Cell lineage and cis-regulation for a unique GABAergic/glycinergic neuron type in the larval nerve cord of the ascidian Ciona intestinalis

The tunicate Ciona intestinalis larva has a simple central nervous system (CNS), consisting of fewer than 400 cells, which is homologous to the vertebrate CNS. Recent studies have revealed neuronal types and networks in the larval CNS of C. intestinalis, yet their cell lineage and the molecular mechanism by which particular types of neurons are specified and differentiate remain poorly understood. Here, we report cell lineage origin and a cis‐regulatory module for the anterior caudal inhibitory neurons (ACINs), a putative component of the central pattern generator regulating swimming locomotion. The vesicular GABA/glycine transporter gene Ci‐VGAT, a specific marker for GABAergic/glycinergic neurons, is expressed in distinct sets of neurons, including ACINs of the tail nerve cord and others in the brain vesicle and motor ganglion. Comparative genomics analysis between C. intestinalis and Ciona savignyi and functional analysis in vivo identified the cis‐regulatory module responsible for Ci‐VGAT expression in ACINs. Our cell lineage analyses inferred that ACINs derive from A11.116 cells, which have been thought to solely give rise to glial ependymal cells of the lateral wall of the nerve cord. The present findings will provide a solid basis for future studies addressing the molecular mechanism underlying specification of ACINs, which play a critical role in controlling larval locomotion.     

Pilot testing of a bioremediation system for water and soils contaminated with heavy metals: vegetable depuration module

Abstract

We present a novel constructed wetland called a vegetable depuration module (VDM) as a pilot test of a bioremediation system (BS) for decontaminating water and soil polluted with heavy metals. The VDM consisted of a pool filled with stones of different granulometry and a substrate top layer composed of a mixture of soil and volcanic ash (50:50, v/v) supplemented with 350?ppm Zn. The BS of sunflower plants colonized by the arbuscular mycorrhizal fungus Rhizophagus intraradices was planted in the VDM. Initially, the substrate registered high concentrations of Zn, Cr, Mn, Cu, and Sr, and had Eh > +500?mV and pH 8.4. Irrigation with a Cu solution by vertical flow was carried out. After 3?months, bioaccumulation factors ranged from 1.00 to 8.90, and translocation rates were >1 for Sr and Cu. Total metals extracted by the BS and percolation were 31%, 34%, 50%, 45%, and 57% for Zn, Cu, Mn, Cr, and Sr, respectively. Only the BS was capable of extracting 94% of Cu and 38% of Zn. VDM allowed us to calibrate the extractive performance of the studied elements in BS. This biotechnological development holds great potential for phytoremediation of polluted areas.     

A Cost Analysis of Fully Solution‐Processed ITO‐Free Organic Solar Modules

Organic photovoltaics (OPVs) have become a potential candidate for clean and renewable photovoltaic productions. This work examines the current cost drivers and potential avenues to reduce costs for organic solar modules by constructing a comprehensive bottom‐up cost model. The direct manufacturing cost (MC) and the minimum sustainable price (MSP) for an opaque single solar module (SSM) (MC = 187 ¥ m^?2, MSP = 297 ¥ m^?2) and for a tandem solar module (MC = 224 ¥ m^?2, MSP = 438 ¥ m^?2) are analyzed in detail. Within this calculation, the most expensive layers and processing steps are identified and highlighted. Importantly, the low levelized cost of energy (LCOE) value for an SSM with a 10% power conversion efficiency in a 20‐year range from 0.185 to 0.486 ¥ kWh^?1, with a national average of 0.324 ¥ kWh^?1 in China under an average solar irradiance of 1200 kWh m^?2 year^?1. Moreover, the impact on the cost of alternative materials and constructions, process throughputs, module efficiency, and module lifetime, etc., is presented and avenues to further reduce the MSP and LCOE values are indicated. The analysis shows that OPVs can emerge as a competitive alternative to established power generation technologies if the remaining issues (e.g., active layer material cost, module efficiency, and lifetime) can be resolved.     

Enhancement of hydrolytic activity of thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 through optimization of amino acid residues surrounding the substrate binding site

The hydrolytic activity of a thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A, I509A, and K549A showed notably higher activity than the wild type at 162–254% of wild-type activity. Tyr426, His431, and Ile509 were predicted to be located near subsite −2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M.     

An Unusual Mode of Galactose Recognition by a Family 32 Carbohydrate-Binding Module

Carbohydrate-binding modules (CBMs) are ancillary modules commonly associated with carbohydrate-active enzymes (CAZymes) that function to mediate the adherence of the parent enzyme to its carbohydrate substrates. CBM family 32 (CBM32) is one of the most diverse CBM families, whose members are commonly found in bacterial CAZymes that modify eukaryotic glycans. One such example is the putative μ-toxin, CpGH84A, of the family 84 glycoside hydrolases, which comprises an N-terminal putative β-N-acetylglucosaminidase catalytic module and four tandem CBM32s. Here, we report a unique mode of galactose recognition by the first CBM32, CBM32-1 from CpGH84A. Solution NMR-based analyses of CpGH84A CBM32-1 indicate a divergent subset of residues, located in ordered loops at the apex of the CBM, conferring specificity for the galacto-configured sugars galactose, GalNAc, and LacNAc that differs from those of the canonical galactose-binding CBM32s. This study showcases the impressive variability in ligand binding by this CBM family and offers insight into the growing role of these modules in the interaction of CAZymes with eukaryotic glycans.     

A CBM20 low-affinity starch-binding domain from glucan, water dikinase

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.     

Molecular evolution of ankyrin: gain of function in vertebrates by acquisition of an obscurin/titin-binding-related domain

Ankyrins form a family of modular adaptor proteins that link between integral membrane proteins and the cytoskeleton. They evolved within the Metazoa as an adaptation for organizing membrane microstructure and directing membrane traffic. Molecular cloning has identified one Caenorhabditis elegans (unc-44), two Drosophila (Dank1, Dank2), and three mammalian (Ank1, Ank2, Ank3) genes. We have previously identified a 76-amino acid (aa) alternatively spliced sequence that is present in muscle polypeptides encoded by the rat Ank3 gene. A closely related sequence in a muscle Ank1 product binds the cytoskeletal muscle proteins obscurin and titin. This obscurin/titin-binding-related domain (OTBD) contains repeated modules of 18 aa: three are encoded by Ank1 and Ank2, two by Ank3; this pattern is conserved throughout vertebrate ankyrin genes. The C. elegans ankyrin, UNC-44, contains one 18-aa module as does the ankyrin gene in the urochordate Ciona intestinalis, but the insect ankyrins contain none. Our data indicate that an ancestral ankyrin acquired an 18-aa module which was preserved in the Ecdysozoa/deuterostome divide, but it was subsequently lost from arthropods. Successive duplications of the module led to a gain of function in vertebrates as it acquired obscurin/titin-binding activity. We suggest that the OTBD represents an adaptation of the cytoskeleton that confers muscle cells with resilience to the forces associated with vertebrate life.     

木聚糖酶碳水化合物结合结构域研究进展

木聚糖酶含有催化活性结构域,有时还含有非催化活性结构域,促进酶与底物结合,特别是与不溶性底物的结合及降解,称为碳水化合物结合结构域(CBM),它们在木聚糖降解过程中有重要作用。以下从CBM来源,所属家族类型、对不溶性底物结合特性、与底物结合的特定氨基酸、与催化结构域间的连接肽、特别是对影响木聚糖酶稳定性的5个方面进行了综述,说明CBM对木聚糖酶性质有很大影响。自然界中碳水化合物结构复杂、难以降解,所以认识CBM相关性质对研究其与木聚糖酶的协同作用、提高木聚糖酶活性有重要意义,并根据CBM属性用于改造木聚糖酶相关性质进行了展望。     

华北平原罗布麻种群生殖分株数量特征及变化规律

通过大样本取样调查,研究了华北平原罗布麻种群生殖分株的数量特征和生殖分配规律．结果表明,在籽粒完熟期,罗布麻种群单个生殖分株重为10．38±7．78g,蒴果生物量和蒴果数分别为1．57±1．59g和7．60±6．56个;种子生物量和种子数分别为0．34±0．33g和582．3±558．7个;生殖分配Ⅰ和生殖分配Ⅱ分别为12．98±7．94和2．75±1．69％．蒴果和种子形成分别需要分株积累1．42和1．77g以上的生物量．蒴果生物量、葫果数、种子生物量、种子数分别与分株总生物量呈极显著的（P〈0．01）线性正相关关系;蒴果数、种子数与茎叶生物量分配呈极显著的（P〈0．01）线性负相关关系;生殖分配Ⅰ、生殖分配Ⅱ和叶生物量分配呈极显著的（P〈0．01）线性负相关关系．单葫果的重量、种子重、种子数、种子毛重、胎座重分别和葫果的长度呈极显著（P〈0．01）的幂函数正相关关系．罗布麻种群生殖分株同时具有同速和异速2种不同增减过程的表型可塑性调节．     

CBM21 starch-binding domain: A new purification tag for recombinant protein engineering

The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.