A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a “blade to sheath transformation” phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::ΔOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.     

Aberrant Expression of Osteopontin and E-Cadherin Indicates Radiation Resistance and Poor Prognosis for Patients with Cervical Carcinoma

Radiotherapy is the first-line treatment for all stages of cervical cancer, whether it is used for radical or palliative therapy. However, radioresistance of cervical cancer remains a major therapeutic problem. Consequently, we explored if E-cadherin (a marker of epithelial-mesenchymal transition) and osteopontin could predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). Patients were retrospectively reviewed and 111 patients divided into two groups (radiation-resistant and radiation-sensitive groups) according to progression-free survival (PFS). In pretreated paraffin-embedded tissues, we evaluated E-cadherin and osteopontin expression using immunohistochemical staining. The percentage of patients with high osteopontin but low E-cadherin expression in the radiation-resistant group was significantly higher than those in the radiation-sensitive group (p<0.001). These patients also had a lower 5-year PFS rate (p<0.001). Our research suggests that high osteopontin but low E-cadherin expression can be considered as a negative, independent prognostic factor in patients with LACSCC ([Hazard ratios (95% CI) 6.766 (2.940, 15.572)], p<0.001).     

High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA

Little information is available on the presence of viable Toxoplasma gondii in tissues of lambs worldwide. The prevalence of T. gondii was determined in 383 lambs (<1 year old) from Maryland, Virginia and West Virginia, USA. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100 and 1:200 dilutions, and hearts were selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. For bioassays in cats, the entire myocardium or 500g was chopped and fed to cats, one cat per heart and faeces of the recipient cats were examined for shedding of T. gondii oocysts. For bioassays in mice, 50g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. In total, 53 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 53 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed 57 strains with 15 genotypes. Four lambs had infections with two T. gondii genotypes. Twenty-six (45.6%) strains belong to the clonal Type II lineage (these strains can be further divided into two groups based on alleles at locus Apico). Eight (15.7%) strains belong to the Type III lineage. The remaining 22 strains were divided into 11 atypical genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in lambs, which has important implications in public health. We believe this is the first in-depth genetic analysis of T. gondii isolates from sheep in the USA.     

Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast

A Chlamydomonas reinhardtii chloroplast expression vector, pACTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed and transfered to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by PCR, Southern blot, Western blot and ELISA assays after selection on resistant medium and incubation in the dark. The CTBVP1 fusion protein was expressed in C. reinhardtii chloroplast and accounted for up to 3% of the total soluble protein. The fusion protein also retained both GM1-ganglioside binding affinity and antigenicity of the FMDV VP1 and CTB proteins. These experimental results support the possibility of using transgenic chloroplasts of green alga as a mucosal vaccine source.     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.     

Ameloblastin peptide encoded by exon 5 interacts with amelogenin N-terminus

Interactions between enamel matrix proteins are important for enamel biomineralization. In recent in situ studies, we showed that the N-terminal proteolytic product of ameloblastin co-localized with amelogenin around the prism boundaries. However, the molecular mechanisms of such interactions are still unclear. Here, in order to determine the interacting domains between amelogenin and ameloblastin, we designed four ameloblastin peptides derived from different regions of the full-length protein (AB1, AB2 and AB3 at N-terminus, and AB6 at C-terminus) and studied their interactions with recombinant amelogenin (rP172), and the tyrosine-rich amelogenin polypeptide (TRAP). A series of amelogenin Trp variants (rP172(W25), rP172(W45) and rP172(W161)) were also used for intrinsic fluorescence spectroscopy. Fluorescence spectra of rP172 titrated with AB3, a peptide encoded by exon 5 of ameloblastin, showed a shift in λ_max in a dose-dependent manner, indicating molecular interactions in the region encoded by exon 5 of ameloblastin. Circular dichroism (CD) spectra of amelogenin titrated with AB3 showed that amelogenin was responsible for forming α-helix in the presence of ameloblastin. Fluorescence spectra of amelogenin Trp variants as well as the spectra of TRAP titrated with AB3 showed that the N-terminus of amelogenin is involved in the interaction between ameloblastin and amelogenin. We suggest that macromolecular co-assembly between amelogenin and ameloblastin may play important roles in enamel biomineralization.     

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.