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During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.

Background: The AR-V7 splice variant is a cause of castration-resistant prostate cancer (CRPC). However, testing for the presence of AR-V7 by real-time polymerase chain reaction (RT-PCR) shows AR-V7 positivity in healthy individuals. We hypothesized that the positivity reflects contamination by hematopoietic cells. We tried a novel circulating tumor cell (CTC) enrichment instrument, using Celsee, to clear hematopoietic cells. Methods: We tested whole blood or Celsee-enriched samples for AR-V7 by RT-PCR, and included samples from 41 CRPC patients undergoing sequential therapy. We evaluated the associations between AR-V7 status and clinical factors. We evaluated factors affecting AR-V7 positivity. Results: AR-V7 positivity was lower in Celsee-enriched than in whole blood specimens. AR-V7 and clinical factors did not predict the therapy effectiveness. We found no significant differences in the effectiveness of enzalutamide/abiraterone (Enz/Abi) upon AR-V7 evaluation. All AR-V7 positive patients had resistance to Enz/Abi. Docetaxel (DTX), cabazitaxel (CBZ), and Radium223 treatment showed no significant difference in the treatment effectiveness, regardless of AR-V7 presence. AR-V7 was more frequently positive than Extent of disease (EOD) 2 in cases with bone metastases. Conclusion: Celsee CTC enrichment suppresses AR-V7 false positivity. All AR-V7 positive patients presented resistance to Enz/Abi. DTX, CBZ, and Radium223 were effective and remain treatment options. AR-V7 positivity should progressively appear in patients with advanced bone metastases.

Background: The AR-V7 splice variant is a cause of castration-resistant prostate cancer (CRPC). However, testing for the presence of AR-V7 by RT-PCR shows AR-V7 positivity in healthy individuals. We hypothesized that the positivity reflected contamination by hematopoietic cells. We tried a novel CTC enrichment instrument using Celsee® to clear hematopoietic cells. Methods: We tested whole blood or Celsee-enriched samples for AR-V7 by RT-PCR and included samples from 41 CRPC patients undergoing sequential therapy. We evaluated the associations between AR-V7 status and clinical factors. We evaluated factors affecting AR-V7 positivity. Results: AR-V7 positivity was lower in Celsee-enriched than in whole blood specimens. AR-V7 and clinical factors did not predict the therapy effectiveness. We found no significant differences in the effectiveness of Enz/Abi upon AR-V7 evaluation. All AR-V7 positive patients had resistance to Enz/Abi. DTX, CBZ, and Radium223 treatment showed no significant difference in the treatment effectiveness, regardless of AR-V7 presence. AR-V7 was more frequently positive than EOD2 in cases with bone metastases. Conclusion: Celsee® CTC enrichment suppresses the AR-V7 false positivity. All AR-V7 positive patients presented resistance to Enz/Abi. DTX, CBZ, and Radium223 were effective and remained treatment options. AR-V7 positivity should progressively appear in patients with advanced bone metastases.

Diplonemids were recently found to be the most species-rich group of marine planktonic protists. Based on phylogenetic analysis of 18S rRNA gene sequences and morphological observations, we report the description of new members of the genus Rhynchopus - R. humris sp. n. and R. serpens sp. n., and the establishment of two new genera - Lacrimia gen. n. and Sulcionema gen. n., represented by L. lanifica sp. n. and S. specki sp. n., respectively. In addition, we describe the organism formerly designated as Diplonema sp. 2 (ATCC 50224) as Flectonema neradi gen. n., sp. n. The newly described diplonemids share a common set of traits. Cells are sac-like but variable in shape and size, highly metabolic, and surrounded by a naked cell membrane, which is supported by a tightly packed corset of microtubules. They carry a single highly reticulated peripheral mitochondrion containing a large amount of mitochondrial DNA, with lamellar cristae. The cytopharyngeal complex and flagellar pocket are contiguous and have separate openings. Two parallel flagella are inserted sub-apically into a pronounced flagellar pocket. Rhynchopus species have their flagella concealed in trophic stages and fully developed in swimming stages, while they permanently protrude in all other known diplonemid species.

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

The deep-sea clam Calyptogena okutanii possesses a large gill containing vertically transmitted symbiotic sulfur-oxidizing bacteria. It produces large amounts of highly viscoelastic mucus from the gill, which is thought to be a physical and chemical barrier. The mucus collected from the gill was shown to be composed of glycoproteins having the following sugar composition: Man (17.4%), GlcNAc (16.6%), GalNAc (15%), Glc (1.1%), Gal (29.9%), Xyl (3.0%), Fuc (14.4%), and unknown (2.6%), indicating that it contained mucin-like glycoproteins. In a monoclonal antibody library against the gill tissue, we found a monoclonal antibody (mAb), CokG-B3C10, reacting to the mucus. Western blot analysis using the mAb showed that it reacted to several glycoproteins in the mucus from the gill tissue, but not with those of other tissues such as the mantle, foot, and ovary, where mucus production has been reported in bivalves. Further, immunohistochemical analysis showed the CokG-B3C10 mAb reacting to glycoproteins was detected in the inner area of the gill, which was occupied by many bacteriocytes in the row of gill filaments. Strong mAb signals were found on the outer surface of the bacteriocytes facing the interfilamental space, and in the interfilamental spaces between filaments. Weaker signals were also observed in the bacteriocyte cells. These results indicate that the CokG-B3C10 mAbbinding mucus glycoproteins were produced from cells including bacteriocytes and nonbacteriocyte cells in the inner area of the gill filaments.

Diplonemid mitochondria are considered to have very eccentric structural genes. Coding regions of individual diplonemid mitochondrial genes are fragmented into small pieces and found on different circular DNAs. Short RNAs transcribed from each DNA molecule mature through a unique RNA maturation process involving assembly and three types of RNA editing (i.e., U insertion and A-to-I and C-to-U substitutions), although the molecular mechanism(s) of RNA maturation and the evolutionary history of these eccentric structural genes still remain to be understood. Since the gene fragmentation pattern is generally conserved among the diplonemid species studied to date, it was considered that their structural complexity has plateaued and further gene fragmentation could not occur. Here, we show the mitochondrial gene structure of Hemistasia phaeocysticola, which was recently identified as a member of a novel lineage in diplonemids, by comparison of the mitochondrial DNA sequences with cDNA sequences synthesized from mature mRNA. The genes of H. phaeocysticola are fragmented much more finely than those of other diplonemids studied to date. Furthermore, in addition to all known types of RNA editing, it is suggested that a novel processing step (i.e., secondary RNA insertion) is involved in the RNA maturation in the mitochondria of H. phaeocysticola. Our findings demonstrate the tremendous plasticity of mitochondrial gene structures.

Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin-podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3-atypical protein kinase C (aPKC)-PAR6beta cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKClambda in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC-PAR3 complex associates with the nephrin-podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.