Toolkits for detailed and high-throughput interrogation of synapses in C. elegans.

Visualizing synaptic connectivity has traditionally relied on time-consuming electron microscopy-based imaging approaches. To scale the analysis of synaptic connectivity, fluorescent protein-based techniques have been established, ranging from the labeling of specific pre- or post-synaptic components of chemical or electrical synapses to transsynaptic proximity labeling technology such as GRASP and iBLINC. In this paper, we describe WormPsyQi, a generalizable image analysis pipeline that automatically quantifies synaptically localized fluorescent signals in a high-throughput and robust manner, with reduced human bias. We also present a resource of 30 transgenic strains that label chemical or electrical synapses throughout the nervous system of the nematode Caenorhabditis elegans, using CLA-1, RAB-3, GRASP (chemical synapses), or innexin (electrical synapse) reporters. We show that WormPsyQi captures synaptic structures in spite of substantial heterogeneity in neurite morphology, fluorescence signal, and imaging parameters. We use these toolkits to quantify multiple obvious and subtle features of synapses - such as number, size, intensity, and spatial distribution of synapses - in datasets spanning various regions of the nervous system, developmental stages, and sexes. Although the pipeline is described in the context of synapses, it may be utilized for other 'punctate' signals, such as fluorescently tagged neurotransmitter receptors and cell adhesion molecules, as well as proteins in other subcellular contexts. By overcoming constraints on time, sample size, cell morphology, and phenotypic space, this work represents a powerful resource for further analysis of synapse biology in C. elegans.

The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34.

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. elegans homolog of the Drosophila Sine oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine oculis homolog, our findings invite speculations about the early evolution of nervous systems.

The F-box protein Bard (CG14317) targets the Smaug RNA-binding protein for destruction during the Drosophila maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. Here, we show that an F-box protein, which we name Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT.

Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition.

In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.

The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs.

G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.

The maternal-to-zygotic transition revisited.

The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

[Construction and application of an onboard absorption analyzer device for CDOM].

Colored dissolved organic matter (CDOM) plays an important role in marine ecosystems. In order to solve the current problems in measurement of CDOM absorption, an automated onboard analyzer based on liquid core waveguides (Teflon AF LWCC/LCW) was constructed. This analyzer has remarkable characteristics including adjusted optical pathlength, wide measurement range, and high sensitivity. The model of filtration and injection can implement the function of automated filtration, sample injection, and LWCC cleaning. The LabVIEW software platform can efficiently control the running state of the analyzer and acquire real time data including light absorption spectra, GPS data, and CTW data. By the comparison experiments and shipboard measurements, it was proved that the analyzer was reliable and robust.

[Variations in the optical absorption and attenuation properties of cultured phytoplankton and their relationships with cell size].

The spectral absorption and attenuation coefficients of 16 phytoplankton species were measured in the laboratory using acs instrument. Ancillary measurements included particle size distribution and chlorophyll a concentration (Chl a). The results indicated that both algal cell size and Chl a were the two major factors dominating the magnitudes of the spectral absorption and attenuation coefficients. The spectral behaviors of attenuation spectra were dominated by algal cell size, the relationship of them didn't follow the monotonic function. Both the ratio of absorption in blue and red waveband and the spectral slope of absorption coefficient were influenced by the product of algal cell density and squares of cell size rather than algal cell size alone. The relationship between algal cell size and both absorption and attenuation spectra would be interpreted by Mie theory for homogenous sphere, which imply that the heterogeneity and non-spherical shape in algal cell morphology and internal structure have little effect on the inherent association among them.

[Validation and analysis of water column correction algorithm at Sanya Bay].

Water column correction has been a substantial challenge for remote sensing. In order to improve the accuracy of coastal ocean monitoring where optical properties are complex, optical property of shallow water at Sanya Bay and the suitable water column correction algorithms were studies in the present paper. The authors extracted the bottom reflectance without water column effects by using a water column correction algorithm which is based on the simulation of the underwater light field in idealized water. And we compared the results which were calculated by the model and Christian's model respectively. Based on a detailed analysis, we concluded that: Because the optical properties of Sanya Bay are complex and vary greatly with location, Christian's model lost its advantage in the area. Conversely, the bottom reflectance calculating by the algorithm based on the simulation of the underwater light field in idealized water agreed well with in situ measured bottom reflectance, although the reflectance was lower than in situ measured reflectance value between 400 and 500 nm. So, it is reasonable to extract bottom information by using the water column correction algorithm in local bay area where optical properties are complex.

[Progress in Teflon AF LWCC/LCW applications].

Teflon AF is chemically very inert, quite physically and optically stable, a highly vapor-permeable polymer with optical transparency through much of the UV-Vis region and with an RI lower than that of water, so Teflon AF LWCC/LCW (Long path-length liquid waveguide capillary cell/liquid core waveguides) has been used with a range of different detection techniques, including absorbance spectroscopy, fluorescence spectroscopy, Raman spectroscopy, and gas sensor. The present article describes the properties and the aspects of Teflon AF LWCC/LCW instrumentation and applications. And finally,the future prospect and outlook of Teflon AF LWCC/LCW is also discussed.

[Spectrum simulation based on data derived from red tide].

The present paper utilizes the absorption data of red tide water measured during the growing and dying course to retrieve imaginary part of the index of refraction based on Mie theory, carries out the simulation and analysis of average absorption efficiency factors, average backscattering efficiency factors and scattering phase function. The analysis of the simulation shows that Mie theory can be used to reproduce the absorption property of Chaetoceros socialis with an average error of 11%; the average backscattering efficiency factors depend on the value of absorption whose maximum value corresponds to the wavelength range from 400 to 700 nanometer; the average backscattering efficiency factors showed a maximum value on 17th with a low value during the outbreak of red tide and the minimum on 21th; the total scattering, weakly depending on the absorption, is proportional to the size parameters which represent the relative size of cell diameter with respect to the wavelength, while the angle scattering intensity is inversely proportional to wavelength.

[Reflectance of sea ice in Liaodong Bay].

In the present study, the relationships between sea ice albedo and the bidirectional reflectance distribution in Liaodong Bay were investigated. The results indicate that: (1) sea ice albedo alpha(lambda) is closely related to the components of sea ice, the higher the particulate concentration in sea ice surface is, the lower the sea ice albedo alpha(lambda) is. On the contrary, the higher the bubble concentration in sea ice is, the higher sea ice albedo alpha(lambda) is. (2) Sea ice albedo alpha(lambda) is similar to the bidirectional reflectance factor R(f) when the probe locates at nadir. The R(f) would increase with the increase in detector zenith theta, and the correlation between R(f) and the detector azimuth would gradually increase. When the theta is located at solar zenith 63 degrees, the R(f) would reach the maximum, and the strongest correlation is also shown between the R(f) and the detector azimuth. (3) Different types of sea ice would have the different anisotropic reflectance factors.

[Design and application of hyperspectral radiation system for sea ice observation].

Sea ice plays an important role in the global climate systems. In the present article, a hyperspectral radiation system for the observation of optical properties of sea ice was designed. The system consists of three optical channels, which can operate simultaneously. Two kinds of optical detectors were designed, and the problem relevant to the water-tightness was resolved. The system can be used to measure the solar radiation beneath the sea ice by an "L" bracket. Another bracket for detecting bidirectional reflectance was designed, which can fix the optical detector at any angle ranging over 0-180 measured with an angle detector. In order to make a most suitable and automatic integrated time, the system can adjust the integrated time intelligently by itself. The system can work stably under extremely low temperature. Furthermore, the system was equipped with four thermistors and one GPS. The system was validated showing a good stability and veracity in situ in the Liaodong Bay.

[Design and experimentation of marine optical buoy].

Marine optical buoy is of important value in terms of calibration and validation of ocean color remote sensing, scientific observation, coastal environment monitoring, etc. A marine optical buoy system was designed which consists of a main and a slave buoy. The system can measure the distribution of irradiance and radiance over the sea surface, in the layer near sea surface and in the euphotic zone synchronously, during which some other parameters are also acquired such as spectral absorption and scattering coefficients of the water column, the velocity and direction of the wind, and so on. The buoy was positioned by GPS. The low-power integrated PC104 computer was used as the control core to collect data automatically. The data and commands were real-timely transmitted by CDMA/GPRS wireless networks or by the maritime satellite. The coastal marine experimentation demonstrated that the buoy has small pitch and roll rates in high sea state conditions and thus can meet the needs of underwater radiometric measurements, the data collection and remote transmission are reliable, and the auto-operated anti-biofouling devices can ensure that the optical sensors work effectively for a period of several months.

[Decomposing total suspended particle absorption based on the spectral correlation relationship].

A model for estimating the contributions of phytoplankton and nonalgal particles to the total particulate absorption coefficient was developed based on their separate spectral relationships, and a constrained nonlinear optimization code was used to realize the spectral decomposition. The spectral absorption of total particulate matter including phytoplankton and nonalgal particles was measured using the filter-pad method during two cruises in autumn in Northern South China Sea. Using the dataset collected in 2004, the spectral relationships of particle absorption coefficients were examined and the results showed that the phytoplankton absorption coefficients at various wavebands could be well expressed by aph (443) as the second-order quadratic equations; and the nonalgal particle absorption (aNAP(lambda)) could be successfully modeled with the simple exponential function. Based on these spectral relationships, we developed this partition model. The model was tested using the independently measured absorption by phytoplankton and nonalgal materials which were obtained in 2005 from the same area. The test results showed that the computed spectral absorption coefficients of phytoplankton and nonalgal particles were consistent with in situ measurement. Good correlations were fo und between the comput ed phytoplankton absorption coefficient and the measured value,with the determination coefficients (r2) being higher than 0.97 and slopes being around 1.0; and the RMSE values could be controlled within 17% over the main absorption wavebands such as 443, 490 and 683 nm. Compared with the other two existing models from Bricaud et al. and Oubelkheir et al., this method shows many advantages for local applications. Moreover, this model does not need any information about pigment concentrations and the selected spectral bands are consistent with the ocean color satellite sensor. This method could also be used in the total absorption coefficient decomposition which provides much insight into the phytoplankton absorption retrieval from in situ measurement and ocean color remote sensing data.

[Construction and test of long pathlength automated analyzer of trace nutrients in seawater].

Nutrients are routine aspects for marine environment monitoring. With low concentration in oceans, nutrients are difficult to be measured with a routine spectrophotometer. Based on liquid core waveguides, a new high-sensitivity analyzer was constructed to measure trace nutrients in oceans. The analyzer can provide continuous measurement of spectral absorbance of water sample in the spectral range from 350 to 900 nm with a spectral resolution of 1.02 nm. The processes such as sample injection, data acquisition of absorbance and data analysis are automatically controlled by a built-in PC104 micro computer. The results of test proved that, with the reaction time of about three minutes, the analyzer could provide a detection limit of the order of nmol x L(-1), which is about 1 000 times lower than that of routine spectrophotometer. The analyzer is amenable to trace nutrients monitoring in oceans.