Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

A questionnaire survey on patients' willingness to pay with reference to the waiting time of public in-vitro fertilization treatment in Hong Kong.

OBJECTIVE: To evaluate patients' willingness to pay (WTP) with reference to the waiting time of public in-vitro fertilisation (IVF) treatment in order to improve the public IVF service in Hong Kong. STUDY DESIGN: A prospective multi-centred questionnaire survey. Infertile women attending infertility clinics of nine public hospitals in Hong Kong between October 2017 and August 2018 were asked to complete a questionnaire in their first clinic visit. RESULTS: Out of 1092 respondents, 10.4 % had private IVF cycles prior to their first visit at public hospitals. In general, patients were willing to pay more for a shorter waiting time for public IVF service. The proportion of respondents who were willing to pay more than HK$10,000 (US$1282) for one IVF cycle increased from 54.6% to 80.7% if the waiting time for public IVF service were hypothetically shortened from four years to one year. Likewise, 22.5 % versus 45.5 % were willing to pay more than HK$ 25,000 (US$3205) with a waiting time of four versus one year respectively. Assuming the cost per IVF cycle was HK$ 25,000 (US$3205), 23.4 % of respondents could afford one IVF cycle, 40.0 % of them could afford two IVF cycles and 31.5 % could afford three IVF cycles. A multivariate regression model demonstrated that only family income and presence of existing child(ren) were significant independent determinants of the maximum amount that an individual was willing to pay for IVF (p < 0.05). Those with family monthly income below HK$100,000 ($12,820) were less than half as likely, and those without existing child(ren) were more than double as likely, to be willing to pay higher for IVF. CONCLUSION: Patients were willing to pay more for a shorter waiting time for public IVF service. Those with family income below HK$100,000 (US$ 12,820) were less than half as likely, and those without existing children were more than double as likely, to be willing to pay higher for IVF.

Randomized, controlled, double-blind trial of topical lidocaine gel and intrauterine lidocaine infusion for pain relief during saline contrast sonohysterography.

OBJECTIVES: To evaluate the efficacy of topical lidocaine gel and intrauterine lidocaine infusion administered prior to saline contrast sonohysterography (SCSH) in reducing pain level during the procedure. METHODS: This was a randomized, double-blind, placebo controlled trial. We recruited 120 women scheduled to undergo SCSH and randomized them into one of three groups according to administration of gel and intrauterine infusion immediately prior to the procedure: (1) the 'lidocaine gel' group received 3 mL 2% lidocaine gel applied to the cervix and intrauterine infusion, using an infant feeding tube without balloon, of 5 mL normal saline; (2) the 'lidocaine infusion' group received 3 mL gel lubricant applied to the cervix and intrauterine infusion of 5 mL 2% lidocaine; (3) the placebo group received 3 mL gel lubricant applied to the cervix and intrauterine infusion of 5 mL normal saline. The tube was left in place for the SCSH procedure. The primary outcome measure was the overall pain level (on a scale of 0-100) reported by the women during the SCSH procedure. Women also rated their pain levels at various other time points and an observer assessed visible signs of the women's discomfort during the procedure, producing a distress score. RESULTS: There were no significant differences among the three groups in baseline characteristics, volume of saline solution infused, tenaculum use and duration and difficulty level of the SCSH procedure. The median (range) pain scores during normal saline infusion for the SCSH procedure were 0 (0-65) in the placebo group, 2.5 (0-80) in the lidocaine gel group, and 0 (0-70) in the lidocaine infusion group. The pain scores at other time points, the overall pain score and the distress score were also comparable for the three groups. No significant adverse events were reported. CONCLUSIONS: SCSH performed with an infant feeding tube without balloon is associated with very low pain levels. Topical lidocaine gel application and intrauterine lidocaine infusion do not further reduce pain levels during SCSH.

Factors influencing the mode of delivery and associated pregnancy outcomes for twins: a retrospective cohort study in a public hospital.

OBJECTIVES: To determine current trends for different modes of delivery in twin pregnancies, factors affecting the mode of delivery, and associated outcomes. DESIGN: Retrospective cohort study. SETTING: A public hospital in Hong Kong. PARTICIPANTS: All twin pregnancies booked at Kwong Wah Hospital during a 3-year period from 1 April 2006 to 31 March 2009. RESULTS: Of 197 sets of twins, 35 (18%) were delivered vaginally and 162 (82%) by caesarean section (47% were emergencies and 53% elective). In all, 32 (37%) of the elective and 21 (28%) of the emergency caesarean sections were in response to maternal requests. Vaginal delivery was more common in mothers with a history of vaginal delivery and monochorionic diamniotic twins. Women who conceived by assisted reproduction or those who had a tertiary education were more likely to deliver by caesarean section. The type of conception and the presentation of the second twin were statistically significant factors affecting maternal choice on the mode of delivery. Maternal age did not affect the choice of delivery mode. Except for the higher frequency of sepsis and cord blood acidosis in second twins delivered vaginally, there were no significant differences in neonatal morbidity between the groups that attempted vaginal delivery or requested caesarean sections. All the women who had compression sutures or hysterectomy to control massive postpartum haemorrhage were delivered by caesarean section. CONCLUSION: A high caesarean section rate observed in our cohort was associated with maternal requests for this mode of delivery. The type of conception and the presentation of the second twin were statistically significant factors affecting maternal choice on mode of delivery. Women's requests for caesarean delivery out of the concern for their babies are not supported by current evidence. In response to a woman with a twin pregnancy requesting caesarean delivery, the pros and cons of vaginal deliveries and caesarean sections should be fully explained before the woman's autonomy is respected.

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.

Herpes simplex infection in acute myelogenous leukemia and other hematologic malignancies: a prospective study.

To better define the frequency and clinical characteristics of herpes simplex virus (HSV) infection in adult patients with acute myelogenous leukemia (AML), the authors prospectively studied 29 patients undergoing remission induction chemotherapy with twice weekly throat wash cultures for an average of 25.3 days. Ten seropositive patients (34.5%) shed HSV at least once. Eight patients were asymptomatic. Two episodes of herpes labialis were severe and persistent, but no visceral dissemination was observed. Reactivation of HSV infections in AML patients presumably with marked immunosuppression occurs, but less frequently and more benignly than has been suggested. Daunomycin and cytosine arabinoside, which can inhibit HSV replication, may have accounted for this lower frequency of reactivation.

Invasive pituitary adenoma with abscess formation: case report.

A case is presented and 10 cases are reviewed in which abscess formation developed in an intrasellar tumor. The diagnosis was made preoperatively or before autopsy in only 1 patient. The mortality rate was greater than 50%. The most common presentation was headache, fever, and visual disturbances with an abnormal sella and sterile cerebrospinal fluid (CSF). Nonconstant findings included meningeal signs and leukocytosis of the CSF. Trans-sphenoidal removal of the tumor and drainage of the abscess with appropriate antibiotic therapy is recommended. (Neurosurgery, 5: 718--722, 1979).

Prevention of reactivated herpes simplex infection by human leukocyte interferon after operation on the trigeminal root.

Microneurosurgical procedures on the trigeminal-nerve root are often followed by reactivation of herpes simplex virus infection, manifested by herpes labialis or oropharyngeal herpesvirus shedding or both. In a double-blind study of the ability of human leukocyte interferon to prevent this reactivation, patients with a history of herpes labialis were given 7 x 10(4) U of interferon per kilogram of body weight per day or placebo for five days beginning on the day before operation. In 18 patients treated with placebo, herpes labialis developed in 10, and virus shedding in the oropharynx in 15. In 19 patients treated with interferon, lesions developed in five, and shedding in eight. The frequency of reactivation as measured by lesions or positive throat cultures or both was significantly reduced by interferon (P less than 0.05). Of 127 daily throat-wash cultures in the placebo group, 42 per cent were positive for herpesvirus, but of 134 in the interferon group, only 9 per cent were positive (P less than 0.001). We conclude that interferon at a well-tolerated dosage reduces reactivation of latent herpes simplex virus infection after a potent operative stimulus.