E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.     

More to it than meets the eye: Photo‐Eli citation amongst the Batak of Sumatra

Photo‐elicitation was utilized by the author in the Batak area of North Sumatra, Indonesia, to obtain information on Batak textiles stored in European museums. This article examines the kinds of interaction which occurred between the anthropologist, owner of the photographs, and the viewers of the photographs, from whom responses were being elicited. The data collected demonstrate that, regardless of the edge on reality that photographic documents appear to hold, the kind of information they elicit is subject to the same interpretive pitfalls as information collected in the field in other ways. While hardly a new insight, it holds important implications for salvage projects, such as the documentation of material culture, in which photo‐elicitation is frequently made to serve.     

Evaluation of Alternate Isotope-Coded Derivatization Assay (AIDA) in the LC–MS/MS analysis of aldehydes in exhaled breath condensate

We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively “light” and “heavy”, by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d₃-DNPH) has been synthesized and used as “heavy” reagent in combination with commercial “light” DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d₃-DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2–40 nM for MDA and 0.5–10 nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d₃-DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d₃-DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2–8% for MDA and 4.5–11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98–108% for MDA and 93–114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples.     

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.     

Altered Synaptic Plasticity in Tourette's Syndrome and Its Relationship to Motor Skill Learning

Gilles de la Tourette syndrome is a neuropsychiatric disorder characterized by motor and phonic tics that can be considered motor responses to preceding inner urges. It has been shown that Tourette patients have inferior performance in some motor learning tasks and reduced synaptic plasticity induced by transcranial magnetic stimulation. However, it has not been investigated whether altered synaptic plasticity is directly linked to impaired motor skill acquisition in Tourette patients. In this study, cortical plasticity was assessed by measuring motor-evoked potentials before and after paired associative stimulation in 14 Tourette patients (13 male; age 18–39) and 15 healthy controls (12 male; age 18–33). Tic and urge severity were assessed using the Yale Global Tic Severity Scale and the Premonitory Urges for Tics Scale. Motor learning was assessed 45 minutes after inducing synaptic plasticity and 9 months later, using the rotary pursuit task. On average, long-term potentiation-like effects in response to the paired associative stimulation were present in healthy controls but not in patients. In Tourette patients, long-term potentiation-like effects were associated with more and long-term depression-like effects with less severe urges and tics. While motor learning did not differ between patients and healthy controls 45 minutes after inducing synaptic plasticity, the learning curve of the healthy controls started at a significantly higher level than the Tourette patients'' 9 months later. Induced synaptic plasticity correlated positively with motor skills in healthy controls 9 months later. The present study confirms previously found long-term improvement in motor performance after paired associative stimulation in healthy controls but not in Tourette patients. Tourette patients did not show long-term potentiation in response to PAS and also showed reduced levels of motor skill consolidation after 9 months compared to healthy controls. Moreover, synaptic plasticity appears to be related to symptom severity.     

Pseudomonas syringae pv. syringae Uses Proteasome Inhibitor Syringolin A to Colonize from Wound Infection Sites

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.     

Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids

All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein α/β-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.     

Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors

Serine hydrolases are a diverse enzyme class representing ～1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.