Investigating Late-Onset Pompe Prevalence in Neuromuscular Medicine Academic Practices: The IPaNeMA Study.

Background and Objectives: We investigated the prevalence of late-onset Pompe disease (LOPD) in patients presenting to 13 academic, tertiary neuromuscular practices in the United States and Canada. Methods: All successive patients presenting with proximal muscle weakness or isolated hyperCKemia and/or neck muscle weakness to these 13 centers were invited to participate in the study. Whole blood was tested for acid alpha-glucosidase (GAA) assay through the fluorometric method, and all cases with enzyme levels of ≤10 pmoL/punch/h were reflexed to molecular testing for mutations in the GAA gene. Clinical and demographic information was abstracted from their clinical visit and, along with study data, entered into a purpose-built REDCap database, and analyzed at the University of California, Irvine. Results: GAA enzyme assay results were available on 906 of the 921 participants who consented for the study. LOPD was confirmed in 9 participants (1% prevalence). Another 9 (1%) were determined to have pseudodeficiency of GAA, whereas 19 (1.9%) were found to be heterozygous for a pathogenic GAA mutation (carriers). Of the definite LOPD participants, 8 (89%) were Caucasian and were heterozygous for the common leaky (IVS1) splice site mutation in the GAA gene (c -32-13T>G), with a second mutation that was previously confirmed to be pathogenic. Discussion: The prevalence of LOPD in undiagnosed patients meeting the criteria of proximal muscle weakness, high creatine kinase, and/or neck weakness in academic, tertiary neuromuscular practices in the United States and Canada is estimated to be 1%, with an equal prevalence rate of pseudodeficiency alleles. Trial Registration Information: Clinical trial registration number: NCT02838368.

Chemical activation of lipoplexes formed from DNA and a redox-active, ferrocene-containing cationic lipid.

We recently reported that the ferrocene-containing cationic lipid BFDMA [bis(11-ferrocenylundecyl)dimethylammonium bromide] can be used to mediate cell transfection, and that levels of transfection depend critically upon the oxidation state of the ferrocenyl groups of the lipid. Here, we report that the redox activity of BFDMA can be exploited to transform lipoplexes formed from oxidized BFDMA (which do not transfect cells) to lipoplexes that are "active" (and thus mediate high levels of transgene expression) by treatment with the chemical reducing agent glutathione (GSH). We demonstrate that GSH can be used to reduce the ferrocenium groups of oxidized BFDMA rapidly both (i) in solution and (ii) in lipoplexes formed by mixing oxidized BFDMA and DNA. Lipoplexes transformed in this manner mediate levels of cell transfection in vitro that are comparable to levels of transfection mediated by lipoplexes prepared by mixing DNA and reduced BFDMA. We demonstrate further that the chemical reduction of oxidized BFDMA leads to changes in the zeta potentials of these lipoplexes (e.g., from negative to positive). Characterization of lipoplex internalization using confocal microscopy demonstrated that these changes in zeta potential correlate to differences in the extents to which these lipoplexes are internalized by cells. These results provide a framework from which to interpret differences in cell transfection mediated by reduced and oxidized BFDMA. When combined, the results of this study suggest the basis of an approach that could be used to transform lipoplexes actively or "on-demand" and provide spatial and/or temporal control over the transfection of cells in a range of different fundamental and applied contexts.

Diagnostic accuracy of an initial azoospermic reading compared with results of post-centrifugation semen analysis after vasectomy.

PURPOSE: Semen analysis is a common laboratory procedure but few data are available to support recommendations as to whether centrifugation is necessary in the post-vasectomy context. We evaluated the accuracy of a pre-centrifugation determination of azoospermia compared with post-centrifugation results. MATERIALS AND METHODS: We conducted a secondary analysis of data from 3,205 semen analyses performed during a randomized clinical trial of 2 vasectomy techniques--ligation and excision with fascial interposition vs ligation and excision without fascial interposition. We performed brief, initial microscopic examinations to categorize sperm numbers per high power field to decide whether centrifugation or dilution was needed before estimation of sperm concentration. For specimens initially categorized as azoospermic, we reviewed the post-centrifugation semen analysis results to estimate the accuracy of the initial finding. RESULTS: Of 2,104 samples categorized as azoospermic before centrifugation, post-centrifugation analysis demonstrated that all but 4 (99.8%) were azoospermic or had a sperm concentration of less than 100,000 sperm per ml. Four samples from 1 study site had counts between 104,000 and 315,000 sperm per ml. Of 1,610 apparently azoospermic samples obtained at 10 weeks or later after vasectomy there were 12 (0.7%) that had some motile sperm identified after centrifugation but the numbers of motile sperm were low (mean 1,124 motile sperm per ml, range 238 to 3,710). CONCLUSIONS: Microscopic examination of uncentrifuged specimens is a reliable method for identifying semen samples after vasectomy with more than 100,000 sperm per ml.

Characterization of the nanostructure of complexes formed by a redox-active cationic lipid and DNA.

We report characterization of the nanostructures of complexes formed between the redox-active lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA) and DNA using small-angle neutron scattering (SANS) and cryogenic transmission electron microscopy (cryo-TEM). A particular focus was directed to the influence of lipid oxidation state (where reduced BFDMA has a net charge of +1 and oxidized BFDMA has a charge of +3) on the nanostructures of the solution aggregates formed. Complexes were characterized over a range of charge ratios of reduced BFDMA to DNA (1.1:1, 2.75:1, and 4:1) in solutions of 1 mM Li2SO4. For these complexes, a single peak in the SANS data at 1.2 nm(-1) indicated that a nanostructure with a periodicity of 5.2 nm was present, similar to that observed with complexes of the classical lipids DODAB/DOPE and DNA (multilamellar spacing of 7.0 nm). The absence of additional Bragg peaks in all the SANS data indicated that the periodicity did not extend over large distances. Both inverse Fourier transform analysis and form factor fitting suggested formation of a multilamellar vesicle. These results were confirmed by cryo-TEM images in which multilamellar complexes with diameters between 50 and 150 nm were observed with no more than seven lamellae per aggregate. In contrast to complexes of reduced BFDMA and DNA, Bragg peaks were absent in SANS spectra of complexes formed by oxidized BFDMA and DNA at all charge ratios investigated. The low-q behavior of the SANS data obtained using oxidized BFDMA and DNA complexes suggested that large, loose aggregates were formed, consistent with complementary cryo-TEM images showing predominantly loose disordered aggregates. Some highly ordered spongelike and cubic phase nanostructures were also detected in cryo-TEM images. We conclude that control of BFDMA oxidation state can be used to manipulate the nanostructures of lipid-DNA complexes formed using BFDMA.

Pharmacokinetic interactions between depot medroxyprogesterone acetate and combination antiretroviral therapy.

OBJECTIVE: To evaluate the effect of an antiretroviral (ARV) therapy regimen containing zidovudine (AZT), lamivudine (3TC), and efavirenz (EFV) on the pharmacokinetics of depot medroxyprogesterone acetate (DMPA). DESIGN: Open-label, nonrandomized, clinical trial. SETTING: University hospital clinic. PATIENT(S): Thirty HIV-infected women; 15 using ARV therapy (AZT, 3TC, and EFV) and 15 non-users of ARV therapy, followed biweekly for 12 weeks. INTERVENTION(S): Single injection of DMPA (150 mg IM) for both groups. MAIN OUTCOME MEASURE(S): Pharmacokinetic parameters of DMPA by liquid chromatography with mass spectrometry, and ovulation by serum P. RESULT(S): Maximum serum concentrations of DMPA were reached at 14 days after injection. The area under the curve was similar in both groups, as were the minimum concentration, half-life, and clearance. Only 1 woman, not using ARV therapy, ovulated at 11 weeks after DMPA. CONCLUSION(S): Pharmacokinetics of DMPA were similar in HIV-infected women, regardless of ARV therapy use, suggesting that triple therapy with AZT, 3TC, and EFV is not likely to interfere with the contraceptive effectiveness of DMPA.

Lipoplexes formed by DNA and ferrocenyl lipids: effect of lipid oxidation state on size, internal dynamics, and zeta-potential.

The effect of lipid oxidation state on the physical properties of complexes formed by plasmid DNA and the redox-active lipid bis-(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) is reported. With increasing concentration of BFDMA, the hydrodynamic sizes of complexes formed by BFDMA and DNA (in the presence of 1 mM Li(2)SO(4)) pass through a maximum and the zeta-potential changes monotonically from -40 mV to +40 mV. In contrast, complexes formed by oxidized BFDMA and DNA exhibit a minimum in size and maintain a negative zeta-potential with increasing concentration of BFDMA. Angle-dependent dynamic light scattering measurements also reveal the presence of relaxation processes within complexes formed by DNA and oxidized BFDMA that are absent for complexes formed by DNA and reduced BFDMA. These results, when combined, reveal that the amphiphilic nature of reduced BFDMA leads to lipoplexes with physical properties resembling those formed by classical cationic lipids, whereas the interaction of oxidized BFDMA with DNA is similar to that of nonamphiphilic cationic molecules bearing multiple charges (e.g., spermidine). In particular, the negative zeta-potential and measurable presence of DNA chain dynamics within complexes formed by oxidized BFDMA and DNA indicate that these complexes are loosely packed with excess charge due to DNA in their outer regions. These results, when combined with additional measurements performed in OptiMEM reduced-serum cell culture medium, lead to the proposition that the strong dependence of transfection efficiency on the oxidation state of BFDMA, as reported previously, is largely a reflection of the substantial change in the zeta-potentials of these complexes with changes in the oxidation state of BFDMA.

Reversible condensation of DNA using a redox-active surfactant.

We report characterization of aqueous solutions of dilute Lambda phage DNA containing the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) as a function of the oxidation state of the FTMA. FTMA undergoes a reversible one-electron oxidation from a reduced state that forms micelles in aqueous solution to an oxidized state (containing the ferrocenium cation) that does not self-associate in solution. This investigation sought to test the hypothesis that FTMA can be used to achieve reversible control over the conformation of DNA-surfactant complexes in solution. Whereas DNA adopts extended coil conformations in aqueous solutions, our measurements revealed that addition of reduced FTMA (2-5 microM) to aqueous solutions of DNA (5 microM in nucleotide units) resulted in coexistence of extended coils and compact globules in solution. At higher concentrations of reduced FTMA (up to 30 microM), the DNA was present as compact globules only. In contrast, oxidized FTMA had no measurable effect on the conformation of DNA, allowing DNA to maintain an extended coil state up to a concentration of 75 microM oxidized FTMA. We further demonstrate that it is possible to chemically or electrochemically transform the oxidation state of FTMA in preformed complexes of FTMA and DNA, thus achieving in situ control over the conformations of the DNA in solution. These results provide guidance for the design of surfactant systems that permit active control of DNA-surfactant interactions.

Frequency and patterns of early recanalization after vasectomy.

BACKGROUND: Our understanding of early post-vasectomy recanalization is limited to histopathological studies. The objective of this study was to estimate the frequency and to describe semen analysis patterns of early recanalization after vasectomy. METHODS: Charts displaying serial post-vasectomy semen analyses were created using the semen analysis results from 826 and 389 men participating in a randomized trial of fascial interposition (FI) and an observational study of cautery, respectively. In the FI trial, participants were randomly allocated to vas occlusion by ligation and excision with or without FI. In the cautery study, sites used their usual cautery occlusion technique, two with and two without FI. Presumed early recanalization was based on the assessment of individual semen analysis charts by three independent reviewers. Discrepancies were resolved by consensus. RESULTS: Presumed early recanalization was characterized by a very low sperm concentration within two weeks after vasectomy followed by return to large numbers of sperm over the next few weeks. The overall proportion of men with presumed early recanalization was 13% (95% CI 12%-15%). The risk was highest with ligation and excision without FI (25%) and lowest for thermal cautery with FI (0%). The highest proportion of presumed early recanalization was observed among men classified as vasectomy failures. CONCLUSION: Early recanalization, occurring within the first weeks after vasectomy, is more common than generally recognized. Its frequency depends on the occlusion technique performed.

Bleeding patterns after use of levonorgestrel emergency contraceptive pills.

OBJECTIVE: The objective of this study was to describe bleeding after use of an emergency contraceptive pill (ECP) regimen consisting of 1.5 mg of levonorgestrel in a single dose. METHODS: We asked 120 women who had been treated with the regimen to keep daily bleeding diaries for 9 weeks. We compared bleeding patterns observed after treatment with usual patterns reported by the participants and with patterns observed in a prior study on women who had not taken ECPs. RESULTS: Treatment in the first 3 weeks of the menstrual cycle significantly shortened that cycle as compared both with the usual cycle length and with the cycle duration in a comparison group. The magnitude of this effect was greater the earlier the pills were taken. In contrast, the duration of the first menstrual period after treatment increased significantly with cycle week of treatment and was longer in women who used the treatment than in those who did not. Intermenstrual bleeding occurred in only 5% of women in the first cycle after treatment. CONCLUSIONS: The effect of the single-dose levonorgestrel ECP regimen on the timing and duration of the next menstrual period depends on when during the cycle the pills are taken. Intermenstrual bleeding following treatment is uncommon.

Ferrocene-containing cationic lipids for the delivery of DNA: oxidation state determines transfection activity.

The ability of two redox-active, ferrocene-containing cationic lipids [11-(ferrocenylundecyl)trimethylammonium bromide (FTMA) and bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA)] to transfect mammalian cells was investigated. This study sought to determine the range of conditions over which these lipids were capable of transfecting cells and whether the oxidation state of the ferrocenyl groups in these materials could be used to influence the extent of transfection. Experiments conducted in the COS-7 cell line demonstrated that reduced and oxidized FTMA were substantially cytotoxic and did not transfect cells. Subsequent experiments conducted using BFDMA and reporter plasmids encoding enhanced green fluorescent protein (EGFP) and firefly luciferase demonstrated that BFDMA was able to transfect cells. However, the extent of transfection depended significantly upon both the concentration of BFDMA and the oxidation state of the lipid. Quantitative characterization of cytotoxicity and gene expression demonstrated that a window of concentration existed over which reduced BFDMA was non-cytotoxic and yielded high levels of transfection, but over which electrochemically oxidized BFDMA yielded very low (background) levels of transfection. Characterization of lipoplexes using dynamic light scattering demonstrated that reduced and oxidized BFDMA formed small aggregates (ca. 90 to 250nm) at concentrations of lipid ranging from 2 to 10 microM. Taken together, these results demonstrate that the oxidation state of BFDMA, which can be controlled electrochemically, can be used to control the extent of cell transfection. These results could form the basis of transfection procedures that exploit the redox behavior of ferrocene-containing lipids to achieve active spatial and temporal control over transfection using electrochemical methods.

Electrochemical control of the interactions of polymers and redox-active surfactants.

We report the characterization of aqueous solutions (0.1 M Li(2)SO(4)) of dilute ethyl(hydroxyethyl) cellulose (EHEC) mixed with the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) by measurements of clouding temperatures and dynamic light scattering. The investigation sought to test the hypothesis that FTMA, which forms micelles in aqueous solution in its reduced state but not in its oxidized state, would permit reversible control over the formation of polymer-surfactant complexes in solution. Our measurements revealed that low concentrations of reduced FTMA enhance aggregation, whereas high concentrations of reduced FTMA disperse polymer-surfactant aggregates. This behavior is qualitatively similar to both dodecyltrimethylammonium bromide and sodium dodecyl sulfate and reflects cooperative interactions between FTMA and EHEC. In contrast, oxidized FTMA was found not to promote EHEC aggregation at low concentrations of oxidized FTMA nor disperse EHEC aggregates at high concentrations. Measurements of dynamic light scattering revealed that the reduction of oxidized FTMA in solutions of EHEC containing 0.1-0.3 mmolal FTMA causes an increase in the sizes of polymer-surfactant aggregates of more than 1 order of magnitude. Our cloud point measurements also revealed that clouding can be induced isothermally via the electrochemical reduction of oxidized FTMA at room temperature at FTMA concentrations between 0.4 and 10 mmolal. In contrast, at concentrations of FTMA greater than 15 mmolal, the reduction of oxidized FTMA induces the clearing of EHEC solutions. We conclude that aggregation of EHEC in dilute solutions can be controlled by the manipulation of the oxidation state of FTMA.

Ferrocene-containing cationic lipids: influence of redox state on cell transfection.

A ferrocene-containing, redox-active cationic lipid that can be transformed using electrochemical methods yields large differences in cell transfection depending on the oxidation state of the lipid. Expression of enhanced green fluorescent protein and firefly luciferase occurs at very high levels when DNA lipoplexes are formulated using the lipid in the reduced state. In contrast, transfection is negligible when oxidized lipid is used. These observations suggest the basis of a general method that could be used to transform inactive lipoplex formulations to an active form through the application of externally applied electrical potentials. The ability to activate lipoplexes toward transfection electrochemically and "on demand" could create new opportunities to deliver DNA in vitro and in vivo with both spatial and temporal control.

Vasectomy by ligation and excision, with or without fascial interposition: a randomized controlled trial [ISRCTN77781689].

BACKGROUND: Randomized controlled trials comparing different vasectomy occlusion techniques are lacking. Thus, this multicenter randomized trial was conducted to compare the probability of the success of ligation and excision vasectomy with, versus without, fascial interposition (i.e. placing a layer of the vas sheath between two cut ends of the vas). METHODS: The trial was conducted between December 1999 and June 2002 with a single planned interim analysis. Men requesting vasectomies at eight outpatient clinics in seven countries in North America, Latin America, and Asia were included in the study. The men were randomized to receive vasectomy with versus without fascial interposition. All surgeons performed the vasectomies using the no-scalpel approach to the vas. Participants had a semen analysis two weeks after vasectomy and then every four weeks up to 34 weeks. The primary outcome measure was time to azoospermia. Additional outcome measures were time to severe oligozoospermia (<100 000 sperm/mL) and vasectomy failure based on semen analyses. RESULTS: We halted recruitment after the planned interim analysis, when 841 men had been enrolled. Fascial interposition decreased time to azoospermia (hazard ratio [HR], 1.35; P < 0.0001) and time to severe oligozoospermia (HR, 1.32; P < 0.0001) and reduced failures based on semen analysis by about half, from 12.7% (95% confidence interval [CI], 9.7 to 16.3) to 5.9% (95% CI, 3.8 to 8.6) (P < 0.0001). Older men benefited less from fascial interposition than younger men in terms of the speed of achieving azoospermia. However, the number of vasectomy failures was reduced to a similar degree in all age groups. Slightly more adverse events occurred in the fascial interposition group, but the difference was not significant. These failure rates may appear high to practitioners in countries such as the USA, but they are similar to results from other careful studies of ligation and excision techniques. CONCLUSION: Fascial interposition significantly improves vasectomy success when ligation and excision is the method of vas occlusion. A limitation of this study is that the correlation between postvasectomy sperm concentrations and risk of pregnancy is not well quantified.

Vasectomy effectiveness in Nepal: a retrospective study.

The main purpose of this retrospective, cross-sectional study was to evaluate the effectiveness of vasectomy in an ongoing public sector program in Nepal. We evaluated semen samples from men who had previously had a vasectomy, and asked about the occurrence of pregnancies in the men's partners. In addition, the surgeons who performed the vasectomies completed a questionnaire about their techniques. A two-stage stratified sampling procedure was used to select 1263 men from among over 30,000 men, who had previously undergone a no-scalpel vasectomy, mostly by ligation and excision, in 32 districts between July 1996 and June 1999. Semen samples were preserved and analyzed at a central laboratory. A US andrology laboratory validated the lab results. Twenty-three men (2.3%, 95% confidence interval [CI] 1.1-3.6) had >/=500,000 sperm/mL in their semen. Fifteen of those men reported pregnancies conceived after their vasectomy. In addition, six men with azoospermia reported pregnancies for which conception occurred within 3 months after vasectomy. Eleven men with azoospermia reported pregnancies for which conception occurred more than 3 months after vasectomy. Reported pregnancy was more likely in younger partners. The life table pregnancy rates for all men interviewed were 0.7 (95% CI 0.2-1.1), 1.7 (95% CI 1.4-2.1) and 4.2% (95% CI 3.2-5.2) at 3, 12 and 36 months, respectively. In low-resource, programmatic settings, vasectomy failure rates may be higher than commonly cited rates, especially in younger populations. Additional research is needed to determine if other occlusion techniques could reduce failure rates. Counseling on vasectomy should always convey the possibility of failure and partner pregnancy.

Termination of a randomized controlled trial of two vasectomy techniques.

Family Health International (FHI) and EngenderHealth conducted a multicenter, randomized controlled trial to evaluate a fascial interposition (FI) component of a vas occlusion procedure for male sterilization. A data monitoring committee (DMC) was established to provide independent review of the interim report. The DMC met to review the interim report after enrollment of 552 men. As planned, an estimate of the hazard ratio (HR) for successful vasectomy for the FI group versus the no-FI group controlling for age of the participant and level of experience of the surgeon was obtained for the interim report using Cox's proportional hazards regression. The main analysis comparing time to vasectomy success, defined as two consecutive azoospermic semen samples, was highly significant and met the prespecified level for stopping enrollment (HR=1.54, p<0.01). However, a troublesome age by vasectomy technique interaction became apparent in the analysis. FI was clearly beneficial for younger males. However, the trend for older men was not as clear. The DMC recommended continuing the trial to better study this interaction and obtain more data to clarify the effect of FI for older men. After consulting further with the DMC, the study statisticians presented the interim report to senior management at FHI. A meeting between FHI senior management and the DMC followed. After much deliberation, trial enrollment was terminated. Follow-up of participants already enrolled in the study continued as planned. This paper presents the issues involved in the decision to terminate the study.