cDNA and gene sequence of Manduca sexta arylphorin, an aromatic amino acid-rich larval serum protein. Homology to arthropod hemocyanins

The serum (storage) proteins produced by insect larvae at the end of the feeding cycle are hexameric blood proteins with one or more type of subunits. The cDNA and gene structure of the aromatic amino acid-rich larval serum protein arylphorin from the tobacco hornworm, Manduca sexta, has been determined. In M. sexta arylphorin there are two subunits alpha and beta, which have 686 and 687 amino acids, respectively, and whose amino acid sequences are 68% identical. The two genes, separated by 7.1 kilobases of chromosomal DNA, are transcribed in the same direction. Based on the alignment of the amino acid sequence, the rate of nucleotide substitution between the two coding regions predicts that the two genes diverged about 100 million years ago. Both genes contain 5 exons and the upstream region contains a sequence, TGATAAA, which is similar to a sequence found in all other storage protein genes for which information is available. When the National Biomedical Research Foundation protein sequence data base was searched, it was found that the arylphorin subunits showed significant similarity to the arthropod hemocyanins, which are hexameric oxygen-carrying proteins. Based on the alignment of the sequence of M. sexta arylphorin and the hemocyanin from the spiny lobster (Panulirus interruptus), for which a 3.2 A structure has been determined, it was observed that the highest concentration of conserved residues were found in those regions of the sequence which are involved in subunit interactions in the hexameric protein. It is suggested that the insect storage proteins and the arthropod hemocyanins have evolved from a common ancestor.     

Systematic analysis of multigene predictors in gastric cancer exploiting gene expression signature

Gastric cancer (GC) is the second most common cause of cancer death worldwide but could be more curable if diagnosed at an earlier stage. At present, the capability to predict the efficaciousness of molecular diagnosis for GC for each patient remains elusive. The purpose of this study was to identify tumor biomarkers through systems analysis of multigene predictors exploiting the available data resource. In this study, we investigated the top 10% overexpressed genes in GC from five data sets of the Oncomine platform, with 265 GC samples versus 174 normal gastric mucosa samples. Sixteen candidate genes were identified as predictors of GC, of which 14 genes were verified through the comparison of expression levels in specimens from normal (chronic gastritis, 21 samples) and GC groups (38 samples). In addition, unique molecular portraits of diffuse adenocarcinoma (DA), intestinal adenocarcinoma (IA), and mixed adenocarcinoma (MA) were studied through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, where DA showed higher extracellular matrix alteration while IA and MA showed higher cell-cycle alteration than other types. We also found that the elevated expressions of genes during GC progression were independent of gene mutations, and high core-binding factor subunit β expression is correlated with a high overall survival rate in GC patients. Our research may provide an efficient clinical diagnosis of GC at an early stage with high accuracy and thus help improve the overall survival rate through early therapeutic interventions.     

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 10³ CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5–25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.     

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis

• Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology.
• We used knock‐down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana.
• Knock‐down of RPS9M in the rps9m‐3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m‐3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m‐3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS.
• Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

     

The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity

Hydroxyacid dehydrogenases are responsible for the conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally verified to have both the D-LDH and glutamate dehydrogenase (GDH) activities (reversible deamination). The catalytic mechanism was demonstrated by identification of key residues from the crystal structure analysis and site-directed mutagenesis. The Arg²³⁴ and Gly⁷⁹ residues of this enzyme play a significant role in both D-LDH and GDH activities. His²⁹⁵ and Phe²⁹⁸ in DLDH744 were identified to be key residues for lactate dehydrogenase (LDH) activity only whereas Tyr¹⁰¹ is a unique residue that is critical for GDH activity. Characterization of the biochemical properties contributes to understanding of the catalytic mechanism of this novel D-lactate dehydrogenase enzyme.     

Epigenetic Regulation of Elf5 Is Associated with Epithelial-Mesenchymal Transition in Urothelial Cancer

E74-like factor 5 (Elf5) has been associated with tumor suppression in breast cancer. However, its role in urothelial cancer (UC) is completely unknown. Immunohistochemistry (IHC) and methylation specific PCR (MSP) were done to detect Elf5 expression level and its promoter methylation. Results revealed that low expression of Elf5 on protein and mRNA levels were associated with tumor progression, early relapse and poor survival. In vitro, down-regulation of Elf5 can increase epithelial-mesenchymal transition (EMT). Aberrant Elf5 methylation was identified as major mechanism for Elf5 gene silence. Accordingly, restoration of Elf5 by infection or demethylating treatment effectively reversed EMT processes. In conclusion, we identified Elf5 as a novel biomarker of UC on several biological levels and established a causative link between Elf5 and EMT in UC.     

Optimal Sequence of Irinotecan and Oxaliplatin-Based Regimens in Metastatic Colorectal Cancer: A Population-Based Observational Study

The optimal sequence of irinotecan and oxaliplatin-based regimens for metastatic colorectal cancer remains unclear. We conducted a population-based observational study by retrospectively reviewing records from Taiwan’s National Health Insurance Research Database to explore this issue. Patients aged ≥20 years with metastatic colorectal cancer newly diagnosed between 2004 and 2008 (n = 9490) were enrolled in current study. Among these 9490 patients, 3895 patients (41.04%) did not receive any chemotherapy within the first three months after catastrophic illness registration. Patients who received best supportive care were older and had higher Charlson comorbidity indexes and incidences of comorbidities than those who received irinotecan-based regimens, oxaliplatin-based regimens, and 5-fluorouracil/capecitabine alone. Patients who received irinotecan followed by oxaliplatin-based regimens and those who received the reverse sequence were further stratified into arm A (n = 542) and arm B (n = 1156), respectively. The median first time to next treatment was not significantly different between arm A and arm B (210 days vs. 196 days; p = 0.17). However, the median second time to next treatment was longer in arm A than in arm B (155 days vs. 123 days; p = 0.006), which translated into a better overall survival (487 days vs. 454 days; p = 0.02). The crossover rate was higher in arm A than in arm B (47.84% vs. 41.61%; p<0.001). Multivariate Cox regression analyses showed that overall survival was comparable between the two chemotherapy sequences (p = 0.27). Our study suggested that irinotecan followed by oxaliplatin-based regimens might be a better chemotherapy treatment option for metastatic colorectal cancer than the reverse sequence given the higher crossover rate and potential overall survival benefit.     

Effects of SOX2 on Proliferation,Migration and Adhesion of Human Dental Pulp Stem Cells

As a key factor for cell pluripotent and self-renewing phenotypes, SOX2 has attracted scientists’ attention gradually in recent years. However, its exact effects in dental pulp stem cells (DPSCs) are still unclear. In this study, we mainly investigated whether SOX2 could affect some biological functions of DPSCs. DPSCs were isolated from the dental pulp of human impacted third molar. SOX2 overexpressing DPSCs (DPSCs-SOX2) were established through retroviral infection. The effect of SOX2 on cell proliferation, migration and adhesion ability was evaluated with CCK-8, trans-well system and fibronectin-induced cell attachment experiment respectively. Whole genome expression of DPSCs-SOX2 was analyzed with RNA microarray. Furthermore, a rescue experiment was performed with SOX2-siRNA in DPSC-SOX2 to confirm the effect of SOX2 overexpression in DPSCs. We found that SOX2 overexpression could result in the enhancement of cell proliferation, migration, and adhesion in DPSCs obviously. RNA microarray analysis indicated that some key genes in the signal pathways associated with cell cycle, migration and adhesion were upregulated in different degree, and the results were further confirmed with qPCR and western-blot. Finally, DPSC-SOX2 transfected with SOX2-siRNA showed a decrease of cell proliferation, migration and adhesion ability, which further confirmed the biological effect of SOX2 in human DPSCs. This study indicated that SOX2 could improve the cell proliferation, migration and adhesion ability of DPSCs through regulating gene expression about cell cycle, migration and adhesion, and provided a novel strategy to develop seed cells with strong proliferation, migration and adhesion ability for tissue engineering.     

Pre-cold acclimation promotes cryoprotectant accumulation and enhances freezing tolerance of Meloidogyne incognita

Strong evidence suggests that cryoprotectant accumulation during pre-cold acclimation protects cells against freezing injuries caused by cellular dehydration. In this study, the concentrations of trehalose and glycerol were measured in Meloidogyne incognita and it was found that both cryoprotectants were significantly accumulated in second-stage juveniles (J2) of M. incognita after acclimation at 4°C. However, compared with non-acclimated samples, only a higher level of trehalose was induced in the egg masses of M. incognita in response to cold treatment. Further characterizations indicated that pre-cold acclimation efficiently accelerated the speed of larvae hatching from egg masses that were subjected to freezing at −1°C. In addition, the survival rate and pathogenicity of M. incognita J2 that had been acclimated prior to freezing were significantly enhanced when compared with non-acclimated J2 individuals. As far as we know, this is the first time that this phenomenon has been reported in M. incognita.