Application of High-Resolution DNA Melting for Genotyping in Lepidopteran Non-Model Species: Ostrinia furnacalis (Crambidae)

Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest

Targeting GSTP1-dependent ferroptosis in lung cancer radiotherapy: Existing evidence and future directions

Radiotherapy is applied in about 70% patients with tumors, yet radioresistance of tumor cells remains a challenge that limits the efficacy of radiotherapy. Ferroptosis, an iron-dependent lipid peroxidation regulated cell death, is involved in the development of a variety of tumors. Interestingly, there is evidence that ferroptosis inducers in tumor treatment can significantly improve radiotherapy sensitivity. In addition, related studies show that Glutathione S-transferase P1 (GSTP1) is closely related to the development of ferroptosis. The potential mechanism of targeting GSTP1 to inhibit tumor cells from evading ferroptosis leading to radioresistance has been proposed in this review, which implies that GSTP1 may play a key role in radiosensitization of lung cancer via ferroptosis pathway

Critical contributions of protein cargos to the functions of macrophage‑derived extracellular vesicles

Background Macrophages are highly plastic innate immune cells that play key roles in host defense, tissue repair, and homeostasis maintenance. In response to divergent stimuli, macrophages rapidly alter their functions and manifest a wide polarization spectrum with two extremes: M1 or classical activation and M2 or alternative activation. Extracellular vesicles (EVs) secreted from differentially activated macrophages have been shown to have diverse functions, which are primarily attributed to their microRNA cargos. The role of protein cargos in these EVs remains largely unexplored. Therefore, in this study, we focused on the protein cargos in macrophage-derived EVs. Results Naïve murine bone marrow-derived macrophages were treated with lipopolysaccharide or interlukin-4 to induce M1 or M2 macrophages, respectively. The proteins of EVs and their parental macrophages were subjected to quantitative proteomics analyses, followed by bioinformatic analyses. The enriched proteins of M1-EVs were involved in proinflammatory pathways and those of M2-EVs were associated with immunomodulation and tissue remodeling. The signature proteins of EVs shared a limited subset of the proteins of their respective progenitor macrophages, but they covered many of the typical pathways and functions of their parental cells, suggesting their respective M1-like and M2-like phenotypes and functions. Experimental examination validated that protein cargos in M1- or M2-EVs induced M1 or M2 polarization, respectively. More importantly, proteins in M1-EVs promoted viability, proliferation, and activation of T lymphocytes, whereas proteins in M2-EVs potently protected the tight junction structure and barrier integrity of epithelial cells from disruption. Intravenous administration of M2-EVs in colitis mice led to their accumulation in the colon, alleviation of colonic inflammation, promotion of M2 macrophage polarization, and improvement of gut barrier functions. Protein cargos in M2-EVs played a key role in their protective function in colitis. Conclusion This study has yielded a comprehensive unbiased dataset of protein cargos in macrophage-derived EVs, provided a systemic view of their potential functions, and highlighted the important engagement of protein cargos in the pathophysiological functions of these EVs

Genotyping of SNP <i>rs84</i> by UP-HRM.

<p><b>A:</b> raw melting curve data. <b>B:</b> derivative melting curves. <b>C:</b> normalization of melting curve data. <b>D:</b> normalized derivative melting curves. Three groups are well distinguished: A/A in blue, G/G in red, and G/A in gray.</p

Primer and probe sequences for HRM assays.

1<p>The SNPs were identified within <i>Tpi</i> gene by direct sequencing in this study. Details of the SNPs are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029664#pone-0029664-g001" target="_blank">Figure 1</a>.</p>2<p>F, forward primer; R, reverse primer.</p>3<p>T_a, optimized annealing temperature.</p>4<p>The underlined base was C3-blocked to prevent the 3′ end of the oligonucleotide from extending.</p

Genotyping of SNP <i>rs84</i> by SA-HRM.

<p><b>A, D.</b> normalized melting curves. <b>B, E:</b> normalized derivative melting curves. <b>C, F:</b> normalized difference curves. Unknown samples were successfully genotyped by prior mixing with known controls. Arrows link genotypes with corresponding same color curves.</p

Genotyping of SNP <i>rs577</i> by SA-HRM.

<p><b>A, D.</b> normalized melting curves. <b>B, E:</b> normalized derivative melting curves. <b>C, F:</b> normalized difference curves. Unknown samples were genotyped by prior mixing with known controls. Arrows link genotypes with corresponding same color curves.</p

Silkworm genetic sexing through W chromosome-linked, targeted gene integration.

Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems