The conventional method for producing embryonic stem (ES) cell-derived knockout or transgenic mice involves injection of ES cells into normal, diploid blastocysts followed by several rounds of breeding of resultant chimeras and thus is a timeconsuming and inefficient procedure. F0 ES cell pups can also be derived directly from tetraploid embryo complementation, which requires fusion of two-cell embryos. Recently, F0 ES cell pups have been produced by injection of ES cells into eight-cell embryos using a laser-assisted micromanipulation system. We report a simple method for producing F0 ES cell germlinecompetent mice by piezo injection of ES cells into four-or eight-cell embryos. The efficiency of producing live, transgenic mice by this method is higher than that with the tetraploid blastocyst complementation method. This efficient and economical technique for directly producing F0 ES cell offspring can be applicable in many laboratories for creating genetically manipulated mice using ES cell technology and also for stringent testing of the developmental potency of new ES cell or other types of pluripotent stem cell lines.

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Authentic bovine embryonic stem (ES) cell lines have not been established despite progress made for more than two decades. Isolation and culture of primary ES cell colonies are the first critical step towards establishment of stable ES cell lines. Here we report a novel method designated as "Separate and Seed" that contributes remarkably to efficient derivation of bovine primary ES-like cell colonies from blastocysts. These primary cultured bovine ES-like cells exhibit morphology typical of ES cells and express pluripotent molecular markers including Oct4, Nanog and alkaline phosphatase. Interestingly, bovine primary ES-like cell colonies distinctively express both stage-specific embryonic antigens 1 and 4 (SSEA1 and SSEA4), unlike mouse and human ES cells. These pluripotent markers may be used for characterization of authentic bovine ES cell lines in later studies. In contrast, whole embryos or inner cell mass (ICM) used for primary culture by conventional methods fails to produce primary bovine ES cell colonies that express all pluripotent stem cell markers shown above. Furthermore, bFGF improves growth and maintained undifferentiated state of bovine ES-like cells for several passages, whereas LIF and ERK inhibitor PD98059 known to promote pluripotency of mouse ES cells are unable to sustain bovine ES-like cells. Although continued efforts are required for improving long-term culture of bovine ES cells, this novel "Separate and Seed" method provides an initial effective step that may eventually lead to derivation of authentic bovine ES cell lines.

Smoking is one of the most common addictions in the world. Nicotine inhalation could increase the risk of cardiorespiratory diseases. However, the solution that improved postoperative analgesia for highly nicotine-dependent patients undergoing thoracic surgery has not been specifically addressed.This CONSORT-prospective, randomized, double-blinded, controlled trial investigated the efficacy of combination of dexmedetomidine and sufentanil for highly nicotine (Fagerstrom test of nicotine dependence ≥6)-dependent patients after thoracic surgery.One hundred seventy-four male patients who underwent thoracic surgery were screened between February 2014 and November 2014, and a total of forty-nine were excluded. One hundred thirty-two highly nicotine-dependent male patients who underwent thoracic surgery and received postoperative patient-controlled intravenous analgesia were divided into 3 groups after surgery in this double-blind, randomized study: sufentanil (0.02 μg/kg/h, Group S), sufentanil plus dexmedetomidine (0.02 μg/kg/h each, Group D1), or sufentanil (0.02 μg/kg/h) plus dexmedetomidine (0.04 μg/kg/h) (Group D2). The patient-controlled analgesia (PCA) program was programmed to deliver a bolus dose of 2 ml, with background infusion of 2 ml/h and a lockout of 5 min, 4-hour limit of 40 ml, as our retrospective study. The primary outcome measure was the cumulative amount of self-administered sufentanil; the secondary outcome measures were pain intensity (numerical rating scale, NRS), level of sedation (LOS), Bruggrmann comfort scale (BCS), functional activity score (FAS), and concerning adverse effects.The amount of self-administered sufentanil were lower in group D2 compared with S and D1 groups during the 72 hours after surgery (P < 0.05), whereas the total dosage and dosage per body weight of sufentanil were significantly lower in D1 group than that of S group only at 4, 8, and 16 hours after surgery (P < 0.05). Compared with S group, the NRS scores at rest at 1, 4, and 8 hours after surgery and with coughing at 4, 8, 16, and 24 hours after surgery were significantly lower in D2 group (P < 0.05). However, compared with D1 group, the NRS scores both at rest and with coughing at 4 and 8 hours after surgery were significantly lower in D2 group (P < 0.05). The NRS scores both at rest and with coughing show that there were no significant differences between D1 group and S group at each time point after surgery (P > 0.05). LOS of group D2 was higher than S and D1 groups at 1 hour after surgery (P < 0.05), BCS of group D2 was higher than S and D1 groups at 4, 8, and 16 hours after surgery (P < 0.05), and FAS of group D2 was higher than S and D1 groups at 48 and 72 hours after surgery (P < 0.05). The number of rescue analgesia during 72 hours after surgery in D2 group was lower than S and D1 groups (P < 0.05). There were no significant differences among the 3 groups in terms of baseline clinical characteristics and postoperative adverse effects except for itching (P > 0.05).Among the tested patient-controlled a...

Identification of maternal MTHFR C677T mutation may play a key role for primary prevention of PTB as well as LBW and screening pregnant women of high risk in developing countries.