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1.  Prenatal stress changes the glycoprotein GPM6A gene expression and induces epigenetic changes in rat offspring brain 
Epigenetics  2013;9(1):152-160.
Prenatal stress (PS) exerts strong impact on fetal brain development and on adult offspring brain functions. Previous work demonstrated that chronic stress alters the mRNA expression of GPM6A, a neuronal glycoprotein involved in filopodium extension. In this work, we analyzed the effect of PS on gpm6a expression and the epigenetic mechanisms involved. Pregnant Wistar rats received restraint stress during the last week of gestation. Male offspring were sacrificed on postnatal days 28 and 60. Hippocampus and prefrontal cortex samples were analyzed for gene expression (qPCR for mRNAs and microRNAs), methylation status (bisulfite conversion) and protein levels. Hippocampal neurons in culture were used to analyze microRNA overexpression effects. Prenatal stress induced changes in gpm6a levels in both tissues and at both ages analyzed, indicating a persistent effect. Two CpG islands in the gpm6a gene were identified. Variations in the methylation pattern at three specific CpGs were found in hippocampus, but not in PFC samples from PS offspring. microRNAs predicted to target gpm6a were identified in silico. qPCR measurements showed that PS modified the expression of several microRNAs in both tissues, being microRNA-133b the most significantly altered. Further studies overexpressing this microRNA in neuronal cultures showed a reduction in gmp6a mRNA and protein level. Moreover filopodium density was also reduced, suggesting that GPM6A function was affected. Gestational stress affected gpm6a gene expression in offspring likely through changes in methylation status and in posttranscriptional regulation by microRNAs. Thus, our findings propose gpm6a as a novel target for epigenetic regulation during prenatal stress.
PMCID: PMC3928178  PMID: 23959066
hippocampus; prefrontal cortex; DNA methylation pattern; CpG island; microRNA; target site blocker
2.  Impact of Ventilatory Modes on the Breathing Variability in Mechanically Ventilated Infants 
Objectives: Reduction of breathing variability is associated with adverse outcome. During mechanical ventilation, the variability of ventilatory pressure is dependent on the ventilatory mode. During neurally adjusted ventilatory assist (NAVA), the support is proportional to electrical activity of the diaphragm (EAdi), which reflects the respiratory center output. The variability of EAdi is, therefore, translated into a similar variability in pressures. Contrastingly, conventional ventilatory modes deliver less variable pressures. The impact of the mode on the patient’s own respiratory drive is less clear. This study aims to compare the impact of NAVA, pressure-controlled ventilation (PCV), and pressure support ventilation (PSV) on the respiratory drive patterns in infants. We hypothesized that on NAVA, EAdi variability resembles most of the endogenous respiratory drive pattern seen in a control group.
Methods: Electrical activity of the diaphragm was continuously recorded in 10 infants ventilated successively on NAVA (5 h), PCV (30 min), and PSV (30 min). During the last 10 min of each period, the EAdi variability pattern was assessed using non-rhythmic to rhythmic (NRR) index. These variability profiles were compared to the pattern of a control group of 11 spontaneously breathing and non-intubated infants.
Results: In control infants, NRR was higher as compared to mechanically ventilated infants (p < 0.001), and NRR pattern was relatively stable over time. While the temporal stability of NRR was similar in NAVA and controls, the NRR profile was less stable during PCV. PSV exhibited an intermediary pattern.
Perspectives: Mechanical ventilation impacts the breathing variability in infants. NAVA produces EAdi pattern resembling most that of control infants. NRR can be used to characterize respiratory variability in infants. Larger prospective studies are necessary to understand the differential impact of the ventilatory modes on the cardio-respiratory variability and to study their impact on clinical outcomes.
PMCID: PMC4242927  PMID: 25505779
pediatric intensive care; mechanical ventilation; neurally adjusted ventilatory support; diaphragm; children
3.  Online Detection of Fetal Acidemia during Labour by Testing Synchronization of EEG and Heart Rate: A Prospective Study in Fetal Sheep 
PLoS ONE  2014;9(9):e108119.
Severe fetal acidemia during labour can result in life-lasting neurological deficits, but the timely detection of this condition is often not possible. This is because the positive predictive value (PPV) of fetal heart rate (FHR) monitoring, the mainstay of fetal health surveillance during labour, to detect concerning fetal acidemia is around 50%. In fetal sheep model of human labour, we reported that severe fetal acidemia (pH<7.00) during repetitive umbilical cord occlusions (UCOs) is preceded ∼60 minutes by the synchronization of electroencephalogram (EEG) and FHR. However, EEG and FHR are cyclic and noisy, and although the synchronization might be visually evident, it is challenging to detect automatically, a necessary condition for bedside utility. Here we present and validate a novel non-parametric statistical method to detect fetal acidemia during labour by using EEG and FHR. The underlying algorithm handles non-stationary and noisy data by recording number of abnormal episodes in both EEG and FHR. A logistic regression is then deployed to test whether these episodes are significantly related to each other. We then apply the method in a prospective study of human labour using fetal sheep model (n = 20). Our results render a PPV of 68% for detecting impending severe fetal acidemia ∼60 min prior to pH drop to less than 7.00 with 100% negative predictive value. We conclude that this method has a great potential to improve PPV for detection of fetal acidemia when it is implemented at the bedside. We outline directions for further refinement of the algorithm that will be achieved by analyzing larger data sets acquired in prospective human pilot studies.
PMCID: PMC4182309  PMID: 25268842
4.  Acceleration and Deceleration Capacity of Fetal Heart Rate in an In-Vivo Sheep Model 
PLoS ONE  2014;9(8):e104193.
Fetal heart rate (FHR) variability is an indirect index of fetal autonomic nervous system (ANS) integrity. FHR variability analysis in labor fails to detect early hypoxia and acidemia. Phase-rectified signal averaging (PRSA) is a new method of complex biological signals analysis that is more resistant to non-stationarities, signal loss and artifacts. It quantifies the average cardiac acceleration and deceleration (AC/DC) capacity.
The aims of the study were: (1) to investigate AC/DC in ovine fetuses exposed to acute hypoxic-acidemic insult; (2) to explore the relation between AC/DC and acid-base balance; and (3) to evaluate the influence of FHR decelerations and specific PRSA parameters on AC/DC computation.
Repetitive umbilical cord occlusions (UCOs) were applied in 9 pregnant near-term sheep to obtain three phases of MILD, MODERATE, and SEVERE hypoxic-acidemic insult. Acid-base balance was sampled and fetal ECGs continuously recorded. AC/DC were calculated: (1) for a spectrum of T values (T = 1÷50 beats; the parameter limits the range of oscillations detected by PRSA); (2) on entire series of fetal RR intervals or on “stable” series that excluded FHR decelerations caused by UCOs.
AC and DC progressively increased with UCOs phases (MILD vs. MODERATE and MODERATE vs. SEVERE, p<0.05 for DC  = 2–5, and AC  = 1–3). The time evolution of AC/DC correlated to acid-base balance (0.4<<0.9, p<0.05) with the highest for . PRSA was not independent from FHR decelerations caused by UCOs.
This is the first in-vivo evaluation of PRSA on FHR analysis. In the presence of acute hypoxic-acidemia we found increasing values of AC/DC suggesting an activation of ANS. This correlation was strongest on time scale dominated by parasympathetic modulations. We identified the best performing parameters (), and found that AC/DC computation is not independent from FHR decelerations. These findings establish the basis for future clinical studies.
PMCID: PMC4139279  PMID: 25141131
5.  Putative Role of AMPK in Fetal Adaptive Brain Shut-Down: Linking Metabolism and Inflammation in the Brain 
PMCID: PMC4127551  PMID: 25157238
fetus; neuroinflammation; EEG; microglia; neurons; metabolism; sheep
6.  Adaptive Brain Shut-Down Counteracts Neuroinflammation in the Near-Term Ovine Fetus 
Objective: Repetitive umbilical cord occlusions (UCOs) in ovine fetus leading to severe acidemia result in adaptive shut-down of electrocortical activity [electrocorticogram (ECoG)] as well as systemic and brain inflammation. We hypothesized that the fetuses with earlier ECoG shut-down as a neuroprotective mechanism in response to repetitive UCOs will show less brain inflammation and, moreover, that chronic hypoxia will impact this relationship.
Methods: Near-term fetal sheep were chronically instrumented with ECoG leads, vascular catheters, and a cord occluder and then underwent repetitive UCOs for up to 4 h or until fetal arterial pH was <7.00. Eight animals, hypoxic prior to the UCOs (SaO2 <55%), were allowed to recover 24 h post insult, while 14 animals, 5 of whom also were chronically hypoxic, were allowed to recover 48 h post insult, after which brains were perfusion-fixed. Time of ECoG shut-down and corresponding pH were noted, as well as time to then reach pH <7.00 (ΔT). Microglia (MG) were counted as a measure of inflammation in gray matter layers 4–6 (GM4–6) where most ECoG activity is generated. Results are reported as mean ± SEM for p < 0.05.
Results: Repetitive UCOs resulted in worsening acidosis over 3–4 h with arterial pH decreasing to 6.97 ± 0.02 all UCO groups’ animals, recovering to baseline by 24 h. ECoG shut-down occurred 52 ± 7 min before reaching pH <7.00 at pH 7.23 ± 0.02 across the animal groups. MG counts were inversely correlated to ΔT in 24 h recovery animals (R = −0.84), as expected. This was not the case in normoxic 48 h recovery animals, and, surprisingly, in hypoxic 48 h recovery animals, this relationship was reversed (R = 0.90).
Conclusion: Adaptive brain shut-down during labor-like worsening acidemia counteracts neuroinflammation in a hypoxia- and time-dependent manner.
PMCID: PMC4074896  PMID: 25071698
fetus; microglia; ECoG; EEG; hypoxia; acidemia; labor; sheep
7.  Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila 
The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles.
Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis.
Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.
PMCID: PMC4068974  PMID: 24935095
Extracellular matrix; Heart tube; Alary muscles; Laminin; Collagen
8.  Sampling Rate of Heart Rate Variability Impacts the Ability to Detect Acidemia in Ovine Fetuses Near-Term 
Background: To evaluate the impact of sampling rate on the predictive capability of continuous fetal heart rate (FHR) variability (fHRV) monitoring for detecting fetal acidemia during labor, we tested the performance of the root mean square of successive differences (RMSSD) in R–R intervals from the ECG when acquired with the sampling rate of 4 Hz currently available in FHR monitors, in comparison to the gold standard of 1000 Hz.
Methods: Near-term ovine fetuses (N = 9) were chronically prepared with precordial electrodes for recording ECG, vascular catheters for blood sampling, and an umbilical cord occluder. For 1 min every 2.5 min, animals underwent mild partial umbilical cord occlusions (UCO) × 1 h, moderate partial UCO × 1 h, then complete UCO × 2 h, or until arterial pH reached <7.00. Arterial blood samples were drawn at baseline and every 20 min during the UCO series. RMSSD was calculated continuously in 5 min windows using an automated, standardized system ( Results are presented as mean ± SEM with significance assumed for p < 0.05.
Results: Repetitive UCO resulted in pH decreasing from 7.35 ± 0.01 to 7.00 ± 0.03. In all nine animals, RMSSD increased from 16.7 ± 1.0 ms at baseline to 44.4 ± 2.3 ms, 70 ± 15 min prior to reaching the pH nadir when sampled at 1000 Hz. When sampled at 4 Hz, RMSSD at baseline measured 36.1 ± 6.0 ms and showed no significant increase during the UCO series until the pH nadir was reached. Consequently, early detection of severe hypoxic-acidemia would have been missed in all fetuses.
Conclusion: RMSSD as a measure of fHRV when calculated from FHR sampled at 1000 Hz allowed for the early detection of worsening hypoxic-acidemia in each fetus. However, when calculated at the low sampling rate of 4 Hz used clinically, RMSSD remained unchanged until terminally when the nadir pH was reached. For early detection of fetal acidemia during labor, more sensitive means of acquiring FHR are therefore recommended than currently deployed, e.g., trans-abdominal fetal ECG.
PMCID: PMC4017161  PMID: 24829897
fetus; fHRV; monitoring; acidosis; asphyxia; hypoxia; sampling rate
9.  Org-1 is required for the diversification of circular visceral muscle founder cells and normal midgut morphogenesis 
Developmental biology  2013;376(2):245-259.
The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut.
PMCID: PMC3602240  PMID: 23380635
visceral mesoderm patterning; midgut morphogenesis; T-box transcription factor; combinatorial enhancer binding
10.  Impaired Mouse Mammary Gland Growth and Development is Mediated by Melatonin and its MT1 G Protein-Coupled Receptor via Repression of ERα, Akt1, and Stat5 
Journal of pineal research  2012;53(3):307-318.
To determine if melatonin, via its MT1 G protein-coupled receptor (GPCR), impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT1-Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud (TEB) formation. Elevated MT1 receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT1 expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha (ERα), progesterone receptors (PR) A and B, and milk proteins β-casein and whey acidic protein (WAP). Estrogen and progesterone stimulated mammary gland development was repressed by elevated MT1 receptor expression and exogenous melatonin administration. These studies demonstrate that the MT1 melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.
PMCID: PMC3422609  PMID: 22582905
Melatonin; MT1 Receptor; AKT; Stat5; Mammary Gland Development
11.  Improved bioavailability of inhibitors of Trypanosoma cruzi trans-sialidase: PEGylation of lactose analogs with multiarm polyethyleneglycol 
Glycobiology  2012;22(10):1363-1373.
The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal β-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing β-d-Galp as the non-reducing unit. (1) Amide formation between benzyl β-d-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-d-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40 kDa) were used for each sugar. Substitution of all arms was proved by 1H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5 kDa linear PEG conjugates retaining their inhibitory properties.
PMCID: PMC3425324  PMID: 22653661
inhibitors; multiarm conjugates; PEGylation; trans-sialidase; Trypanosoma cruzi
12.  Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36 
PLoS ONE  2013;8(8):e72772.
Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.
Methodology/Principal Findings
In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.
Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.
PMCID: PMC3732218  PMID: 23936544
13.  Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: differences and similarities with its orthologue in Trypanosoma cruzi 
Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0–7.8 resembling the T. cruzi enzyme. S everal divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns.
PMCID: PMC3383389  PMID: 22575602
Trypanosoma brucei; carboxypeptidase; M32 family; FRET peptides; Trypanosoma cruzi; peptidase
14.  The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells 
Developmental biology  2012;368(1):28-43.
Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.
PMCID: PMC3589571  PMID: 22609944
Cell migration; Cell survival; Fibroblast growth factor; Visceral mesoderm; Gut muscle development
15.  Genome-wide analysis of 3′-untranslated regions supports the existence of post-transcriptional regulons controlling gene expression in trypanosomes 
PeerJ  2013;1:e118.
In eukaryotic cells, a group of messenger ribonucleic acids (mRNAs) encoding functionally interrelated proteins together with the trans-acting factors that coordinately modulate their expression is termed a post-transcriptional regulon, due to their partial analogy to a prokaryotic polycistron. This mRNA clustering is organized by sequence-specific RNA-binding proteins (RBPs) that bind cis-regulatory elements in the noncoding regions of genes, and mediates the synchronized control of their fate. These recognition motifs are often characterized by conserved sequences and/or RNA structures, and it is likely that various classes of cis-elements remain undiscovered. Current evidence suggests that RNA regulons govern gene expression in trypanosomes, unicellular parasites which mainly use post-transcriptional mechanisms to control protein synthesis. In this study, we used motif discovery tools to test whether groups of functionally related trypanosomatid genes contain a common cis-regulatory element. We obtained conserved structured RNA motifs statistically enriched in the noncoding region of 38 out of 53 groups of metabolically related transcripts in comparison with a random control. These motifs have a hairpin loop structure, a preferred sense orientation and are located in close proximity to the open reading frames. We found that 15 out of these 38 groups represent unique motifs in which most 3′-UTR signature elements were group-specific. Two extensively studied Trypanosoma cruzi RBPs, TcUBP1 and TcRBP3 were found associated with a few candidate RNA regulons. Interestingly, 13 motifs showed a strong correlation with clusters of developmentally co-expressed genes and six RNA elements were enriched in gene clusters affected after hyperosmotic stress. Here we report a systematic genome-wide in silico screen to search for novel RNA-binding sites in transcripts, and describe an organized network of several coordinately regulated cohorts of mRNAs in T. cruzi. Moreover, we found that structured RNA elements are also conserved in other human pathogens. These results support a model of regulation of gene expression by multiple post-transcriptional regulons in trypanosomes.
PMCID: PMC3728762  PMID: 23904995
RNA-binding protein; Trypanosomes; Cis-element; Post-transcriptional control; RNA regulon
16.  Emerging roles for lysophosphatidylserine in resolution of inflammation 
Progress in Lipid Research  2012;51(3):199-207.
Despite overlapping structural aspects with other phospholipids, lysophosphatidylserine (lysoPS), the monoacyl derivative of phosphatidylserine (diacylPS), appears to exert unique signaling characteristics important in both the early stages of initiating acute inflammation and in the orchestration of its resolution. LysoPS has long been known as a signaling phospholipid in mast cell biology, markedly enhancing stimulated histamine release and eicosanoid production. More recently, there has been a resurgence of interest in lysoPS as new roles in the promotion of phagocytosis of apoptotic cells, so-called efferocytosis, and resolution of inflammation have been identified. With regard to the latter, lysoPS generated in/on activated or aged apoptotic neutrophils enhances their clearance by macrophages via signaling through the macrophage G-protein coupled receptor G2A. In macrophages, this early acting pathway results in PKA-dependent augmentation of Rac1 activity via increased production of PGE2 and cAMP. As such, macrophages stimulated with lysoPS demonstrate significantly increased efferocytic capacity necessary to clear large numbers of recruited neutrophils typical of acute inflammation. Given that clearance of these cells is critical for restoration of tissue function, lysoPS, as a pro-resolving lipid mediator, is hypothesized to play a key role in promoting timely resolution of inflammation. This article will review our current knowledge of lysoPS biology including receptor signaling and mechanisms of generation as well as summarize the more recent evidence of its expanding roles in inflammation.
PMCID: PMC3365616  PMID: 22465125
17.  Description and Nomenclature of Neisseria meningitidis Capsule Locus 
Emerging Infectious Diseases  2013;19(4):566-573.
Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.
PMCID: PMC3647402  PMID: 23628376
Neisseria meningitidis; capsule; serogroup; bacteria; nomenclature
18.  Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures 
PLoS Genetics  2013;9(1):e1003195.
The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ∼50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites.
Author Summary
The Drosophila homeodomain protein Tinman was the first transcription factor found to control the development and differentiation of the heart in any species. In spite of that, our knowledge of the number, identities, and mode of regulation of the downstream target genes of Tinman that are necessary to exert its cardiogenic functions is still very incomplete. To address these issues, we have performed a genome-wide analysis of DNA regions associated with Tinman-binding in embryos and the genes linked to them. The combined data from our in-depth in vivo assays of sequence elements with high Tinman occupancy allow the following general conclusions: (1) The majority of such sequences are active as regulatory elements (called enhancers) in mesodermal tissues that include Tinman-expressing cells. (2) The enhancers active in the heart progenitor cells and the heart generally are dependent on tinman gene activity, whereas those active in non-cardiac mesoderm are often bound neutrally by Tinman. (3) Tinman binding motifs in most cases are essential for cardiac enhancer activity, but in some cases they can be functionally-redundant with those of other cardiogenic factors. (4) Tinman-occupied cardiac enhancers are enriched for a newly discovered binding motif for an unknown factor that is functional in vivo.
PMCID: PMC3542182  PMID: 23326246
19.  Fetal cholinergic anti-inflammatory pathway and necrotizing enterocolitis: the brain-gut connection begins in utero 
Necrotizing enterocolitis (NEC) is an acute neonatal inflammatory disease that affects the intestine and may result in necrosis, systemic sepsis and multisystem organ failure. NEC affects 5–10% of all infants with birth weight ≤ 1500 g or gestational age less than 30 weeks. Chorioamnionitis (CA) is the main manifestation of pathological inflammation in the fetus and is strong associated with NEC. CA affects 20% of full-term pregnancies and upto 60% of preterm pregnancies and, notably, is often an occult finding. Intrauterine exposure to inflammatory stimuli may switch innate immunity cells such as macrophages to a reactive phenotype (“priming”). Confronted with renewed inflammatory stimuli during labour or postnatally, such sensitized cells can sustain a chronic or exaggerated production of proinflammatory cytokines associated with NEC (two-hit hypothesis). Via the cholinergic anti-inflammatory pathway, a neurally mediated innate anti-inflammatory mechanism, higher levels of vagal activity are associated with lower systemic levels of proinflammatory cytokines. This effect is mediated by the α7 subunit nicotinic acetylcholine receptor (α7nAChR) on macrophages. The gut is the most extensive organ innervated by the vagus nerve; it is also the primary site of innate immunity in the newborn. Here we review the mechanisms of possible neuroimmunological brain-gut interactions involved in the induction and control of antenatal intestinal inflammatory response and priming. We propose a neuroimmunological framework to (1) study the long-term effects of perinatal intestinal response to infection and (2) to uncover new targets for preventive and therapeutic intervention.
PMCID: PMC3737662  PMID: 23964209
necrotizing enterocolitis; chorioamnionitis; preterm birth; inflammation; neuroimmunology; vagus nerve; intestines; prevention
20.  Time scales of autonomic information flow in near-term fetal sheep 
Autonomic information flow (AIF) characterizes fetal heart rate (FHR) variability (fHRV) in the time scale dependent complexity domain and discriminates sleep states [high voltage/low frequency (HV/LF) and low voltage/high frequency (LV/HF) electrocortical activity (ECoG)]. However, the physiologic relationship of AIF time scales to the underlying sympathetic and vagal rhythms is not known. Understanding this relationship will enhance the benefits derived from using fHRV to monitor fetal health non-invasively. We analyzed AIF measured as Kullback–Leibler entropy (KLE) in fetal sheep in late gestation as function of vagal and sympathetic modulation of fHRV, using atropine and propranolol, respectively (n = 6), and also analyzed changes in fHRV during sleep states (n = 12). Atropine blockade resulted in complexity decrease at 2.5 Hz compared to baseline HV/LF and LV/HF states and at 1.6 Hz compared to LV/HF. Propranolol blockade resulted in complexity increase in the 0.8–1 Hz range compared to LV/HF and in no changes when compared to HV/LF. During LV/HF state activity, fHRV complexity was lower at 2.5 Hz and higher at 0.15–0.19 Hz than during HV/LF. Our findings show that in mature fetuses near term vagal activity contributes to fHRV complexity on a wider range of time scales than sympathetic activity. Related to sleep, during LV/HF we found lower complexity at short-term time scale where complexity is also decreased due to vagal blockade. We conclude that vagal and sympathetic modulations of fHRV show sleep state-dependent and time scale-dependent complexity patterns captured by AIF analysis of fHRV. Specifically, we observed a vagally mediated and sleep state-dependent change in these patterns at a time scale around 2.5 Hz (0.2 s). A paradigm of state-dependent non-linear sympathovagal modulation of fHRV is discussed.
PMCID: PMC3457070  PMID: 23055982
fetal heart rate; HRV; autonomic nervous system; permutation entropy; complexity; sleep; atropine; propranolol
21.  Oscillation of Clock and Clock Controlled Genes Induced by Serum Shock in Human Breast Epithelial and Breast Cancer Cells: Regulation by Melatonin 
This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells.
PMCID: PMC3448497  PMID: 23012497
melatonin; clock genes; circadian; serum shock; breast cancer
22.  A role for the membrane protein M6 in the Drosophila visual system 
BMC Neuroscience  2012;13:78.
Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system.
The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light.
Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.
PMCID: PMC3438117  PMID: 22762289
Myelin PLP family; Gpm6a; Eye development; Phototactic behavior; Lifespan; Protrusion/filopodium formation; Cell remodeling
23.  The Impact of Intermittent Umbilical Cord Occlusions on the Inflammatory Response in Pre-Term Fetal Sheep 
PLoS ONE  2012;7(6):e39043.
Fetal hypoxic episodes may occur antepartum with the potential to induce systemic and cerebral inflammatory responses thereby contributing to brain injury. We hypothesized that intermittent umbilical cord occlusions (UCOs) of sufficient severity but without cumulative acidosis will lead to a fetal inflammatory response. Thirty-one chronically instrumented fetal sheep at ∼0.85 of gestation underwent four consecutive days of hourly UCOs from one to three minutes duration for six hours each day. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1β and IL-6 levels. Animals were euthanized at the end of experimental study with brain tissue processed for subsequent counting of microglia and mast cells. Intermittent UCOs resulted in transitory fetal hypoxemia with associated acidemia which progressively worsened the longer umbilical blood flow was occluded, but with no cumulative blood gas or pH changes over the four days of study. Fetal arterial IL-1β and IL-6 values showed no significant change regardless of the severity of the UCOs, nor was there any evident impact on the microglia and mast cell counts for any of the brain regions studied. Accordingly, intermittent UCOs of up to three minutes duration with severe, but limited fetal hypoxemia and no cumulative acidemia, do not result in either a systemic or brain inflammatory response in the pre-term ovine fetus. However, fetal IL-1B and IL-6 values were found to be well correlated with corresponding maternal values supporting the placenta as a primary source for these cytokines with related secretion into both circulations. Female fetuses were also found to have higher IL-1β levels than males, indicating that gender may impact on the fetal inflammatory response to various stimuli.
PMCID: PMC3380034  PMID: 22745702
24.  Padlock probe-mediated qRT-PCR for DNA computing answer determination 
Natural computing  2011;10(2):947-959.
Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.
PMCID: PMC3116661  PMID: 21691417
DNA computing; Padlock probe; Quantitative real-time PCR; TaqMan chemistry; Ordered node pair abundance; Traveling salesman problem
25.  Evaluation of a Recombinant Trypanosoma cruzi Mucin-Like Antigen for Serodiagnosis of Chagas' Disease ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(11):1850-1855.
Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.
PMCID: PMC3209031  PMID: 21880857

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