Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (>10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

Intrauterine fluid instillation to confirm tubal occlusion after transcervical permanent contraception: A pilot study.

OBJECTIVES: To determine if women with tubal patency experience more fluid loss compared to those with bilateral tubal occlusion following intrauterine instillation of fluid via a balloon catheter. STUDY DESIGN: In this prospective cohort pilot study, we enrolled women with prior Essure® procedures and healthy controls from September 2016 to July 2017. We excluded women using an implant or intrauterine device, or with a prior cesarean delivery or permanent contraception procedures other than Essure®. An infusion pump delivered saline via balloon catheter under continuous pressure monitoring. After one minute, we withdrew the fluid and recorded volumes in and out. Subjects then underwent hysterosalpingogram for evaluation of tubal patency. We conducted crude analyses with t-tests and sensitivity analyses. RESULTS: We recruited 23 participants; ten provided analyzable data in each group. Hysterosalpingogram confirmed patency in all control and occlusion in all post-Essure® subjects in the analysis group. We found the median volume of saline lost among control subjects [7.8 mL (7.4, 8.4)] larger than post-Essure® participants [2.2 mL (2.0, 3.8), p < 0.01]. While 50% of control subjects tolerated the full 10 mL of fluid instillation, none of the post-Essure® subjects tolerated this volume (p = 0.03). A combination of saline loss ≤4 mL and participant intolerance of the full 10 mL volume yielded sensitivity of 0.80 (95% CI: 0.57, 1.00) and specificity of 1.00 for bilateral tubal occlusion. CONCLUSION: Instillation of a fixed volume into the uterus may discriminate between women with tubal patency and occlusion following permanent contraception procedures with high specificity and adequate sensitivity. These findings should be validated in larger, more diverse study populations. IMPLICATIONS: Confirmation of tubal occlusion following permanent contraception with an office-based approach could improve acceptability of transcervical approaches. The recent removal of Essure® from the U.S. market increases the need for novel transcervical procedures and occlusion verification methods.

C-H Bond Cleavage Is Rate-Limiting for Oxidative C-P Bond Cleavage by the Mixed Valence Diiron-Dependent Oxygenase PhnZ.

PhnZ utilizes a mixed valence diiron(II/III) cofactor and O2 to oxidatively cleave the carbon-phosphorus bond of (R)-2-amino-1-hydroxyethylphosphonic acid to form glycine and orthophosphate. The active site residues Y24 and E27 are proposed to mediate induced-fit recognition of the substrate and access of O2 to one of the active site Fe ions. H62 is proposed to deprotonate the C1-hydroxyl of the substrate during catalysis. Kinetic isotope effects (KIEs), pH-rate dependence, and site-directed mutagenesis were used to probe the rate-determining transition state and the roles of these three active site residues. Primary deuterium KIE values of 5.5 ± 0.3 for D(V) and 2.2 ± 0.4 for D(V/K) were measured with (R)-2-amino[1-2H1]-1-hydroxyethylphosphonic acid, indicating that cleavage of the C1-H bond of the substrate is rate-limiting. This step is also rate-limiting for PhnZ Y24F, as shown by a significant deuterium KIE value of 2.3 ± 0.1 for D(V). In contrast, a different reaction step appears to be rate-limiting for the PhnZ E27A and H62A variants, which exhibited D(V) values near unity. A solvent KIE of 2.2 ± 0.3 for D2O(V) is observed for PhnZ. Significant solvent KIE values are also observed for the PhnZ Y24F and E27A variants. In contrast, the PhnZ H62A variant does not show a significant solvent KIE, suggesting that H62 is mediating proton transfer in the transition state. A proton inventory study with PhnZ indicates that 1.5 ± 0.6 protons are in flight in the rate-determining step. Overall, the rate-determining transition state for oxidative C-P bond cleavage by PhnZ is proposed to involve C-H bond cleavage that is coupled to deprotonation of the substrate C1-hydroxyl by H62.

Pressure dynamics in the non-gravid uterus: intrauterine pressure cannot confirm tubal occlusion after non-surgical permanent contraception.

OBJECTIVE: The objective was to determine if intrauterine pressure can distinguish bilateral tubal occlusion (BTO) from unilateral or bilateral tubal patency (TP) in women following a permanent contraception procedure. STUDY DESIGN: We used a small inline pressure sensor to continuously monitor intrauterine pressure during hysterosalpingogram (HSG) in a cross-sectional study that enrolled women having HSGs for any indication. The primary outcome was the peak intrauterine pressure compared between women with BTO and TP as verified by HSG. RESULTS: We enrolled 150 subjects, of which 111 (74.0%) provided usable pressure readings. Of these, 98/111 (88.3%) had TP, and 13 (11.7%) had BTO. There was no difference in peak intrauterine pressure for subjects with TP (mean 293.8±58.7 mmHg) compared to those with BTO (292.7±71.3 mmHg, p=.95). Among parous women, peak intrauterine pressure in subjects with BTO (311.9±78.0 mmHg) was higher but not significantly different from subjects with TP (282.7±49.2 mmHg, p=.20). In linear regression analysis, peak intrauterine pressure was not associated with age, body mass index, gravidity or having at least one prior live birth. CONCLUSIONS: Measurement of peak intrauterine pressure does not distinguish between women with patent and blocked fallopian tubes. This approach would not be clinically useful to verify occlusion following permanent contraception. IMPLICATIONS: Peak intrauterine pressure does not differ between women with patent and occluded fallopian tubes and cannot be used to confirm tubal occlusion after nonsurgical permanent contraception.

Tubal patency during the menstrual cycle and during treatment with hormonal contraceptives: a pilot study in women.

Background Hysterosalpingram (HSG) evaluation of tubal patency is typically performed in the follicular phase, but data to support this timing are lacking. Purpose To determine whether menstrual cycle phase or hormonal treatments affect observation of tubal patency during HSG. Material and Methods Ten participants underwent repeated HSG examinations: during the follicular and luteal phase of a natural menstrual cycle; 30 days following continuous administration of a combined oral contraceptive (COC); and 30 days after an intramuscular injection of depo medroxyprogesterone (DMPA) acetate. Participants with tubal blockade following DMPA had a fifth HSG 30 days following a second course of COCs. The primary outcome was tubal patency. Results All 10 participants demonstrated bilateral tubal patency (BTP) on at least one HSG examination during the study. One participant showed bilateral functional occlusion (FO) during the follicular phase examination, but BTP with the luteal phase, COC cycle, and DMPA exams. One participant with BTP discontinued participation and nine completed the COC HSG exam with BTP in seven, and one each with bilateral or unilateral FO. Seven participants completed the DMPA HSG with BTP in six and unilateral FO in one; BTP was seen in the final HSG after restarting the COC. Conclusion This pilot study supports the luteal phase of natural cycles as the optimum time for evaluation of tubal patency. The occurrence of functional occlusion of the fallopian tube on HSG examination performed during the follicular phase and following contraceptive steroid treatment supports a role of hormonal action on the utero-tubal junction.

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates.

The enzymes PhnY and PhnZ comprise an oxidative catabolic pathway that enables marine bacteria to use 2-aminoethylphosphonic acid as a source of inorganic phosphate. PhnZ is notable for catalyzing the oxidative cleavage of a carbon-phosphorus bond using Fe(II) and dioxygen, despite belonging to a large family of hydrolytic enzymes, the HD-phosphohydrolase superfamily. We have determined high-resolution structures of PhnZ bound to its substrate, (R)-2-amino-1-hydroxyethylphosphonate (2.1 Å), and a buffer additive, l-tartrate (1.7 Å). The structures reveal PhnZ to have an active site containing two Fe ions coordinated by four histidines and two aspartates that is strikingly similar to the carbon-carbon bond cleaving enzyme, myo-inositol-oxygenase. The exception is Y24, which forms a transient ligand interaction at the dioxygen binding site of Fe2. Site-directed mutagenesis and kinetic analysis with substrate analogs revealed the roles of key active site residues. A fifth histidine that is conserved in the PhnZ subclade, H62, specifically interacts with the substrate 1-hydroxyl. The structures also revealed that Y24 and E27 mediate a unique induced-fit mechanism whereby E27 specifically recognizes the 2-amino group of the bound substrate and toggles the release of Y24 from the active site, thereby creating space for molecular oxygen to bind to Fe2. Structural comparisons of PhnZ reveal an evolutionary connection between Fe(II)-dependent hydrolysis of phosphate esters and oxidative carbon-phosphorus or carbon-carbon bond cleavage, thus uniting the diverse chemistries that are found in the HD superfamily.