at 1 h post-incubation) and prolonged retention (5.47 ± 0.36 fold, at 1 h of cell efflux) of radioiodine (125I) were observed in hNIS and hTPO co-expressed PC-3 cells as compared to non-transfected PC-3 cells. We concluded that the new virus vector displayed favorable radioiodine uptake and retention properties in hNIS-hTPO transfected PC-3 cells. Our study will provide valuable information on improving the efficacy of hNIS-hTPO co-mediated radioiodine gene therapy.
Gene therapy; prostate cance; hNIS; hTPO; gateway cloning system
In eukaryotic cells, the bidirectional trafficking of proteins and genetic materials across the double-membrane nuclear envelope is mediated by nuclear pore complexes (NPCs). A highly selective barrier formed by the phenylalanine–glycine (FG)-nucleoporin (Nup) in the NPC allows for two transport modes: passive diffusion and transport receptor-facilitated translocation. Strict regulation of nucleocytoplasmic transport is crucial for cell survival, differentiation, growth and other essential activities. However, due to the limited knowledge of the native configuration of the FG-Nup barrier and the interactions between the transiting molecules and the barrier in the NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics of individual fluorescent molecules through the NPC and to map the interactions between transiting molecules and the FG-Nup barrier. Important characteristics of nucleocytoplasmic transport, such as transport time, transport efficiency and spatial distribution of single transiting molecules in the NPC, have been obtained that could not be measured by either ensemble average methods or conventional electron microscopy. In this critical review, we discuss the development of various single-molecule techniques and their application to nucleocytoplasmic transport in vitro and in vivo. In particular, we highlight a recent advance from one-dimensional to three-dimensional single-molecule characterization of transport through the NPC and present a comprehensive understanding of the nucleocytoplasmic transport mechanism obtained by this new technical development (105 references).
Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calciumregulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule superresolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.
nuclear envelope; nuclear pore complex; nucleocytoplasmic transport; calcium stores; single-molecule fluorescence microscopy
Cerenkov luminescence tomography (CLT) provides the three-dimensional (3D) radiopharmaceutical biodistribution in small living animals, which is vital to biomedical imaging. However, existing single-spectral and multispectral methods are not very efficient and effective at reconstructing the distribution of the radionuclide tracer. In this paper, we present a semi-quantitative Cerenkov radiation spectral characteristic-based source reconstruction method named the hybrid spectral CLT, to efficiently reconstruct the radionuclide tracer with both encouraging reconstruction results and less acquisition and image reconstruction time.
We constructed the implantation mouse model implanted with a 400 µCi Na131I radioactive source and the physiological mouse model received an intravenous tail injection of 400 µCi radiopharmaceutical Iodine-131 (I-131) to validate the performance of the hybrid spectral CLT and compared the reconstruction results, acquisition, and image reconstruction time with that of single-spectral and multispectral CLT. Furthermore, we performed 3D noninvasive monitoring of I-131 uptake in the thyroid and quantified I-131 uptake in vivo using hybrid spectral CLT. Results showed that the reconstruction based on the hybrid spectral CLT was more accurate in localization and quantification than using single-spectral CLT, and was more efficient in the in vivo experiment compared with multispectral CLT. Additionally, 3D visualization of longitudinal observations suggested that the reconstructed energy of I-131 uptake in the thyroid increased with acquisition time and there was a robust correlation between the reconstructed energy versus the gamma ray counts of I-131 (). The ex vivo biodistribution experiment further confirmed the I-131 uptake in the thyroid for hybrid spectral CLT.
Results indicated that hybrid spectral CLT could be potentially used for thyroid imaging to evaluate its function and monitor its treatment for thyroid cancer.
The aim of this study was to investigate the predictive role of the orbital somatostatin receptor scintigraphy with 99mTc-EDDA/HYNIC-TOC (99mTc-TOC) to detect clinical stage of Graves’ ophthalmopathy and the response to corticosteroid therapy. The subjects of the experiment were 46 patients with Graves’ ophthalmopathy (GO) and four volunteers without eye disease or GO as the normal group (NG). Single photon emission computed tomography (SPECT), computed tomography (CT) and the left and right lateral position planar imaging of the heads of the all subjects were obtained 4 h after the intravenous injection of 555 MBq of 99mTc-TOC. The 99mTc-TOC SPECT/CT was repeated 3 months later. 35 (35/46) patients were received corticosteroid therapy (prednisolone, 10 mg po tid ) for 3 months, however, the other 11 patients as control groups did not receive any treatment. The treatment effect was evaluated both by the orbital 99mTc-TOC uptake and NOSPECS. A significant decrease in the O/OC ratio was observed in 22 GO patients between pre- and post-treatment (1.64 ± 0.13 vs. 1.21 ± 0.09, P < 0.05). There were neither significant difference of the O/OC ratio in 13 GO patients between pre- and post-treatment periods, nor significant difference in the 9 (9/11) patients before and after three months. Orbital 99mTc-TOC scintigraphy is a feasible technique to estimate the Graves’ ophthalmopathy activity and predict the response to subsequent corticosteroid therapy in GO patients. The technique could be a useful tool for physicians not familiar with CAS determination.
Graves’ ophthalmopathy; single photon emission computed tomography (SPECT); somatostatin receptor; 99mTc-TOC
The nuclear pore complex (NPC) acts as a selective gate that mediates the bidirectional transport of macromolecules between the cytoplasm and the nucleus of eukaryotic cells. ‘Natively unfolded’ nucleoporins (Nups) with domains rich in phenylalanine-glycine (FG) repeats form the selective permeability barrier and provide binding sites for mobile transport receptors in the NPC. Understanding the structure and function of the FG-Nups barrier under real-time trafficking conditions is still a formidable challenge due to the dynamic nature of a channeled membranous environment. Recently, we have shown that three-dimensional (3D) density maps of transient interactions between the FG-Nups barrier and a cargo-free or a cargo-bound transport receptor in native NPCs can be obtained by an advanced single-molecule fluorescence microscopy approach. Moreover, we found that these interaction sites are spatially clustered into distinct groups in the periphery around a central axial channel with a diameter of approximately 10–20 nm in the NPC. The 3D distribution of interaction sites may indicate some native properties of the FG-Nups barrier. Here we speculate that the selective permeability barrier in the NPC could be formed by clustered FG-Nups.
nucleocytoplasmic transport; single-molecule tracking; three-dimensional distribution; real-time trafficking; super-resolution microscopy; single-molecule fluorescence
We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein 2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP-P1) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP-P1 exhibited significantly (p<0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP-P1, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP-P1 exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP-P1 stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP-P1 to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.
perlecan domain I; hyaluronic acid; hydrogel particles; controlled release; bone morphogenetic protein-2; chondrogenic differentiation; mesenchymal stem cells
Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri–miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri–miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.