DDBJ Data Analysis Challenge: a machine learning competition to predict Arabidopsis chromatin feature annotations from DNA sequences.

Recently, the prospect of applying machine learning tools for automating the process of annotation analysis of large-scale sequences from next-generation sequencers has raised the interest of researchers. However, finding research collaborators with knowledge of machine learning techniques is difficult for many experimental life scientists. One solution to this problem is to utilise the power of crowdsourcing. In this report, we describe how we investigated the potential of crowdsourced modelling for a life science task by conducting a machine learning competition, the DNA Data Bank of Japan (DDBJ) Data Analysis Challenge. In the challenge, participants predicted chromatin feature annotations from DNA sequences with competing models. The challenge engaged 38 participants, with a cumulative total of 360 model submissions. The performance of the top model resulted in an area under the curve (AUC) score of 0.95. Over the course of the competition, the overall performance of the submitted models improved by an AUC score of 0.30 from the first submitted model. Furthermore, the 1st- and 2nd-ranking models utilised external data such as genomic location and gene annotation information with specific domain knowledge. The effect of incorporating this domain knowledge led to improvements of approximately 5%-9%, as measured by the AUC scores. This report suggests that machine learning competitions will lead to the development of highly accurate machine learning models for use by experimental scientists unfamiliar with the complexities of data science.

A prospective compound screening contest identified broader inhibitors for Sirtuin 1.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Comparison of biological features between severely immuno-deficient NOD/Shi-scid Il2rgnull and NOD/LtSz-scid Il2rgnull mice.

Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.

QEX: target-specific druglikeness filter enhances ligand-based virtual screening.

Druglikeness is a useful concept for screening drug candidate compounds. We developed QEX, which is a new druglikeness index specific to individual targets. QEX is an improvement of the quantitative estimate of druglikeness (QED) method, which is a popular quantitative evaluation method of druglikeness proposed by Bickerton et al. QEX models the physicochemical properties of compounds that act on each target protein based on the concept of QED modeling physicochemical properties from information on US Food and Drug Administration-approved drugs. The result of the evaluation of PubChem assay data revealed that QEX showed better performance than the original QED did (the area under the curve value of the receiver operating characteristic curve improved by 0.069-0.236). We also present the c-Src inhibitor filtering results of the QEX constructed using Src family kinase inhibitors as a case study. QEX distinguished the inhibitors and non-inhibitors better than QED did. QEX works efficiently even when datasets of inactive compounds are unavailable. If both active and inactive compounds are present, QEX can be used as an initial filter to enhance the screening ability of conventional ligand-based virtual screenings.

Background data on NOD/Shi-scid IL-2Rγnull mice (NOG mice).

To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immunedeficient mice, a total of 120 animals were examined at 7, 26 and 52 weeks-old (20 mice/sex/group). The survival rate at 52 weeks-old was 95% (19/20) in both sexes. Clinically, circling behavior in one direction along the cage wall was observed in males after 8 weeks and females after 47 weeks-old, and hunchback position was found in males after 32 weeks-old. Hematologically, lymphocyte count markedly decreased at all ages, while white blood cell count increased in several mice at 52 weeks-old. Blood chemistry results revealed high values of aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase in some females at 26 weeks-old, without any related histological change. Histologically, lymphoid hypoplasia characterized by severe lymphocyte depletion with poorly developed tissue architectures was observed. In addition, spongiotic change in the nerve tissue was observed in both sexes at 7 and 26 weeks-old, and intracytoplasmic materials known as tubular aggregates in the skeletal muscles were found in males terminated at 26 and 52 weeks-old and in females at 52 weeks-old. Malignant lymphoma was found in one female euthanized at 20 weeks-old. Further, small intestinal adenoma, hepatocellular adenoma, leukemia, cerebral lipomatous hamartoma, Harderian gland adenoma and uterine polyp were also observed, and their incidences were low except for that of uterine polyp. This study provided detailed background data on NOG mice up to 52 weeks-old and provided information on appropriate use of NOG mice in the various research fields.

An iterative compound screening contest method for identifying target protein inhibitors using the tyrosine-protein kinase Yes.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-TΨC domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 Å resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies' results.

Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats.

This study measured blood parameters, particularly those related to coagulation, and alterations in the expression levels of blood-coagulation-related genes in lactating Sprague-Dawley rats. The day of delivery was designated as lactation day 0 (LD 0). On the day after delivery (LD 1), prothrombin time and overall activity of vitamin-K-dependent coagulation factors were decreased, whereas fibrinogen contents, platelet counts and antithrombin III concentrations were increased as compared with those in nonpregnant rats. In addition, hepatic expression of blood-coagulation-related genes in the liver was increased at LD 0 as compared with that in nonpregnant rats. These changes may be physiologic responses to prevent prolonged bleeding at delivery. Except for fibrinogen content, which remained elevated, the described changes returned to baseline on and after LD 7. Activities of AST, ALT, and ALP were increased on LD 7, 14, and 21 as compared with nonpregnant rats. In contrast, total protein, albumin, Cl, and Ca were consistently lower on LD 7, 14, or 21 as compared with levels in nonpregnant rats. These results provide background data for evaluation of nursing rats.

Analysis of chimeric ribosomal stalk complexes from eukaryotic and bacterial sources: structural features responsible for specificity of translation factors.

Ribosomal protein P0 forms a pentameric complex with two heterodimers of the flexible stalk proteins P1•P2 and plays a role in the functional interaction of eukaryotic ribosomes with translational factors. To investigate the functionality of domains of P0 characteristic to eukaryotes, we constructed various chimeras between silkworm P0 and Escherichia coli counterpart L10. Replacement of the C-terminal region of L10 with that of P0 allowed the binding of two P1•P2 heterodimers, which supported ribosomal activity dependent on eukaryotic elongation factors eEF-2/eEF-1α, but not activity dependent on bacterial factors EF-G/EF-Tu. Conversely, replacement of the C-terminal region of P0 with that of L10 allowed binding of two bacterial L12 homodimers, which resulted in a low level of activity dependent on bacterial factors. Insertion of the extended region of P0 that is absent in the bacterial counterpart into L10 did not affect L12 binding or bacterial factor-dependent activity, but deletion of this region from P0 resulted in a 40% reduction in eukaryotic factor-dependent activity. The results indicate that the C-terminal regions of P0 and L10 are responsible for binding of the cognate stalk dimers and ribosomal specificity for translation factors and suggest that the extended region participates in accessibility only for eukaryotic factors.

Background Data for General Toxicology Parameters in RccHan:WIST Rats at 8, 10, 19 and 32 Weeks of Age.

Recently, RccHan(TM):WIST (Wistar Hannover) rats were introduced to toxicity studies in Japan. The present study was performed to obtain control data for general toxicological parameters as an aid for interpretation of results in toxicity studies using this strain of rats. Four test groups comprising of 25 male and 25 female RccHan(TM):WIST rats were housed for 2, 4, 13 or 26 weeks from 6 weeks of age and observed and examined for clinical observation, body weight, food consumption, urinalysis, hematology, blood chemistry, organ weight, necropsy and/or histopathology. Ophthalmological examination was not conducted in this study, and the data in this report were obtained from an ongoing 104-week background study in RccHan(TM):WIST rats. These data were compared with the historical control data of CD(SD) (Sprague-Dawley) and/or F344 (Fischer) rats. The body weights of RccHan(TM):WIST rats were lower than those of CD(SD) rats and higher than those of F344 rats. The ophthalmological examination revealed a greater incidence of focal corneal opacity. Histopathology revealed focal mineralization of the cornea and Berlin blue-positive pigmentation in the epididymal interstitium as well as hepatocytes. Other than the above, some minor differences were found in urinalysis, hematology, blood chemistry and organ weights as compared with CD(SD) rats.

Omnipotent role of archaeal elongation factor 1 alpha (EF1α in translational elongation and termination, and quality control of protein synthesis.

The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

Effects of pregnancy and lactation on warfarin-induced changes in blood coagulation-related parameters in rats.

Effects of pregnancy and lactation on warfarin-induced changes in blood coagulation-related parameters were examined in rats. Warfarin (0.5 mg/kg/day) was given orally to pregnant and non-pregnant rats for 3 days from gestation day (GD) 17 to 19 or to lactating and non-pregnant rats for 3 days from post partum day (PPD) 10 to 12. Blood samples were collected from the rats on the day following the last administration (GD 20 or PPD 13) to measure prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombotest (TBT), factor VII and X activities and anti-thrombin III concentration (ATIII). Administration of warfarin to non-pregnant rats resulted in significant prolongation of APTT and TBT and significant decreases in factor VII and X activities. On the other hand, similar but not significant changes were observed in pregnant rats and similar significant but less prominent changes were observed in lactating rats. The reduction of the anticoagulant effects of warfarin may partially be related to high plasma 17beta-estradiol concentration in pregnant rats and to high plasma prolactin concentration in lactating rats, respectively.

Structural basis for translation factor recruitment to the eukaryotic/archaeal ribosomes.

The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)(2)(P1)(2)(P1)(2)) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.

Phenobarbital (PB)-induced changes in blood coagulationrelated parameters in pregnant rats, lactating rats and pups.

Effects of repeated administration of phenobarbital (PB) on blood coagulation-related parameters were examined in non-pregnant, pregnant and lactating rats, and also in pups born to PB-treated lactating dams. PB was orally administered at a dose level of 80 mg/kg/day to pregnant (from gestation day (GD) 13), postpartum (from postpartum day (PPD) 7) and non-pregnant rats (from 13 weeks of age) for 7 days. Blood was collected on GD20 or PPD14 to perform blood coagulation examination. Concurrently, the blood coagulation parameters were examined in the pups. Increases in liver weight and/or hepatic cytochrome P450 content were observed in the PB-treated non-pregnant, pregnant and lactating rats. Activated partial thromboplastin time (APTT) was prolonged and anti-thrombin III (ATIII) concentration was increased in the lactating rats, while there were no changes in prothrombin time (PT) or APTT in the non-pregnant and pregnant rats. Moreover, prolongation of PT and APTT and decreases in factors VII and IX activities were observed in their pups. Thus, prolongation of blood coagulation time was confirmed in both dams and their pups following PB-administration to lactating dams. Effects of vitamin K(2) (VK(2)) on PB-induced changes in blood coagulation-related parameters of both dams and their pups were examined by co-administration with PB and VK(2) to lactating dams. PT and APTT were comparable to the control and PB-induced prolongation of blood coagulation time was improved in the pups while APTT was prolonged in dams, suggesting that VK(2) was beneficial to pups but not to dams.

Structural basis of novel interactions between the small-GTPase and GDI-like domains in prokaryotic FeoB iron transporter.

The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe(2+) uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe(2+) uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)-like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe(2+) uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe(2+) uptake.

Changes in blood coagulation-related parameters in phenobarbital-treated rabbits.

The effects of repeated administration of phenobarbital (PB) on blood coagulation time were examined using male Japanese white SPF rabbits, which are widely used for toxicological studies. PB was administered to the rabbits by oral gavage for 2 weeks, at dose levels of 0, 12.5, 25 and 50 mg/kg/day. Blood was collected on Days 8 and 14 after each day's dosing to perform blood coagulation examination. The liver was excised, weighed and examined histopathologically. Activated partial thromboplastin time (APTT) was prolonged at dose levels of 12.5 mg/kg/day or more and Thrombotest (TBT) was prolonged at 50 mg/kg/day on Day 8. APTT was prolonged at dose levels of 12.5 mg/kg/day or more, TBT was prolonged at 25 mg/kg/day or more and factor IX activity decreased at 50 mg/kg/day on Day 14. At pathological examination, liver weight increased at dose levels of 25 mg/kg/day or more, and a ground-glass appearance of the hepatocytes was observed in the central and middle parts of lobules at 12.5 mg/kg/day or more. However, changes in factor VII or X activity or prolongation of prothrombin time (PT) were not observed. Therefore, prolongation of blood coagulation time by PB administration in rabbits was considered to be due to PB's effect on the endogenous pathway alone. Moreover, an increase in anti-thrombin III (ATIII) concentration was noted at 50 mg/kg/day; however, no change was noted at dose levels of 25 mg/kg/day or less. This suggests that the contribution of ATIII to the PB-induced prolongation of coagulation time in rabbits was small.

Changes in blood parameters and coagulation-related gene expression in pregnant rats.

The present study examined changes in maternal blood parameters, particularly those related to blood coagulation, as well as alterations in blood coagulation-related gene expression in the liver during gestation in rats. Fibrinogen concentration and platelet count increased as pregnancy progressed whereas prothrombin time and overall activity of vitamin-K-dependent coagulation factors decreased before delivery, suggesting a physiologic response to prevent prolonged bleeding at parturition. Conversely, compared with values for nonpregnant rats, activated partial thromboplastin time was prolonged before delivery and antithrombin time was significantly higher during fetal organogenesis and thereafter, indicating a mechanism to prevent the development of deep tissue thrombosis in dams. DNA microarray analysis revealed no differences in coagulation-related gene expression in the liver on gestation day 13 between pregnant and nonpregnant rats, whereas the gene expression of various fibrinogen-related factors, coagulation factors II and X, and the anticoagulation factor-related factor leuserpin 2 were increased on gestational day 19. In addition, changes similar to those reported previously in pregnant rats were confirmed. The data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rats, and they suggest that the rat is a useful animal model for investigating the mechanisms of disorders in the blood coagulation system that can occur during late pregnancy in women.

Carbon tetrachloride-induced hepatotoxicity in pregnant and lactating rats.

Carbon tetrachloride (CCl4) is well known to induce hepatotoxicity after being metabolized to trichloromethyl free radical ((.)CCl3) by CYP2E1. In the present study, the hepatotoxicity induced by a single oral dose (2,000 mg/kg) of CCl4 was compared between pregnant (gestation days (GD) 13 and 19) or postpartum (postpartum days (PPD) 1, 13 and 27) and non-pregnant rats. Hepatotoxicity in CCl4-treated pregnant rats evaluated by blood chemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities) and histopathological finding (area of damaged hepatocytes) was minimal on GD19, being weaker than that in non-pregnant rats. CYP2E1 expression in non-treated pregnant rats decreased as pregnancy progressed and reached minimum level on GD19. Thus, the degree of CCl4-induced hepatotoxicity roughly corresponded to CYP2E1 levels during pregnancy. After delivery, hepatotoxicity in CCl4-treated lactating rats was maximal on PPD13, being stronger than that in non-pregnant rats, and then it decreased slightly on PPD27. The CYP2E1 level in the non-treated lactating rats tended to increase but remained at lower levels until PPD13 compared with that in non-pregnant rats. Thus, the degree of CCl4-induced hepatotoxicity did not correspond to CYP2E1 levels during lactation. This suggests that during lactation, there may be certain factors other than CYP2E1 expression responsible for the degree of CCl4-induced hepatotoxicity.

Studies of the toxicological potential of capsinoids: I. Single-dose toxicity study and genotoxicity studies of CH-19 Sweet extract.

A single-dose oral toxicity lethal-dose study was conducted to examine the toxicity of capsinoids contained in CH-19 Sweet extract. CH-19 Sweet extract was administered once by gavage to SPF (Crl:CD(SD)) Sprague-Dawley male and female rats at dose levels of 0 (vehicle), 5, 10, or 20 ml/kg of body weight (BW). The concentration of capsinoids in the CH-19 Sweet extract was 71.25 mg/ml; this resulted in administered dose levels of capsinoids of 356.25, 712.5, and 1425 mg/kg BW, respectively. The toxicity of CH-19 Sweet extract by single oral administration was low; only transient salivation or decreased spontaneous movement was observed on the day of administration at > or =10 ml/kg BW. It was concluded that the lethal dose of CH-19 Sweet extract was estimated to be higher than 20 ml/kg (1425 mg/kg as capsinoids) for both males and females since no deaths were observed at any dose in this study. A bacterial reverse mutation test of CH-19 Sweet extract was performed employing Salmonella typhimurium and Escherichia coli and using the preincubation method. Treatment with CH-19 Sweet extract did not increase the number of revertant colonies compared with negative controls either in the presence (+S9) or absence (-S9) of metabolic activation. An in vitro chromosome aberration test was conducted using Chinese hamster lung cultured cells (CHL/IU). Treatment with CH-19 Sweet extract failed to induce chromosome aberrations in either short-term or continuous treatment scenarios, with or without metabolic activation (-S9, +S9). In an in vivo micronucleus test using BDF(1) male mice, CH-19 Sweet extract failed to increase the incidence of micronucleated polychromatic erythrocytes (MNPCEs) or decrease the ratio of polychromatic erythrocytes (PCEs) in any of the treatment groups. These results suggest the absence of mutagenicity as well as in vitro and in vivo clastogenicity of capsinoids contained in CH-19 Sweet extract.