A loss of function mutation in CLDN25 causing Pelizaeus-Merzbacher-like leukodystrophy.

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key scaffolding protein ZO-1. However, CLDN-25 does not contain this motif. Here, we show that the C-terminal end of CLDN-25 is required for its junctional localization in a ZO-1 independent manner. The A249P mutant protein as well as a deletion mutant lacking its last 5 C-terminal amino acids also failed to localize to the cell-cell border in vitro. Intriguingly, cellular knockout of CLDN25, in vitro, appeared to increase the integrity of the tight junction between 2 contacting cells, while driving highly unusual increased movement of solutes between cells. We propose that the barrier function of CLDN-25 is akin to a decoy claudin, whereby decreasing its expression in "leaky" epithelial cells and endothelial cells will drive dynamic changes in the adhesion and interaction capacity of cell-cell contact points. While it remains unclear how this de novo CLDN-25 mutant induces leukodystrophy, our findings strongly suggest that this mutation induces haploinsufficiency of CLDN-25. Elucidating the function of this uncharacterized claudin protein will lead to a better understanding of the role of claudin proteins in health and disease.

Recurrent de novo mutations in CLDN5 induce an anion-selective blood-brain barrier and alternating hemiplegia.

Claudin-5 is the most enriched tight junction protein at the blood-brain barrier. Perturbations in its levels of expression have been observed across numerous neurological and neuropsychiatric conditions; however, pathogenic variants in the coding sequence of the gene have never been reported previously. Here, we report the identification of a novel de novo mutation (c.178G>A) in the CLDN5 gene in two unrelated cases of alternating hemiplegia with microcephaly. This mutation (G60R) lies within the first extracellular loop of claudin-5 and based on protein modelling and sequence alignment, we predicted it would modify claudin-5 to become an anion-selective junctional component as opposed to a purely barrier-forming protein. Generation of stably transfected cell lines expressing wild-type or G60R claudin-5 showed that the tight junctions could still form in the presence of the G60R mutation but that the barrier against small molecules was clearly attenuated and displayed higher Cl- ion permeability and lower Na+ permeability. While this study strongly suggests that CLDN5 associated alternating hemiplegia is a channelopathy, it is also the first study to identify the conversion of the blood-brain barrier to an anion-selective channel mediated by a dominant acting variant in CLDN5.

Dissipative Kerr soliton microcombs for FEC-free optical communications over 100 channels.

The demand for high-speed and highly efficient optical communication techniques has been rapidly growing due to the ever-increasing volume of data traffic. As well as the digital coherent communication used for core and metro networks, intensity modulation and direct detection (IM-DD) are still promising schemes in intra/inter data centers thanks to their low latency, high reliability, and good cost performance. In this work, we study a microresonator-based frequency comb as a potential light source for future IM-DD optical systems where applications may include replacing individual stabilized lasers with a continuous laser driven microresonator. Regarding comb line powers and spectral intervals, we compare a modulation instability comb and a soliton microcomb and provide a quantitative analysis with regard to telecom applications. Our experimental demonstration achieved a forward error correction (FEC) free operation of bit-error rate (BER) <10-9 with a 1.45 Tbps capacity using a total of 145 lines over the entire C-band and revealed the possibility of soliton microcomb-based ultra-dense wavelength division multiplexing (WDM) with a simple, cost-effective IM-DD scheme, with a view to future practical use in data centers.

On-chip photon-pair generation in a silica microtoroidal cavity.

Microcavities with high Q factor and small mode volume have the potential to be efficient and compact sources of photon pairs. Here, we demonstrate on-chip photon-pair generation by spontaneous four-wave mixing in a silica microtoroidal cavity and obtain a coincidence-to-accidental ratio of 7.4 ± 0.1 with a pump power of 46 µW. The heralded photons also exhibit antibunching characterized by autocorrelation function values of gc(2)(0)=0.57±0.03<1. Comparing with a scaling model, the main noise source is found to be spontaneous Raman scattering in the cavity. This work opens a new possible means for realizing integrated nonclassical photon sources based on silica photonic circuits toward scalable quantum technologies.

Claudin-5: A Pharmacological Target to Modify the Permeability of the Blood-Brain Barrier.

Claudin-5 is the dominant tight junction protein in brain endothelial cells and exclusively limits the paracellular permeability of molecules larger than 400 Da across the blood-brain barrier (BBB). Its pathological impairment or sustained down-regulation has been shown to lead to the progression of psychiatric and neurological disorders, whereas its expression under physiological conditions prevents the passage of drugs across the BBB. While claudin-5 enhancers could potentially act as vascular stabilizers to treat neurological diseases, claudin-5 inhibitors could function as delivery systems to enhance the brain uptake of hydrophilic small-molecular-weight drugs. Therefore, the effects of claudin-5 manipulation on modulating the BBB in different neurological diseases requires further examination. To manipulate claudin-5 expression levels and function, several claudin-5 modulating molecules have been developed. In this review, we first describe the molecular, cellular and pathological aspects of claudin-5 to highlight the mechanisms of claudin-5 enhancers/inhibitors. We then discuss recently developed claudin-5 enhancers/inhibitors and new methods to discover these molecules.

Phase Locking between Two All-Optical Quantum Memories.

Optical approaches to quantum computation require the creation of multimode photonic quantum states in a controlled fashion. Here we experimentally demonstrate phase locking of two all-optical quantum memories, based on a concatenated cavity system with phase reference beams, for the time-controlled release of two-mode entangled single-photon states. The release time for each mode can be independently determined. The generated states are characterized by two-mode optical homodyne tomography. Entanglement and nonclassicality are preserved for release-time differences up to 400 ns, confirmed by logarithmic negativities and Wigner-function negativities, respectively.

A Novel Platform for Cancer Vaccines: Antigen-Selective Delivery to Splenic Marginal Zone B Cells via Repeated Injections of PEGylated Liposomes.

Treating cancer with vaccines has been a challenge. In this study, we introduce a novel Ag delivery platform for cancer vaccines that delivers an encapsulated Ag to splenic marginal zone B (MZ-B) cells via the aid of a PEGylated liposome (PL) system. Splenic MZ-B cells have recently attracted interest as alternative APCs. In mice, preimmunization with empty (no Ag encapsulation) PLs triggered the efficient delivery of a subsequent dose of Ag-containing PLs, injected 3 d later, to the spleen compared with a single dose of Ag-containing PLs. In addition, immunization with empty PLs allowed three subsequent sequential injections of OVA-PLs to efficiently induce a CTL response against OVA-expressing murine thymoma (EG7-OVA) cells and resulted in in vivo growth inhibition of subsequently inoculated EG7-OVA cells. However, these sequential treatments require repeated immunizations to achieve their antitumor effect. Therefore, to improve the antitumor effect of our novel vaccine system, an adjuvant, α-galactosylceramide (αGC), was incorporated into the OVA-PLs (αGC/OVA-PLs). As expected, the incorporation of αGC reduced the required number of immunizations with OVA-PLs to the point that a single immunization treatment with empty PLs and an injection of αGC/OVA-PL efficiently triggered a potent CTL induction, resulting in a rejection of the development and a suppression of the growth of tumors that had already developed s.c. Results of this study indicate that a novel Ag delivery platform that grants efficient Ag delivery to splenic MZ-B cells shows promise as a therapeutic modality for conquering tumor growth and/or progression.

Anti-Claudin Antibodies as a Concept for Development of Claudin-Directed Drugs.

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.

All-Optical Storage of Phase-Sensitive Quantum States of Light.

We experimentally demonstrate storage and on-demand release of phase-sensitive, photon-number superposition states of the form α|0⟩+ße^{iθ}|1⟩ for an optical quantized oscillator mode. For this purpose, we newly developed a phase-probing mechanism compatible with a storage system composed of two concatenated optical cavities, which was previously employed for storage of phase-insensitive single-photon states [Phys. Rev. X 3, 041028 (2013)PRXHAE2160-330810.1103/PhysRevX.3.041028]. This is the first demonstration of all-optically storing highly nonclassical and phase-sensitive quantum states of light. The strong nonclassicality of the states after storage becomes manifest as a negative region in the corresponding Wigner function shifted away from the origin in phase space. This negativity is otherwise, without the phase information of the memory system, unobtainable. While our scheme includes the possibility of optical storage, on-demand release and synchronization of arbitrary single-rail qubit states, it is not limited to such states. In fact, our technique is extendible to more general phase-sensitive states such as multiphoton superposition or entangled states, and thus it represents a significant step toward advanced optical quantum information processing, where highly nonclassical states are utilized as resources.

Associations of coronary plaque characteristics by integrated backscatter intravascular ultrasound with detectability of vessel external elastic lamina using optical frequency domain imaging in human coronary arteries: A sub-analysis of the MISTIC-1 trial.

OBJECTIVES: We sought to examine associations between plaque characteristics by intravascular ultrasound (IVUS) and detectability of external elastic lamina (EEL) by optical frequency domain imaging (OFDI) in human coronary arteries. BACKGROUND: It is often challenging to detect EEL which represents vessel size by light-based imaging modalities due to light intensity attenuation through atherosclerotic plaque. METHODS: IVUS and OFDI prior to stent implantation were sequentially investigated per protocol. We identified corresponding cross-sections by minimum lumen area (MLA) or just distally to side branches as anatomical landmarks. Plaque characterization was determined by integrated backscatter IVUS analysis. We categorized detectable EEL arc by OFDI into four groups: 0≤ and <1 quadrant (group 1), 1≤ and <2 quadrants (group 2), 2≤ and <3 quadrants (group 3), or 3≤ and <4 quadrants (group 4). RESULTS: We prospectively studied 103 vessels in 93 patients with stable coronary artery disease. Corresponding 711 cross-sections were analyzed. Cross-sections with detectable EEL arc <2 quadrants (group 1 or 2) were observed in 86.1% of MLA sites but only in 29.3% of non-MLA sites (p < .05). Percentage plaque area (%PA) appeared to be the strongest predictor to detect EEL arc <2 quadrants with the cut-off of 60.3% (AUC 0.90; sensitivity 79.8%, specificity 85.5%). Lipid pool and calcification remained statistically significant in predicting detectable EEL arc <2 quadrants after adjustment with %PA. CONCLUSIONS: Presence of large plaque burden, lipid pool, and calcification significantly predicts the detectability of EEL by OFDI assessment. Locations with detectable EEL arc <2 quadrants should thus be avoided for optimal stent landing zone.

Potential for Tight Junction Protein-Directed Drug Development Using Claudin Binders and Angubindin-1.

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood-brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development.

A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics.

PURPOSE: Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry. METHODS: The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics. RESULTS: The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA. CONCLUSIONS: PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.

Roles of the first-generation claudin binder, Clostridium perfringens enterotoxin, in the diagnosis and claudin-targeted treatment of epithelium-derived cancers.

Given that most malignant tumors are derived from epithelium, developing a strategy for treatment of epithelium-derived cancers (i.e., carcinomas) is a pivotal issue in cancer therapy. Carcinomas, including ovarian, breast, prostate, and pancreatic cancers, are known to overexpress various claudins (CLDNs); in particular, CLDN-3 and -4 are frequently overexpressed in malignant case. The generation of CLDN binders is a key for expanding CLDN-targeted cancer therapy but has been delayed due to the small size of CLDN extracellular domains (approximately 50 amino acids for the first domain and 15 amino acids for the second) and their high homology among species. Interestingly, however, the receptors for Clostridium perfringens enterotoxin (CPE), a foodborne toxin in humans, happen to be identical to CLDN-3 and -4. Thus, the first CLDN binder, CPE, has provided us CLDN-targeted cancer therapy from a concept into a potential reality. In this review, we describe roles of CPE technology in cancer therapy and discuss future directions in the CLDN-targeting concept-to-therapy process.

Claudin-5-Binders Enhance Permeation of Solutes across the Blood-Brain Barrier in a Mammalian Model.

A current bottleneck in the development of central nervous system (CNS) drugs is the lack of drug delivery systems targeting the CNS. The intercellular space between endothelial cells of the blood-brain barrier (BBB) is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. In the present study, to investigate whether CLDN-5 binders can be used for delivery of drugs to the CNS, we generated monoclonal antibodies (mAbs) specific to the extracellular domains of CLDN-5. In an in vitro model of the BBB, the anti-CLDN-5 mAbs attenuated trans-epithelial/endothelial electrical resistance and enhanced solute permeation. These anti-CLDN-5 mAbs are potential leads for the development of novel drug delivery systems targeting the CNS.

Generation and characterization of a human-mouse chimeric antibody against the extracellular domain of claudin-1 for cancer therapy using a mouse model.

Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced epidermal absorption of a drug and prevented hepatitis C virus infection in human hepatocytes. Here, we investigated anti-tumor activity of a human-mouse chimeric IgG1, xi-3A2, from one of the anti-CLDN-1 mAbs, clone 3A2. Xi-3A2 accumulated in the tumor tissues in mice bearing with human CLDN-1-expressing tumor cells. Xi-3A2 activated Fcγ receptor IIIa-expressing reporter cells in the presence of human CLDN-1-expressing cells, suggesting xi-3A2 has a potential to exhibit antibody-dependent cellular cytotoxicity against CLDN-1 expressing tumor cells. We also constructed a mutant xi-3A2 antibody with Gly, Ser, and Ile substituted with Ala, Asp, and Arg at positions 236, 239, and 332 of the Fc domain. This mutant antibody showed greater activation of Fcγ receptor IIIa and in vivo anti-tumor activity in mice bearing human CLDN-1-expressing tumors than xi-3A2 did. These findings indicate that the G236A/S239D/I332E mutant of xi-3A2 might be a promising lead for tumor therapy.

Anti-PEG IgM Is a Major Contributor to the Accelerated Blood Clearance of Polyethylene Glycol-Conjugated Protein.

Limited therapeutic efficacy of polyethylene glycol-conjugated (PEGylated) protein drugs has been recently reported in animals and human following repeat injections. Since there are reports that an accelerated blood clearance (ABC) phenomenon is caused by repeated injection of PEGylated liposome, there is an assumption that PEGylated proteins lose their long circulating property when they are injected repeatedly due to the induction of anti-PEG antibody. Although induction of anti-PEG antibody by PEGylated protein has been reported, there is little evidence of accelerated blood clearance of PEGylated protein upon repeated injection. Herein, we investigated the blood concentration of PEGylated ovalbumin (PEG-OVA), a model PEGylated protein, upon its repeated injection. A single intravenous administration of PEG-OVA elicited an anti-PEG IgM response but not anti-PEG IgG response, while the administration did not elicit antibody against OVA. At 24 h postinjection of test PEG-OVA, although control mice showed 41.6% dose of PEG-OVA in blood, the mice pretreated with PEG-OVA showed rapid clearance of test PEG-OVA from blood and undetectable level of PEG-OVA. Interestingly, the anti-PEG IgM induced by PEGylated liposome did not affect the blood concentration of subsequent dose of PEG-OVA. Our result suggests that anti-PEG IgM is a major contributor to the accelerated blood clearance of PEG-conjugated protein, but the presence of anti-PEG IgM in blood circulation does not necessarily affect circulating property of entire PEGylated materials.

Comprehensive analysis of PEGylated liposome-associated proteins relating to the accelerated blood clearance phenomenon by combination with shotgun analysis and conventional methods.

PEGylated liposome, sterically stabilized by polyethylene glycol (PEG), results in reduced recognition of the liposome by the mononuclear phagocyte system. Recently, we reported regarding the accelerated blood clearance (ABC) phenomenon that PEGylated liposome is cleared very rapidly from blood circulation upon repeated injection. Anti-PEG IgM production and subsequent complement activation were crucial in causing the ABC phenomenon. However, there still remains the possibility that unknown plasma factors might affect the fate of PEGylated liposome that is subjected to the ABC phenomenon. A label-free approach to shotgun analysis is a great tool for characterizing proteins in a biological system. In this study, therefore, a shotgun analysis was employed to identify plasma protein bound on PEGylated liposome after the ABC phenomenon was induced in the mouse model. The analysis revealed that immunoglobulin and complement components (C1 and C3) are the major proteins. Subsequent analysis with enzyme-linked immunosorbent assay and Western blotting showed that the immunoglobulin was IgM and that the complement system was mainly activated via an anti-PEG IgM-mediated classical pathway. These results support our earlier assumptions-anti-PEG IgM and complement activation were the major causes of the ABC phenomenon. Our proposed analytical strategy would be expected to provide useful information for the development and design of the nanocarrier drug delivery system.

Relationship between the concentration of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) and the intensity of the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes in mice.

PEGylation, which is the surface modification of nanocarriers with polyethylene glycol (PEG), has increased the circulation time and reduced the immunogenic responses to nanocarriers. However, many reports have demonstrated that the intravenous injection of sterically stabilized PEGylated liposome (SL) causes an accelerated blood clearance (ABC) of subsequent doses via anti-PEG immunoglobulin M (IgM)-mediated complement activation. In the present study, the relationships between serum anti-PEG IgM concentration, the intensity of complement activation and the hepatic clearance of SL were quantitatively investigated for their role in the ABC phenomenon. Interestingly, with increasing serum anti-PEG IgM concentrations, the intensity of complement activation increased linearly, while the intensity of the hepatic clearance of SL was increased and then saturated. In addition, only 15-17% of anti-PEG IgM in blood circulation induced by SL at different doses was associated with a second dose SL. The present results indicate that it is the hepatic uptake of SL that is the limiting step in the ABC phenomenon, rather than the association of anti-PEG IgM to the SL and a subsequent complement activation.

Synthesis and evaluation of fatty acid amides on the N-oleoylethanolamide-like activation of peroxisome proliferator activated receptor α.

A series of fatty acid amides were synthesized and their peroxisome proliferator-activated receptor α (PPAR-α) agonistic activities were evaluated in a normal rat liver cell line, clone 9. The mRNAs of the PPAR-α downstream genes, carnitine-palmitoyltransferase-1 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) as PPAR-α agonistic activities. We prepared nine oleic acid amides. Their PPAR-α agonistic activities were, in decreasing order, N-oleoylhistamine (OLHA), N-oleoylglycine, Oleamide, N-oleoyltyramine, N-oleoylsertonin, and Olvanil. The highest activity was found with OLHA. We prepared and evaluated nine N-acylhistamines (N-acyl-HAs). Of these, OLHA, C16:0-HA, and C18:1Δ(9)-trans-HA showed similar activity. Activity due to the different chain length of the saturated fatty acid peaked at C16:0-HA. The PPAR-α antagonist, GW6471, inhibited the induction of the PPAR-α downstream genes by OLHA and N-oleoylethanolamide (OEA). These data suggest that N-acyl-HAs could be considered new PPAR-α agonists.