Development of a novel monoclonal antibody that binds to most HLA-A allomorphs in a conformation-dependent yet peptide-promiscuous fashion.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months.

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

Novel method for evaluating the health condition of mice in space through a video downlink.

Clarification of the criteria for managing animal health is essential to increase the reliability of experiments and ensure transparency in animal welfare. For experiments performed in space, there is no consensus on how to care for animals owing to technical issues, launch mass limitation, and human resources. Some biological processes in mammals, such as musculoskeletal or immune processes, are altered in the space environment, and mice in space can be used to simulate morbid states, such as senescence acceleration. Thus, there is a need to establish a novel evaluation method and evaluation criteria to monitor animal health. Here, we report a novel method to evaluate the health of mice in space through a video downlink in a series of space experiments using the Multiple Artificial-gravity Research System (MARS). This method was found to be more useful in evaluating animal health in space than observations and body weight changes of the same live mice following their return to Earth. We also developed criteria to evaluate health status via a video downlink. These criteria, with "Fur condition" and "Respiratory" as key items, provided information on the daily changes in the health status of mice and helped to identify malfunctions at an early stage. Our method and criteria led to the success of our missions, and they will help establish appropriate rules for space experiments in the future.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Identification and characterization of coding single-nucleotide polymorphisms within human protocadherin-alpha and -beta gene clusters.

The human protocadherin (Pcdh) gene clusters are located on chromosome 5q31. Single-nucleotide polymorphisms (SNPs) were detected in the Pcdh-alpha and -beta variable exons, and in the Pcdh-alpha constant exon, in samples from 104 individuals. Among coding SNPs (cSNPs), nonsynonymous (amino acid exchange) SNPs were 2.2 times more common than synonymous (silent) changes in the Pcdh-alpha variable exons, but only 1.2 times more common in the Pcdh-beta variable exons. The nonsynonymous SNPs were high in the ectodomain (EC) 1 encoding region of Pcdh-alpha but not of Pcdh-beta. One 48-kb region of extensive linkage disequilibrium (LD) is reported that has two haplotypes extending from the alpha1 to alpha7 genes in the Pcdh-alpha cluster. Here we identified 15 amino acid exchanges in these two major haplotypes; therefore, the two haplotypes encode different sets of Pcdh-alpha proteins in the brain. The distribution of cSNPs was different for each EC region of Pcdh-alpha or -beta. The frequency of cSNPs was negatively correlated with the paralogous sequence diversity. These results suggested that gene conversion events in homologous regions of the Pcdh-alpha and Pcdh-beta clusters generated the cSNPs. Within the cSNPs, gene conversions were found in Pcdh-alpha4 in the major haplotype, and in Pcdh-beta9. These gene conversions were caused by the unequal crossing-over of homologous sequence regions. Thus, nonsynonymous variations in the Pcdh-alpha and -beta genes are possible contributors to the variations in human brain function.

Evolutionary and developmental understanding of the spinal accessory nerve.

The vertebrate spinal accessory nerve (SAN) innervates the cucullaris muscle, the major muscle of the neck, and is recognized as a synapomorphy that defines living jawed vertebrates. Morphologically, the cucullaris muscle exists between the branchiomeric series of muscles innervated by special visceral efferent neurons and the rostral somitic muscles innervated by general somatic efferent neurons. The category to which the SAN belongs to both developmentally and evolutionarily has long been controversial. To clarify this, we assessed the innervation and cytoarchitecture of the spinal nerve plexus in the lamprey and reviewed studies of SAN in various species of vertebrates and their embryos. We then reconstructed an evolutionary sequence in which phylogenetic changes in developmental neuronal patterning led towards the gnathostome-specific SAN. We hypothesize that the SAN arose as part of a lamprey-like spinal nerve plexus that innervates the cyclostome-type infraoptic muscle, a candidate cucullaris precursor.

Genomic organization and transcripts of the zebrafish Protocadherin genes.

We have examined the protocadherin (Pcdh) gene clusters of the zebrafish (Danio rerio). At least three sets of the Pcdh gene cluster were found in the zebrafish genome. Here, we describe the complete organization of the DrPcdh2 gene clusters. Classification by phylogenetic and transcript analyses revealed 7 DrPcdh2omicron, 20 DrPcdh2alphaa, 12 DrPcdh2alphab, and 1 DrPcdh2alphac variable exons upstream of the DrPcdh2alpha constant region exons in the DrPcdh2 gene cluster. The constant regions of the DrPcdh1alpha and DrPcdh2alpha genes in zebrafish were orthologs of those of the mammalian Pcdhalpha. These exons all encoded plural PXXP motifs in their cytoplasmic tails. The sequences of the variable exons were highly conserved within each family: DrPcdh2omicron, DrPcdh2alphaa, and DrPcdh2alphab. Transcript analysis revealed that zebrafish Pcdhs had alternatively spliced variants in the constant region that were not found in mammals. More gene clusters, more variable exons, and more alternative splicing variants were found in zebrafish than in mammals. Thus, although the Pcdhalpha families were common to diverse vertebrates, their gene number, structure, and transcripts were different between teleosts and mammals.