Biomimetic nanocarriers loaded with temozolomide by cloaking brain-targeting peptides for targeting drug delivery system to promote anticancer effects in glioblastoma cells.

Glioma is the leading cancer of the central nervous system (CNS). The efficacy of glioma treatment is significantly hindered by the presence of the blood-brain barrier (BBB) and blood-brain tumour barrier (BBTB), which prevent most drugs from entering the brain and tumours. Hence, we established a novel drug delivery nanosystem of brain tumour-targeting that could self-assemble the method using an amphiphilic Zein protein isolated from corn. Zein's amphiphilicity prompted it to self-assembled into NPs, efficiently containing TMZ. This allowed us to investigate temozolomide (TMZ) for glioblastoma (GBM) treatment. To construct TMZ-encapsulated NPs (TMZ@RVG-Zein NPs), the NPs' Zein was clocked to rabies virus glycoprotein 29 (RVG29). To verify that the NPs could penetrate the BBB and precisely target and kill the GBM cancer cell line, in vitro studies were performed. The process of NPs penetrating cancer cell membranes was investigated using enzyme-linked immunosorbent assays (ELISAs) to measure the expressions of nicotinic acetylcholine receptors (nAChRs) on the U87 cell line. Therefore, effective targeted brain cancer treatment is possible by forming NP clocks, a cell-penetrating natural Zein protein with an RVG29. These NPs can penetrate the blood-brain barrier (BBB) and enter the glioblastoma (U87) cell line to release TMZ. These NPs have a distinct cocktail of biocompatibility and brain-targeting abilities, making them ideal for involving brain diseases.

Safety and Efficacy of Stereotactic Ablative Radiotherapy for Ultra-Central Lung Cancer.

Background: Ultra-central lung cancer (UCLC) is difficult to achieve surgical treatment. Over the past few years, stereotactic ablative radiotherapy (SABR) or stereotactic body radiotherapy (SBRT) obviously improved the clinical efficacy and survival of UCLC patients. However, the adapted scheme of radiation therapy is still controversial. For this, a single arm retrospective analysis was performed on UCLC patients treated with SBRT. Material and Methods: We retrospectively studied primary UCLC patients who were treated with SBRT of 56 Gy/6-8f between 2010 and 2018. UCLC was defined as planning target volume (PTV) touching or overlapping the proximal bronchial tree, trachea, esophagus, heart, pulmonary vein, or pulmonary artery within 2 cm around the bronchial tree in all directions. Results: A total of 58 patients whose median age was 68 years (range, 46-85) were included in our study, 79.3% of whom did not undergo any previous therapy. The median dose of the PTV was 77.8 Gy (range, 43.3-91.8), and the median PTV of tumors was 6.2 cm3 (range, 12.9-265.0). With a median follow-up of 57 months (range, 6-90 months), the median cumulative overall survival (OS) rate was 58 months (range, 2-105). In addition, the 1-year, 2-year and 5-year OS rates were 94.7%, 75.0% and 45.0%, respectively. In our univariable analysis (p=0.020) and multivariate analysis (p=0.004), the OS rate was associated with the PTV. The 5-year OS rates for PTV <53.0 cm3 and PTV ≥53.0 cm3 were 61.6% and 37.4%, respectively. Regarding toxicity after SBRT, there were two cases (3.5%) with grade ≥3 adverse events, of which 1 case died of sudden severe unexplained hemoptysis. Conclusions: Patients with UCLC can benefit from SBRT at a dose of 56 Gy/6-8f. On the other hand, smaller PTV was associated with superior outcomes, and the cure difference needs to be validated by prospective comparative trials.

Risk-adapted stereotactic body radiation therapy for central and ultra-central early-stage inoperable non-small cell lung cancer.

To determine the therapeutic efficacy and safety of risk-adapted stereotactic body radiation therapy (SBRT) schedules for patients with early-stage central and ultra-central inoperable non-small cell lung cancer. From 2006 to 2015, 80 inoperable T1-2N0M0 NSCLC patients were treated with two median dose levels: 60 Gy in six fractions (range, 48-60 Gy in 4-8 fractions) prescribed to the 74% isodose line (range, 58%-79%) for central lesions (ie within 2 cm of, but not abutting, the proximal bronchial tree; n = 43), and 56 Gy in seven fractions (range, 48-60 Gy in 5-10 fractions) prescribed to the 74% isodose line (range, 60%-80%) for ultra-central lesions (ie abutting the proximal bronchial tree; n = 37) on consecutive days. Primary endpoint was overall survival (OS); secondary endpoints included progression-free survival (PFS), tumor local control rate (LC), and toxicity. Median OS and PFS were 64.47 and 32.10 months (respectively) for ultra-central patients, and not reached for central patients. Median time to local failure, regional failure, and any distant failures for central versus ultra-central lesions were: 27.37 versus 26.07 months, 20.90 versus 12.53 months, and 20.85 versus 15.53 months, respectively, all P < .05. Multivariate analyses showed that tumor categorization (ultra-central) and planning target volume ≥52.76 mL were poor prognostic factors of OS, PFS, and LC, respectively (all P < .05). There was one grade 5 toxicity; all other toxicities were grade 1-2. Our results showed that ultra-central tumors have a poor OS, PFS, and LC compared with central patients because of the use of risk-adapted SBRT schedules that allow for equal and favorable toxicity profiles.

The complex architecture and epigenomic impact of plant T-DNA insertions.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

A Second Course of Stereotactic Image-Guided Robotic Radiosurgery for Patients with Cerebral Metastasis.

OBJECTIVES: The purpose of this research was to study the outcome of brain metastases in a cohort of patients undergoing a second course of stereotactic image-guided robotic radiosurgery and to identify predictors corelated with survival. METHODS: A total of 63 patients with primary malignancies underwent a second course of CyberKnife radiosurgery for intracranial progression, including recurrence and new metastases after initial stereotactic radiosurgery (SRS). Overall survival (OS) and control rate were calculated by the Kaplan-Meier method. A Cox proportional hazards model was used to analyze predictive factors for survival. RESULTS: With a median follow-up duration of 12 months after second SRS, the median OS of the second course of radiosurgery was 18 months. On multivariate analysis, the sum of total planned target volume (hazard ratio, 2.112; 95% confidence interval, 1.069-4.173) and minimum dose (hazard ratio, 1.990, 95% confidence interval, 1.017-3.892) were significantly associated with OS. Median intracranial progression-free survival was 23 months. The 6-month and 12-month local control rates of the targets were 97.0% and 94.4%, respectively. Univariate analysis showed that only tumor number significantly influenced intracranial progression-free survival (P = 0.012). Nine patients (14.2%) developed brain necrosis. Median time to brain necrosis in regions in which brain necrosis occurred after a single course of SRS was not reached, compared with 16 months for those treated with repeat SRS (P = 0.041). CONCLUSIONS: A second course of CyberKnife radiosurgery seems to be an effective salvage option for brain progression after initial SRS. The total planned target volume shows prediction for OS. Tumor volume of initial SRS may influence selection of the potential population that may benefit from salvage radiosurgery.

Analysis of clinical efficacy of CyberKnife(®) treatment for locally advanced pancreatic cancer.

OBJECTIVE: To evaluate the efficacy and safety of CyberKnife(®) treatment for locally-advanced pancreatic cancer (LAPC). METHODS: The efficacy of CyberKnife(®) treatment was analyzed in 59 LAPC patients treated between October 2006 and September 2014. The median tumor volume was 27.1 mL (13.0-125.145 mL). The median prescribed dose was 45 Gy (35-50 Gy), delivered in 5 fractions (3-8 fractions). The overall survival (OS) rates and freedom from local progression (FFLP) rates were estimated using the Kaplan-Meier survival curve. RESULTS: The median follow-up for all patients was 10.9 months (3.2-48.7 months) and 15.6 months (3.9-37.6 months) among surviving patients. The median OS was 12.5 months, and the 1-year and 2-year survival rates were 53.9% and 35.1%, respectively. The 1-year FFLP rate was 90.8% based on the computed tomography (CT) evaluation. Grade 1-2 acute and late-stage gastrointestinal (GI) reactions were observed in 61% of the patients. One patient experienced grade 3 toxicity. CONCLUSION: Excellent clinical efficacy was obtained after treatment of LAPC using CyberKnife(®), with minimal toxicity.

Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis.

The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways. DOI:http://dx.doi.org/10.7554/eLife.00675.001.

Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Atovaquone derivatives as potent cytotoxic and apoptosis inducing agents.

2-Piperazinyl naphthoquinones (2) and 2-piperidinyl naphthoquinones (3) were designed and synthesized as new cytotoxic and apoptosis inducing agents by utilizing the anti-parasite drug atovaquone as lead compound. Several compounds displayed significantly improved cytotoxic activities against a panel of cancer cell lines than that of atovaquone. These compounds also induced apoptosis through activating pro-apoptotic caspases 9 and 3.

Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.

Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.

Characteristics of airway inflammation and bronchodilator reversibility in COPD: a potential guide to treatment.

STUDY OBJECTIVES: The management of stable patients with COPD depends on the severity of symptoms and airflow limitation. Regarding inflammation, corticosteroids are the only medications that are recommended for use, and only under restricted circumstances. Corticosteroids tend to undertreat or overtreat patients with COPD when only clinical manifestations and the findings of simple spirometry are considered. Accordingly, our aim was to survey the characteristics of airway inflammation in stable COPD patients, and to assess the interrelations among inflammatory cells, inflammatory mediators, bronchodilator reversibility, and pulmonary function. Factors related to airway inflammation and bronchodilator reversibility may be important in the management of stable COPD patients. METHODS: A total of 88 stable patients with smoking-related COPD were recruited into the study. All patients were steroid-free, and had been treated with theophylline, oral beta(2)-agonist agents, anticholinergic agents, and possibly mucolytic agents. Bronchodilator tests and sputum induction were performed to evaluate bronchodilator reversibility, and numbers of inflammatory cells and mediators (eg, interleukin [IL]-8, eotaxin, and regulated on activation, normal T cells expressed and secreted [RANTES]). RESULTS: Thirty-one of 48 patients (64.6%) who had bronchodilator reversibility, and 19 of 40 patients (47.5%) without bronchodilator reversibility had sputum eosinophilia (median, 8.0% and 7.0%, respectively). FEV(1) showed a significant inverse correlation with the number of sputum neutrophils. The correlation coefficient for postbronchodilator FEV(1) vs the percentage of neutrophils in patients with nonreversible COPD was higher than that in those with reversible COPD. The levels of IL-8 were closely associated with the percentage of neutrophils. The sputum concentrations of IL-8 and albumin were significantly higher in patients with nonreversible COPD than in those with reversible COPD. A significant inverse correlation was found between bronchodilator response (ie, DeltaFEV(1) and DeltaFVC) and prebronchodilator FEV(1). CONCLUSIONS: Eosinophilic inflammation may play a substantial role in COPD, while neutrophils and IL-8 may have a great influence on nonreversible obstructive airways. The assessment of airway inflammation and bronchodilator responses can help the selection of specific therapies and the prediction of clinical outcomes for COPD patients.

Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.