Uncovering changes in proteomic signature of rat pelvic floor muscles in pregnancy.

BackgroundStructural and functional changes of the rat pelvic floor muscles during pregnancy, specifically, sarcomerogenesis, increase in extracellular matrix content, and higher passive tension at larger strains protect the integral muscle components against birth injury. The mechanisms underlying these antepartum alterations are unknown. Quantitative proteomics is an unbiased method of identifying protein expression changes in differentially conditioned samples. Therefore, proteomics analysis provides an opportunity to identify molecular mechanisms underlying antepartum muscle plasticity.ObjectiveTo elucidate putative mechanisms accountable for pregnancy-induced adaptations of the pelvic floor muscles, and to identify other novel antepartum alterations of the pelvic floor muscles.Materials and methodsPelvic floor muscles, comprised of coccygeus, iliocaudalis, and pubocaudalis, and nonpelvic limb muscle, tibialis anterior, were harvested from 3-month-old nonpregnant and late-pregnant Sprague-Dawley rats. After tissue homogenization, trypsin-digested peptides were analyzed by ultra-high-performance liquid chromatography coupled with tandem mass spectroscopy using nano-spray ionization. Peptide identification and label free relative quantification analysis were carried out using Peaks Studio 8.5 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada). Proteomics data were visualized using the Qlucore Omics Explorer (New York, NY). Differentially expressed peptides were identified using the multi-group differential expression function, with q-value cutoff set at <0.05. Proteomic signatures of the pelvic floor muscles were compared to nonpelvic limb muscle and between nonpregnant and pregnant states.ResultsUnsupervised clustering of the data showed clear separation between samples from nonpregnant and pregnant animals along principal component 1 and between pelvic and nonpelvic muscles along principal component 2. Four major gene clusters were identified segregating proteomic signatures of muscles examined in nonpregnant vs pregnant states: (1) proteins increased in the pelvic floor muscles only; (2) proteins increased in the pelvic floor muscles and tibialis anterior; (3) proteins decreased in the pelvic floor muscles and tibialis anterior; and (4) proteins decreased in the pelvic floor muscles alone. Cluster 1 included proteins involved in cell cycle progression and differentiation. Cluster 2 contained proteins that participate in mitochondrial metabolism. Cluster 3 included proteins involved in transcription, signal transduction, and phosphorylation. Cluster 4 comprised proteins involved in calcium-mediated regulation of muscle contraction via the troponin tropomyosin complex.ConclusionPelvic floor muscles gain a distinct proteomic signature in pregnancy, which provides a mechanistic foundation for the antepartum physiological alterations acquired by these muscles. Variability in genes encoding these proteins may alter plasticity of the pelvic floor muscles and therefore the extent of the protective pregnancy-induced adaptations. Furthermore, pelvic floor muscles' proteome is divergent from that of the nonpelvic skeletal muscles

Comparison of reproducibility, accuracy, sensitivity, and specificity of miRNA quantification platforms

Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use

Unraveling Epigenetic Regulation in Embryonic Stem Cells

Embryonic stem (ES) cells can replicate indefinitely while retaining the capacity to differentiate into functionally distinct cell types. ES cells proliferate and differentiate without detectable genetic changes, indicating that these processes are controlled by epigenetic factors. Here we describe what is known about the epigenetics of ES cells and speculate that a dynamic balance among at least three epigenetic elements (chromatin structure, DNA methylation, and microRNAs), in conjunction with transcription factors, contributes to the maintenance of pluripotence. Understanding the interactions among these factors will be critical to the development of improved strategies to reprogram differentiated cells or direct differentiation of pluripotent cells

Developing a risk profile for spontaneous preterm birth and short interval to delivery among patients with threatened preterm labor

BACKGROUND: Threatened preterm birth is the most common reason for antepartum hospitalization in the United States, accounting for approximately 50% of these admissions. However, fewer than 10% of patients with inpatient evaluation for signs or symptoms of preterm labor ultimately deliver before term. OBJECTIVE: This study aimed to generate predictive models to assess the risk of preterm delivery and time to delivery based on clinical signs and symptoms of patients evaluated in our institution for preterm labor concerns. STUDY DESIGN: This was a retrospective cohort study of singleton pregnancies evaluated for signs and/or symptoms of preterm labor, including contractions, abdominal pain, vaginal bleeding, and short cervix, between 22 0/7 and 33 6/7 weeks of gestation. Inpatient evaluations were classified by patient presentation: (1) symptomatic with cervical findings (transvaginal cervical length of \u3c2.5 cm or cervical dilation of \u3e= 2.0 cm), (2) asymptomatic with cervical findings, and (3) symptomatic without cervical findings. The primary outcomes included incidence of spontaneous preterm birth and interval from presentation to delivery, compared between groups. The risk of preterm delivery was evaluated using logbinomial regression, and presentation to delivery timing was assessed by survival analysis and Cox proportional hazards modeling. RESULTS: Of 631 patients with preterm labor concerns, 96 (16%) were symptomatic with cervical findings on evaluation, 51 (8%) were asymptomatic with cervical findings, and 466 (76%) were symptomatic without cervical findings. The occurrence of preterm birth was significantly higher among symptomatic patients with cervical findings (49%) than among those with cervical findings alone (31%) or symptoms alone (11%) (P\u3c.0001). In addition, symptomatic patients with cervical findings were significantly more likely to deliver within 48 hours (20%), 1 week (30%), 2 weeks (33%), and 1 month (43%) of presentation than patients with cervical findings alone (2%, 2%, 6%, and 10%, respectively) or symptoms alone (0.4%, 1%, 1.5%, and 5%, respectively) (P value for trend\u3c.0001). Adjusted for gestational age at presentation and previous preterm birth, the overall risk of preterm delivery was significantly higher among patients with symptoms and cervical findings than among patients with cervical findings alone (relative risk, 2.81; 95% confidence interval, 1.74-4.54) or symptoms alone (relative risk, 4.39; 95% confidence interval, 3.16 -6.09). Adjusted for the same variables, symptomatic patients with cervical findings were also at higher risk of delivery over time after assessment than patients with cervical findings alone (hazard ratio, 2.06; 95% confidence interval, 1.47-2.90) or symptoms alone (hazard ratio, 2.16; 95% confidence interval, 1.74-2.70). The negative predictive value of these models suggested that only 1% of patients with isolated symptoms or cervical findings are at risk of preterm delivery within 1 week of initial presentation. CONCLUSION: Symptomatic patients with cervical findings suggestive of preterm labor were at the greatest risk of preterm birth and a shorter interval from presentation to delivery. The study findings supported a risk profile that may facilitate the selection of patients most appropriate for admission and targeted management. Nonetheless, as nearly 50% of patients meeting this risk profile subsequently deliver at term, future research is needed to identify which of these patients will require intervention

Residential proximity to traffic and female pubertal development

Traffic-related air pollution (TRAP) has been linked with several adverse health outcomes, including preterm birth and low birth weight, which are both related to onset of puberty. No studies to date have investigated the association between TRAP and altered pubertal timing

Mechanisms of nuclear content loading to exosomes.

Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development

Extracellular RNAs: development as biomarkers of human disease

Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth