Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants

Background: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. Methods: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. Results: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. Conclusions: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations

Physiological recovery among workers in long-distance sleddog race: a case study on female veterinarians in Finnmarksløpet.

During Finnmarksløpet (FL, one of the longest distance sleddog races in the world), veterinarians are exposed to extreme environmental conditions and tight working schedules, with little and fragmented sleep. OBJECTIVE: The aim of this case study was to examine cardiovascular parameters and sleep-wake patterns among veterinarians working within FL, during and after (for a month) the end of the race. METHODS: Six female veterinarians volunteered for the study. The participants wore a wrist device for a total of eight weeks in order to passively and semi-continuously record physiological responses throughout the day (i.e., heart rate, heart rate variability, number of steps, and sleep quality). Moreover, perceived sleep quality was assessed by Pittsburgh Sleep Quality Index (PSQI). RESULTS: During and for one month after completion of the FL, most veterinarians presented an alteration of cardiovascular parameters and sleep quality. The heart rate circadian rhythm returned to pre-race values within about two weeks. CONCLUSIONS: The long-lasting alteration of the veterinarians’ cardiovascular parameters and sleep-wake patterns might have negative consequences for their health in the long-term, especially if similar experiences are repeated more times though the course of a year or season. More research is needed in order to understand the health risks, as well as how to prevent them, among veterinarians in long-distance sleddog races or other similar events.acceptedVersio

Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress

Background Maternal exposure to environmental stressors poses a risk to fetal development. Oxidative stress (OS), microglia activation, and inflammation are three tightly linked mechanisms that emerge as a causal factor of neurodevelopmental anomalies associated with prenatal ethanol exposure. Antioxidants such as glutathione (GSH) and CuZnSOD are perturbed, and their manipulation provides evidence for neuroprotection. However, the cellular and molecular effects of GSH alteration in utero on fetal microglia activation and inflammation remain elusive. Methods Ethanol (EtOH) (2.5 g/kg) was administered to pregnant mice at gestational days 16–17. One hour prior to ethanol treatment, N-acetylcysteine (NAC) and L-buthionine sulfoximine (BSO) were administered to modulate glutathione (GSH) content in fetal and maternal brain. Twenty-four hours following ethanol exposure, GSH content and OS in brain tissues were analyzed. Cytokines and chemokines were selected based on their association with distinctive microglia phenotype M1-like (IL-1β, IFN γ, IL-6, CCL3, CCL4, CCL-7, CCL9,) or M2-like (TGF-β, IL-4, IL-10, CCL2, CCL22, CXCL10, Arg1, Chi1, CCR2 and CXCR2) and measured in the brain by qRT-PCR and ELISA. In addition, Western blot and confocal microscopy techniques in conjunction with EOC13.31 cells exposed to similar ethanol-induced oxidative stress and redox conditions were used to determine the underlying mechanism of microglia activation associated with the observed phenotypic changes. Results We show that a single episode of mild to moderate OS in the last trimester of gestation causes GSH depletion, increased protein and lipid peroxidation and inflammatory responses inclined towards a M1-like microglial phenotype (IL-1β, IFN-γ) in fetal brain tissue observed at 6–24 h post exposure. Maternal brain is resistant to many of these marked changes. Using EOC 13.31 cells, we show that GSH homeostasis in microglia is crucial to restore its anti-inflammatory state and modulate inflammation. Microglia under oxidative stress maintain a predominantly M1 activation state. Additionally, GSH depletion prevents the appearance of the M2-like phenotype, while enhancing morphological changes associated with a M1-like phenotype. This observation is also validated by an increased expression of inflammatory signatures (IL-1β, IFN-γ, IL-6, CCL9, CXCR2). In contrast, conserving intracellular GSH concentrations eliminates OS which precludes the nuclear translocation and more importantly the phosphorylation of the NFkB p105 subunit. These cells show significantly more pronounced elongations, ramifications, and the enhanced expression of M2-like microglial phenotype markers (IL-10, IL-4, TGF-β, CXCL10, CCL22, Chi, Arg, and CCR2). Conclusions Taken together, our data show that maintaining GSH homeostasis is not only important for quenching OS in the developing fetal brain, but equally critical to enhance M2 like microglia phenotype, thus suppressing inflammatory responses elicited by environmental stressors

Evaluation of polyneuropathy markers in type 1 diabetic kidney transplant patients and effects of islet transplantation: Neurophysiological and skin biopsy longitudinal analysis

OBJECTIVE - The purpose of this study was to evaluate whether islet transplantation may stabilize polyneuropathy in uremic type 1 diabetic patients (end-stage renal disease [ESRD] and type 1 diabetes), who received a successful islet-after-kidney transplantation (KI-s). RESEARCH DESIGN AND METHODS - Eighteen KI-s patients underwent electroneurographic tests of sural, peroneal, ulnar, and median nerves: the nerve conduction velocity (NCV) index and amplitudes of both sensory action potentials (SAPs) and compound motor action potentials (CMAPs) were analyzed longitudinally at 2, 4, and 6 years after islet transplantation. Skin content of advanced glycation end products (AGEs) and expression of their specific receptors (RAGE) were also studied at the 4-year follow-up. Nine patients with ESRD and type 1 diabetes who received kidney transplantation alone (KD) served as control subjects. RESULTS - The NCV score improved in the KI-s group up to the 4-year time point (P = 0.01 versus baseline) and stabilized 2 years later, whereas the same parameter did not change significantly in the KD group throughout the follow-up period or when a cross-sectional analysis between groups was performed. Either SAP or CMAP amplitudes recovered in the KI-s group, whereas they continued worsening in KD control subjects. AGE and RAGE levels in perineurium and vasa nervorum of skin biopsies were lower in the KI-s than in the KD group (P < 0.01 for RAGE). CONCLUSIONS - Islet transplantation seems to prevent long-term worsening of polyneuropathy in patients with ESRD and type 1 diabetes who receive islets after kidney transplantation. No statistical differences between the two groups were evident on cross-sectional analysis. A reduction in AGE/RAGE expression in the peripheral nervous system was shown in patients receiving islet transplantation. © 2007 by the American Diabetes Association

Feedback topology and XOR-dynamics in Boolean networks with varying input structure

We analyse a model of fixed in-degree Random Boolean Networks in which the fraction of input-receiving nodes is controlled by a parameter gamma. We investigate analytically and numerically the dynamics of graphs under a parallel XOR updating scheme. This scheme is interesting because it is accessible analytically and its phenomenology is at the same time under control, and as rich as the one of general Boolean networks. Biologically, it is justified on abstract grounds by the fact that all existing interactions play a dynamical role. We give analytical formulas for the dynamics on general graphs, showing that with a XOR-type evolution rule, dynamic features are direct consequences of the topological feedback structure, in analogy with the role of relevant components in Kauffman networks. Considering graphs with fixed in-degree, we characterize analytically and numerically the feedback regions using graph decimation algorithms (Leaf Removal). With varying gamma, this graph ensemble shows a phase transition that separates a tree-like graph region from one in which feedback components emerge. Networks near the transition point have feedback components made of disjoint loops, in which each node has exactly one incoming and one outgoing link. Using this fact we provide analytical estimates of the maximum period starting from topological considerations

Energetic metabolic profile of ewes presenting low body condition score induced to subclinical hypocalcemia in early postpartum

The aim of the present study is to assess plasma concentrations of metabolites related to energy balance in ewes showing low body condition score (BCS) induced to subclinical hypocalcemia in early postpartum. Sixteen crossbred ewes (Ideal x Corriedale) presenting BCS &lt;3 were divided in two groups: 1) control group (n = 9), which received no treatment, and 2) hypocalcemia group (n = 7), which was subjected to twelve hours of induced subclinical hypocalcemia through intravenous Na2EDTA infusion six hours postpartum. Ionized calcium levels were monitored and kept between 0.62 and 0.87 mmol/L. All ewes were subjected to daily blood sampling for five days in order to set the postpartum metabolic profile. There was no interaction between day and treatment in total and ionized calcium concentrations (P&gt;0.05), whereas the mean concentrations of these marks after five days were lower in the hypocalcemia group (P&lt;0.05). However, neither the mean group were different at glucose, non-esterified fatty acids, beta-hydroxybutyrate and insulin (P&gt;0.05) level. These results evidence that, despite the subclinical hypocalcemia induction at early postpartum, ewes presenting low body condition do not change the concentrations of energy balance-related metabolites in the following five days

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificialtransmission to grapevine plants and the resulting symptoms and cytopathological alteration

Prepartum body condition score changes and the secretion of acute phase proteins in dairy cows.

O objetivo deste estudo foi investigar o efeito da condição corporal (BCS) durante o período de pré-parto, sobre a concentração de proteínas de fase aguda e parâmetros metabólicos em vacas leiteiras

autologous pancreatic islet transplantation in human bone marrow diabetes 2013 62 3523 3531

In the article listed above, there is an error in Fig. 3. In panel B , the immunohistochemical staining of insulin ( top middle panel

Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients

OBJECTIVE: Our aim was to evaluate the long-term effects of transplanted islets on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients. RESEARCH DESIGN AND METHODS: A total of 34 type 1 diabetic kidney-transplanted patients underwent islet transplantation and were divided into two groups: successful islet-kidney transplantation (SI-K; 21 patients, fasting C-peptide serum concentration &gt;0.5 ng/ml for &gt;1 year) and unsuccessful islet-kidney transplantation (UI-K; 13 patients, fasting C-peptide serum concentration &lt;0.5 ng/ml). Patients cumulative survival, cardiovascular death rate, and atherosclerosis progression were compared in the two groups. Skin biopsies, endothelial dependent dilation (EDD), nitric oxide (NO) levels, and atherothrombotic risk factors [von Willebrand factor (vWF) and D-dimer fragment (DDF)] were studied cross-sectionally. RESULTS: The SI-K group showed a significant better patient survival rate (SI-K 100, 100, and 90% vs. UI-K 84, 74, and 51% at 1, 4, and 7 years, respectively, P = 0.04), lower cardiovascular death rate (SI-K 1/21 vs. UI-K 4/13, chi(2) = 3.9, P = 0.04), and lower intima-media thickness progression than the UI-K group (SI-K group: delta1-3 years -13 +/- 30 micro m vs. UI-K group: delta1-3 years 245 +/- 20 micro m, P = 0.03) with decreased signs of endothelial injuring at skin biopsy. Furthermore, the SI-K group showed a higher EDD than the UI-K group (EDD: SI-K 7.8 +/- 4.5% vs. UI-K 0.5 +/- 2.7%, P = 0.02), higher basal NO (SI-K 42.9 +/- 6.5 vs. UI-K 20.2 +/- 6.8 micro mol/l, P = 0.02), and lower levels of vWF (SI-K 138.6 +/- 15.3 vs. UI-K 180.6 +/- 7.0%, P = 0.02) and DDF (SI-K 0.61 +/- 0.22 vs. UI-K 3.07 +/- 0.68 micro g/ml, P &lt; 0.01). C-peptide-to-creatinine ratio correlated positively with EDD and NO and negatively with vWF and DDF. CONCLUSIONS: Successful islet transplantation improves survival, cardiovascular, and endothelial function in type 1 diabetic kidney-transplanted patient