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1.  Knockdown of Both Mitochondrial Isocitrate Dehydrogenase Enzymes In Pancreatic Beta Cells Inhibits Insulin Secretion 
Biochimica et biophysica acta  2013;1830(11):5104-5111.
There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.
With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.
With knockdown of both mitochondrial IDH’s mRNA, enzyme activity and protein level, but not with knockdown of one mitochondrial IDH, glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.
The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.
General Significance
As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.
PMCID: PMC3923500  PMID: 23876293
shRNA; Mitochondrial isocitrate dehydrogenase; cytosolic isocitrate dehydrogenase; Stable knockdown of isocitrate dehydrogenase; Insulin secretion; INS-1 832/13 cell line
2.  Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators 
Nature  2014;508(7497):494-499.
The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three percent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was non-random, and in two cases, convergent across placental and marsupial mammals. We conclude that the Y chromosome's gene content became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and plays unappreciated roles in Turner syndrome and in phenotypic differences between the sexes in health and disease.
PMCID: PMC4139287  PMID: 24759411
3.  Elevated plasma norepinephrine inhibits insulin secretion, but adrenergic blockade reveals enhanced β-cell responsiveness in an ovine model of placental insufficiency at 0.7 of gestation 
Journal of developmental origins of health and disease  2013;4(5):10.1017/S2040174413000093.
In pregnancies complicated by placental insufficiency (PI), fetal hypoglycemia and hypoxemia progressively worsen during the third trimester, which increases circulating norepinephrine (NE). Pharmacological adrenergic blockade (ADR-block) at 0.9 gestation revealed that NE inhibits insulin secretion and enhanced β-cell responsiveness in fetuses with PI-induced intrauterine growth restriction (IUGR). NE concentrations in PI fetuses at 0.7 gestation were 3-fold greater compared to age-matched controls but the levels were similar to near-term controls. Therefore, our objective was to determine whether elevations in plasma NE concentrations inhibit insulin secretion and produce compensatory β-cell responsiveness in PI fetuses at 0.7 gestation. Fetal insulin was measured under basal, glucose-stimulated (GSIS), and glucose potentiated arginine-stimulated (GPAIS) conditions in the absence and presence of an ADR-block. Placental weights were 38% lower (P<0.05) in PI fetus than in controls, but fetal weights were not different. PI fetuses had lower (P<0.05) basal blood oxygen content, plasma glucose, IGF-1, and insulin concentrations and greater plasma NE concentrations (891±211 vs. 292±65 pg/ml; P<0.05) than controls. GSIS was lower in PI fetuses than in controls (0.34±0.03 vs. 1.08±0.06 ng/ml; P<0.05). ADR-block increased GSIS in PI fetuses (1.19±0.11 ng/ml; P<0.05) but decreased GSIS in controls (0.86±0.02 ng/ml; P<0.05). Similarly, GPAIS was 44% lower (P<0.05) in PI fetuses than in controls, and ADR-block increased (P<0.05) GPAIS in PI fetuses but not in controls. Insulin content per islet was not different between treatments. We conclude that elevations in fetal plasma NE suppress insulin concentrations and that compensatory β-cells stimulus-secretion responsiveness is present before IUGR.
PMCID: PMC3864835  PMID: 24358443
pancreas; fetus; adrenergic receptors; intrauterine growth restriction
4.  Endocrine regulation of fetal skeletal muscle growth: impact on future metabolic health 
The Journal of endocrinology  2014;221(2):R13-R29.
Establishing sufficient skeletal muscle mass is essential for lifelong metabolic health. The intrauterine environment is a major determinant of the muscle mass that is present for the life course of an individual, because muscle fiber number is set at the time of birth. Thus, a compromised intrauterine environment from maternal nutrient restriction or placental insufficiency that restricts development of muscle fiber number can have permanent effects on the amount of muscle an individual will live with. Reduced muscle mass due to fewer muscle fibers persists even after compensatory or “catch up” postnatal growth occurs. Furthermore, muscle hypertrophy can only partially compensate for this limitation in fiber number. Compelling associations link low birth weight and decreased muscle mass to future insulin resistance, which can drive the development of the metabolic syndrome and type 2 diabetes, and risk for cardiovascular events later in life. There are gaps in knowledge about the origins of reduced muscle growth at the cellular level and how these patterns are set during fetal development. By understanding the nutrient and endocrine regulation of fetal skeletal muscle growth and development, we can direct research efforts towards improving muscle growth early in life in order to prevent the development of chronic metabolic disease later in life.
PMCID: PMC4004098  PMID: 24532817
muscle; insulin; IGF1; amino acids; protein synthesis; myogenesis
6.  A physiological increase in insulin suppresses muscle‐specific ubiquitin ligase gene activation in fetal sheep with sustained hypoglycemia 
Physiological Reports  2014;2(6):e12045.
Decreased glucose transfer to the fetus is characteristic of pregnancies complicated by maternal under nutrition and placental insufficiency. Chronic experimental restriction of glucose transfer to the sheep fetus for the final 40% of gestation with a maternal insulin infusion (HG fetuses) results in fetal hypoglycemia, hypoinsulinemia, and decreased rates of fetal growth and protein accretion compared to controls (CON). Lower rates of fetal protein accretion are due to increased fetal protein breakdown and not decreased protein synthesis. However, the specific skeletal muscle pathways responsible for increased protein breakdown have not been determined. Nor has it been determined if low fetal glucose or insulin concentrations are more important for regulating these skeletal muscle protein breakdown pathways. We tested whether chronic restriction of glucose transfer to the fetus increased the ubiquitin–proteosome pathway or autophagy‐lysosome pathway in fetal sheep skeletal muscle and found no evidence for an increase in the autophagy‐lysosome pathway. However, HG fetuses had increase mRNA expression of MaFBx1 (twofold, P <0.01) and a trend for increased mRNA expression of MuRF1 (P =0.08) compared to CON. A subset of chronically hypoglycemic fetuses received an isoglycemic insulin infusion for the final 7 days of the maternal insulin infusion (HG + INS fetuses) and had MaFBx1 and MuRF1 mRNA concentrations similar to CON fetuses. These results demonstrate that fetuses exposed to sustained hypoglycemia have decreased protein accretion due to activation of the skeletal muscle ubiquitin–proteosome pathway and that a fetal hyperinsulinemic clamp can suppress this pathway even in the context of continued hypoglycemia.
This study shows the importance of the ubiquitin–proteosome pathway for regulating protein accretion during chronic fetal hypoglycemia. Furthermore, we show that this pathway can be suppressed by physiological concentrations for fetal insulin.
PMCID: PMC4208658  PMID: 24944291
Autophagy‐Lysosome; MaFBx1; MuRF1; pregnancy; ubiquitin‐proteosome
7.  Changes in fetal mannose and other carbohydrates induced by a maternal insulin infusion in pregnant sheep 
The importance of non-glucose carbohydrates, especially mannose and inositol, for normal development is increasingly recognized. Whether pregnancies complicated by abnormal glucose transfer to the fetus also affect the regulation of non-glucose carbohydrates is unknown. In pregnant sheep, maternal insulin infusions were used to reduce glucose supply to the fetus for both short (2-wk) and long (8-wk) durations to test the hypothesis that a maternal insulin infusion would suppress fetal mannose and inositol concentrations. We also used direct fetal insulin infusions (1-wk hyperinsulinemic-isoglycemic clamp) to determine the relative importance of fetal glucose and insulin for regulating non-glucose carbohydrates.
A maternal insulin infusion resulted in lower maternal (50%, P < 0.01) and fetal (35-45%, P < 0.01) mannose concentrations, which were highly correlated (r2 = 0.69, P < 0.01). A fetal insulin infusion resulted in a 50% reduction of fetal mannose (P < 0.05). Neither maternal nor fetal plasma inositol changed with exogenous insulin infusions. Additionally, maternal insulin infusion resulted in lower fetal sorbitol and fructose (P < 0.01).
Chronically decreased glucose supply to the fetus as well as fetal hyperinsulinemia both reduce fetal non-glucose carbohydrates. Given the role of these carbohydrates in protein glycosylation and lipid production, more research on their metabolism in pregnancies complicated by abnormal glucose metabolism is clearly warranted.
PMCID: PMC4051387  PMID: 24917928
Fructose; Glucose; Inositol; Insulin; Mannose; Pregnancy
8.  Independent specialization of the human and mouse X chromosomes for the male germline 
Nature genetics  2013;45(9):1083-1087.
We compared the human and mouse X chromosomes to systematically test Ohno’s law, which states that the gene content of X chromosomes is conserved across placental mammals1. First, we improved the accuracy of the human X-chromosome reference sequence through single-haplotype sequencing of ampliconic regions. This closed gaps in the reference sequence, corrected previously misassembled regions, and identified new palindromic amplicons. Our subsequent analysis led us to conclude that the evolution of human and mouse X chromosomes was bimodal. In accord with Ohno’s law, 94–95% of X-linked single-copy genes are shared between human and mouse; most are expressed in both sexes. Strikingly, most X-ampliconic genes are exceptions to Ohno’s law: only 31% of human and 22% of mouse X-ampliconic genes share orthologs. X-ampliconic genes are expressed predominantly in testicular germ cells, and many were independently acquired since the common ancestor of humans and mice, specializing portions of their X chromosomes for sperm production.
PMCID: PMC3758364  PMID: 23872635
9.  Increased Hepatic Glucose Production in Fetal Sheep With Intrauterine Growth Restriction Is Not Suppressed by Insulin 
Diabetes  2012;62(1):65-73.
Intrauterine growth restriction (IUGR) increases the risk for metabolic disease and diabetes, although the developmental origins of this remain unclear. We measured glucose metabolism during basal and insulin clamp periods in a fetal sheep model of placental insufficiency and IUGR. Compared with control fetuses (CON), fetuses with IUGR had increased basal glucose production rates and hepatic PEPCK and glucose-6-phosphatase expression, which were not suppressed by insulin. In contrast, insulin significantly increased peripheral glucose utilization rates in CON and IUGR fetuses. Insulin robustly activated AKT, GSK3β, and forkhead box class O (FOXO)1 in CON and IUGR fetal livers. IUGR livers, however, had increased basal FOXO1 phosphorylation, nuclear FOXO1 expression, and Jun NH2-terminal kinase activation during hyperinsulinemia. Expression of peroxisome proliferator–activated receptor γ coactivator 1α and hepatocyte nuclear factor-4α were increased in IUGR livers during basal and insulin periods. Cortisol and norepinephrine concentrations were positively correlated with glucose production rates. Isolated IUGR hepatocytes maintained increased glucose production in culture. In summary, fetal sheep with IUGR have increased hepatic glucose production, which is not suppressed by insulin despite insulin sensitivity for peripheral glucose utilization. These data are consistent with a novel mechanism involving persistent transcriptional activation in the liver that seems to be unique in the fetus with IUGR.
PMCID: PMC3526037  PMID: 22933111
10.  Chronic Late Gestation Hypoglycemia Up-Regulates Hepatic PEPCK Associated with Increased PGC1α mRNA and pCREB in Fetal Sheep 
American journal of physiology. Endocrinology and metabolism  2007;294(2):10.1152/ajpendo.00639.2007.
Hepatic glucose production is normally activated at birth, but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 weeks of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin signaling pathway, transcription factors, and coactivators which regulate gluconeogenesis. Hepatic PEPCK and glucose-6-phosphatase mRNA increased 12-fold and 7-fold respectively following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon, but increased plasma cortisol 3.5-fold. PGC1 mRNA and phosphorylation of CREB at serine 133 were both increased, with no change in Akt, FOXO1, HNF4α, or C/EBPβ. These results demonstrate that chronic fetal hypoglycemia triggers signals which can activate gluconeogenesis in the fetal liver.
PMCID: PMC3857025  PMID: 18056789
glucose; gluconeogenesis; cortisol; CREB; PGC1
11.  ClinicalKey 
PMCID: PMC3794697
12.  Extended Longitudinal Analysis of Arterial Pressure and Heart Rate Control in Unanesthetized Rats with Type 1 Diabetes 
We recorded arterial pressure (BP) and heart rate (HR) in type-1 diabetic rats versus controls for ≥ 6 months. Diabetic rats (DIAB) were maintained on insulin from the day glucose > 250 mg/dl (“Day 0”). Weight was similar between groups until ~3 weeks before Day 0 when the weight in DIAB transiently lagged the controls (CONT); this difference was maintained throughout the study, but both groups otherwise gained weight in parallel. Plasma glucose attained 371 ± 109 (SD) mg/dl by day 1 in DIAB. Mean BP was similar across groups, and declined through the initial 4–6 months in both the CONT (at −0.06 ± 0.04 mm Hg/day) and in the DIAB (at −0.14 ± 0.21 mm Hg/day; NS vs. CONT). HR in the CONT (Month 1: 341 ± 13 bpm) exceeded DIAB (325 ± 25 bpm) through ~6 months after Day 0, and also decreased progressively over this period in CONT (−0.19 ± 0.14 beats/day) and DIAB (−0.29 ± 0.23 bpm/day; NS vs. CONT) before leveling. The BP power within 0.35–0.45 Hz changed during the 90 minutes before vs. after the transition from dark to light, and light to dark; there were no between group differences. The slope of the log-log linear portion of the BP power spectrum between 1.0/hr to 1/min was similar across groups, and increased in both from month 1 to month 6. Regulatory mechanisms maintain similar profiles in BP and HR in diabetic vs. control animals through the initial half year of the disease.
PMCID: PMC3433651  PMID: 22809731
Cardiovascular system; autonomic nervous system; weight gain; plasma glucose; dysautonomia; slope β (beta)
13.  Quantifying the Effects of Normal Ageing on White Matter Structure using Unsupervised Tract Shape Modelling 
NeuroImage  2010;51(1):1-10.
Quantitative tractography may provide insights into regional heterogeneity of changes in white matter structure in normal ageing. Here we examine how brain atrophy and white matter lesions affect correlations between tract shape, tract integrity and age in a range of frontal and non-frontal tracts in 90 non-demented subjects aged over 65 years using an enhanced version of probabilistic neighbourhood tractography. This novel method for automatic single seed point placement employs unsupervised learning and streamline selection to provide reliable and accurate tract segmentation, whilst also indicating how the shape of an individual tract compares to that of a predefined reference tract. There were significant negative correlations between tract shape similarity to reference tracts derived from a young brain white matter atlas and age in genu and splenium of corpus callosum. Controlling for intracranial and lateral ventricle volume, the latter of which increased significantly with age, attenuated these correlations by 40 and 84 % respectively, indicating that this age-related change in callosal tract topology is significantly mediated by global atrophy and ventricular enlargement. In accordance with the ‘frontal ageing’ hypothesis, there was a significant positive correlation between mean diffusivity (〈D〉) and age, and a significant negative correlation between fractional anisotropy (FA) and age in corpus callosum genu; correlations not seen in splenium. Significant positive correlations were also observed between 〈D〉 and age in bilateral cingulum cingulate gyri, uncinate fasciculi and right corticospinal tract. This pattern of correlations was not, however, reproduced when those subjects with significant white matter lesion load were analyzed separately from those without. These data therefore suggest that brain atrophy and white matter lesions play a significant role in driving regional patterns of age-related changes in white matter tract shape and integrity.
PMCID: PMC3763188  PMID: 20171285
Ageing; white matter; magnetic resonance imaging; water diffusion tensor; tractography
14.  Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota 
PLoS Biology  2013;11(7):e1001610.
A common human gut bacterium, Bacteroides fragilis, produces a sphingolipid ligand for the conserved host receptor CD1d and can modulate natural killer T cell activity.
While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCerBf), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCerBf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.
Author Summary
While human gut bacteria are thought to produce diffusible molecules that influence host biology, few of these molecules have been identified. Species of Bacteroides, a Gram-negative bacterial genus whose members often comprise >50% of the gut community, are unusual in that they produce sphingolipids, signaling molecules that play a key role in modulating the host immune response. Sphingolipid production is ubiquitous among eukaryotes but present in only a few bacterial genera. We set out to construct a Bacteroides strain that is incapable of producing sphingolipids, knocking out a gene predicted to encode the first enzymatic step in the Bacteroides sphingolipid biosynthetic pathway. The resulting mutant is indeed deficient in sphingolipid production, and we purified and solved the structures of three sphingolipids that are present in the wild-type strain but absent in the mutant. To our surprise, one of these molecules is a close chemical relative of a sponge sphingolipid that is the prototypical ligand for a host receptor that controls the activity of natural killer T cells. Like the sponge sphingolipid, the Bacteroides sphingolipid can modulate natural killer T cell activity, suggesting a novel mechanism by which Bacteroides in the gut might influence the host immune response.
PMCID: PMC3712910  PMID: 23874157
15.  Accurate preterm labor diagnosis using a CD55-TLR4 combination biomarker model 
We previously demonstrated immune activation in the maternal peripheral circulation associated with preterm labor (PTL). There was an elevation in WBC mRNA of anti-inflammatory complement decay-accelerating factor (CD55) and the innate-immune response activating toll-like receptor 4 (TLR4). These findings suggested that collectively, these two molecules might serve as useful biomolecules to aid in the diagnosis of PTL. In this study, we used a combined marker approach to determine whether a dual marker model utilizing both CD55 and TLR4 mRNA levels to classify PTL would increase diagnostic accuracy compared to either molecule alone. Two methods were evaluated; a linear discriminant (LD) method and a distribution free (DF) method, in order to find the optimal linear combination of TLR4 and CD55 data to diagnose PTL accurately. Our results indicated that a combined CD55-TLR4 dual marker model could provide statistically significant improvements compared to CD55 or TLR4 single marker models for PTL classification performance.
PMCID: PMC3649766  PMID: 23667715
Preterm Labor (PTL); CD55; Decay Accelerating Factor (DAF); Toll-Like Receptor 4 (TLR4)
16.  Isodicentric Y Chromosomes and Sex Disorders as Byproducts of Homologous Recombination that Maintains Palindromes 
Cell  2009;138(5):855-869.
Massive palindromes in the human Y chromosome harbor mirror-image gene pairs essential for spermatogenesis. During evolution, these gene pairs have been maintained by intrapalindrome, arm-to-arm recombination. The mechanism of intrapalindrome recombination and risk of harmful effects are unknown. We report 51 patients with isodicentric Y (idicY) chromosomes formed by homologous crossing-over between opposing arms of palindromes on sister chromatids. These ectopic recombination events occur at nearly all Y-linked palindromes. Based on our findings, we propose that intrapalindrome sequence identity is maintained via noncrossover pathways of homologous recombination. DNA double-strand breaks that initiate these pathways can be alternatively resolved by crossing over between sister chromatids to form idicY chromosomes, with clinical consequences ranging from spermatogenic failure to sex reversal and Turner syndrome. Our observations imply that crossover as well as noncrossover pathways are active in nearly all Y-linked palindromes, exposing an Achilles' heel in the mechanism that preserves palindrome-borne genes.
PMCID: PMC3616640  PMID: 19737515
17.  A biomechanical study on the effects of rib head release on thoracic spinal motion 
European Spine Journal  2011;21(4):606-612.
Idiopathic scoliosis is generally treated by surgical derotation of the spine. A secondary goal of surgery is minimization of the “rib hump” deformity. Previous studies have evaluated the effects of surgical releases such as diskectomy, costo-vertebral joint release, facetectomy, and costoplasty on spine mobilization and overall contribution to thoracic stability. The present study was designed to evaluate the biomechanical effects of the rib head joints alone on axial rotation, lateral bending, and segmental rotation, without diskectomy or disruption of anterior or posterior elements.
Four female cadaver thoracic spines with intact sternums and rib cages were mounted in an Instron servo-hydraulic bi-axial MTS. In a 12-step sequence, the costo-vertebral and costo-transverse ligaments were released, first unilaterally from T10–T7, then bilaterally until complete disarticulation between the rib heads and the vertebral bodies. After each release, biomechanical testing, including axial rotation and lateral bending, was performed. Vertebral body displacement was also measured using electromagnetic trackers.
We found that rib displacement during axial rotation was significantly increased by unilateral rib head release, and torque was decreased with each successive cut. We also found increased vertebral displacement with sequential rib head release.
Our results show that sequential costo-vertebral joint releases result in a decrease in the force required for axial rotation and lateral bending, coupled with an increase in the displacement of vertebral bodies. These findings suggest that surgical release of the costo-transverse and costo-vertebral ligaments can facilitate segmental correction in scoliosis by decreasing the torso’s natural biomechanical resistance to this correction.
PMCID: PMC3326120  PMID: 21989737
Rib head release; Scoliosis; Thoracic spine; Vertebrae; Costo-vertebral release
18.  A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals 
eLife  2012;1:e00013.
Bacterially-produced small molecules exert profound influences on animal health, morphogenesis, and evolution through poorly understood mechanisms. In one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta, we find that rosette colony development is induced by the prey bacterium Algoriphagus machipongonensis and its close relatives in the Bacteroidetes phylum. Here we show that a rosette inducing factor (RIF-1) produced by A. machipongonensis belongs to the small class of sulfonolipids, obscure relatives of the better known sphingolipids that play important roles in signal transmission in plants, animals, and fungi. RIF-1 has extraordinary potency (femtomolar, or 10−15 M) and S. rosetta can respond to it over a broad dynamic range—nine orders of magnitude. This study provides a prototypical example of bacterial sulfonolipids triggering eukaryotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals.
eLife digest
All animals, including humans, evolved in a world filled with bacteria. Although bacteria are most familiar as pathogens, some bacteria produce small molecules that are essential for the biology of animals and other eukaryotes, although the details of the ways in which these bacterial molecules are beneficial are not well understood.
The choanoflagellates are water-dwelling organisms that use their whip-like flagella to move around, feeding on bacteria. They can exist as one cell or a colony of multiple cells and, perhaps surprisingly, are the closest known living relatives of animals. This means that experiments on these organisms have the potential to improve our understanding of animal development and the transition from egg to embryo to adult.
Alegado et al. have explored how the morphology of Salpingoeca rosetta, a colony-forming choanoflagellate, is influenced by its interactions with various species of bacteria. In particular, they find that the development of multicellularity in S. rosetta is triggered by the presence of the bacterium Algoriphagus machipongonensis as well as its close relatives. They also identify the signaling molecule produced by the bacteria to be C32H64NO7S; this lipid molecule is an obscure relative of the sphingolipid molecules that have important roles in signal transmission in animals, plants, and fungi. Moreover, Alegado et al. show that S. rosetta can respond to this molecule – which they call rosette-inducing factor (RIF-1) – over a wide range of concentrations, including concentrations as low as 10−17 M.
The work of Alegado et al. suggests that interactions between S. rosetta and Algoriphagus bacteria could be a productive model system for studying the influences of bacteria on animal cell biology, and for investigating the mechanisms of signal delivery and reception. Moreover, the molecular mechanisms revealed by this work leave open the possibility that bacteria might have contributed to the evolution of multicellularity in animals.
PMCID: PMC3463246  PMID: 23066504
Salpingoeca rosetta; Algoriphagus; bacterial sulfonolipid; multicellular development; Other
19.  Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes 
Nature  2012;483(7387):82-86.
The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200–300 million years1–3. Due to genetic decay, the human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes’ genes4,5. This evolutionary decay was driven by a series of five “stratification” events. Each event suppressed X-Y crossing over within a chromosome segment or “stratum”, incorporated that segment into the MSY, and subjected its genes to the erosive forces that attend the absence of crossing over2,6. The last of these events occurred 30 million years ago (mya), or 5 million years before the human and Old World monkey (OWM) lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome7–10, remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the OWM lineage. To explore this question, we sequenced the MSY of the rhesus macaque, an OWM, and compared it to the human MSY. We discovered that, during the last 25 million years, MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. Within the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 mya. Likewise, the rhesus MSY has not lost any older genes (from strata 1–4) during the past 25 million years, despite major structural differences from the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection.
PMCID: PMC3292678  PMID: 22367542
20.  Prediction of On-Road Driving Performance in Patients with Early Alzheimer’s Disease 
Physicians and family members frequently are asked to provide information about driving ability in patients with Alzheimer’s disease (AD), yet there has been little research on the validity of their assessments of driving performance.
Participants were recruited from the neurology department of a community hospital affiliated with Brown Medical School.
Participants included 75 older adults (17 with mild AD, 33 with very mild AD, and 25 elderly controls).
The participant him/herself, an informant, and an experienced neurologist rated each participant’s driving ability on a 3-point rating scale (safe, marginal, unsafe). A professional driving instructor also completed a standardized 108-point on-road driving assessment of each participant and then rated driving ability on the 3-point scale. Ratings were compared with the on-road driving score and with each other.
Only the neurologist’s rating of the participants’ driving abilities was significantly related to on-road driving score. When related to the instructor’s safety rating, the neurologist’s ratings were the most sensitive and specific. Mini-Mental State Examination score was a borderline covariate for the neurologist’s rating. Overall, the instructor was the most stringent rater of participant driving ability, followed by the neurologist, the informant, and the participant.
An experienced neurologist’s assessment of driving competence may be a valid predictor of driving performance of patients with early AD.
PMCID: PMC3292197  PMID: 15667383
dementia; driving; assessment; Alzheimer’s disease
21.  Driving and Dementia: A Review of the Literature 
The purpose of this article is to review the literature on the ability of individuals with dementia to drive an automobile. Based on a review of the literature, several factors were identified that may be useful in differentiating between people with dementia who presently remain safe drivers from those who have progressed to impaired driving. These factors include disease duration and severity, sex, patient self-assessment, family assessment, neuropsychological measures, findings on road evaluations, and driving simulator testing. The approach of the physician to driving and dementia is addressed, including in-office screening, referral for on-road driving assessments, and the potential for physician reporting to state agencies.
PMCID: PMC3292210  PMID: 15533995
dementia; driving; competence; impairment
22.  Driving Scenes test of the Neuropsychological Assessment Battery (NAB) and on-road driving performance in aging and very mild dementia 
The Driving Scenes test of the new Neuropsychological Assessment Battery (NAB; [Stern, R.A., & White, T. (2003a). Neuropsychological Assessment Battery. Lutz, FL: Psychological Assessment Resources, Inc.]) measures several aspects of visual attention thought to be important for driving ability. The current study examined the relationship between scores on the Driving Scenes test and on-road driving performance on a standardized driving test. Healthy participants performed significantly better on the Driving Scenes test than did very mildly demented participants. A correlation of 0.55 was found between the brief, office-based Driving Scenes test and the 108-point on-road driving score. Furthermore, the Driving Scenes test scores differed significantly across the driving instructor’s three global ratings (safe, marginal, and unsafe), and results of a discriminant function analysis indicated that the Driving Scenes test correctly classified 66% of participants into these groups. Thus, the new NAB Driving Scenes test appears to have good ecological validity for real-world driving ability in normal and very mildly demented older adults.
PMCID: PMC3292213  PMID: 15708731
Driving; Aging; Dementia; Neuropsychology; Attention; Visual
23.  The Intrauterine Growth Restriction Phenotype: Fetal Adaptations and Potential Implications for Later Life Insulin Resistance and Diabetes 
Seminars in reproductive medicine  2011;29(3):225-236.
The intrauterine growth restricted (IUGR) fetus develops unique metabolic adaptations in response to exposure to reduced nutrient supply. These adaptations provide survival value for the fetus by enhancing the capacity of the fetus to take up and use nutrients, thereby reducing the need for nutrient supply. Each organ and tissue in the fetus adapts differently, with the brain showing the greatest capacity for maintaining nutrient supply and growth. Such adaptations, if persistent, also have the potential in later life to promote nutrient uptake and storage, which directly lead to complications of obesity, insulin resistance, reduced insulin production, and type 2 diabetes.
PMCID: PMC3216466  PMID: 21710398
Fetus; placenta; pregnancy; intrauterine growth restriction (IUGR); nutrition
24.  Maternal amino acid supplementation for intrauterine growth restriction 
Maternal dietary protein supplementation to improve fetal growth has been considered as an option to prevent or treat intrauterine growth restriction. However, in contrast to balanced dietary supplementation, adverse perinatal outcomes in pregnant women who received high amounts of dietary protein supplementation have been observed. The responsible mechanisms for these adverse outcomes are unknown. This review will discuss relevant human and animal data to provide the background necessary for the development of explanatory hypotheses and ultimately for the development therapeutic interventions during pregnancy to improve fetal growth. Relevant aspects of fetal amino acid metabolism during normal pregnancy and those pregnancies affected by IUGR will be discussed. In addition, data from animal experiments which have attempted to determine mechanisms to explain the adverse responses identified in the human trials will be presented. Finally, we will suggest new avenues for investigation into how amino acid supplementation might be used safely to treat and/or prevent IUGR.
PMCID: PMC3181118  PMID: 21196387
amino acids; taurine; leucine; arginine; pregnancy; intrauterine growth restriction; insulin; metabolism; protein; dietary supplementation
25.  Longitudinal Analysis of Arterial Blood Pressure and Heart Rate Response to Acute Behavioral Stress in Rats with Type 1 Diabetes Mellitus and in Age-Matched Controls 
We recorded via telemetry the arterial blood pressure (BP) and heart rate (HR) response to classical conditioning following the spontaneous onset of autoimmune diabetes in BBDP/Wor rats vs. age-matched, diabetes-resistant control (BBDR/Wor) rats. Our purpose was to evaluate the autonomic regulatory responses to an acute stress in a diabetic state of up to 12 months duration. The stress was a 15-s pulsed tone (CS+) followed by a 0.5-s tail shock. The initial, transient increase in BP (i.e., the “first component,” or C1), known to be derived from an orienting response and produced by a sympathetic increase in peripheral resistance, was similar in diabetic and control rats through ∼9 months of diabetes; it was smaller in diabetic rats 10 months after diabetes onset. Weakening of the C1 BP increase in rats that were diabetic for >10 months is consistent with the effects of sympathetic neuropathy. A longer-latency, smaller, but sustained “second component” (C2) conditional increase in BP, that is acquired as a rat learns the association between CS+ and the shock, and which results from an increase in cardiac output, was smaller in the diabetic vs. control rats starting from the first month of diabetes. A concomitant HR slowing was also smaller in diabetic rats. The difference in the C2 BP increase, as observed already during the first month of diabetes, is probably secondary to the effects of hyperglycemia upon myocardial metabolism and contractile function, but it may also result from effects on cognition. The small HR slowing concomitant with the C2 pressor event is probably secondary to differences in baroreflex activation or function, though parasympathetic dysfunction may contribute later in the duration of diabetes. The nearly immediate deficit after disease onset in the C2 response indicates that diabetes alters BP and HR responses to external challenges prior to the development of structural changes in the vasculature or autonomic nerves.
PMCID: PMC3163305  PMID: 21904530
cardiovascular system; autonomic nervous system; dysautonomia; Pavlovian (classical) conditioning; anxiety; telemetry

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