Association between Social Skills and Motor Skills in Individuals with Autism Spectrum Disorder: A Systematic Review.

Social communication and motor skill deficits are prevalent characteristics of individuals with autism spectrum disorder (ASD). This systematic research review investigates whether and how broad social skills and motor skills may be related among individuals with ASD. We performed a PubMed search of articles written in English, using these study inclusion criteria: (a) an association between social and motor and skills among individuals previously diagnosed with autism; (b) one or more social skills measures were used; and (c) one or more measures of gross or fine motor skills were used. We classified data into two categories, and we based the association of these variables on correlation coefficients, p-values, coefficients of determination, and authors' description of "may be associated" and "may not be associated." Despite heterogeneity among these relevant studies, a highly likely association between social and motor skills emerged. Of a total of 16 studies reviewed, 12 reported associations between these skill sets. Three studies reported that fine motor skills had a stronger relationship with social skills than did gross motor skills. Among the gross motor skills associated with social skills, object control skills seemed most closely linked to social skills. Among fine motor skills, manual dexterity seemed to most closely related to social skills.

Competitive reaction kinetics of sulfate-reducing bacteria and methanogenic bacteria in anaerobic filters.

A kinetic model for the anaerobic filter (AF) that takes into account the mass fractions of sulfate-reducing bacteria (SRB) (fSRB) and methanogenic bacteria (MB) (fMB) and an inhibiting effect of H2S on bacterial groups is proposed. When the acetate-fed AFs were maintained at the low organic loading rate of 2.5kg COD/m3d, variations of the influent COD/SO4(2-) ratio (0.5-3.0) does not materially affect the acetate removal efficiency (all varying between 98.1% and 99.7%). With an increase in influent COD/SO4(2-) ratio, both the biofilm thickness and the specific substrate utilization rate decreased slightly but f(SRB) decreased markedly. The estimated results of fSRB and fMB showed that SRB out-competed MB for bacterial growth if the influent COD/SO4(2-) ratio was maintained at less than 1.3, whereas MB out-competed SRB for bacterial growth if the influent COD/SO4(2-) ratio was maintained at greater than 2.0. The specific substrate utilization rate of SRB (0.19-0.24mg acetate/mg VSSd) was lower than that of MB (0.31-0.59mg acetate/mg VSSd). The estimated kinetic parameters disclosed that the affinity of acetate to MB was higher and unionized H2S imposed a greater inhibiting effect on MB. The model simulation results (acetate and sulfate removal) agreed well with the experimental results.

Influencing effect of intra-granule mass transfer in expanded granular sludge-bed reactors treating an inhibitory substrate.

Two expanded granular sludge-bed (EGSB) reactors (superficial velocity u s=6.0 and 9.0m/h) were used to treat an inhibitory substrate phenol. The granule diameter (dp) increased with increasing organic loading rate (OLR) and u(s). At the OLRs of 1.67-4.44 kg phenol/m3 d, the accumulation of volatile fatty acids (VFAs) was insignificant; whereas at the OLR of 5.11 kg phenol/m3 d, both the accumulation of VFAs and the washout of large hollow granules (average dp=2.90-3.12 mm) occurred. The comparative experimental and simulated results showed that the proposed kinetic model is suitable for design and predicting purposes. The calculation results of mass transfer parameters (Thiele modulus, Biot number, diffusion layer thickness, and overall effectiveness factor) and parametric sensitivity analysis results (half-saturation constant Ks and dp) showed that the intra-granule mass transfer would lead to a more influencing effect than the external mass transfer on the overall substrate removal rate in EGSB reactors.

Consecutive reaction kinetics involving a layered structure of the granule in UASB reactors.

A consecutive-reaction kinetic model for the sucrose-fed upflow anaerobic sludge bed (UASB) reactor that accounts for a layered structure of the granule and the mass fraction of methanogens (f) is proposed. When the UASB reactor was maintained at the volumetric loading rates (VLR) of 7.9-13.8 kg chemical oxygen demand (COD)/m(3) d, the accumulated volatile fatty acids (VFAs) increased with increasing VLR, whereas the experimental f decreased with increasing VLR. This was primarily because methanogenesis was the rate-limiting step and the sucrose-fed granule was a layered structure. The calculated residual concentrations of sucrose and the intermediates VFAs using the layered-structure model are less deviated from the experimental measurements than those using the homogeneous-structure model. The calculated effectiveness factors for sucrose uptake and intermediates VFAs uptake (eta(1); eta(2)) ranged from 0.18 to 0.35 and 0.65 to 0.96, respectively, indicating that the overall substrate (sucrose or intermediates VFAs) removal in the UASB reactor was diffusion-controlled, especially at the VLRs of 7.9-10.6. kg COD/m(3) d. This finding was also confirmed by the simulated concentration profiles of sucrose and VFAs in the UASB-granule. From the simulation results, the effect of internal mass transfer resistance on overall substrate (sucrose) removal should not be neglected, especially for a granule size of greater than 2.0 mm.

CDNA library construction from a small amount of RNA: adaptor-ligation approach for two-round cRNA amplification using T7 and SP6 RNA polymerases.

In this study, we developed a method that allows cDNA library construction from a small amount of RNA without causing serious size bias in the resulting cDNA population. For this purpose, we adopted two-round cRNA amplification by T7 and SP6 RNA polymerases. The first-round cDNAs, flanked by the promoter sequences of T7 and SP6 RNA polymerases, were synthesized from 1 microg total RNA and then subjected to two rounds of cRNA amplification. Comparison of the sizes of the first-round and the second-round cRNAs indicated that the size-bias effect of the second-round cRNA synthesis was not serious. The resultant double-stranded cDNAs were cloned into a plasmid by in vitro lambda phage recombination with an efficiency of 1.2 x 10(11) colony-forming unit/microgram of starting total RNA. Characterization of the resultant cDNA library in terms of the insert size, clone redundancy, and integrity of 3' ends of cDNAs indicated that the amplified library was comparable to a library constructed by a conventional method, although large cDNAs tend to be slightly truncated in the amplified library. This method enables the construction of a library from a small amount of RNA, and calculations suggest that the strategy would be efficient enough to use even a single cell as starting material.

Prediction of the coding sequences of mouse homologues of KIAA gene: I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes. A primary aim of this study was to prepare a set of mouse. KIAA-homologous cDNAs that could be used to analyze the physiological roles of KIAA genes in mice. In addition, comparison of the structures of mouse and human KIAA cDNAs might enable us to evaluate the integrity of KIAA cDNAs more convincingly. In this study, we selected mouse KIAA-homologous cDNA clones to be sequenced by screening a library of terminal sequences of mouse cDNAs in size-fractionated libraries. We present the entire sequences of 100 cDNA clones thus selected and predict their protein-coding sequences. The average size of the 100 cDNA sequences reached 5.1 kb and that of mouse KIAA-homologous proteins predicted from these cDNAs was 989 amino acid residues.

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins II: public release of inaugural version of InGaP database containing gene/protein expression profiles for 127 mouse KIAA genes/proteins.

The inaugural version of the InGaP database (Integrative Gene and Protein expression database; http://www.kazusa.or.jp/ingap/index.html) is a comprehensive database of gene/protein expression profiles of 127 mKIAA genes/proteins related to hypothetical ones obtained in our ongoing cDNA project. Information about each gene/protein consists of cDNA microarray analysis, subcellular localization of the ectopically expressed gene, and experimental data using anti-mKIAA antibody such as Western blotting and immunohistochemical analyses. KIAA cDNAs and their mouse counterparts, mKIAA cDNAs, were mainly isolated from cDNA libraries derived from brain tissues, thus we expect our database to contribute to the field of neuroscience. In fact, cDNA microarray analysis revealed that nearly half of our gene collection is predominantly expressed in brain tissues. Immunohistochemical analysis of the mouse brain provides functional insight into the specific area and/or cell type of the brain. This database will be a resource for the neuroscience community by seamlessly integrating the genomic and proteomic information about the mouse KIAA genes/proteins.

Method for systematic targeted isolation of homologous cDNA fragments in a multiplex format.

In this study, a two-step method for systematic multiplex cloning of homologous cDNAs from related species was developed. The first step, called MUCH (multiplex cloning of homologous genes), is cloning of partial but authentic cDNA fragments of homologous cDNAs by hybridization to arrayed cRNA probes of specified genes on a nylon membrane, followed by PCR amplification of the hybridized fragments. The second step is PCR-based screening of a library that contains longer cDNA inserts based on the sequences obtained in the first step. To evaluate this method, we tried to isolate mouse counterparts of 53 human large cDNAs by MUCH and could successfully isolate 32 mouse counterpart cDNAs from a single library. Complete sequencing of two mouse cDNAs isolated by PCR-based screening further demonstrated that this method enabled us to isolate multiple homologous cDNAs in parallel. We thus expect that this method could be applied to high-throughput cloning of homologous cDNAs in related species.

[Improvement of carbon monoxide analysis in fish meat].

Carbon monoxide (CO) treatment of fish meat of tuna, yellowtail, tilapia etc. is not allowed in Japan, since it can maintain the red color for a longer period than the microbiological shelf life of fish meat. The official method for quantification of CO has a problem, in that a part of the CO is lost during the preparation of the fish sample. To solve this problem, we modified the official method in this study. We also applied this modified method to survey the contents of CO in tuna, yellowtail, young yellowtail, and tilapia. As a result, the modified method was found to be more suitable for CO quantification than the official method. An inter-laboratory study by 4 laboratories confirmed that the CO content of many samples of tilapia exceeded the regulation value, apparently due to the higher recovery of CO, compared to the official method. Therefore, it was suggested that the regulation value in the case of tilapia should be changed if this method is introduced as an official method.

The novel protein complex with SMARCAD1/KIAA1122 binds to the vicinity of TSS.

The SMARCAD1/KIAA1122 protein is structurally classified into the SWI2/SNF2 superfamily of DNA-dependent ATPases that are catalytic subunits of chromatin-remodeling complexes. Although the importance of other members of the SWR1-like subfamily in chromatin remodeling (EP400, INOC1, and SRCAP) has already been elucidated, the biological function of SMARCAD1/KIAA1122 in transcriptional regulation remains to be clarified. To gain insight into the role of this protein, we generated a specific antibody against SMARCAD1/KIAA1122 and used it for chromatin and protein immunoprecipitation assays. We employed high-resolution genome tiling microarrays in chromatin immunoprecipitation and found the binding sites of SMARCAD1/KIAA1122 in the vicinity of the transcriptional start site of 69 candidate target genes. In the protein immunoprecipitation assay, we found that endogenous SMARCAD1/KIAA1122 binds with TRIM28, a recently highlighted transcriptional regulator in the cancer field. From these findings, we propose a novel model for gene regulation via the SMARCAD1/KIAA1122 protein complex.

Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology.

Antibodies play a pivotal role in studying the expression and function of proteins. Proteomics studies require the generation of specific and high-affinity antibodies against large numbers of proteins. While traditional animal-based antibody generation is laborious, difficult to automate, and therefore less suited to keep up with the requirements of proteomics research, the use of recombinant in vitro antibody technology might offer a solution to this problem. However, it has not been demonstrated yet that such antibodies are at least as useful as conventional antibodies for typical proteomics applications. Here we generated novel recombinant Fab antibody fragments from the naïve HuCAL GOLD library against a number of targets derived from a mouse cDNA library. We compared these antibodies with polyclonal antisera produced against the same targets and show that these recombinant antibodies are useful reagents for typical applications like Western blotting or immunohistochemistry.

Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method.

Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS-polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.

Influence of the 3'-UTR-length of mKIAA cDNAs and their sequence features to the mRNA expression level in the brain.

We have previously described the sequence features of approximately 1500 mouse KIAA (mKIAA) genes in comparison with those of human KIAA genes (Okazaki, N., Kikuno, R., Inamoto, S., Hara, Y., Nagase, T., Ohara, O., and Koga, H. 2002, DNA Res., 9, 179-188; Okazaki, N., Kikuno, R., Ohara, R., Inamoto, S., Aizawa, H., Yuasa, S., Nakajima, D., Nagase, T., Ohara, O., and Koga, H. 2003, DNA Res., 10, 35-48; Okazaki, N., Kikuno, R., Ohara, R., Inamoto, S., Koseki, H., Hiraoka, S., Saga, Y., Nagase, T., Ohara, O., and Koga, H. 2003, DNA Res., 10, 167-180; and Okazaki, N., F-Kikuno, R., Ohara, R., Inamoto, S., Koseki, H., Hiraoka, S., Saga, Y., Seino, S., Nishimura, M., Kaisho, T., Hoshino, K., Kitamura, H., Nagase, T., Ohara, O., and Koga, H. 2004, DNA Res., 11, 205-218). To validate the orthologous relationship between mKIAA and KIAA genes in detail, we examined their chromosomal positions and evolutionary rate of synonymous substitutions and confirmed that >93% of the mKIAA/KIAA gene pairs are orthologous. During the sequence analysis of mKIAA genes, we found that 3'-untranslated region (3'-UTR) lengths of mKIAA and KIAA genes are extremely long. In the meanwhile, we have also examined the tissue-specific expression of approximately 1700 mKIAA genes using cDNA microarray and verified predominantly their expression in adult brain (Koga, H., Yuasa, S., Nagase, T., Shimada, K., Nagano, M., Imai, K., Ohara, R., Nakajima, D., Murakami, M., Kawai, M., Miki, F., Magae, J., Inamoto, S., Okazaki, N., Ohara, O. 2004, DNA Res., 11, 293-304). To connect these two evidences, we statistically analysed the relationship between them by using the mKIAA genes. Consequently, a positive correlation was observed between the 3'-UTR lengths and the relative expression intensities in adult brain. Furthermore, we searched sequence elements in the 3'-UTR possibly related with their expression and found some candidates regarding the brain-specific expression.