Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic <Emphasis Type="Italic">Halomonas</Emphasis> sp. Y2

Na⁺/H⁺ antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na⁺/H⁺ antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na⁺ and Li⁺ translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0–8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na⁺ and Li⁺, while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.     

Optimising Drug Solubilisation in Amorphous Polymer Dispersions: Rational Selection of Hot-melt Extrusion Processing Parameters

The aim of this article was to construct a T–ϕ phase diagram for a model drug (FD) and amorphous polymer (Eudragit® EPO) and to use this information to understand the impact of how temperature–composition coordinates influenced the final properties of the extrudate. Defining process boundaries and understanding drug solubility in polymeric carriers is of utmost importance and will help in the successful manufacture of new delivery platforms for BCS class II drugs. Physically mixed felodipine (FD)–Eudragit^® EPO (EPO) binary mixtures with pre-determined weight fractions were analysed using DSC to measure the endset of melting and glass transition temperature. Extrudates of 10 wt% FD–EPO were processed using temperatures (110°C, 126°C, 140°C and 150°C) selected from the temperature–composition (T–ϕ) phase diagrams and processing screw speed of 20, 100 and 200rpm. Extrudates were characterised using powder X-ray diffraction (PXRD), optical, polarised light and Raman microscopy. To ensure formation of a binary amorphous drug dispersion (ADD) at a specific composition, HME processing temperatures should at least be equal to, or exceed, the corresponding temperature value on the liquid–solid curve in a F–H T–ϕ phase diagram. If extruded between the spinodal and liquid–solid curve, the lack of thermodynamic forces to attain complete drug amorphisation may be compensated for through the use of an increased screw speed. Constructing F–H T–ϕ phase diagrams are valuable not only in the understanding drug–polymer miscibility behaviour but also in rationalising the selection of important processing parameters for HME to ensure miscibility of drug and polymer.KEY WORDS: DSC, Flory–Huggins theory, hot-melt extrusion, thermal processing     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Effects of tobacco–peanut relay intercropping on soil bacteria community structure

A reasonable cultivation pattern is beneficial to maintain soil microbial activity and optimize the structure of the soil microbial community. To determine the effect of tobacco−peanut (Nicotiana tabacum−Arachis hypogaea) relay intercropping on the microbial community structure in soil, we compared the effects of relay intercropping and continuous cropping on the soil bacteria community structure. We collected soil samples from three different cropping patterns and analyzed microbial community structure and diversity using high-throughput sequencing technology. The number of operational taxonomic units (OTU) for bacterial species in the soil was maximal under continuous peanut cropping. At the phylum level, the main bacteria identified in soil were Proteobacteria, Actinobacteria, and Acidobacteria, which accounted for approximately 70% of the total. The proportions of Actinobacteria and Firmicutes increased, whereas the proportion of Proteobacteria decreased in soil with tobacco–peanut relay intercropping. Moreover, the proportions of Firmicutes and Proteobacteria among the soil bacteria further shifted over time with tobacco–peanut relay intercropping. At the genus level, the proportions of Bacillus and Lactococcus increased in soil with tobacco–peanut relay intercropping. The community structure of soil bacteria differed considerably with tobacco–peanut relay intercropping from that detected under peanut continuous cropping, and the proportions of beneficial bacteria (the phyla Actinobacteria and Firmicutes, and the genera Bacillus and Lactococcus) increased while the proportion of potentially pathogenic bacteria (the genera Variibacter and Burkholderia) decreased. These results provide a basis for adopting tobacco–peanut relay intercropping to improve soil ecology and microorganisms, while making better use of limited cultivable land.     

High‐Performance and Stable Perovskite Solar Cells Based on Dopant‐Free Arylamine‐Substituted Copper(II) Phthalocyanine Hole‐Transporting Materials

A power conversion efficiency (PCE) as high as 19.7% is achieved using a novel, low‐cost, dopant‐free hole transport material (HTM) in mixed‐ion solution‐processed perovskite solar cells (PSCs). Following a rational molecular design strategy, arylamine‐substituted copper(II) phthalocyanine (CuPc) derivatives are selected as HTMs, reaching the highest PCE ever reported for PSCs employing dopant‐free HTMs. The intrinsic thermal and chemical properties of dopant‐free CuPcs result in PSCs with a long‐term stability outperforming that of the benchmark doped 2,2′,7,7′‐Tetrakis‐(N,N‐di‐p‐methoxyphenylamine)‐9,9′‐Spirobifluorene (Spiro‐OMeTAD)‐based devices. The combination of molecular modeling, synthesis, and full experimental characterization sheds light on the nanostructure and molecular aggregation of arylamine‐substituted CuPc compounds, providing a link between molecular structure and device properties. These results reveal the potential of engineering CuPc derivatives as dopant‐free HTMs to fabricate cost‐effective and highly efficient PSCs with long‐term stability, and pave the way to their commercial‐scale manufacturing. More generally, this case demonstrates how an integrated approach based on rational design and computational modeling can guide and anticipate the synthesis of new classes of materials to achieve specific functions in complex device structures.     

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post‐translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large‐scale phosphoproteome profiling is performed using IMAC and TiO₂ enrichment. In total, 13 835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis‐specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase‐substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.     

RSBP15 interacts with and stabilizes dRSPH3 during sperm axoneme assembly in Drosophila

Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans.Here, we identified a novel Drosophila protein radial spoke binding protein 15(RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles(SVs), asynchronous individualization complex(IC) and defective "9 + 2"structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover,loss of dRSPH3, as well as dRSPH1, dRSPH4 a and dRSPH9, showed similar phenotypes to rsbp15 KO mutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella.     

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.