A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

Garlic extract induces intestinal P-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans

Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein and cytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to study the effects of garlic on pre-systemic drug elimination, healthy volunteers were administered garlic extract for 21 days. Prior to and at the end of this period, expression of duodenal P-glycoprotein and cytochrome P450 3A4 protein were assayed and normalized to villin, while hepatic cytochrome P450 3A4 function and simvastatin, pravastatin and saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extract increased expression of duodenal P-glycoprotein to 131% (95% CI, 105-163%), without increasing the expression of cytochrome P450 3A4 which amounted to 87% (95% CI, 67-112%), relative to baseline in both cases. For the erythromycin breath test performed, the average result was 96% (95% CI, 83-112%). Ingestion of garlic extract had no effect on drug and metabolite AUCs following a single dose of simvastatin or pravastatin, although the average area under the plasma concentration curve (AUC) of saquinavir decreased to 85% (95% CI, 66-109%), and changes in intestinal P-glycoprotein expression negatively correlated with this change. In conclusion, garlic extract induces intestinal expression of P-glycoprotein independent of cytochrome P450 3A4 in human intestine and liver.

Mechanisms of osteoclastogenesis inhibition by a novel class of biphenyl-type cannabinoid CB(2) receptor inverse agonists

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Citizen science reveals unexpected continental-scale evolutionary change in a model organism

Organisms provide some of the most sensitive indicators of climate change and evolutionary responses are becoming apparent in species with short generation times. Large datasets on genetic polymorphism that can provide an historical benchmark against which to test for recent evolutionary responses are very rare, but an exception is found in the brown-lipped banded snail (Cepaea nemoralis). This species is sensitive to its thermal environment and exhibits several polymorphisms of shell colour and banding pattern affecting shell albedo in the majority of populations within its native range in Europe. We tested for evolutionary changes in shell albedo that might have been driven by the warming of the climate in Europe over the last half century by compiling an historical dataset for 6,515 native populations of C. nemoralis and comparing this with new data on nearly 3,000 populations. The new data were sampled mainly in 2009 through the Evolution MegaLab, a citizen science project that engaged thousands of volunteers in 15 countries throughout Europe in the biggest such exercise ever undertaken. A known geographic cline in the frequency of the colour phenotype with the highest albedo (yellow) was shown to have persisted and a difference in colour frequency between woodland and more open habitats was confirmed, but there was no general increase in the frequency of yellow shells. This may have been because snails adapted to a warming climate through behavioural thermoregulation. By contrast, we detected an unexpected decrease in the frequency of Unbanded shells and an increase in the Mid-banded morph. Neither of these evolutionary changes appears to be a direct response to climate change, indicating that the influence of other selective agents, possibly related to changing predation pressure and habitat change with effects on micro-climate.

SiRNA inhibits replication of Langat virus, a member of the tick-borne encephalitis virus complex in organotypic rat brain slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000-3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3' untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

Quantitative assessment of soil parameters in Western Tajikistan using a soil spectral library approach

Soil degradation is a major problem in the agriculturally dominated country of Tajikistan, which makes it necessary to determine and monitor the state of soils. For this purpose a soil spectral library was established as it enables the determination of soil properties with relatively low costs and effort. A total of 1465 soil samples were collected from three 10x10 km test sites in western Tajikistan. The diffuse reflectance of the samples was measured with a FieldSpec PRO FR from ASD in the spectral range from 380 to 2500 nm in laboratory. 166 samples were finally selected based on their spectral information and analysed on total C and N, organic C, pH, CaCO₃, extractable P, exchangeable Ca, Mg and K, and the fractions clay, silt and sand. Multiple linear regression was used to set up the models. Two third of the chemically analysed samples were used to calibrate the models, one third was used for hold-out validation. Very good prediction accuracy was obtained for total C (R² = 0.76, RMSEP = 4.36 g kg⁻¹), total N (R² = 0.83, RMSEP = 0.30 g kg⁻¹) and organic C (R² = 0.81, RMSEP = 3.30 g kg⁻¹), good accuracy for pH (R² = 0.61, RMSEP = 0.157) and CaCO3(R² = 0.72, RMSEP = 4.63 %). No models could be developed for extractable P, exchangeable Ca, Mg and K, and the fractions clay, silt and sand. It can be concluded that the spectral library approach has a high potential to substitute standard laboratory methods where rapid and inexpensive analysis is required.