Defining an adequate sample of earlywood vessels for retrospective injury detection in diffuse-porous species

Vessels of broad-leaved trees have been analyzed to study how trees deal with various environmental factors. Cambial injury, in particular, has been reported to induce the formation of narrower conduits. Yet, little or no effort has been devoted to the elaboration of vessel sampling strategies for retrospective injury detection based on vessel lumen size reduction. To fill this methodological gap, four wounded individuals each of grey alder (Alnus incana (L.) Moench) and downy birch (Betula pubescens Ehrh.) were harvested in an avalanche path. Earlywood vessel lumina were measured and compared for each tree between the injury ring built during the growing season following wounding and the control ring laid down the previous year. Measurements were performed along a 10 mm wide radial strip, located directly next to the injury. Specifically, this study aimed at (i) investigating the intra-annual duration and local extension of vessel narrowing close to the wound margin and (ii) identifying an adequate sample of earlywood vessels (number and intra-ring location of cells) attesting to cambial injury. Based on the results of this study, we recommend analyzing at least 30 vessels in each ring. Within the 10 mm wide segment of the injury ring, wound-induced reduction in vessel lumen size did not fade with increasing radial and tangential distances, but we nevertheless advise favoring early earlywood vessels located closest to the injury. These findings, derived from two species widespread across subarctic, mountainous, and temperate regions, will assist retrospective injury detection in Alnus, Betula, and other diffuse-porous species as well as future related research on hydraulic implications after wounding.     

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 ¹. Because of the ease of genetic manipulation in mice, these models have also been developed in this species ^2-3.Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume ⁴. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.     

A Rapid and Efficient Method for Isolating High Quality DNA from Leaves of Carnivorous Plants from the Drosera Genus

Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A???/A??? ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.     

Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes

Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.     

Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa

ABSTRACT: Bluetongue (BT) is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV), the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd -immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.     

Tolerance without clonal expansion: self-antigen-expressing B cells program self-reactive T cells for future deletion

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.     

Regulation of the high affinity IgE receptor (Fc epsilonRI) in human neutrophils: role of seasonal allergen exposure and Th-2 cytokines

The high affinity IgE receptor, Fc epsilonRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional Fc epsilonRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of Fc epsilonRI-alpha chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, Fc epsilonRI-alpha chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/Fc gammaRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced Fc epsilonRI-alpha chain surface expression. In conclusion, these results suggest that enhanced Fc epsilonRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma.