Dauer larva-derived extracellular vesicles extend the life of Caenorhabditis elegans.

There is growing evidence that extracellular vesicles (EVs) play a functional role in tissue repair and anti-aging by transferring the contents of donor cells to recipient cells. We hypothesized that Dauer (C. elegans), known as "ageless" nematodes, can also secrete extracellular vesicles and influence the lifespan of C. elegans. Here, we isolated EVs of dauer larvae (dauer EVs). Dauer EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA), and Western blot analysis. Wild-type C. elegans were fed in the presence or absence of dauer EVs and tested for a range of phenotypes, including longevity, mobility and reproductive capacity. Results showed that dauer EVs increased the average lifespan of nematodes by 15.74%, improved mobility, slowed age-related pigmentation as well as body length, and reduced the accumulation of reactive oxygen species and lipids, while not impairing nematode reproductive capacity. These findings suggest that dauer EVs can extend the lifespan of C. elegans as well as the healthy lifespan by reducing ROS accumulation, with potential anti-aging capacity.

Partial ablation of the pancreas of Sprague Dawley® rats by focused ultrasound reveals no significant adverse effects on glycometabolism function.

To investigate the safety of focused ultrasound (FUS) partial ablation on the pancreas of Sprague Dawley® (SD) rats by histopathological examination of the outcome and investigation of glycometabolism function changes after local ablation. A total of 135 healthy SD rats were randomly divided into three groups (n=45 of each): FUS ½ group, FUS » group, and control group. Levels of serum amylase was measured using the enzyme dynamics method, fasting blood glucose was measured by the glucose oxidase-peroxidase method, fasting serum insulin was measured by direct chemiluminescence assay, and an ELISA was used to measure fasting serum glucagon immediately after treatment, and at 2h, 3days, 1, 2, 3 and 4weeks, 3 and 6months after FUS ablation. Pancreatic tissue was stained with hematoxylin and eosin and the pathology of the ablation area was examined under an optical microscope; additionally, the expression of insulin and glucagon was investigated by immunohistochemistry. Compared with the control group, serum amylase and fasting blood glucose levels in the ablation groups rose significantly immediately after operation; fasting blood glucose, serum amylase, serum insulin and glucagon levels in the ablation groups were significantly different at 2h after treatment, and serum amylase levels in the ablation groups remained significantly different on day 3. Histological findings showed that the coagulation necrosis area gradually shrank, with formation of new blood vessels observed at week 3, and new ducts observable in the ablation area at the 3rd month after FUS ablation, but no formation of islets was observed. Expression of insulin and glucagon in the ablation groups were significantly higher than in the control group at 2h after FUS ablation. There were no significant adverse effects on the glycometabolic function of SD rats after FUS ablation, and the influence of FUS treatment on pancreatic functions were minimal.

Lactation-Related MicroRNA Expression in Microvesicles of Human Umbilical Cord Blood.

BACKGROUND The complex process by which lactation is initiated upon neonate delivery remains incompletely understood. Microvesicles (MVs) can transmit microRNAs (miRNAs) into recipient cells to influence cell function, and recent studies have identified miRNAs essential for mammary gland development and lactation. This study aimed to investigate the expression of lactation-related miRNAs in MVs isolated from human umbilical cord blood immediately after delivery. MATERIAL AND METHODS Umbilical cord blood samples were collected from 70 healthy pregnant women, and MVs were isolated through differential centrifugation and characterized by transmission electron microscopy, Western blotting, and nanoparticle tracking analysis. Lactation-related miRNAs were screened using bioinformatics tools for miRNA target prediction, gene ontology, and signaling pathway analyses. miRNA PCR arrays were used for miRNA expression analysis, and the results were validated by real-time PCR. Upon exposure of HBL-100 human mammary epithelial cells to MVs, MV uptake was examined by fluorescence confocal microscopy and b-casein secretion was detected by ELISA. RESULTS Spherical MVs extracted from umbilical cord blood expressed CD63 and had an average diameter of 167.0±77.1 nm. We profiled 337 miRNAs in human umbilical cord blood MVs and found that 85 were related to lactation by bioinformatics analysis. The 25 most differentially expressed lactation-related miRNAs were validated by real-time PCR. MV uptake by HBL-100 cells was after 4 h in culture, and significantly increased secretion of ß-casein was observed after 96 h from cells exposed to MVs (P<0.05). CONCLUSIONS Umbilical cord blood MVs contain many lactation-related miRNAs and can induce ß-casein production by HBL-100 cells in vitro. Thus, umbilical cord blood MVs may mediate secretion of ß-casein through miRNAs, thereby playing an important role in fetal-maternal crosstalk.

RNAi-mediated MMP-9 silencing inhibits mouse melanoma cell invasion and migration in vitro and in vivo.

MMP-9 participates in tumour growth, invasion, metastasis and vascularisation. Thus, inhibition of MMP-9 may be involved in the process of tumourigenesis. We have investigated the effect of RNAi-mediated MMP-9 silencing on inhibiting invasion and migration of mouse melanoma cell B16. A specific and optimised siRNA vector was used to target MMP-9 mRNA synthesis in B16 cells. Changes of invasion and migration capability of B16 cell were examined after transfection at different time, and a footpad tumour model was performed to measure the effect of MMP-9 siRNA on melanoma tumourigenesis in vivo. In vitro, down-regulation of MMP-9 expression significantly inhibited B16 cell invasion and migration. In vivo, intratumoural injection of plasmid DNA expressing MMP-9 siRNA every 3 days for three times remarkably inhibited melanoma growth and also suppressed tumour metastasis. The results indicate that RNA-mediated targeting of MMP-9 may have promising applications for the treatment of melanoma.

Preliminary ex vivo feasibility study on targeted cell surgery by high intensity focused ultrasound (HIFU).

High intensity focused ultrasound (HIFU) has become a new noninvasive surgical modality in medicine. A portion of tissue seated inside a patient's body may experience coagulative necrosis after a few seconds of insonification by high intensity focused ultrasound (US) generated by an extracorporeal focusing US transducer. The region of tissue affected by coagulative necrosis (CN) usually has an ellipsoidal shape when the thermal effect due to US absorption plays the dominant role. Its long and short axes are parallel and perpendicular to the US propagation direction respectively. It was shown by numerical computations using a nonlinear Gaussian beams model to describe the sound field in a focal zone and ex vivo experiments that the dimension of the short and long axes of the tissue which experiences CN can be as small as 50µm and 250µm respectively after one second exposure of US pulse (the spatial and pulse average acoustic power is on the order of tens of Watts and the local acoustic spatial and temporal pulse averaged intensity is on the order of 3×10(4)W/cm(2)) generated by a 1.6MHz HIFU transducer of 12cm diameter and 11cm geometric focal length (f-number=0.92). The concept of thermal dose of cumulative equivalent minutes was used to describe the possible tissue coagulative necrosis generated by HIFU. The numbers of cells which suffered CN were estimated to be on the order of 40. This result suggests that HIFU is able to interact with tens of cells at/near its focal zone while keeping the neighboring cells minimally affected, and thus the targeted cell surgery may be achievable.

Mouse trophoblastic cells exhibit a dominant invasiveness phenotype over cancer cells.

Invasion process occurs both in mammalian embryo implantation during development and malignant cancer cell metastasis. We investigated the interactions between trophoblasts and metastatic cancer cells and found the phenomenon that mouse trophoblastic cells invaded the monolayer of malignant cancer cells in vitro and appeared the general trait of invasiveness to more than 30 types of malignant cancer cell lines which were derived from different histological origins and with different invasive or metastatic potential. We further investigated the cellular and molecular changes in the process of mouse trophoblastic cells invading human ovarian cancer HO-8910 cells. The results show that the invasion of trophoblastic cells lead HO-8910 cells near mouse embryo to apoptosis, and expression of cell-cycle-related protein cyclinD1 and Ki-67 mRNA were steadily remained both in mouse blastocysts and human ovarian cancer HO-8910 cells, which in part explain the proliferation activities of these cells. Our study also shows that expression of some proteins including MMP-9, FAK and Integrinαvß3 was changeable in trophoblastic cells and HO-8910 cells in the process of blastocyst invasion, which suggested temporal expression of these molecules may involved in the invasive behavior of trophoblasts cells to cancer cells.

Effect of mid-late mouse fetus' microenvironment on the growth of tumor cells after intrauterine transplantation.

Successful intrauterine transplantation (IUT) of stem cells for treatment of fetal defects in some animal models of human diseases has prompted us to study the mechanisms of transplantation, immunological tolerance and embryonic environment. The objective of this study was to determine whether intrauterine transplantation of tumor cells would affect the survival and growth of the tumor cells themselves as well as fetus development. A total of 2 x 10(6) H(22) cells or S(180) cells were transplanted into the amniotic or abdominal cavity of NIH mice on D9-D12 or D13-D18 of gestation. The adult and newborn NIH mice which were inoculated with the same number of H(22) cells and S(180) cells by intraperitoneal injection were used as positive controls for the cancer bearing control group while undisrupted fetuses of the same gestation were used as negative controls (i.e. for the normal development) group. The development of fetuses transplanted with tumor cells in utero was monitored by several developmental indices, and the tumor growth of them were observed by some distinctive bearing cancer index. The H(22) transplanted group was further assessed for minimal cancer bearing by detection of alpha-fetoprotein (AFP) using radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). In addition, tumor burden and the development of the F1 generation of the mice by IUT were also investigated. Protein kinase C (PKC) and proliferating cell nuclear antigen (PCNA) of GFP-expressing H(22) cells transplanted in the uterus were analyzed under laser confocal microscopy. There was no significant difference in the developmental indices between the experimental and control groups. HE staining of the major organs, including liver, kidney, and lung, showed that these organs properly developed. No tumor ascites were found in those delivered mice after intrauterine transplantation with H(22) cells and S(180) cells. Furthermore, as minimal bearing cancer index for H(22) cells, AFP expression analyzed by RIA and RT-PCR indicated that no tumor cells were detected in the experimental groups. The F1 progenies developed normally without any signs of tumor development. Fluorescence analysis revealed that expression of PKC and PCNA was markedly reduced in the H(22) cells after injection for 24, 48, and 72 h. Our study showed that the tumor cells did not grow in the mice by intrauterine transplantation, whereas transplantation of the same number of tumor cells resulted in obvious ascites tumor in the adult and newborn mice. Furthermore, the differentiation and proliferation of H(22) cells changed dramatically after injection. Our results suggest that, while the embryonic transplantation of tumor cells does not affect fetal development, the survival and growth of implanted tumor cells may be significantly inhibited in the embryonic microenvironment.