Helical micro-swimmer: hierarchical tail design and propulsive motility.

Helical micro-swimmers have markedly extended the reach of human beings in numerous fields, ranging from in vitro tasks in lab-on-a-chip to in vivo applications for minimally invasive medicine. The previous studies on the propulsive motility optimization of the micro-swimmers mainly focused on the distinct actuation principles (e.g., chemically powered, magnetic- or ultrasound energy-driven) and paid little attention to the structural design of these swimming machines themselves. The improvements of the structures can assist the externally powered motors in providing propulsion in a tiny scale and satisfy the agile locomotion demands. This paper presents the design, mechanics modeling and available experiments of a novel type of hierarchical helical swimming robot that significantly enhances the motility of the helix-based swimmers. Validated by the resistive force theory, our numerical model can well analyze the mechanical properties with a variety of geometric parameters. The motion performance of the hierarchical and conventional helical structures in low Reynolds regimes is presented, highlighting the advantages of hierarchical swimmers over the existing typical swimmers. In addition, the stability and resilience of the hierarchical swimmers can be maintained at a decent level. Moreover, the variable forward velocity resulting from the combined hierarchical structures is investigated here, which can thereby serve as a reliable design strategy. The proposed hierarchical helical design enables enticing opportunities for various device systems of medical robots and bio-integrated electronics.

Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting.

Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies.

Optimizing electrical activation of porcine oocytes by adjusting pre- and post-activation mannitol exposure times.

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre- and post-activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre- or post-electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3 min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2 min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3 min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1 min pre- and 3 min post-activation showed significantly higher blastocyst development than 0 min pre- and 0 min post-activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0 min pre- and 0 min post-activation. In conclusion, exposure of oocytes to mannitol for 1 min pre- and 3 min post-activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1 min pre- and 3 min post-activation.

Effect of roscovitine-treated donor cells on development of porcine cloned embryos.

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 µM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p < 0.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.

Influence of ovulation status, seasonality and embryo transfer method on development of cloned porcine embryos.

To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of somatic cell nuclear transfer. In pre-ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively. In post-ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When ET was performed during winter (December-February), spring (March-May), summer (June-August) and autumn (September-November), the PRs were 13.4%, 37.3%, 24.6% and 51.0%, while DRs were 0%, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets were born in winter group, which might be because of the effect of low temperature during ET. To overcome the low PR in winter group, 0.25 ml straws were used for ET to minimize exposure time of embryos to ambient temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter-loading group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In addition, alternative method to reduce cold shock during ET in winter is necessary.

Electrical activation induces reactive oxygen species in porcine embryos.

The objectives were to determine factors affecting generation of reactive oxygen species (ROS) in porcine embryos after electrical activation of oocytes, and the effects of an antioxidant and chemical agent on ROS generation. Greater ROS were induced by electrical activation compared to IVF (mean+/-S.E.M., 14.6+/-0.8 vs. 9.2+/-0.4, P<0.05). Furthermore, ROS generation in embryos after electrical activation was significantly increased by higher intensity and longer duration electrical pulses and by higher exogenous Ca(2+) concentrations. Cleavage rate and blastocyst formation rate were not directly related to the level of ROS. Supplementation of the IVC medium with 0.5mM glutathione (GSH) reduced ROS (9.2+/-0.4 vs. 14.7+/-0.9, P<0.05). Treatment with the chemical activation agent, 6-dimethylaminopurine (6-DMAP) for 3h did not induce further ROS generation in combination with electrical activation, but it improved blastocyst formation rate (53.8+/-1.1 vs. 23.7+/-3.5, P<0.05). We concluded that generation of ROS should be considered for optimizing electrical activation and that supplementing an antioxidant or combining electrical and chemical activation induced lower ROS generation in electrically activated porcine embryos.

[Factors related to the choice of clinic between Chinese traditional medicine and Western medicine].

The study applied Andersen's health-service utilization model to analyze the basic demographic, enabling and need factors related to the choice of traditional Chinese medicine clinic or modern Western medicine clinic by single-method-treatment (i.e. traditional Chinese medicine or modern Western medicine only) patients. During the period from August 1989 to October 1989, systemic sampling was done and a structured questionnaire survey was carried out among patients from the Out-patient Departments of 13 teaching hospitals accepting reimbursement by Labor Medical Insurance in Taiwan. The total number of valid respondents was 579: 378 (65.3%) were visiting modern Western medicine clinics and 201 (34.7%), traditional Chinese medicine clinics. There were 339 (58.6%) males and 240 (41.4%) females, aged from 15 to 85 years old, with a mean of 40.7 years. Under univariate analysis, the significant variables (p < 0.05) related to visiting the two types of clinics were: nativity, religious belief, career, general health condition, severity of illness of this episode, types of disorder as neuromusculoskeletal, digestive, circulatory, endocrine-metabolic and sense-and-skin. By logistic regression analysis, the significant variables (p < 0.05) relating to visiting two types of clinics were religion, career, and two kinds of disorders. Folk-religion believers, farmers and businessmen favored traditional Chinese medicine; and patients who suffered from musculoskeletal, sense organs or skin disorders were also likely to visit traditional Chinese medicine clinics.

Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs.

To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P < 0.05) and not different between nulliparous dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P < 0.05) and in nulliparous dogs than multiparous dogs (3.0 vs. 1.7%, P < 0.05). Even though SY showed increased pregnancy and delivery rate (20.0% and 3.0%) compared to AA (15.0% and 2.0%) and RA (0.0% and 0.0%), there was no significant difference. In conclusion, we recommend non-operated dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.

Birth of viable puppies derived from breeding cloned female dogs with a cloned male.

Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo-matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.

Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts.

The aim of this study was to investigate whether roscovitine (the cyclin-dependent kinase 2 inhibitor) effectively induces synchronization of the donor cell cycle at G0/G1 and to examine the effect of donor cell cycle synchronization protocols on canine somatic cell nucleus transfer. Canine fibroblasts were obtained from skin biopsy cultures taken from a 7-yr-old retriever. The donor cell cycle was synchronized either by culturing cells to reach confluency or by treating cells with 15 microg/mL roscovitine for 24h. Cell cycle stages and apoptosis were analyzed by flow cytometry. After synchronization of the donor cell cycle, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into 18 naturally estrus-synchronized surrogates. There was no significant difference in cell cycle synchronization and apoptosis rates between the confluent and roscovitine groups. After transfer of reconstructed embryos, pregnancy was detected in three of nine surrogates that received cloned embryos reconstructed with roscovitine-treated cells, whereas only one of nine surrogates was pregnant after transfer of cloned embryos reconstructed with confluent cells. One pregnant female from the confluent cell group delivered one live and one dead pup, but the live one died within 5 days after birth. Three pregnant females from the roscovitine-treated cell group delivered eight live pups and one dead pup, and one of eight live pups died within 6 days after birth. In conclusion, the current results demonstrated that reconstructing embryos with roscovitine-treated cells resulted in increased efficiency of canine somatic cell nucleus transfer.

[Factors related to the choice of different hospitals providing Chinese traditional medicine].

A structured questionnaire was answered by patients who used the Outpatient Departments of two types of Chinese traditional medicine clinics: clinics associated with western-style teaching hospitals, and regular Chinese medicine clinics which were area-matched with, but not located in the teaching hospitals. The study applied the Andersen behavior model and discussed the factors related to the choice of one type or the other clinic by one-method-treatment (i.e. traditional only, not mixed with western, medicine) patients. The total number of valid questionnaires was 362. Under the analysis of logistic regression, the variables which were significantly related to the choice of the types of the hospitals were age, insurance status, first visit or follow-up, general health condition, severity of illness, musculoskeletal and neurological disorders. The older the patients, the more likely they were to visit teaching hospitals. First-visit or uninsured patients also favored teaching hospitals. Patients with perceived stable conditions or more serious illness were likely to use teaching hospitals. Patients with musculoskeletal or neurological disorders favored regular clinics.

Factors related to the choice between traditional Chinese medicine and modern Western medicine among patients with two-method treatment.

BACKGROUND: The aim of this paper is to investigate the different factors influencing patients' choice of traditional Chinese medicine or modern western medicine, applying Andersen's health-service utilization model to analyze the basic demographic, enabling and need factors related to the choice of clinics by patients who use two-method treatment (i.e. both Chinese medicine and western medicine). METHODS: Systemic sampling was done and a structured questionnaire survey was carried out among patients from the outpatient departments of 13 teaching hospitals accepting reimbursement by Labor Medical Insurance in Taiwan. RESULTS: The total number of valid respondents was 549. Of them 181 (33%) were visiting western medicine clinics and 368 (67%) visiting Chinese medicine clinics. There were 279 (51%) males and 270 (49%) females, whose age distribution was in the range from 16 to 87 years old, with a mean of 42.7 years. Under univariate analysis, the significant variables (p < 0.05) related to visiting the two types of clinics were: applicability of medical insurance, bed rest from discomfort in recent years, the amount of discomfort from this disease episode, respiratory disease, circulatory disease, endocrine or metabolic disease, and sense organ and skin disorders. By logistic regression analysis, the significant variables (p < 0.05) related to visiting the two types of clinics were religion, bed rest during the past year, discomfort associated with the episode, respiratory disease, and endocrine or metabolic diseases. CONCLUSIONS: Patients with folk-religion beliefs or respiratory diseases favored Chinese medicine; patients with illness requiring bed rest in the past year, who experienced discomfort in this episode, or who suffered from endocrine or metabolic diseases were likely to visit western medicine clinics.