The aim of the present study was to investigate the expression, distribution and function of dendritic cells (DCs) and to study their role in nasal polyps. The study involved 55 participants, 45 of whom had nasal polyps and were the study group and 10 who had normal inferior turbinates and were the control group. Immunohistochemical staining was used to visualize the expression and distribution of the S-100 protein. A double immunostaining method was used to visualize the CD1a and CD40 expression and the images were analyzed with Axioplan 2 microscopy. The expression level of the S-100 protein in the nasal polyps was higher than that in the normal inferior turbinates with a significant difference (P<0.01). The distribution area, number and density of the double stained cells in the nasal polyps were all greater than in the normal inferior turbinates (P<0.01). The S-100 protein and double stained cells were mainly located in the lamina propria below the mucous membrane. The present study demonstrates that DCs are involved in the pathogenesis of nasal polyps and the presence of CD40-positive DCs suggests that this was related to the reciprocal interaction between the DCs and T lymphocytes.
nasal polyps; dendritic cells; S-100 protein; CD1a; CD40; pathogenesis
Although adjuvants are critical vaccine components, their modes of action are poorly understood. Here, we investigated the mechanisms by which the heat-killed mycobacteria in complete Freund’s adjuvant (CFA) promote T helper 17 (Th17) CD4+ T cell responses. We found that IL-17 secretion by CD4+ T cells following CFA immunization requires MyD88 and IL-1β/IL-1 receptor (IL-1R) signaling. Through measurement of antigen-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase 1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule CARD9 plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase 1-and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.
Photodynamic therapy is an emerging treatment modality that is under intensive preclinical and clinical investigations for many types of disease including cancer. Despite the promise, there is a lack of a reliable drug delivery vehicle that can transport photosensitizers (PSs) to tumors in a site-specific manner. Previous efforts have been focused on polymer- or liposome-based nanocarriers, which are usually associated with a suboptimal PS loading rate and a large particle size. We report herein that a RGD4C-modified ferritin (RFRT), a protein-based nanoparticle, can serve as a safe and efficient PS vehicle. Zinc hexadecafluorophthalocyanine (ZnF16Pc), a potent PS with a high 1O2 quantum yield but poor water solubility, can be encapsulated into RFRTs with a loading rate as high as ~60 wt % (i.e., 1.5 mg of ZnF16Pc can be loaded on 1 mg of RFRTs), which far exceeds those reported previously. Despite the high loading, the ZnF16Pc-loaded RFRTs (P-RFRTs) show an overall particle size of 18.6 ± 2.6 nm, which is significantly smaller than other PS–nanocarrier conjugates. When tested on U87MG subcutaneous tumor models, P-RFRTs showed a high tumor accumulation rate (tumor-to-normal tissue ratio of 26.82 ±4.07 at 24 h), a good tumor inhibition rate (83.64% on day 12), as well as minimal toxicity to the skin and other major organs. This technology can be extended to deliver other metal-containing PSs and holds great clinical translation potential.
photodynamic therapy; photosensitizer; targeted delivery; ferritin; nanoparticle
Tissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells.
hESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis.
We first found that TF was expressed only in trophoblasts and granulocyte–monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway.
The miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.
Established ARDS is often refractory to treatment. Clinical trials have demonstrated modest treatment effects, and mortality remains high. Ventilator strategies must be developed to prevent ARDS.
Early ventilatory intervention will block progression to ARDS if the ventilator mode: 1) maintains alveolar stability and 2) reduces pulmonary edema formation.
Yorkshire Pigs (38–45kg) were anaesthetized and subjected to "2-hit" Ischemia-Reperfusion and Peritoneal Sepsis. Following injury, animals were randomized into two groups: Early Preventative Ventilation (Airway Pressure Release Ventilation- APRV) vs. Non-Preventative Ventilation (NPV) and followed for 48hr. All animals received anesthesia, antibiotics, and fluid/vasopressor therapy per Surviving Sepsis Campaign. Ventilation parameters: 1) NPV Group - Tidal volume (Vt): 10cc/kg + PEEP- 5 cm/H2O volume-cycled mode, 2) APRV Group - Vt: 10–15 cc/kg; Phigh, Plow, Thigh, Tlow were titrated for optimal alveolar stability. Physiologic data and plasma were collected throughout the 48hr study period, followed by BAL and necropsy.
APRV prevented development of ARDS (p<0.001 vs NPV) by PaO2/FiO2 ratio. Quantitative histological scoring showed APRV prevented lung tissue injury (p<0.001 vs. NPV). BALF showed APRV lowered total protein and IL-6, while preserving surfactant proteins A & B (p<0.05 vs. NPV). APRV significantly lowered lung water (p<0.001 vs. NPV). Plasma IL-6 concentrations were similar between groups.
Early preventative mechanical ventilation with APRV blocked ARDS development, preserved surfactant proteins, and reduced pulmonary inflammation and edema, despite systemic inflammation similar to NPV. These data suggest early preventative ventilation strategies stabilizing alveoli and reducing pulmonary edema can attenuate ARDS after ischemia-reperfusion-sepsis.
Sepsis; Shock; ARDS; ALI; Ventilator Induced Lung Injury; Airway Pressure Release Ventilation
Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.
The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbors an ortholog (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in S. coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. S. avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in E. coli catalyzed the Mg2+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16. The resulting transformants accumulated epi-isozizaene after 24-hr cultivation. The previously characterized oxidized epi-isozizaene metabolites, (5R)- and (5S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was co-expressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-7-epi-zizaan-3β-ol which is most likely formed by oxidation of (5S)-albaflavenol.
gene expression/regulation; metabolic design; metabolism; natural products; secondary metabolites
Warming and drought pose a serious threat to tropical forest. Yet the extent of this threat is uncertain, given the lack of methods to evaluate the forest tree cover changes under future climate predicted by complex dynamic vegetation models. Here we develop an empirical approach based on the observed climate space of tropical trees to estimate the maximum potential tropical tree cover (MPTC) in equilibrium with a given climate. We show that compared to present-day (2000–2009) conditions, MPTC will be reduced by 1 to 15% in the tropical band under equilibrium future (2090–2099) climate conditions predicted by 19 IPCC climate models. Tropical forests are found to regress or disappear mainly in the current transition zones between forest and savanna ecosystems. This climate pressure on tropical forests, added to human-caused land use pressure, poses a grand challenge to the sustainability of the world's largest biomass carbon pool.
Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor induced lymphangiogensis with a tumor lymphatics-specific peptide Lyp-1. The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. At days 3, 7, 14 and 21 after subcutaneous 4T1 tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities of the tumor-bearing mice. At 45 min and 24 h postinjection, brachial LN fluorescence imaging was performed. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. Tumor induced lymphangiogenesis was confirmed by LYVE-1 immunostaining and increased size of tumor side brachial LNs. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, suggesting lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at days 7, 14, and 21, but not day 3 after tumor inoculation. At day 21 after tumor inoculation, the average signal of tumor-draining LNs was 78.0 ± 2.44, 24.3 ± 5.43, 25.6 ± 0.25 (×103 photon/cm2/s) using Cy5.5-LyP-1, Cy5.5-LyP-1 with blocking, and Cy5.5 only, respectively. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Use of LyP-1 based imaging probes with optical imaging offers a useful tool for the study of tumor-induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis useful in detecting “high risk” lymph nodes before tumor metastasis and after micro-metastasis, as well as for screening potential anti-lymphatic therapies.
Lymphangiogenesis; lymph node; LyP-1 peptide; optical imaging
Evidence suggests that cytoglobin (Cygb) may function as a tumor suppressor gene.
We immunohistochemically evaluated the expression of Cygb, phosphatidylinositol-3 kinase (PI-3K), phosphorylated (p)-Akt, Interleukin-6 (IL-6), tumor necrosis factor-α (TNFα) and vascular endothelial growth factor (VEGF) in 88 patients with 41 high-grade gliomas and 47 low-grade gliomas. Intratumoral microvessel density (IMD) was also determined and associated with clinicopathological factors.
Low expression of Cygb was significantly associated with the higher histological grading and tumor recurrence. A significant negative correlation emerged between Cygb expression and PI3K, p-Akt, IL-6, TNFα or VEGF expression. Cygb expression was negatively correlated with IMD. There was a positive correlation between PI3K, p-Akt, IL-6, TNFα and VEGF expression with IMD.High histologic grade, tumor recurrence, decreased Cygb expression, increased PI3K expression, increased p-Akt expression and increased VEGF expression correlated with patients’ overall survival in univariate analysis. However, only histological grading and Cygb expression exhibited a relationship with survival of patients as independent prognostic factors of glioma by multivariate analysis.
Cygb loss may contribute to tumor recurrence and a worse prognosis in gliomas. Cygb may serve as an independent predictive factor for prognosis of glioma patients.
Glioma; Cytoglobin; Phosphatidylinositol-3 kinase; Recurrence; Prognosis
KR-31831 ((2R,3R,4S)-6-amino-4-[N-(4-chloropheyl)-N-(1H-imidazol-2ylmethyl)amino]-3-hydroxyl-2-methyl-2-dimethoxymethyl-3,4-dihydro-2H-1-benzopyran), an angiogenesis inhibitor, was evaluated in tumor-bearing mice using molecular imaging technology. Pre-treatment microPET images were acquired on SKOV-3 cell-implanted nude mice after injection with 64Cu-DOTA-VEGF121. KR-31831 (50 mg/kg) was then injected intraperitoneally into the treatment group (n=3), while injection vehicle was injected into the control (n=4) and blocking (n=3) groups. After injections occurred daily for 28 days, all groups of mice underwent post-treatment microPET imaging after injection with 64Cu-DOTA-VEGF121. The post-treatment images showed high tumor uptake in the control group and reduced tumor uptake in both the blocking and treatment groups. ROI analysis of the tumor images revealed 6.25%±1.18% ID/g at 1 h, 6.55%±0.69% ID/g at 2 h, and 4.68%±0.63% ID/g at 16 h in the control group; 3.87%±0.45% ID/g at 1 h, 4.50%±0.44% ID/g at 2 h, and 3.63%±0.25% ID/g at 16 h in the blocking group; and 4.03%±0.74% ID/g at 1 h, 4.37%±0.67% ID/g at 2 h, and 3.83%±0.90% ID/g at 16 h in the treatment group. Biodistribution obtained after the post-treatment microPET imaging also demonstrated high tumor uptake (3.74%±0.27% ID/g) in the control group and reduced uptakes in both the blocking group (2.69%±0.73% ID/g, P<.05) and the treatment group (3.11%±0.25% ID/g, P<.05), which correlated well with microPET imaging data. Immunofluorescence analysis showed higher levels of VEGFR2 and CD31 expressions in tumor tissues of the control and blocking groups than in tumor tissues of the treatment group. These results suggest that the antiangiogenic activity of KR-31831 is mediated through VEGFR2 and microPET serves as a useful molecular imaging tool for evaluation of a newly developed angiogenesis inhibitor, KR-31831.
KR-31831; 64Cu-DOTA-VEGF121; MicroPET; VEGFR2; Angiogenesis
ferritin; fluorescent probes; imaging agents; metalloenzymes; nanoparticles
Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging.
Tumor targeting; Antibody; Human epidermal growth factor receptor (HER); Vascular endothelial growth factor (VEGF)
Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3β to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.
The scaffold protein CARMA1 is required for the T-cell receptor (TCR)-induced lymphocyte activation. Here, we show that CARMA1 also plays an essential role in T-cell differentiation. We have found that the adoptive transfer of bone-marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5 and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 prefferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4+ splenocytes and subsequently production of GATA3-dependent cytokines, IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.
CARD11; Th2 cytokine; JunB; GATA3
Scaffold proteins play pivotal roles in the regulation of signal transduction pathways by connecting upstream receptors to downstream effector molecules. During last decade, many scaffold proteins that contain caspase-recruitment domain (CARD) have been identified. Investigating the roles of CARD proteins has revealed that many play crucial roles in signaling cascades that lead to activation of nuclear factor-κB (NF-κB). In this review, we discuss the contributions of CARD proteins to NF-κB activation in various signaling cascades. In particular, we share some of our personal experiences during the initial investigation of the functions of the CARMA family of CARD proteins and then summarize the roles of these proteins in signaling pathways induced by antigen receptors, G protein-coupled receptors, receptor tyrosine kinase, and C-type lectin receptors in the context of recent progress in the field.
signal transduction; adapter proteins; signaling proteins
In an era when global biodiversity is increasingly impacted by rapidly changing climate, efforts to conserve global biodiversity may be compromised if we do not consider the uneven distribution of climate-induced threats. Here, via a novel application of an aggregate Regional Climate Change Index (RCCI) that combines changes in mean annual temperature and precipitation with changes in their interannual variability, we assess multi-dimensional climate changes across the “Global 200” ecoregions – a set of priority ecoregions designed to “achieve the goal of saving a broad diversity of the Earth’s ecosystems” – over the 21st century. Using an ensemble of 62 climate scenarios, our analyses show that, between 1991–2010 and 2081–2100, 96% of the ecoregions considered will be likely (more than 66% probability) to face moderate-to-pronounced climate changes, when compared to the magnitudes of change during the past five decades. Ecoregions at high northern latitudes are projected to experience most pronounced climate change, followed by those in the Mediterranean Basin, Amazon Basin, East Africa, and South Asia. Relatively modest RCCI signals are expected over ecoregions in Northwest South America, West Africa, and Southeast Asia, yet with considerable uncertainties. Although not indicative of climate-change impacts per se, the RCCI-based assessment can help policy-makers gain a quantitative and comprehensive overview of the unevenly distributed climate risks across the G200 ecoregions. Whether due to significant climate change signals or large uncertainties, the ecoregions highlighted in the assessment deserve special attention in more detailed impact assessments to inform effective conservation strategies under future climate change.
In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.
Phaeodactylum tricornutum; Thalassiosira pseudonana; Histone modifications; Chromatin immunoprecipitation; Epigenomics
The Encyclopedia of DNA Elements (ENCODE) consortium aims to identify all functional elements in the human genome including transcripts, transcriptional regulatory regions, along with their chromatin states and DNA methylation patterns. The ENCODE project generates data utilizing a variety of techniques that can enrich for regulatory regions, such as chromatin immunoprecipitation (ChIP), micrococcal nuclease (MNase) digestion and DNase I digestion, followed by deeply sequencing the resulting DNA. As part of the ENCODE project, we have developed a Web-accessible repository accessible at http://factorbook.org. In Wiki format, factorbook is a transcription factor (TF)-centric repository of all ENCODE ChIP-seq datasets on TF-binding regions, as well as the rich analysis results of these data. In the first release, factorbook contains 457 ChIP-seq datasets on 119 TFs in a number of human cell lines, the average profiles of histone modifications and nucleosome positioning around the TF-binding regions, sequence motifs enriched in the regions and the distance and orientation preferences between motif sites.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute respiratory failure and are associated with diverse disorders, such as pulmonary edema, pneumonia, sepsis, trauma, shock and lung contusion. Gene therapy is a potentially powerful approach to treat a variety of diseases related to ALI/ARDS. Numerous viral and non-viral methods for gene delivery to the lung have been developed, although pulmonary architecture and immune activation represent barriers to successful gene transfer. In this review, recent advances in the development of more efficient viral and non-viral gene transfer systems are discussed. In addition, the current status of gene therapy applied to ALI/ARDS-associated pulmonary diseases is reviewed. With the development of more efficient gene therapy vectors, gene therapy is a promising strategy for clinical application in the not too distant future.
Gene therapy; acute lung injury; viral vectors; non-viral vectors
An ongoing effort in the field of nanomedicine is to develop nanoplatforms with both imaging and therapeutic functions, the “nano-theranostics”. We have previously developed a human serum albumin (HSA) coated iron oxide nanoparticle (HINP) formula and used multiple imaging modalities to validate its tumor targeting attributes. In the current study, we sought to impart doxorubicin (Dox) onto the HINPs and to assess the potential of the conjugates as theranostic agents. In a typical preparation, we found that about 0.5 mg of Dox and 1 mg of iron oxide nanoparticles (IONPs, Fe content) could be loaded into 10 mg of HSA matrices. The resulting D-HINPs (Dox loaded HINPs) have a hydrodynamic size of 50 nm and are able to release Dox in a sustained fashion. More impressively, the HINPs can assist the translocation of Dox across cell membrane and even its accumulation in the nucleus. In vivo, D-HINPs retained a tumor targeting capability of HINPs, as manifested by both in vivo MRI and ex vivo immunostaining results. In a follow-up therapeutic study on a 4T1 murine breast cancer xenograft model, D-HINPs showed a striking tumor suppression effect that was comparable to Doxil and greatly outperformed free Dox. Such a strategy can be readily extended to load other types of small molecules, making HINP a promising theranostic nanoplatform.
Iron oxide nanoparticle; theranostic nanomedicine; magnetic resonance imaging; doxorubicin; drug delivery; breast cancer
Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy.
A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models.
A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-18F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection).
QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.
Tumor endothelial marker 8 (TEM8); Small-animal PET; 18F; Peptide
What determines large-scale patterns of species richness remains one of the most controversial issues in ecology. Using the distribution maps of 11 405 woody species in China, we compared the effects of habitat heterogeneity, human activities and different aspects of climate, particularly environmental energy, water–energy dynamics and winter frost, and explored how biogeographic affinities (tropical versus temperate) influence richness–climate relationships. We found that the species richness of trees, shrubs, lianas and all woody plants strongly correlated with each other, and more strongly correlated with the species richness of tropical affinity than with that of temperate affinity. The mean temperature of the coldest quarter was the strongest predictor of species richness, and its explanatory power for species richness was significantly higher for tropical affinity than for temperate affinity. These results suggest that the patterns of woody species richness mainly result from the increasing intensity of frost filtering for tropical species from the equator/lowlands towards the poles/highlands, and hence support the freezing-tolerance hypothesis. A model based on these results was developed, which explained 76–85% of species richness variation in China, and reasonably predicted the species richness of woody plants in North America and the Northern Hemisphere.
freezing-tolerance hypothesis; niche conservatism; species richness patterns; water–energy dynamics; winter temperature; woody plants of China and North America
Direct or ex vivo BMP9 adenoviral gene therapy can induce massive bone formation at the injection sites and clearly promote spinal fusion. A comprehensive analysis of the osteogenic activity indicated that BMP9 was one of the most potent inducers of osteogenic differentiation both in vitro and in vivo among 14 types of human BMPs. However, genetic variations and whether they correlated with OPLL were not considered. We have sequenced the complete BMP9 gene in 450 patients with OPLL and in 550 matched controls. Analyses were performed on single markers and haplotypes. Single marker tests identified 6 SNPs, among which the minor alleles of rs7923671 (T>C; P = 0.0026; OR: 1.33, CI: 1.10–1.60), rs75024165 (C>T, Thr304Met; P<0.001; OR: 1.76, CI: 1.47–2.12) and rs34379100 (A>C; P<0.001; OR: 1.52, CI: 1.27–1.82) were associated with OPLL. Logistic regression analysis showed that the additive model of rs75024165 (TT vs. CT vs. CC; P<0.001; OR: 1.74) and rs34379100 (CC vs. AC vs. AA; P = 0.003; OR: 1.95) retained statistical significance when adjusted for clinical and demographic characteristics. Linkage disequilibrium (LD) analysis identified one 3 kb block of intense LD in BMP9 and one specific haplotype, CTCA (P<0.001; OR: 2.37), that contained the OPLL-associated risk alleles and was a risk factor for OPLL. This haplotype is associated with increased severity of OPLL, as shown by the distribution of ossified vertebrae in patients with OPLL (P = 0.001). In summary, in the Chinese population studied, SNPs in the BMP9 gene appear to contribute to the risk of OPLL in association with certain clinical and demographic characteristics. The severity of OPLL seems to be mediated predominantly by genetic variations in a 3kb BMP9 locus with the specific haplotype CTCA.
Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C6, C12, C16, C18:1, and C24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe2+. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.
ceramidase; Tncer; activity; biochemical properties; plasma membrane; mRNA levels