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1.
Hye-Young An Hyun-Soo Shin Jeong-Seok Choi Hun Jung Kim Jae-Yol Lim Young-Mo Kim 《PloS one》2015,10(11)
Background and Purpose
This study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model.Materials and Methods
Human adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O2 < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 μL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects.Results
The conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia-cultured hAdMSC secretome.Conclusion
These results show that the hAdMSC secretome from hypoxic-conditioned medium may provide radioprotection and tissue remodeling via release of paracrine mediators. 相似文献2.
Simon D. Tran Younan Liu Dengsheng Xia Ola M. Maria Saeed Khalili Renee Wan-Jou Wang Vu-Hung Quan Shen Hu Jan Seuntjens 《PloS one》2013,8(4)
Background
There are reports that bone marrow cell (BM) transplants repaired irradiated salivary glands (SGs) and re-established saliva secretion. However, the mechanisms of action behind these reports have not been elucidated.Methods
To test if a paracrine mechanism was the main effect behind this reported improvement in salivary organ function, whole BM cells were lysed and its soluble intracellular contents (termed as “BM Soup”) injected into mice with irradiation-injured SGs. The hypothesis was that BM Soup would protect salivary cells, increase tissue neovascularization, function, and regeneration. Two minor aims were also tested a) comparing two routes of delivering BM Soup, intravenous (I.V.) versus intra-glandular injections, and b) comparing the age of the BM Soup’s donors. The treatment-comparison group consisted of irradiated mice receiving injections of living whole BM cells. Control mice received irradiation and injections of saline or sham-irradiation. All mice were followed for 8 weeks post-irradiation.Results
BM Soup restored salivary flow rates to normal levels, protected salivary acinar, ductal, myoepithelial, and progenitor cells, increased cell proliferation and blood vessels, and up-regulated expression of tissue remodeling/repair/regenerative genes (MMP2, CyclinD1, BMP7, EGF, NGF). BM Soup was as an efficient therapeutic agent as injections of live BM cells. Both intra-glandular or I.V. injections of BM Soup, and from both young and older mouse donors were as effective in repairing irradiated SGs. The intra-glandular route reduced injection frequency/dosage by four-fold.Conclusion
BM Soup, which contains only the cell by-products, can be advantageously used to repair irradiation-damaged SGs rather than transplanting whole live BM cells which carry the risk of differentiating into unwanted/tumorigenic cell types in SGs. 相似文献3.
4.
Nienke Roescher Jelle L. Vosters Hongen Yin Gabor G. Illei Paul P. Tak John A. Chiorini 《PloS one》2011,6(5)
Introduction
Intercellular adhesion molecule-1 (ICAM-1) is involved in migration and co-stimulation of T and B cells. Membrane bound ICAM-1 is over expressed in the salivary glands (SG) of Sjögren''s syndrome (SS) patients and has therefore been proposed as a potential therapeutic target. To test the utility of ICAM-1 as a therapeutic target, we used local gene therapy in Non Obese Diabetic (NOD) mice to express soluble (s)ICAM-1 to compete with membrane bound ICAM-1 for binding with its receptor. Therapy was given prior to and just after the influx of immune cells into the SG.Methods
A recombinant serotype 2 adeno associated virus (rAAV2) encoding ICAM-1/Fc was constructed and its efficacy tested in the female NOD mice after retrograde instillation in SG at eight (early treatment) and ten (late treatment) weeks of age. SG inflammation was evaluated by focus score and immunohistochemical quantification of infiltrating cell types. Serum and SG tissue were analyzed for immunoglobulins (Ig).Results
Early treatment with ICAM-1/Fc resulted in decreased average number of inflammatory foci without changes in T and B cell composition. In contrast, late treated mice did not show any change in focus scores, but immunohistochemical staining showed an increase in the overall number of CD4+ and CD8+ T cells. Moreover, early treated mice showed decreased IgM within the SGs, whereas late treated mice had increased IgM levels, and on average higher IgG and IgA.Conclusions
Blocking the ICAM-1/LFA-1 interaction with sICAM-1/Fc may result in worsening of a SS like phenotype when infiltrates have already formed within the SG. As a treatment for human SS, caution should be taken targeting the ICAM-1 axis since most patients are diagnosed when inflammation is clearly present within the SG. 相似文献5.
Kazuhiro Ikeura Tetsuya Kawakita Kazuyuki Tsunoda Taneaki Nakagawa Kazuo Tsubota 《PloS one》2016,11(1)
Purpose
Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs).Methods
SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis.Results
SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype.Conclusions
Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. 相似文献6.
Boivin A Hanot M Malesys C Maalouf M Rousson R Rodriguez-Lafrasse C Ardail D 《PloS one》2011,6(1):e14558
Background
Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation.Methodology/Principal Findings
Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10% of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria.Conclusions
This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials of endogenous redox modulation to enhance the cytotoxic effects of radiotherapy. 相似文献7.
Background
Bone marrow cell extract (termed as BM Soup) has been demonstrated to repair irradiated salivary glands (SGs) and restore saliva secretion in our previous study. In the present study, we aim to investigate if the function of damaged SGs in non-obese diabetic (NOD) mice can be restored by BM Soup treatment and the molecular alterations associated with the treatment.Methods
Whole BM cells were lysed and soluble intracellular contents (“BM Soup”) were injected I.V. into NOD mice. Tandem mass tagging with 2-D liquid chromatography-mass spectrometry was used to quantify proteins in the submandibular glands (SMGs) between untreated and BM Soup-treated mice. Quantitative PCR was used to identify genes with altered expression in the treated mice.Results BM Soup
restored salivary flow rates to normal levels and significantly reduced the focus scores of SMGs in NOD mice. More than 1800 proteins in SMG cells were quantified by the proteomic approach. Many SMG proteins involved in inflammation and apoptosis were found to be down-regulated whereas those involved in salivary gland biology and development/regeneration were up-regulated in the BM Soup-treated mice. qPCR analysis also revealed expression changes of growth factors and cytokines in the SMGs of the treated NOD mice.Conclusion
BM Soup treatment is effective to restore the function of damaged SGs in NOD mice. Through gene/protein expression analysis, we have found that BM Soup treatment might effectuate via inhibiting apoptosis, focal adhesion and inflammation whereas promoting development, regeneration and differentiation of the SG cells in NOD mice. These findings provide important insights on the potential mechanisms underlying the BM Soup treatment for functional restoration of damaged SGs in NOD mice. Additional studies are needed to further confirm the identified target genes and their related signaling pathways that are responsible for the BM Soup treatment. 相似文献8.
Nienke Roescher Jelle L. Vosters Zhenan Lai Toshimitsu Uede Paul P. Tak John A. Chiorini 《PloS one》2012,7(12)
Objective
CD40–CD154 (CD40 ligand) interaction in the co-stimulatory pathway is involved in many (auto)immune processes and both molecules are upregulated in salivary glands of Sjögren’s syndrome (SS) patients. Interference within the CD40 pathway has ameliorated (auto)inflammation in a number of disease models. To test the potential role of the CD40 pathway in loss of gland function and inflammation in SS, an inhibitor of CD40-CD154 interaction was overexpressed in the salivary glands (SGs) of a spontaneous murine model of SS; the Non-Obese Diabetic (NOD) mouse.Materials and Methods
At different disease stages an adeno associated viral vector encoding CD40 coupled to a human Fc domain (CD40:Fc) was injected locally into the SGs of NOD mice. Delivery was confirmed by PCR. The overall effect on local inflammation was determined by assessment of the focus score (FS), quantification of infiltrating cell types, immunoglobulin levels, and microarray analysis. The effect on SG function was determined by measuring stimulated salivary flow.Results
CD40:Fc was stably expressed in the SG of NOD mice, and the protein was secreted into the blood stream. Microarray analysis revealed that expression of CD40:Fc affected the expression of many genes involved in regulation of the immune response. However, FS, infiltrating cell types, immunoglobulin levels, and salivary gland output were similar for treated and control mice.Discussion
Although endogenous CD40 is expressed in SG inflammatory foci in the SG of NOD mice, the expression of soluble CD40:Fc did not lead to reduced overall inflammation and/or improved salivary gland function. These data indicate possible redundancy of the CD40 pathway in the SG and suggests that targeting CD40 alone may not be sufficient to alter the disease phenotype. 相似文献9.
Kariithi HM Ince IA Boeren S Abd-Alla AM Parker AG Aksoy S Vlak JM Oers MM 《PLoS neglected tropical diseases》2011,5(11):e1371
Background
The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly''s salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.Methodology/Principal Findings
After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.Conclusions/Significance
SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides. 相似文献10.
11.
Napapat Amornwichet Takahiro Oike Atsushi Shibata Hideaki Ogiwara Naoto Tsuchiya Motohiro Yamauchi Yuka Saitoh Ryota Sekine Mayu Isono Yukari Yoshida Tatsuya Ohno Takashi Kohno Takashi Nakano 《PloS one》2014,9(12)
Background and Purpose
To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.Materials and Methods
DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.Results
The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.Conclusions
Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment. 相似文献12.
Toru Tanaka Sachiyo OhashiShunsuke Kobayashi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.Method
Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.Results
Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.Conclusion
In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.General significance
Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes. 相似文献13.
Context
Randomized controlled trails have identified online cognitive behavioral therapy as an efficacious intervention in the management of common mental health disorders.Objective
To assess the effectiveness of online CBT for different mental disorders in routine clinical practice.Design
An uncontrolled before-after study, with measurements at baseline, posttest, 6-week follow-up, and 1-year follow-up.Participants & Setting
1500 adult patients (female: 67%; mean age: 40 years) with a GP referral for psychotherapy were treated at a Dutch online mental health clinic for symptoms of depression (n = 413), panic disorder (n = 139), posttraumatic stress (n = 478), or burnout (n = 470).Interventions
Manualized, web-based, therapist-assisted CBT, of which the efficacy was previously demonstrated in a series of controlled trials. Standardized duration of treatment varied from 5 weeks (online CBT for Posttraumatic stress) to 16 weeks (online CBT for Depression).Main Outcome Measures
Validated self-report questionnaires of specific and general psychopathology, including the Beck Depression Inventory, the Impact of Event Scale, the Panic Disorder Severity Scale-Self Report, the Oldenburg Burnout Inventory, and the Depression Anxiety Stress Scales.Results
Treatment adherence was 71% (n = 1071). Study attrition was 21% at posttest, 33% at 6-week FU and 65% at 1-year FU. Mixed-model repeated measures regression identified large short-term reductions in all measures of primary symptoms (d = 1.9±0.2 to d = 1.2±0.2; P<.001), which sustained up to one year after treatment. At posttest, rates of reliable improvement and recovery were 71% and 52% in the completer sample (full sample: 55%/40%). Patient satisfaction was high.Conclusions
Results suggest that online therapist-assisted CBT may be as effective in routine practice as it is in clinical trials. Although pre-treatment withdrawal and long-term outcomes require further study, results warrant continued implementation of online CBT. 相似文献14.
A Alkhalaf N Kleefstra KH Groenier HJ Bilo RO Gans P Heeringa JL Scheijen CG Schalkwijk GJ Navis SJ Bakker 《PloS one》2012,7(7):e40427
Background
Formation of advanced glycation endproducts (AGEs), endothelial dysfunction, and low-grade inflammation are intermediate pathways of hyperglycemia-induced vascular complications. We investigated the effect of benfotiamine on markers of these pathways in patients with type 2 diabetes and nephropathy.Methods
Patients with type 2 diabetes and urinary albumin excretion in the high-normal and microalbuminuric range (15–300 mg/24h) were randomized to receive benfotiamine (n = 39) or placebo (n = 43). Plasma and urinary AGEs (N ε-(carboxymethyl) lysine [CML], N ε-(Carboxyethyl) lysine [CEL], and 5-hydro-5-methylimidazolone [MG-H1]) and plasma markers of endothelial dysfunction (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble intercellular adhesion molecule-1 [sICAM-1], soluble E-selectin) and low-grade inflammation (high-sensitivity C-reactive protein [hs-CRP], serum amyloid-A [SAA], myeloperoxidase [MPO]) were measured at baseline and after 6 and 12 weeks.Results
Compared to placebo, benfotiamine did not result in significant reductions in plasma or urinary AGEs or plasma markers of endothelial dysfunction and low-grade inflammation.Conclusions
Benfotiamine for 12 weeks did not significantly affect intermediate pathways of hyperglycemia-induced vascular complications.Trial Regristration
ClinicalTrials.gov NCT00565318 相似文献15.
Background
To study the long-term effects of low-dosage strontium-90 (Sr90) irradiation on the recurrence of pterygium.Methodology/Principal Findings
One hundred twenty eyes from 104 patients with primary or recurrent pterygia were treated with surgery followed by Sr90 irradiation. In brief, starting on the sixth day after surgery, patients were treated with irradiation three times every other day at a total combined dosage of 2000 cGy to 3000 cGy. Corneal topography was used to evaluate ocular surface regularity before and after treatment. Patient follow-up was performed 2 days, 5 days, 2 weeks, 1 month, 3 months, 1 year, 5 years, and 10 years after surgery. Recurrence of pterygium was not observed in any of the patients in this study. Obvious cataract progression was observed in 6 eyes, which may be due to aging. During follow-up studies, only one eye was reported with dryness and foreign-body sensation. Significant pterygium-induced astigmatism was observed in corneal topography, which decreased after surgery.Conclusions/Significance
Sr90 irradiation is effective in preventing the recurrence of primary and recurrent pterygia. We recommend delivering a total combined dosage of 2000 cGy to 3000 cGy of Sr90 irradiation administered in three batches every other day starting from the sixth day after surgery. Surgery is important in the rapid recovery of the cornea from pterygium-induced astigmatism. 相似文献16.
Irena Auersperger Branko ?kof Bojan Lesko?ek Bojan Knap Ale? Jerin Mitja Lainscak 《PloS one》2013,8(3)
Background and Aims
Exercise-induced iron deficiency is a common finding in endurance athletes. It has been suggested recently that hepcidin may be an important mediator in this process.Objective
To determine hepcidin levels and markers of iron status during long-term exercise training in female runners with depleted and normal iron stores.Methods
Fourteen runners were divided into two groups according to iron status. Blood samples were taken during a period of eight weeks at baseline, after training and after ten days’ recovery phase.Results
Of 14 runners, 7 were iron deficient at baseline and 10 after training. Hepcidin was lower at recovery compared with baseline (p<0.05). The mean cell haemoglobin content, haemoglobin content per reticulocyte and total iron binding capacity all decreased, whereas soluble transferrin receptor and hypochromic red cells increased after training and recovery (p<0.05 for all).Conclusion
The prevalence of depleted iron stores was 71% at the end of the training phase. Hepcidin and iron stores decreased during long-term running training and did not recover after ten days, regardless of baseline iron status. 相似文献17.
18.
19.
Xin-Yi Wang Shenghong Ju Cong Li Xin-Gui Peng Alex F. Chen Hui Mao Gao-Jun Teng 《PloS one》2012,7(11)
Objective
Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.Methods
The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.Results
Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.Conclusions
This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization. 相似文献20.
Ai Kawana-Tachikawa Josep M. Llibre Isabel Bravo Roser Escrig Beatriz Mothe Jordi Puig Maria C. Puertas Javier Martinez-Picado Julia Blanco Christian Manzardo Jose M. Miro Aikichi Iwamoto Anton L. Pozniak Jose M. Gatell Bonaventura Clotet Christian Brander the MARAVIBOOST investigators 《PloS one》2014,9(1)