The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice

Psoriasis is a chronic inflammatory skin disorder, characterised by epidermal hyperplasia (acanthosis) and leukocyte infiltration of the skin. Current therapies are inadequate, highlighting the need for new therapeutic targets. The P2X7 receptor is implicated in the pathogenesis of psoriasis. This study investigated the role of P2X7 in imiquimod (IMQ)-induced psoriasis-like inflammation. Topically applied IMQ caused twofold greater ear swelling in BALB/c mice compared to C57BL/6 mice, which encode a partial loss-of-function missense mutation in the P2RX7 gene. However, there was no difference in histological skin pathology (acanthosis and leukocyte infiltration) between the two strains. IMQ treatment up-regulated P2X7 expression in skin from both mouse strains. Additionally, IMQ induced ATP release from cultured human keratinocytes, a process independent of cell death. Injection of the P2X7 antagonist Brilliant Blue G (BBG) but not A-804598 partly reduced ear swelling compared to vehicle-injected control mice. Neither antagonist altered skin pathology. Moreover, no difference in ear swelling or skin pathology was observed between C57BL/6 and P2X7 knock-out (KO) mice. Flow cytometric analysis of IMQ-treated skin from C57BL/6 and P2X7 KO mice demonstrated similar leukocyte infiltration, including neutrophils, macrophages and T cells. In conclusion, this study demonstrates that P2X7 is not essential for development of IMQ-induced psoriasis-like inflammation but does not exclude a role for this receptor in psoriasis development in humans or other mouse models of this disease.     

Solving the maximum representation problem to prioritize areas for the conservation of terrestrial mammals at risk in Oaxaca

Oaxaca, located in south‐west México within the Mesoamerican biodiversity hotspot, holds exceptionally high biodiversity for several taxa, including mammals. It has four decreed natural protected areas (NPAs) covering 5% of its total area, but only three of these, covering only 0.2% of the area, are strictly protected as National Parks. The current study develops ecological niche models for 183 terrestrial mammals for use as biodiversity surrogates in a systematic conservation planning exercise. Forty‐five of these species were selected on the basis of their being either endangered or threatened or otherwise listed under the Mexican Red List or because they were endemic to either Oaxaca or to Mexico. The niche models were constructed with a machine‐learning algorithm (GARP, Genetic Algorithm for Rule‐Set Prediction) and refined by restricting each model to sites with suitable vegetation and habitat patches contiguous with known occurrences of the species. If the entire predicted geographical distribution of each of the 45 species listed above is put under protection, the entire state of Oaxaca gets included. Therefore, we imposed different constraints on the maximum area that can be put under protection (5–30% of the area of Oaxaca) and selected nominal conservation area networks based on different percentage representation targets for the species’ modelled distributions based on their conservation status (10–100%). The area selection utilized a rarity‐ and complementarity‐based algorithm (in the ResNet software package). The goal was to have as many as possible of the 45 species at risk meet their specified representation targets in the budgeted area. The methods developed here combine ecological niche modelling and area prioritization algorithms for integrated conservation planning in a protocol that is suitable for other highly biodiverse regions.     

Increased alpha-defensins as a blood marker for schizophrenia susceptibility

Schizophrenia is a severe psychotic illness affecting 1% of the general population. There are no consistent pathological features, and the disorder is defined by a complex symptomatology, which overlaps with other psychiatric illnesses. Diagnosis is based on a clinical interview, relying on the patient meeting criteria according to diagnosis manuals, including Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. and International Statistical Classification of Diseases, 10th Revision. Because of the ambiguous symptoms, the diagnostic process can take many months and often years. Rapid and effective treatment has been shown to impact positively on disease progression and outcome, and it is therefore important to identify disease-associated biomarkers allowing early diagnosis. Reliable biomarkers can be used for the development of diagnostic tests and may also help us understand the underlying pathology of this disorder. In the present study, proteins from anti-CD3 stimulated and unstimulated peripheral blood T cell lysates from 15 minimally medicated and unmedicated patients and 15 age-, sex-, race-, and smoking-matched controls were profiled on cation exchange (CM10) chips using SELDI-TOF. Partial least squares discriminate analysis was used to separate patient and control groups according to the expression of 108 detected peaks, and two peaks of 3,374 and 3,450 Da, corresponding to alpha-defensins based on masses and cationic properties, were found to contribute significantly to the separation of patient and control groups. Reduction of T cell lysates with DTT resulted in a 6-Da shift in the mass of these peaks consistent with the presence of three cysteine bonds in the structure, confirming them as alpha-defensins. Quantification of alpha-defensins in T cell lysates from six patients and 18 healthy controls was carried out by ELISA, which also showed that alpha-defensin levels were significantly increased in patient lysates when compared with matched controls (p = 0.0197). Plasma from 21 monozygotic twins discordant for schizophrenia and eight healthy unaffected twin pairs was also analyzed for the expression of alpha-defensins by ELISA. Notably both affected and unaffected twins were found to have significantly elevated alpha-defensin levels compared with healthy control twin pairs (p = 0.0014 and p = 0.0115, respectively). Increased expression of alpha-defensins in unaffected as well as affected discordant monozygotic twins is of particular interest as monozygotic twins share genes and usually environmental upbringing. The unaffected twin therefore represents the biological and environmental risk of developing schizophrenia in the absence of overt symptomatology and therapeutic medication. These findings suggest that alpha-defensins could be an important early indicator of the risk of schizophrenia.     

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 × 10⁻⁸ ± 0.87 × 10⁻⁸ per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry

During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes.     

Nematode resistance

Plant-parasitic nematodes are major pests of both temperate and tropical agriculture. Many of the most damaging species employ an advanced parasitic strategy in which they induce redifferentiation of root cells to form specialized feeding structures able to support nematode growth and reproduction over several weeks. Current control measures, particularly in intensive agriculture systems, rely heavily on nematicides but alternative strategies are required as effective chemicals are withdrawn from use. Here, we review the different approaches that are being developed to provide resistance to a range of nematode species. Natural, R gene-based resistance is currently exploited in traditional breeding programmes and research is ongoing to characterize the molecular basis for the observed resistant phenotypes. A number of transgenic approaches hold promise, the best described being the expression of proteinase inhibitors to disrupt nematode digestion. The application of plant-delivered RNA interference (RNAi) to silence essential nematode genes has recently emerged as a potentially valuable resistance strategy.     

Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.

Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (integralPhr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial PO(2) = 40 +/- 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [1) control (n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia (n = 7 each)]. Ketanserin transiently decreased integralPhr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, integralPhr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.