A bioluminogenic HDAC activity assay: validation and screening

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z'), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 μL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Long-term climate forcing in loggerhead sea turtle nesting

The long-term variability of marine turtle populations remains poorly understood, limiting science and management. Here we use basin-scale climate indices and regional surface temperatures to estimate loggerhead sea turtle (Caretta caretta) nesting at a variety of spatial and temporal scales. Borrowing from fisheries research, our models investigate how oceanographic processes influence juvenile recruitment and regulate population dynamics. This novel approach finds local populations in the North Pacific and Northwest Atlantic are regionally synchronized and strongly correlated to ocean conditions--such that climate models alone explain up to 88% of the observed changes over the past several decades. In addition to its performance, climate-based modeling also provides mechanistic forecasts of historical and future population changes. Hindcasts in both regions indicate climatic conditions may have been a factor in recent declines, but future forecasts are mixed. Available climatic data suggests the Pacific population will be significantly reduced by 2040, but indicates the Atlantic population may increase substantially. These results do not exonerate anthropogenic impacts, but highlight the significance of bottom-up oceanographic processes to marine organisms. Future studies should consider environmental baselines in assessments of marine turtle population variability and persistence.     

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.     

Pollen production and circulation patterns along an elevation gradient in Mt Olympos (Greece) National Park

We studied airborne pollen along an elevation gradient of Mt Olympos (Greece). Samples were collected on a regular basis, over the period March–October 2009, in eight elevation-different stations, by use of a portable Hirst-type volumetric sampler. Concurrently, we studied pollen production in Quercus coccifera, Q. ilex, Pinus heldreichii and P. nigra, which are dominant species in the main vegetation types of the mountain. Of the 35 pollen taxa detected in the air, 18 account for 99.1 % of the total airborne pollen. These are the main pollen taxa each contributing by at least 0.5 %. Pinaceae (32 %) followed by Quercus (24 %) and Urticaceae (18 %) are the most abundantly represented taxa. Duration of the pollen season decreases with elevation by on average 3 days for every 100 m of elevation increase or by 5 days for every Celsius-degree of temperature decrease. Pollen concentration in the air decreases with elevation for the lowland taxa; with the exception of Cupressaceae, no pattern is observed for the other main taxa. The pine and oak species studied carry comparable amounts of pollen, approximately 10⁴ grains per flower, 10⁸–10⁹ per m² of crown surface and 10¹⁰–10¹¹ per individual; pollen production, primarily of the two Quercus species, is not responsive to environmental changes associated with elevation. Results provide evidence that, within a margin of error, airborne pollen reflects the distributions of pollen taxa on the mountain; regarding abundance, airborne pollen is representative of Quercus, but under-representative of Pinaceae. Ambrosia pollen is found at all elevations examined, although plants producing it have not been recorded on or around Mt Olympos.     

Effects of environmental factors on pollen production in anemophilous woody species

The aim of this study was to estimate the amount of pollen produced by anemophilous woody taxa with allergenic properties and with considerable contribution in the concentration of pollen in the air of a Mediterranean city (Thessaloniki, Greece). The taxa selected are Corylus avellana, Cupressus sempervirens var. horizontalis and var. pyramidalis, Olea europaea and Platanus orientalis; each was studied in more than one sampling stations differing in elevation, direction or both. O. europaea produced the highest number of pollen grains per flower (1.3 × 10⁵ ± 0.1 × 10⁵) and P. orientalis the highest per inflorescence (3.3 × 10⁶ ± 0.2 × 10⁶). At the level of crown, pollen grains produced were of the order of 10⁹ per surface/volume unit for O. europaea and the two C. sempervirens varieties; for the other two taxa, they were of the order of 10⁶. Pollen production was lower at higher elevation and northern direction and depended on the size of the floral unit sampled (flower for O. europaea, inflorescence for all other species): the bigger the floral unit, the more pollen it contained. Our results and reports from other areas, where C. sempervirens and O. europaea grow, show that these two Mediterranean species produce comparable amounts of pollen at the levels of inflorescence or flower, respectively, wherever they occur.     

Development and use of a gene deletion strategy for Flavobacterium johnsoniae to identify the redundant gliding motility genes remF, remG, remH, and remI

Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmid restored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type rpsL and used streptomycin for counterselection. This approach was used to delete the region spanning remF, remG, and fjoh_0982. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had no effect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The rpsL counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of F. johnsoniae physiology.     

<Emphasis Type="Italic">Parietaria judaica</Emphasis> flowering phenology,pollen production,viability and atmospheric circulation,and expansive ability in the urban environment: impacts of environmental factors

Parietaria judaica (Urticaceae) grows abundantly in urban areas of the Mediterranean region. Its pollen is a major allergy source. We studied the species’ distribution and abundance in and around Thessaloniki (Greece), pollen production and pollen season. We also examined how urban pollution affects pollen viability. Our ultimate goal was to obtain an estimate of the species’ performance and ability to expand under different environmental conditions related to climate change. We mapped P. judaica and the other Urticaceae species. In a north- and a south-facing population, we recorded the progress of P. judaica flowering and estimated the pollen content per flower, shoot and surface unit. We concurrently assessed atmospheric circulation of Urticaceae pollen. We estimated P. judaica pollen viability and Cu, Pb and Zn concentrations in plants collected from sites differing in traffic intensity. P. judaica is the most abundant Urticaceae species in the area; its occurrence has increased dramatically over the last 100 years. Production of flowers is intense in spring and autumn. Flowering started 12 days earlier in the south-facing population in spring, and 3 days later in autumn. Pollen production was higher in spring and in the south-facing population. Flower and pollen production were positively correlated with the size of the plant and the flower, respectively. Copper and lead concentrations in plants were positively correlated with pollen viability, which was higher for plants collected from high-traffic sites. P. judaica has a high phenotypic plasticity; this is a feature that promotes success of expansive and invasive species. It is also well adapted to warm and polluted urban environments. The climatic change forecast for the Mediterranean region could provoke earlier, longer, and more pronounced flowering and, consequently, more P. judaica pollen in the air. In return, this would result in increased severity of Parietaria pollinosis.     

Signals of Climate Change in Butterfly Communities in a Mediterranean Protected Area

The European protected-area network will cease to be efficient for biodiversity conservation, particularly in the Mediterranean region, if species are driven out of protected areas by climate warming. Yet, no empirical evidence of how climate change influences ecological communities in Mediterranean nature reserves really exists. Here, we examine long-term (1998–2011/2012) and short-term (2011–2012) changes in the butterfly fauna of Dadia National Park (Greece) by revisiting 21 and 18 transects in 2011 and 2012 respectively, that were initially surveyed in 1998. We evaluate the temperature trend for the study area for a 22-year-period (1990–2012) in which all three butterfly surveys are included. We also assess changes in community composition and species richness in butterfly communities using information on (a) species’ elevational distributions in Greece and (b) Community Temperature Index (calculated from the average temperature of species'' geographical ranges in Europe, weighted by species'' abundance per transect and year). Despite the protected status of Dadia NP and the subsequent stability of land use regimes, we found a marked change in butterfly community composition over a 13 year period, concomitant with an increase of annual average temperature of 0.95°C. Our analysis gave no evidence of significant year-to-year (2011–2012) variability in butterfly community composition, suggesting that the community composition change we recorded is likely the consequence of long-term environmental change, such as climate warming. We observe an increased abundance of low-elevation species whereas species mainly occurring at higher elevations in the region declined. The Community Temperature Index was found to increase in all habitats except agricultural areas. If equivalent changes occur in other protected areas and taxonomic groups across Mediterranean Europe, new conservation options and approaches for increasing species’ resilience may have to be devised.     

Copper chelator induced efficient episodic memory recovery in a non-transgenic Alzheimer's mouse model

Alzheimer's disease (AD) is a neurodegenerative syndrom involving many different biological parameters, including the accumulation of copper metal ions in Aβ amyloid peptides due to a perturbation of copper circulation and homeostasis within the brain. Copper-containing amyloids activated by endogenous reductants are able to generate an oxidative stress that is involved in the toxicity of abnormal amyloids and contribute to the progressive loss of neurons in AD. Since only few drugs are currently available for the treatment of AD, we decided to design small molecules able to interact with copper and we evaluated these drug-candidates with non-transgenic mice, since AD is mainly an aging disease, not related to genetic disorders. We created a memory deficit mouse model by a single icv injection of Aβ(1-42) peptide, in order to mimic the early stage of the disease and the key role of amyloid oligomers in AD. No memory deficit was observed in the control mice with the antisense Aβ(42-1) peptide. Here we report the capacity of a new copper-specific chelating agent, a bis-8-aminoquinoline PA1637, to fully reverse the deficit of episodic memory after three weeks of treatment by oral route on non-transgenic amyloid-impaired mice. Clioquinol and memantine have been used as comparators to validate this fast and efficient mouse model.