Efficient and Precise Processing of the Optimized Primary Artificial MicroRNA in a Huntingtin-Lowering Adeno-Associated Viral Gene Therapy In Vitro and in Mice and Nonhuman Primates.

Huntington's disease is a fatal neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene comprising an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat sequence that results in a pathogenic huntingtin protein. Adeno-associated viral (AAV) gene therapy containing a primary artificial microRNA (pri-amiRNA) specifically targeting HTT messenger RNA (mRNA) has the potential to provide long-lasting therapeutic benefit, through durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri-amiRNA precursor to the mature guide (G) strand by transduced cells are critical for specific and potent HTT mRNA lowering. The selection of the optimized pri-amiRNA comprised a series of in vitro studies followed by in vivo studies in small and then large mammals. Our studies demonstrate the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all key features of pri-amiRNA processing. In addition, our results show that the processing of pri-amiRNAs to the mature guide strand can differ greatly across different scaffolds and sequences while providing the same levels of target lowering. Importantly, our data demonstrate that there is a combinatorial effect of guide and passenger (P) strand sequences, together with the scaffold, on pri-amiRNA processing, with different guide and passenger strand sequences within the same scaffold dramatically altering pri-amiRNA processing. Taken together, our results highlight the importance of optimizing not only target lowering but also the efficiency and precision of pri-amiRNA processing in vitro, in rodents and in large mammals to identify the most potent and selective AAV gene therapy that harnesses the endogenous microRNA (miRNA) biogenesis pathway for target lowering without perturbing the endogenous cellular miRNA profile. The optimized pri-amiRNA was selected with this focus on efficiency and precision of pri-amiRNA processing in addition to its pharmacological activity on HTT mRNA lowering and general tolerability in vivo.

A study of pregnancy rates in "cleared" male factor couples.

BACKGROUND: Among couples with male factor infertility, the natural pregnancy rates associated with classic male factor treatments are well described. In couples with unexplained infertility, the proportion due to occult male factor is unclear. We hypothesized that many men diagnosed with unexplained infertility are actually fertile. We describe the 1-year natural pregnancy rates among couples in whom the male partner has been "cleared" of infertility on urologic evaluation. METHODS: Consecutive infertile couples were recruited from a single practice (PJT) over a 3-year period. A thorough male factor evaluation was performed, including a history, physical examination and 2 semen analyses. Based on this assessment, male partners in whom bulk semen parameters were normal were "cleared" from further evaluation. Lifestyle modifications were allowed, but no medical or surgical treatments were offered. The presence or absence of a female factor evaluation was not required for study inclusion. Subjects were followed for 12 months or until a pregnancy was achieved. Subjects were contacted via telesurvery 1-year later and pregnancy status ascertained. Simple descriptive statistics were used to evaluate the significance of observations. RESULTS: Fifty-four men were enrolled in the study. The mean duration of infertility was 1.5 years (range, 0.4 to 4.0 years) and the mean male and female partner ages were 38.6 and 35.1 years, respectively. On evaluation, 40% of men were noted to have significant fertility risks that included a clinical varicocele, exposures, and androgen altering medications. Among n=31 couples with known pregnancy outcomes, 20/31 (65%) conceived naturally at a mean of 9 months after evaluation (range, 3-30 mos). Another 1/31 (3%) couples conceived with intrauterine insemination (IUI) and 4/31 (13%) conceived with IVF-ICSI. CONCLUSIONS: A significant proportion of men diagnosed with unexplained infertility have lifestyle risk factors on urologic evaluation. Care in the form of counseling at-risk patients regarding lifestyle issues, in the absence of formal treatment, may have value in improving the fertility potential in this population. Indeed, natural conception rates among men identified with unexplained infertility are substantial and suggest that many of these men are truly fertile.

In crystallo screening for proline analog inhibitors of the proline cycle enzyme PYCR1.

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ1-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N-formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 µm The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RASV12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.

Methylmercury promotes breast cancer cell proliferation.

CONTEXT: Metalloestrogens are small ionic metals that activate the estrogen receptor (ER). Studies have shown that when metalloestrogens bind to the ER, there is an increase in transcription and expression of estrogen-regulated genes, which induces proliferation of estrogen-dependent breast cancer. Methylmercury (MeHg), a metalloestrogen, is present in the environment and is toxic at moderate to high concentrations. However, at lower concentrations MeHg may promote the proliferation of ER-positive breast cancers and protect cells against pro-apoptotic signals. OBJECTIVE: To investigate the effects of MeHg treatment on breast cancer cells in vitro. MATERIALS AND METHODS: MCF7 breast cancer cells were treated with concentrations of MeHg ranging from 1 nM to 100 mM. Hg analysis was used to quantify intracellular mercury concentrations. Cell proliferation and apoptosis were determined by cell counting and Annexin-V staining, respectively. RESULTS: We defined a protocol that maximizes cellular exposure to mercury. Treatment of human ER-positive breast cancer cells with 1 nM MeHg promoted proliferation, while treatment with a concentration of 100 nM induced apoptosis. DISCUSSION AND CONCLUSIONS: Clarifying the effects of MeHg on breast cancer will improve our understanding of how environmental toxins affect tumor progression and may lead to the development of future therapeutic strategies.

A systematic review of pediatric self-report symptom measures: Congruence with the theory of unpleasant symptoms.

PURPOSE: Many children and adolescents experience unpleasant symptoms due to acute and chronic illnesses. Several symptom measures specific to children and adolescents are available to assess unpleasant symptoms. What is unclear is how well these measures assess all dimensions of the symptom experience. Using the Theory of Unpleasant Symptoms (TOUS) as a guide, the purpose of this systematic review of self-report symptom measures was to examine the comprehensiveness of current symptom measures designed to assess children's and adolescents' symptom experiences. CONCLUSIONS: The TOUS did not guide the development of any of the measures studied; hence, exact parallels were not expected. Currently, no pediatric self-report symptom measures were found that assessed all dimensions of the symptom experience. Four measures captured three dimensions: intensity, timing, and distress. Only two measures assessed quality. PRACTICE IMPLICATIONS: Practitioners need to be aware of the dimensions reflected in commonly used symptom measures. Augmentation of these measures is needed in order to assess the entire symptom experience in children and adolescents.

Effectiveness of message framing on women's intention to perform cytomegalovirus prevention behaviors: a cross-sectional study.

BACKGROUND: The purpose of this study was to evaluate the effect of message framing on women's intention to perform cytomegalovirus (CMV) prevention behaviors involving handwashing, not sharing food and eating utensils, not kissing a child on the lips and not placing a pacifier in the mouth after it was in a child's mouth. METHODS: An online panel of women 18-40 years, who were pregnant or planning a pregnancy were randomized in a 2 × 2 factorial design to receive 1 of 4 CMV fact sheets. The fact sheets were framed as either what could be gained or be lost by following (or not) the recommendations and the likelihood of being affected by CMV (i.e., small chance or one of the most common infections in infants). The questionnaire measured CMV knowledge, participation in CMV risk or prevention behaviors, perceived severity of and susceptibly to CMV, and the perceived control over and the efficacy of recommended prevention behaviors. The dependent variable, intention to modify behavior, was an index score that ranged from 0 to 16 with higher values indicating greater intention. Linear regression was used to evaluate the association between all independent variables and overall behavioral intention. RESULTS: The sample included 840 women; 15.5% were familiar with CMV. Behavioral intention was high (M = 10.43; SD = 5.13) but did not differ across the message frames (p = 0.23). Overall, behavioral intention was predicted by CMV knowledge, message credibility, perceived severity of CMV, perceived behavioral control and response efficacy. Significant interactions with gain vs. loss frame were observed for perceived behavioral control (p = 0.03) and response efficacy (p = .003). CONCLUSIONS: Framing CMV messages by what women stand to gain or lose interacts with perceived behavioral control and response efficacy to influence behavioral intention. Perceived behavioral control and response efficacy were most predictive of behavioral intention overall regardless of frame. Messaging that focuses on these two variables, particularly for avoiding kissing a child on the lips and sharing food, cups and utensils, may result in greater gains in intention to participate in CMV prevention behaviors.

Soluble interleukin-13rα1: a circulating regulator of glucose.

Soluble IL-13 receptor-α1, or sIL13rα1, is a soluble protein that binds to interleukin-13 (IL-13) that has been previously described in mice. The function of sIL13rα1 remains unclear, but it has been hypothesized to act as a decoy receptor for IL-13. Recent studies have identified a role for IL-13 in glucose metabolism, suggesting that a decoy receptor for IL-13 might increase circulating glucose levels. Here, we report that delivery of sIL13rα1 to mice by either gene transfer or recombinant protein decreases blood glucose levels. Surprisingly, the glucose-lowering effect of sIL13rα1 was preserved in mice lacking IL-13, demonstrating that IL-13 was not required for the effect. In contrast, deletion of IL-4 in mice eliminated the hypoglycemic effect of sIL13rα1. In humans, endogenous blood levels of IL13rα1 varied substantially, although there were no differences between diabetic and nondiabetic patients. There was no circadian variation of sIL13rα1 in normal human volunteers. Delivery of sIL13rα1 fused to a fragment crystallizable (Fc) domain provided sustained glucose lowering in mice on a high-fat diet, suggesting a potential therapeutic strategy. These data reveal sIL13rα1 as a circulating human protein with an unexpected role in glucose metabolism.

Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1.

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination.

Proline: Mother Nature's cryoprotectant applied to protein crystallography.

L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.