Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4–5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III₂ and Apo C-III₁ (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r²>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III₀ showed no correlation (r² = 0.01–0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.The maternal physiology during pregnancy is characterized by inflammation and hyperlipidemia. Plasma protein composition fluctuates dynamically throughout gestation to reflect these physiological changes. Apolipoproteins, a diverse subset of triglyceride transport proteins, contribute to the hyperlipidemia of pregnancy by modulating lipid homeostasis in maternal plasma (1–3). Exaggerated hyperlipidemia and peripheral apolipoprotein burden are associated with inflammatory insult and signal obstetric complications (4–5). Numerous post-translationally modified apolipoprotein isoforms are reported in plasma, but it is unclear how these modifications affect apolipoprotein function and plasma distribution. For example, changes in the glycosylation status of apolipoprotein variants predate the onset of clinical symptoms in patients with preeclampsia, a hypertensive disorder of pregnancy with clinical features in common with cardiovascular disease (6–8). The identification and functional characterization of plasma apolipoprotein isoforms and their post-translationally modified subtypes may reveal important diagnostic and/or therapeutic targets for hypertensive disorders of pregnancy (6).Mass spectrometry and targeted proteomics analyses afford unprecedented sensitivity and specificity for detecting apolipoproteins and their numerous isoforms and subtypes (9–12). Mass spectrometry approaches overcome limitations inherent in biochemical approaches (e.g. ELISA [enzyme-linked immunosorbant assays] and Western blot analysis), especially the lack of specificity of antibodies for post-translationally modified variants of plasma proteins. The objective of this study was to longitudinally evaluate maternal plasma apolipoprotein profile over gestational time by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)¹ analysis of intact proteins and a complementary targeted LTQ-Orbitrap XL MS approach. We evaluate changes in 13 post-translationally modified subtypes of the plasma apolipoproteins A-II, C-I, C-II, and C-III over gestational time.     

Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland,the gland of the third eyelid and the knee joint

Background aimsMesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs.MethodsAllogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation.ResultsClinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations.ConclusionsThis is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.     

Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.     

Effects of Maternal Obstructive Sleep Apnoea on Fetal Growth: A Prospective Cohort Study

Objective

The objective of this study is to determine whether obstructive sleep apnea (OSA) is associated with reduced fetal growth, and whether nocturnal oxygen desaturation precipitates acute fetal heart rate changes.

Study Design

We performed a prospective observational study, screening 371 women in the second trimester for OSA symptoms. 41 subsequently underwent overnight sleep studies to diagnose OSA. Third trimester fetal growth was assessed using ultrasound. Fetal heart rate monitoring accompanied the sleep study. Cord blood was taken at delivery, to measure key regulators of fetal growth.

Results

Of 371 women screened, 108 (29%) were high risk for OSA. 26 high risk and 15 low risk women completed the longitudinal study; 14 had confirmed OSA (cases), and 27 were controls. The median (interquartile range) respiratory disturbance index (number of apnoeas, hypopnoeas or respiratory related arousals/hour of sleep) was 7.9 (6.1–13.8) for cases and 2.2 (1.3–3.5) for controls (p<0.001). Impaired fetal growth was observed in 43% (6/14) of cases, vs 11% (3/27) of controls (RR 2.67; 1.25–5.7; p = 0.04). Using logistic regression, only OSA (OR 6; 1.2–29.7, p = 0.03) and body mass index (OR 2.52; 1.09–5.80, p = 0.03) were significantly associated with impaired fetal growth. After adjusting for body mass index on multivariate analysis, the association between OSA and impaired fetal growth was not appreciably altered (OR 5.3; 0.93–30.34, p = 0.06), although just failed to achieve statistical significance. Prolonged fetal heart rate decelerations accompanied nocturnal oxygen desaturation in one fetus, subsequently found to be severely growth restricted. Fetal growth regulators showed changes in the expected direction- with IGF-1 lower, and IGFBP-1 and IGFBP-2 higher- in the cord blood of infants of cases vs controls, although were not significantly different.

Conclusion

OSA may be associated with reduced fetal growth in late pregnancy. Further evaluation is warranted to establish whether OSA may be an important contributor to adverse perinatal outcome, including stillbirth.     

Genetic and Non-Genetic Determinants of Raltegravir Penetration into Cerebrospinal Fluid: A Single Arm Pharmacokinetic Study

Background

Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C→T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C→T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.

Methods

Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.

Results

The 4-hour CSF concentration correlated more strongly with 2-hour (r²=0.76, P=1.12x10^-11) than 4-hour (r²=0.47, P=6.89x10^-6) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC_0-4h r²=0.86, P=5.15x10^-16). There was no significant association between ABCB1 3435C→T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.

Conclusions

Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C→T genotype but strongly correlate with plasma exposure.

Trial Registration

ClinicalTrials.gov NCT00729924 http://clinicaltrials.gov/show/NCT00729924     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.     

Task-Based Core-Periphery Organization of Human Brain Dynamics

As a person learns a new skill, distinct synapses, brain regions, and circuits are engaged and change over time. In this paper, we develop methods to examine patterns of correlated activity across a large set of brain regions. Our goal is to identify properties that enable robust learning of a motor skill. We measure brain activity during motor sequencing and characterize network properties based on coherent activity between brain regions. Using recently developed algorithms to detect time-evolving communities, we find that the complex reconfiguration patterns of the brain''s putative functional modules that control learning can be described parsimoniously by the combined presence of a relatively stiff temporal core that is composed primarily of sensorimotor and visual regions whose connectivity changes little in time and a flexible temporal periphery that is composed primarily of multimodal association regions whose connectivity changes frequently. The separation between temporal core and periphery changes over the course of training and, importantly, is a good predictor of individual differences in learning success. The core of dynamically stiff regions exhibits dense connectivity, which is consistent with notions of core-periphery organization established previously in social networks. Our results demonstrate that core-periphery organization provides an insightful way to understand how putative functional modules are linked. This, in turn, enables the prediction of fundamental human capacities, including the production of complex goal-directed behavior.