Magnetite nanoparticles coated with citric acid are not phytotoxic and stimulate soybean and alfalfa growth.

In this work, the internalization and distribution of citric acid-coated magnetite nanoparticles (here, Fe3O4-NPs) in soybean and alfalfa tissues and their effects on plant growth were studied. Both legumes were germinated in pots containing an inert growing matrix (vermiculite) to which Hoagland solution without (control, C), with Fe3O4-NPs (50 and 100 mgironL-1, NP50 and NP100), or with the same amount of soluble iron supplied as Fe-EDTA (Fe50, Fe100) was added once before sowing. Then, plants were watered with the standard nutrient solution. The observation of superparamagnetic signals in root tissues at harvest (26 days after emergence) indicated Fe3O4-NPs uptake by both legumes. A weak superparamagnetic signal was also present in the stems and leaves of alfalfa plants. These findings suggest that Fe3O4-NPs are readily absorbed but not translocated (soybean) or scarcely translocated (alfalfa) from the roots to the shoots. The addition of both iron sources resulted in increased root weight; however, only the addition of Fe3O4-NPs resulted in significantly higher root surface; shoot weight also increased significantly. As a general trend, chlorophyll content enhanced in plants grown in vermiculite supplemented with extra iron at pre-sowing; the greatest increase was observed with NP50. The only antioxidant enzyme significantly affected by our treatments was catalase, whose activity increased in the roots and shoots of both species exposed to Fe3O4-NPs. However, no symptoms of oxidative stress, such as increased lipid peroxidation or reactive oxygen species accumulation, were evidenced in any of these legumes. Besides, no evidence of cell membrane damage or cell death was found. Our results suggest that citric acid-coated Fe3O4-NPs are not toxic to soybean and alfalfa; instead, they behave as plant growth stimulators.

Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

A phase 2 trial of standard-dose cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) and rituximab plus bevacizumab for patients with newly diagnosed diffuse large B-cell non-Hodgkin lymphoma: SWOG 0515.

S0515 was a phase 2 trial to determine whether the addition of bevacizumab to cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) plus rituximab (R-CHOP) would improve progression-free survival (PFS) without adding significant toxicity in patients with newly diagnosed advanced diffuse large B-cell lymphoma. A total of 73 patients were enrolled. For the 64 eligible patients, median age was 68 years, and 60% had International Prognostic Index scores more than or equal to 3. The observed 1- and 2-year PFS estimates were 77% and 69%, respectively. These PFS estimates were not statistically different from the expected PFS for this population if treated with R-CHOP alone. Grade 3 or higher toxicities were observed in 81% of patients, including 2 grade 5 events. The majority of serious toxicities were hematologic but also included 5 patients with gastrointestinal perforations, 4 patients with thrombotic events, and 11 patients who developed grade 2 or 3 left ventricular dysfunction. Higher baseline urine VEGF and plasma VCAM levels correlated with worse PFS and overall survival. In conclusion, the addition of bevacizumab to R-CHOP chemotherapy was not promising in terms of PFS and resulted in increased serious toxicities, especially cardiac and gastrointestinal perforations. This study is registered at www.clinicaltrials.gov as #NCT00121199.

Defining an optimal stromal derived factor-1 presentation for effective recruitment of mesenchymal stem cells in 3D.

In "situ" tissue engineering is a promising approach in regenerative medicine, envisaging to potentiate the physiological tissue repair processes by recruiting the host's own cellular progenitors at the lesion site by means of bioactive materials. Despite numerous works focused the attention in characterizing novel chemoattractant molecules, only few studied the optimal way to present signal in the microenvironment, in order to recruit cells more effectively. In this work, we have analyzed the effects of gradients of stromal derived factor-1 (SDF-1) on the migratory behavior of human mesenchymal stem cells (MSCs). We have characterized the expression of the chemokine-associated receptor, CXCR4, using cytofluorimetric and real-time PCR analyses. Gradients of SDF-1 were created in 3D collagen gels in a chemotaxis chamber. Migration parameters were evaluated using different chemoattractant concentrations. Our results show that cell motion is strongly affected by the spatio-temporal features of SDF-1 gradients. In particular, we demonstrated that the presence of SDF-1 not only influences cell motility but alters the cell state in terms of SDF-1 receptor expression and productions, thus modifying the way cells perceive the signal itself. Our observations highlight the importance of a correct stimulation of MSCs by means of SDF-1 in order to implement on effective cell recruitment. Our results could be useful for the creation of a "cell instructive material" that is capable to communicate with the cells and control and direct tissue regeneration. Biotechnol. Bioeng. 2014;111: 2303-2316. © 2014 Wiley Periodicals, Inc.

Quantitative phase maps denoising of long holographic sequences by using SPADEDH algorithm.

We propose a denoising method for digital holography mod 2π wrapped phase map by using an adaptation of the SPArsity DEnoising of Digital Holograms (SPADEDH) algorithm. SPADEDH is a l(1) minimization algorithm able to suppress the noise components on digital holograms without any prior knowledge or estimation about the statistics of noise. We test our algorithm with either general numerical simulated wrapped phase, quantifying the performance with different efficiency parameters and comparing it with two popular denoising strategies, i.e., median and Gaussian filters, and specific experimental tests, by focusing our attention on long-sequence wrapped quantitative phase maps (QPMs) of in vitro cells, which aim to have uncorrupted QPMs. In addition, we prove that the proposed algorithm can be used as a helper for the typical local phase unwrapping algorithms.

On the holographic 3D tracking of in vitro cells characterized by a highly-morphological change.

Digital Holography (DH) in microscopic configuration is a powerful tool for the imaging of micro-objects contained into a three dimensional (3D) volume, by a single-shot image acquisition. Many studies report on the ability of DH to track particle, microorganism and cells in 3D. However, very few investigations are performed with objects that change severely their morphology during the observation period. Here we study DH as a tool for 3D tracking an osteosarcoma cell line for which extensive changes in cell morphology are associated to cell motion. Due to the great unpredictable morphological change, retrieving cell's position in 3D can become a complicated issue. We investigate and discuss in this paper how the tridimensional position can be affected by the continuous change of the cells. Moreover we propose and test some strategies to afford the problems and compare it with others approaches. Finally, results on the 3D tracking and comments are reported and illustrated.

Polyamines modulate nitrate reductase activity in wheat leaves: involvement of nitric oxide.

In the present work, the effect of polyamines (PAs) on nitrate reductase (NR) activity was studied in wheat leaves exposed to exogenously added PAs while assessing the nitric oxide (NO) involvement in the regulation of the enzyme activity. A biphasic response was observed along the time of treatment using 0.1 mM of putrescine (Put), spermidine (Spd) or spermine (Spm). At 3 h, Spd and Spm significantly reduced NR activity by 29 or 35%, respectively, whereas at 6 h, the activity of the enzyme decreased by an average of 25%. At 21 h, Put increased NR activity by 63%, while Spd and Spm elevated the enzyme activity by 114%. NR activity, that was reduced by 0.1 mM Spm at 3 and 6 h, returned almost to control values when c-PTIO (an NO scavenger) was used, confirming that NO was involved in the inhibition of NR activity. Nitric oxide was also mediating the PA-increase of the enzyme activity at longer incubation times, evidenced when the raise in NR activity produced by 0.1 mM Spm at the longest incubation time returned to the value of the control in the presence of cPTIO. Neither the protein expression nor the nitrate content were modified by PAs treatments. The involvement of PAs and NO in the regulation of NR activity is discussed.

Beneficial effects of magnetite nanoparticles on soybean-Bradyrhizobium japonicum and alfalfa-Sinorhizobium meliloti associations.

Nanoparticles (NPs)-based growth stimulators have promising usage in agriculture. This research analyzed the impact of citric acid-coated magnetite nanoparticles (Fe3O4-NPs; 50 mg Fe L-1) added once at pre-sowing on soybean and alfalfa seedlings growing in association with their corresponding microsymbiont partners, Bradyrhizobium japonicum and Sinorhizobium meliloti; also on the in vitro growth rate of these microorganisms. Fe-EDTA (50 mg Fe L-1) was used as a comparator. Fe3O4-NPs significantly augmented the growth rate constant (7-17%) and extracellular polysaccharides production of both microsymbionts (B. japonicum: 2-fold; S. meliloti: 43%), which probably favored bacterial adhesion to the root hairs. In both legumes, Fe3O4-NPs increased chlorophyll content (up to 56% in soybean) and improved plant growth, evidenced by a greater root biomass system (80-90% higher than the control), and increased shoot biomass (30-40%). Besides, Fe3O4-NPs addition resulted in earlier nodule formation and enhanced nodule biomass (about 2.5-fold in both species). Nodules were mainly located in the crown of the root in the NP50 treatment, while they were evenly distributed along lateral roots in the control and the comparator. Fe3O4-NPs also augmented significantly nodule leghemoglobin content (∼50-70%) and total N in legumes' shoots (ca. 20%). CAT activity increased only under NP50 treatment and no symptoms of oxidative damage were evidenced. In this work, we found that besides not being toxic neither to soybean and alfalfa plants nor to their microsymbiont partners, Fe3O4-NPs do not exert adverse effects on the symbioses establishment; oppositely, a more efficient nodulation pattern was verified in both plant species.

Surface investigation on biomimetic materials to control cell adhesion: the case of RGD conjugation on PCL.

The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(epsilon-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.

Circulating Carbonic Anhydrase IX and Antiangiogenic Therapy in Breast Cancer.

INTRODUCTION: Carbonic anhydrase IX (CAIX) is a hypoxia regulated metalloenzyme integral to maintaining cellular pH. Increased CAIX expression is associated with poor prognosis in breast cancer. To explore CAIX as a biomarker for breast cancer therapies, we measured plasma CAIX levels in healthy control subjects and in breast cancer patients. METHODS: In control subjects we evaluated plasma CAIX stability via commercially available ELISA. We then similarly quantified plasma CAIX levels in (1) locally advanced breast cancer (LABC) patients treated with neoadjuvant paclitaxel + sunitinib (T + S) followed by doxorubicin and cyclophosphamide (AC); (2) metastatic breast cancer (MBC) patients treated with systemic chemotherapy. RESULTS: Plasma CAIX levels were stable at room temperature for at least 48 hours in control subjects. Mean baseline plasma CAIX levels were lower in controls compared to patients with LABC or MBC. In LABC, CAIX levels rose significantly in response to administration of antiangiogenic therapy (T + S) (p = 0.02) but not AC (p = 0.37). In patients with MBC treated without an antiangiogenic agent CAIX levels did not change with therapy. CONCLUSIONS: Our results suggest that CAIX may be an easily obtained, stable measure of tumor associated hypoxia as well as a useful pharmacodynamic biomarker for antiangiogenic therapy.

A phase I clinical trial of bavituximab and paclitaxel in patients with HER2 negative metastatic breast cancer.

Bavituximab is a chimeric monoclonal antibody that targets phosphatidylserine (PS). PS is externalized on cells in the tumor microenvironment when exposed to hypoxia and/or other physiological stressors. On attaching to PS, bavituximab is thought to promote antitumor immunity through its effects on PS receptors in monocytes, and myeloid-derived suppressor cells, as well as trigger antitumor effects by inducing an antibody-dependent cellular cytotoxicity on tumor-associated endothelial cells. We conducted a phase I clinical trial of bavituximab in combination with paclitaxel in patients with HER2-negative metastatic breast cancer. Patients were treated with weekly paclitaxel (80 mg/m(2) for 3/4 weeks) and weekly bavituximab (3 mg/kg for 4/4 weeks). Correlative studies included the measurement of circulating microparticles, endothelial cells, and apoptotic tumor cells by flow cytometry. Fourteen patients with metastatic breast cancer were enrolled; all were evaluable for toxicity and 13 were evaluable for response. Treatment resulted in an overall response rate (RR) of 85% with a median progression-free survival (PFS) of 7.3 months. Bone pain, fatigue, headache, and neutropenia were the most common adverse effects. Infusion-related reactions were the most common adverse event related to bavituximab therapy. Correlative studies showed an increase in the PS-expressing apoptotic circulating tumor cells in response to bavituximab, but not with paclitaxel. No changes in the number of circulating endothelial cells or apoptotic endothelial cells were observed with therapy. Platelet and monocyte-derived microparticles decreased after initiation of bavituximab. Bavituximab in combination with paclitaxel is well tolerated for treatment of patients with metastatic breast cancer with promising results observed in terms of clinical RRs and PFS. The toxicity profile of bavituximab is notable for manageable infusion-related reactions with no evidence for increased thrombogenicity. Recent preclinical data suggest that bavituximab can also promote antitumor immune activity that should be explored in future clinical trials.

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf discs exposed to paraquat.

In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O (2) (.-) ) formation was inhibited at 3 or 21 h of exposure, but H(2)O(2) production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.

Nitric oxide inhibits nitrate reductase activity in wheat leaves.

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, is involved in the generation of nitric oxide (NO) in plants. In wheat leaf segments exposed to sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO), NR activity was significantly reduced to different degrees between 3 and 21 h, whereas its activity was partially recovered when the NO scavenger cPTIO was used. At 21 h, NR activity decreased from 38% with 10 µM SNP to 91% with 500 µM SNP, respect to the C values. S-nitrosoglutathione reduced NR activity between 18% and 26% only at 3 h. When added directly to the incubation solution, NR activity was quickly and strongly inhibited more than 90% by 10 or 50 µM SNP, whereas 10 µM GSNO reduced the enzyme activity an average of 50%, at 30 min of incubation. l-NAME and d-arginine (nitric oxide synthase (NOS) inhibitors) increased NR activity by 14% and 52% respectively, at 21 h of exposure, leading us to suppose that endogenous NOS-dependent NO formation could also be modulating NR activity. NR protein expression was not affected by 10 or 100 µM SNP at 3 or 21 h of incubation, whereas nitration of tyrosines was not detected in the NR protein. Nitrates, which content increased along the time in the tissues, could be exerting a role in this regulation.

Effect of micro- and macroporosity of bone tissue three-dimensional-poly(epsilon-caprolactone) scaffold on human mesenchymal stem cells invasion, proliferation, and differentiation in vitro.

The design of porous scaffolds able to promote and guide cell proliferation, colonization, and biosynthesis in three dimensions is key determinant in bone tissue engineering (bTE). The aim of this study was to assess the role of the micro-architecture of poly(epsilon-caprolactone) scaffolds in affecting human mesenchymal stem cells' (hMSCs) spatial organization, proliferation, and osteogenic differentiation in vitro. Poly(epsilon-caprolactone) scaffolds for bTE and characterized by mono-modal and bi-modal pore size distributions were prepared by the combination of gas foaming and selective polymer extraction from co-continuous blends. The topological properties of the pore structure of the scaffolds were analyzed and the results correlated with the ability of hMSCs to proliferate, infiltrate, and differentiate in vitro in three dimensions. Results showed that the micro-architecture of the pore structure of the scaffolds plays a crucial role in defining cell seeding efficiency as well as hMSCs' three-dimensional colonization, proliferation, and osteogenic differentiation. Taken all together, our results indicated that process technologies able to allow a fine-tune of the topological properties of biodegradable porous scaffolds are essential for bTE strategies.

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium.

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 microM CdCl(2) increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O (2)(-) formation in a dose dependent manner in SR1 very strongly at 500 microM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 microM CdCl(2) almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.

A phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma: Southwest oncology group study S0108.

This is the first report of the Southwest oncology group phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma (NHL). Fifty-two patients in first or second relapse with diffuse large B-cell or mantle cell lymphoma were enrolled. Patients were treated with bevacizumab at 10 mg/kg every 2 weeks. Therapy was well tolerated with no unexpected toxicities observed. Six-month progression-free survival (PFS) was 16% with a response rate of 2% and median duration of response or stable disease of 5.2 months (range 3.5-72.7). Vascular endothelial growth factor A (VEGF) and VEGF receptor expression was observed in 70% and 65% of specimens, respectively. In an exploratory subgroup analysis, baseline urine VEGF and plasma vascular cell adhesion molecule-1 (VCAM) levels correlated with survival. Prolonged PFS in several patients as well as biomarker studies suggest the VEGF pathway plays an important role in aggressive NHL. Clinical trials combining active chemotherapy regimens with VEGF targeted agents are currently in progress.

A phase 1 trial of 2 dose schedules of ABT-510, an antiangiogenic, thrombospondin-1-mimetic peptide, in patients with advanced cancer.

BACKGROUND: ABT-510 is a substituted nonapeptide that mimics the antiangiogenic activity of the endogenous protein thrombospondin-1 (TSP-1). The current study was designed to establish the safety of ABT-510 in the treatment of patients with advanced malignancies on a once-daily (QD) and twice-daily dosing schedule. METHODS: Patients were randomly assigned to 1 of 6 dosing regimens: 20 mg, 50 mg, or 100 mg QD or 10 mg, 25 mg, or 50 mg twice daily. ABT-510 was administered by subcutaneous bolus injection in cycles of 28 days. Tumor response and disease progression were monitored at 8-week intervals by computed tomography scan or magnetic resonance imaging. RESULTS: Thirty-six patients were randomly assigned in equal numbers to the 6 study regimens, with an additional 13 patients randomized to the 10-mg-twice-daily and 50-mg-twice-daily ABT-510 regimens. The expected pharmacokinetic target was achieved at all dose levels tested. The majority of adverse events were grade 1 or 2 (according to National Cancer Institute Common Toxicity Criteria [version 2]) and were not found to be dose related. The most frequently reported adverse events that were possibly related to ABT-510 included injection site reactions, asthenia, headache, and nausea. Grade 3 events considered to possibly be related included nausea, dyspnea, bone pain, constipation, vomiting, asthenia, and chills and tremors. One partial response was observed in a patient with carcinosarcoma who received 20 mg QD. The 6-month progression-free survival rate was 6%. Approximately 42% of patients (21 of 50 patients) had stable disease for > or =3 months. CONCLUSIONS: ABT-510 can be administered at doses of 20 mg/day to 100 mg/day without significant toxicity. In the current study, minimal antitumor activity was observed, which was similar to observations in other single-agent antiangiogenic trials.

Coronary flow velocity pattern assessed by transthoracic Doppler echocardiography predicts adverse clinical events and myocardial recovery after successful primary angioplasty.

BACKGROUND: Doppler guidewire studies demonstrated that specific velocity patterns in the left anterior descending coronary artery (LAD) after primary percutaneous coronary intervention (PCI) predict myocardial recovery and clinical outcome. The present study assessed whether similar results can be achieved by transthoracic Doppler echocardiography (TTDE). METHODS: Coronary flow velocities of LAD were evaluated by TTDE in 35 consecutive patients with anterior acute myocardial infarction who were treated with successful primary PCI plus stenting, performed within 6 h after the onset of symptoms or within 6-12 h if there was evidence of continuing ischaemia. Coronary-flow velocity of the LAD was achieved after 12 h and within 48 h after the PCI; TTDE standard examination was repeated after 2 months of follow-up. RESULTS: Three patterns were found: (i) 'pattern A' with good antegrade systolic flow and slow diastolic deceleration rate (63.7%); (ii) 'pattern B' with reduced or absent systolic flow and rapid diastolic deceleration rate (9.1%); and (iii) 'pattern C' with protosystolic retrograde flow and rapid diastolic deceleration rate (27.2%). The clinical characteristics and echocardiographic data were compared: wall-motion-score-index (WMSI), ejection fraction, end-diastolic volume (EDV) after PCI (T1) and after 2 months (T2). Patients with pattern A demonstrated recovery of contractile function (WMSI-T1 1.48 + or - 0.42/WMSI-T2 1.29 + or - 0.29, P < 0.05) and better clinical outcome; patients with patterns B and C ran into ventricular remodelling (EDV-T1 89 + or - 6.3 ml/EDV-T2 123 + or - 25 ml, P = 0.002) and more early and late complications. CONCLUSIONS: TTDE is a reliable method to achieve coronary flow velocities in LAD after an anterior acute myocardial infarction and it could be useful to evaluate no-reflow phenomenon at bedside and thus clinical outcome.