Showing papers by "Agilent Technologies published in 2014"

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study

Abstract: MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer

Abstract: Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohyd...

A Permanent Mesoporous Organic Cage with an Exceptionally High Surface Area

Abstract: Recently, porous organic cage crystals have become a real alternative to extended framework materials with high specific surface areas in the desolvated state. Although major progress in this area has been made, the resulting porous compounds are restricted to the microporous regime, owing to the relatively small molecular sizes of the cages, or the collapse of larger structures upon desolvation. Herein, we present the synthesis of a shape-persistent cage compound by the reversible formation of 24 boronic ester units of 12 triptycene tetraol molecules and 8 triboronic acid molecules. The cage compound bears a cavity of a minimum inner diameter of 2.6 nm and a maximum inner diameter of 3.1 nm, as determined by single-crystal X-ray analysis. The porous molecular crystals could be activated for gas sorption by removing enclathrated solvent molecules, resulting in a mesoporous material with a very high specific surface area of 3758 m2 g−1 and a pore diameter of 2.3 nm, as measured by nitrogen gas sorption.

The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology

Abstract: The synthetic biology research community describes a standard language for exchanging designs of biological 'parts'. The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a community-driven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.

Evidence for the biogenesis of more than 1,000 novel human microRNAs

Abstract: Background: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. Results: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. Conclusions: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.

Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome

Abstract: Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients' phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics.

Toward Structurally Defined Carbon Dots as Ultracompact Fluorescent Probes

Abstract: There has been much discussion on the need to develop fluorescent quantum dots (QDs) as ultracompact probes, with overall size profiles comparable to those of the genetically encoded fluorescent tags. In the use of conventional semiconductor QDs for such a purpose, the beautifully displayed dependence of fluorescence color on the particle diameter becomes a limitation. More recently, carbon dots have emerged as a new platform of QD-like fluorescent nanomaterials. The optical absorption and fluorescence emissions in carbon dots are not bandgap in origin, different from those in conventional semiconductor QDs. The absence of any theoretically defined fluorescence color - dot size relationships in carbon dots may actually be exploited as a unique advantage in the size reduction toward having carbon dots serve as ultracompact QD-like fluorescence probes. Here we report on carbon dots of less than 5 nm in the overall dot diameter with the use of 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) molecules for the carbo...

A Shape-Persistent Quadruply Interlocked Giant Cage Catenane with Two Distinct Pores in the Solid State

Abstract: Discrete interlocked three-dimensional structures are synthetic targets that are sometimes difficult to obtain with "classical" synthetic approaches, and dynamic covalent chemistry has been shown to be a useful method to form such interlocked structures as thermodynamically stable prod- ucts. Although interlocked and defined hollow structures are found in nature, for example, in some viruses, similar structures have rarely been synthesized on a molecular level. Shape- persistent interlocked organic cage compounds with dimen- sions in the nanometer regime are now accessible in high yields during crystallization through the formation of 96 covalent bonds. The interlocked molecules form an unprecedented porous material with intrinsic and extrinsic pores both in the micropore and mesopore regime. The synthesis of mechanically interlocked molecules has evolved from being a scientific curiosity to a renowned subtopic in supramolecular chemistry. (1) Interlocked mole- cules, such as catenanes, rotaxanes, or trefoil knots, are "molecules" by definition because at least one covalent bond has to be broken to decompose two or more molecular parts, which are mechanically interlocked and interact only through noncovalent interactions. (1) After catenanes had been pre- dicted theoretically, their first statistical syntheses suffered from low yields. (2) Later on, Sauvage and co-workers intro- duced a remarkable synthetic improvement by using metal templates to significantly increase the yields. (3) The groups of Hunter, Vcgtle, Leigh as well as Stoddart developed methods that are based on weak supramolecular interactions, for example, hydrogen bonding or p-p stacking, to construct catenanes. (4, 5) Even multiple interlocked rings, including

Utilizing Ethnic-Specific Differences in Minor Allele Frequency to Recategorize Reported Pathogenic Deafness Variants

Abstract: Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) >0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Association of N-Glycosylation with Breast Carcinoma and Systemic Features Using High-Resolution Quantitative UPLC

Abstract: An improved separation of the human serum N-glycome using hydrophilic interaction chromatography technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique...

Calibrated complex impedance and permittivity measurements with scanning microwave microscopy.

Abstract: We present a procedure for calibrated complex impedance measurements and dielectric quantification with scanning microwave microscopy. The calibration procedure works in situ directly on the substrate with the specimen of interest and does not require any specific calibration sample. In the workflow tip‐sample approach curves are used to extract calibrated complex impedance values and to convert measured S11 reflection signals into sample capacitance and resistance images. The dielectric constant of thin dielectric SiO2 films were determined from the capacitance images and approach curves using appropriate electrical tip‐sample models and the"r value extracted at fD 19:81 GHz is in good agreement with the nominal value of"r 4. The capacitive and resistive material properties of a doped Si semiconductor sample were studied at different doping densities and tip‐sample bias voltages. Following a simple serial model the capacitance‐voltage spectroscopy curves are clearly related to the semiconductor depletion zone while the resistivity is rising with falling dopant density from 20 to 20 k. The proposed procedure of calibrated complex impedance measurements is simple and fast and the accuracy of the results is not affected by varying stray capacitances. It works for nanoscale samples on either fully dielectric or highly conductive substrates at frequencies between 1 and 20 GHz.

Deregulation of cancer-related miRNAs is a common event in both benign and malignant human breast tumors

Abstract: MicroRNAs (miRNAs) are endogenous non-coding RNAs, which play an essential role in the regulation of gene expression during carcinogenesis. The role of miRNAs in breast cancer has been thoroughly investigated, and although many miRNAs are identified as cancer related, little is known about their involvement in benign tumors. In this study, we investigated miRNA expression profiles in the two most common types of human benign tumors (fibroadenoma/fibroadenomatosis) and in malignant breast tumors and explored their role as oncomirs and tumor suppressor miRNAs. Here, we identified 33 miRNAs with similar deregulated expression in both benign and malignant tumors compared with the expression levels of those in normal tissue, including breast cancer-related miRNAs such as let-7, miR-21 and miR-155. Additionally, messenger RNA (mRNA) expression profiles were obtained for some of the same samples. Using integrated mRNA/miRNA expression analysis, we observed that overexpression of certain miRNAs co-occurred with a significant downregulation of their candidate target mRNAs in both benign and malignant tumors. In support of these findings, in vitro functional screening of the downregulated miRNAs in non-malignant and breast cancer cell lines identified several possible tumor suppressor miRNAs, including miR-193b, miR-193a-3p, miR-126, miR-134, miR-132, miR-486-5p, miR-886-3p, miR-195 and miR-497, showing reduced growth when re-expressed in cancer cells. The finding of deregulated expression of oncomirs and tumor suppressor miRNAs in benign breast tumors is intriguing, indicating that they may play a role in proliferation. A role of cancer-related miRNAs in the early phases of carcinogenesis and malignant transformation can, therefore, not be ruled out.

A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation

Abstract: Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood. Here using a high-coverage genome-wide shRNA library, we identify the uncharacterized protein TMEM129 and the ubiquitin-conjugating E2 enzyme UBE2J2 to be essential for US11-mediated HLA class I downregulation. TMEM129 is an unconventional C4C4-type RING finger E3 ubiquitin ligase that resides within a complex containing various other ERAD components, including Derlin-1, Derlin-2, VIMP and p97, indicating that TMEM129 is an integral part of the ER-resident dislocation complex mediating US11-induced HLA class I degradation.

An iron–dopamine index predicts risk of parkinsonian neurodegeneration in the substantia nigra pars compacta

Abstract: The co-localization of iron and dopamine raises the risk of a potentially toxic reaction. Disturbance of the balance in this unique chemical environment makes neurons in the substantia nigra pars compacta (SNc) particularly vulnerable to parkinsonian neurodegeneration in the aging brain. In Parkinson's disease, these neurons degenerate coincident with an elevation in brain iron levels, yet relatively little is known about specific regional iron distribution with respect to dopamine. To directly appraise the iron–dopamine redox couple, we applied immuno-assisted laser ablation-inductively coupled plasma-mass spectrometry imaging to co-localize iron with the dopamine-producing enzyme tyrosine hydroxylase at the coronal level of the substantia nigra. We found that in the healthy brain the SNc does not contain the greatest concentration of iron within the midbrain, while the dopamine-rich environment in this region reflects an increased oxidative load. The product of iron and dopamine was significantly greater in the SNc than the adjacent ventral tegmental area, which is less susceptible to neuron loss in Parkinson's disease. Accordingly, this ‘risk factor’ was elevated further following 6-hydroxydopamine (6-OHDA) lesioning. Considering mounting evidence that brain iron increases with age, this measurable iron–dopamine index provides direct experimental evidence of a relationship between these two redox-active chemicals in degenerating dopaminergic neurons.

Graphene/fly ash geopolymeric composites as self-sensing structural materials

Abstract: The reduction of graphene oxide during the processing of fly ash-based geopolymers offers a completely new way of developing low-cost multifunctional materials with significantly improved mechanical and electrical properties for civil engineering applications such as bridges, buildings and roads. In this paper, we present for the first time the self-sensing capabilities of fly ash-based geopolymeric composites containing in situ reduced graphene oxide (rGO). Geopolymeric composites with rGO concentrations of 0.0, 0.1 and 0.35% by weight were prepared and their morphology and conductivity were determined. The piezoresistive effect of the rGO-geopolymeric composites was also determined under tension and compression. The Fourier transform infrared spectroscopy (FTIR) results indicate that the rGO sheets can easily be reduced during synthesis of geopolymers due to the effect of the alkaline solution on the functional groups of GO. The scanning electron microscope (SEM) images showed that the majority of pores and voids within the geopolymers were significantly reduced due to the addition of rGO. The rGO increased the electrical conductivity of the fly ash-based rGO-geopolymeric composites from 0.77 S m−1 at 0.0 wt% to 2.38 S m−1 at 0.35 wt%. The rGO also increased the gauge factor by as much as 112% and 103% for samples subjected to tension and compression, respectively.

Vibrational spectroscopic methods for cytology and cellular research

Abstract: The use of vibrational spectroscopy, FTIR and Raman, for cytology and cellular research has the potential to revolutionise the approach to cellular analysis. Vibrational spectroscopy is non-destructive, simple to operate and provides direct information. Importantly it does not require expensive exogenous labels that may affect the chemistry of the cell under analysis. In addition, the advent of spectroscopic microscopes provides the ability to image cells and acquire spectra with a subcellular resolution. This introductory review focuses on recent developments within this fast paced field and highlights potential for the future use of FTIR and Raman spectroscopy. We particularly focus on the development of live cell research and the new technologies and methodologies that have enabled this.

Optimisation of laser parameters for the analysis of sulphur isotopes in sulphide minerals by laser ablation ICP-MS

Abstract: The effects of laser type (Nd:YAG and excimer lasers) and their analytical parameters on 34S/32S isotopic fractionation during LA-ICP-MS analysis were investigated. Laser fluence has a larger fractionation effect when ablating pyrite with the New Wave Nd:YAG 193 nm laser, compared to the Resonetics 193 nm excimer laser which did not produce significant fractionation over the same range of fluence (1.3–3.7 J cm−2). Matrix effects occurred between pyrite and bornite on both laser systems, especially at low fluence. However, matrix effects can be reduced with increasing fluence lessening the need for matrix matched reference materials. The effects of interface tubing configuration were also investigated and the addition of a ‘squid’ mixing device, a coil of small diameter Tygon tubing and a small volume glass bulb, was found to improve signal precision and reproducibility and decrease the washout time of the S signal between analyses. The degassing of air from the inner surfaces of the interface tubing can produce significant isotopic drift (8‰ h−1), hence flushing the tubing prior to analyses is crucial for reproducible analyses. The isotopic composition and homogeneity of a range of sulphide minerals were characterised for use as potential reference materials. We present preliminary data for a large, isotopically homogeneous pyrite crystal (PPP-1) which could be considered as a new isotopic reference material (δ34SV-CDT = 5.3 ± 0.2‰).

Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells

Abstract: A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133+ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with 64Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2–3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133+ cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133+ tumor stem cells.

Companion Diagnostics for Targeted Cancer Drugs - Clinical and Regulatory Aspects

Abstract: Companion diagnostics (CDx) holds the promise of improving the predictability of the oncology drug development process and become an important tool for the oncologist in relation to the choice of treatment for the individual patient. A number of drug-diagnostic co-development programs have already been completed successfully, and in the clinic, the use of several targeted cancer drugs is now guided by a CDx. This central role of the CDx assays has attracted the attention of the regulators, and especially the US FDA has been at the forefront in relation to developing regulatory strategies for CDx and the drug-diagnostic co-development project. For an increasing number of cancer patients the treatment selection will depend on the result generated by a CDx assay, and consequently this type of assay has become critical for the care and safety of the patients. In order to secure that the CDx assays have a high degree of analytical and clinical validity they must undergo an extensive non-clinical and clinical testing before release for routine patient management. This review will give a brief introduction to some of the scientific and medical challenges related to the CDx development with specific emphasis on the regulatory requirements in different regions of the world.

Nucleic acid enrichment using cas9

Abstract: A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

Urban WiFi characterization via mobile crowdsensing

Abstract: We present a mobile crowdsensing approach for urban WiFi characterization that leverages commodity smartphones and the natural mobility of people. Specifically, we report measurement results obtained for Edinburgh, a representative European city, on detecting the presence of deployedWiFi APs via the mobile crowdsensing approach. They show that few channels in 2.4GHz are heavily used; in contrast, there is hardly any activity in the 5GHz band even though relatively it has a greater number of available channels. Spatial analysis of spectrum usage reveals that mutual interference among nearby APs operating in the same channel can be a serious problem with around 10 APs contending with each other in many locations. We find that the characteristics of WiFi deployments at city-scale are similar to that of WiFi deployments in public spaces of different indoor environments. We validate our approach in comparison with wardriving, and also show that our findings generally match with previous studies based on other measurement approaches. As an application of the mobile crowdsensing based urban WiFi monitoring, we outline a cloud based WiFi router configuration service for better interference management with global awareness in urban areas.

Lipidomic “Deep Profiling” : An Enhanced Workflow to Reveal New Molecular Species of Signaling Lipids

Abstract: Current mass spectrometry-based lipidomics aims to comprehensively cover wide ranges of lipid classes. We introduce a strategy to capture phospho-monoester lipids and improve the detection of long-chain base phosphates (LCB-Ps, e.g., sphingosine-1-phosphate). Ten novel LCB-Ps (d18:2, t20:1, odd carbon forms) were discovered and characterized in tissues from human and mouse, as well in D. melanogaster and S. cerevisiae. These findings have immediate relevance for our understanding of sphingosine-1-phosphate biosynthesis, signaling, and degradation.

Methods of in vivo engineering of large sequences using multiple crispr/cas selections of recombineering events

Abstract: The present invention provides a method for making a large nucleic acid having a defined sequence in vivo. The method combines recombineering techniques with a CRISPR/Cas system to permit multiple insertions of defined sequences into a target nucleic acid at one time, double stranded cleavage of target nucleic acids in which the defined sequences were not successfully inserted, and selection of successful recombinant cells. The method further includes repeating the process one or more times, using a successful recombinant from one round as the host cell for the next round.

Dynamic FET model - DynaFET - for GaN transistors from NVNA active source injection measurements

Abstract: A complete nonlinear characterization and modeling flow for modern GaN transistors is presented. Features include a new active-source injection based waveform measurement HW/SW system built around an NVNA, an extended artificial neural network (ANN) training infrastructure for coupled electro-thermal and trap-dependent model constitutive relations, and the native implementation in a commercial simulator. The model is validated by detailed comparisons to measured data for an advanced mm-wave 150 nm 6×60μm GaN HFET manufactured by Raytheon Integrated Defense Systems. Excellent results are achieved for DC, S-parameters, harmonic and intermodulation distortion, and load-pull figures of merit, over a very wide range of bias conditions, complex loads, powers, and frequencies.

Pathways of Toxicity.

Abstract: Despite wide-spread consensus on the need to transform toxicology and risk assessment in order to keep pace with technological and computational changes that have revolutionized the life sciences, there remains much work to be done to achieve the vision of toxicology based on a mechanistic foundation. A workshop was organized to explore one key aspect of this transformation – the development of Pathways of Toxicity (PoT) as a key tool for hazard identification based on systems biology. Several issues were discussed in depth in the workshop: The first was the challenge of formally defining the concept of a PoT as distinct from, but complementary to, other toxicological pathway concepts such as mode of action (MoA). The workshop came up with a preliminary definition of PoT as “A molecular definition of cellular processes shown to mediate adverse outcomes of toxicants”. It is further recognized that normal physiological pathways exist that maintain homeostasis and these, sufficiently perturbed, can become PoT. Second, the workshop sought to define the adequate public and commercial resources for PoT information, including data, visualization, analyses, tools, and use-cases, as well as the kinds of efforts that will be necessary to enable the creation of such a resource. Third, the workshop explored ways in which systems biology approaches could inform pathway annotation, and which resources are needed and available that can provide relevant PoT information to the diverse user communities.