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Summary During murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity through the repression of Rheb in a post-transcriptional manner., Graphical abstract, Highlights • Single-cell RNA-seq data recapitulated sexual differentiation processes • Cell cycle state of developing germ cells was revealed by single-cell RNA-seq data • NANOS2 suppresses mTORC1 activators to induce cell cycle arrest of germ cells, Biological sciences; Molecular biology; Developmental biology

Summary The genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies., Graphical Abstract, Highlights • Genome-wide CRISPR screening identifies naive pluripotency regulators in mouse ESCs • mTORC1-negative regulators from two axes show opposing phenotypes • Gator1 is required for proper self-renewal and differentiation via Gsk3 regulation • Tsc2 loss causes Akt-dependent, mTORC1-dependent Gsk3 suppression, Li et al. conducted genome-wide CRISPR screens in mouse ESCs to identify genes affecting maintenance of and exit from naive pluripotency using a Rex1GFP reporter. They show that loss of two mTORC1-negative regulators, Tsc1/2 and Gator1, can cause opposing phenotypes through differential regulation of Gsk3 activity.

Summary The Palaeognathae comprise the flightless ratites and the volant tinamous, and together with the Neognathae constitute the extant members of class Aves. It is commonly believed that Palaeognathae originated in Gondwana since most of the living species are found in the Southern Hemisphere [1–3]. However, this hypothesis has been questioned because the fossil paleognaths are mostly from the Northern Hemisphere in their earliest time (Paleocene) and possessed many putative ancestral characters [4]. Uncertainties regarding the origin and evolution of Palaeognathae stem from the difficulty in estimating their divergence times [1, 2] and their remarkable morphological convergence. Here, we recovered nuclear genome fragments from extinct elephant birds, which enabled us to reconstruct a reliable phylogenomic time tree for the Palaeognathae. Based on the tree, we identified homoplasies in morphological traits of paleognaths and reconstructed their morphology-based phylogeny including fossil species without molecular data. In contrast to the prevailing theories, the fossil paleognaths from the Northern Hemisphere were placed as the basal lineages. Combined with our stable divergence time estimates that enabled a valid argument regarding the correlation with geological events, we propose a new evolutionary scenario that contradicts the traditional view. The ancestral Palaeognathae were volant, as estimated from their molecular evolutionary rates, and originated during the Late Cretaceous in the Northern Hemisphere. They migrated to the Southern Hemisphere and speciated explosively around the Cretaceous-Paleogene boundary. They then extended their distribution to the Gondwana-derived landmasses, such as New Zealand and Madagascar, by overseas dispersal. Gigantism subsequently occurred independently on each landmass.

Prehistoric hunter–gatherers in Northeast Asia lived along coastlines and made abundant use of coastal resources in their subsistence strategies. However, the extent to which they operated only along the coastline or sailed out into deeper waters for hunting and fishing remains rather uncertain. In this case-study, we reconstruct past subsistence strategies through analysis of albatross (Diomedeidae) remains recovered from two hunter–gatherer archaeological sites in Hokkaido, Northern Japan (the Funadomari site, from the late Jomon period on Rebun Island and the Bentenjima site, from the Okhotsk period on Bentenjima Island). Three questions are examined: (1) were the birds hunted for meat or for feathers; (2) were the birds procured at coastal breeding grounds or out on the open water; (3) was hunting conducted in the shallow waters of the continental shelf or out in the deeper ocean? Analysis of the bone assemblages, and species determination by ancient DNA, indicate that people must have been hunting the bird in the open waters at the edge of the continental shelf. This provides strong, although indirect, evidence that Jomon and Okhotsk Culture communities must have possessed ocean-going boats and suitable sea-faring skills in order to undertake these kinds of operations. These insights, in turn, hint at the existence of sophisticated maritime traditions in prehistoric Northeast Asia.

Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.

In this study, 14C analysis by accelerator mass spectrometry (AMS) was applied to age estimation based on temporal variations in bomb-produced-14C contents of a full elephant tusk registered at Kyushu University. The tusk measured 175 cm long and 13.8 cm in diameter at the root. Thirty tusk-fragment samples were used for 14C analysis with AMS to estimate the formation ages of different positions according to catalogued global 14C contents (F14C). The F14C value of the tip of the tusk suggested that the elephant was born around 1980, while that of the root suggested death around 1994, a lifespan of at least 14 years, rather shorter period than the average lifetime of an elephant (ca. 80 years). In addition, the F14C values of fragments collected along a cross-sectional line suggested that the outer part of the tusk formed first with inner parts being deposited gradually with growth.

Greater White-fronted Goose Anser albifrons has a holarctic breeding distribution and is polymorphic. Three subspecies winter in the Palaearctic region, one of which also winters in the Nearctic region: European White-fronted Goose A. a. albifrons breeds in the far north of Europe and Asia and winters in the south and west of Europe; Pacific White-fronted Goose A. a. frontalis breeds in east Siberia and Arctic Canada and winters in East Asia and United States; and Greenland White-fronted Goose A. a. flavirostris breeds in Greenland and winters in Ireland and western Scotland. The phylogenetic relationships among these three subspecies are unclear. We determined the mitochondrial DNA control region sequences of Pacific Whitefronted Goose, using 66 shed feathers collected from wintering sites in Japan, and compared the sequences with those previously published for Greater White-fronted Goose subspecies. Phylogenetic trees and networks revealed that there are three clades within the species. The seq...

SummaryIn 2011 the Black-faced Spoonbill Platalea minor census recorded a 22% drop in numbers from 2010, particularly at the known large coastal wintering sites. During this period, we discovered two new inland wintering sites for the species using satellite telemetry data, one located in China, where the individual followed the Yangtze river as far as Wuhan (500 km inland), and the other across the Vietnam-Cambodia border (70–200 km inland). Long periods of concentrated use of various freshwater habitats were in evidence for these two tagged individuals (China: 79 days, Vietnam/Cambodia: 91 Days) and in the latter case visual confirmation indicated a larger group of at least five individuals. The importance and potential of inland freshwater environments is highlighted towards the further conservation of the recovering population.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer., Author Summary The mutation load of human tissues is unknown and represents the genetic divergence from the fertilised egg. Reprogramming of somatic cells generates induced pluripotent stem cells (iPSCs), a cell type being considered for clinical applications. We generated iPSCs from tissues of healthy individuals and used whole genome sequencing to identify in vivo mutations accrued in a somatic cell during the lifetime of the individual. Next we identified in vitro mutations introduced during reprogramming and cell culture. Each has a unique mutation signature suggesting different mutagenic processes. Our study demonstrates the use of reprogramming as a tool to elucidate mutational processes within normal cells and highlights the importance of genetic characterisation of iPSCs prior to clinical translation.

The horseshoe crab Tachypleus tridentatus is critically endangered in Japan due to rapidly decreasing numbers resulting from the loss of tidal flats and sandy beaches, and the deterioration of coastal environments. We monitored the year-round migratory patterns and residency of this species in a coastal embayment at Tsuyazaki, Japan, using acoustic telemetry. Total 20 adult crabs (15 males and 5 females) were tagged with ultrasonic transmitters and tracked during two periods (2006–2008; n = 10 and 2007–2009; n = 10). Adult crabs were more active during periods of higher water temperatures and their activity peaked in July, during the spawning period. Water temperature appeared to be one of the key factors influencing the movement patterns for the species. Moreover, the crabs tended to be more active at night than in the day. The nocturnal activity pattern was clearly evident before and during the reproductive period (May–August). Tracking data also showed that one pair-bond was maintained for a maximum of 17 days after the pair-bonded female had spawned. Overall, 11 males (73% of 15 individuals) remained in the bay area over winter, whereas three females (60% of 5 individuals) overwintered outside of the bay. Telemetry data showed that over 60% (13 of 20) of tagged crabs overwintered within the bay where there are sandy beaches, mudflats, and scattered seagrass beds. This year-round residence by adult T. tridentatus in the bay area identifies it as a critical habitat for the management of this species, regardless of life-stage. Not only is it a comprehensive management strategy that effectively reflects this species’ habitat use patterns but also its implementation, such as the establishment of a protected area, would contribute to its conservation.

The history of population structure is a key to effective wildlife management and conservation. However, inferring the history of population structure using present genetic structures is problematic when the method is applied to species that have experienced severe population bottlenecks. Ancient DNA analysis seemed to be a promising, direct method for inferring ancient population structures. However, the usual methods for inferring modern population structure, i.e. the phylogeographic approach using mitochondrial DNA and the Bayesian approach using microsatellite DNA, are often unsuitable for ancient samples. In this study, we combined ancient DNA obtained from zooarchaeological bones with carbon/nitrogen stable isotope ratios and morphological variations to infer ancient population structure of the short-tailed albatross Phoebastria albatrus. The results showed that the bird existed in two populations, between which the genetic distance was greater than that of distinct sister albatross species, although no subspecies of P. albatrus have been proposed. Our results suggest that the birds at the present two breeding regions (Torishima in the Izu Islands and two islets of the Senkaku Islands) are descended from these two ancient populations, and that reevaluation of the status and conservation strategy for the species is required. Our results also indicate that lineage breeding on the Senkaku Islands has drastically reduced genetic diversity, while that on Torishima has not. The approach proposed in this study would be useful for inferring ancient population structure, using samples of highly mobile animals and/or samples from archaeological sites, and the reconstructed ancient population structure would be useful for conservation and management recommendations.

Banded iron formations are the most characteristic of Archean–Paleoproterozoic sediment records. Laminated textures resembling banded iron formations can be observed in modern hot-spring environments. Using sedimentological and microbiological techniques, we investigated the processes of laminar formation and considered the origin of lamination textures. An iron-rich deposit at the Okuoku-hachikurou hot spring in Japan exhibits sub-millimeter laminations consisting of bacteria-induced ferrihydrite and aragonite. The ferrihydrite particles are spherical and exhibit fine lamination, up to 100 µm thick in ferrihydrite-rich parts. In aragonite-rich parts, ferrihydrite particles form filamentous textures with diameters of 10–30 µm, but not laminations. Textural analysis using scanning electron microscopy and phylotype analysis using 16S rRNA indicated the bacterial contribution to ferrihydrite precipitation. A sheath-like fabric showing a meshwork of nanometer-order organic filaments, and sheath-forming bacteria were observed in the deposit specimen etched by citric acid. Phylotype analysis detected in the iron-rich deposits some bacterial types related to cyanobacteria, purple bacteria, and iron-oxidizing bacteria. Iron-oxidizing bacteria probably were responsible for precipitation of the ferrihydrite. Chemolithoautotrophic iron-oxidizing bacteria are microaerophilic and thrive on Fe(II) in a redox gradient, but dissolved oxygen was not detected in the Okuoku-hachikurou hot spring. Thus, a certain supply of oxygen is needed for metabolism of the microaerophilic iron-oxidizing bacteria. The distribution of photosynthetic pigments in the iron-rich parts indicates that the most likely source of oxygen is photosynthesis by cyanobacteria. This symbiotic relationship between cyanobacteria and iron-oxidizing bacteria can explain the laminated texture of iron-rich deposits in the Okuoku-hachikurou hot spring. These laminations may reflect changes in photosynthetic intensity. There is presently some debate about the bacterial groups that may have played roles in precipitation of banded iron formations. This study presents a new bacterial model for iron precipitation and may provide a mechanism for sub-millimeter laminations in banded iron formations deposited in shallow water.

An aragonite travertine at Nagano-yu hot spring, SW Japan, exhibits clear sub-millimeter-order lamination that resembles ancient ministromatolites. Thirty-three hours of continuous observation showed that the lamination is formed daily with no changes in physicochemical properties except light intensity. Phylotype analysis and fluorescence in situ hybridization indicate that Hydrogenophaga sp. is dominant and concentrated in diurnal layers containing abundant extracellular polymeric substances. Growth of Hydrogenophaga sp. is activated in the daytime, likely due to extracellular polymeric substance production by cyanobacterial photosynthesis. Daytime development of Hydrogenophaga-dominant biofilms, and the concurrent inhibiting effect on aragonite precipitation, explains the daily lamination observed.

ヒシクイAnser fabalisは北極圏で夏季に繁殖し，温帯域で越冬する．形態学的観点から，日本には東北地方の太平洋側で主に亜種ヒシクイが，北陸地方の日本海側と霞ヶ浦周辺で主にオオヒシクイA. f. middendorffii が越冬し，出雲平野で越冬する個体群の亜種は不明とされてきた．本研究では，4ヶ所の越冬地（宮城県・化女沼，新潟県・朝日池，福井県・坂井平野，島根県・出雲平野）で脱落羽毛を採集し，ミトコンドリアDNA・制御領域の塩基配列を決定して，先行研究と比較した．その結果，化女沼の試料はすべて亜種ヒシクイなどの系統に，朝日池の試料はすべてオオヒシクイの系統に含まれ，形態学的観点からの亜種区分を支持する結果となった．一方，亜種不明とされてきた出雲平野の試料は遺伝的にはすべてオオヒシクイの系統に含まれ，また，従来亜種ヒシクイが殆ど確認されていなかった坂井平野でも亜種ヒシクイの系統に含まれる試料が認められた．

Genetic diversity estimation of the major histocompatibility complex (MHC) gene may be an important tool in the assessment of immune response ability against infectious disease. We were able to identify a near full-length expressed DQB sequence by RACE-PCR method from the Asiatic black bear, Ursus thibetanus in Japan. This is the first such full length expression in the Ursidae. The bear had at least one functional DQB locus. In phylogenetic tree analysis its DQB amino acid sequence formed a monophyletic group with DQB sequences from members of the order Carnivora and had a 90% nucleotide sequence similarity with the DQB allele of the California sea lion, Zalophus californianus. We compared the DQB amino acid composition of U. thibetanus with those of several other mammalian species including Homo sapiens. Amino acid residues known to be functionally important for human MHC genes, tended to be also conserved among other mammalian species while PBRs in the beta1 domain were heterogeneous among mammalian species. The DQB sequence obtained from the bear had not only no putative frameshifts or deletions but also no abnormal amino acid mutations such as had been observed in human DQB molecules. This suggests that the bear DQB sequence was an apparently functional DQB allele. As a preliminary study, we sequenced the exon 2 region of DQB alleles from genomic DNA, and succeeded to amplify the exon 2 of DQB loci. Our study will provide useful information for conservation genetics of the U. thibetanus as well as more generally regarding the mammalian MHC region.

In the late part of the nineteenth century and the early part of the last century, the short-tailed albatross Phoebastria albatrus was in danger of extinction owing to feather hunting. In the middle of the last century, the total number of this species was inferred to be approximately 50-60 with breeding occurring only on Torishima Island of the Izu Islands. Recently, the number of individuals has increased to more than 2,000 and that of their breeding islands to three, namely, Torishima Island, and Minami- and Kita-kojima Islands of the Senkaku Islands. Here, we show that the 44 short-tailed albatrosses we examined represent 29 haplotypes in the control region of mitochondrial DNA, and have a considerably higher genetic diversity than most avian species, but not very high in albatross species; the h and π were 0.96 and 0.013, respectively. However, the parsimony network clearly showed that many intermediate haplotypes were lost. It was concluded that the majority of the haplotypes in the founder population have been maintained. Judging from these findings and the exponential increase in the number of individuals, the present population of the short-tailed albatross seems not to be affected by inbreeding depression through a severe bottleneck. The conservation and expansion of their breeding grounds, and effective protection from bycatch mortality in foraging areas are important for the future survival of this species.

The present study was designed to investigate whether octacalcium phosphate (OCP)-precipitated alginate (Alg) promotes osteoblastic cell proliferation and bone regeneration in comparison with Alg itself. Alg, known to lack mammal cell attachment capability, was used as the matrix to test the distribution effect of OCP in a three-dimensional environment. A series of Alg/OCP scaffolds with different pore sizes was prepared by centrifuging Alg gels precipitated by OCP crystals. The scaffolds had a bimodal distribution of pores (ultrafine pores: approximately 100 nm; relatively large pores: from 6.0 to 51.7 microm) and over 86% porosity. The osteoconductive capability of Alg/OCP was determined by examining mouse bone marrow stromal ST-2 cell proliferation after 3 days in vitro and bone regeneration in mouse calvaria critical-sized defect after 21 days. The analyses showed that ST-2 cell proliferation and bone regeneration increased with an increase in the pore size and reached the highest level in the 51.7 microm pore scaffold. The results suggest that OCP-precipitated Alg provides a better scaffold for osteoblasts to attach and proliferate in a three-dimensional environment and promotes bone regeneration, indicating that OCP is a candidate material to modify the surface of non-cell-interactive polymeric scaffolds, such as Alg, into an osteogenic condition.

The genetic structure of the Asiatic black bear (Ursus thibetanus) in Japan was studied to understand the events that occurred during its evolution. The left domain of the mitochondrial control region (about 240 bp) was sequenced, defining 27 haplotypes that consisted of 23 haplotypes from 333 bears in Japan and 22 bears in the Asian continent. The network tree of the control region indicated that the Japanese population formed a distinct clade from the continental population. The phylogeographic analysis of the haplotypes indicated that the Shikoku and Kii Hanto populations had diverged during the initial phase from the ancestral population. After the 3 dominant haplotypes were rapidly distributed throughout Japan in the early stage of the population dispersal, the Japanese population diverged into eastern and western populations. Using the entire mitochondrial cytochrome b sequence, divergence time between the Japanese and the Continental populations suggested that the Japanese population might have colonized into Japan through the land bridge from the Korean Peninsula around 500 ka, which is consistent with paleontological evidence. Our finding that bears in western Japan exhibit lower genetic diversity and higher levels of genetic differentiation than bears in eastern Japan provides a vital contribution to conservation policy for these isolated populations.

The estimated population of the Tsushima leopard cat Prionailurus bengalensis euptilura is only 80–110 individuals. However, the cause of the population decline is not clear. We investigated temporal changes in the food habits of the cat and two other species of Carnivora (marten and weasel) inhabiting the Tsushima Islands by measuring δ13C and δ15N values in hair samples. Hair samples of the cat were collected not only from specimens and furs, but also from feces. The gathering of hair from cat feces was most efficient when the feces were collected in the spring. The food habit of male cats seemed to be more diverse and tended to comprise prey of higher trophic levels than the food habits of the females. The δ13C and δ15N measurements suggested that the trophic level of the food sources has been decreasing over the last several decades for the cat and weasel, but not for the marten. Increased consumption of prey from lower trophic levels in the food habit of the cat seems to be related to the decline of the cat population because these phenomena occurred simultaneously.

The Black-footed Albatross Diomedea nigripes is an endangered seabird that is endemic to the North Pacific. The genetic structure of Black-footed Albatross populations on three of the Hawaiian Islands and on Izu-Torishima Island, Japan, has been studied previously, using the mitochondrial cytochrome b region. Hawaiian and Japanese breeding groups are genetically different, and the genetic diversity of the birds on Izu-Torishima is lower than that of birds in the Hawaiian Islands. We analyzed 50 Black-footed Albatrosses from the Bonin Islands, where a relatively stable population persisted throughout the twentieth century. Although albatrosses in the Bonin Islands do not differ significantly from those on Izu-Torishima in their cytochrome b region sequences, they do exhibit higher genetic diversity (as high as those from the Hawaiian colonies). A statistical parsimony network revealed two clades, one primarily in the western North Pacific colonies and the other primarily in eastern North Pacific c...

Although angiotensin II (Ang II) type 1 receptor antagonist ameliorates proteinuria, its pharmacological mechanism and the differential roles of Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R) are not well understood. We analyzed the effect of Ang II type 1 receptor antagonist on proteinuria caused by antibody against nephrin, a functional molecule of glomerular slit diaphragm and dysfunction of which is involved in the development of proteinuria in several glomerular diseases. We show here that AT1R antagonist ameliorated proteinuria by preventing a reduction in the functional molecules of the slit diaphragm. We also analyzed the role of AT1R- or AT2R-mediated actions on the expression of the slit diaphragm molecules in an in vivo study of normal rat and in an in vitro study of cultured podocytes. AT1R-mediated action hampered the mRNA expression of the slit diaphragm molecules, whereas AT2R-mediated action enhanced it. These findings indicate that Ang II receptor subtypes play opposite roles in regulating the barrier function of glomerular capillary wall and that the enhancement of AT2R stimulation may serve as a potential therapeutic strategy for proteinuria.

The tri-spine horseshoe crab Tachypleus tridentatus, known as a living fossil and endangered, is distributed from coastal Southeast Asia to western Japan. The Japanese population represents the northernmost range for this species. To examine the genetic structure and diversity of the Japanese population, over 600 samples from ten localities were analyzed using mitochondrial DNA AT-rich region sequences as a maternal inheritance marker and over 250 samples using nucleic compound microsatellite loci as biparental markers. Genetic subdivisions were detected in Japanese populations using both markers. Two genetically distinct groups, the western and the eastern group, were detected and six tentative local management units were defined. Locality and genetic units should be taken into account for conservation of Japanese populations.

Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glomerular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.

The Major Histocompatibility Complex (MHC) is a large multigene coding for glycoproteins that play a key role in the initiation of immune responses in vertebrates. The exon 2 region of the MHC DQB locus was analyzed using 160 finless porpoises from 5 populations in Japanese waters. The 5 populations were based on a previous mitochondrial DNA control region analysis, which showed distinct geographical separation. Eight DQB alleles were detected, and the geographical distribution of the alleles indicated that most of them are shared among the populations. Heterozygosity of the DQB alleles in each population ranged from 0.55 to 0.78, and for all 5 populations was 0.78. Low MHC variability is not a common feature in marine mammals, but the finless porpoise populations inhabiting coastal waters had a relatively high MHC heterozygosity. Balancing selection in the MHC DQB alleles of the finless porpoise was indicated by the higher rate of nonsynonymous than synonymous substitutions for PBR; however, an excess of hetrozygotes compared to expectation was not observed. This suggests that the MHC DQB locus in the finless porpoise may have been under balancing selection for a long evolutionary time period, and is influenced by genetic drift beyond the effect of balancing selection for short time periods in small local populations.

Recent investigations have revealed the importance of glomerular podocytes with its diaphragm as the major filtration barrier. Junctional adhesion molecule 4 (JAM4) has been identified as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 and is reported to be expressed on podocytes. To elucidate the role of JAM4 on podocytes, we examined the expression of JAM4 and MAGI-1 in normal and two different proteinuric rat models: puromycin aminonucleoside (PAN) nephropathy and anti-nephrin antibody-induced (ANA) nephropathy, one model with and one without effacement of podocyte foot processes. JAM4 was detected by immunomicroscopy at the apical membrane of normal podocytes. JAM4 immunostaining was focally increased in the podocytes in PAN nephropathy but not in ANA nephropathy. In proteinuric podocytes, the expression of JAM4 was distinct from that of MAGI-1 or other slit diaphragm molecules such as nephrin and ZO-1. Close colocalization of JAM4 and ezrin was maintained in PAN nephropathy. By immunoelectron microscopy, the signals for JAM4 were detected at the free apical membrane of the podocytes with effaced foot processes. Studies with selective detergent extract revealed that the subcellular localization of JAM4 was altered in PAN nephropathy. Thus the altered expression of JAM4 appears to be associated with morphological changes in podocytes and can be a useful marker of injured podocytes. JAM4 may have a different role at the apical membrane besides the role as a junctional molecule and is likely associated with the unique structure of this epithelium.

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was reported recently to have several additional biologic activities. In this study, the pathophysiologic role of IP-10 in the glomerular visceral epithelial cell (podocyte) was investigated. In cultured podocytes subjected to recombinant IP-10 treatment, the expression of slit-diaphragm (SD) components nephrin and podocin clearly was heightened. Rats that had puromycin aminonucleoside nephropathy and anti-nephrin antibody-induced nephropathy and were subjected to anti-IP-10 function-blocking antibody (anti-IP-10 mAb) treatment displayed a decrease in the protein level of SD components, as well as exacerbated proteinuria. For exploration of the mechanisms of this process, the interaction between IP-10 and the cell-cycle regulatory proteins was investigated. Cultured podocytes subjected to recombinant IP-10 treatment displayed an increase in the protein level of p27(Kip1), whereas the levels of cyclins E and A decreased. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A, whereas these expressions were lowered by the treatment of siRNA of p27(Kip1). Proteinuric rats subjected to anti-IP-10 mAb treatment displayed a heightened expression of cyclin A from the early phase of the disease, which indicates that the anti-IP-10 mAb treatment exacerbates podocyte injury by disturbing the cell-cycle balance. These results raise the possibility that IP-10 could become a novel therapeutic target in nephrotic syndrome and several diseases with altered cell-cycle balance.

Background/Aims: Multi-glycoside from Tripterygium wilfordii Hook f. (GTW) is used for various immune and inflammatory diseases including renal diseases represented by mesangial proliferative glomerulonephritis (MsPGN) in China. However, there have been no fundamental studies on the operating mechanism of GTW on MsPGN. The aim of this study is to examine as the first step the effects of GTW on acute injurious process such as mesangial injury and proteinuria in an acute and reversible Thy.1.1 glomerulonephritis (Thy1.1GN) model and then to clarify the action mechanism of GTW at molecular level by examining its effects on various injurious factors in this model. Methods: Thy1.1 GN was induced in rats by a single intravenous injection with 500 µg of anti-Thy1.1 mAb 1-22-3. Daily oral administration of GTW and vehicle as a control was started from 3 days before injection of mAb to the day of sacrifice in each experiment. Fourteen rats were randomly divided into 2 groups, GTW-treated and vehicle-treated groups, and sacrificed on day 14 in experiment 1 or on day 7 in experiment 2 after induction of Thy1.1 GN. Proteinuria was determined on days 1, 3, 5, 7, 10 and 14 in experiment 1 or on 1, 3, 5 and 7 in experiment 2. From blood and kidneys taken at sacrifice, blood biochemical parameters, mesangial morphological changes, glomerular macrophage infiltration, and glomerular mRNA expression of cytokines were examined. Results: In experiment 1, proteinuria and mesangial matrix expansion were significantly attenuated by GTW treatment. In experiment 2, GTW treatment significantly ameliorated proteinuria, mesangial lesions and macrophage accumulation in glomerulus. In addition, it significantly reduced the glomerular expression of mRNA for PDGF, MCP-1 and IL-2. Conclusion: GTW ameliorated not only proteinuria but also mesangial alterations in Thy1.1 GN most likely by reducing expression of injurious cytokines, indicating that GTW has suppressive effects on acute inflammatory changes in glomeruli.

A technique of accelerator mass spectrometry (AMS) has made it possible to directly measure radiocarbon ages of pottery by isolating organic materials sealed in the pottery when the pottery was formed. We analyzed the carbon contents and 14C ages for black pottery from the Philippines and fiber pottery from Japan using the relevant carbonaceous materials extracted from the pottery samples, i.e., adhered chaff or grass fibers that were incorporated in the pottery matrix, respectively. The carbon yield of the pottery sample varied largely depending on the pottery types, the preservation conditions, as well as the chemical pretreatment methods to purify carbonaceous materials for 14C dating. We will discuss criteria for sample selection of well-preserved pottery, and a modified method, instead of the standard alkali treatment, to obtain sufficient material for precise 14C dating.

Putative nucleotide sequences for DQB exon 2 alleles of the major histocompatibility complex (MHC) were amplified and determined in the 16 cetacean species using the PCR technique. The 172 bp sequences amplified showed no multiple alleles more than two in each of the examined individuals. The sequences of the 31 cetacean DQB alleles detected were monophyletic with the HLA-DQB1, and were separated from the lineages of HLA- DQB2 and DQB3. These results suggest that the locus is the homolog to the human DQB1 gene. The observed frequency of nonsynonymous substitutions in the cetacean DQB sequences was significantly higher than that of synonymous substitutions. The amino acid variation at the putative peptide binding region (PBR) was considerably high. These results imply that positive selection has promoted its variability at the cetacean DQB gene as other mammalian MHC. The DQB gene tree showed that four Mysticeti alleles branched off from the clade consisted of Mysticeti alleles only and were included in the clade consisted of Odontoceti alleles. This suggests trans-species polymorphism in the cetacean MHC gene.

The SRY (sex determining region on the Y chromosome) gene has an important role in sex determination and shows a strict paternal mode of inheritance. To better understand evolutionary changes in the gene structure and their phylogenetic utility, complete sequences of the SRY region (591-651 bp) spanning from the initiation codon (ATG) to the stop codon of TAG were determined in 20 cetacean species, covering two families of suborder Mysticeti and six families of suborder Odonticeti. The HMG (high mobility group) box (231 bp), a DNA binding motif, was highly conserved in the cetacean SRY sequences. In contrast, seven synonymous substitutions and 12 nonsynonymous substitutions were detected in the N-terminal region (156 bp). The C-terminal region was heterogeneous in length (216-261 bp). One base insertion at the end of the HMG box, which resulted in a frame-shift in the C-terminal region, was detected in superfamily Delphinoidea. Further sequence determination (171-175 bp) in the 5' flanking region of the SRY gene revealed the presence of an element of "GGGGGCGG", the consensus motif of Sp1 (specificity protein 1), in the predicted promoter region. Paternal lineage relationships inferred from these SRY sequences were concordant with previous phylogenetic inferences in the literature, using other gene markers.

Atotal of 62 mitochondrial haplotypes were detected from 174 samples of hazel grouse Bonasa bonasia from Hokkaido, Primorskii, Sakhalin, Magadan, Siberia and Bohemia using 428 bp of the mitochondrial DNA control region. Haplotype diversity for four populations in Hokkaido was more than 0.8, suggesting that a reasonable population size had been maintained throughout their history. Haplotypes from Hokkaido and haplotypes from Primorskii were clearly separated from other Eurasian continent haplotypes, not only in the phylogenetic tree but also in the network tree. Haplotypes from Hokkaido, Sakhalin and Magadan radiated from the hypothetical root composed of a double cubic network of parallel substitutions. Most of the haplotypes were separated by three substitutions from the root, or within a maximum of five substitutions. Pairwise sequence differences for most Eurasian haplotypes had a bimodal curve consisting of the first peak at 0-1 substitution differences and the second peak at 3-4 substitution differences, whereas those for Hokkaido haplotypes had only a peak at around 4-6 substitution differences. These observations most likely indicate that the populations analysed were differentiated about 40, 000 years ago, and have expanded to the present distribution during the climatic optimum over the last 10, 000 years.

It is well established that the glomerular capillary wall consists of three layers: endothelial cell, glomerular basement membrane, and the slit diaphragm bridging foot processes of glomerular epithelial cell. Which structure in the glomerular capillary wall represents the primary filter for retaining plasma proteins is not clearly elucidated. An anti-slit diaphragm monoclonal antibody (mAb) 5-1-6 causes massive proteinuria in rats by single intravenous injection, which clearly indicates that the slit diaphragm plays a critical role for maintaining the barrier function of the glomerular capillary wall. Recently, we concluded that mAb 5-1-6 recognized a rat homolog of nephrin, a gene product of NPHS1. The expression of nephrin decreased in puromycin aminonucleoside nephropathy and adriamycin nephropathy as well as mAb 5-1-6-induced nephropathy, which suggested that nephrin was involved in the development of proteinuria in these proteinuric states. In mAb 5-1-6 nephropathy, the slit diaphragm was maintained morphologically normal, although nephrin expression dramatically decreased. The finding suggested that nephrin was not a sole component of the slit diaphragm. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin.

The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.

Podocyte injuries exacerbate mesangial proliferative glomerulonephritis. Background From the observations of morphology seen in early phases of the experimental models of the irreversible mesangial proliferative glomerulonephritis, we hypothesized that podocyte injury is one of the important factors in bringing upon irreversible glomerular alterations. To verify this hypothesis, we investigated whether podocyte injury induced by puromycin aminonucleoside (PAN) injection affects the mesangial alterations of anti-Thy 1.1 glomerulonephritis. Methods Female Wistar rats were injected with 0.5mg monoclonal antibody (mAb) 1-22-3 five days after the injection of 10mg or 5 mg/100g body weight (BW) of puromycin aminonucleoside (PAN), and sacrificed at 7 days or 8 weeks after the mAb 1-22-3 injection. Results Consecutive injections of 10 mg/100g BW of PAN and mAb 1-22-3 caused the irreversible mesangial alteration with persistent proteinuria (at week 8, proteinuria 100.3 ± 57.8 mg/24 h, matrix score 1.13 ± 0.52, collagen type I score 2.04 ± 0.53, mRNA for collagen type I 227 ± 79% to the group with a single injection of 1-22-3). Although single injection of 5 mg/100g BW of PAN was not capable of inducing abnormal proteinuria, consecutive injections of 5 mg/100g BW of PAN and mAb 1-22-3 also caused irreversible mesangial alteration and persistent proteinuria. Conclusions Podocyte injury might be an important factor that exacerbates mesangial proliferation and mesangial matrix expansion. The irreversible mesangial alterations caused by consecutive injections of PAN and mAb 1-22-3 may be a novel model that could be used to analyze the mechanism of progressive mesangial alteration.

Background: The crescent-forming mechanism has not yet been fully clarified and a cell which constitutes a crescent still remains controversial. This study was undertaken to analyze the crescent-forming mechanism in an irreversible Thy-1 model by applying a new marker-recognizing monoclonal antibody (mAb) OS-3. Methods: An irreversible Thy-1 model was induced by an intravenous injection of 500 µg of anti-Thy-1 mAb 1-22-3 to unilaterally nephrectomized Wistar rats. Seven rats were sacrificed 3, 7 and 14 days after the mAb injection respectively and the renal tissues were examined histologically and immunohistochemically. Results: Inflammatory cells were demonstrated mostly in the interstitium, but they were located within advanced cellular crescents in later stages. OS-3, which stained parietal glomerular epithelial cell (PGEC) only partly in a normal rat kidney section, reacted to PGEC more extensively at day 3 and also with cellular crescents at day 7. During the course of this model the podocytes lost their characteristic to be stained by anti-podocalyxcin Ab and obtained a new marker of a diseased state, i.e. to be positively stained by OS-3. Conclusion: Glomerular epithelial cells, but not inflammatory cells, are suggested to directly participate in the crescent formation in early stages, and podocytes with phenotypic changes might be partly involved in the formation of the crescents.

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

The tri-spine horseshoe crab, Tachypleus tridentatus, known as “a living fossil”, is distributed from coastal Southeast Asia to western Japan. The Japanese population represents the northernmost range for this species. As is probably the case for almost of all the Asian T. tridentatus populations, in Japan it is facing near extinction and lies in urgent need of protective conservation measures. To examine the genetic structure and the genetic diversity of the Japanese population, eight novel compound microsatellite loci were developed by the intercompound microsatellite method. The number of alleles ranged from 2 to 4 per locus. The observed and expected heterozygosities were between 0.38 and 0.66, and between 0.36 and 0.67, respectively. All 32 individuals analyzed had unique genotype with 8 loci and were considered useful as conservation genetic markers for this endangered species.