Discovery and characterization of novel TRPML1 agonists.

Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.

Discovery of quinuclidine modulators of cellular progranulin.

Phenotypic screening of an annotated small molecule library identified the quinuclidine tetrahydroisoquinoline solifenacin (1) as a robust enhancer of progranulin secretion with single digit micromolar potency in a murine microglial (BV-2) cell line. Subsequent SAR development led to the identification of 29 with a 38-fold decrease in muscarinic receptor antagonist activity and a 10-fold improvement in BV-2 potency.

Cleavage and reduced CD36 ectodomain density on heart and spleen macrophages in the spontaneously hypertensive rat.

Metabolic disease is accompanied by a range of cellular defects ("comorbidities") whose origin is uncertain. To investigate this pathophysiological phenomenon we used the Spontaneously Hypertensive Rat (SHR), which besides an elevated arterial blood pressure also has many other comorbidities, including a defective glucose and lipid metabolism. We have shown that this model of metabolic disease has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of membrane receptors. We hypothesize here that the increased MMP activity also leads to abnormal cleavage of the scavenger receptor and fatty acid transporter CD36. To test this idea, chronic pharmaceutical MMP inhibition (CGS27023A) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to determine if inhibition of MMP activity serves to maintain CD36 receptor density and function. Surface density of CD36 on macrophages from the heart, spleen, and liver was determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry with an antibody against the CD36 ectodomain. The extracellular CD36 density was lower in SHR heart and spleen macrophages compared to that in the WKY. MMP inhibition by CGS served to restore the reduced CD36 density on SHR cardiac and splanchnic macrophages to levels of the WKY. To examine CD36 function, culture assays with murine macrophages (RAW 264.7) after incubation in fresh WKY or SHR plasma were used to test for adhesion of light-weight donor red blood cell (RBC) by CD36. This form of RBC adhesion to macrophages was reduced after incubation in SHR compared WKY plasma. Analysis of the supernatant macrophage media by Western blot shows a higher level of CD36 extracellular protein fragments following exposure to SHR plasma compared to WKY. MMP inhibition in the SHR plasma compared to untreated plasma, served to increase the RBC adhesion to macrophages and decrease the number of receptor fragments in the macrophage media. In conclusion, these studies bring to light that plasma in the SHR model of metabolic disease has an unchecked MMP degrading activity which causes cleavage of a variety of membrane receptors, including CD36, which attenuates several cellular functions typical for the metabolic disease, including RBC adhesion to the scavenger receptor CD36. In addition to other cell dysfunctions chronic MMP inhibition restores CD36 in the SHR.

Evaluating the frequency of crisis pregnancy center visits among a population of patients seeking abortions in Los Angeles.

OBJECTIVES: Studies have illustrated crisis pregnancy centers' (CPC) disingenuous practices regarding abortion services. We evaluated the proportion of patients who visited a CPC before their abortion in Los Angeles. STUDY DESIGN: Patients attending one of two abortion clinics between July 2019 and March 2020 were recruited for a survey that examined patient characteristics and locations of care. RESULTS: Five hundred eleven respondents participated (62% response rate). Less than 1% of individuals visited a CPC before their abortion. Fifty-three percent of survey respondents obtained their abortion within one visit, 39% within two unique clinic visits, and eight percent visited three or more clinics. CONCLUSION: CPC visits before abortion were uncommon in our patient population.

Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.

Receptor cleavage reduces the fluid shear response in neutrophils of the spontaneously hypertensive rat.

Physiological fluid shear stress evokes pseudopod retraction in normal leukocytes by a mechanism that involves the formyl peptide receptor (FPR) as mechanosensor. In hypertensives, such as the spontaneously hypertensive rat (SHR), leukocytes lack the normal fluid shear response. The increased activity of matrix metalloproteinases (MMPs, including MMP-9) in SHR plasma is associated with cleavage of several cell membrane receptors. We hypothesize that the attenuated fluid shear response in leukocytes (neutrophils) of the SHR is due to extracellular proteolytic cleavage of the FPR. We show that suspended SHR neutrophils in whole blood sheared in a cone-and-plate device or individual neutrophils adherent to a glass surface and subject to fluid shear exhibited reduced pseudopod retractions compared with neutrophils of control Wistar-Kyoto (WKY) rats. SHR neutrophils and naïve Wistar rat neutrophils exposed to SHR plasma also exhibited impaired fluid shear responses as shown by their inability to project pseudopods with fluid shear. Labeling of extracellular FPR revealed that the FPR density in SHR neutrophils is on average 27% reduced compared with those of the WKY rats. Exposure of Wistar rat neutrophils to the gelatinase MMP-9 (final concentration 5 nM) led to attenuation of fluid shear response and decrease in extracellular FPR density. Chronic treatment of the SHR with a broad-acting MMP inhibitor, doxycycline, significantly improved the fluid shear response and increased the FPR extracellular density of SHR neutrophils. These results suggest that proteolytic cleavage of the FPR may interfere with normal fluid shear-induced pseudopod retractions in SHR neutrophils.

Comparison of complications associated with induction by misoprostol versus dilation and evacuation for second-trimester abortion.

OBJECTIVE: To compare the frequency of complications between medical induction with misoprostol and dilation and evacuation (D&E) for second-trimester uterine evacuation. METHODS: In a retrospective cohort study, records were reviewed from all women who underwent second-trimester uterine evacuation between 14 and 24 weeks of pregnancy at an academic hospital in the USA from January 2007 to December 2008. Total complications and serious complications were compared between medical induction and D&E, and clinical covariates associated with complications were assessed. RESULTS: Overall, 465 women were included. At least one complication occurred in 28 (23.0%) of 122 women in the medical induction group, and 24 (7.0%) of 343 women in the D&E group (P<0.001). One or more serious complications occurred in 4 (3.3%) women undergoing medical induction and 5 (1.5%) undergoing D&E (P=0.251). Logistic regression analysis demonstrated that increasing pregnancy length (P=0.003) and medical induction (as compared with D&E; P=0.004) were associated with complications. CONCLUSION: Although D&E resulted in fewer overall complications as compared with medical induction, the rate of serious complications did not differ between the two procedures.

Reproductive healthcare experiences of women with cerebral palsy.

BACKGROUND: Little is known about pregnancy rates in women with disabilities in general and even less is known about women with child-onset disabilities such as cerebral palsy (CP). HYPOTHESIS: We hypothesized that discussions about pregnancy with healthcare providers and pregnancy rates for woman with CP would be related to their functional levels. METHODS: Survey methodology was used to gather information about demographics, function, whether women were asked about their desire for children, pregnancy outcomes, and services offered during pregnancy and postpartum. RESULTS: Of the 375 women with CP who participated in the survey, 76 (20%) reported 149 pregnancies resulting in 100 live births. Using Chi square statistics, mobility, manual dexterity, and communication function were significantly higher in women who were queried about or who experienced pregnancy. More than half of the women experienced a loss of mobility during pregnancy but few received referrals for physical or occupational therapy. Few reported screening for postpartum depression. A higher rate of Cesarean sections (50.4%), preterm births (12.1%), low birth weight infants (15.7%), and very low birth weight infants (7.1%) was reported by women with CP compared to national statistics. CONCLUSIONS: Pregnancy rates and discussions were related to functional levels. As 20% of women with CP surveyed experienced pregnancy, there is a need to increase awareness, education, support, and advocacy for achievement of optimal reproductive health. More research is needed to identify factors contributing to maternal and infant health in women with CP.

Bleeding after medication-induced termination of pregnancy with two dosing schedules of mifepristone and misoprostol.

OBJECTIVE: This study examines bleeding pattern following medication-induced termination of pregnancy, comparing two different dosing schedules of mifepristone and misoprostol. STUDY DESIGN: Diary information was analyzed from a randomized, multicenter trial in which women used vaginal misoprostol 800 mug either 6-8 or 24 h following 200 mg of oral mifepristone. PARTICIPANTS AND METHODS: One thousand eighty women with pregnancies up to 63 days' gestation were recruited for the study; 540 were randomized to the 6- to 8-h dosing schedule, and 540 were randomized to the 24-h dosing schedule. Subjects recorded daily bleeding in a diary over 5 weeks. RESULTS: Total duration of bleeding ranged from 1 to 54 days, with a median of 7 days. Duration of spotting ranged from 1 to 80 days, with a median of 5 and 6 days (NS) in each of the two groups. Neither duration of bleeding nor duration of spotting were related to interval between mifepristone and misoprostol. Bleeding and spotting durations were not correlated with maternal age or smoking. Increased gestational age was correlated with longer bleeding and spotting times. Nulliparity was associated with longer bleeding time. CONCLUSION: Varying the interval between mifepristone and misoprostol in medication abortion does not affect duration or quantity of bleeding.

A randomized clinical trial of the addition of laminaria to misoprostol and hypertonic saline for second-trimester induction abortion.

OBJECTIVE: To compare the outcomes of second-trimester induction abortion with misoprostol and hypertonic saline, with and without use of laminaria. METHOD: Fifty-eight women, between 17.5 and 22.5 weeks' gestation, were randomly assigned to receive or omit laminaria in conjunction with other procedures for induction abortion. All women received a fetocidal dose of 60 cc intra-amniotic hypertonic saline. If the woman was to receive laminaria, they were inserted next. This was followed by vaginal misoprostol 200 mug, which was repeated every 6 h. RESULT: Women with laminaria inserted before misoprostol administration had longer intervals from start of misoprostol to delivery of the fetus (induction times) than women without laminaria. Induction time was 14.4 vs. 11.4 h, respectively (p=.04, Wilcoxon rank sum test). Total misoprostol use was higher in the laminaria group, 628 mug (95% CI, 516-738) vs. 496 mug (95% CI, 419-573) (p=.05). Total analgesic use was also higher in the laminaria group, 41 mg of morphine (95% CI, 32-50) vs. 26 mg of morphine (95% CI, 18-32) (p=.02). CONCLUSION: Laminaria use, in conjunction with misoprostol and hypertonic saline, significantly prolongs induction time and increases narcotic analgesia usage.

Medical Compared With Surgical Abortion for Effective Pregnancy Termination in the First Trimester.

OBJECTIVE: To compare efficacy between medical and surgical abortion at 9 weeks of gestation or less. METHODS: We performed a retrospective cohort study comparing efficacy of medical compared with surgical abortion before 64 days of gestation at Planned Parenthood, Los Angeles, from November 2010 to August 2013. Electronic medical records were reviewed for ongoing pregnancies after the initial abortion procedure. Data were also collected on complications occurring within the immediate postabortal period (8 weeks postabortal) including unanticipated aspiration and major adverse events (emergency department presentation, hospitalization, perforation, transfusion, infection). Chi square test and logistic regression were used to compare the primary outcomes between cohorts. RESULTS: Data were collected from 30,146 women with pregnancies seeking termination before 64 days of gestation. Sociodemographic and clinical characteristics were similar in the medication and surgical abortion groups. Efficacy of pregnancy termination was 99.6% for medication abortions and 99.8%% for surgical abortions (P<.001). The medication abortion group was more likely to undergo an unanticipated aspiration, for ongoing pregnancy or persistent pain, bleeding, or both (2.1% compared with 0.6%, respectively, odds ratio 1.6, 95% confidence interval 1.1-2.3). These rates were unchanged after controlling for gravidity, parity, and body mass index. There was no difference in major adverse events between the two groups. CONCLUSION: Medication abortion and surgical abortion before 64 days of gestation are both highly effective with low complication rates. LEVEL OF EVIDENCE: II.

Circulating cell-free fetal nucleic acid analysis may be a novel marker of fetomaternal hemorrhage after elective first-trimester termination of pregnancy.

Analysis of cell-free fetal DNA (fDNA) and RNA in maternal plasma could be useful in the diagnosis and management of complications of pregnancy. In this review, we discuss our studies to investigate the potential of fetal nucleic acid measurement in maternal plasma as a marker of fetomaternal hemorrhage (FMH) after elective first-trimester termination of pregnancy (TOP). Using quantitative real-time PCR amplification of the DYS1 sequence, elevation of plasma fDNA levels after TOP was observed, especially in the late first trimester. This corresponds with the functional development of the placental vascular structure and fetal hematopoiesis. This Y sequence-based PCR amplification assay, however, limits the analysis to pregnant women carrying male fetuses. Therefore, we also developed a real-time quantitative reverse-transcriptase PCR assay of the gamma-globin transcript as a marker of fetal erythroid cells. Although plasma gamma-globin mRNA levels were decreased after TOP in many patients, an elevation was observed in some patients at greater than 9 weeks' gestation, which is consistent with the increase in plasma fDNA levels. Our data suggest that fetal hematopoietic cells contribute to the pool of fetal nucleic acids in the maternal circulation. Measurement of cell-free fetal nucleic acid levels in maternal plasma may have clinical application as a novel marker of FMH after 9 weeks of gestation.

Cell-free fetal DNA levels in maternal plasma after elective first-trimester termination of pregnancy.

OBJECTIVE: To determine if first-trimester elective termination of pregnancy affects cell-free fetal DNA (fDNA) levels in maternal plasma. DESIGN: Prospective cohort study. SETTING: Clinical and academic research centers. PATIENT(S): One hundred thirty-four women who underwent first-trimester elective termination procedures. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Real-time polymerase chain reaction (PCR) amplification and measurement of DYS1, a Y-chromosome sequence, was used as a marker of fDNA. RESULT(S): We detected fDNA in pretermination samples from 27 out of 71 patients in the surgical arm, and 29 out of 63 patients in the medical arm. Based on confirmation of male gender in placental tissue, the sensitivity of fDNA detection is 92.6%. We detected fDNA as early as 32 days of gestation, which increased 4.2 genome equivalents/mL/week. In the surgical arm, the mean level of posttermination fDNA, adjusted for the clearance of fDNA in maternal blood, was higher than projected based on an expected increase with gestational age. In the medical arm, six patients had increased fDNA levels up to 11 days following termination. CONCLUSION(S): We found that fDNA can be reliably quantified in the early first trimester; fDNA elevation that occurs shortly after surgical termination may reflect fetomaternal hemorrhage or destruction of trophoblastic villi. Continued elevation of fDNA for several days may occur following medical termination.

Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat.

The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression.

mTORC1 signaling and regulation of pancreatic ß-cell mass.

The capacity of ß cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of ß cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for ß-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of ß-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in ß cells. This review will discuss recent advances in the understanding of how this pathway regulates ß-cell mass and present data on the role of TSC1 in modulation of ß-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic ß cells results in improved glucose tolerance, hyperinsulinemia and expansion of ß-cell mass that persists with aging.

Proteolytic Cleavage of the Red Blood Cell Glycocalyx in a Genetic Form of Hypertension.

Recent evidence suggests that the spontaneously hypertensive rat (SHR) has an elevated level of proteases, including matrix metalloproteinases (MMPs), involved in cell membrane receptor cleavage. We hypothesize that SHR red blood cells (RBCs) may be subject to an enhanced glycocalyx cleavage compared to the RBCs of the normotensive Wistar-Kyoto (WKY) rats. By direct observation of RBC rouleaux, we found no significant difference in RBC aggregation for unseparated SHR and WKY RBCs. However, lighter SHR RBCs have a greater tendency to aggregate than WKY RBCs when separated by centrifugation. When SHR plasma was mixed with WKY RBCs, SHR plasma proteases cleaved the glycocalyx of WKY RBCs, a process that can be blocked by MMP inhibition. When treated with MMPs, WKY RBCs showed strong aggregation in dextran but not in fibrinogen, indicating that RBC membrane glycoproteins from the inner core of the glycocalyx were cleaved and that dextran was able to bind to the lipid portion of the RBC membrane. In contrast, treatment with amylases produced fibrinogen-induced aggregation with fibrinogen binding to the protein core. MMP cleavage of RBC glycocalyx reduces RBC adhesion to macrophages as a mechanism to remove old RBCs from the circulation.

Microfabricated arrays of cylindrical wells facilitate single-molecule enzymology of alpha-chymotrypsin.

Single-molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single-molecule enzymatic kinetics and provide comparisons to ensemble-averaged kinetics. To isolate our model enzyme, alpha-chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 microm in diameter and 1.35 microm in height. Inside the wells, a protease assay with a profluorescent substrate detects alpha-chymotrypsin activity. We hold the concentration of alpha-chymotrypsin at 0.39 nM in a given well with an enzyme-to-substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge-coupled device. This method allows for the simultaneous real-time characterization of hundreds of individual enzymes. We analyze single-molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity.

Plasma gamma-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy.

BACKGROUND: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for gamma-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma. METHODS: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma gamma-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample. RESULTS: gamma-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P <0.01). The concentrations of gamma-globin mRNA were decreased in most women after TOP, but gamma-globin mRNA was increased in some patients when TOP was performed later than 9 weeks of gestation. CONCLUSIONS: gamma-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma gamma-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination gamma-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of gamma-globin mRNA as a marker for fetomaternal hemorrhage.