Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities.

Our laboratory and others previously showed that Annexin A2 knockout (A2KO) mice had impaired blood-brain barrier (BBB) development and elevated pro-inflammatory response in macrophages, implying that Annexin A2 (AnxA2) might be one of the key endogenous factors for maintaining homeostasis of the neurovascular unit in the brain. Traumatic brain injury (TBI) is an important cause of disability and mortality worldwide, and neurovascular inflammation plays an important role in the TBI pathophysiology. In the present study, we aimed to test the hypothesis that A2KO promotes pro-inflammatory response in the brain and worsens neurobehavioral outcomes after TBI. TBI was conducted by a controlled cortical impact (CCI) device in mice. Our experimental results showed AnxA2 expression was significantly up-regulated in response to TBI at day three post-TBI. We also found more production of pro-inflammatory cytokines in the A2KO mouse brain, while there was a significant increase of inflammatory adhesion molecules mRNA expression in isolated cerebral micro-vessels of A2KO mice compared with wild-type (WT) mice. Consistently, the A2KO mice brains had a significant increase in leukocyte brain infiltration at two days after TBI. Importantly, A2KO mice had significantly worse sensorimotor and cognitive function deficits up to 28 days after TBI and significantly larger brain tissue loss. Therefore, these results suggested that AnxA2 deficiency results in exacerbated early neurovascular pro-inflammation, which leads to a worse long-term neurologic outcome after TBI.

Lactoferrin (LF) has demonstrated stimulation of osteogenic differentiation of mesenchymal stem cells (MSCs). Long non-coding RNAs (lncRNAs) participate in regulating the osteogenic differentiation processes. However, the impact of LF on lncRNA expression in MSC osteogenic differentiation is poorly understood. Our aim was to investigate the effects of LF on lncRNAs expression profiles, during osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), by RNA sequencing. A total number of 1331 putative lncRNAs were identified in rBMSCs during osteogenic differentiation in the study. LF influenced the expression of 120 lncRNAs (differentially expressed lncRNAs [DELs], Fold change > 1.5 or < -1.5; p < 0.05) in rBMSCs on day 14 of osteogenic differentiation, consisted of 60 upregulated and 60 down-regulated. Furthermore, the potential functions of DELs were of prediction by searching their target cis- and trans-regulated protein-coding genes. The bioinformatic analysis of DELs target gene revealed that LF led to the disfunction of transforming growth factor beta stimulus (TGF-β) and positive regulation of I-κappa B kinase/NF-κappa B signaling pathway, which may relate to osteogenic differentiation of rBMSCs. Our work is the first profiling of lncRNA in osteogenic differentiation of rBMSCs induced by LF, and provides valuable insights into the potential mechanisms for LF promoting osteogenic activity.

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, involved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

Warm temperatures induce plant bolting accompanied by flower initiation, where endogenous auxin is dynamically associated with accelerated growth. Auxin signaling is primarily regulated by a family of plant-specific transcription factors, AUXIN RESPONSE FACTORS (ARFs), which either activate or repress the expression of downstream genes in response to developmental and environmental cues. However, the relationship between ARFs and bolting has not been completely understood in lettuce yet. Here, we identified 24 LsARFs (Lactuca sativa ARFs) in the lettuce genome. The phylogenetic tree indicated that LsARFs could be classified into three clusters, which was well supported by the analysis of exon-intron structure, consensus motifs, and domain compositions. RNA-Seq analysis revealed that more than half of the LsARFs were ubiquitously expressed in all tissues examined, whereas a small number of LsARFs responded to UV or cadmium stresses. qRT-PCR analysis indicated that the expression of most LsARFs could be activated by more than one phytohormone, underling their key roles as integrative hubs of different phytohormone signaling pathways. Importantly, the majority of LsARFs displayed altered expression profiles under warm temperatures, implying that their functions were tightly associated with thermally accelerated bolting in lettuce. Importantly, we demonstrated that silencing of LsARF8a, expression of which was significantly increased by elevated temperatures, resulted in delayed bolting under warm temperatures, suggesting that LsARF8a might conduce to the thermally induced bolting. Together, our results provide molecular insights into the LsARF gene family in lettuce, which will facilitate the genetic improvement of the lettuce in an era of global warming.

Neuroglobin is an endogenous neuroprotective protein, but the underlying neuroprotective mechanisms remain to be elucidated. Our previous yeast two-hybrid screening study identified that Dishevelled-1, a key hub protein of Wnt/β-Catenin signaling, is an interaction partner of Neuroglobin. In this study, we further examined the role of Neuroglobin in regulating Dishevelled-1 and the downstream Wnt/β-Catenin and NFκB signaling pathway. We found that Neuroglobin directly interacts with Dishevelled-1 by co-immunoprecipitation, and the two proteins are co-localized in both cytoplasma and nucleus of SK-N-SH cells. Moreover, the ectopic expression of Neuroglobin promotes the degradation of exogenous and endogenous Dishevelled-1 through the proteasomal degradation pathway. Furthermore, our results showed that Neuroglobin significantly inhibits the luciferase activity of Topflash reporter and the expression of β-Catenin mediated by Dishevelled-1 in SK-N-SH cells. In addition, we also documented that Neuroglobin enhances TNF-α-induced NFκB activation via down-regulating Dishevelled-1. Finally, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays showed that Neuroglobin is an important neuroprotectant that protects SK-N-SH cells from TNF-α-induced decrease in cell viability. Taken together, these findings demonstrated that Neuroglobin functions as an important modulator of the Wnt/β-Catenin and NFκB signaling pathway through regulating Dishevelled-1.

In the present study, hematopoietic progenitor kinase 1 (HPK1)-interacting protein of 55 kDa (HIP-55) protein was over-expressed in HEK293 cells, which was genetically attached with 6x His tag. The protein was purified by nickel-charged resin and was then subjected to tryptic digestion. The phosphorylated peptides within the HIP-55 protein were enriched by TiO2 affinity chromatography, followed by mass spectrometry analysis. Fourteen phosphorylation sites along the primary structure of HIP-55 protein were identified, most of which had not been previously reported. Our results indicate that bio-mass spectrometry coupled with manual interpretation can be used to successfully identify the phosphorylation modification in HIP-55 protein in HEK293 cells.

Salmonella enterica serovar Infantis (S. Infantis) is an intracellular bacterial pathogen. It is prevalent but resistant to antibiotics. Therefore, the therapeutic effect of antibiotics on Salmonella infection is limited. In this study, we used the piglet diarrhea model and the Caco2 cell model to explore the mechanism of probiotic Lactobacillus johnsonii L531 (L. johnsonii L531) against S. Infantis infection. L. johnsonii L531 attenuated S. Infantis-induced intestinal structural and cellular ultrastructural damage. The expression of NOD pathway-related proteins (NOD1/2, RIP2), autophagy-related key proteins (ATG16L1, IRGM), and endoplasmic reticulum (ER) stress markers (GRP78, IRE1) were increased after S. Infantis infection. Notably, L. johnsonii L531 pretreatment not only inhibited the activation of the above signaling pathways but also played an anti-S. Infantis infection role in accelerating autophagic degradation. However, RIP2 knockdown did not interfere with ER stress and the activation of autophagy induced by S. Infantis in Caco2 cells. Our data suggest that L. johnsonii L531 pretreatment alleviates the intestinal damage caused by S. Infantis by inhibiting NOD activation and regulating ER stress, as well as promoting autophagic degradation.

Intersectin-2Long (ITSN2L) is a multi-domain protein participating in endocytosis and exocytosis. In this study, RABEP1 was identified as a novel ITSN2L interacting protein using a yeast two-hybrid screen from a human brain cDNA library and this interaction, specifically involving the ITSN2L CC domain and RABEP1 CC3 regions, was further confirmed by in vitro GST (glutathione-S-transferase) pull-down and in vivo co-immunoprecipitation assays. Corroboratively, we observed that these two proteins co-localize in the cytoplasm of mammalian cells. Furthermore, over-expression of ITSN2L promotes RABEP1 degradation and represses RABEP1-enhanced endosome aggregation, indicating that ITSN2L acts as a negative regulator of RABEP1. Finally, we showed that ITSN2L and RABEP1 play opposite roles in regulating endocytosis. Taken together, our results indicate that ITSN2L interacts with RABEP1 and stimulates its degradation in regulation of endocytosis.

Recombinant fibroblast growth factor 21 (rFGF21) has been shown to be potently beneficial for improving long-term neurological outcomes in type 2 diabetes mellitus (T2DM) stroke mice. Here, we tested the hypothesis that rFGF21 protects against poststroke blood-brain barrier (BBB) damage in T2DM mice via peroxisome proliferator-activated receptor gamma (PPARγ) activation in cerebral microvascular endothelium. We used the distal middle cerebral occlusion (dMCAO) model in T2DM mice as well as cultured human brain microvascular endothelial cells (HBMECs) subjected to hyperglycemic and inflammatory injury in the current study. We detected a significant reduction in PPARγ DNA-binding activity in the brain tissue and mRNA levels of BBB junctional proteins and PPARγ-targeting gene CD36 and FABP4 in cerebral microvasculature at 24 h after stroke. Ischemic stroke induced a massive BBB leakage two days after stroke in T2DM mice compared to in their lean controls. Importantly, all abnormal changes were significantly prevented by rFGF21 administration initiated at 6 h after stroke. Our in vitro experimental results also demonstrated that rFGF21 protects against hyperglycemia plus interleukin (IL)-1β-induced transendothelial permeability through upregulation of junction protein expression in an FGFR1 activation and PPARγ activity elevation-dependent manner. Our data suggested that rFGF21 has strong protective effects on acute BBB leakage after diabetic stroke, which is partially mediated by increasing PPARγ DNA-binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

Obesity, along with type 2 diabetes mellitus (T2DM), is a major contributor to hypertension. The renin-angiotensin-aldosterone system is involved in the occurrence of diabetes and hypertension. However, the mechanism by which obesity is related to T2DM induced hypertension is unclear. In this study, we observed that blood pressure and serum renin content were increased in patients with diabetes and hypertension. Hydrogen sulfide (H2S), as an endogenous bioactive molecule, has been shown to be a vasodilator. Db/db mice, characterized by obesity and T2DM, and juxtaglomerular (JG) cells, which line the afferent arterioles at the entrance of the glomeruli to produce renin, treated with glucose, palmitic acid (PA) and oleic acid (OA), were used as animal and cellular models. NaHS, the H2S donor, was administered to db/db mice through intraperitoneal injection. NaHS significantly alleviated blood pressure in db/db mice, decreased the renin content in the serum of db/db mice and reduced renin secretion from JG cells. NaHS modulated renin release via cAMP and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2), which mediate renin exocytosis. Furthermore, NaHS increased the levels of autophagy-related proteins and colocalization with EGFP-LC3 puncta with renin-containing granules and VAMP2 to consume excessive renin to maintain intracellular homeostasis. Therefore, exogenous H2S attenuates renin release and promotes renin-vesicular autophagy to relieve diabetes-induced hypertension.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH2) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP), one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1), which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.

The present study found that ricin toxicity did not only manifest itself as inhibition of protein synthesis, but also induced apoptosis of immune cells and played an extremely significant role in intestinal injury. In this report, we describe a novel method to estimate binding events occurring on intestinal brush border membranes (BBM) based on SPR technology in an attempt to mimic the real intestinal surface capable of interacting physically and/or actively with certain biological molecules. Combined with HPCE-ESI-MS indentification, we obtained 28 kinds of proteins in BBM that interacted with ricin.

Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-κB and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.

PIMT/NCOA6IP, a transcriptional coactivator PRIP/NCOA6 binding protein, enhances nuclear receptor transcriptional activity. Germline disruption of PIMT results in early embryonic lethality due to impairment of development around blastocyst and uterine implantation stages. We now generated mice with Cre-mediated cardiac-specific deletion of PIMT (csPIMT-/-) in adult mice. These mice manifest enlargement of heart, with nearly 100% mortality by 7.5 months of age due to dilated cardiomyopathy. Significant reductions in the expression of genes (i) pertaining to mitochondrial respiratory chain complexes I to IV; (ii) calcium cycling cardiac muscle contraction (Atp2a1, Atp2a2, Ryr2); and (iii) nuclear receptor PPAR- regulated genes involved in glucose and fatty acid energy metabolism were found in csPIMT-/- mouse heart. Elevated levels of Nppa and Nppb mRNAs were noted in csPIMT-/- heart indicative of myocardial damage. These hearts revealed increased reparative fibrosis associated with enhanced expression of Tgfβ2 and Ctgf. Furthermore, cardiac-specific deletion of PIMT in adult mice, using tamoxifen-inducible Cre-approach (TmcsPIMT-/-), results in the development of cardiomyopathy. Thus, cumulative evidence suggests that PIMT functions in cardiac energy metabolism by interacting with nuclear receptor coactivators and this property could be useful in the management of heart failure.

Phosphodiesterase 1C (PDE1C) is expressed in mammalian heart and regulates cardiac functions by controlling levels of second messenger cyclic AMP and cyclic GMP (cAMP and cGMP, respectively). However, molecular mechanisms of cardiac Pde1c regulation are currently unknown. In this study, we demonstrate that treatment of wild type mice and H9c2 myoblasts with Wy-14,643, a potent ligand of nuclear receptor peroxisome-proliferator activated receptor alpha (PPARα), leads to elevated cardiac Pde1C mRNA and cardiac PDE1C protein, which correlate with reduced levels of cAMP. Furthermore, using mice lacking either Pparα or cardiomyocyte-specific Med1, the major subunit of Mediator complex, we show that Wy-14,643-mediated Pde1C induction fails to occur in the absence of Pparα and Med1 in the heart. Finally, using chromatin immunoprecipitation assays we demonstrate that PPARα binds to the upstream Pde1C promoter sequence on two sites, one of which is a palindrome sequence (agcTAGGttatcttaacctagc) that shows a robust binding. Based on these observations, we conclude that cardiac Pde1C is a direct transcriptional target of PPARα and that Med1 may be required for the PPARα mediated transcriptional activation of cardiac Pde1C.

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation-reduction process, response to stress, plant-pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.

In eukaryotic cells, nucleocytoplasmic trafficking of macromolecules is largely mediated by Karyopherin β/Importin (KPNβ or Impβ) nuclear transport factors, and they import and export cargo proteins or RNAs via the nuclear pores across the nuclear envelope, consequently effecting the cellular signal cascades in response to pathogen attack and environmental cues. Although achievements on understanding the roles of several KPNβs have been obtained from model plant Arabidopsis thaliana, comprehensive analysis of potato KPNβ gene family is yet to be elucidated. In our genome-wide identifications, a total of 13 StKPNβ (Solanum tuberosum KPNβ) genes were found in the genome of the doubled monoploid S. tuberosum Group Phureja DM1-3. Sequence alignment and conserved domain analysis suggested the presence of importin-β N-terminal domain (IBN_N, PF08310) or Exporin1-like domain (XpoI, PF08389) at N-terminus and HEAT motif at the C-terminal portion in most StKPNβs. Phylogenetic analysis indicated that members of StKPNβ could be classified into 16 subgroups in accordance with their homology to human KPNβs, which was also supported by exon-intron structure, consensus motifs, and domain compositions. RNA-Seq analysis and quantitative real-time PCR experiments revealed that, except StKPNβ3d and StKPNβ4, almost all StKPNβs were ubiquitously expressed in all tissues analyzed, whereas transcriptional levels of several StKPNβs were increased upon biotic/abiotic stress or phytohormone treatments, reflecting their potential roles in plant growth, development or stress responses. Furthermore, we demonstrated that silencing of StKPNβ3a, a SA- and H2O2-inducible KPNβ genes led to increased susceptibility to environmental challenges, implying its crucial roles in plant adaption to abiotic stresses. Overall, our results provide molecular insights into StKPNβ gene family, which will serve as a strong foundation for further functional characterization and will facilitate potato breeding programs.