High-efficiency protein delivery into transfection-recalcitrant cell types

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.     

Dielectric Properties of Normal and Metastatic Lymph Nodes Ex Vivo From Lung Cancer Surgeries

The dielectric properties of normal and tumor human tissues have been widely reported in recent years. However, the dielectric properties of intrathoracic lymph nodes (LNs) have not been reported. In this communication, we measured the dielectric properties (i.e., permittivity and conductivity) of ex vivo intrathoracic LNs obtained from lung cancer surgeries. Results show that the permittivity and conductivity of metastatic LNs are higher than those of normal LNs over the frequency range of 1 MHz–4 GHz. Statistically significant differences are observed at single specific frequencies (64, 128, 298, 433, and 915 MHz and 2.45 GHz). Our study provides the basic data to support future-related research and fills the research gap on the dielectric properties of LNs in the lungs. Bioelectromagnetics. 2020;41:148–155. © 2020 Bioelectromagnetics Society.     

Sequence verification of synthetic DNA by assembly of sequencing reads

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org.     

Identification of genetic associations of SP110/MYBBP1A/RELA with pulmonary tuberculosis in the Chinese Han population

Genetic factors play important roles in the development of tuberculosis (TB). SP110 is a promising candidate target for controlling TB infections. However, several studies associating SP110 single nucleotide polymorphisms (SNPs) with TB have yielded conflicting results. This may be partly resolved by studying other genes associated with SP110, such as MYBBP1A and RELA. Here, we genotyped 6 SP110 SNPs, 8 MYBBP1A SNPs and 5 RELA SNPs in 702 Chinese pulmonary TB patients and 425 healthy subjects using MassARRAY and SNaPshot methods. Using SNP-based analysis with Bonferroni correction, rs3809849 in MYBBP1A [Pcorrected (cor) = 0.0038] and rs9061 in SP110 (Pcor = 0.019) were found to be significantly associated with TB. Furthermore, meta-analysis of rs9061 in East Asian populations showed that the rs9061 T allele conferred significant risk for TB [P = 0.002, pooled odds ratio (OR), 1.24, 95 % confidence interval (CI) = 1.08–1.43]. The MYBBP1A GTCTTGGG haplotype and haplotypes CGACCG/TGATTG within SP110 were found to be markedly and significantly associated with TB (P = 2.00E?06, 5.00E?6 and 2.59E?4, respectively). Gene-based analysis also demonstrated that SP110 and MYBBP1A were each associated with TB (Pcor = 0.011 and 0.035, respectively). The logistic regression analysis results supported interactions between SP110 and MYBBP1A, indicating that subjects carrying a GC/CC genotype in MYBBP1A and CC genotype in SP110 possessed the high risk of developing TB (P = 1.74E?12). Our study suggests that a combination of SP110 and MYBBP1A gene polymorphisms may serve as a novel marker for identifying the risk of developing TB in the Chinese Han population.     

适当浓度的过氧化氢诱导人支气管上皮细胞发生钙振荡

包括过氧化氢（Hzoz）在内的活性氧通过引起细胞内钙的变化而造成细胞损伤。然而,不同浓度的H202可以导致细胞内不同的钙变化,并激活不同的信号通路。细胞内钙振荡是其中的一种钙信号变化形式,钙振荡可以调控转录因子NF—KB的活性。该研究探讨可以诱导支气管上皮细胞内钙振苏发生的H2o2浓度。体外培养人支气管上皮细胞,采取钙离子荧光探针Fura_2标记细胞。并使用离子成像系统,观测不同浓度的H：0：（0～1000μmol／L）作用下细胞内钙浓度的变化。结果发现,低于50μmol／L的H202仅仅引起“钙火花”;50～500μmol／L的H202导致细胞内钙振荡的发生;而1000μmol／L的H202引起细胞内持续的高钙;同时也证实150μmol／L的H202诱发明显的钙振荡,而钙振荡随后引起了NF—KB活性的升高。该研究提示,适当浓度的H：0：可以诱发支气管上皮细胞内钙振荡的发生,推测可能是活性氧导致慢性气道炎症损伤的一个机制。     

ER stress and ASK1-JNK activation contribute to oridonin-induced apoptosis and growth inhibition in cultured human hepatoblastoma HuH-6 cells

Workers who are exposed to extreme heat or work in hot environments may be at risk of heat stress. Exposure to extreme heat can result in occupational illnesses and injuries. On the other hand, local and regional heat therapy has been used for the treatment of some cancers, such as liver cancer, lung cancer, and kidney cancer. Although heat stress has been shown to induce the accumulation of p53 protein, a key regulator of cell cycle, apoptosis, DNA repair, and autophagy, how it regulates p53 protein accumulation and what the p53 targets are remain unclear. Here, we show that, among various genotoxic stresses, including ionizing radiation (IR) and ultraviolet (UV) radiation, heat stress contributes significantly to increase p53 protein levels in normal liver cells and liver cancer cells. Heat stress did not increase p53 mRNA expression as well as p53 promoter activity. However, heat stress enhanced the half-life of p53 protein. Moreover, heat stress increased the expression of puma and light chain 3 (LC-3), which are associated with the apoptotic and autophagic function of p53, respectively, whereas it did not change the expression of the cell cycle regulators p21, 14-3-3δ, and GADD45α, suggesting that heat-triggered alteration of p53 selectively modulates the downstream targets of p53. Our study provides a novel mechanism by which heat shock stimulates p53 protein accumulation, which is different from common DNA damages, such as IR and UV, and also provides new molecular basis for heat injuries or heat therapy.     

Loss of microRNA-132 predicts poor prognosis in patients with primary osteosarcoma

??₂-glycoprotein I (??₂-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of ??₂-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of ??₂-GPI to endothelial cells, we investigated the effect of ??₂-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of ??₂-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that ??₂-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, ??₂-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, ??₂-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that ??₂-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.     

Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer

Dysregulation in the expression of miRNAs contributes to the occurrence and development of many human cancers. We herein attempted to obtain the potential association between miRNA expression profile and breast cancer by applying high-throughput sequencing technology. Small RNAs from seven paired tumor and adjacent normal tissue samples were sequenced. To determine the miRNA expression profiles in tissues and sera, another five equally pooled serum samples from 20 patients and 30 normal women were sequenced. Despite a similar number in abundantly expressed miRNAs across samples, we detected varying miRNA expression profiles. Some miRNAs showed inconsistent or opposite dysregulation trends across different tumor tissues, including some abundantly expressed miRNA gene clusters and gene families. Wilcoxon sign-rank test for paired samples analysis revealed that abnormal miRNAs showed a higher level of variation across the seven tumor samples. We also completely surveyed abnormal miRNAs expressed in tumor and serum tissues in the mixed datasets based on the relative expression levels. Most of these miRNAs were significantly down-regulated in tumor samples, but nine abnormal miRNAs (miR-18a, 19a, 20a, 30a, 103b, 126, 126*, 192, 1287) were consistently expressed in tumor tissues and serum samples. Based on experimentally validated target mRNAs, functional enrichment analysis indicated that these abnormal miRNAs and miRNA groups (miRNA gene clusters and gene families) have important roles in multiple biological processes. Dynamic miRNA expression profiles, various abnormal miRNA profiles and complexity of the miRNA regulatory network reveal that the miRNA expression profile is a potential biomarker for classifying or detecting human disease.     

Depletion interaction in colloid/polymer mixtures: application of density functional theory

Insert-route density functional approach (IRDFT), modified fundamental measure theory (MFMT) and thermodynamic perturbation theory (TPT1 and TPT2) are combined to study the depletion force between colloidal particles in hard sphere/hard sphere chain mixtures which represent a model of systems containing colloids dispersed in an athermal polymer solution. The predicted results are compared to simulations showing the reliability of the method used which captures the main characteristics of depletion interaction between colloids induced by polymers. Results of TPT2 are slightly more repulsive and better than that of TPT1 especially when the inter-particle distance is small than the diameter of polymer segment indicating the essential influence of the three-body correlations. Effects of the polymer density, polymer chain length and size ratio of colloid to polymer segment on the depletion force are studied in detail. Due to a little deterioration of the prediction in the high density region, further improvement is anticipated to better balance the competition between the excluded-volume effect and the chain connectivity.     

Rit subfamily small GTPases: Regulators in neuronal differentiation and survival

Ras family small GTPases serve as binary molecular switches to regulate a broad array of cellular signaling cascades, playing essential roles in a vast range of normal physiological processes, with dysregulation of numerous Ras-superfamily G-protein-dependent regulatory cascades underlying the development of human disease. However, the physiological function for many “orphan” Ras-related GTPases remain poorly characterized, including members of the Rit subfamily GTPases. Rit is the founding member of a novel branch of the Ras subfamily, sharing close homology with the neuronally expressed Rin and Drosophila Ric GTPases. Here, we highlight recent studies using transgenic and knockout animal models which have begun to elucidate the physiological roles for the Rit subfamily, including emerging roles in the regulation of neuronal morphology and cellular survival signaling, and discuss new genetic data implicating Rit and Rin signaling in disorders such as cancer, Parkinson's disease, autism, and schizophrenia.