Effect of MicroRNA-150 (miR-150) on the Biological Activity of Breast Cancer Cells and Its Correlation with Notch Signal

Our study explores miR-150’s effect on the biological activity of breast cancer cells and its correlation with Notch signaling. Human breast cancer cells MCF-7 were divided into WZ group (MCF-7 cells); KZ group (transfected with miR-150-NC); and group II (transfected with miR-150inhibitor) followed by analysis of miR-150 expressio,n cell replication, apoptosis, invasion, migration ability and Notch1 and Notch3 expression by qRT-PCR, cloning, Hoechst33258 fluorescent staining, Transwell chamber, cell scratch test, dual luciferase report and Western blot. Lowest miR-150 expression in MCF-7 cells indicated a successful transfection (P < 0.05). Compared with KZ and WZ groups, Notch1 and Notch3 mRNA levels in group II were decreased (P <0.05); and the number of cell clones in group II was reduced (P <0.05) without difference between WZ and KZ group (P >0.05); miR-150 inhibitor reduced Notch1 and Notch3 expression (P <0.05). The fluorescence intensity of MCF-7 cells in group II was highest among three groups (P <0.05). The number of cell invasion and migration as well as Notch1 and Notch 3 expression in group II was reduced (P <0.05) without difference between group KZ and WZ (P >0.05). miR-150 expression is increased in MCF-7 cells. The miR-150 inhibitor can inhibit cell apoptosis, migration and other biological behaviors, which is related to target Notch signaling pathway.

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

Hereditary diffuse gastric cancer (HDGC) caused by CDH1 variants predisposes to early-onset diffuse gastric (DGC) and lobular breast cancer (LBC). In Northern Portugal, the unusually high number of HDGC cases in unrelated families carrying the c.1901C>T variant (formerly known as p.A634V) suggested this as a CDH1-founder variant. We aimed to demonstrate that c.1901C>T is a bona fide truncating variant inducing cryptic splicing, to calculate the timing of a potential founder effect, and to characterize tumour spectrum and age of onset in carrying families. The impact in splicing was proven by using carriers’ RNA for PCR-cloning sequencing and allelic expression imbalance analysis with SNaPshot. Carriers and noncarriers were haplotyped for 12 polymorphic markers, and the decay of haplotype sharing (DHS) method was used to estimate the time to the most common ancestor of c.1901C>T. Clinical information from 58 carriers was collected and analysed. We validated the cryptic splice site within CDH1-exon 12, which was preferred over the canonical one in 100% of sequenced clones. Cryptic splicing induced an out-of-frame 37bp deletion in exon 12, premature truncation (p.Ala634ProfsTer7), and consequently RNA mediated decay. The haplotypes carrying the c.1901C>T variant were found to share a common ancestral estimated at 490 years (95% Confidence Interval 445–10,900). Among 58 carriers (27 males (M)–31 females (F); 13–83 years), DGC occurred in 11 (18.9%; 4M–7F; average age 33 ± 12) and LBC in 6 females (19.4%; average age 50 ± 8). Herein, we demonstrated that the c.1901C>T variant is a loss-of-function splice-site variant that underlies the first CDH1-founder effect in Portugal. Knowledge on this founder effect will drive genetic testing of this specific variant in HDGC families in this geographical region and allow intrafamilial penetrance analysis and better estimation of variant-associated tumour risks, disease age of onset, and spectrum.

Mitochondrial genome of Geomydoecus aurei, a pocket-gopher louse

Parasitic lice demonstrate an unusual array of mitochondrial genome architectures and gene arrangements. We characterized the mitochondrial genome of Geomydoecus aurei, a chewing louse (Phthiraptera: Trichodectidae) found on pocket gophers (Rodentia: Geomyidae) using reads from both Illumina and Oxford Nanopore sequencing coupled with PCR, cloning, and Sanger sequencing to verify structure and arrangement for each chromosome. The genome consisted of 12 circular mitochondrial chromosomes ranging in size from 1,318 to 2,088 nucleotides (nt). Total genome size was 19,015 nt. All 37 genes typical of metazoans (2 rRNA genes, 22 tRNA genes, and 13 protein-coding genes) were present. An average of 26% of each chromosome was composed of non-gene sequences. Within the non-gene region of each chromosome, there was a 79-nt nucleotide sequence that was identical among chromosomes and a conserved sequence with secondary structure that was always followed by a poly-T region. We hypothesize that these regions may be important in the initiation of transcription and DNA replication, respectively. The G. aurei genome shares 8 derived gene clusters with other chewing lice of mammals, but in G. aurei, genes on several chromosomes are not contiguous.

Parasites, Drugs and Captivity: Blastocystis-Microbiome Associations in Captive Water Voles

(1) Background: Blastocystis is a microbial eukaryote inhabiting the gastrointestinal tract of a broad range of animals including humans. Several studies have shown that the organism is associated with specific microbial profiles and bacterial taxa that have been deemed beneficial to intestinal and overall health. Nonetheless, these studies are focused almost exclusively on humans, while there is no similar information on other animals. (2) Methods: Using a combination of conventional PCR, cloning and sequencing, we investigated presence of Blastocystis along with Giardia and Cryptosporidium in 16 captive water voles sampled twice from a wildlife park. We also characterised their bacterial gut communities. (3) Results: Overall, alpha and beta diversities between water voles with and without Blastocystis did not differ significantly. Differences were noted only on individual taxa with Treponema and Kineothrix being significantly reduced in Blastocystis positive water voles. Grouping according to antiprotozoal treatment and presence of other protists did not reveal any differences in the bacterial community composition either. (4) Conclusion: Unlike human investigations, Blastocystis does not seem to be associated with specific gut microbial profiles in water voles.

Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

Characterization of nematode mitochondrial genomes.

This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.

Characterization of nematode mitochondrial genomes.

This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.

Epiphytic bet-mannanase producing bacterial strains

Dry remains of the herbal species of the plantain (Plantago major), the wormwood (Artemisia vulgaris) and the reed grass (Calamagrostis acutiflora) were used as a natural source for isolation of β- mannanase producing strains. They were isolated by using the carob gum as a single source of carbon and energy. Each chosen plant species was found to be colonized with a single dominant epiphytic group of microorganism, although the plants had been collected in the same location. Bacillus circulans was only found in P. major, Bacillus subtilis on A. vulgaris, whereas Pantoea sp. was found in C. acutiflora. Identification of the taxonomy affiliation of the isolated β-mannanase producers allowed using the formerly proposed primers for PCR cloning of β-mannanase genes previously occurred in the respective bacterial species. This approach let us cloning 330 bp fragment of β-mannanase genes from B. circulans and B. subtilis and 1000 bp fragment of β-mannanase gene from Pantoea sp. Testing the enzymatic activity of the isolated strains by staining the carob gum hydrolysis zones on the plates with Congo Red was carried out. As a result, the maximum activity was found in Pantoea sp.

Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (azadirachta indica)

Background Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.