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Methods for constructing and maintaining knowledge representation systems are disclosed herein. The knowledge representation system is initially organized and populated using knowledge engineers. After the initial organization, scientific domain experts digest and structure source texts for direct entry into the knowledge representation system using templates created by the knowledge engineers. These templates constrain both the form and content of the digested information, allowing it to be entered directly into the knowledge representation system. Although knowledge engineers are available to evaluate and dispose of those instances when the digested information cannot be entered in the form required by the templates, their role is much reduced from conventional knowledge representation system construction methods. The methods disclosed herein permit the construction and maintenance of a much larger knowledge representation system than could be constructed and maintained using known methods.

Ingenuity Systems | Date: 2013-06-10

Methods for identifying disease-related pathways that can used to identify drug discovery targets, to identify new uses for known drugs, to identify markers for drug response, and related purposes.

Ingenuity Systems | Date: 2014-07-28

Methods for identifying disease-related pathways that can be used to identify drug discovery targets, to identify new uses for known drugs, to identify markers for drug response, and related purposes.

All studies with mice were reviewed and approved by the Institutional Animal Use and Care Committee of Beth Israel Deaconess Medical Center (BIDMC). Pgc1α−/− (stock no. 008597), Pax8-rtTA (no. 007176) and TRE-PGC1α (no. 012387) mice were all obtained from Jackson Laboratories and bred at BIDMC. The parental strains were generated on a mixed C57 background with further backcrossing into C57BL/6J as described by the manufacturer, except for the TRE-PGC1α mouse, which was generated on and is maintained on FVB. Primers for genotyping have been described elsewhere31,32. All experiments were performed with genetically appropriate littermate controls. Ischaemia-reperfusion injury (IRI) was performed on 8–12-week-old males through two small paramedial dorsal incisions by applying a microvascular clamp to each renal pedicle for 20 min. Mice were anaesthetized with isoflurane for the duration of surgery and warmed to 37 °C using a servo-controlled heating pad. Incisions were closed in two layers and mice were revived with 1 ml warm saline injected intraperitoneally. All chemicals were purchased from Sigma-Aldrich unless otherwise noted. NAM was given by intraperitoneal injection of 400 mg−1 kg−1 day−1 for 4 days in saline, with the final dose an hour before IRI surgery. In rescue experiments, the same dose was administered once 18 h after reperfusion. Indomethacin was given by intraperitoneal injection of 10 mg kg−1 in 0.1 M sodium carbonate/saline an hour before IRI. The HCAR2 inhibitor, mepenzolate bromide, was given by intraperitoneal injection of 10 mg kg−1 in saline an hour before IRI33, 34, 35. LPS (E.coli serotype O111:B4) was given by intraperitoneal injection of 25 mg kg−1 in saline. Cisplatin was given by intraperitoneal injection of 25 mg kg−1 as previously described36. Unless otherwise stated, blood and kidneys were collected 24 h after the AKI model. The experiments were not randomized. All measurements were performed in a blinded fashion by an independent facility. Creatinine was analysed by LC/MS-MS at the University of Alabama Birmingham O’Brien Core Center for Acute Kidney Injury Research (NIH P30-DK079337). This method adds the accuracy of MS to the LC method of creatinine measurement endorsed by a renal investigative consortium (diacomp.org). The coefficient of variation was 6% indicating high assay precision. For metabolomics measurements, snap frozen kidneys were cut to equal weights (20 mg per specimen) and mechanically homogenized into four volumes of ice-cold water. Metabolites were assayed as previously described37. In brief, amino acids, amines, acylcarnitines, nucleotides, and other cationic polar metabolites were measured in 10 μl of kidney homogenate using hydrophilic interaction liquid chromatography coupled with non-targeted, positive ion mode MS analysis on an Exactive Plus Orbitrap MS (Thermo Scientific). Polar and non-polar lipids were measured in 10 μl of kidney homogenate using C8 chromatography and non-targeted, positive ion mode MS analysis on a Q Exactive MS (Thermo Scientific). Identification of known metabolites was achieved by matching retention times and mass-to-charge ratio (m/z) to synthetic mixtures of reference compounds and characterized pooled plasma reference samples. Results were analysed in MetaboAnalyst (http://www.metaboanalyst.ca). LC–MS assays were developed for multiplex quantification of NAM, NAD, and β-OHB from cellular experiments. NAD measurements reflect total NAD+ plus NADH. In brief, conditioned medium was extracted with methanol (80% methanol final concentration) spiked with isotopic standards for NAM and β-OHB (CDN Isotopes, Inc.). Precipitated proteins were removed by centrifugation, and supernatants were analysed directly. For analysis of cell lysates, cells were washed with ice-cold PBS, scraped and lysed on dry ice into methanol containing isotopic standards. After extraction, cell and media supernatants were analysed by LC–MS/MS using reverse-phase chromatography (NAM and NAD/NADH) or hydrophilic interaction chromatography (β-OHB) coupled to tandem mass spectrometry using an API 5000 triple quadruple mass spectrometer. Analytes were quantified by multiple reaction monitoring using the following m/z transitions: β-OHB 103.1 > 59, β-OHB IS 105.1 > 60, NAM 123.3 > 80.2, NAM IS 127.3 > 84.2, NAD/NADH 664.2 > 542.0. Eluting peaks were quantified by area under the curve (AUC). Raw AUC values were divided by the mean value of the control group for each experiment, thus the results are presented as relative concentrations to the control group. All assays were performed in triplicate and replicate measurements demonstrated a CV < 5%. Poly(A)-enriched RNA was isolated from whole kidneys and checked for quality by denaturing agarose gel as well as an Agilent Bioanalyzer. Sequencing libraries were generated from the double-stranded cDNA using the Illumina TruSeq kit according to the manufacturer’s protocol. Library quality control was checked using the Agilent DNA High Sensitivity Chip and qRT–PCR. High quality libraries were sequenced on an Illumina HiSeq 2000. To achieve comprehensive coverage for each sample, we generated ~25–30 million single-end reads. Raw results were passed through quality controls steps and alig ned to the mouse genome. Gene expression measurement was performed from aligned reads by counting the unique reads. The read count based gene expression data was normalized on the basis of library complexity and gene variation. The normalized count data was compared among groups using a negative binomial model to identify differentially expressed genes. The differentially expressed genes were identified on the basis of raw P value and fold change. Genes were considered significantly differentially expressed if the multiple test corrected P value was <0.05 and absolute fold change >2. Ingenuity Pathway Analysis (IPA 8.0, Qiagen) was used to identify the functions that are significantly affected by significantly differentially expressed genes from different comparisons. The knowledge base of this software consists of functions, pathways, and network models derived by systematically exploring the peer reviewed scientific literature. A detailed description of IPA analysis is available at the Ingenuity Systems’ website (http://www.ingenuity.com). A P value is calculated for each function according to the fit of the user’s data to the IPA database using one-tailed Fisher exact test. The functions with multiple-test-corrected P values <0.01 were considered significantly affected. Kidney lysate preparation, gel electrophoresis, transfer, immunoblotting, detection, and image acquisition were performed as previously described31. Antibodies against PGC1α (Cayman Chemical), cytochrome c oxidase subunit IV (Cell Signaling Technology), and Transcription Factor A Mitochondrial, TFAM (Abcam) were used as previously described31, 38. Total RNA extraction and cDNA synthesis were performed as previously described31. PCR reactions were performed in duplicate using the ABI 7500 Fast Real-Time PCR and TaqMan gene expression assays (Applied Biosystems). The following TaqMan gene probes were used: Ppargc1a, Ndufs1, Cycs, Atp5o, Nrf1, Tfam, Vegfa, Nos1, Nos3 and Hcar2. Of the four known Ppargc1a transcripts (1–4), Ppargc1a1 (Taqman Mm00447183_m1) was studied in all gene expression analyses39. Mouse Ido2, Afmid, Kynu, Kmo, Haao, Qprt, Naprt and Nmnat1 for SYBR Green PCR have been described elsewhere40, 41. Mouse Nampt SYBR primers were designed using PrimerQuest Tool (Integrated DNA Technologies). Relative expression levels were determined using the comparative threshold method. Total DNA was extracted from mouse kidneys using the DNeasy Blood and Tissue Kit (Qiagen) with on-column RNase digestion per manufacturer’s instructions. Gene expression of mitochondrial-encoded NADH dehydrogenase 1 (mt-Nd1) relative to nuclear 18S rRNA was used to determine mitochondrial DNA copy number as previously described42. Formalin-fixed, paraffin-embedded blocks were sectioned and stained with H & E, PAS, and Masson trichrome. Ten random high-power fields in the cortex and ten random high-power fields in the outer stripe of the outer medulla were viewed and graded for tubular necrosis—defined as the loss of the proximal tubular brush border, blebbing of apical membranes, tubular necrosis/apoptosis and epithelial cell detachment from the basement membrane or intraluminal aggregation of necrotic debris. Each high-power field was separately scored on a scale (0, no necrosis; 1, rare single necrotic cells; 2, frequent single necrotic cells; 3, groups of necrotic cells; and 4, confluent tubular necrosis) and the average score was compiled for each specimen and then used for between-group comparisons. All scoring was performed by a single operator blinded to genotype and experimental model (IES). Enzyme histochemistry to detect cytochrome c oxidase (COX) activity was performed on 6-μm snap-frozen sagittal sections as previously described31. Functional electron microscopy used in the cisplatin kidney injury model was described earlier36. The complete method is previously described31. In brief, kidneys were fixed with 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and cut into 1-μm sections in both sagittal and transverse planes for image analysis. After drying the sections, slides were stained at 65 °C for 20 min in 0.1% Toluidine blue in 1% sodium borate, cooled to room temperature, washed in distilled water, cleaned in xylene, and mounted in Permount sections for light microscopy. Subsequent ultrathin sections (0.5 μm) were examined by transmission electron microscopy (JEOL 1011, JEOL Corp.) with Orca-HR Digital Camera (Hamamatsu Corp.), and Advanced Microscopy Technique Corporation image capture system. Oil-Red-O solution was prepared by dissolving 0.5 g Oil-Red-O (Poly Scientific) in 100 ml isopropanol. Frozen sections were cut to 5 μm and natively stained in Oil-Red-O solution for 20 min at room temperature, then rinsed in running tap water for 2 min. Haematoxylin counter-staining was performed without differentiation in HCl–ethanol and sections were rinsed with water, then mounted with VectaMount AQ Aqueous Mounting medium (Vector Labs). All studies were approved by the Institutional Review Board at BIDMC. Control specimens came from normal tissue sections of nephrectomies. CKD diagnoses included focal segmental glomerulosclerosis, chronic allograft nephropathy, chronic interstitial nephritis, and chronic IgA nephropathy. AKI diagnoses included acute ischaemic injury, post-transplant delayed graft function attributable to ischaemia-reperfusion injury, and acute interstitial nephritis. PGC1α antibody (Abcam ab54481) was used at a dilution of 1:100 and developed with horseradish peroxidase (ImmPRESS polymer staining kit, Vector Labs). The peptide immunogen SKYDSLDFDSLLKEAQRSLRR (synthesized by the Biopolymers Lab, Koch Institute at MIT) was pre-incubated in 100-fold excess of the PGC1α antibody to confirm antibody specificity in human IHC studies. Ten randomly selected high-powered fields were viewed per specimen, with each field scored on a 4-point scale (1, weakest; 4, strongest) based on the intensity of staining, specifically in non-necrotic areas and unscarred areas and avoiding obvious collecting ducts. The average score of each specimen was then used for between-group comparisons. All scoring was performed by a single operator blinded to the underlying diagnosis (IES). The full method is previously described31. In brief, mice were lightly anaesthetized, secured to a heat-controlled stage, and continuously monitored for respiration, ECG, and core temperature. A high-frequency, high-resolution digital imaging platform with linear array technology and equipped with a high-frequency linear array probe MS550D (22–55 MHz) was used throughout the study (Vevo 2100 Visual Sonics). The flow volume was modelled as a circular cylinder of length equal to the average velocity time integral and diameter measured empirically (n = 3 cardiac cycles), then multiplied by the heart rate (b.p.m.), then converted from mm3 min−1 to ml min−1. All measurements and analyses were performed by a single blinded operator (EVK). Mouse inner medullary collecting duct (IMCD3) cells were obtained from ATCC. Please refer to their website for validation and mycoplasma testing (http://www.atcc.org/Products/All/CRL-2123.aspx). Cells were transfected with siRNA targeting mouse PGC1α, HCAR2 or a negative control siRNA (Qiagen) for 24 h. Niacin, mepenzolate bromide, β-hydroxybutyrate, the NAMPT inhibitor FK866 (ref. 43), and NAM were diluted to the indicated concentrations in serum-free cell culture medium. Prostaglandin E2 (PGE ) was measured in the conditioned media 24–72 h after treatment. Cystatin C in mouse serum (1:200 dilution) was measured by ELISA (R&D Systems). The full method is described elsewhere44. In brief, male C57BL/6J mice (Jackson Laboratories) were given a single bolus injection of 5%-FITC-inulin (3.74 μl per g body weight). Clearance kinetics of FITC-inulin post-injection was measured by serial blood collection at specified time points from 3 through 70 min post-injection. Blood samples were centrifuged and resulting plasma was buffered to pH 7.4 with 500 mM HEPES. Fluorescence in the buffered plasma samples was determined with 485 nm excitation, 538 nm emission. Glomerular filtration rate (GFR) was calculated from the two-phase exponential decay model outlined previously. PGE was measured in mouse kidney tissue by ELISA (Cayman Chemical). β-OHB (Cayman Chemical) and total NAD (BioVision) were measured in mouse kidney tissue by colorimetric assays. These assays were performed on kidneys used for metabolomics and lipidomics in order to compare coordinated changes in metabolism and downstream signalling. NAD measurements reflect total NAD+ plus NADH. Comparisons between continuous characteristics of subject groups were analysed with Mann–Whitney U-tests or Student’s t-test. Survival was analysed by log-rank test. For comparisons among more than two groups, ANOVA with Bonferroni’s correction was used where indicated. Associations between micro-ultrasound measurements and other functional parameters were analysed with Spearman’s rank correlation coefficients. Sample size determination was guided by power calculations and prior experience. The following sample calculation was used to guide creatinine studies in mice: serum creatinine of 1.6 (±0.3 s.d.) mg dl−1 versus 1.0 (±0.2 s.d.) requires n = 5 mice per condition to achieve an α-error <5% and power 96%. Mice were randomized to experimental intervention versus control. Two-tailed P values < 0.05 were considered significant. Results are presented as mean ± s.e.m. and were prepared in GraphPad Prism.

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