Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.

Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influences the Nterm conformation. To explore the latter, we used sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm-ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with single-stranded RNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm-ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.

Randomized trial of intermittent intraputamenal glial cell line-derived neurotrophic factor in Parkinson's disease.

We investigated the effects of glial cell line-derived neurotrophic factor (GDNF) in Parkinson's disease, using intermittent intraputamenal convection-enhanced delivery via a skull-mounted transcutaneous port as a novel administration paradigm to potentially afford putamen-wide therapeutic delivery. This was a single-centre, randomized, double-blind, placebo-controlled trial. Patients were 35-75 years old, had motor symptoms for 5 or more years, and presented with moderate disease severity in the OFF state [Hoehn and Yahr stage 2-3 and Unified Parkinson's Disease Rating Scale motor score (part III) (UPDRS-III) between 25 and 45] and motor fluctuations. Drug delivery devices were implanted and putamenal volume coverage was required to exceed a predefined threshold at a test infusion prior to randomization. Six pilot stage patients (randomization 2:1) and 35 primary stage patients (randomization 1:1) received bilateral intraputamenal infusions of GDNF (120 µg per putamen) or placebo every 4 weeks for 40 weeks. Efficacy analyses were based on the intention-to-treat principle and included all patients randomized. The primary outcome was the percentage change from baseline to Week 40 in the OFF state (UPDRS-III). The primary analysis was limited to primary stage patients, while further analyses included all patients from both study stages. The mean OFF state UPDRS motor score decreased by 17.3 ± 17.6% in the active group and 11.8 ± 15.8% in the placebo group (least squares mean difference: -4.9%, 95% CI: -16.9, 7.1, P = 0.41). Secondary endpoints did not show significant differences between the groups either. A post hoc analysis found nine (43%) patients in the active group but no placebo patients with a large clinically important motor improvement (≥10 points) in the OFF state (P = 0.0008). 18F-DOPA PET imaging demonstrated a significantly increased uptake throughout the putamen only in the active group, ranging from 25% (left anterior putamen; P = 0.0009) to 100% (both posterior putamina; P < 0.0001). GDNF appeared to be well tolerated and safe, and no drug-related serious adverse events were reported. The study did not meet its primary endpoint. 18F-DOPA imaging, however, suggested that intermittent convection-enhanced delivery of GDNF produced a putamen-wide tissue engagement effect, overcoming prior delivery limitations. Potential reasons for not proving clinical benefit at 40 weeks are discussed.

Racial and ethnic disparities in postpartum care and contraception in California's Medicaid program.

BACKGROUND: Considerable racial and ethnic disparities have been identified in maternal and infant health in the United States, and access to postpartum care likely contributes to these disparities. Contraception is an important component of postpartum care that helps women and their families achieve optimal interpregnancy intervals and avoid rapid repeat pregnancies and preterm births. National quality measurements to assess postpartum contraception are being developed and piloted. OBJECTIVE: To assess racial/ethnic variation in receipt of postpartum care and contraception among low-income women in California. STUDY DESIGN: We conducted a prospective cohort study of 199,860 Californian women aged 15-44 with a Medicaid-funded delivery in 2012. We examined racial/ethnic variation of postpartum care and contraception using multivariable logistic regression to control for maternal age, language, cesarean delivery, Medicaid program, and residence in a primary care shortage area (PCSA). RESULTS: Only one-half of mothers attended a postpartum visit (49.4%) or received contraception (47.5%). Compared with white women, black women attended postpartum visits less often (adjusted odds ratio [aOR], 0.73; 95% confidence interval [CI], 0.71-0.76), were less likely to receive any contraception (aOR, 0.83; 95% CI, 0.78-0.89) and were less likely to receive highly effective contraception (aOR, 0.64; 95% CI, 0.58-0.71). Women with Spanish as their primary language were more likely to get any contraception (aOR, 1.15; 95% CI, 1.11-1.19) but had significantly lower odds of receiving a highly effective method (aOR, 0.94; 95% CI, 0.90-0.99) compared with women with English as their primary language. Similarly, women in PCSAs had a greater odds of getting any contraception (aOR, 1.06; 95% CI, 1.03-1.09), but 24% lower odds of getting highly effective contraception than women not living in PCSAs (aOR, 0.76; 95% CI, 0.73-0.79). CONCLUSION: Significant racial/ethnic disparities exist among low-income Californian mothers' likelihood of attending postpartum visits and receiving postpartum contraception as well as receiving highly effective contraception.

Interpregnancy intervals: impact of postpartum contraceptive effectiveness and coverage.

OBJECTIVE: The purpose of this study was to determine the use of contraceptive methods, which was defined by effectiveness, length of coverage, and their association with short interpregnancy intervals, when controlling for provider type and client demographics. STUDY DESIGN: We identified a cohort of 117,644 women from the 2008 California Birth Statistical Master file with second or higher order birth and at least 1 Medicaid (Family Planning, Access, Care, and Treatment [Family PACT] program or Medi-Cal) claim within 18 months after index birth. We explored the effect of contraceptive method provision on the odds of having an optimal interpregnancy interval and controlled for covariates. RESULTS: The average length of contraceptive coverage was 3.81 months (SD = 4.84). Most women received user-dependent hormonal contraceptives as their most effective contraceptive method (55%; n = 65,103 women) and one-third (33%; n = 39,090 women) had no contraceptive claim. Women who used long-acting reversible contraceptive methods had 3.89 times the odds and women who used user-dependent hormonal methods had 1.89 times the odds of achieving an optimal birth interval compared with women who used barrier methods only; women with no method had 0.66 times the odds. When user-dependent methods are considered, the odds of having an optimal birth interval increased for each additional month of contraceptive coverage by 8% (odds ratio, 1.08; 95% confidence interval, 1.08-1.09). Women who were seen by Family PACT or by both Family PACT and Medi-Cal providers had significantly higher odds of optimal birth intervals compared with women who were served by Medi-Cal only. CONCLUSION: To achieve optimal birth spacing and ultimately to improve birth outcomes, attention should be given to contraceptive counseling and access to contraceptive methods in the postpartum period.

Extended Treatment with Glial Cell Line-Derived Neurotrophic Factor in Parkinson's Disease.

BACKGROUND: Intraputamenal glial cell line-derived neurotrophic factor (GDNF), administered every 4 weeks to patients with moderately advanced Parkinson's disease, did not show significant clinical improvements against placebo at 40 weeks, although it significantly increased [18F]DOPA uptake throughout the entire putamen. OBJECTIVE: This open-label extension study explored the effects of continued (prior GDNF patients) or new (prior placebo patients) exposure to GDNF for another 40 weeks. METHODS: Using the infusion protocol of the parent study, all patients received GDNF without disclosing prior treatment allocations (GDNF or placebo). The primary outcome was the percentage change from baseline to Week 80 in the OFF state Unified Parkinson's Disease Rating Scale (UPDRS) motor score. RESULTS: All 41 parent study participants were enrolled. The primary outcome decreased by 26.7±20.7% in patients on GDNF for 80 weeks (GDNF/GDNF; N = 21) and 27.6±23.6% in patients on placebo for 40 weeks followed by GDNF for 40 weeks (placebo/GDNF, N = 20; least squares mean difference: 0.4%, 95% CI: -13.9, 14.6, p = 0.96). Secondary endpoints did not show significant differences between the groups at Week 80 either. Prespecified comparisons between GDNF/GDNF at Week 80 and placebo/GDNF at Week 40 showed significant differences for mean OFF state UPDRS motor (-9.6±6.7 vs. -3.8±4.2 points, p = 0.0108) and activities of daily living score (-6.9±5.5 vs. -1.0±3.7 points, p = 0.0003). No treatment-emergent safety concerns were identified. CONCLUSIONS: The aggregate study results, from the parent and open-label extension suggest that future testing with GDNF will likely require an 80- rather than a 40-week randomized treatment period and/or a higher dose.

Postpartum care and contraception provided to women with gestational and preconception diabetes in California's Medicaid program.

OBJECTIVES: To compare rates of postpartum care and contraception provided to women with gestational or preconception diabetes mellitus to women with no known diabetes mellitus. METHODS: A retrospective cohort study of 199,860 women aged 15-44 years who were continuously enrolled in California's Medicaid program, Medi-Cal, from 43 days prior to 99 days after delivering in 2012. Claims for postpartum clinic visits and contraceptive supplies were compared for 11,494 mothers with preconception diabetes, 17,970 mothers with gestational diabetes, and 170,396 mothers without diabetes. Multivariable logistic regression was used to control for maternal age, race/ethnicity, primary language, residence in a primary care shortage area, state-funded healthcare program and Cesarean delivery, when examining the effects of diabetes on postpartum care and contraception. RESULTS: Although postpartum clinic visits were more common with diabetes (55% preconception, 55% gestational, 48% no diabetes, p=<.0001), almost half did not receive any postpartum care within 99 days of delivery. Women with pregnancies complicated by diabetes were more likely to receive permanent contraception than women without diabetes (preconception diabetes, aOR: 1.39, 95% CI: 1.31-1.47; gestational diabetes, aOR: 1.20, 95% CI: 1.14-1.27). However, among women without permanent contraception, less than half received any reversible contraception within 99 days of delivery (44% preconception, 43% gestational, 43% no diabetes) and less effective, barrier contraceptives were more commonly provided to women with preconception diabetes than women without diabetes (aOR: 1.24, 95% CI:1.16-1.33). CONCLUSIONS: Low-income Californian women with pregnancies complicated by diabetes do not consistently receive postpartum care or contraception that may prevent complication of future pregnancies. IMPLICATIONS: Efforts are needed to improve rates of provision of postpartum care and high quality contraceptive services to low income women in California, particularly following pregnancies complicated by diabetes.

Chromosomal instability selects gene copy-number variants encoding core regulators of proliferation in ER+ breast cancer.

Chromosomal instability (CIN) is associated with poor outcome in epithelial malignancies, including breast carcinomas. Evidence suggests that prognostic signatures in estrogen receptor-positive (ER(+)) breast cancer define tumors with CIN and high proliferative potential. Intriguingly, CIN induction in lower eukaryotic cells and human cells is context dependent, typically resulting in a proliferation disadvantage but conferring a fitness benefit under strong selection pressures. We hypothesized that CIN permits accelerated genomic evolution through the generation of diverse DNA copy-number events that may be selected during disease development. In support of this hypothesis, we found evidence for selection of gene amplification of core regulators of proliferation in CIN-associated cancer genomes. Stable DNA copy-number amplifications of the core regulators TPX2 and UBE2C were associated with expression of a gene module involved in proliferation. The module genes were enriched within prognostic signature gene sets for ER(+) breast cancer, providing a logical connection between CIN and prognostic signature expression. Our results provide a framework to decipher the impact of intratumor heterogeneity on key cancer phenotypes, and they suggest that CIN provides a permissive landscape for selection of copy-number alterations that drive cancer proliferation.

¹H, ¹³C and ¹5N resonance assignment of the N-terminal domain of human lysyl aminoacyl tRNA synthetase.

Human lysyl aminoacyl tRNA synthetase (hLysRS) is integral to a variety of different functions ranging from protein biosynthesis, initiation of a proinflammatory response as well as signal transduction. Another important, non-canonical function of hLysRS is that it chaperones tRNA(Lys,3), the HIV-1 reverse transcription primer molecule into new HIV-1 particles. Since the N-terminal domain of hLysRS has been shown to be essential for such primer uptake, NMR studies of this domain are being conducted to obtain a better understanding of how hLysRS interacts with the primer tRNA. In order to study the RNA binding behavior of this domain, we are studying its complex with a fragment of the cognate tRNA corresponding to the tRNA anticodon loop. We report herein the backbone and side chain NMR resonance assignments of uniformly (15)N-, (13)C-labeled hLysRS N-terminal domain alone, as well as complexed to RNA.

Postpartum contraception in publicly-funded programs and interpregnancy intervals.

OBJECTIVE: To assess the extent to which women received contraceptive services within 90 days after birth at their first or subsequent visits and whether contraceptive provision was associated with optimal interpregnancy intervals. METHOD: We linked California's 2008 Birth Statistical Master File with Medicaid databases to build a cohort of women aged 15-44 years who had given birth in 2008 and received publicly-funded health care services in the 18 months after their previous live birth (N=117,644). We determined whether provision of contraception within 90 days after birth was associated with optimal interpregnancy intervals when controlling for covariates. RESULT: Only 41% (n=48,775) of women had a contraceptive claim within 90 days after birth. To avoid short interpregnancy intervals, 6 women would need to receive contraception to avoid one additional short interval (number needed to treat=6.38). Receipt of a contraceptive method, receiving contraception at the first clinic visit, and being seen by Medi-Cal and its family planning expansion program were significantly associated with avoidance of short interpregnancy intervals. Receiving contraception at the first postpartum clinic visit had an additional independent effect on avoiding short interpregnancy intervals when controlling for the other variables. Although foreign-born women had 47% higher odds of avoiding short interpregnancy intervals than U.S.-born women, women of Asian and Pacific Islander ethnicity had 24% lower odds of avoiding short interpregnancy intervals than white women. CONCLUSION: Findings of this study suggest that closer attention to provision of postpartum contraception in publicly-funded programs has the potential to improve optimal interpregnancy intervals among low-income women. LEVEL OF EVIDENCE: II.