Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes

Molecular Biology - When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be...     

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.     

A chloride efflux transporter,BIG RICE GRAIN 1, is involved in mediating grain size and salt tolerance in rice

Grain size is determined by the size and number of cells in the grain. The regulation of grain size is crucial for improving crop yield; however, the genes and molecular mechanisms that control grain size remain elusive. Here, we report that a member of the detoxification efflux carrier /Multidrug and Toxic Compound Extrusion (DTX/MATE) family transporters, BIG RICE GRAIN 1 (BIRG1), negatively influences grain size in rice (Oryza sativa L.). BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the outer parenchyma layer cells of spikelet hulls are larger than in wild-type (WT) grains, but the cell number is unaltered. When expressed in Xenopus laevis oocytes, BIRG1 exhibits chloride efflux activity. Consistent with this role of BIRG1, the birg1 mutant shows reduced tolerance to salt stress at a toxic chloride level. Moreover, grains from birg1 plants contain a higher level of chloride than those of WT plants when grown under normal paddy field conditions, and the roots of birg1 accumulate more chloride than those of WT under saline conditions. Collectively, the data suggest that BIRG1 in rice functions as a chloride efflux transporter that is involved in mediating grain size and salt tolerance by controlling chloride homeostasis.     

Transmission of synovial sarcoma from a single multi-organ donor to three transplant recipients: case report

Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination. Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant. Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.     

Construction of a robust prognostic model for adult adrenocortical carcinoma: Results from bioinformatics and real-world data

This study aims to construct a robust prognostic model for adult adrenocortical carcinoma (ACC) by large-scale multiomics analysis and real-world data. The RPPA data, gene expression profiles and clinical information of adult ACC patients were obtained from The Cancer Proteome Atlas (TCPA), Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Integrated prognosis-related proteins (IPRPs) model was constructed. Immunohistochemistry was used to validate the prognostic value of the IPRPs model in Fudan University Shanghai Cancer Center (FUSCC) cohort. 76 ACC cases from TCGA and 22 ACC cases from GSE10927 in NCBI’s GEO database with full data for clinical information and gene expression were utilized to validate the effectiveness of the IPRPs model. Higher FASN (P = .039), FIBRONECTIN (P < .001), TFRC (P < .001), TSC1 (P < .001) expression indicated significantly worse overall survival for adult ACC patients. Risk assessment suggested significantly a strong predictive capacity of IPRPs model for poor overall survival (P < .05). IPRPs model showed a little stronger ability for predicting prognosis than Ki-67 protein in FUSCC cohort (P = .003, HR = 3.947; P = .005, HR = 3.787). In external validation of IPRPs model using gene expression data, IPRPs model showed strong ability for predicting prognosis in TCGA cohort (P = .005, HR = 3.061) and it exhibited best ability for predicting prognosis in GSE10927 cohort (P = .0898, HR = 2.318). This research constructed IPRPs model for predicting adult ACC patients’ prognosis using proteomic data, gene expression data and real-world data and this prognostic model showed stronger predictive value than other biomarkers (Ki-67, Beta-catenin, etc) in multi-cohorts.     

Allelopathic effects of switchgrass on redroot pigweed and crabgrass growth

Non-native invasive plant species influence plant community composition and competitively eradicate native species. However, there is doubt regarding how global invasive species increase and explosively interfere with native plants. Invasive plants always have strong allelopathic potential. In this study, allelopathic effects of switchgrass on redroot pigweed and crabgrass growth were investigated by field and laboratory experiments. Within a 0.4-m distance of switchgrass, density and shoot biomass of native species were significantly suppressed in the field, with 95.1% and 93.0% inhibition on density of redroot pigweed and crabgrass and with 99.0% and 97.7% inhibition on shoot biomass, respectively, during the third growing season. Significant inhibitory effects on shoot and root biomass were observed at the 5:5 (switchgrass–native species) proportion in glass bottles, by 41.57% and 51.21% for shoot and root biomass of redroot pigweed and by 33.42% and 56.95% for shoot and root biomass of crabgrass, respectively. Results of a glass bottle experiment showed that shoot and root biomass of redroot pigweed and crabgrass could be significantly inhibited by contact with switchgrass root. Results of a Petri dish experiment showed that aqueous extracts of switchgrass significantly inhibited germination process of both species at high concentrations, with 90.74% and 18.62% inhibition on germination rate and plumule length of redroot pigweed and with 63.59%, 16.38%, and 19.92% inhibition on germination rate, plumule, and radicle lengths of crabgrass, respectively, at the concentration of 0.1 g·mL^?1. This report demonstrated that switchgrass had allelopathic effects on redroot pigweed and crabgrass growth.

     

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

A Single Nucleotide Polymorphism of Chicken Acetyl-CoA Carboxylase A Gene Associated with Fatness Traits

Acetyl-CoA carboxylase α (ACCα) is a major rate-limiting enzyme in the biogenesis of long-chain fatty acids. It can catalyze the carboxylation of acetyl-CoA to form malonyl-CoA that plays a key role in the regulation of fatty acid metabolism. The objective of the present study was to investigate the associations of ACCα gene polymorphisms with chicken growth and body composition traits. The Northeast Agricultural University broiler lines divergently selected for abdominal fat content and the Northeast Agricultural University F₂ Resource Population were used in the current study. Body weight and body composition traits were measured in the aforementioned two populations. A synonymous mutation was detected in the exon 19 region of ACCα gene, then polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to genotype all the individuals derived from the aforementioned populations. Association analysis revealed that the polymorphism was associated with abdominal fat weight and percentage of abdominal fat in the two populations. The results suggested that ACCα gene could be a candidate locus or linked to a major gene that affects abdominal fat content in the chicken.