Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore- induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore- induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.      

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

Artificial insemination and in-vitro fertilization using donor spermatozoa: a report on 15 years of experience

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.      

Expression and characterization of Gag protein of HIV-1（CN） in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain （HIV-1 （CN）） in Pichia pastoris, the fulllength gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by Sail was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA （1.3 kb） was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kD, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13 % of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows： BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85 %. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.     

柱前手性衍生化反相高效液相色谱法拆分地佐西平对映异构体的研究

用乙酰葡萄糖异硫氰酸酯(GITC)作柱前手性衍生化试剂,以反相高效液相色谱法成功地拆分了地佐西平对映异构体。在此基础上建立了地佐西平对映异构体纯度和血药浓度测定方法。已用于测定右旋地佐西平的光学纯度和小鼠血药浓度。     

Ceramide changes in abdominal subcutaneous and visceral adipose tissue among diabetic and nondiabetic patients

BackgroundThis study profiles ceramides extracted from visceral and subcutaneous adipose tissue of human subjects by liquid chromatography‐mass spectrometry to determine a correlation with status of diabetes and gender.MethodsSamples of visceral and abdominal wall subcutaneous adipose tissue (n = 36 and n = 31, respectively) were taken during laparoscopic surgery from 36 patients (14 nondiabetic, 22 diabetic and prediabetic) undergoing bariatric surgery with a body mass index (BMI) >35 kg/m² with ≥1 existing comorbidity or BMI ≥40 kg/m². Sphingolipids were extracted and analyzed using liquid chromatography‐mass spectrometry.ResultsAfter logarithm 2 conversion, paired analysis of visceral to subcutaneous tissue showed differential accumulation of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) in visceral tissue of prediabetic/diabetic female subjects, but not in males. Within‐tissue analysis showed higher mean levels of ceramide species linked to insulin resistance, such as Cer(d18:1/18:0) and Cer(d18:1/16:0), in visceral tissue of prediabetic/diabetic patients compared with nondiabetic subjects and higher content of Cer(d18:1/14:0) in subcutaneous tissue of insulin‐resistant female patients compared with prediabetic/diabetic males. Statistically significant differences in mean levels of ceramide species between insulin‐resistant African American and insulin‐resistant Caucasian patients were not evident in visceral or subcutaneous tissue.ConclusionsAnalysis of ceramides is important for developing a better understanding of biological processes underlying type 2 diabetes, metabolic syndrome, and obesity. Knowledge of the accumulated ceramides/dihydroceramides may reflect on the prelipolytic state that leads the lipotoxic phase of insulin resistance and may shed light on the predisposition to insulin resistance by gender.     

A ventilation strategy during general anaesthesia to reduce postoperative atelectasis

Background. Atelectasis is common during and after general anaesthesia. We hypothesized that a ventilation strategy, without recruitment manoeuvres, using a combination of continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP) and a reduced end-expiratory oxygen fraction (F_ETO₂) before ending mask ventilation with CPAP after extubation would reduce the area of postoperative atelectasis.

Methods. Thirty patients were randomized into three groups. During induction and emergence, inspiratory oxygen fractions (F_IO₂) were 1.0 in the control group and 1.0 or 0.8 in the intervention groups. No CPAP/PEEP was used in the control group, whereas CPAP/PEEP of 6 cmH₂O was used in the intervention groups. After extubation, F_IO₂ was set to 0.30 in the intervention groups and CPAP was applied, aiming at F_ETO₂ < 0.30. Atelectasis was studied by computed tomography 25 min postoperatively.

Results. The median area of atelectasis was 5.2 cm² (range 1.6–12.2 cm²) and 8.5 cm² (3–23.1 cm²) in the groups given F_IO₂ 1.0 with or without CPAP/PEEP, respectively. After correction for body mass index the difference between medians (2.9 cm²) was statistically significant (confidence interval 0.2–7.6 cm², p = 0.04). In the group given F_IO₂ 0.8, in which seven patients were ex- or current smokers, the median area of atelectasis was 8.2 cm² (1.8–14.7 cm²).

Conclusion. Compared with conventional ventilation, after correction for obesity, this ventilation strategy reduced the area of postoperative atelectasis in one of the intervention groups but not in the other group, which included a higher proportion of smokers.     

Perspectives of Dutch Physiotherapists on Self-Management Support: A Q-Methodology Study

Self-management support programs are reported to have positive effects on the health and care of people with physical disabilities. It is unclear how physiotherapists (PTs) view self-management support. A cross-sectional study with mixed-methods design using Q-methodology was conducted to determine the perspectives of Dutch PTs on self-management support. PTs (n = 39) rank-ordered 37 validated statements about self-management support from “most disagree (?3)” to “most agree (+3).” Differences in perspectives on self-management support were explored in a by-person centroid factor analysis on the basis of the explained variance (R²). After having sorted the statements, PTs explained their ranking of ?3 and +3 statements in semi-structured interviews. All PTs recognized self-management support as intrinsic part of physiotherapy practice. Nevertheless, four particular perspectives could be identified: (1) the “externally driven educator” (R² = 14%), (2) “internally driven educator” (R² = 9%), (3) “client-centered coach” (R² = 13%), and (4) “client-initiated coach” perspective (R² = 15%). Each perspective reflects particular goals and perceptions of self-management support. Differences were identified with respect to the PTs’ role perception (educating or coaching), drive (external or internal), and collaboration with the patient (as partner or not). The identified perspectives can be used in the physiotherapy education curriculum to create awareness about the various ways of practicing self-management support and the skills needed to be able to tailor support to patient needs and to switch between perspectives.