Delivery of Nucleic Acids, Proteins, and Nanoparticles by Arginine-Rich Cell-Penetrating Peptides in Rotifers

Cell-penetrating peptides (CPPs) are a group of short, membrane-permeable cationic peptides that represent a nonviral technology for delivering nanomaterials and macromolecules into live cells. In this study, two arginine-rich CPPs, HR9 and IR9, were found to be capable of entering rotifers. CPPs were able to efficiently deliver noncovalently associated with cargoes, including plasmid DNAs, red fluorescent proteins (RFPs), and semiconductor quantum dots, into rotifers. The functional reporter gene assay demonstrated that HR9-delivered plasmid DNAs containing the enhanced green fluorescent protein and RFP coding sequences could be actively expressed in rotifers. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay further confirmed that CPP-mediated cargo delivery was not toxic to rotifers. Thus, these two CPPs hold a great potential for the delivery of exogenous genes, proteins, and nanoparticles in rotifers.     

Crp‐dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis

Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (c yclic AMP r eceptor p rotein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp‐independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.     

Molecular Characterization and Alternative Splicing of a Sodium Channel and DSC1 Ortholog Genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels.     

Circulating Matrix Metalloproteinase-2 and -9 Enzyme Activities in the Children with Ventricular Septal Defect

Ventricular septal defect (VSD) is the most common form of congenital heart diseases. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in causal cardiac tissue remodeling. We studied the changes of circulating MMP-2 and MMP-9 activities in the patients with VSD severity and closure. There were 96 children with perimembranous VSD enrolled in this study. We assigned the patients into three groups according to the ratio of VSD diameter/diameter of aortic root (Ao). They were classified as below: Trivial (VSD/Ao ratio ≤ 0.2), Small (0.2 < VSD/Ao ≤ 0.3) and Median (0.3 < VSD/Ao) group. Plasma MMP-2 and MMP-9 activities were assayed by gelatin zymography.There was a significant higher MMP-2 activity in the VSD (Trivial, Small and Median) groups compared with that in Control group. The plasma MMP-9 activity showed a similar trend as the findings in MMP-2 activity. After one year follow-up, a significant difference in the MMP-9 activity was found between VSD spontaneous closure and non-closure groups. In conclusion, a positive trend between the severity of VSD and activities of MMP-2 and MMP-9 was found. Our data imply that MMP-2 and MMP-9 activities may play a role in the pathogenesis of VSD.     

Regulation of ICAM‐1 expression in gingival fibroblasts infected with high‐glucose‐treated P. gingivalis

Porphyromonas gingivalis is a major pathogen in the initiation and progression of periodontal disease, which is recognized as a common complication of diabetes. ICAM‐1 expression by human gingival fibroblasts (HGFs) is crucial for regulating local inflammatory responses in inflamed periodontal tissues. However, the effect of P. gingivalis in a high‐glucose situation in regulating HGF function is not understood. The P. gingivalis strain CCUG25226 was used to study the mechanisms underlying the modulation of HGF ICAM‐1 expression by invasion of high‐glucose‐treated P. gingivalis (HGPg). A high‐glucose condition upregulated fimA mRNA expression in P. gingivalis and increased its invasion ability in HGFs. HGF invasion with HGPg induced increases in the expression of ICAM‐1. By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of p38 MAPK and Akt pathways is critical for HGPg‐induced ICAM‐1 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that HGPg invasion increases NF‐κB‐ and Sp1‐DNA‐binding activities in HGFs. Inhibition of NF‐κB and Sp1 activations blocked the HGPg‐induced ICAM‐1 promoter activity and expression. The effect of HGPg on HGF signalling and ICAM‐1 expression is mediated by CXC chemokine receptor 4 (CXCR4). Our findings identify the molecular pathways underlying HGPg‐dependent ICAM‐1 expression in HGFs, providing insight into the effect of P. gingivalis invasion in HGFs.     

Priming of the Arabidopsis pattern‐triggered immunity response upon infection by necrotrophic Pectobacterium carotovorum bacteria

Boosted responsiveness of plant cells to stress at the onset of pathogen‐ or chemically induced resistance is called priming. The chemical β‐aminobutyric acid (BABA) enhances Arabidopsis thaliana resistance to hemibiotrophic bacteria through the priming of the salicylic acid (SA) defence response. Whether BABA increases Arabidopsis resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) is not clear. In this work, we show that treatment with BABA protects Arabidopsis against the soft‐rot pathogen Pcc. BABA did not prime the expression of the jasmonate/ethylene‐responsive gene PLANT DEFENSIN 1.2 (PDF1.2), the up‐regulation of which is usually associated with resistance to necrotrophic pathogens. Expression of the SA marker gene PATHOGENESIS RELATED 1 (PR1) on Pcc infection was primed by BABA treatment, but SA‐defective mutants demonstrated a wild‐type level of BABA‐induced resistance against Pcc. BABA primed the expression of the pattern‐triggered immunity (PTI)‐responsive genes FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1 (FRK1), ARABIDOPSIS NON‐RACE SPECIFIC DISEASE RESISTANCE GENE (NDR1)/HAIRPIN‐INDUCED GENE (HIN1)‐LIKE 10 (NHL10) and CYTOCHROME P450, FAMILY 81 (CYP81F2) after inoculation with Pcc or after treatment with purified bacterial microbe‐associated molecular patterns, such as flg22 or elf26. PTI‐mediated callose deposition was also potentiated in BABA‐treated Arabidopsis, and BABA boosted Arabidopsis stomatal immunity to Pcc. BABA treatment primed the PTI response in the SA‐defective mutants SA induction deficient 2‐1 (sid2‐1) and phytoalexin deficient 4‐1 (pad4‐1). In addition, BABA priming was associated with open chromatin configurations in the promoter region of PTI marker genes. Our data indicate that BABA primes the PTI response upon necrotrophic bacterial infection and suggest a role for the PTI response in BABA‐induced resistance.     

Expression and role of Toll-like receptors on human umbilical cord mesenchymal stromal cells

Background aimsToll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs).MethodsIn the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined.ResultsAt the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon β, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs.ConclusionsTaken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs.     

RTK/ERK Pathway under Natural Selection Associated with Prostate Cancer

Prostate cancer (PCa) is a global disease causing large numbers of deaths every year. Recent studies have indicated the RTK/ERK pathway might be a key pathway in the development of PCa. However, the exact association and evolution-based mechanism remain unclear. This study was conducted by combining genotypic and phenotypic data from the Chinese Consortium for Prostate Cancer Genetics (ChinaPCa) with related databases such as the HapMap Project and Genevar. In this analysis, expression of quantitative trait loci (eQTLs) analysis, natural selection and gene-based pathway analysis were involved. The pathway analysis confirmed the positive relationship between PCa risk and several key genes. In addition, combined with the natural selection, it seems that 4 genes (EGFR, ERBB2, PTK2, and RAF1) with five SNPs (rs11238349, rs17172438, rs984654, rs11773818, and rs17172432) especially rs17172432, might be pivotal factors in the development of PCa. The results indicate that the RTK/ERK pathway under natural selection is a key link in PCa risk. The joint effect of the genes and loci with positive selection might be one reason for the development of PCa. Dealing with all the factors simultaneously might give insight into prevention and aid in predicting the success of potential therapies for PCa.     

The Composition of Microbiome in Larynx and the Throat Biodiversity between Laryngeal Squamous Cell Carcinoma Patients and Control Population

The throat is an ecological assemblage involved human cells and microbiota, and the colonizing bacteria are important factors in balancing this environment. However, this bacterial community profile has thus been poorly investigated. The purpose of this study was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. Barcoded pyrosequencing analysis of the 16S rRNA gene was used. We collected tissue samples from 29 patients with laryngeal carcinoma and 31 control patients with vocal cord polyps. The findings of high-quality sequence datasets revealed 218 genera from 13 phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx were Firmicutes (54%), Fusobacteria (17%), Bacteroidetes (15%), Proteobacteria (11%), and Actinobacteria (3%). The leading genera were Streptococcus (36%), Fusobacterium (15%), Prevotella (12%), Neisseria (6%), and Gemella (4%). The throat bacterial compositions were highly different between laryngeal carcinoma subjects and control population (p = 0.006). The abundance of the 26 genera was significantly different between the laryngeal cancer and control groups by metastats analysis (p<0.05). Fifteen genera may be associated with laryngeal carcinoma by partial least squares discriminant analysis (p<0.001). In summary, this study revealed the microbiota profiles in laryngeal mucosa from tissue specimens. The compositions of bacteria community in throat were different between laryngeal cancer patients and controls, and probably were related with this carcinoma. The disruption of this bio-ecological niche might be a risk factor for laryngeal carcinoma.