How to deal with ground truthing affected by human‐induced habitat change?: Identifying high‐quality habitats for the Critically Endangered Red Siskin

Species distribution models (SDM) can be valuable for identifying key habitats for conservation management of threatened taxa, but anthropogenic habitat change can undermine SDM accuracy. We used data for the Red Siskin (Spinus cucullatus), a critically endangered bird and ground truthing to examine anthropogenic habitat change as a source of SDM inaccuracy. We aimed to estimate: (1) the Red Siskin's historic distribution in Venezuela; (2) the portion of this historic distribution lost to vegetation degradation; and (3) the location of key habitats or areas with both, a high probability of historic occurrence and a low probability of vegetation degradation. We ground‐truthed 191 locations and used expert opinion as well as landscape characteristics to classify species' habitat suitability as excellent, good, acceptable, or poor. We fit a Random Forest model (RF) and Enhanced Vegetation Index (EVI) time series to evaluate the accuracy and precision of the expert categorization of habitat suitability. We estimated the probability of historic occurrence by fitting a MaxLike model using 88 presence records (1960–2013) and data on forest cover and aridity index. Of the entire study area, 23% (20,696 km²) had a historic probability of Red Siskin occurrence over 0.743. Furthermore, 85% of ground‐truthed locations had substantial reductions in mean EVI, resulting in key habitats totaling just 976 km², in small blocks in the western and central regions. Decline in Area of Occupancy over 15 years was between 40% and 95%, corresponding to an extinction risk category between Vulnerable and Critically Endangered. Relating key habitats with other landscape features revealed significant risks and opportunities for proposed conservation interventions, including the fact that ongoing vegetation degradation could limit the establishment of reintroduced populations in eastern areas, while the conservation of remaining key habitats on private lands could be improved with biodiversity‐friendly agri‐ and silviculture programs.     

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6

Diabetic retinopathy (DR ) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose‐induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT 6, a NAD ‐dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT 6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT 6 in modulating glycolysis, we aimed to analyze SIRT 6 participation in the molecular machinery that regulates the development of experimental DR . Using non‐obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF ), and the loss of a neuroprotective factor (brain‐derived neurotrophic factor, BDNF) associated with reduced levels of SIRT 6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT 6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF . Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT 6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over‐expression of SIRT 6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT 6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

     

Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants

Plants synthesize N-glycans containing the antigenic sugars α(1,3)-fucose and β(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of α(1,3)-fucose and β(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of α1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.     

N,N′-bis-(2,3-methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea interact with auxin in enhancing root formation of M26 apple (Malus pumila Mill.) stem slices

Stem slices cut from micropropagated cuttings of apple rootstock M26 were cultured in the presence of indole-3-butyric acid (IBA) plus N,N-bis-(2,3-methylenedioxyphenyl)urea or N,N-bis-(3,4-methylenedioxyphenyl)urea, to verify if there was an interaction between them in enhancing root formation. The N,N-bis-(methylenedioxyphenyl)ureas were supplemented after, before and in the simultaneous presence of auxin. Our data demonstrate that only the simultaneous presence of auxin and N,N-bis-(methylenedioxyphenyl)ureas in the culture medium enhanced root formation on M26 stem slices. The percentage of rooted slices obtained in the presence of the mixtures was significantly different from that obtained in the presence of low auxin concentration alone (1µM). Moreover both the percentage of rooted slices and the number of roots per slice obtained in these culture conditions was not significantly different to that of the optimal auxinic treatment in which the auxin concentration was threefold higher.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.     

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.