The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 μmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between β-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue-specific and cold shock-induced gene expression. We identified the promoter sequences of Am-actin5c, elp2l, Am-hsp83 and Am-hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron-specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.

We would like to call your attention to a publication by promotes an aggressive phenotype in human breast cancer cells") 1 in Tissue Barriers. The authors show that high claudin-20 expression correlates with the aggressiveness of breast cancer and poor survival rates for affected patients. They discuss a connection between the sensitivity of cancer cells to Gefitinib and claudin-20 content and suggest that claudin-20 might serve as a biomarker for Gefitinib resistance. However, this conclusion is based on a misconception on the part of the authors. They claim that claudin-20 is the same protein as epithelial membrane protein 1 (EMP-1), and to substantiate this claim, they misleadingly refer to a prior publication demonstrating that EMP-1 is a biomarker for Gefitinib-resistance in an adenocarcinoma model. However, Claudin-20 and EMP-1 are 2 distinct proteins. EMP-1 (NCBI reference sequence accession number NP_001414.1) has been described as a tight junction protein in the blood-brain barrier 3 as well as in the human airway epithelium, where it regulates tight junction formation. 4 To our knowledge, the paper of Martin et al. 1 is the first publication that presents functional data regarding human claudin-20 (NP_001001346.1). Their paper will be immediately found (and likely cited) by other scientists working with either of the 2 proteins. Therefore, it is crucial to avoid misrepresentations through a careful review of the existing literature.According to Price et al. 5 (also cited by Martin and colleagues 1 ) both claudin-20 and EMP-1 belong to the Pfam00822 superfamily, which includes claudins, peripheral myelin protein 22 (PMP-22), lens intrinsic membrane protein (LIM-2/MP20), EMP-1, ¡2, ¡3 and voltage dependent calcium channel gamma subunits (CACNGs). The study places claudin-20 within the claudin family (where it is most closely related to the classic claudins 2 and 14), while EMP-1 is placed in the LIM2/PMP22/EMP family.5 This classification was confirmed in an independent investigation, which came to the same results and confirmed the separate identities of claudin-20 and EMP-1. 6 Nevertheless, the phylogeny of the Pfam00822 family has not yet been completely resolved. TMEM114 and TMEM235 were first assigned to the claudin family using PMP-22 as an outgroup.7 Later, these TMEMs were renamed claudin-26 and ¡27, respectively. 8 Subsequent work that also included CACNGs, EMPs and LIM-2 and used clarin-1 as an outgroup revealed that TMEM114 and TMEM235 were more closely related to the CACNG family than with the claudin family. 6 Thus, Pfam00822 superfamily nomenclature has not yet been conclusively determined.The members of this superfamily are tetraspanning membrane proteins and share a WX 9-44 GLWXXC(X 9-26 C) signature motif in the first extracellular loop (whereas the second cysteine is not conserved in the LIM2/PMP22/EMP family). To illustrate the differences between EMP-1 and claudin-20, we modeled their structures using I-TASSER. 9 The recently published crystal structure of claudin-15 10 was applied a...

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