Dynamic of centromere associated RNAs and the centromere loading of DNA repair proteins in growing oocytes.

Mammalian centromeres are generally composed of dispersed repeats and the satellites such as α-satellites in human and major/minor satellites in mouse. Transcription of centromeres by RNA polymerase II is evolutionary conserved and critical for kinetochore assembly. In addition, it has been found that the transcribed satellite RNAs can bind DNA repair proteins such as MRE11 and PRKDC, and excessively expressed satellite RNAs could induce genome instability and facilitate tumorigenesis. During the maturation of female oocyte, centromeres are critical for accurate segregation of homologous chromosomes and sister chromatids. However, the dynamics of oocyte centromere transcription and whether it associated with DNA repair proteins are unknown. In this study, we found the transcription of centromeres is active in growing oocytes but it is silenced when oocytes are fully grown. DNA repair proteins like Mlh1, Mre11 and Prkdc are found associated with the minor satellites and this association can be interfered by RNA polymerase II inhibitor α-amanitin. When the growing oocyte is in vitro matured, Mlh1/Mre11/Prkdc foci would release from centromeres to the ooplasm. If the oocytes are treated with Mre11 inhibitor Mirin, the meiosis resumption of growing oocytes with Mre11 foci can be suppressed. These data revealed the dynamic of centromeric transcription in oocytes and its potential association with DNA repair proteins, which provide clues about how oocytes maintain centromere stability and assemble kinetochores.

Distribution of thrombus predicts severe reperfusion pulmonary edema after pulmonary endarterectomy.

OBJECTIVES: Patients underwent pulmonary endarterectomy (PEA) for chronic thromboembolic pulmonary hypertension (CTEPH). This study aimed to investigate the effect of thrombus distribution on the occurrence of severe reperfusion pulmonary edema (RPE) and identify specific parameters for predicting severe RPE. METHODS: Patients with CTEPH who underwent PEA surgery were retrospectively analyzed. The thrombus in pulmonary arteries were evaluated through computed tomography pulmonary angiography. Based on presence of prolonged artificial ventilation, extracorporeal membrane oxygenation required, or perioperative death due to RPE, the patients were divided into the severe RPE and without severe RPE groups. MAIN RESULTS: Among the 77 patients (29 women), 16 (20.8%) patients developed severe RPE. The right major pulmonary artery (RPA) (0.64[0.58, 0.73] vs 0.58[0.49, 0.64]; p = 0.008) and pulmonary artery trunk (PAT) thrombus ratios (0.48[0.44, 0.61] vs 0.42[0.39, 0.50]; p = 0.009) (the PAT ratio is expressed as the sum of the right middle lobe clot burden and right lower lobe clot burden divided by the total clot burden multiplied by 100) of the severe RPE group was significantly higher than that of the without severe RPE group. Receiver operator characteristics curve identified a PAT ratio of 43.4% as the threshold with areas under the curve = 0.71(95%CI 0.582; 0.841) for the development of severe RPE (sensitivity 0.875, specificity 0.541). The logistic regression analysis demonstrated that age, period from symptom onset to PEA, NT-pro BNP, preoperative mPAP, preoperative PVR, RPA ratio, and PAT ratio were associated with the development of severe RPE. Multivariable logistic regression analysis revealed PAT ratio (odds ratio = 10.2; 95% confidence interval 1.87, 55.53, P = 0.007) and period from symptom onset to PEA (OR = 1.01; 95% CI = 1.00-1.02, P = 0.015) as independent risk factors for the development of severe RPE. CONCLUSIONS: The thrombus distribution could be a key factor in the severity of RPE. PAT ratio and medical history could predict the development of severe RPE.

Continuous light exposure influences luteinization and luteal function of ovary in ICR mice.

With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.

Preparation and cellular uptake behaviors of uniform fiber-like micelles with length controllability and high colloidal stability in aqueous media.

Fragmentation/disassembly of fiber-like micelles generated by living crystalline-driven self-assembly (CDSA) is usually encountered in aqueous media, which hinders the applications of micelles. Herein, we report the generation of uniform fiber-like micelles consisting of a π-conjugated oligo(p-phenylenevinylene) core and a cross-linking silica shell with grafted poly(ethylene glycol) (PEG) chains by the combination of living CDSA, silica chemistry and surface grafting-onto strategy. Owing to the presence of crosslinking silica shell and the outmost PEG chains, the resulting micelles exhibit excellent dispersity and colloidal stability in PBS buffer, BSA aqueous solution and upon heating at 80 °C for 2 h without micellar fragmentation/disassembly. The micelles also show negligible cytotoxicity toward both HeLa cervical cancer and HEK239T human embryonic kidney cell lines. Interestingly, micelles with L n of 156 nm show the "stealth" property with no significant uptake by HeLa cells, whereas some certain amounts of micelles with L n of 535 nm can penetrate into HeLa cells, showing length-dependent cellular uptake behaviors. These results provide a route to prepare uniform, colloidally stable fiber-like nanostructures with tunable length and functions derived for biomedical applications.

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes.

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

Cell-sorting centrifugal microfluidic chip with a flow rectifier.

Centrifugal microfluidic chips offer rapid, highly integrable and simultaneous multi-channel microfluidic control without relying on external pressure pumps and pipelines. Current centrifugal microfluidic chips mainly separate particles of differing density based on the sedimentation method. However, in some biological cells, the volume difference is more notable than the density difference. In particular, cancer cells are generally larger than normal cells. The instability of particle velocity caused by the non-steady flow of the fluid in the centrifugal microfluidic chip leads to low separation purity of particles of different sizes. Thus, we propose herein a centrifugal microfluidic chip with a flow rectifier that transforms the centrifugal non-steady flow into locally steady flow with continuous flow. This chip resolves the problems caused by particle sedimentation in the sample chamber and non-steady flow and greatly improves the recovery ratio and separation purity of target particles. Therefore, it can be used to separate particles of differing size. The experimental results show that the chip can separate an equal-volume mixture of 25 µm and 12 µm polystyrene particles diluted 50 times with a ratio of 1 : 6 and obtain a recovery ratio and separation purity better than 95% for the 25 µm particles. In addition, rare tumour cells are separated from high-concentration white blood cells (ratio 1 : 25) with a recovery ratio of 90.4% ± 2.4% and separation purity of 83.0% ± 3.8%. In conclusion, this chip is promising for sorting of various biological cells and has significant potential for use in biomedical and clinical applications.

Comprehensive analysis of AGPase genes uncovers their potential roles in starch biosynthesis in lotus seed.

BACKGROUND: Starch in the lotus seed contains a high proportion of amylose, which endows lotus seed a promising property in the development of hypoglycemic and low-glycemic index functional food. Currently, improving starch content is one of the major goals for seed-lotus breeding. ADP-glucose pyrophosphorylase (AGPase) plays an essential role in regulating starch biosynthesis in plants, but little is known about its characterization in lotus. RESULTS: We describe the nutritional compositions of lotus seed among 30 varieties with starch as a major component. Comparative transcriptome analysis showed that AGPase genes were differentially expressed in two varieties (CA and JX) with significant different starch content. Seven putative AGPase genes were identified in the lotus genome (Nelumbo nucifera Gaertn.), which could be grouped into two subfamilies. Selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of AGPase genes. Expression analysis revealed that lotus AGPase genes have varying expression patterns, with NnAGPL2a and NnAGPS1a as the most predominantly expressed, especially in seed and rhizome. NnAGPL2a and NnAGPS1a were co-expressed with a number of starch and sucrose metabolism pathway related genes, and their expressions were accompanied by increased AGPase activity and starch content in lotus seed. CONCLUSIONS: Seven AGPase genes were characterized in lotus, with NnAGPL2a and NnAGPS1a, as the key genes involved in starch biosynthesis in lotus seed. These results considerably extend our understanding on lotus AGPase genes and provide theoretical basis for breeding new lotus varieties with high-starch content.

Tributyltin oxide exposure impairs mouse oocyte maturation and its possible mechanisms.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Removal of mouse ovary fat pad affects sex hormones, folliculogenesis and fertility.

As a fat storage organ, adipose tissue is distributed widely all over the body and is important for energy supply, body temperature maintenance, organ protection, immune regulation and so on. In humans, both underweight and overweight women find it hard to become pregnant, which suggests that appropriate fat storage can guarantee the female reproductive capacity. In fact, a large mass of adipose tissue distributes around the reproductive system both in the male and female. However, the functions of ovary fat pad (the nearest adipose tissue to ovary) are not known. In our study, we found that the ovary fat pad-removed female mice showed decreased fertility and less ovulated mature eggs. We further identified that only a small proportion of follicles developed to antral follicle, and many follicles were blocked at the secondary follicle stage. The overall secretion levels of estrogen and FSH were lower in the whole estrus cycle (especially at proestrus); however, the LH level was higher in ovary fat pad-removed mice than that in control groups. Moreover, the estrus cycle of ovary fat pad-removed mice showed significant disorder. Besides, the expression of FSH receptor decreased, but the LH receptor increased in ovary fat pad-removed mice. These results suggest that ovary fat pad is important for mouse reproduction.

Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus).

Dairy goats are one of the most utilized domesticated animals in China. Here, we selected extreme populations based on differential fecundity in two Laoshan dairy goat populations. Utilizing deep sequencing we have generated 68.7 and 57.8 giga base of sequencing data, and identified 12,458,711 and 12,423,128 SNPs in the low fecundity and high fecundity groups, respectively. Following selective sweep analyses, a number of loci and candidate genes in the two populations were scanned independently. The reproduction related genes CCNB2, AR, ADCY1, DNMT3B, SMAD2, AMHR2, ERBB2, FGFR1, MAP3K12 and THEM4 were specifically selected in the high fecundity group whereas KDM6A, TENM1, SWI5 and CYM were specifically selected in the low fecundity group. A sub-set of genes including SYCP2, SOX5 and POU3F4 were localized both in the high and low fecundity selection windows, suggesting that these particular genes experienced strong selection with lower genetic diversity. From the genome data, the rare nonsense mutations may not contribute to fecundity, whereas nonsynonymous SNPs likely play a predominant role. The nonsynonymous exonic SNPs in SETDB2 and CDH26 which were co-localized in the selected region may take part in fecundity traits. These observations bring us a new insights into the genetic variation influencing fecundity traits within dairy goats.

N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis.

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of the oocyte is silenced at the fully grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in the oocyte. Recently, RNA methylation has been found to exist in various systems. In this study, we sequenced the N6-methyladenosine (m6A) modified mRNAs in fully grown germinal vesicle-stage and metaphase II-stage oocytes. As a result, we identified 4207 mRNAs with m6A peaks in germinal vesicle-stage or metaphase II-stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with protein phosphorylation. Our results suggest that oocyte mRNA methylation can regulate cellular translation and cell division during oocyte meiotic maturation and early embryo development.

[The prognostic value of serum procalcitonin on severity of illness in non-sepsis critically ill patients].

OBJECTIVE: To evaluate the correlation between serum procalcitonin (PCT) level and severity of diseases caused by different kinds of stress factors, and to identify the prognostic value of PCT on the prognosis in non-sepsis critically ill patients. METHODS: A retrospective case control study was conducted. The clinical data of non-sepsis critically ill patients with age of ≥ 18 years admitted to surgery intensive care unit (ICU) of China-Japan Friendship Hospital from August 2013 to December 2015 and stayed for more than 3 days were enrolled. The PCT level in the first 24 hours, acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score and 28-day mortality were recorded. Patients were divided into different groups by the original injury, including trauma stress group, stroke stress group and non-infection inflammation stress group. According to PCT level, patients were divided into PCT normal group, low level group, medium level group and high level group. Furthermore, patients were divided into survival group and non-survival group according to 28-day prognosis. The clinical data of patients were compared among the groups, and the correlations among different markers were analyzed with Pearson or Spearman correlation analysis. The predictive value of PCT on prognosis of non-sepsis critically ill patients was evaluated with receiver operating characteristic curve (ROC). RESULTS: Ninety-four non-sepsis critical ill patients were enrolled, with 28 patients in trauma stress group, 30 in stroke stress group, and 36 in non-infection inflammation stress group, as well as 32 patients in PCT normal group, 18 in low level group, 18 in medium level group, and 26 in high level group. Of them, 78 survivors and 16 non-survivors were found. (1) The PCT level of non-sepsis critically ill patients was significantly positively correlated with APACHE II score and SOFA score (r1 = 0.688, r2 = 0.771, both P = 0.000). (2) The PCT level in trauma stress group was significantly higher than that in stroke stress group and non-infection inflammation stress group [µg/L: 4.43 (0.86, 11.72 ) vs. 0.28 (0.16, 5.85), 2.39 (0.13, 4.11), both P < 0.01]. APACHE II score (13.9±7.5, 13.9±7.0 vs. 9.4±4.4), SOFA score [7.0 (4.0, 9.0), 5.0 (3.0, 8.0) vs. 4.0 (2.0, 6.0)], and 28-day mortality [21.4% (6/28), 33.3% (10/30) vs. 0 (0/36)] in trauma stress group and stroke stress group were significantly higher than those of non-infection inflammation stress group (all P < 0.05). The abnormal rate of PCT in trauma stress group was significantly higher than that of stroke stress group and non-infection inflammation stress group [100.0% (28/28) vs. 33.3% (10/30), 66.7% (24/36), both P < 0.01]. (3) Non-survivors had significantly higher PCT level [µg/L: 6.02 (4.43, 18.34) vs. 0.76 (0.16, 4.11)], APACHE II score (22.5±3.8 vs. 10.1±5.1) and SOFA score [9.0 (7.0, 11.0) vs. 4.0 (2.0, 8.0)] as compared with those of survivors (all P < 0.01). (4) APACHE II score (7.8±2.8, 9.3±4.3, 13.7±6.2, 18.7±5.8, F = 22.495, P = 0.000), SOFA score [3.0 (1.2, 4.8), 4.0 (3.5, 4.5), 6.0 (3.5, 8.0), 10.0 (8.8, 12.0), Z = 51.040, P = 0.000], and 28-day mortality [0 (0/32), 11.1% (2/18), 22.2% (4/18), 38.5% (10/26), χ (2) = 15.816, P = 0.001] were gradually increased as PCT level elevated. (5) The area under ROC curve (AUC) of PCT for evaluating prognosis of non-sepsis critically ill patients was 0.799 [95% confidence interval (95%CI) = 0.709-0.889, P = 0.000], when the cut-off value was 4.2 µg/L, the sensitivity was 87.5%, and the specificity was 77.6%. CONCLUSIONS: Serum PCT level was positively correlated with severity of illness in non-sepsis critically ill patients, which had predicted value on prognosis. Trauma stress can lead to higher PCT level than stroke stress and non-infection inflammation stress can.

On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance.

Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ∼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.

Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks.

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

The influence of N-acetyl-l-cysteine on damage of porcine oocyte exposed to zearalenone in vitro.

Zearalenone (ZEA), one of the mycotoxins produced by Fusarium fungi, impacts porcine reproduction by interfering with the estrogen signaling pathway. Previous studies have shown that ZEA inhibits porcine oocyte maturation through the formation of aberrant spindle. To explore the effect of ZEA on porcine oocyte meiotic maturation, the extent of both nuclear and cytoplasmic maturation was examined in this study. Compared with control group, presence of ZEA (3 µM) during oocyte maturation, significantly inhibited the polar body extrusions from 71% to 51%, and significantly increased intracellular reactive oxygen species (ROS) level (12.01 vs. 5.89). Intracellular glutathione (GSH) content in ZEA treatment group was lower than in the control group (1.08 pmol/oocyte vs. 0.18 pmol/oocyte), and cortical granules of cortical area distributed oocytes were reduced (88% vs. 62%). ZEA decreases cumulus expansion in both morphology and mRNA level (HAS2, PTX3, TNFAIP6 and CX43). Addition of N-acetyl-l-cysteine (NAC) to the oocyte maturation media reversed the ZEA-induced inhibition of polar body extrusion (from 69% to 81%), up-regulated ROS (from 7.9 to 6.5), down-regulated GSH content (from 0.16 to 0.82 pmol/oocyte) and recovered cumulus cells expansion in morphology and mRNA level. It is concluded that ZEA affects both oocyte nucleus and cytoplasmic maturation during in vitro maturation, and NAC can reverse these damages to some extent.

Selective targeting of alveolar type II respiratory epithelial cells by anti-surfactant protein-C antibody-conjugated lipoplexes.

Alveolar type II (ATII) respiratory epithelial cells are essential to normal lung function. They may be also central to the pathogenesis of diseases such as acute lung injury, pulmonary fibrosis, and pulmonary adenocarcinoma. Hence, ATII cells are important therapeutic targets. However, effective ATII cell-specific drug delivery in vivo requires carriers of an appropriate size, which can cross the hydrophobic alveolar surfactant film and polar aqueous layer overlying ATII cells, and be taken up without inducing ATII cell dysfunction, pulmonary inflammation, lung damage, or excessive systemic spread and side-effects. We have developed lipoplexes as a versatile nanoparticle carrier system for drug/RNA delivery. To optimize their pulmonary localization and ATII cell specificity, lipoplexes were conjugated to an antibody directed against the ATII cell-specific antigen surfactant protein-C (SP-C) then administered to C57BL/6 mice via the nares. Intranasally-administered, anti-SP-C-conjugated lipoplexes targeted mouse ATII cells with >70% specificity in vivo, were retained within ATII cells for at least 48h, and did not accumulate at significant levels in other lung cell types or viscera. 48h after treatment with anti-SP-C-conjugated lipoplexes containing the test microRNA miR-486, expression of mature miR-486 was approximately 4-fold higher in ATII cells than whole lung by qRT-PCR, and was undetectable in other viscera. Lipoplexes induced no weight loss, hypoxemia, lung dysfunction, pulmonary edema, or pulmonary inflammation over a 6-day period. These findings indicate that ATII cell-targeted lipoplexes exhibit all the desired characteristics of an effective drug delivery system for the treatment of pulmonary diseases that result primarily from ATII cell dysfunction.

Transgenerational inheritance of ovarian development deficiency induced by maternal diethylhexyl phthalate exposure.

Diethylhexyl phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. It disrupts the physiological functions of endogenous hormones and induces abnormal development of mammals. The objectives of the present study were to evaluate the effects of DEHP exposure on ovarian development of pregnant mice and whether the effects are inheritable. We found that the synthesis of oestradiol in pregnant mice after DEHP exposure was significantly decreased, and that the first meiotic progression of female fetal germ cells was delayed. Furthermore, the DNA methylation level of Stra8 was increased and the expression levels of Stra8 were significantly decreased. An accelerated rate of follicle recruitment in F1 mice was responsible for the depletion of the primordial-follicle pool. Maternal DEHP exposure also significantly accelerated the recruitment of primordial follicles in F2 mice. In conclusion, our results indicated that maternal DEHP exposure induced ovarian development deficiency, which was transgenerational in mice.

Targeted delivery of tumor suppressor microRNA-1 by transferrin-conjugated lipopolyplex nanoparticles to patient-derived glioblastoma stem cells.

OBJECTIVE: Among heterogeneous glioblastoma multiforme (GBM) cells, glioblastoma stem cells (GSCs) is a subpopulation having a critical role in tumor initiation and therapy resistance. Thus targeting GSCs would be an essential step to completely eradicate this lethal disease. MicroRNA-1 (miR-1) expression is deregulated in GBM patients and restoration of miR-1 by viral-vector in GBM cells has been demonstrated to inhibit tumor initiation and attenuate cell migration. Here, we show that a transferrin-targeting non-invasive nanoparticle delivery system (Tf-NP) can efficiently deliver miR-1 to GBM patient-derived GSC-enriched sphere cultures (GBM spheres). METHODS: Delivery efficiency of the transferrin- targeting non-invasive nanoparticle was investigated by flow cytometry and further confirmed by confocal microscopy. The levels of miR-1 and its target molecules in GBM spheres were measured by qRT-PCR and immunoblotting. Migration capacity of Tf-NP-miR-1 treated GBM spheres were evaluated by transwell migration assay. RESULTS: Tf-NPmiR- 1 treatment resulted in an over 200-fold increase of mature miR-1 compared to free miR-1 and Tf-NP-miR negative control (Tf-NP-miR-NC). Transferrin-mediated NP delivery resulted in a 3-fold higher delivery efficiency compared to NP without transferrin modification. Tf-NP-miR-1 treatment on GBM spheres significantly inhibited migration of GBM spheres by 30-50% with associated decline of MET and EGFR expression. Our data supported that Tf-NP could be used as an efficient and effective delivery system which has high potential to benefit the development of miR-based therapeutics for GBM treatment.

Detection of extracellular RNAs in cancer and viral infection via tethered cationic lipoplex nanoparticles containing molecular beacons.

Noninvasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick isolation kit were first used to isolate exosomes from the cell culture medium and human serum, respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here we demonstrate this concept using lentivirus and serum from lung cancer patients.