High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

IgA Vasculitis as a Potential Complication of Fourth-Line Chemotherapy with Tegafur/Gimeracil/Oteracil (S-1) in Advanced Non-Small Cell Lung Cancer: A Case Report.

BACKGROUND Immunoglobulin A (IgA) vasculitis is a systemic vasculitis that involves the small vessels. It is mainly characterized by skin symptoms such as purpura, arthritis/arthralgia, abdominal symptoms, and nephropathy, which are caused by IgA adherence to the vessel walls. Herein, we report the case of an advanced non-small cell lung cancer (NSCLC) and a purpuric skin rash of the legs that developed during fourth-line chemotherapy with tegafur/gimeracil/oteracil (S-1). CASE REPORT A 68-year-old man diagnosed with NSCLC 2 years ago was undergoing S-1 as fourth-line chemotherapy when he developed purpura and edema on the lower extremities. Biopsy renal specimens were consistent with IgA vasculitis. Considering his medical history, both IgA vasculitis induced by S-1 and a paraneoplastic syndrome were considered, although the exact cause could not be identified. Subsequently, chemotherapy was discontinued because of his deteriorating general condition, and he received optimal supportive care. The purpura spontaneously disappeared; however, his ascites and renal function deteriorated. Systemic steroids improved renal function, but the ascites did not resolve. One month after being diagnosed with IgA vasculitis, the patient died due to deterioration of his general condition. CONCLUSIONS This case emphasizes the occurrence of IgA vasculitis during lung cancer treatment and its potential impact on the disease course of lung cancer. Moreover, the possible causes of IgA vasculitis in this case were paraneoplastic syndrome or S-1 adverse effects, but further case series are needed to gain a more comprehensive understanding. Refractory, steroid-unresponsive ascites may occur as an abdominal manifestation of IgA vasculitis.

In vivo nanoscopic landscape of neurexin ligands underlying anterograde synapse specification.

Excitatory synapses are formed and matured by the cooperative actions of synaptic organizers, such as neurexins (Nrxns), neuroligins (Nlgns), LRRTMs, and Cbln1. Recent super-resolution nanoscopy developments have revealed that many synaptic organizers, as well as glutamate receptors and glutamate release machinery, exist as nanoclusters within synapses. However, it is unclear how such nanodomains interact with each other to organize excitatory synapses in vivo. By applying X10 expansion microscopy to epitope tag knockin mice, we found that Cbln1, Nlgn1, and LRRTM1, which share Nrxn as a common presynaptic receptor, form overlapping or separate nanodomains depending on Nrxn with or without a sequence encoded by splice site 4. The size and position of glutamate receptor nanodomains of GluD1, NMDA, and AMPA receptors were regulated by Cbln1, Nlgn1, and LRRTM1 nanodomains, respectively. These findings indicate that Nrxns anterogradely regulate the postsynaptic nanoscopic architecture of glutamate receptors through competition and coordination of Nrxn ligands.

Posterior Vitreous Detachment in Normal Healthy Subjects Younger Than Age Twenty.

Purpose: To describe the initiation of posterior vitreous detachment (PVD) in the eyes of normal individuals, under 20 years of age, using wide-angle optical coherence tomography (OCT). Methods: This is an observational cross-sectional study. Montaged images of horizontal and vertical OCT scans were obtained in 63 healthy eyes of 35 consecutive subjects ranging in age from 4 to 17 years. Results: Forty-five eyes (71.4%) had obvious PVD, defined as a contiguous line of posterior cortical vitreous separated from the surface of the retina. Eighteen eyes (28.6%) had no PVD. The mean age of the individuals without PVD was significantly younger than those with PVD (P = 0.008). The spatial distribution of PVD initiation was highest in the superior quadrants, with the nasal, inferior, septum papillomaculae, and temporal quadrants following in descending order of frequency (P < 0.001). PVD was observed to begin anterior to the premacular liquefied lacuna, where the vitreous gel directly adheres to the vitreoretinal interface. In the majority of subjects (80.6%), PVD was initiated anterior to the vascular arcades. Conclusions: PVD can be observed by OCT to begin in the first and second decade of life. It begins in the mid-peripheral vitreous, most frequently in the superior quadrants anterior to the vascular arcades. In this study, all PVDs originated outside of the macular liquefied lacunae, where the vitreous gel adheres directly to the retina.

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum.

Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.

Retinol Promotes In Vitro Growth of Proximal Colon Organoids through a Retinoic Acid-Independent Mechanism.

Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance.

Serine racemase is involved in d-aspartate biosynthesis.

d-Aspartate is found in the nervous and reproductive system and participates in various physiological roles. While several lines of evidence suggest that this amino acid has an endogenous origin, the enzyme responsible for mammalian d-Asp biosynthesis has not yet been identified. We show that mammalian serine racemase (SRR), the primary enzyme responsible for brain d-Ser production, catalyses Asp racemization via a two-base mechanism. We observed that overexpression of SRR in rat pheochromocytoma PC12 cells resulted in an increase in intracellular d-Asp compared with control cells, demonstrating that SRR functions as an Asp racemase in the cells. To investigate the impact of endogenous SRR on endogenous d-Asp levels in the cells, we generated SRR-knockout (SRR-KO) PC12 cells. The SRR-KO cells exhibited decreased intracellular d-Ser levels, but production levels of d-Asp were unaffected. In contrast, SRR-KO mice showed significantly decreased d-Asp levels in their frontal cortices and hippocampi, where SRR is normally highly expressed, while d-Asp levels in the cerebellum and testes remained unchanged. Our results indicate that SRR indeed acts as a d-Asp biosynthetic enzyme in some organs and/or tissues, and also provide evidences that there should be some additional enzyme for d-Asp synthesis in mammals.

Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan.

Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram-negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N-terminal SmpA/OmlA domain and a C-terminal OmpA_C-like domain with a peptidoglycan (PGN)-binding motif. Cell fractionation and a pull-down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN-associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface-displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA-TpgA-like protein cassette can be found in diverse Gram-negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis.

Deletion of SIRT1 in myeloid cells impairs glucose metabolism with enhancing inflammatory response to adipose tissue hypoxia.

Chronic inflammation is a pathophysiology of insulin resistance in metabolic diseases, such as obesity and type 2 diabetes. Adipose tissue macrophages (ATMs) play important roles in this inflammatory process. SIRT1 is implicated in the regulation of glucose metabolism in some metabolic tissues, such as liver or skeletal muscle. This study was performed to investigate whether SIRT1 in macrophages played any roles in the regulation of inflammation and glucose metabolism. Myeloid cell-specific SIRT1-knockout mice were originally generated and analyzed under chow-fed and high-fat-fed conditions. Myeloid cell-specific SIRT1 deletion impaired insulin sensitivity and glucose tolerance assessed by the glucose- or insulin-tolerance test, which was associated with the enhanced expression of inflammation-related genes in epididymal adipose tissue of high-fat-fed mice. Interestingly, the M1 ATMs from the SIRT1-knockout mice showed more hypoxic and inflammatory phenotypes than those from control mice. The expressions of some inflammatory genes, such as Il1b and Nos2, which were induced by in vitro hypoxia treatment, were further enhanced by SIRT1 deletion along with the increased acetylation of HIF-1α in cultured macrophages. These results suggest that deletion of SIRT1 in myeloid cells impairs glucose metabolism by enhancing the hypoxia and inflammatory responses in ATMs, thereby possibly representing a novel therapeutic target for metabolic diseases, such as type 2 diabetes.

Is D-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase?

D-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, D-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, D-aspartate strongly enhances N-methyl-D-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for D-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially D-aspartate from L-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where D-aspartate is abundant. The D-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed L-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown D-aspartate-synthesizing enzyme.

Increased production of intestinal immunoglobulins in Syntenin-1-deficient mice.

Syntenin-1 is an intracellular PDZ protein that binds multiple proteins and regulates protein trafficking, cancer metastasis, exosome production, synaptic formation, and IL-5 signaling. However, the functions of Syntenin-1 have not yet been clearly characterized in detail, especially in vivo. In this study, we generated a Syntenin-1 knock out (KO) mouse strain and analyzed the role(s) of Syntenin-1 in IL-5 signaling, because the direct interaction of Syntenin-1 with the cytoplasmic domain of the IL-5 receptor α subunit and the regulation of IL-5 signaling by Syntenin-1 have been reported. Unexpectedly, the number of IL-5-responding cells was normal and the levels of fecal immunoglobulins were rather higher in the Syntenin-1 KO mice. We also found that IgA and IgM production of splenic B cells stimulated in vitro was increased in Syntenin-1 KO mice. In addition, we showed that a distribution of intestinal microbial flora was influenced in Syntenin-1 KO mice. Our data indicate that Syntenin-1 negatively regulates the intestinal immunoglobulin production and has a function to maintain the intestinal homeostasis in vivo. The analysis of Syntenin-1 KO mice may provide novel information on not only mucosal immunity but also other functions of Syntenin-1 such as cancer metastasis and neural development.

[Identification methods of terpenoid gum bases using TLC and GC/MS].

Simple chromatographic methods were applied to terpenoid resins used as gum bases. Five triterpenoid resins, mastic, dammar resin, olibanum, benzoin gum and elemi resin, and two diterpenoid resins, rosin and copal resin, were separated with normal-phase TLC. Characteristic patterns were observed for all resins. Different samples of the same resin gave identical patterns. The TLC method is a candidate for a simple identification test for terpenoids resins. Samples were then methyl-esterified and analyzed with GC/MS. All resins exhibited characteristic chromatograms for total ion current. Major constituents of all resins were detected. Unique constituents that can be used as indicators were found in every resin. Therefore, GC/MS of methyl-esterified terpenoid resins is a valuable identification method.

Self-interaction of soluble and surface-bound beta2-glycoprotein I and its enhancement by lupus anticoagulants.

Antiphospholipid antibodies found in antiphospholipid syndrome are autoantibodies to phospholipid-binding proteins, such as beta2-glycoprotein I (beta2GPI). We have previously reported that among these antibodies, the so-called lupus anticoagulants (LAs) augment beta2GPI binding to the phospholipid membrane surface, which is associated with the pathological action of LAs. However, the molecular mechanisms underlying this augmentation are uncertain. Here we show that beta2GPI, which is monomeric in solution, self-interacts at the interface of soluble and surface-bound molecules. In addition, this self-interaction is enhanced by LA-positive, but not LA-negative, anti-beta2GPI monoclonal antibodies. This study suggests that beta2GPI self-interaction upon surface binding could be involved in the LA-induced potentiation of beta2GPI binding to the phospholipid surface.

Unfolding and aggregation of transthyretin by the truncation of 50 N-terminal amino acids.

Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wild-type transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C-terminal fragments starting near amino acid residue 50 as well as full-length transthyretin. Although a number of previous studies suggest the importance of the C-terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C-terminal fragments of transthyretin. To understand the role of C-terminal fragments in SSA, we examined the effects of the truncation of the N-terminal portions on the structure and aggregation properties of wild-type transthyretin. The deletion mutant lacking 50 N-terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self-assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N-terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self-assembly into aggregations in SSA.