Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impact of the zebra mussel Dreissena polymorpha invasion on the budget of suspended material in a shallow lagoon ecosystem

The role of the zebra mussel Dreissena polymorpha in redistribution of total particulate material (TPM) between the water column and bottom sediment was estimated using the TPM budget for a mussel bed in the Curonian lagoon, the Baltic Sea. Seasonal clearance rates were derived from the TPM budget assuming two resuspension scenarios: no resuspension and full resuspension of biodeposits. Estimated clearance rates for both scenarios were compared with the rates calculated from the population clearance rate model. Seasonal clearance rates estimated using the population model (1.1 and 11.8 l g⁻¹ SFDW day⁻¹) fitted well into the interval of seasonal clearance rates calculated from TPM budgets assuming no resuspension of biodeposits (3.2 and 21.4 l g SFDW⁻¹ day⁻¹). In the scenario with biodeposits resuspension clearance rates were much higher (57.4 and 148.9 g SFDW⁻¹ day⁻¹). The ratio of clearance to residence time was highly dependent on the fate of biodeposits. Therefore its use in interpretation of the species impact on TPM was limited. An alternative measure based on the ratio of the amount of TPM biodeposited to TPM transported into the bed was used. It was found that zebra mussels are able to deposit between 10 and 30% of the incoming TPM, and the amount of biodeposited material was correlated with water residence time. Results indicate that the impact of zebra mussels on TPM in the lagoon is small relative to the high transport rates of TPM over the bed. However, annual biosedimentation rate (~590 g m⁻²) in the mussel bed was higher than physical deposition rate (~380 g m⁻²) in accumulation areas devoid of large suspension feeders. We suggest that a local impact due to enhanced availability of organic material to other trophic groups of associated benthic organisms may be more significant than effects on TPM pathways at an ecosystem scale.     

Pathological and immunological evaluation of different regimens of praziquantel treatment in a mouse model of Schistosoma mansoni infection

BackgroundOne of the considerable challenges of schistosomiasis chemotherapy is the inefficacy of praziquantel (PZQ) at the initial phase of the infection. Immature schistosomes are not susceptible to PZQ at the curative dose. Here, we investigated the efficacy of different PZQ regimens administered during the initial stage of Schistosoma mansoni infection in mice.Methodology/Principal findingsTwo months-old mice were individually infected with 80 S. mansoni cercariae and divided into one infected-untreated control group (IC) and four PZQ-treated groups: PZQ at 100 mg/kg/day for five consecutive days (group PZQ1), PZQ at 100 mg/kg/day for 28 days (group PZQ2), PZQ at 18 mg/kg/day for 28 days (group PZQ3) and a single dose of PZQ at 500 mg/kg (group PZQ4). The treatment started on day one post-infection (p.i), and each group of mice was divided into two subgroups euthanized on day 36 or 56 p.i, respectively. We determined the mortality rate, the parasitological burden, the hepatic and intestinal granulomas, the serum levels of Th-1, Th-2, and Th-17 cytokines, and gene expression. The treatment led to a significant (p < 0.001) reduction of worm burden and egg counts in the intestine and liver in groups PZQ2 and PZQ3. On 56^th day p.i, there was a significant reduction (p < 0.001) of the number and volume of the hepatic granulomas in groups PZQ2 and PZQ3 compared to group PZQ1 or PZQ4. Moreover, in group PZQ3, the serum levels of IFN-γ, TNF-α, IL-13, and IL-17 and their liver mRNA expressions were significantly reduced while IL-10 and TGF-β gene expression significantly increased. The highest mortality rate (81.25%) was recorded in group PZQ2.Conclusion/SignificanceThis study revealed that the administration of PZQ at 18 mg/kg/day for 28 consecutive days was the optimal effective posology for treating S. mansoni infection at the initial stage in a murine model.     

Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

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Calibration-in-time versus calibration-in-space (transfer function) to quantitatively infer July air temperature using biological indicators (chironomids) preserved in lake sediments

Calibration-in-space (i.e. modern taxonomic assemblages of biota from many lakes located along a wide temperature gradient calibrated against meteorological data) is generally used to derive species-specific optima and tolerances. This results in transfer functions which then are applied to subfossil assemblages to quantitatively reconstruct environmental variables such as air/water temperature. Developing such transfer functions is time- and money-consuming, thus many biota-inferred temperature records are either based on transfer functions from other regions which might not take into account local characteristics or are only used qualitatively. In varved Lake Silvaplana (Engadine, Switzerland), another way of obtaining quantitative climate reconstructions from taxonomical assemblages preserved in lake sediments was assessed for the past 1000 years. A calibration-in-time (i.e. taxonomic-assemblage-of-biota time series calibrated against meteorological data covering the same time period) was developed for chironomids (non-biting midges) using a weighted-average-partial-least-square (WAPLS) model and compared with a calibration-in-space model. The calibration-in-time had a weaker correlation coefficient (r² = 0.71) than the calibration-in-space (r² = 0.86), but the error of prediction (RMSEP = 0.58 °C) and the maximum bias (Max Bias = 0.73 °C) outperformed the statistics of the calibration-in-space (RMSEP = 1.5 °C; Max Bias = 1.72). This result is probably due to the smaller temperature gradient of the calibration-in-time (6.5 °C) than the calibration-in-space (11.5 °C). For the last 150 years, the Pearson correlation coefficient was significant between the two reconstructions (r_Pearson = 0.52; p < 0.01) suggesting that both models recorded a similar pattern of temperature changes. On the millennium time-scale, both models showed a warm “Medieval Climate Anomaly”, a cold “Little Ice Age” and a warming through the present with significant correlations (rPearson corrected for autocorrelation (corr) = 0.61, p < 0.01) until ca. 1780 AD and between ca. 1937 and 2000 AD (r_{Pearson corr} = 0.90, p < 0.01). The reconstructions using both models significantly diverged between ca. 1780 and 1937 AD (r_{Pearson corr} = ?0.47, p < 0.01). The results of both reconstruction methods were compared with four independent local and regional records of early instrumental and documentary data during the period of divergence. Both reconstructions showed similarities with the early instrumental/documentary records, thus it was inconclusive which of the reconstruction models provides the better estimates. However, these results suggest that a calibration-in-time can be used to reconstruct climate over the last 1000 years when no calibration-in-space is available.     

Energy dissipation is an essential mechanism to sustain the viability of plants: The physiological limits of improved photosynthesis

In bright sunlight photosynthetic activity is limited by the enzymatic machinery of carbon dioxide assimilation. This supererogation of energy can be easily visualized by the significant increases of photosynthetic activity under high CO₂ conditions or other metabolic strategies which can increase the carbon flux from CO₂ to metabolic pools. However, even under optimal CO₂ conditions plants will provide much more NADPH + H⁺ and ATP that are required for the actual demand, yielding in a metabolic situation, in which no reducible NADP⁺ would be available. As a consequence, excited chlorophylls can activate oxygen to its singlet state or the photosynthetic electrons can be transferred to oxygen, producing highly active oxygen species such as the superoxide anion, hydroxyl radicals and hydrogen peroxide. All of them can initiate radical chain reactions which degrade proteins, pigments, lipids and nucleotides. Therefore, the plants have developed protection and repair mechanism to prevent photodamage and to maintain the physiological integrity of metabolic apparatus. The first protection wall is regulatory energy dissipation on the level of the photosynthetic primary reactions by the so-called non-photochemical quenching. This dissipative pathway is under the control of the proton gradient generated by the electron flow and the xanthophyll cycle. A second protection mechanism is the effective re-oxidation of the reduction equivalents by so-called “alternative electron cycling” which includes the water-water cycle, the photorespiration, the malate valve and the action of antioxidants. The third system of defence is the repair of damaged components. Therefore, plants do not suffer from energy shortage, but instead they have to invest in proteins and cellular components which protect the plants from potential damage by the supererogation of energy. Under this premise, our understanding and evaluation for certain energy dissipating processes such as non-photochemical quenching or photorespiration appear in a quite new perspective, especially when discussing strategies to improve the solar energy conversion into plant biomass.