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Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host’s high adult emergence and improved body colour; these were called ‘mutualistic’ E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of ‘mutualistic’ E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host–microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

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.

Immune-mediated necrotizing myopathy (IMNM) is a type of autoimmune myositis typically characterized clinically by proximal muscle weakness with elevated creatine kinase levels, pathologically by myofiber necrosis and regeneration with paucity of lymphocytic cell infiltration, and serologically by the presence of either of two myositis-specific autoantibodies, anti-SRP and anti-HMGCR antibodies. However, the HLA loci and alleles associated with IMNM are still not fully understood at least partly because IMNM was a relatively recently established condition. In this study, we genotyped the six HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) in 250 patients (237 patients over age 18 years and 13 juvenile patients) diagnosed with IMNM based on clinicopathological features and autoantibody information and performed a case control study with Japanese healthy subjects. In the adult patients, specific HLA alleles associated with IMNM were identified at all HLA loci, with DRB1*08:03 showing the strongest association (OR=2.5; p=0.00000017). Furthermore, subgroup analysis with various clinical information showed that C*03:04 (OR=3.7; P=0.00012) was a higher risk allele for collagen disease in adult patients, and B*13:01 (OR=23.2; P=0.021) and C*03:04 (OR=5.8; P=0.0074) were higher risk for juvenile patients with anti-HMGCR antibody-positive IMNM. These findings will help to better understand the HLA genetic background and features of IMNM in designing future studies. This article is protected by copyright. All rights reserved.

Three novel HLA-DRA alleles, DRA*01:03, DRA*01:04, and DRA*01:05 alleles with unique amino acid sequences.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing. © 2020 The American Society of Gene and Cell TherapySuzuki et al. report correction of the CFTR defect in cystic fibrosis airway basal stem cells. They utilized gene-editing strategies either specific for the ΔF508 CFTR mutation or applicable to most CFTR mutations. Both approaches yielded highly efficient correction without selection, restoring CFTR function to therapeutically relevant levels.

HLA sequence-based DNA typing (SBT) by long-range PCR amplification (LR PCR) and next-generation sequencing (NGS) is a high-throughput DNA sequencing method (LR-NGS-SBT) for the efficient and sensitive detection of novel and null HLA alleles to the field-4 level of allelic resolution without phase ambiguity. However, the accuracy and reliability of the HLA typing results using buccal cells (BCs) and saliva as genetic source materials for the LR-NGS-SBT method are dependent largely on the quality of the extracted genomic DNA (gDNA) because a large degree of gDNA fragmentation can result in insufficient PCR amplification with the incorrect assignment of HLA alleles because of allele dropouts. In this study, we developed a new cost-efficient swab storage gel (SSG) for wet swab collection of BCs (BC-SSG) and evaluated its usefulness by performing different DNA analytical parameters including LR-NGS-SBT to compare the quality and quantity of gDNA extracted from BCs (in SSG or air dried), blood and saliva of 30 subjects. The BC-SSG samples after 5 days of storage revealed qualitative and quantitative DNA values equivalent to that of blood and/or saliva and better than swabs that were only air-dried (BC-nSSG). Moreover, all the gDNA extracted from blood, saliva and BC-SSG samples were HLA-typed successfully to an equivalent total of 408 alleles for each sample type. Therefore, the application of BC-SSG collection media for LR-NGS-SBT has benefits over BC dried samples (dry swabs) such as reducing retesting and the number of untestable BC samples because of insufficient DNA amplification.

Three novel HLA-A*02:06:01:04, -DRB1*12:02:01:02, -DQB1*03:01:01:07 and five extended HLA-B*51:01:05, -DRB1*12:02:01:01, -DRB1*14:01:01, -DRB1*14:04:01:01, -DRB1*15:04 alleles.

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.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3718-2) contains supplementary material, which is available to authorized users.

Many significant gene mutations in E. coli have contributed to the development of genetics. Among these, a commonly used recA mutation, Δ(srl-recA)306 has been sequenced by a next-generation sequencer combined with a long PCR. An original report described that Δ(srl-recA)306 cells were deleted from srlR to recA genes in their genome. The next-generation sequencer enables more accurate details to be determined. We ask whether both surrounding genes from hypF to norV for srlR and alaS for recA is there first. The long PCR was carried out with primers, norR and alaS, and amplified DNA fragments differed in length from wild to Δ(srl-recA)306 cells, suggesting that an entire Δ(srl-recA)306 mutation was included. Sequences of those DNA fragments indicated that 9147 bp, from srlR to recA including 10 genes, were replaced by a Tn10 DNA sequence. Junction points at both srlR-Tn10 and Tn10-recA were determined precisely. The results indicate that the first 97% of recA gene sequences were lost with a downstream recX gene remaining intact. The phenotypic difference between Δ(srl-recA)306 and ΔrecA::Km is discussed.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here we report the directed differentiation of human iPSCs into airway basal cells (“iBCs”), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1(GFP);TP63(tdTomato)) we track and purify these cells as they first emerge as developmentally immature NKX2-1(GFP+) lung progenitors and subsequently augment a TP63 program during subsequent proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1(GFP+)/ TP63(tdTomato+) cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multilineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Recent studies have reported loss of heterozygosity in the chromosome 6p arms (6pLOH) of acquired aplastic anemia (AA) patients, and in tumor cells trying to escape the autoimmune system. We thus sought to establish detection methods for LOH to investigate the mechanisms underlying AA and tumor immunity. Herein, we report our evaluation of 6pLOH in a patient with severe AA patient using super-high resolution, single-molecule, sequence-based typing (SS-SBT). The highest ratios of 6pLOH detection were observed during the patient's treatment with granulocyte colony stimulating factor (G-CSF). This result suggested that most of the neutrophil precursor cells stimulated by G-CSF already had LOH in the HLA lesion. The SS-SBT method is a simple NGS method that provides complete HLA allele coverage.

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.

Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome is characterized by congenital absence of the vagina and uterus. We conducted genome-wide SNP analyses and exome sequencing to detect the causes of MRKH syndrome. We identified de novo variants of MYCBP2, NAV3, and PTPN3 in three families and a variant of MYCBP2 in a sporadic case. Here, we demonstrated the partial genetic makeup of Japanese MRKH syndrome.

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. This work was supported by the “Investissements d’Avenir” programs managed by the ANR under reference ANR-11-INBS-0008 funding the Infectious Disease Models and Innovative Therapies (IDMIT, Fontenay-aux-Roses, France) infrastructure, and ANR-10-EQPX-02-01 funding the FlowCyTech facility (IDMIT, Fontenay-aux-Roses, France). We warmly thank all members of ASW and L2I core lab facility from IDMIT center. TMB is supported by MINECO BFU2014-55090-P (FEDER), U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya.

BackgroundAlopecia areata (AA) is a highly heritable multifactorial and complex disease. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a region in the human genome known to be associated with AA as compared to other regions.ResultsBy sequencing MHC risk haplotypes, 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. Using CRISPR/Cas9 for allele-specific genome editing, we then phenocopied AA symptomatic patched hair loss in mice engineered to carry theCchcr1risk allele. Skin biopsies of these alopecic mice showed strong up-regulation of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). Using transcriptomics findings, we further identified CCHCR1 as a novel component of hair shafts and cuticles in areas where the engineered alopecic mice displayed fragile and impaired hair.ConclusionsThese results suggest an alternative mechanism for the aetiology of AA based on aberrant keratinization, in addition to generally well-known autoimmune events.

In adaptive evolution, an increase in fitness to an environment is frequently accompanied by changes in fitness to other environmental conditions, called cross-resistance and sensitivity. Although the networks between fitness changes affect the course of evolution substantially, the mechanisms underlying such fitness changes are yet to be fully elucidated. Herein, we performed high-throughput laboratory evolution of Escherichia coli under various stress conditions using an automated culture system, and quantified how the acquisition of resistance to one stressor alters the resistance to other stressors. We demonstrated that resistance changes could be quantitatively predicted based on changes in the transcriptome of the resistant strains. We also identified several genes and gene functions, for which mutations were commonly fixed in the strains resistant to the same stress, which could partially explain the observed cross-resistance and collateral sensitivity. The integration of transcriptome and genome data enabled us to clarify the bacterial stress resistance mechanisms.

HLA-DPB1 T-cell epitope (TCE) mismatching algorithm and rs9277534 SNP at the 3' untranslated region (3'UTR) in the HLA-DPB1 gene are key factors for transplant-related events in unrelated hematopoietic cell transplantation (UR-HCT). However, the association of these 2 mechanisms has not been elucidated. We analyzed 19 frequent HLA-DPB1 alleles derived from Japanese healthy subjects by next-generation sequencing of the entire HLA-DPB1 gene region and multi-SNP data of the HLA region in 1589 UR-HCT pairs. The risk of acute graft-versus-host disease (aGVHD) was analyzed in 1286 patients with single HLA-DPB1 mismatch UR-HCT. The phylogenetic tree constructed using the entire gene region demonstrated that HLA-DPB1 alleles were divided into 2 groups, HLA-DP2 and HLA-DP5. Although a phylogenetic relationship in the genomic region from exon 3 to 3'UTR (Ex3-3'UTR) obviously supported the division of HLA-DP2 and HLA-DP5 groups, which in exon 2 showed intermingling of HLA-DPB1 alleles in a non-HLA-DP2 and non-HLA-DP5-group manner. Multi-SNP data also showed 2 discriminative HLA-DPB1 groups according to Ex3-3'UTR. Risk of grade 2-4 aGVHD was significantly higher in patient HLA-DP5 group mismatch than patient HLA-DP2 group mismatch (hazard ratio, 1.28; P = .005), regardless of donor mismatch HLA-DP group. Regarding TCE mismatch, increasing risk of aGVHD in patient HLA-DP5 group mismatch and TCE-nonpermissive mismatch were observed only in patients with TCE-permissive mismatch and patient HLA-DP2 group mismatch, respectively. Evolutionary analysis revealed that rs9277534 represented a highly conserved HLA-DPB1 Ex3-3'UTR region and may provoke aGVHD differently to TCE mismatching algorithm, reflecting exon 2 polymorphisms. These findings enrich our understanding of the mechanism of aGVHD in HLA-DPB1 mismatch UR-HCT.

Bacterial cells have a remarkable capacity to adapt and to evolve to environmental changes. Although many mutations contributing to adaptive evolution have been identified, the relationship between the mutations and the phenotypic changes responsible for fitness gain has yet to be fully elucidated. For a better understanding of phenotype-genotype relationship in evolutionary dynamics, we performed high-throughput laboratory evolution ofEscherichia coliunder various stress conditions using an automated culture system. One measure of phenotype, transcriptome analysis, revealed that the expression changes which occurred during the evolution were generally similar among the strains evolved in the same stress environment. We also found several genes and gene functions for which mutations were commonly fixed in the strains resistant to the same stress, and whose effects on resistance were verified experimentally. We demonstrated that the integration of transcriptome and genome data enables us to extract the mechanisms for stress resistance.Author summaryUnderstanding the relationship between phenotypic and genetic changes is a fundamental goal in evolutionary biology, which can provide insights into the past and future evolutionary trajectories. Evolution of microorganisms in a laboratory has been the primary approach to clarify the mappings of phenotypic and genotypic changes. Here, we performed high-throughput laboratory evolution with bacteria using an automated culture system, to quantify phenotypic and genotypic changes occurred under various stress conditions. We identified various stress-specific gene expression changes and mutations, and contributions of them to fitness gain were validated. These results demonstrated that the integration of phenotypic and genotypic changes makes it possible to extract the mechanisms for stress resistance evolution, which will contribute to bioengineering applications.

The common marmoset (Callithrix jacchus) is a New World monkey that is used frequently as a model for various human diseases. However, detailed knowledge about the MHC is still lacking. In this study, we sequenced and annotated a total of 854 kb of the common marmoset MHC region that corresponds to the HLA-A/G/F segment (Caja-G/F) between the Caja-G1 and RNF39 genes. The sequenced region contains 19 MHC class I genes, of which 14 are of the MHC-G (Caja-G) type, and 5 are of the MHC-F (Caja-F) type. Six putatively functional Caja-G and Caja-F genes (Caja-G1, Caja-G3, Caja-G7, Caja-G12, Caja-G13, and Caja-F4), 13 pseudogenes related either to Caja-G or Caja-F, three non-MHC genes (ZNRD1, PPPIR11, and RNF39), two miscRNA genes (ZNRD1-AS1 and HCG8), and one non-MHC pseudogene (ETF1P1) were identified. Phylogenetic analysis suggests segmental duplications of units consisting of basically five (four Caja-G and one Caja-F) MHC class I genes, with subsequent expansion/deletion of genes. A similar genomic organization of the Caja-G/F segment has not been observed in catarrhine primates, indicating that this genomic segment was formed in New World monkeys after the split of New World and Old World monkeys.

Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.

To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88–DLA-12 and DLA-88–DLA-88L, were identified, and the novel haplotype DLA-88–DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. Recently, genotyping of the polymorphisms of MHC genes using targeted next-generation sequencing (NGS) technologies was developed for humans and some nonhuman species. Most species have numerous highly homologous MHC loci so the NGS technologies are likely to replace traditional genotyping methods in the near future for the investigation of human and animal MHC genes in evolutionary biology, ecology, population genetics, and disease and transplantation studies. In this chapter, we provide a short review of the use of targeted NGS for MHC genotyping in humans and nonhuman species, particularly for the class I and class II regions of the Crab-eating Macaque MHC (Mafa).

Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1∼BTN1∼BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers - CjHEP21, CjTRIM39.2 and CjBTN2 - in the TRIM subregion that may be useful for the functional analysis of the MHC-Coja region. We examined MHC-Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes - CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail-specific VNTR (variable number of long tandem repeats, 133-137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

The swine is an important animal model for allo- and xeno-transplantation donor studies, which necessitates an extensive characterization of the expression and sequence variations within the highly polygenic and polymorphic swine leukocyte antigen (SLA) region. Massively parallel pyrosequencing is potentially an effective new 2ndGen method for simultaneous high-throughput genotyping and detection of SLA class I gene expression levels. In this study, we compared the 2ndGen method using the Roche Genome Sequencer 454 FLX with the conventional method using sub-cloning and Sanger sequencing to genotype SLA class I genes in five pigs of the Clawn breed and four pigs of the Landrace breed. We obtained an average of 10.4 SLA class I sequences per pig by the 2ndGen method, consistent with the inheritance data, and an average of only 6.0 sequences by the conventional method. We also performed a correlation analysis between the sequence read numbers obtained by the 2ndGen method and the relative expression values obtained by quantitative real-time PCR analysis at the allele level. A significant correlation coefficient (r = 0.899, P

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.

In the current study, we describe four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. The putative proteins encoded by these quail genes contained motifs considered essential for HSP90 gene function. In addition, the predicted proteins were more similar to HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 proteins expressed in vertebrates than they were to other members of the HSP90 family. Exon numbers of CjHSP90AA1 (11), CjHSP90AB1 (12) or CjTRAP1 (18) are the same as the chicken and mammalian orthologs. Furthermore, gene order in the regions surrounding CjHSP90AB1 and CjTRAP1 has been preserved, providing evidence that the genomic regions were orthologous to HSP90-containing regions in the chicken genome. The promoter regions of the genes also contained conserved motifs identified in related genes of chicken. However, the nucleotide sequences of the 5'-flanking region of these genes were highly polymorphic. We also found that CjHSP90AA1 exhibited a robust response to heat shock treatment. Taken together, the data suggest that CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1 encode orthologs of HSP90AA1, HSP90AB1, HSP90B1 and TRAP1, respectively.

Motivation: High-density DNA microarrays provide us with useful tools for analyzing DNA and RNA comprehensively. However, the background signal caused by the non-specific binding (NSB) between probe and target makes it difficult to obtain accurate measurements. To remove the background signal, there is a set of background probes on Affymetrix Exon arrays to represent the amount of non-specific signals, and an accurate estimation of non-specific signals using these background probes is desirable for improvement of microarray analyses. Results: We developed a thermodynamic model of NSB on short nucleotide microarrays in which the NSBs are modeled by duplex formation of probes and multiple hypothetical targets. We fitted the observed signal intensities of the background probes with those expected by the model to obtain the model parameters. As a result, we found that the presented model can improve the accuracy of prediction of non-specific signals in comparison with previously proposed methods. This result will provide a useful method to correct for the background signal in oligonucleotide microarray analysis. Availability: The software is implemented in the R language and can be downloaded from our website (http://www-shimizu.ist.osaka-u.ac.jp/shimizu_lab/MSNS/). Contact: furusawa@ist.osaka-u.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online.

Background: the acquisition of antibiotic resistance in bacterial cells is often accompanied with a reduction of fitness in the absence of antibiotics, known as the “fitness cost”. The magnitude of this fitness cost is an important biological parameter that influences the degree to which antibiotic resistant strains become widespread. However, the relationship between the fitness cost and comprehensive phenotypic and genotypic changes remains unclear. Here, we quantified the fitness cost of resistant strains obtained by experimental evolution in the presence of various antibiotics, and analyzed how the cost correlated to phenotypic and genotypic changes in the resistant strains. Results: we measured the specific growth rate of the resistant strains in the presence of various concentrations of drugs or in their absence. In the absence of drugs, the resistant strains showed reductions of approximately 20% to 50% in growth rate compared with the parent strain, which corresponded to the fitness cost. We found that the decrease of the specific growth rate was correlated with overall expression changes between the parent and resistant strains, measured by the Euclid distance between expression profiles. We also found that there are a number of genes whose changes in expression levels were significantly correlated with the growth rate, which may account for the observed correlation between the fitness cost and overall expression changes. Conclusions: our analysis provides a basis for quantitative understanding of the mechanism of the fitness cost. This understanding may provide clues on how to influence the fitness cost that accompanies resistance acquisition and consequently how to limit the spread of antibiotic resistant strains.

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., Author Summary Mutations that have little influence on biological function are referred to as neutral mutations and frequently appear in molecular phylogenetic analyses. The fixation of neutral mutations in populations has been attributed to genetic drift in fitness-steady evolutionary processes or hitchhiking in adaptive evolution. We examined the fitness-increasing evolution of Escherichia coli for thermal adaptation to observe the fixation dynamics of genome-wide mutations. In the adaptive evolution, all genomes in the population equally accumulated neutral mutations by replication errors. The infrequent occurrence of an adaptive mutation on one of the genomes by chance resulted in the fixation of the neutral mutations that had pre-accumulated in the same genome by hitchhiking. Via successive hitchhiking events, the neutral mutations were fixed in the population linearly over generations at the same rate as the spontaneous mutation accumulation rate in the genome. The molecular clock of neutral mutations thus functions even in adaptive evolution. The evolutionary period characterized by the accumulation of numerous neutral mutations observed in molecular phylogenetic trees may not be specific to neutral evolution but may occur in adaptive evolution as well.

Although the low polymorphism of the major histocompatibility complex (MHC) transplantation genes in the Filipino cynomolgus macaque (Macaca fascicularis) is expected to have important implications in the selection and breeding of animals for medical research, detailed polymorphism information is still lacking for many of the duplicated class I genes. To better elucidate the degree and types of MHC polymorphisms and haplotypes in the Filipino macaque population, we genotyped 127 unrelated animals by the Sanger sequencing method and high-resolution pyrosequencing and identified 112 different alleles, 28 at cynomolgus macaque MHC (Mafa)-A, 54 at Mafa-B, 12 at Mafa-I, 11 at Mafa-E, and seven at Mafa-F alleles, of which 56 were newly described. Of them, the newly discovered Mafa-A8*01:01 lineage allele had low nucleotide similarities (

We obtained a series of methicillin-resistant Staphylococcus aureus isolates, including both daptomycin-susceptible strain TD1 and daptomycin-resistant strain TD4, from a patient. We determined the complete genome sequences of TD1 and TD4 using next-generation sequencing, and only four single-nucleotide polymorphisms (SNPs) were identified, one each in capB (E58K), rpoB (H481Y), lytN (I16V), and mprF (V351E). We determined that these four SNPs were sufficient to cause the strains to develop daptomycin, vancomycin, and rifampin resistance.

Background HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1. Results We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template. Conclusions In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.

Although many mutations contributing to antibiotic resistance have been identified, the relationship between the mutations and the related phenotypic changes responsible for the resistance has yet to be fully elucidated. To better characterize phenotype–genotype mapping for drug resistance, here we analyse phenotypic and genotypic changes of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution. We demonstrate that the resistances can be quantitatively predicted by the expression changes of a small number of genes. Several candidate mutations contributing to the resistances are identified, while phenotype–genotype mapping is suggested to be complex and includes various mutations that cause similar phenotypic changes. The integration of transcriptome and genome data enables us to extract essential phenotypic changes for drug resistances., The relationship between mutations and phenotypic changes associated with drug resistance in bacteria remains unclear. Here, the authors use antibiotic-resistant E. coli strains, obtained by laboratory evolution, to show that resistance profiles can be predicted by changes in expression of a few genes.