High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage.

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome.

Evaluation of a rail-travel simulator for elderly adults with cognitive disorders. The Grand-Via Project, a travel therapy pilot study.

BACKGROUND AND PURPOSE: Travel therapy can reduce anxiety symptoms in elderly adults with cognitive disorders. The objective of this pilot study was to evaluate the use of a rail-travel simulator in this purpose. MATERIALS AND METHODS: The study was a prospective, single centre cohort survey. Our study population consisted of persons either from the nursing home, the cognitive and behavioural unit or the day-care centre of our university hospital. Participants were accompanied on a virtual trip using a film projection in a replica of train compartment. Participants were interviewed before and after each session using a short questionnaire developed by a multi-disciplinary team. RESULTS: Forty-two participants performed sessions. While only 58.3% of the participants reported being relaxed before the session, this rate increased significantly to 87.5% by the end of the trip. A majority of participants gave their personal impressions and half of the group reported memories evoked by the experience. CONCLUSION: The majority of elderly persons who completed the virtual trip replied positively about the experience. We need now to confirm the efficacy of our simulator using a randomised controlled trial.

Multiplexed Quantitative Assessment of the Fate of Taurine and Sulfoquinovose in the Intestinal Microbiome.

(1) Introduction: Sulfonates, which can be diet- or host-derived, are a class of compounds detected in the gut, are involved in host-microbiome interactions and have several health effects. Our aim was to develop a method to quantify five of the sulfonates in the intestine and apply it in a simplified human microbiome model. These were taurine, its metabolic precursor cysteate and one of its degradation products isethionate, as well as sulfoquinovose and one of its most relevant degradation products 2,3-dihydroxy-1-propanesulfonate. (2) Methods: An extraction and sample preparation method was developed, without the need for derivatization. To detect and quantify the extracted sulfonates, a multiplexed LC-MS/MS-MRM method was established. (3) Results: The accuracy and precision of the method were within GLP-accepted parameters (www.ema.europa.eu). To apply this method in a pilot study, we spiked either taurine or sulfoquinovose into an in vitro simplified human microbiota model with and without Bilophila wadsworthia, a known sulfonate utilizer. The results revealed that only the culture with B. wadsworthia was able to degrade taurine, with isethionate as an intermediate. After spiking the communities with sulfoquinovose, the results revealed that the simplified human microbiome model was able to degrade sulfoquinovose to 2,3-dihydroxypropane-1-sulfonate, which was probably catalyzed by Escherichia coli. In the community with B. wadsworthia, the 2,3-dihydroxypropane-1-sulfonate produced was further degraded by B. wadsworthia to sulfide. (4) Conclusions: We successfully developed a method for sulfonate quantification and applied it in a first pilot study.

Gastric bypass surgery in a rat model alters the community structure and functional composition of the intestinal microbiota independently of weight loss.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery is a last-resort treatment to induce substantial and sustained weight loss in cases of severe obesity. This anatomical rearrangement affects the intestinal microbiota, but so far, little information is available on how it interferes with microbial functionality and microbial-host interactions independently of weight loss. METHODS: A rat model was employed where the RYGB-surgery cohort is compared to sham-operated controls which were kept at a matched body weight by food restriction. We investigated the microbial taxonomy and functional activity using 16S rRNA amplicon gene sequencing, metaproteomics, and metabolomics on samples collected from theileum, the cecum, and the colon, and separately analysed the lumen and mucus-associated microbiota. RESULTS: Altered gut architecture in RYGB increased the relative occurrence of Actinobacteria, especially Bifidobacteriaceae and Proteobacteria, while in general, Firmicutes were decreased although Streptococcaceae and Clostridium perfringens were observed at relative higher abundances independent of weight loss. A decrease of conjugated and secondary bile acids was observed in the RYGB-gut lumen. The arginine biosynthesis pathway in the microbiota was altered, as indicated by the changes in the abundance of upstream metabolites and enzymes, resulting in lower levels of arginine and higher levels of aspartate in the colon after RYGB. CONCLUSION: The anatomical rearrangement in RYGB affects microbiota composition and functionality as well as changes in amino acid and bile acid metabolism independently of weight loss. The shift in the taxonomic structure of the microbiota after RYGB may be mediated by the resulting change in the composition of the bile acid pool in the gut and by changes in the composition of nutrients in the gut. Video abstract.

Mucosal-associated invariant T-Cell (MAIT) activation is altered by chlorpyrifos- and glyphosate-treated commensal gut bacteria.

Mucosal-associated invariant T-cells (MAIT) can react to metabolites of the vitamins riboflavin and folate which are produced by the human gut microbiota. Since several studies showed that the pesticide chlorpyrifos (CPF) and glyphosate (GLP) can impair the gut microbiota, the present study was undertaken to investigate the impact of CPF and GLP treatment on the metabolism of gut microbiota and the resulting bacteria-mediated modulation of MAIT cell activity. Here, Bifidobacterium adolescentis (B. adolescentis), Lactobacillus reuteri (L. reuteri), and Escherichia coli (E. coli) were treated with CPF (50-200 µM) or GLP (75-300 mg/L) and then used in MAIT cell stimulation assays as well as in vitamin and proteome analyses. All three bacteria were nonpathogenic and chosen as representatives of a healthy human gut microflora. The results showed that E. coli activated MAIT cells whereas B. adolescentis and L. reuteri inhibited MAIT cell activation. CPF treatment significantly increased E. coli-mediated MAIT cell activation. Treatment of B. adolescentis and L. reuteri with CPF and GLP weakened the inhibition of MAIT cell activation. Riboflavin and folate production by the test bacteria was influenced by CPF treatment, whereas GLP had only minor effects. Proteomic analysis of CPF-treated E. coli revealed changes in the riboflavin and folate biosynthesis pathways. The findings here suggest that the metabolism of the analyzed bacteria could be altered by exposure to CPF and GLP, leading to an increased pro-inflammatory immune response.

Identification of algal growth inhibitors in treated waste water using effect-directed analysis based on non-target screening techniques.

Growth inhibition of freshwater microalga Pseudokirchneriella subcapitata caused by a waste water treatment plant (WWTP) effluent extract was investigated using an effect directed analysis (EDA) approach. The objective was to identify compounds responsible for the toxicity by combining state-of-the-art sampling, bioanalytical, fractionation and non-target screening techniques. Three fractionation steps of the whole extract were performed and bioactive fractions were analysed with GC (xGC)-MS and LC-HRMS. In total, 383 compounds were tentatively identified, and their toxicity was characterized using US EPA Ecotox database, open scientific literature or modelled by ECOSAR. Among the top-ranking drivers of toxicity were pesticides and their transformation products, pharmaceuticals (barbiturate derivatives and macrolide antibiotics e.g. azithromycin), industrial compounds or caffeine and its metabolites. Several of the top-ranking pesticides are no longer registered for use in plant protection products or biocides in the Czech Republic (e.g. prometryn, atrazine, acetochlor, resmethrin) and some are approved only for use in biocides (e.g. terbutryn, carbendazim, phenothrin), which indicates that their non-agricultural input into aquatic environment via WWTPs should be carefully considered. The study demonstrated a functional strategy of combining biotesting, fractionation and non-target screening techniques in the EDA study focused on the identification of algal growth inhibitors in WWTP effluent.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

Miniaturization of a transthyretin binding assay using a fluorescent probe for high throughput screening of thyroid hormone disruption in environmental samples.

Thyroid hormone (TH) disrupting compounds are potentially important environmental contaminants due to their possible adverse neurological and developmental effects on both humans and wildlife. Currently, the most successful bio-analytical method to detect and evaluate TH disruptors, which target the plasma transport of TH in environmental samples, is the radio-ligand thyroxine-transthyretin (T4-TTR) binding assay. Yet, costly materials and tedious handling procedures prevent the use of this assay in high throughput analysis that is nowadays urgently demanded in environmental quality assessment. For the first time a miniaturized fluorescence T4-TTR binding assay was developed in a 96 well microplate and tested with eight TH disrupting compounds. For most of the compounds, the sensitivity of the newly developed assay was slightly lower than the radio-ligand binding assay, however, throughput was enhanced at least 100-fold, while using much cheaper materials. The TH disrupting potency of 22 herring gull (Larus argentatus) egg extracts, collected from two different locations (Musvær and Reiaren) in Norway, was evaluated to demonstrate the applicability of the assay for environmental samples.

Assessment of a novel device for onsite integrative large-volume solid phase extraction of water samples to enable a comprehensive chemical and effect-based analysis.

The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from -3.6 to 9.4 (at pH7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1ngL-1 to 2400ngL-1). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes.

Identification of petrogenic produced water components as acetylcholine esterase inhibitors.

Effect-directed analysis (EDA) was applied to identify acetylcholine esterase (AChE) inhibitors in produced water. Common produced water components from oil production activities, such as polycyclic aromatic hydrocarbons (PAHs), alkylphenols, and naphthenic acids were tested for AChE inhibition using a simple mixture of PAHs and naphthenic acids. Produced water samples collected from two offshore platforms in the Norwegian sector of the North Sea were extracted by solid phase extraction and fractionated by open-column liquid solid chromatography and high-performance liquid chromatography (HPLC) before being tested using a high-throughput and automated AChE assay. The HPLC fractions causing the strongest AChE inhibition were analysed by gas chromatography coupled to a high-resolution time-of-flight mass spectrometry (GC-HR-ToF-MS). Butylated hydroxytoluene and 4-phenyl-1,2-dihydronaphthalene were identified as two produced water components capable of inhibiting AChE at low concentrations. In order to assess the potential presence of such compounds discharged into aquatic ecosystems, AChE activity in fish tissues was measured. Saithe (Pollachius virens) caught near two offshore platforms showed lower enzymatic activity than those collected from a reference location. Target analysis of saithe did not detected the presence of these two putative AChE inhibitors and suggest that additional compounds such as PAHs, naphthenic acids and yet un-identified compounds may also contribute to the purported AChE inhibition observed in saithe.

Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results.

Visualization of vascular compression of the trigeminal nerve with high-resolution 3T MRI: a prospective study comparing preoperative imaging analysis to surgical findings in 40 consecutive patients who underwent microvascular decompression for trigeminal neuralgia.

BACKGROUND: High-resolution three-dimensional (3D) magnetic resonance imaging (MRI) has demonstrated its ability to predict fine trigeminal neurovascular anatomy. OBJECTIVE: To address the predictive value of 3-Tesla (3T) MRI in detecting and assessing features of neurovascular compression (NVC), particularly regarding the degree of compression exerted on the root, in patients who underwent microvascular decompression (MVD) for classic primary trigeminal neuralgia. METHODS: This prospective study includes 40 consecutive patients who underwent MVD for classic primary trigeminal neuralgia. All patients underwent a preoperative 3T MRI with 3D T2-weighted driven equilibrium (DRIVE), 3D time-of-flight (TOF) magnetic resonance angiography (MRA), and 3D T1-weighted gadolinium-enhanced sequences in combination. Evaluations were performed by 2 independent observers and compared with the operative findings. RESULTS: For prediction of NVC, image analysis corresponded with surgical findings in 39 cases. Of the 3 patients in whom image analysis did not show NVC, 2 did not have NVC at the time of intraoperative observation. MRI sensitivity was 97.4% (37/38), and specificity was 100% (2/2). The kappa coefficients (κ) for predicting the offending vessel, its location, and the site of compression were 0.882, 0.813, and 0.942, respectively. Image analysis correctly defined the severity of the compression in 31 of the 37 cases. The κ coefficients predicting the degree of compression were 0.813, 0.833, and 0.852, respectively, for Grades 1 (simple contact), 2 (distortion), and 3 (marked indentation). CONCLUSION: 3T MRI using 3D T2-weighted DRIVE in combination with 3D TOF-MRA and 3D T1-weighted gadolinium-enhanced sequences proved to be reliable in detecting NVC and in predicting the degree of root compression, the outcome being correlated with the latter.

Preoperative demonstration of the neurovascular compression characteristics with special emphasis on the degree of compression, using high-resolution magnetic resonance imaging: a prospective study, with comparison to surgical findings, in 100 consecutive patients who underwent microvascular decompression for trigeminal neuralgia.

PURPOSE: Surgical outcome after microvascular decompression (MVD) for primary trigeminal neuralgia (TN) has been demonstrated as being related to the characteristics of the neurovascular compression (NVC), especially to the degree of compression exerted on the root. Therefore, preoperative determination of the NVC features could be of great value to the neurosurgeon, for evaluation of conflicting nature, exact localization, direction and degree of compression. This study deals with the predictive value of MRI in detecting and assessing features of vascular compression in 100 consecutive patients who underwent MVD for TN. METHODS: The study included 100 consecutive patients with primary TN who were submitted to a preoperative 3D MRI 1.5 T with T2 high-resolution, TOF-MRA, and T1-Gadolinium. Image analysis was performed by an independent observer blinded to the operative findings and compared with surgical data. FINDINGS: In 88 cases, image analysis showed NVC features that coincided with surgical findings. There were no false-positive results. Among 12 patients that did not show NVC at image analysis, nine did not have NVC at intraoperative observation, resulting in three false-negative cases. MRI sensitivity was 96.7% (88/91) and specificity 100% (9/9). Image analysis correctly identified compressible vessel in 80 of the 91 cases and degree of compression in 77 of the 91 cases. Kappa-coefficient predicting degree of root compression was 0.746, 0.767, and 0.86, respectively, for Grades I (simple contact), II (distortion), and III (marked indentation; p < 0.01). CONCLUSION: 3D T2 high-resolution in combination with 3D TOF-MRA and 3D T1-Gadolinium proved to be reliable in detecting NVC and in predicting the degree of the root compression.

Inflammatory response after ischemic stroke: a USPIO-enhanced MRI study in patients.

BACKGROUND AND PURPOSE: The intensity of the inflammatory response may be related to the volume of acute infarction. Ultra-small superparamagnetic particles of iron oxide (USPIO) may enable assessment of neuroinflammation. We aimed to assess whether the intensity of the inflammatory response might be related to the subacute ischemic lesion volume. METHODS: We enrolled patients who presented with acute anterior circulation stroke. MRI was performed at day 0, day 6, and day 9. The MRI protocol included T1-weighted imaging, gradient-echo T2*-weighted imaging, diffusion-weighted imaging, perfusion-weighted imaging and MR angiography. Blood-brain barrier disruption was defined as post-gadolinium enhancement on T1-weighted images. USPIO was administered after day 6 MRI. USPIO enhancement ratios were defined as the ratio between USPIO-related signal volume on day 9 T1-weighted imaging (respectively T2*-weighted imaging) and day 6 diffusion-weighted imaging infarct volume. The relationship between day 6 infarct volume and the enhancement ratio was assessed using Pearson and Spearman correlation tests. RESULTS: The protocol was completed in 10 patients. Signal alterations after USPIO injection was observed in 9/10 patients on day 9 T1-weighted imaging and in 5/10 patients on day 9 T2*-weighted imaging. USPIO-related MRI enhancement was heterogeneous. Lesion volume on day 6 diffusion-weighted imaging had no impact on USPIO enhancement at day 9 according to the Pearson correlation test (P=0.39) or Spearman test (P=0.25). There was no relationship between blood-brain barrier disruption and USPIO enhancement. CONCLUSIONS: USPIO MRI enhancement is heterogeneous and not clearly related to subacute lesion volume.

Evolution of lesion volume in acute stroke treated by intravenous t-PA.

PURPOSE: To determine the evolution of the ischemic lesion volumes in a population treated with tissue plasminogen activator (t-PA), MRIs were performed before treatment and 24 hours later; final infarct size was evaluated 60 days later. MATERIALS AND METHODS: A total of 42 patients with hemispheric stroke were recruited for a thrombolytic study. Intravenous t-PA was given after MRI within the first seven hours after stroke onset. Volumes were evaluated on day 0 and day 1 with diffusion-weighted imaging (DWI), on day 60 with T2-weighted imaging (T2WI), and recanalization was assessed based on day 1 MR angiography (MRA). RESULTS: Lesion volume increased between day 0 and day 1, and decreased between day 1 and day 60. It was lower in the group of patients with recanalization on day 1 MRA. CONCLUSION: Volume analysis emphasizes the effectiveness of recanalization as a predictive factor for better outcome, based on final infarct size. The decrease in lesion volumes between day 1 and day 60 suggests that other factors leads to overestimation of day 1 abnormal diffusion volume. This could explain the delayed partial reversibility of the DWI abnormality.

Wild animal mortality monitoring and human Ebola outbreaks, Gabon and Republic of Congo, 2001-2003.

All human Ebola virus outbreaks during 2001-2003 in the forest zone between Gabon and Republic of Congo resulted from handling infected wild animal carcasses. After the first outbreak, we created an Animal Mortality Monitoring Network in collaboration with the Gabonese and Congolese Ministries of Forestry and Environment and wildlife organizations (Wildlife Conservation Society and Programme de Conservation et Utilisation Rationnelle des Ecosystemes Forestiers en Afrique Centrale) to predict and possibly prevent human Ebola outbreaks. Since August 2001, 98 wild animal carcasses have been recovered by the network, including 65 great apes. Analysis of 21 carcasses found that 10 gorillas, 3 chimpanzees, and 1 duiker tested positive for Ebola virus. Wild animal outbreaks began before each of the 5 human Ebola outbreaks. Twice we alerted the health authorities to an imminent risk for human outbreaks, weeks before they occurred.

Cranial sutures and craniometric points detected on MRI.

The main goal of the study was to determine on MRI the cranial sutures, the craniometric points and craniometric measurements, and to correlate these results with classical anthropometric measurements. For this purpose, we reviewed 150 cerebral MRI examinations considered as normal (Caucasian population aged 20-49 years). For each examination we individualized 11 craniometric landmarks (Glabella, Bregma, Lambda, Opisthocranion, Opisthion, Basion, Inion, Porion, Infra-orbital, Eurion) and three measurements. Measurements were also calculated independently on 498 dry crania (Microscribe 3-DX digitizer). To validate the MRI procedure, we measured four dry crania by MRI and with compass or digital caliper gauges. Cranial sutures always appeared without signal (black), whatever the MRI sequence used, and they are better visualized with a 5 mm slice thickness (compact bone overlapping). Slice dynamic analysis and multiplanar reformatting allowed the detection of all craniometric points, some of these being more difficult to detect than others (Porion, Infra-orbital). The measurements determined by these points were as follows: Vertex-Basion height=135.66+/-6.56 mm; Eurion-Eurion width=141.17+/-5.19 mm; Glabella-Opisthocranion length=181.94+/-6.40 mm. On the midline T1-weighted sagittal image, all median craniometric landmarks can be individualized and the Glabella-Opisthocranion length, Vertex-Basion height and parenchyma indices can be calculated. Craniometric points and measurements between these points can be estimated with a standard cerebral MRI examination, with results that are similar to anthropometric data.

Influence of pretreatment MRI parameters on clinical outcome, recanalization and infarct size in 49 stroke patients treated by intravenous tissue plasminogen activator.

We hypothesized that pretreatment magnetic resonance imaging (MRI) parameters might predict clinical outcome, recanalization and final infarct size in acute ischemic stroke patients treated by intravenous recombinant tissue plasminogen activator (rt-PA). MRI was performed prior to thrombolysis and at day 1 with the following sequences: magnetic resonance angiography (MRA), T2*-gradient echo (GE) imaging, diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI). Final infarct size was assessed at day 60 by T2-weighted imaging (T2-WI). The National Institutes of Health Stroke Scale (NIHSS) score was assessed prior to rt-PA therapy and the modified Rankin Scale (m-RS) score was assessed at day 60. A poor outcome was defined as a day 60 m-RS score >2. Univariate and multivariate logistic regression analyses were used to identify the predictors of clinical outcome, recanalization and infarct size. Forty-nine patients fulfilled the inclusion criteria. Baseline NIHSS score was the best independent indicator of clinical outcome (p=0.002). A worse clinical outcome was observed in patients with tandem internal carotid artery (ICA)+middle cerebral artery (MCA) occlusion versus other sites of arterial occlusion (p=0.009), and in patients with larger pretreatment PWI (p=0.001) and DWI (p=0.01) lesion volumes. Two factors predict a low rate of recanalization: a proximal site of arterial occlusion (p=0.02) and a delayed time to peak (TTP) on pretreatment PWI (p=0.05). The final infarct size was correlated with pretreatment DWI lesion volume (p=0.025). Recanalization was associated with a lower final infarct size (p=0.003). In conclusion, a severe baseline NIHSS score, a critical level of pretreatment DWI/PWI parameters and a proximal site of occlusion are predictive of a worse outcome after IV rt-PA for acute ischemic stroke.