Temporal Small RNA Expression Profiling under Drought Reveals a Potential Regulatory Role of Small Nucleolar RNAs in the Drought Responses of Maize.

Small RNAs (sRNAs) are short noncoding RNAs that play roles in many biological processes, including drought responses in plants. However, how the expression of sRNAs dynamically changes with the gradual imposition of drought stress in plants is largely unknown. We generated time-series sRNA sequence data from maize ( L.) seedlings under drought stress (DS) and under well-watered (WW) conditions at the same time points. Analyses of length, functional annotation, and abundance of 736,372 nonredundant sRNAs from both DS and WW data, as well as genome copy numbers at the corresponding genomic regions, revealed distinct patterns of abundance and genome organization for different sRNA classes. The analysis identified 6646 sRNAs whose regulation was altered in response to drought stress. Among drought-responsive sRNAs, 1325 showed transient downregulation by the seventh day, coinciding with visible symptoms of drought stress. The profiles revealed drought-responsive microRNAs, as well as other sRNAs that originated from ribosomal RNAs (rRNAs), splicing small nuclear RNAs, and small nucleolar RNAs (snoRNA). Expression profiles of their sRNA derivers indicated that snoRNAs might play a regulatory role through regulating the stability of rRNAs and splicing small nuclear RNAs under drought condition.

Grass carp reovirus-GD108 fiber protein is involved in cell attachment.

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. In this study, structure analysis, neutralization assays, and cell attachment assays were carried out to evaluate the cell attachment functions of the outer capsid fiber protein of grass carp reovirus GD108 strain (GCRV-GD108). The GCRV-GD108 fiber protein contained 512 amino acids encoded by S7 segment and shared sequence similarities with mammalian reovirus cell attachment protein σ1 and adenovirus fiber. Structural analyses predicted the presence of a coiled-coil tail domain, three adenoviral shafts in the body domain, and a globular head domain, similar to other fiber proteins. Neutralization assays showed that polyclonal antibodies against the fiber protein could prevent viral infection in both fish and grass carp snout fibroblast cells (PSF), suggesting that the recombinant fiber protein could induce neutralized antibodies against GCRV-GD108. Cell attachment assays showed that recombinant fiber protein could bind to PSF cells, demonstrating that the fiber protein functioned as the cell attachment protein in GCRV-GD108. These results provided the basis for further studies of the pathogenesis of grass carp reovirus.

Characterization of the calcineurin B-Like (CBL) gene family in maize and functional analysis of ZmCBL9 under abscisic acid and abiotic stress treatments.

In plants, calcineurin B-like proteins (CBLs) play crucial roles in regulating calcium-signaling in response to various abiotic stresses by interacting with specific CBL-interacting protein kinases (CIPKs). However, the identities and functions of CBL gene family members in maize are largely unknown. Here, we identified from the maize genome 12 CBL genes. All 12 CBLs have conserved EF-hand domains, and half harbor myristoylation motifs. We further characterized the function of one CBL gene, ZmCBL9, which can be induced by salt, dehydration, glucose and abscisic acid (ABA) treatments. Overexpression of ZmCBL9 enhanced resistance or tolerance to ABA, glucose, salt and osmotic stress in Arabidopsis and complemented the hypersensitive phenotype of the Arabidopsis cbl9 mutant in response to ABA and abiotic stress. The ZmCBL9 gene negatively regulates the expression of genes in the ABA signaling, biosynthesis and catabolism pathways. Moreover, the ZmCBL9 protein is found to interact with eight maize CIPKs and these ZmCIPK genes were up-regulated by different stress treatments, including salt, dehydration, glucose, low potassium and ABA. These results suggest that ZmCBL9 may interact with various ZmCIPKs to regulate the abiotic stress and ABA response signaling in plants.

Efficient production of sTNFRII-gAD fusion protein in large quantity by use of the modified CHO-S cell expression system.

TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.

Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner.

Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.

Influence of Curing on the Strength Development of Calcium-Containing Geopolymer Mortar.

This paper investigated the curing effects on the mechanical properties of calcium-containing geopolymer mortar. Three precursors are used: Class C fly ash, Class F fly ash plus calcium hydroxide and Class F fly ash plus slag. Curing conditions included: (1) standard curing at 20 ± 3 °C and RH 95% (C); (2) steam curing at 60 °C for 24 h (S); (3) steam curing at 60 °C for 6 h (S6); and (4) oven curing at 60 °C for 24 h (O), then the latter three followed by the standard curing. Under the standard conditions, the flexural strength and compressive strength of Class C fly ash geopolymer mortars developed quickly until the age of 7 days, followed by a gradual increase. Specimens with Class F fly ash plus Ca(OH)2 showed slow increase till the age of 28 days. Under these non-standard conditions (2-4), all specimens showed higher 3-day strength, while later strengths were either higher or lower than those in standard conditions, depending on the type of the precursor.