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The Noto Peninsula Earthquake that occurred on January 1, 2024, caused significant damage to the Noto Peninsula and surrounding areas. This study aims to contribute to evidence-based disaster response by identifying the information needs of the affected areas through a data-driven approach using web search queries. Specifically, the study focuses on the Oku-Noto region in Ishikawa Prefecture, which includes Wajima City, Suzu City, Anamizu Town, and Noto Town. By assigning anomaly scores to web search queries and applying statistical processing such as standardized scores, kurtosis, and skewness, this study elucidates the patterns of changes in information needs in the affected areas. From January 1, 2024, to June 23, 2024, a total of 15,107 search queries were extracted based on anomaly scoring, and 4790 queries were obtained after filtering out irrelevant data. These search queries were classified into five categories: “Hazard and Situation,” “Transportation,” “Critical Infrastructure,” “Coping and Recovery,” and “Daily Life.” The analysis revealed two distinct periods—around 10 days and one month after the disaster—where significant changes in the nature of information needs were observed. Furthermore, information needs related to “Transportation,” particularly road and traffic information, were relatively high, with a notable emphasis on the Noto Satoyama Kaido and Kanazawa Station.

Abstract Background and Aim To date, no randomized trials have compared the efficacy of 7‐day vonoprazan, amoxicillin, and metronidazole triple therapy (VAM) versus 7‐day vonoprazan, amoxicillin, and clarithromycin triple therapy (VAC) as a first‐line treatment for Helicobacter pylori eradication. This study was performed to compare the efficacy of VAM and VAC as first‐line treatments. Methods This prospective multicenter randomized trial was performed in Japan and involved 124 H. pylori‐positive patients without a history of eradication. Patients without antibiotic resistance testing of H. pylori were eligible. The patients were randomized to receive either VAC (vonoprazan 20 mg + amoxicillin 750 mg + clarithromycin 200 or 400 mg twice a day) or VAM (vonoprazan 20 mg + amoxicillin 750 mg + metronidazole 250 mg twice a day) for 7 days, with stratification by age and sex. Eradication success was evaluated using the 13C‐urea breath test. We evaluated safety using patient questionnaires (UMIN000025773). Results The intention‐to‐treat and per‐protocol eradication rates of VAM were 91.3% (95% confidence interval [CI], 82.0–96.7%) and 92.6% (95% CI, 83.7–97.6%), respectively, and those of VAC were 89.1% (95% CI, 77.8–95.9%) and 96.1% (95% CI, 86.5–99.5%), respectively. No significant difference was observed between VAM and VAC in either analysis (P = 0.76 and P = 0.70, respectively). Abdominal fullness was more frequent in patients who received VAM than VAC. Conclusions These findings suggest that VAM as a first‐line treatment in Japan can be categorized as grade B (intention‐to‐treat cure rate of 90–95%) and have potential as a first‐line national insurance ‐approved regimen.

IntroductionParkinson’s disease (PD) is a neurodegenerative and polygenic disorder characterised by the progressive loss of neural dopamine and onset of movement disorders. We previously described eight SINE-VNTR-Alu (SVA) retrotransposon-insertion-polymorphisms (RIPs) located and expressed within the Human Leucocyte Antigen (HLA) genomic region of chromosome 6 that modulate the differential co-expression of 71 different genes including the HLA classical class I and class II genes in a Parkinson’s Progression Markers Initiative (PPMI) cohort.Aims and methodsIn the present study, we (1) reanalysed the PPMI genomic and transcriptomic sequencing data obtained from whole blood of 1521 individuals (867 cases and 654 controls) to infer the genotypes of the transcripts expressed by eight classical HLA class I and class II genes as well as DRA and the DRB3/4/5 haplotypes, and (2) examined the statistical differences between three different PD subgroups (cases) and healthy controls (HC) for the HLA and SVA transcribed genotypes and inferred haplotypes.ResultsSignificant differences for 57 expressed HLA alleles (21 HLA class I and 36 HLA class II alleles) up to the three-field resolution and four of eight expressed SVA were detected at p

Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3’ splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3’ splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3’ splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3’ splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays.

Early child maltreatment, such as child abuse and neglect, is well known to affect the development of social skills. However, the mechanisms by which such an adverse environment interrupts the development of social skills remain unelucidated. Identifying the period and brain regions that are susceptible to adverse environments can lead to appropriate developmental care later in life. We recently reported an excitatory/inhibitory imbalance and low activity during social behavior in the medial prefrontal cortex (mPFC) of the maternal separation (MS) animal model of early life neglect after maturation. Based on these results, in the present study, we investigated how MS disturbs factors related to excitatory and inhibitory neurons in the mPFC until the critical period of mPFC development. Additionally, we evaluated whether the effects of MS could be recovered in an enriched environment after MS exposure. Rat pups were separated from their dams on postnatal days (PDs) 2–20 (twice daily, 3 h each) and compared with the mother-reared control (MRC) group. Gene expression analysis revealed that various factors related to excitatory and inhibitory neurons were transiently disturbed in the mPFC during MS. A similar tendency was found in the sensory cortex; however, decreased parvalbumin (PV) expression persisted until PD 35 only in the mPFC. Moreover, the number of PV+ interneurons decreased in the ventromedial prefrontal cortex (vmPFC) on PD 35 in the MS group. Additionally, perineural net formation surrounding PV+ interneurons, which is an indicator of maturity and critical period closure, was unchanged, indicating that the decreased PV+ interneurons were not simply attributable to developmental delay. This reduction of PV+ interneurons improved to the level observed in the MRC group by the enriched environment from PD 21 after the MS period. These results suggest that an early adverse environment disturbs the development of the mPFC but that these abnormalities allow room for recovery depending on the subsequent environment. Considering that PV+ interneurons in the mPFC play an important role in social skills such as empathy, an early rearing environment is likely a very important factor in the subsequent acquisition of social skills.

Immunity: Crucial cell surface proteins New genetic sequencing and bioinformatic analysis techniques are improving our understanding of the human major histocompatibility complex (MHC), a large and complicated group of genes that encode cell surface proteins crucial to the human immune system. In a review, Jerzy Kulski and co-workers at the Tokai University School of Medicine in Isehara, Japan report that new sequencing technologies have now allowed researchers to map fine-scale and large-scale differences between different subgroups of genes within the MHC that are inherited together, called haplotypes. Study of haplotypes has illuminated the relatedness and evolutionary history of various human ethnic groups. Large-scale bioinformatic studies have revealed associations between MHC genes and disease. These advances will potentially provide better diagnosis or treatment for many diseases, both infectious and autoimmune, and may help improve transplant donor selection.

Abstract Aim The striatum, a main component of the basal ganglia, is a critical part of the motor and reward systems of the brain. It consists of GABAergic and cholinergic neurons and receives projections of dopaminergic, glutamatergic, and serotonergic neurons from other brain regions. Brain‐derived neurotrophic factor (BDNF) plays multiple roles in the central nervous system, and striatal BDNF has been suggested to be involved in psychiatric and neurodegenerative disorders. However, the transcriptomic impact of BDNF on the striatum remains largely unknown. In the present study, we performed transcriptomic profiling of striatal cells stimulated with BDNF to identify enriched gene sets (GSs) and their novel target genes in vitro. Methods We carried out RNA sequencing (RNA‐Seq) of messenger RNA extracted from primary dissociated cultures of rat striatum stimulated with BDNF and conducted Generally Applicable Gene‐set Enrichment (GAGE) analysis on 10599 genes. Significant differentially expressed genes (DEGs) were determined by differential expression analysis for sequence count data 2 (DESeq2). Results GAGE analysis identified significantly enriched GSs that included GSs related to regulation and dysregulation of synaptic functions, such as synaptic vesicle cycle and addiction to nicotine and morphine, respectively. It also detected GSs related to various types of synapses, including not only GABAergic and cholinergic synapses but also dopaminergic and glutamatergic synapses. DESeq2 revealed 72 significant DEGs, among which the highest significance was observed in the apolipoprotein L domain containing 1 (Apold1). Conclusions The present study indicates that BDNF predominantly regulates the expression of synaptic‐function‐related genes and that BDNF promotes synaptogenesis in various subtypes of neurons in the developing striatum. Apold1 may represent a unique target gene of BDNF in the striatum.

Objective Microminipig (MMP) is a miniature pig with an extra small body size for experimental use. In the present study, the occurrence of stillbirths and their genetic association with swine leukocyte antigen (SLA) class II haplotypes were evaluated in a population of MMPs. Methods The occurrences of stillbirth and genetic association with SLA class II haplotypes using 483 stillborn and 2,246 live piglets, and their parents were compared among the three groups of newborn piglet litters; an all stillborn (AS) group consisting of only stillborn piglet litters, a partial stillborn (PS) group consisting of stillborn and live piglet litters, and an all alive (AA) group consisting of only live piglet litters. Results The incidence of stillborn piglets was 483/2,729 (17.7%). Distributions of litter sizes, numbers of stillborn piglets in a litter, parities, and gestation periods were distinct among the three groups. The frequencies of low resolution haplotype (Lr)-0.7 or Lr-0.23 were higher in the AS group than in the PS or AA groups. In sires, the frequency of Lr-0.7 associated with the AS group was significantly higher in the AS group than with the AA group. In dams, the frequency of Lr-0.23 was significantly higher in the AS group than in the PS or AA groups, whereas the frequency of Lr-0.7 was not significantly different. Conclusion The incidence of stillborn piglets in MMPs appears to be higher than those in other pig breeds. Several traits related with stillbirths such as the number of stillborn piglets and parities of the AS group were different from those of the PS and AA groups. Specific SLA class II haplotypes were associated significantly with a high incidence of stillbirths and could be used as genetic markers to adopt breeding strategies to lower the rate of stillbirth in MMPs.

Abstract Background Training for the fundus examination using traditional teaching is challenging, resulting in low generalist physicians’ confidence in performing the funduscopic examination. There is growing evidence suggesting a flexible e-learning video approach’s value in teaching physical examination procedures. However, whether the flexible e-learning video approach is superior to the traditional, face-to-face (F2F) lecture-based teaching for the funduscopic exam and the cognitive processes supporting its effectiveness has not yet been determined. Methods We conducted a sequential explanatory mixed-method study to compare the flexible e-learning video approach’s effectiveness versus the F2F lecture-based approach for teaching the funduscopic exam to medical students at Chiba University in Japan. Medical students were randomly assigned to either a flexible e-learning video approach group or a F2F lecture approach group. We then quantitatively measured the diagnostic accuracy of funduscopic findings before and after attending the specific classrooms. Next, we conducted student focus groups to explore the students’ thinking processes in the flexible e-learning video approach vs. the F2F lecture-based teaching of fundus examination. The qualitative data were analyzed using the qualitative content analysis method. Results The mean diagnostic accuracy scores in the post-test significantly increased from pre-test in the intervention group (36.6 to 63.4%, p

Acute graft-versus-host disease (aGVHD) is defined as a syndrome of an immunological response of graft to the host that occurs early after allogeneic hematopoietic stem cell transplantation (HCT). This disease is frequently observed even in HCT matched for human leukocyte antigen (HLA) alleles at multiple gene loci. Although the HLA region represents complex and diverse genomic characteristics, detailed association analysis is required for the identification of uncharacterized variants that are strongly associated with aGVHD. We genotyped three loci, OR2H2, HLA-F-AS1, and HLA-G, that are located in the 460 kb of HLA telomeric region and statistically analyzed the genotypes including HLA-DPB1 with clinical and transplantation outcomes using 338 unrelated bone marrow transplantation (UR-BMT) patient–donor pairs who were matched for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 (HLA-10/10). Multivariate analyses demonstrated that HLA-F-AS1 and HLA-DPB1 mismatches were associated with grade II–IV aGVHD (hazard ratio (HR), 1.76; 95% CI, 1.07–2.88; p = 0.026; and HR, 1.59; CI, 1.02–2.49; p = 0.042, respectively). There was no confounding between HLA-F-AS1 and HLA-DPB1 (p = 0.512), suggesting that the HLA-F-AS1 mismatch has a strong effect on aGVHD independently of HLA-DPB1. Moreover, a stratified analysis suggested possible associations of HLA-F-AS1, HLA-DPB1, and/or HLA-G mismatches with grade II–IV aGVHD and the more severe grade III–IV aGVHD. These findings provide new insights into understanding the molecular mechanism of aGVHD caused by HLA-matched UR-BMT.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach.Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells.Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Abstract Aim Chromosome 8 open reading frame 46 (C8orf46), a human protein‐coding gene, has recently been named Vexin. A recent study indicated that Vexin is involved in embryonic neurogenesis. Additionally, some transcriptomic studies detected changes in the mRNA levels of patients with psychiatric and neurological diseases. In our previous study, we sought for target genes of brain‐derived neurotrophic factor (BDNF) in cultured rat cortical neurons, finding that BDNF potentially leads to the upregulation of Vexin mRNA. However, its underlying mechanisms are unknown. In the present study, we assessed the regulatory mechanisms of the BDNF‐induced gene expression of Vexin in vitro. Methods We reanalyzed ChIP‐seq data in various human organs provided by the ENCODE project, evaluating acetylation levels of the 27th lysine residue of the histone H3 (H3K27ac) at the Vexin locus. The transcriptomic effects of BDNF on rat Vexin (RGD1561849) were evaluated by real‐time quantitative PCR (RT‐qPCR) in primary cultures of cerebral cortical neurons, in the presence or absence of inhibitors for signaling molecules activated by BDNF. Results The Vexin locus and its promoter region in the brain angular gyrus show higher acetylation levels of the H3K27 than those in other organs. Stimulation of cultured rat cortical neurons, but not astrocyte, with BDNF, led to marked elevations in the mRNA levels of Vexin, which was inhibited in the presence of K252a and U0126. Conclusion The upregulated H3K27ac in the brain may be associated with the enriched gene expression of Vexin in the brain. It is indicated that BDNF induces the gene expression of Vexin in the cortical neurons via the TrkB‐MEK signaling pathway.

Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the CFTR gene locus, of which at least 382 are disease-causing (CFTR2.org). Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all CFTR mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous CFTR locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how in vitro gene insertion methodologies may be adapted for direct in vivo editing.

Behavioral science, the scientific study of human behavior and the elucidation of its laws, is also applied to medicine, and is included in pre‐graduate education.Understanding patient behaviors that correspond to behavior‐based medical diagnosis and interpreting the clinical information suggested by these patient behaviors can be useful in avoiding diagnostic errors in clinical practice.

We have previously reported specific swine leukocyte antigen (SLA) haplotype associations with significant effects on several reproduction performance traits in a highly inbred miniature pig population of Microminipigs (MMPs). In this study, to clarify the effects on farrowing rates of SLA similarity between mating partners in the MMP population, we compared the farrowing rates as a measure of reproductive success after 1063-cumulative matings among the following three groups of mating partners: (1) completely sharing SLA class I or class II haplotypes or alleles between partners (CS), (2) only one sharing the haplotypes or alleles (OS), and (3) non-sharing the haplotypes or alleles (NS). Average farrowing rates in CS groups consisting of completely sharing SLA class II haplotypes or DRBI and DQB1 alleles were lowest in the three groups. Moreover, lower farrowing rates were indicated in mating pairs with smaller amino acid pairwise genetic distances of SLA-1, SLA-3, DRB1 and DQB1 alleles between the pairs. These results suggested that the dissimilarity of SLA class I and class II alleles between mating partners markedly improved reproductive performance; therefore, SLA alleles or haplotypes are potentially useful genetic markers for the selection of mating pairs in breeding programs and epistatic studies of reproductive traits of MMPs.

Summary: Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ‘‘induced basal cells (iBCs)’’ from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine.For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

The major histocompatibility complex (MHC) on chromosome 6p21 is one of the most single-nucleotide polymorphism (SNP)-dense regions of the human genome and a prime model for the study and understanding of conserved sequence polymorphisms and structural diversity of ancestral haplotypes/conserved extended haplotypes. This study aimed to follow up on a previous analysis of the MHC class I region by using the same set of 95 MHC haplotype sequences downloaded from a publicly available BioProject database at the National Center for Biotechnology Information to identify and characterize the polymorphic human leukocyte antigen (HLA)-class II genes, the MTCO3P1 pseudogene alleles, the indels of transposable elements as haplotypic lineage markers, and SNP-density crossover (XO) loci at haplotype junctions in DNA sequence alignments of different haplotypes across the extended class II region (∼1 Mb) from the telomeric PRRT1 gene in class III to the COL11A2 gene at the centromeric end of class II. We identified 42 haplotypic indels (20 Alu, 7 SVA, 13 LTR or MERs, and 2 indels composed of a mosaic of different transposable elements) linked to particular HLA-class II alleles. Comparative sequence analyses of 136 haplotype pairs revealed 98 unique XO sites between SNP-poor and SNP-rich genomic segments with considerable haplotype shuffling located in the proximity of putative recombination hotspots. The majority of XO sites occurred across various regions including in the vicinity of MTCO3P1 between HLA-DQB1 and HLA-DQB3, between HLA-DQB2 and HLA-DOB, between DOB and TAP2, and between HLA-DOA and HLA-DPA1, where most XOs were within a HERVK22 sequence. We also determined the genomic positions of the PRDM9-recombination suppression sequence motif ATCCATG/CATGGAT and the PRDM9 recombination activation partial binding motif CCTCCCCT/AGGGGAG in the class II region of the human reference genome (NC_ 000006) relative to published meiotic recombination positions. Both the recombination and anti-recombination PRDM9 binding motifs were widely distributed throughout the class II genomic regions with 50% or more found within repeat elements; the anti-recombination motifs were found mostly in L1 fragmented repeats. This study shows substantial haplotype shuffling between different polymorphic blocks and confirms the presence of numerous putative ancestral recombination sites across the class II region between various HLA class II genes.

The genomic region (~4 Mb) of the human major histocompatibility complex (MHC) on chromosome 6p21 is a prime model for the study and understanding of conserved polymorphic sequences (CPSs) and structural diversity of ancestral haplotypes (AHs)/conserved extended haplotypes (CEHs). The aim of this study was to use a set of 95 MHC genomic sequences downloaded from a publicly available BioProject database at NCBI to identify and characterise polymorphic human leukocyte antigen (HLA) class I genes and pseudogenes, MICA and MICB, and retroelement indels as haplotypic lineage markers, and single-nucleotide polymorphism (SNP) crossover loci in DNA sequence alignments of different haplotypes across the Olfactory Receptor (OR) gene region (~1.2 Mb) and the MHC class I region (~1.8 Mb) from the GPX5 to the MICB gene. Our comparative sequence analyses confirmed the identity of 12 haplotypic retroelement markers and revealed that they partitioned the HLA-A/B/C haplotypes into distinct evolutionary lineages. Crossovers between SNP-poor and SNP-rich regions defined the sequence range of haplotype blocks, and many of these crossover junctions occurred within particular transposable elements, lncRNA, OR12D2, MUC21, MUC22, PSORS1A3, HLA-C, HLA-B, and MICA. In a comparison of more than 250 paired sequence alignments, at least 38 SNP-density crossover sites were mapped across various regions from GPX5 to MICB. In a homology comparison of 16 different haplotypes, seven CEH/AH (7.1, 8.1, 18.2, 51.x, 57.1, 62.x, and 62.1) had no detectable SNP-density crossover junctions and were SNP poor across the entire ~2.8 Mb of sequence alignments. Of the analyses between different recombinant haplotypes, more than half of them had SNP crossovers within 10 kb of LTR16B/ERV3-16A3_I, MLT1, Charlie, and/or THE1 sequences and were in close vicinity to structurally polymorphic Alu and SVA insertion sites. These studies demonstrate that (1) SNP-density crossovers are associated with putative ancestral recombination sites that are widely spread across the MHC class I genomic region from at least the telomeric OR12D2 gene to the centromeric MICB gene and (2) the genomic sequences of MHC homozygous cell lines are useful for analysing haplotype blocks, ancestral haplotypic landscapes and markers, CPSs, and SNP-density crossover junctions.

It is widely accepted that maternal separation (MS) induces stress in children and disrupts neural circuit formation during early brain development. Even though such disruption occurs transiently early in life, its influence persists after maturation, and could lead to various neurodevelopmental disorders. Our recent study revealed that repeated MS reduces the number of inhibitory neurons and synapses in the medial prefrontal cortex (mPFC) and causes mPFC-related social deficits after maturation. However, how MS impedes mPFC development during early brain development remains poorly understood. Here, we focused on brain-derived neurotrophic factor (BDNF) involved in the development of inhibitory neurons, and examined time-dependent BDNF expression in the mPFC during the pre-weaning period in male rats exposed to MS. Our results show that MS attenuates BDNF expression only around the end of the first postnatal week. Likewise, mRNA expression of activity-regulated cytoskeleton-associated protein (Arc), an immediate-early gene whose expression is partly regulated by BDNF, also decreased in the MS group along with the reduction in BDNF expression. On the contrary, mRNA expression of tropomyosin-related kinase B (TrkB), which is a BDNF receptor, was scarcely altered, while its protein expression decreased in the MS group only during the weaning period. In addition, MS reduced mRNA levels of glutamic acid decarboxylase (GAD) 65, a GABA synthesizing enzyme, only during the weaning period. Our results suggest that repeated MS temporarily attenuates BDNF signaling in the mPFC during early brain development. BDNF plays a crucial role in the development of inhibitory neurons; therefore, transient attenuation of BDNF signaling may cause delays in GABAergic neuron development in the mPFC.

The major histocompatibility complex (MHC) is a highly polymorphic and duplicated genomic region that encodes transplantation and immune regulatory molecules. Although it is well-known that particular MHC allelic polymorphisms and haplotypes are genetically relate to immune-mediated diseases detailed information of the cat MHC (Feline Leukocyte Antigen; FLA) genetic and haplotypic structure and diversity is limited in comparison to humans and many other species. In this study, to better understand the degree and types of allele and allelic haplotype diversity of FLA-class I (FLA-I) and FLA-DRB loci in domestic cats, we identified six expressible FLA-I loci in peripheral white blood cells by in silico estimation of the coding exons and NGS-based amplicon sequencing using five unrelated cats. We then used a newly developed NGS-based genotyping method to genotype and annotate 32 FLA-I and 16 FLA-DRB sequences in two families of 20 domestic cats. A total of 14 FLA-I and seven FLA-DRB were identified as novel polymorphic sequences. Phylogenetic analyses grouped the sequences into six FLA-I (FLA-E/H/K, FLA-A, FLA-J, FLA-L, FLA-O and a tentatively named FLA-E/H/K_Rec) and four FLA-DRB (FLA-DRB1, FLA-DRB3, FLA-DRB4, and FLA-DRB5) lineages. Pedigree analysis of two cat families revealed eight distinct FLA structural haplotypes (Class I – DRB) with five to eight FLA-I and two to three FLA-DRB transcribed loci per haplotype. It is evident that the eight FLA haplotypes were generated by gene duplications and deletions, and rearrangements by genetic recombination with the accumulation and/or inheritance of novel polymorphisms. These findings are useful for further genetic diversity analysis and disease association studies among cat breeds and in veterinary medicine.

ABSTRACT: Background: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. Methods: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. Findings: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). Interpretation: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. Funding: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).

The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10−15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

IntroductionInclusion body myositis (IBM) is an idiopathic inflammatory myopathy, characterized by unique clinical features including finger flexor and quadriceps muscle weakness and a lack of any reliable treatment. The human leukocyte antigen (HLA)-DRB1 allele and autoantibody profiles in Japanese IBM patients have not been fully elucidated.MethodsWe studied 83 Japanese IBM patients with a mean age of 69 years (49 males and 34 females) who participated in the 'Integrated Diagnosis Project for Inflammatory Myopathies' from January 2011 to September 2016. IBM was diagnosed by histological diagnosis. Various autoantibodies were screened by RNA immunoprecipitation and enzyme-linked immunosorbent assays. HLA-DRB1 genotyping was performed using polymerase chain reaction-sequence based typing. A total of 460 unrelated healthy Japanese controls were also studied.ResultsThe allele frequencies of DRB1*01:01, DRB1*04:10, and DRB1*15:02 were significantly higher in the IBM group than in the healthy control group (Corrected P = 0.00078, 0.00038 and 0.0046). There was a weak association between the DRB1*01:01 allele and severe leg muscle weakness and muscle atrophy. While hepatitis type C virus infection and autoantibodies to cytosolic 5'-nucleotidase 1A were found in 18 and 28 patients, respectively, no significant association with HLA-DRB1 alleles was observed.ConclusionJapanese IBM patients had the specific HLA-DRB1 allele and autoantibody profiles.

Abstract In the Mauritian macaque experimentally inoculated with SIV, gene polymorphisms potentially associated with the plasma virus load at a set point, approximately 100 days post inoculation, were investigated. Among the 42 animals inoculated with 50 AID50 of the same strain of SIV, none of which received any preventive or curative treatment, nine individuals were selected: three with a plasma virus load (PVL) among the lowest, three with intermediate PVL values and three among the highest PVL values. The complete genomes of these nine animals were then analyzed. Initially, attention was focused on variants with a potential functional impact on protein encoding genes (non-synonymous SNPs (NS-SNPs) and splicing variants). Thus, 424 NS-SNPs possibly associated with PVL were detected. The 424 candidates SNPs were genotyped in these 42 SIV experimentally infected animals (including the nine animals subjected to whole genome sequencing). The genes containing variants most probably associated with PVL at a set time point are analyzed herein.

Abstract Background The emergence and spread of antibiotic resistance in bacteria is becoming a global public health problem. Combination therapy, i.e., the simultaneous use of multiple antibiotics, is used for long-term treatment to suppress the emergence of resistant strains. However, the effect of the combinatorial use of multiple drugs on the development of resistance remains elusive, especially in a quantitative assessment. Results To understand the evolutionary dynamics under combination therapy, we performed laboratory evolution of Escherichia coli under simultaneous addition of two-drug combinations. We demonstrated that simultaneous addition of a certain combinations of two drugs with collateral sensitivity to each other could suppress the acquisition of resistance to both drugs. Furthermore, we found that the combinatorial use of enoxacin, a DNA replication inhibitor, with Chloramphenicol can accelerate acquisition of resistance to Chloramphenicol. Genome resequencing analyses of the evolved strains suggested that the acceleration of resistance acquisition was caused by an increase of mutation frequency when enoxacin was added. Conclusions Integration of laboratory evolution and whole-genome sequencing enabled us to characterize the development of resistance in bacteria under combination therapy. These results provide a basis for rational selection of antibiotic combinations that suppress resistance development effectively.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

The objective of this study was to develop a prototype of transfemoral prosthesis for swimming focused on the ankle joint motion. This prosthesis has (i) an ankle joint which provides appropriate foot motion, (ii) the capability of walking on land by the poolside, (iii) and a foot whose appearance is sufficiently similar to an actual one. In order to confirm the validity of the developed prosthesis, a swimming experiment, in which a subject swimmer swam in a pool with the developed prosthesis attached, was conducted. The joint angles of the lower limbs were measured and an evaluation with questionnaires was performed. The validity of the proposed prosthesis was experimentally confirmed since the movable ankle joint with an appropriate spring brought a large foot motion, yielding a better swimming performance and more comfort to the swimmer. In addition to the experiment, the simulation of the swimming movement with the prosthesis was conducted. Through the simulation, the general validity of the developed prosthesis was confirmed since the simulation's result exhibited the same tendency as the experiment, in which a spring constant that was too large for the extension spring brought worse swimming performance.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ≃100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

Cystic fibrosis (CF) is the most common lethal inherited disorder and is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The CF lung expresses a profound proinflammatory phenotype that appears to be related to a constitutive hypersecretion of interleukin (IL)-8 from airway epithelial cells in response to microbial infection. Since overproduction of IL-8 in CF contributes to massive bronchial infiltrates of neutrophils, identification of the pathways underlying IL-8 induction could provide novel drug targets for treatment of neutrophil-dominated inflammatory diseases such as CF. Here, we show that IL-17A synergistically increases IL-8 production induced by a toll-like receptor (TLR) 2 agonist, peptidoglycan (PGN), or TLR4 agonist, lipopolysaccharide (LPS), in a human CF bronchial epithelial cell line (CFBE41o-). A strong synergism was also observed in primary human CF bronchial epithelial cells, but not in human non-CF cell lines and primary cells. Notably, despite the induction of nuclear factor-κB and MAP kinases during TLR2 or TLR4 activation in CFBE41o-, IL-17A-dependent synergism appears to be the result of enhanced PGN- or LPS-induced phosphorylation of p38. Taken together, these studies provide evidence that IL-17A is a critical factor in increasing IL-8 expression in bacteria-infected CF airways via a pathway that regulates p38 phosphorylation. Keywords:: interleukin (IL)-17, toll-like receptor 2 and 4 (TLR2/TLR4), IL-8, cystic fibrosis (CF), p38

Abstract Stochastic switching is considered as a cost‐saving strategy for adaptation to environmental challenges. We show here that stochastic switching of a monostable circuit can mediate the adaptation of the engineered OSU12‐hisC Escherichia coli strain to histidine starvation. In this strain, the hisC gene was deleted from the His operon and placed under the control of a monostable foreign promoter. In response to histidine depletion, the OSU12‐hisC population shifted to a higher HisC expression level, which is beneficial under starving conditions but is not favoured by the monostable circuit. The population shift was accompanied by growth recovery and was reversible upon histidine addition. A weak directionality in stochastic switching of hisC was observed in growing microcolonies under histidine‐free conditions. Directionality and fate decision were in part dependent on the initial cellular status. Finally, microarray analysis indicated that OSU12‐hisC reorganized its transcriptome to reach the appropriate physiological state upon starvation. These findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.

The objective of this study was to develop a simulation model for monofin swimming which considers the mechanical interaction between the monofin and swimmer. For this objective, the swimming human simulation model SWUM was extended to the monofin swimming. In order to identify the bending stiffness and damping coefficient of the monofin, the static and dynamic bending tests of a monofin were conducted. From the identification process, it was found that the simulation using the identified parameters reproduced the experiment well. This suggests the validity of the modeling of the monofin itself. The body geometry and joint motion of the swimmer were acquired from the experiment using a subject swimmer. In order to identify the fluid force coefficients of the monofin, the fluid force acting on the monofin was measured in the experiment which was conducted in a circulating water tank. The simulation with the identified parameters reproduced the experiment well. This suggests the validity of the fluid force model for the monofin. From the simulation of monofin swimming using the identified model parameters, it was found that the whole behavior of the swimmer and monofin in the simulation agrees well with that in the experiment, especially for the bending motion of the monofin. The velocities averaged in one kicking cycle in the simulation and experiment also agreed within a 10% error. These results sufficiently suggest the validity of the developed simulation model.

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRβ2. The inhibition by T3-bound TRβ2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHβ gene.

Chloroplasts originated from cyanobacteria through endosymbiosis. The original cyanobacterial endosymbiont evolved to adapt to the biochemically rich intracellular environment of the host cell while maintaining its photosynthetic function; however, no such process has been experimentally demonstrated. Here, we show the adaptation of a model cyanobacterium, Synechocystis sp. PCC 6803, to a biochemically rich environment by experimental evolution. Synechocystis sp. PCC 6803 does not grow in a biochemically rich, chemically defined medium because several amino acids are toxic to the cells at approximately 1 mM. We cultured the cyanobacteria in media with the toxic amino acids at 0.1 mM, then serially transferred the culture, gradually increasing the concentration of the toxic amino acids. The cells evolved to show approximately the same specific growth rate in media with 0 and 1 mM of the toxic amino acid in approximately 84 generations and evolved to grow faster in the media with 1 mM than in the media with 0 mM in approximately 181 generations. We did not detect a statistically significant decrease in the autotrophic growth of the evolved strain in an inorganic medium, indicating the maintenance of the photosynthetic function. Whole-genome resequencing revealed changes in the genes related to the cell membrane and the carboxysome. Moreover, we quantitatively analyzed the evolutionary changes by using simple mathematical models, which evaluated the evolution as an increase in the half-maximal inhibitory concentration (IC50) and estimated quantitative characteristics of the evolutionary process. Our results clearly demonstrate not only the potential of a model cyanobacterium to adapt to a biochemically rich environment without a significant decrease in photosynthetic function but also the properties of its evolutionary process, which sheds light of the evolution of chloroplasts at the initial stage.

High-density DNA microarrays are useful tools for analyzing sequence changes in DNA samples. Although microarray analysis provides informative signals from a large number of probes, the analysis and interpretation of these signals have certain inherent limitations, namely, complex dependency of signals on the probe sequences and the existence of false signals arising from non-specific binding between probe and target. In this study, we have developed a novel algorithm to detect the single-base substitutions by using microarray data based on a thermodynamic model of hybridization. We modified the thermodynamic model by introducing a penalty for mismatches that represent the effects of substitutions on hybridization affinity. This penalty results in significantly higher detection accuracy than other methods, indicating that the incorporation of hybridization free energy can improve the analysis of sequence variants by using microarray data.

We made an H1N1 vaccine candidate from a virus library consisting of 144 ( = 16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naïve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.

Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR.

To understand how two organisms that have not previously been in contact can establish mutualism, it is first necessary to examine temporal changes in their phenotypes during the establishment of mutualism. Instead of tracing back the history of known, well-established, natural mutualisms, we experimentally simulated the development of mutualism using two genetically-engineered auxotrophic strains of Escherichia coli, which mimic two organisms that have never met before but later establish mutualism. In the development of this synthetic mutualism, one strain, approximately 10 hours after meeting the partner strain, started oversupplying a metabolite essential for the partner's growth, eventually leading to the successive growth of both strains. This cooperative phenotype adaptively appeared only after encountering the partner strain but before the growth of the strain itself. By transcriptome analysis, we found that the cooperative phenotype of the strain was not accompanied by the local activation of the biosynthesis and transport of the oversupplied metabolite but rather by the global activation of anabolic metabolism. This study demonstrates that an organism has the potential to adapt its phenotype after the first encounter with another organism to establish mutualism before its extinction. As diverse organisms inevitably encounter each other in nature, this potential would play an important role in the establishment of a nascent mutualism in nature.

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate the performance of these sequencing technologies, investigation of the quality of sequence reads obtained from these methods is important. In this study, we analyzed the quality of sequence reads and SNP detection performance using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Genome Analyzer (GA), and Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample obtained from Escherichia coli strain DH1 was applied to these sequencers. The obtained sequence reads were aligned to the complete genome sequence of E. coli DH1, to evaluate the accuracy and sequence bias of these sequence methods. We found that the fraction of "junk" data, which could not be aligned to the reference genome, was largest in the data set of SOLiD, in which about half of reads could not be aligned. Among data sets after alignment to the reference, sequence accuracy was poorest in GA data sets, suggesting relatively low fidelity of the elongation reaction in the GA method. Furthermore, by aligning the sequence reads to the E. coli strain W3110, we screened sequence differences between two E. coli strains using data sets of three different next-generation platforms. The results revealed that the detected sequence differences were similar among these three methods, while the sequence coverage required for the detection was significantly small in the FLX data set. These results provided valuable information on the quality of short sequence reads and the performance of SNP detection in three next-generation sequencing platforms.

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression.

It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2°C or 4°C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9°C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.