RatDEGdb: a knowledge base of differentially expressed genes in the rat as a model object in biomedical research

The animal models used in biomedical research cover virtually every human disease. RatDEGdb, a knowledge base of the differentially expressed genes (DEGs) of the rat as a model object in biomedical research is a collection of published data on gene expression in rat strains simulating arterial hypertension, age-related diseases, psychopathological conditions and other human afflictions. The current release contains information on 25,101 DEGs representing 14,320 unique rat genes that change transcription levels in 21 tissues of 10 genetic rat strains used as models of 11 human diseases based on 45 original scientific papers. RatDEGdb is novel in that, unlike any other biomedical database, it offers the manually curated annotations of DEGs in model rats with the use of independent clinical data on equal changes in the expression of homologous genes revealed in people with pathologies. The rat DEGs put in RatDEGdb were annotated with equal changes in the expression of their human homologs in affected people. In its current release, RatDEGdb contains 94,873 such annotations for 321 human genes in 836 diseases based on 959 original scientific papers found in the current PubMed. RatDEGdb may be interesting first of all to human geneticists, molecular biologists, clinical physicians, genetic advisors as well as experts in biopharmaceutics, bioinformatics and personalized genomics. RatDEGdb is publicly available at https://www.sysbio.ru/RatDEGdb.


Introduction
The animal models required for understanding the physio logical, genetic and epigenetic mechanisms regulating evolu tionarily fixed phenotypic traits of an organism are supposed to perfectly mimic the symptoms of the pathology being studied and to conform to strict criteria (Gryksa et al., 2023).The most popular animal models are rats and mice, with do zens of thousands of laboratory strains in use (Gayday E.A., Gayday D.S., 2019).
The first inbred rat strain was developed in 1906 in the Wistar Institute (Philadelphia, USA), about the time that mice came to the laboratory settings.Nevertheless, the mouse has become the model of choice for research into mammalian genetics, and the rat, into physiology and biomedicine.Labo ratory rats have certain advantages over mice: rats are larger and therefore submit more tissue for analyses.Large organs make surgical procedures more manageable and rather small anatomical structures easier to dissect.
A low maintenance and cheap species, the rat (Rattus nor vegicus) has become a convenient object in numerous bio medical research studies (Carter et al., 2020;Modlinska, Pi sula, 2020).Rats are recommended for use as model animals in studying aging, hypertension, catalepsy etc. (Carter et al., 2020;MartínCarro et al., 2023).
There are generally acknowledged differences between wild and laboratory rats.For example, laboratory rats are noted for smaller adrenals and preputial glands, earlier puberty, lack of seasonality of reproduction and higher fertility than have their wild conspecifics.In addition, the rat and human genomes share a 90 % identity (Gibbs et al., 2004).Thus, the genetic strains of laboratory rats simulating human patholo gies have been developed: for example, the Zucker strain for human obesity, hypertension, type II diabetes and heart disease (Schmidt, 2002); the reelin-deficient shaking rat Kawasaki for schizophrenia and autism (Aikawa et al., 1988); and the Brattleboro strain for hypothalamic diabetes insipidus (Ideno et al., 2003).To date, there are about 1,000 inbred strains of laboratory rats developed by genetic breeding that have "fixed" alleles for natural diseases (Greenhouse et al., 1990), such as mental disorders (Taylor et al., 2002), depression (Bay et al., 2020) and chronic renal failure (Zhang H.F. et al., 2019).The Wistar and SpragueDawley strains are the most commonly used laboratory rats (Sengupta, 2013).At present, the search of PubMed (Lu, 2011) with "rats biomedical model" as a search string returns the annotations of 19,555 original scientific papers, which lends support to the relevance of the subject.
To contribute to the effort, several rat strains simulating hu man diseases have been developed in the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences.Thus, the ISIAH rats are characterized by an increased arterial blood pressure and used for studying the causes and treatments of hypertension in humans (Markel, 1992;Markel et al., 1999;Fedoseeva et al., 2016aFedoseeva et al., , 2019;;Klimov et al., 2016;Ryazanova et al., 2016), the OXYS rats represent a unique selectionbased model of premature ageing and associated diseases (Kozhevnikova et al., 2013;Kolosova et al., 2014;Perepechaeva et al., 2014;Stefanova et al., 2018Stefanova et al., , 2019;;Stefanova, Kolosova, 2023), rats with pendulum-like movements (the PM strain) with stereotypies and audiogenic epilepsy, and rats with genetic catatonia (the GC rats), a syn drome observed in patients with mental disorders, including schizophrenia (Barykina et al., 1983;Kolpakov et al., 2004;Ryazanova et al., 2017Ryazanova et al., , 2023)).
Changes in the expression of the genes associated with a disease of interest have been studied in the model rats by semiquantitative realtime PCR of separate key genes or by profiling transcriptomes by next-generation sequencing or by use of microarrays.This effort has created a large body of data on the differentially expressed genes (DEGs) signifi cantly associated with diseases, and it has become possible to collect, perform comparative analyses on and systematize the results obtained from these or similar experiments with the use of bioinformatics technologies.This has enabled the development of specialized databases and knowledge bases.
The aim of this work was to create a knowledge base con taining information on DEGs of various rat strains developed, first of all, in the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences as well as those developed in a range of Russia's and other scientific organizations.This knowledge base is freely available at https://www.sysbio.ru/RatDEGdb.
RatDEGdb: knowledge base of rat differentially expressed genes in biomedical research

Materials and methods
Experimental animals.We performed in vivo experiments on 12 adult male gray rat (Rattus norvegicus) from two out bred strains resulting from genetic breeding for more than 90 generations in two directions (Belyaev, Borodin, 1982): one for increased aggressive behavior towards humans (the aggressive strain) and one for decreased (the tame strain).The animals were kept in standard conditions at the Conventional Animal Facility of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (No vosibirsk, Russia) as groups by four in 50×33×20 cm cages at an adjustable light/dark cycle (12 light:12 dark) and had free access to water and complete feed.The test subjects were twomonthold individuals, each weighing 250-270 g, from unrelated litters.Within the first 4 hours of the light phase of the diurnal lightdark cycle, each animal's level of tameness/aggression was measured in the "glove" test as the reaction to a gloved hand and was scored from "-4" (most aggressive) to "+4" (most friendly), accord ing to Plyusnina and Oskina (1997).Upon the completion of this test, the animals were put back to their home cages and kept in standard conditions for one week, to reduce possible effects that the "glove" test might have on gene expression, at which point the animals were euthanized and hypothala mus specimens were prepared according to the brain atlas of Paxinos and Watson (2013).Samples were placed in liquid nitrogen for transportation and further storage at -70 °C until use.The protocol of experiments was approved by the Com mission on Bioethics at the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (resolution No. 97 as of October 28, 2021).
Measurement of the hypothalamic mRNA levels of the Asmtl gene in tame and aggressive male gray rats by semi-quantitative PCR.To measure mRNA levels by semi quantitative realtime polymerase chain reaction, hypotha lamic RNA was isolated from six aggressive rats (n = 6) and six tame rats (n = 6), each specimen weighing ~100 mg.Total RNA was isolated using TRIzol ™ (Invitrogen, #15596018) and purified using magnetic beads in the Agencourt RNAClean XP Kit (Beckman, #A63987).Purified RNA was quantified using a Qubit ™ 2.0 fluorimeter (Invitrogen/Life Technologies) and a Qubit ™ RNA High-Sensitivity Assay Kit (Invitrogen #In=Q32852).Next, we synthesized cDNA using the Reverse Transcription Kit (Syntol, #OT-1).
The oligonucleotide primers for each gene in question were designed using the web service PrimerBLAST (Ye et al., 2012) (Table 1).Realtime PCR was carried out using the EVA Green I kit in three technical replicates in a LightCycler ® 96 operated in the automatic mode, according to the manufac turer's instruction (Roche, Switzerland).The efficiency of the polymerase chain reaction was determined by serial cDNA dilutions (standards).
The human gene ASMT encodes acetylserotonin O-methyl transferase, a key enzyme in the synthesis of melatonin, one of the hormones that regulate the molecular and genetic processes in the entire organism, including circadian rhythms as well as cancer protective (Lv et al., 2019), anti-inflammatory, and immunomediatory mechanisms (Li G. et al., 2021).That is why the mRNA level of its rat homolog, Asmtl, in the hypo thalamus of adult tame and aggressive male rats used as model animals in the biomedical studies of increased aggression was heuristically chosen as the quantity to be found by semi quantitative real-time PCR (real-time PCR) in its first run.As was recommended by Bustin and the coworkers (2009), the Asmtl mRNA values were normalized to the mRNA levels of two comparison genes, Ppia (Gholami et al., 2017) and Rpl30 (Penning et al., 2007).The relevance of Ppia and Rpl30 as the comparison genes in the experimental identification of DEGs in the hypothalamus of these aggressive and tame rat strains by realtime PCR was demonstrated in one of our previous works (Chadaeva et al., 2021).
RatDEGdb: the knowledge base.The observed lower hypothalamic levels of the Asmtl gene in the adult aggressive and tame male rats were checked against clinical data sug gesting that lower levels of the protein encoded by its human homologs ASMT and ASMTL were in patients with various diseases than in otherwise healthy individuals.The results of this comparison were presented in an Excel-compatible, flat text format and then converted to RatDEGdb containing information about differential gene expression in the rat used as a model animal in biomedical research (URL=https:// www.sysbio.ru/RatDEGdb).The conversion was performed using MariaDB 10.2.12, a freely available database (MariaDB Corp AB, Finland).
Likewise, Lu (2011) submitted a representative selection of PubMed publications telling about the current diversity of laboratory rat strains used as biomedical models simu lating human diseases and about experimental methods to    1. Rat strain: A, aggressive rats (n = 6); T, tame rats (n = 6).Tests: "glove" test, in which each rat was scored from "-4" (most aggressive) to "+4" (most friendly), according to a work by Plyusnina and Oskina (1997); Asmtl expression levels, M 0 ± SEM, estimates of the mean ± standard error of the mean from three technical replicates, with a LightCycler ® 96 operated in the automatic mode (Roche, Switzerland).assess differential gene expression with.Next, all rat DEGs in this selection of papers were documented and uploaded to RatDEGdb together with their supervised annotations, using an algorithm similar to the one described above for hypothalamic deficiency of Asmtl in aggressive rats.The lists of homologous genes were taken from the paralogs section of the GeneCards database (Stelzer et al., 2016).RatDEGdb includes the statistical significance of each DEG according to the estimates provided in the papers as referenced.
Statistical analysis of the differential expression of the Asmtl gene in the hypothalamus of the tame and aggressive rats used as an animal model of human aggressive behavior was performed using the menu "Statistics → Nonparametric → Mann-Whitney test" in STATISTICA (StatSoft ™ , USA), when two independent statistical criteria are being as sessed at once: the nonparametric Mann-Whitney U test and the parametric test Fisher's Z, to assess the sustainability of results.

Lower hypothalamic Asmtl mRNA levels in aggressive than in tame rats
Asmtl mRNA levels in the hypothalamus as measured and compared between the aggressive and tame rats are presented in Table 2.As can be seen from Figure 1, significantly lower Asmtl mRNA levels were in the aggressive than in tame rats in the settings of this experiment ( p < 0.05; the Mann-Whitney U test and Fisher's Z).

Clinical manifestations of human ASMTL and ASMT deficiency
Table 3 presents the PubMed search results, with search terms (Lu, 2011) relating to human diseases associated with low expression levels of the ASMTL gene and its human paralog, ASMT.Line 1: the Asmtdeleted mouse models of human diseases (Trent et al., 2013) suggest a neurodevelopmental problem in the form of attention-deficit/hyperactivity disorder in combination with externalization symptoms (aggressive behavior) in children (Kang et al., 2023).
Line 2: ASMT deficiency is a molecular marker of autism, according to Melke and coworkers (2008), while a recent sur vey of teenagers above 12 years of age with autism spectrum disorders and epilepsy in past medical history revealed their inclination to aggression (Gaitanis et al., 2023).
These two examples are in favor of rather than against the low expression levels of the human genes ASMTL and ASMT representing, at least, combined molecular characteristics of the predisposition to some forms of aggressive behavior.

RatDEGdb: the knowledge base
Figure 2 shows how RatDEGdb compares the hypothalamic level of Asmtl in the aggressive rat strain with that in the tame.
Here aggression is considered to be a comorbid symptom in human diseases such as thalassemia, obesity and carci noma (for review, see Chadaeva et al., 2016).Consequently,

799
СИСТЕМНАЯ КОМПЬЮТЕРНАЯ БИОЛОГИЯ / SYSTEMS COMPUTATIONAL BIOLOGY Fig. 2. A sample entry in RatDEGdb documents original experimental data on Asmtl deficiency in the hypothalamus of aggressive rats compared to the tame rats as a biomedical model of aggressive behavior in human diseases (see Fig. 1 and Table 2) together with their annotation (see Table 3: first row) using independent data on low expression levels of its human homolog ASMT in patients with hyperactivity disorders according to an Asmt-deleted mouse model of human disease (Trent et al., 2013).
RatDEGdb (see Fig. 1 and Table 2) integrated data on low hypothalamic levels of the Asmtl gene in the aggressive rats and low levels of its human homolog ASMTL as found in patients with neurodevelopmental problems in the form of attention-deficit/hyperactivity disorder using an Asmtdeleted mouse model (Trent et al., 2013) (see Table 3).
The current release contains information on DEGs in ten genetic rat strains used as models of 11 human pathologies (Tables 4-6).As can be seen in the bottom lines of these tables, RatDEGdb now contains information on 25,101 DEGs representing 14,320 unique rat genes that change transcription levels in 21 tissues of 10 genetic rat strains used as models of 11 human diseases based on 45 original scientific papers refer enced in the rightmost column of Tables 4-6.These rat DEGs were annotated with information about equal changes in the expression levels of their human homologs in affected people.In total, the current release contains 94,873 such annotations for 321 human genes in 836 diseases based on 959 PubMed publications (Lu, 2011).Thus, RatDEGdb is unique in that the manual curation of the annotation of DEGs of the rat as a model object simulating human pathology uses indepen dent clinical data, which none of other biomedical databases does.

Discussion
The elementary step in filling RatDEGdb with data can be seen in Tables 1-3 and Figures 1-2, with the Asmtl (acetylserotonin O-methyltransferase like) gene as an example.The hypo thalamic expression of this gene was and compared between aggressive and tame rats used as model animals in human aggression research.Results of the analysis of this gene by realtime PCR are provided.These results were an notated using PubMed papers (Lu, 2011) about equal changes in the expression levels of its human homologs ASMTL and ASMT in patients.Then this annotation of the Asmtl gene dif ferentially expressed in the hypothalamus of the aggressive and tame rats was supplemented with PCR, RNAseq and microarraybased information on all DEGs in the rat used as a model object in biomedical research.Next, the uncharacterized, unannotated, predicted, and not proteinencoding genes were dropped.Finally, we annotated the remaining rat DEGs with publicly available works about the clinical manifesta tions of equal changes in the expression levels of their human homologs in patients, put these annotations together as the RatDEGdb the knowledge base, and made it freely available at https://www.sysbio.ru/RatDEGdb.
Figures 1 and 2 show how RatDEGdb characterizes the DEGs of various breedingbased rat strains primarily deve loped in the Institute of Cytology and Genetics of the Sibe rian Branch of the Russian Academy of Sciences (Novosibirsk, Russia).The ISIAH rats were used as model animals in the biomedical studies of stressinduced arterial hypertension, as summarized in Tables 4 and 5.The same tables show that that OXYS rats were used for studying age-related diseases and ageing processes; and GC rats, for studying psychopathologi cal conditions (see Table 4).In addition, tame and aggres sive rat strains were used for studying animal domestication (Plyusnina, Oskina, 1997;Gulevich et al., 2019;Chadaeva et al., 2021) and aggression (Popova et al., 2010) as symptoms of obesity and thalassemia (Chadaeva et al., 2016(Chadaeva et al., , 2019)).As can seen from Tables 4-6, whole-genome sequencing was performed on each of these models, except for the GC strain, in which only the expression levels of the gluta mate receptor genes and the catecholamine system genes were measured.
The existing biomedical databases intended for studying human diseases are normally focused on the information on the human genome (Stenson et al., 2014;Singh et al., 2018;

Conclusion
The RatDEGdb knowledge base is a collection of experimental data and a toolkit for interactive analyses in genomic research into diseases, such as Alzheimer's disease, autism, hyperten sion and some others.We are planning to continue updating RatDEGdb by adding new information on gene expression in rats as model objects of human diseases and annotating the DEGs with pieces of works on equal changes in the expression levels of their human homologs in patients.

UFig. 1 .
Fig.1.Statistically significant differences in hypothalamic Asmtl expression levels between tame and aggressive adult male rats.* Significance level p < 0.05 according to two independent statistical criteria: the nonparametric Mann-Whitney U test and the parametric test Fisher's Z, which reflects the sustainability of assessment results for Asmtl as a differentially expressed gene (DEG) in aggressive versus tame rats.

Table 1 .
Primers for quantitative real-time polymerase chain reaction (qPCR)

Table 2 .
Experimental data on "glove" test behavior and Asmtl mRNA levels for 12 adult male rats

Table 3 .
Clinical manifestation of deficiencies in ASMTL and in its human paralogue ASMT in human diseases according to the current release of the RatDEGdb knowledge base

Table 4 .
Characterization of the qPCR-inferred DEGs of the rat as a model animal in biomedicine documented in the RatDEGdb knowledge base Sun et al., 2022)and contain primary transcriptome informa tion.RatDEGdb is novel in that it supplements biomedicine based whole-genome experimental data on rat DEGs with clinical data on equal changes in the expression levels of their human homologs in patients, for further use of all these data in personalized medicine.With a new capability that enables the researcher to compare pathogenic changes in gene expression in humans and model animals, RatDEGdb can be useful in addressing problems in systems biology and clinical medicine.СИСТЕМНАЯ КОМПЬЮТЕРНАЯ БИОЛОГИЯ / SYSTEMS COMPUTATIONAL BIOLOGY

Table 6 .
Characterization of the microarray-inferred DEGs of the rat as a model animal in biomedicine documented in the RatDEGdb knowledge base Notе.Models: wo, weeks old.