Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

Mosaic arthroplasty of the medial femoral condyle in horses - an experimental study.

One Arabian and 5 Hungarian half-bred horses were used to study the macroscopic and microscopic survival of autologous osteochondral grafts in the weight-bearing surface of the medial femoral condyle (MFC). Grafts were harvested from the cranial surface of the medial femoral trochlea (MFT) under arthroscopic control. Three of them were transplanted into the weight-bearing surface of the contralateral MFC using an arthrotomy approach. Three months later this transplantation procedure was repeated on the opposite stifle joints in the same animals, but at that time transplantation was performed arthroscopically. Follow-up arthroscopy was carried out 12 months after the first operations, and biopsies were taken from both the recipient and the donor sites for histological examination. During follow-up arthroscopy, the transplanted areas looked congruent and smooth. Microscopically, the characteristics of hyaline cartilage were present in 5 out of the 10 biopsies examined; however, in the other half of biopsies glycosaminoglycan (GAG) loss and change in the architecture of the transplanted cartilage was observed. In a 16-year-old horse, all grafts broke during harvesting, and thus transplantation was not performed. No radiological signs of osteoarthritic changes were detected 9 to 12 months after the operations in the donor and recipient joints. Clinically, no lameness or effusion was present three months after the transplantations.

Method for the determination of specific molecular markers of biomass burning in lake sediments

Fire has an influence on regional to global atmospheric chemistry and climate. Molecular markers of biomass burning archived in lake sediments are becoming increasingly important in paleoenvironmental reconstruction and may help determine the interaction between climate and fire activity. Here, we present a high performance anion exchange chromatography–mass spectrometry method to allow separation and analysis of levoglucosan, mannosan and galactosan in lake sediments, with implications for reconstructing past biomass burning events. Determining mannosan and galactosan in Lake Kirkpatrick, New Zealand (45.03°S, 168.57°E) sediment cores and comparing these isomers with the more abundant biomass burning markers levoglucosan and charcoal represents a significant advancement in our ability to analyze past fire activity. Levoglucosan, mannosan and galactosan concentrations correlated significantly with macroscopic charcoal concentration. Levoglucosan/mannosan and levoglucosan/(mannosan + galactosan) ratios may help determine not only when fires occurred, but also if changes in the primary burned vegetation occurred.

Contrasting long-term records of biomass burning in wet and dry savannas of equatorial East Africa

Rainfall controls fire in tropical savanna ecosystems through impacting both the amount and flammability of plant biomass, and consequently, predicted changes in tropical precipitation over the next century are likely to have contrasting effects on the fire regimes of wet and dry savannas. We reconstructed the long-term dynamics of biomass burning in equatorial East Africa, using fossil charcoal particles from two well-dated lake-sediment records in western Uganda and central Kenya. We compared these high-resolution (5 years/sample) time series of biomass burning, spanning the last 3800 and 1200 years, with independent data on past hydroclimatic variability and vegetation dynamics. In western Uganda, a rapid (<100 years) and permanent increase in burning occurred around 2170 years ago, when climatic drying replaced semideciduous forest by wooded grassland. At the century time scale, biomass burning was inversely related to moisture balance for much of the next two millennia until ca. 1750 ad, when burning increased strongly despite regional climate becoming wetter. A sustained decrease in burning since the mid20th century reflects the intensified modern-day landscape conversion into cropland and plantations. In contrast, in semiarid central Kenya, biomass burning peaked at intermediate moisture-balance levels, whereas it was lower both during the wettest and driest multidecadal periods of the last 1200 years. Here, burning steadily increased since the mid20th century, presumably due to more frequent deliberate ignitions for bush clearing and cattle ranching. Both the observed historical trends and regional contrasts in biomass burning are consistent with spatial variability in fire regimes across the African savanna biome today. They demonstrate the strong dependence of East African fire regimes on both climatic moisture balance and vegetation, and the extent to which this dependence is now being overridden by anthropogenic activity.

Self-burning – A rare suicide method in switzerland and other industrialised nations – A review

News items reporting self-immolation by Tibetans have been on the increase in recent years. After examining the corpse of a Swiss man who had committed suicide by deliberate self-burning, we wondered how often this occurs in Switzerland. The Federal Statistics Office (FSO) does not register self-burning specifically so no official national data on this form of suicide are available. However, we had access to the data from a Swiss National Science Foundation (SNSF) project Suicides in Switzerland between 2000 and 2010, which collected information on all (4885) cases of suicide investigated by the various institutes of forensic medicine. From this data pool we extracted 50 cases (1.02%) of suicide by selfburning, in order to determine the details and to identify the possible reasons for choosing this method. To look at our results in the light of studies from other countries, we searched the literature for studies that had also retrospectively examined suicide by self-immolation based on forensic records. Our results showed that, on the whole, personal aspects of selfburning in Switzerland do not differ from those in other industrialised nations. Some data, including religious and sociocultural background, were unfortunately missing – not only from our study but also from the similar ones. In our opinion, the most important prevention strategy is to make healthcare professionals more aware of this rare method of suicide.

Late cardiac sodium current can be assessed using automated patch-clamp

The cardiac late Na (+) current is generated by a small fraction of voltage-dependent Na (+) channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na (+) component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

Mosaic pattern of sucrase isomaltase deficiency in two brothers

The pathophysiology of mucosal changes observed in infants with chronic protracted diarrhea is poorly understood. We report on two brothers suffering from a special form of sucrase isomaltase (SI) deficiency. The children presented with weight loss and dyspepsia after sucrose exposition. We performed an H respiration test, which showed a pathologic result in the younger brother. Analysis of the brush border enzyme activities showed low expression of lactase and SI. Immunoelectron microscopy of duodenal biopsies showed an isolated SI deficiency in a mosaic pattern [e.g., 42% (14%) crypt enterocytes and 64% (59%) villus enterocytes with decreased amounts of SI on microvilli], whereas lactase and aminopeptidase n (ApN) were present at the apical membrane of all cells in a normal range. The SI mosaic pattern of these patients shows that the enterocytes contain low amounts of SI on the apical membrane but express normal quantities of other disaccharidases. These findings suggest the existence of different clonal expressions or specific (posttranslational) mechanisms of postGolgi transportation for individual brush border enzymes. It remains unresolved whether the mosaic distribution is part of a normal maturation process or caused by a lack of an overall control mechanism in the expression of brush border hydrolases.

Holocene biomass burning and global dynamics of the carbon cycle

Fire regimes have changed during the Holocene due to changes in climate, vegetation, and in human practices. Here, we hypothesise that changes in fire regime may have affected the global CO2 concentration in the atmosphere through the Holocene. Our data are based on quantitative reconstructions of biomass burning deduced from stratified charcoal records from Europe, and South-, Central- and North America, and Oceania to test the fire-carbon release hypothesis. In Europe the significant increase of fire activity is dated ≈6000 cal. yr ago. In north-eastern North America burning activity was greatest before 7500 years ago, very low between 7500–3000 years, and has been increasing since 3000 years ago. In tropical America, the pattern is more complex and apparently latitudinally zonal. Maximum burning occurred in the southern Amazon basin and in Central America during the middle Holocene, and during the last 2000 years in the northern Amazon basin. In Oceania, biomass burning has decreased since a maximum 5000 years ago. Biomass burning has broadly increased in the Northern and Southern hemispheres throughout the second half of the Holocene associated with changes in climate and human practices. Global fire indices parallel the increase of atmospheric CO2 concentration recorded in Antarctic ice cores. Future issues on carbon dynamics relatively to biomass burning are discussed to improve the quantitative reconstructions.

Linking livelihood strategies with a regional level landscape mosaic to explore trade-offs between forest conservation and human wellbeing in a Madagascar biodiversity hotspot

The north-eastern escarpment of Madagascar contains the island’s last remaining large-scale humid forest massifs surrounded by diverse small-scale agricultural mosaics. There is high deforestation mainly caused by shifting cultivation practiced by local land users to produce upland rice for subsistence. Today, large protected areas restrict land users’ access to forests to collect wood and other forest products. Moreover, they are no more able to expand their cultivated land, which leads to shorter shifting cultivation cycles and decreasing plot sizes for irrigated rice and cash crop cultivation. Cash crop production of clove and vanilla is exposed to risks such as extreme inter-annual price fluctuations, pests and cyclones. In the absence of work opportunities, agricultural extension services and micro-finance schemes people are stuck in a poverty trap. New development strategies are needed to mitigate the trade-offs between forest conservation and human well-being. As landscape composition and livelihood strategies vary across the region, these strategies need to be spatially differentiated to avoid implementing generic solutions, which do not fit the local context. However, up to date, little is known about the spatial patterns of shifting cultivation and other land use systems at the regional level. This is mainly due to the high spatial and temporal dynamics inherent to shifting cultivation, which makes it difficult to monitor the dynamics of this land use system with remote sensing methods. Furthermore, knowledge about land users’ livelihood strategies and the risks and opportunities they face stems from very few local case studies. To overcome this challenge, firstly, we used remote sensing data and a landscape mosaic approach to delineate the main landscape types at the regional level. Secondly, we developed a land user typology based on socio-ecological data from household surveys in 45 villages spread throughout the region. Combining the land user typology with the landscape mosaic map allowed us to reveal spatial patterns of the interaction between landscapes and people and to better understand the trade-offs between forest conservation and local wellbeing. While shifting cultivation systems are being transformed into more intensive permanent agricultural systems in many countries around the globe, Madagascar seems to be an exception to this trend. Linking land cover information to human-environmental interactions over large areas is crucial to designing policies and to inform decision making for a more sustainable development of this resource-rich but poverty-prone context.

Comparison of several wood smoke markers and source apportionment methods for wood burning particulate mass

Residential wood combustion has only recently been recognized as a major contributor to air pollution in Switzerland and in other European countries. A source apportionment method using the aethalometer light absorption parameters was applied to five winter campaigns at three sites in Switzerland: a village with high wood combustion activity in winter, an urban background site and a highway site. The particulate mass from traffic (PMtraffic) and wood burning (PMwb) emissions obtained with this model compared fairly well with results from the 14C source apportionment method. PMwb from the model was also compared to well known wood smoke markers such as anhydrosugars (levoglucosan and mannosan) and fine mode potassium, as well as to a marker recently suggested from the Aerodyne aerosol mass spectrometer (mass fragment m/z 60). Additionally the anhydrosugars were compared to the 14C results and were shown to be comparable to literature values from wood burning emission studies using different types of wood (hardwood, softwood). The levoglucosan to PMwb ratios varied much more strongly between the different campaigns (4–13%) compared to mannosan to PMwb with a range of 1–1.5%. Possible uncertainty aspects for the various methods and markers are discussed.

Beyond deforestation monitoring in conservation hotspots: Analysing landscape mosaic dynamics in north-eastern Madagascar

Due to its extraordinary biodiversity and rapid deforestation, north-eastern Madagascar is a conservation hotspot of global importance. Reducing shifting cultivation is a high priority for policy-makers and conservationists; however, spatially explicit evidence of shifting cultivation is lacking due to the difficulty of mapping it with common remote sensing methods. To overcome this challenge, we adopted a landscape mosaic approach to assess the changes between natural forests, shifting cultivation and permanent cultivation systems at the regional level from 1995 to 2011. Our study confirmed that shifting cultivation is still being used to produce subsistence rice throughout the region, but there is a trend of intensification away from shifting cultivation towards permanent rice production, especially near protected areas. While large continuous forest exists today only in the core zones of protected areas, the agricultural matrix is still dominated by a dense cover of tree crops and smaller forest fragments. We believe that this evidence makes a crucial contribution to the development of interventions to prevent further conversion of forest to agricultural land while improving local land users' well-being.

The role of patch testing in the management of oral lichenoid reactions.

BACKGROUND AND OBJECTIVES The distinction of oral lichenoid reactions from oral lichen planus may be difficult in a clinical setting. Our aims were to ascertain the utility of patch testing to confirm the association of oral lichenoid reactions with dental restorations and to identify the benefits of replacement of restorations, primarily made of amalgam. METHODS Patients seen in an oral medicine unit over a 10-year period diagnosed with oral lichenoid reactions, with oral lichen planus resistant to treatment or with atypical lichenoid features were included in this study. All had been subjected to skin patch testing. Histopathology reports blinded to patch test results were scrutinized. Patch-test-positive subjects were advised to have their restorations replaced. All were followed up to determine disease resolution for at least 3 months thereafter. RESULTS Among 115 patients, 67.8% patients reacted positive to a dental material and nearly a quarter to mercury or amalgam. No correlation was found between pathology and skin patch testing results (P = 0.44). A total of 87 patients were followed up in clinic, and among 26 patch-test-positive patients who had their amalgam fillings replaced, moderate to complete resolution was noted in 81%. CONCLUSIONS Skin patch testing is a valuable tool to confirm clinically suspected oral lichenoid reactions. Pathology diagnoses of oral lichenoid reactions did not correlate with patch test results. Prospective studies are needed to ascertain that a clinically suspected oral lichenoid reaction with a positive patch test result may resolve after the replacement of amalgam fillings.