De-oncogenic HPV E6/E7 vaccine gets enhanced antigenicity and promotes tumoricidal synergy with cisplatin

In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus （HPV） infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of ET- and E6-containing vaccines. It has been shown recently that PE（AIII）-E7-KDEL3 （E7）, a fusion protein containing the HPVI6 oncoprotein E7 and the trans- location domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, there- fore, we engineered PE（AIII）-E6-CRL-KDEL3 （E6）, the deoncogenic versions of the E7 and E6 fusion proteins [i.e. PE（AIII）-E7（d）-KDEL3, E7（d）, and PE（AIII）-E6（d）-CRL- KDEL3, E6（d）] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7（d） get higher immunogenicity than its wild type and the E6 fusion proteins augmented the im- munogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7（d） plus E6（d）, in the presence of cisplatin, showed the best tumori- static and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas.     

Cloning and Characterization of miRNAs and Their Targets,Including a Novel miRNA-Targeted NBS-LRR Protein Class Gene in Apple （Golden Delicious）

MicroRNA （miRNA） has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple （Malus domestica cv. Golden Delicious） by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 （Malus domestica microRNA Ln11）, targeted an apple nucleotide-binding site （NBS）-Ieucine-rich repeat （LRR） class protein coding gene （Md-NBS）. 5/ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ＇JiGuan＇ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ＇FuJi＇ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.     

Chaperone function and mechanism of small heat-shock proteins

Small heat-shock proteins （sHSPs） are ubiquitous ATP-independent molecular chaperones that play crucial roles in protein quality control in cells. They are able to prevent the aggregation and/or inactivation of various non-native sub- strate proteins and assist the refolding of these substrates independently or under the help of other ATP-dependent chaperones. Substrate recognition and binding by sHSPs are essential for their chaperone functions. This review focuses on what natural substrate proteins an sHSP pro- tects and how it binds the substrates in cells under fluctuat- ing conditions. It appears that sHSPs of prokaryotes, although being able to bind a wide range of cellular pro- teins, preferentially protect certain classes of functional proteins, such as translation-related proteins and metabolic enzymes, which may well explain why they could increase the resistance of host cells against various stresses. Mechanistically, the sHSPs of prokaryotes appear to possess numerous multi-type substrate-binding residues and are able to hierarchically activate these residues in a temperature-dependent manner, and thus act as tempera- ture-regulated chaperones. The mechanism of hierarchical activation of substrate-binding residues is also discussed regarding its implication for eukaryotic sHSPs.     

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.     

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.     

Interaction between Rice stripe virus Disease- Specific Protein and Host PsbP Enhances Virus Symptoms

Rice stripe virus （RSV） causes severe diseases in Oryza sativa （rice） in many Eastern Asian countries. Diseasespecific protein （SP） of RSV is a non-structural protein and its accumulation level in rice plant was shown to determine the severity of RSV symptoms. Here, we present evidence that expression of RSV SP alone in rice or Nicotiana benthamiana did not produce visible symptoms. Expression of SP in these two plants, however, enhanced RSV- or Potato virus X （PVX）- induced symptoms. Through yeast two-hybrid screening, GST pull-down, and bimolecular fluorescence complementation assays, we demonstrated that RSV SP interacted with PsbP, a 23-kDa oxygen-evolving complex protein, in both rice and N. benthamiana. Furthermore, our investigation showed that silencing of PsbP expression in both plants increased disease symptom severity and virus accumulation. Confocal microscopy using N, benthamiana protoplast showed that PsbP accu- mulated predominantly in chloroplast in wild-type N. benthamiana cells. In the presence of RSV SP, most PsbP was recruited into cytoplasm of the assayed cells. In addition, accumulation of SP during RSV infection resulted in alterations of chloroplast structure and function. Our findings shed light on the molecular mechanism underlying RSV disease symptom development.     

The key residue for SSB-RecO interaction is dispensable for Deinococcus radiodurans DNA repair in vivo

The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein （SSB） coated single-stranded DNA （ssDNA）. However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO （drRecO-R121） as the key residue for RecO-SSB interaction. The substitution of drRecO-R121 with alanine greatly abolished the binding of RecO to SSB but not the binding to RecR. Meanwhile, SSB-coated ssDNA annealing activity was also compromised by the mutation of the residue of drRecO. However, the drRecO-R121A strain showed only modest sensitivity to DNA damaging agents. Taking these data together, arginine-121 of drRecO is the key residue for SSB-RecO interaction, which may not play a vital role in the SSB displacement and RecA loading process of RecFOR DNA repair pathway in vivo.     

The Zα domain of fish PKZ converts DNA hairpin with d（GC）n inserts to Z-conformation

PKZ, protein kinase containing Z-DNA domains, is a novel member of the vertebrate eIF2α kinase family. Containing a catalytic domain in C-terminus and two Z-DNA binding domains （Zαl and Zα2） in N-terminus, PKZ can be acti- vated through the binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα remains to be established. Here, to understand the impact of PKZ Zα on DNA con- formational transition, wild-type ZαdZα2 and 11 mutant proteins were expressed and purified. At the same time, several different lengths of DNA hairpins-d（GC）nT4（GC）, （n = 2-6） and an RNA hairpin-r（GC）6T4（GC）6 were synthesized. The effects of ZαdZα2 and mutant proteins on the conformation of these synthetic DNA or RNA hairpins were investigated by using circular dichroism spectrum and gel mobility shift assays. The results showed that DNA hair- pins retained a conventional B-DNA conformation in the absence of ZαdZα2, while some of the DNA hairpins （n 〉 3） were converted to Z-conformation under Zαd Zα2 induction. The tendency was proportionally associated with the increas- ing amount of GC repeat. In comparison with ZodZα2, ZαdZαd rather than Zα2ZαL2 displayed a higher ability in converting d（GC）6T4（GC）6 from B- to Z-DNA. These results demonstrated that Zcd sub-domain played a more essential role in the process of B-Z conformational transition than Zα2 sub-domain did. Mutant proteins （K34A, N38A, R39A, Y42A, P57A, P58A, and W60A） could not convert d（GC）6T4（GC）6 into Z-DNA, whereas S35A or K56A retained some partial activities. Interestingly, ZαlZα2 was also able to induce r（GC）6T4（GC）6 RNA from A-conform- ation to Z-conformation under appropriate conditions.     

A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice

We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein （MOMP） multi-epitope of Chlamydia trachomatis. A short gene of muiti-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a（＋） containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His- MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multiepitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies （lgG1 and IgG2a）, but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi- epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Thl response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi- epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.     

C57/BL6癫痫小鼠海马齿状回DCX和GFAP表达的时程变化

目的：观察C57/BL6癫痫小鼠海马神经元DCX和GFAP表达的时程变化,为神经元发生,发育和星形胶质细胞的变化提供理论基础。方法：应用海人藻酸建立小鼠癫痫模型,应用免疫荧光组织化学方法检测海马齿状回不同时间点双皮质醇,胶质纤维酸性蛋白的表达。结果：与对照组相比,癫痫发生后3天7、天,海马齿状回DCX阳性神经元的免疫荧光强度明显增强;GFAP阳性神经元的免疫荧光强度在癫痫发生后持续的一周内较对照组均见明显持续表达增强。结论：小鼠癫痫后会引起海马星形胶质细胞的活化,同时发病早期神经元已经出现再生标记物的增加。     

Decipher the mechanisms of rabbit＇s low susceptibility to prion infection

Rabbits have low susceptibility to prion infection. Studies on prion protein （PrP） from animal species of different sus- ceptibility to prion diseases identified key amino acid resi- dues, specific motif, and special features in rabbit prion protein （RaPrPc） that contribute to the stability of rabbit PrP~ and low susceptibility to prion infection. However, there is no evidence showing that rabbits are completely re- sistant to prion diseases. It has been reported that the rabbit prion could be generated in vitro through protein misfolding cyclic amplification and proved to be infectious and transmissible. Here, we reviewed studies on rabbit- specific PrP structures and features in relation to rabbit＇s low susceptibility to prion infection.     

Salt Stress in Arabidopsis： Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant＇s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase （MAPK） cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein （LTP）-related hybrid proline-rich protein （HyPRP）, as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azil are hypersensitive to salt stress, while AZIl-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZIl-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.     

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

Fibroblast activation protein alpha （FAPα） is a 95-kDa serine protease of post-prolyl peptidase family on cell surface. FAPoL is widely expressed in tumor microenviron- ment. The wide spread association of FAPα expression with cancer suggests that it has important functions in the disease. However, the nature of FAPα＇s roles in cancer cell activity is not well-determined. It has been showed that FAPα silencing in SKOV3 cells induces ovarian tumors but significantly reduces tumor growth in a xenograft mouse model. To further determine the role of FAPoL in epithelial ovarian cancer cells, SKOV3-FAPα and HO8910-FAPα cell lines, which over-expressed FAPα stably, were con- structed and then their biological behaviors were investi- gated. It was found that FAPoL promoted ovarian cancer cell proliferation, drug resistance, invasiveness, and migra- tion in vitro. Immunochemistry assay showed that FAPα significantly facilitated tumor growth in xenograft tumor tissues. These results suggested that FAPα might directly promote tumor growth and invasiveness in ovarian cancer cells.     

14-3-3 Binding to Cyclin Y contributes to cyclin Y/CDK14 association

Cyclin Y is a highly conserved cyclin among eumetazoans, yet its function and regulation are poorly understood. To search for Cyclin Y-interacting proteins, we screened a yeast two-hybrid library using human Cyclin Y （CCNY） as a bait and identified the following interactors： CDK14 and four members of the 14-3-3 family （ε,β,η,τ）. The interaction between CCNY and 14-3-3 proteins was confirmed both in vitro and in vivo. The results showed that Ser-100 and Ser-326 residues in CCNY were crucial for 14-3-3 binding. Interestingly, binding of CCNY to 14-3-3 significantly enhanced the association between CCNY and CDK14. Our findings may add a new layer of regulation of CCNY binding to its kinase partner.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-lnduced Volatile Oil Production Through Protein Phosphorylation Pathways in Atractylodes lancea Plantlets

Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide （NO） and brassinolide （BL） in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. ALl2 enhanced the activities of total protein phosphorylation, Ca2~-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte- induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.     

Control of Rice Embryo Development,Shoot Apical Meristem Maintenance,and Grain Yield by a Novel Cytochrome P450

Angiosperm seeds usually consist of two major parts： the embryo and the endosperm. However, the molec- ular mechanism（s） underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO （GE） gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scuteUum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem （SAM） mainte- nance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78AlO, a GE homolog, also produced bigger seeds, implying a con- served role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.