Calcium-binding protein 7 expressed in muscle negatively regulates age-related degeneration of neuromuscular junctions in mice.

The neuromuscular junction (NMJ) forms centrally in myotubes and, as the only synapse between motor neuron and myotube, are indispensable for motor activity. The midmuscle formation of NMJs, including midmuscle-restricted expression of NMJ-related genes, is governed by the muscle-specific kinase (MuSK). However, mechanisms underlying MuSK-mediated signaling are unclear. Here, we find that the Calcium-binding protein 7 (Cabp7) gene shows midmuscle-restricted expression, and muscle-specific depletion of Cabp7 in mice accelerated age-related NMJ degeneration, muscle weakness/atrophy, and motor dysfunction. Surprisingly, forced expression in muscle of CIP, an inhibitory peptide of the negative regulator of NMJ formation cyclin-dependent kinase 5 (Cdk5), restored NMJ integrity and muscle strength, and healed muscle atrophy in muscle-specific Cabp7-deficient mice, which showed increased muscle expression of the Cdk5 activator p25. These findings together demonstrate that MuSK-mediated signaling induces muscle expression of Cabp7, which suppresses age-related NMJ degeneration likely by attenuating p25 expression, providing insights into prophylactic/therapeutic intervention against age-related motor dysfunction.

Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes.

PURPOSE: Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina's inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. METHODS: We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. RESULTS: One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. CONCLUSIONS: Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice.

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells.

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.

MYCL-mediated reprogramming expands pancreatic insulin-producing cells.

ß cells have a limited capacity for regeneration, which predisposes towards diabetes. Here, we show that, of the MYC family members, Mycl plays a key role in proliferation of pancreatic endocrine cells. Genetic ablation of Mycl causes a reduction in the proliferation of pancreatic endocrine cells in neonatal mice. By contrast, the expression of Mycl in adult mice stimulates the proliferation of ß and α cells, and the cells persist after withdrawal of Mycl expression. A subset of the expanded α cells give rise to insulin-producing cells after this withdrawal. Transient Mycl expression in vivo is sufficient to normalize the hyperglycaemia of diabetic mice. In vitro expression of Mycl similarly provokes active replication in islet cells, even in those from aged mice. Finally, we show that MYCL stimulates the division of human adult cadaveric islet cells. Our results demonstrate that the induction of Mycl alone expands the functional ß-cell population, which may provide a regenerative strategy for ß cells.

Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus.

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.

Mapping membrane lipids in the developing and adult mouse retina under physiological and pathological conditions using mass spectrometry.

Membrane phospholipids play pivotal roles in various cellular processes, and their levels are tightly regulated. In the retina, phospholipids had been scrutinized because of their distinct composition and requirement in visual transduction. However, how lipid composition changes during retinal development remains unclear. Here, we used liquid chromatography-mass spectrometry (LC-MS) to assess the dynamic changes in the levels of two main glycerophospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in the developing mouse retina under physiological and pathological conditions. The total levels of PC and PE increased during retinal development, and individual lipid species exhibited distinct level changes. The amount of very-long-chain PC and PE increased dramatically in the late stages of retinal development. The mRNA levels of Elovl2 and Elovl4, genes encoding enzymes essential for the synthesis of very-long-chain polyunsaturated fatty acids, increased in developing photoreceptors. Cell sorting based on CD73 expression followed by LC-MS revealed distinct changes in PC and PE levels in CD73-positive rod photoreceptors and CD73-negative retinal cells. Finally, using the NaIO3-induced photoreceptor degeneration model, we identified photoreceptor-specific changes in PC and PE levels from 1 day after NaIO3 administration, before the outer segment of photoreceptors displayed morphological impairment. In conclusion, our findings provide insight into the dynamic changes in PC and PE levels in the developing and adult mouse retina under physiological and pathological conditions. Furthermore, we provide evidence that cell sorting followed by LC-MS is a promising approach for investigating the relevance of lipid homeostasis in the function of different retinal cell types.

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

BACKGROUND: Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. METHODS: We selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells). RESULTS: Our results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2ß1 and α11ß1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization. CONCLUSION: The use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission.

Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8-week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.

Sall1 plays pivotal roles for lens fiber cell differentiation in mouse.

Mammals possess four Sall transcription factors that play various roles in organogenesis. Previously, we found that Sall1 is expressed in microglia in the central nervous system, and it plays pivotal roles in microglia maturation. In the eye, Sall1 was also expressed in the developing lens, and we examined its role in lens development. A knock-in mouse harboring the EGFP gene in the Sall1 locus (Sall1-gfp) was used to analyze the Sall1 expression pattern. In Sall1-gfp/wild, EGFP was expressed throughout the presumptive lens at E11.5, and subsequently the expression in the lens epithelium became weaker. After birth, signals were observed in the equator region. The effects of Sall1 knockout on lens development were examined in Sall1-gfp/gfp. Lens sections revealed small vacuole-like holes and gaps in the center of the lens fibers at E14.5. Subsequently, the vacuoles appeared in most regions of the fiber cells. Electron microscopic analysis indicated that the vacuoles were between the fiber cells, leading to huge gaps. In addition, contact between the lens epithelium and apical end of the fiber cell was disrupted, and there were gaps between the adjoining lens epithelial cells. However, gap junction structure was observed by electron microscopic analysis, and immunostaining of Zo1 showed rather appropriate expression pattern. Immunohistochemistry indicated that the major lens transcription factors Prox1 and Pax6 were expressed in relatively normal patterns. However, although the expression of Prox1 and Pax6 decreased in nuclei in the control lens, it remained in Sall1-gfp/gfp. In addition, lower expression level of c-Maf protein was observed. Therefore, Sall1 is strongly expressed in the lens from the early developmental stage and plays an essential role in the maintenance of fiber cell and lens epithelium adhesion.

Molecular basis for governing the morphology of type-I collagen fibrils by Osteomodulin.

Small leucine-rich repeat proteoglycan (SLRP) proteins have an important role in the organization of the extracellular matrix, especially in the formation of collagen fibrils. However, the mechanism governing the shape of collagen fibrils is poorly understood. Here, we report that the protein Osteomodulin (OMD) of the SLRP family is a monomeric protein in solution that interacts with type-I collagen. This interaction is dominated by weak electrostatic forces employing negatively charged residues of OMD, in particular Glu284 and Glu303, and controlled by entropic factors. The protein OMD establishes a fast-binding equilibrium with collagen, where OMD may engage not only with individual collagen molecules, but also with the growing fibrils. This weak electrostatic interaction is carefully balanced so it modulates the shape of the fibrils without compromising their viability.

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina.

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with ß-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.

Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions.

Helicobacter pylori (H. pylori), a gram-negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral-shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis-induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin-associated gene A (CagA), components of the cag-Type IV secretion system (TFSS), the blood group antigen-binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis-induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis-induced fragmented state cells could be converted to proliferation-competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.

Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes.

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging.

The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes (RNPs) arranged in a specific "1+7" pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same "1+7" pattern as wild-type virions. Next-generation sequencing reveals that the virions specifically incorporate host-derived 18S and 28S ribosomal RNAs (rRNAs) seemingly as the eighth RNP in place of the HA vRNA. These findings highlight the importance of the assembly of eight RNPs into a specific "1+7" configuration for genome packaging in progeny virions and suggest a potential role for cellular RNAs in viral genome packaging.

MiR-199a Inhibits Secondary Envelopment of Herpes Simplex Virus-1 Through the Downregulation of Cdc42-specific GTPase Activating Protein Localized in Golgi Apparatus.

Because several studies have shown that exogenous miR-199a has antiviral effects against various viruses, including herpesviruses, we examined how miR-199a exerts its antiviral effects using epithelial tumour cell lines infected with herpes simplex virus-1 (HSV-1). We found that both miR-199a-5p and -3p impair the secondary envelopment of HSV-1 by suppressing their common target, ARHGAP21, a Golgi-localized GTPase-activating protein for Cdc42. We further found that the trans-cisternae of the Golgi apparatus are a potential membrane compartment for secondary envelopment. Exogenous expression of either pre-miR-199a or sh-ARHGAP21 exhibited shared phenotypes i.e. alteration of Golgi function in uninfected cells, inhibition of HSV-1 secondary envelopment, and reduction of trans-Golgi proteins upon HSV-1 infection. A constitutively active form of Cdc42 also inhibited HSV-1 secondary envelopment. Endogenous levels of miR-199a in epithelial tumour cell lines were negatively correlated with the efficiency of HSV-1 secondary envelopment within these cells. These results suggest that miR-199a is a crucial regulator of Cdc42 activity on Golgi membranes, which is important for the maintenance of Golgi function and for the secondary envelopment of HSV-1 upon its infection.

Herpes Simplex Virus 1 Small Capsomere-Interacting Protein VP26 Regulates Nucleocapsid Maturation.

VP26 is a herpes simplex virus 1 (HSV-1) small capsomere-interacting protein. In this study, we investigated the function of VP26 in HSV-1-infected cells with the following results. (i) The VP26 null mutation significantly impaired incorporation of minor capsid protein UL25 into nucleocapsids (type C capsids) in the nucleus. (ii) The VP26 mutation caused improper localization of UL25 in discrete punctate domains containing multiple capsid proteins (e.g., the VP5 major capsid protein) in the nucleus; these domains corresponded to capsid aggregates. (iii) The VP26 mutation significantly impaired packaging of replicated viral DNA genomes into capsids but had no effect on viral DNA concatemer cleavage. (iv) The VP26 mutation reduced the frequency of type C capsids, which contain viral DNA but not scaffolding proteins, and produced an accumulation of type A capsids, which lack both viral DNA and scaffold proteins, and had no effect on accumulation of type B capsids, which lack viral DNA but retain cleaved scaffold proteins. Collectively, these results indicated that VP26 was required for efficient viral DNA packaging and proper localization of nuclear capsids. The phenotype of the VP26 null mutation was similar to that reported previously of the UL25 null mutation and of UL25 mutations that preclude UL25 binding to capsids. Thus, VP26 appeared to regulate nucleocapsid maturation by promoting incorporation of UL25 into capsids, which is likely to be required for proper capsid nuclear localization.IMPORTANCE HSV-1 VP26 has been reported to be important for viral replication and virulence in cell cultures and/or mouse models. However, little is known about the function of VP26 during HSV-1 replication, in particular, in viral nucleocapsid maturation although HSV-1 nucleocapsids are estimated to contain 900 copies of VP26. In this study, we present data suggesting that VP26 promoted packaging of HSV-1 DNA genomes into capsids by regulating incorporation of capsid protein UL25 into capsids, which was reported to increase stability of the capsid structure. We also showed that VP26 was required for proper localization of capsids in the infected cell nucleus. This is the first report showing that HSV-1 VP26 is a regulator for nucleocapsid maturation.

Docosahexaenoic acid preserves visual function by maintaining correct disc morphology in retinal photoreceptor cells.

Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction.

A subset of cerebrovascular pericytes originates from mature macrophages in the very early phase of vascular development in CNS.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRß/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.