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SELDI ProteinChip(R) Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications.

Predictive medicine, utilizing the ProteinChip(R) Array technology, will develop through the implementation of novel biomarkers and multimarker patterns for detecting disease, determining patient prognosis, monitoring drug effects such as efficacy or toxicity, and for defining treatment options. These biomarkers may also serve as novel protein drug candidates or protein drug targets. In addition, the technology can be used for discovering small molecule drugs or for defining their mode of action utilizing protein-based assays. In this review, we describe the following applications of the ProteinChip Array technology: (1) discovery and identification of novel inhibitors of HIV-1 replication, (2) serum and tissue proteome analysis for the discovery and development of novel multimarker clinical assays for prostate, breast, ovarian, and other cancers, and (3) biomarker and drug discovery applications for neurological disorders.

Journal Article↗

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice.

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.

Animals↗

The rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides.

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.

Animals↗

Nova regulates brain-specific splicing to shape the synapse.

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2-/- mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.

Alternative Splicing↗

Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE).

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC). RESULTS: We report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation. CONCLUSION: This first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Cervix Uteri↗

Automated acquisition of stained tissue microarrays for high-throughput evaluation of molecular targets.

At present, limiting factors in the use of tissue microarrays (TMAs) for high-throughput analysis relate to the visual evaluation of the staining patterns of each of the individual cores in the array and to the subsequent input of the results into a database. Such a database is essential to correlate the data with tumor type and outcome, and to evaluate the performance against other markers achieved in separate experiments. So far, these steps are mostly performed by hand, and consequently are time-consuming and potentially prone to bias and errors, respectively. This paper describes the use of a high-resolution flat-bed scanner for digitization of TMAs with a resolution of about 5 x 5 micro m(2). The arrays are acquired, the positions of the tissue cores are automatically determined, and measurement data including the images of the individual cores are archived. The program provides digital zooming of arrays for interactive verification of the results and rapid linkage of individual core images to data sets of other markers derived from the same array. Performance of the system was compared to manual classification for a representative set of arrays containing colorectal tumors stained with different markers.

Automation↗

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.

Adolescent↗

Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus.

Postnatal development of the mouse uterus involves differentiation and development of the endometrial glands as well as the myometrium. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in extracellular matrix breakdown and morphogenesis of many epitheliomesenchymal organs. As a first step to understanding their roles in postnatal mouse uterine development, MMPs and TIMPs found to be expressed in the neonatal mouse uterus by microarray analysis were localized by in situ hybridization. The MMP-2 mRNA was detected only in the uterine stroma, whereas the MMP-10 mRNA was present only in the uterine epithelium from Postnatal Day (PND) 3 to PND 9. All other MMPs (MMP-11, MMP-14, and MMP-23) as well as TIMP-1, TIMP-2, and TIMP-3 were detected in both epithelial and stromal cells of the endometrium, but not in the myometrium. Uterine extracts were then analyzed by gelatin and casein gel zymography to detect active gelatinases and stromelysins, respectively. Five major gelatinase bands of activity were detected and inhibited by the MMP inhibitors, EDTA or 1,10-phenanthroline, but not by PMSF, a serine protease inhibitor. Western blot analysis confirmed the presence of MMP-2 and MMP-9 proteins in the uterus. Immunoreactive MMP-9 protein was detected only in the endometrial stroma, whereas immunoreactive MMP-2 protein was detected in both the stroma and epithelium of the uterus. Casein zymography detected three major bands of activity ( approximately 54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.

Aging↗

Hemoglobin is expressed in alveolar epithelial type II cells.

Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin alpha- and beta-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry revealed that hemoglobin protein was specifically localized in type II cells of a lung cell mixture and rat lung tissue. The endogenous synthesis of hemoglobin in alveolar epithelial cells suggests that hemoglobin may have unidentified functions other than oxygen transport in the lung.

Animals↗

Microarray analysis of developing Arabidopsis seeds.

To provide a broad analysis of gene expression in developing Arabidopsis seeds, microarrays have been produced that display approximately 2,600 seed-expressed genes. DNA for genes spotted on the arrays were selected from >10,000 clones partially sequenced from a cDNA library of developing seeds. Based on a series of controls, sensitivity of the arrays was estimated at one to two copies of mRNA per cell and cross hybridization was estimated to occur if closely related genes have >70% to 80% sequence identity. These arrays have been hybridized in a series of experiments with probes derived from seeds, leaves, and roots of Arabidopsis. Analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approximately 25% of the 2, 600 genes were expressed at ratios > or =2-fold higher in seeds than leaves or roots and 10% at ratios > or =10. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases, and transcription factors. The Arabidopsis arrays were also found to be useful for transcriptional profiling of mRNA isolated from developing oilseed rape (Brassica napus) seeds and expression patterns correlated well between the two species.

Arabidopsis↗

Protein array technology. Potential use in medical diagnostics.

The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogs and functional catalogs. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.

Diagnosis↗

Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesity-related genes.

Susceptibility genes present in both mother and fetus most likely contribute to the risk of pre-eclampsia. Placental biopsies were therefore investigated by high-density DNA microarray analysis to determine genes differentially regulated within chorionic villous tissue in pre-eclampsia and normal pregnancy. The pooled RNAs of pre-eclamptic and normotensive subjects were hybridized to the HuGeneFL array representing sequences from approximately 5600 full-length human cDNAs. The differentially expressed genes that were detected could be categorized into nine groups: adhesion molecules, obesity-related genes, transcription factors/signalling molecules, immunological factors, neuromediators, oncogenic factors, protease inhibitors, hormones and growth factor-binding proteins. Among those, the obesity-related genes included putative candidate genes associated with the pathogenesis of pre-eclampsia. One of the most up-regulated transcripts was the obese gene (43.6-fold change), and this was reflected by elevated leptin protein levels. In the case of feto-maternal contribution of polymorphic genes to pre-eclampsia, expression analysis of placental tissue has lead to numerous target genes waiting for large scale genetic linkage analyses.

Female↗

Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.

Despite several advances in our basic understanding and in the clinical management of pancreatic cancer, virtually all patients who will be diagnosed with pancreatic cancer will die from this disease. The high mortality of pancreatic cancer is predominantly because of diagnosis at an advanced stage of disease and a lack of effective treatments. We used the Gene Logic Inc. BioExpress platform and Affymetrix GeneChip arrays to identify genes differentially expressed in pancreatic cancer. cDNA was prepared from samples of normal pancreas (n = 11), normal gastrointestinal mucosa (n = 22), resected pancreas cancer tissues (n = 14), and pancreas cancer cell lines (n = 8), and was hybridized to the complete Affymetrix Human Genome U95 GeneChip set (arrays U95 A, B, C, D, and E) for simultaneous analysis of 60,000 cDNA fragments, with 12,000 fragments covering full-length genes and 48,000 fragments covering expressed sequence tags (ESTs). Genes expressed at levels at least fivefold greater in the pancreatic cancers ascompared to normal tissues were identified. Serial analysis of gene expression (SAGE) libraries (http://www.ncbi.nlm.nih.gov/SAGE/) of two normal pancreatic ductal cell cultures (HX and H126) were used to exclude genes expressed in the normal ducts (more than five tags per library). Differential expression of selected candidate genes was validated by immunohistochemical analysis (n = 3), by in situ hybridization (n = 1), and by reverse transcriptase-polymerase chain reaction (n = 8). One hundred eighty fragments were identified as having fivefold or greater expression levels in pancreas cancer specimens as compared to normal tissue, of which 124 corresponded to known genes and 56 to ESTs. Of these 124 fragments, 10 genes were represented by two or more fragments, resulting in 107 known genes identified as differentially expressed in pancreatic cancer. An additional 10 genes were expressed in the SAGE libraries of normal pancreatic duct epithelium, and were excluded from further analysis. A literature search indicated that 28 of the remaining 97 genes have been reported in association with pancreatic cancer, validating this approach. The remaining 69 genes have not been implicated in pancreatic cancer before, and have immediate potential as novel therapeutic targets and tumor markers of pancreatic cancer.

Biomarkers, Tumor↗

Gene expression in blood changes rapidly in neutrophils and monocytes after ischemic stroke in humans: a microarray study.

Ischemic brain and peripheral white blood cells release cytokines, chemokines and other molecules that activate the peripheral white blood cells after stroke. To assess gene expression in these peripheral white blood cells, whole blood was examined using oligonucleotide microarrays in 15 patients at 2.4+/-0.5, 5 and 24 h after onset of ischemic stroke and compared with control blood samples. The 2.4-h blood samples were drawn before patients were treated either with tissue-type plasminogen activator (tPA) alone or with tPA plus Eptifibatide (the Combination approach to Lysis utilizing Eptifibatide And Recombinant tPA trial). Most genes induced in whole blood at 2 to 3 h were also induced at 5 and 24 h. Separate studies showed that the genes induced at 2 to 24 h after stroke were expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes. These genes included: matrix metalloproteinase 9; S100 calcium-binding proteins P, A12 and A9; coagulation factor V; arginase I; carbonic anhydrase IV; lymphocyte antigen 96 (cluster of differentiation (CD)96); monocarboxylic acid transporter (6); ets-2 (erythroblastosis virus E26 oncogene homolog 2); homeobox gene Hox 1.11; cytoskeleton-associated protein 4; N-formylpeptide receptor; ribonuclease-2; N-acetylneuraminate pyruvate lyase; BCL6; glycogen phosphorylase. The fold change of these genes varied from 1.6 to 6.8 and these 18 genes correctly classified 10/15 patients at 2.4 h, 13/15 patients at 5 h and 15/15 patients at 24 h after stroke. These data provide insights into the inflammatory responses after stroke in humans, and should be helpful in diagnosis, understanding etiology and pathogenesis, and guiding acute treatment and development of new treatments for stroke.

Adult↗

Microarray analysis of genes expressed in the frontal cortex of rats chronically treated with morphine and after naloxone precipitated withdrawal.

Opioid dependence may be associated with adaptive changes in gene expression in the brain. In the present study we used DNA microarrays (U34A; Affymetrix) to analyze the expression of about 8000 genes in the frontal cortex of rats chronically treated with morphine and in rats after naloxone precipitated withdrawal. Chronic treatment for 10 days with ascending doses of morphine (10-50 mg/kg twice daily) resulted in a more than twofold induction of 14 genes after the last injection of morphine. The majority of these genes code for heat shock proteins (hsp70, hsp 27, hsp 40, hsp105, GRP78, etc.). The expression of the heat shock genes in the morphine-treated animals was reversed by naloxone (10 mg/kg). The opioid antagonist, in turn, precipitated withdrawal and increased the expression of a set of genes which are predominantly transcription factors (krox20, CREM, NGFI-B, IkappaB, etc). Only a few genes remained increased after naloxone application. Such persistently changed genes code for arc, a cytoskeleton-associated protein which is induced by synaptic activity, ania-3, a splice variant of the Homer 1 protein which is critically involved in activity-dependent alterations of synaptic function and rPer2, a protein regulating circadian rhythms. For selected genes the changes in gene expression were confirmed by real time PCR and by in situ hybridization. These findings indicate that the persistent changes in long-lasting plasticity during opiate dependence do not primarily depend on the increased expression levels of genes encoding for neurotransmitter, receptor and/or ion channel proteins, but rather on altered pattern of synaptic connectivity.

Animals↗

Correlation between [5-3H]glucose and [U-14C]deoxyglucose as markers of glycolysis in reperfused myocardium.

Studies were conducted in extracorporeally perfused, intact, working pig hearts to determine whether, in heart muscle, trace-labeled deoxyglucose serves as an accurate marker of glycolytic flux in reperfusion after exposures to mild to moderate regional ischemia. In the main study, two groups of hearts were compared, as distinguished by levels of glucose in the whole-blood perfusate (euglycemic hearts [group I], blood glucose of 7.4 +/- 0.2 mumol/ml, n = 7; hyperglycemic hearts [group II], blood glucose of 12.9 +/- 0.5 mumol/ml, n = 8). Both groups were subjected to a 60% reduction in anterior descending coronary flow for 30 minutes followed by reperfusion for 40 minutes. Modest and comparable regional mechanical stunning during reflow was noted in both groups. Glucose utilization, as estimated from the release of 3H2O from the steady-state infusion of [5-3H]glucose during aerobic perfusion, was modest but during reperfusion was noted to increase significantly above aerobic values in each of the two groups, with a doubling of rates in group II hearts compared with group I hearts (p less than 0.041 or p less than 0.090). Net lactate extraction was comparable in reflow in both groups, suggesting in this specific instance a preferential enhancement of glucose oxidation in hyperglycemic group II hearts. Shifts in accumulation of tissue radioactivity of [U-14C]2-deoxyglucose in reperfused myocardium were not able to track these trends. The variability of 14C-labeled radioactivity among animals was marked and essentially masked any ability to discern trends in glycolysis as described by tritiated glucose between the aerobic and reperfusion intervals. When the data were arrayed by linear regression analysis, the slopes derived from 14C-labeled deoxyglucose were either discordant or insensitive to those described by 3H-labeled glucose. Tissue glycogen levels were slow to recover in early reflow and at end reperfusion were still significantly depressed from aerobic levels. The present data indicate that coronary reperfusion and hyperglycemia have influence in determining glycolytic flux in myocardium. Labeled deoxyglucose, considered solely as a marker of exogenous glucose utilization, appears to be an insensitive agent in describing these events at conditions of relatively low glucose flux.

Animals↗

Tissue profiling by mass spectrometry: a review of methodology and applications.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. For example, protein profiles from tumors may allow accurate prediction of tumor behavior, diagnosis, and prognosis and uncover etiologies underlying idiopathic diseases. MALDI MS, in conjunction with laser capture microdissection, is able to produce protein expression profiles from a relatively small number of cells from specific regions of heterogeneous tissue architectures. Imaging mass spectrometry enables the investigator to assess the spatial distribution of proteins, drugs, and their metabolites in intact tissues. This article provides an overview of several tissue profiling and imaging applications performed by MALDI MS, including sample preparation, matrix selection and application, histological staining prior to MALDI analysis, tissue profiling, imaging, and data analysis. Several applications represent direct translation of this technology to clinically relevant problems.

Animals↗

Failure of decidual arteriolar remodeling in the CBA/J x DBA/2 murine model of recurrent pregnancy loss is linked to increased expression of tissue inhibitor of metalloproteinase 2 (TIMP-2).

OBJECTIVE: Uterine vascular remodeling at mid gestation includes the thinning of the vessel walls and, typically, an increase in lumen diameter. This study aimed to elucidate any differences in structural remodeling in normal murine pregnancies versus those differences that resulted from the crossing of CBA/J female mice by DBA/2 male mice, a combination that is known to exhibit recurrent resorption/pregnancy loss. STUDY DESIGN: CBA/J female mice that were pregnant by DBA/2 male mice (abnormals) and DBA/2 female mice that were pregnant by CBA/J male mice (normals) were killed at mid gestation, which is a time when fetal resorption can be identified. Tissues were collected for permanent fixation and gene expression studies with complementary DNA macroarrays that were specific for extracellular matrix proteins. A 2-fold increase in expression or a 50% decline was considered significant. Expression changes were confirmed by real-time reverse transcriptase-polymerase chain reaction. RESULTS: The vessel-to-lumen diameter ratios were found to be significantly greater for the CBA/J implantation sites (1.50 +/- 0.05 vs 1.22 +/- 0.02, respectively; P < .0001), which indicates a lack of vascular remodeling. There was also a trend towards smaller lumen diameters for the CBA/J vessels, but this was not statistically significant (78.2 +/- 4.4 microm vs 93.5 +/- 6.8 microm, respectively; P = .22). The mean coefficient of variation for lumen measurements was 0.8% and for vessel diameter was 0.3%. The ranges were 0 to 3.2% and 0 to 1.4%, respectively. Tissue inhibitor of metalloproteinase 2 expression was up-regulated in the placentas of the group with higher resorption rates when compared with normals. This was confirmed with reverse transcriptase-polymerase chain reaction, where abnormals exhibited 2.6-fold greater tissue inhibitor of metalloproteinase 2 protein quantities when compared with normal controls (P = .03). CONCLUSION: The expansive vascular remodeling of decidual vessels that is characteristic of normal murine pregnancy is attenuated significantly in the CBA/J x DBA/2 mating combination, which is known for its tendency to recurrent fetal resorption. This has been correlated with a relative overexpression of tissue inhibitor of metalloproteinase 2 protein in placentas of this strain combination and compared with normals.

Animals↗