Measurement of Subcellular Force Generation in Neurons

Forces are important for neuronal outgrowth during the initial wiring of the nervous system and after trauma, yet subcellular force generation over the microtubule-rich region at the rear of the growth cone and along the axon has never, to our knowledge, been directly measured. Because previous studies have indicated microtubule polymerization and the microtubule-associated proteins Kinesin-1 and dynein all generate forces that push microtubules forward, a major question is whether the net forces in these regions are contractile or expansive. A challenge in addressing this is that measuring local subcellular force generation is difficult. Here we develop an analytical mathematical model that describes the relationship between unequal subcellular forces arranged in series within the neuron and the net overall tension measured externally. Using force-calibrated towing needles to measure and apply forces, in combination with docked mitochondria to monitor subcellular strain, we then directly measure force generation over the rear of the growth cone and along the axon of chick sensory neurons. We find the rear of the growth cone generates 2.0 nN of contractile force, the axon generates 0.6 nN of contractile force, and that the net overall tension generated by the neuron is 1.3 nN. This work suggests that the forward bulk flow of the cytoskeletal framework that occurs during axonal elongation and growth-cone pauses arises because strong contractile forces in the rear of the growth cone pull material forward.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Coelomocytes of earthworms: the T-cell-like rosette.

The coelomocytes forming spontaneous rosettes with sheep red blood cells (SRBC) were studied under various experimental conditions in lumbricus terrestris. The percentage of rosettes did vary with the pH, increased with the incubation period and remained constant at 4°C and at room temperature. The viability of the coelomocytes was a prerequisite for the formation of rosettes.The phenomenon was found to be stable, repeatable, and specific, thus suggesting rosettes are probably not an artefact.The coelomocytes forming rosettes were basophilic, nongranulated, and nonadhering cells with a large nucleus and little cytoplasm closely resembling vertebrate T-lymphocytes. The adhering cells did not form rosette.These results suggest that the coelomocytes forming rosettes could well be the evolutionary precursors or analogs of immunocyte rosettes forming cells of the vertebrates. Whether or not the coelomocyte receptors for the SRBC are similar to those of the T-cells of the vertebrate remains to be established.     

Proteomic Screen of Brain Glycoproteome Reveals Prion Specific Marker of Pathogenesis

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE‐infected and aged‐matched control mice using two‐dimensional fluorescence difference gel electrophoresis (2D‐DIGE). Since the brain proteome is too complex to resolve all proteins using 2D‐DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat‐germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin‐3 (CNN3), peroxiredoxin‐6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE‐infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.     

The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.).

We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16,700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M x s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.     

The Impact of Macronutrients on Retinal Microvasculature among Singapore Pregnant Women during the Mid-Late Gestation

BackgroundImbalanced macronutrient intakes can induce impairment of endothelial and vascular function, and further lead to metabolic and cardiovascular disease. However, little is known about the influence of such diets on endothelial and vascular dysfunction in pregnant women, even though high-fat diet is a known risk for pregnancy complications such as gestational diabetes and pre-eclampsia.ObjectiveWe aimed to assess the association between maternal macronutrient intakes (protein, fat and carbohydrates), dietary quality and retinal microvascular changes in a multi-ethnic Asian mother-offspring cohort.MethodsPregnant women (n = 614) with singleton pregnancies were recruited during their first trimester from June 2009 to Sep 2010. Maternal diet quality and macronutrient intakes, expressed as a percentage of total energy during pregnancy, were ascertained using 24 hr recalls and 3 d food diaries at 26–28 weeks gestation. Retinal examination was completed at the same clinic visit. Dietary quality was assessed and scored using the Health Eating Index in Asian Pregnant women (HEI-AP), while macronutrients intakes ware expressed as percentages of total energy and further log transformed for analysis. Associations were examined cross-sectionally by substitution models with the use of multiple linear regression.ResultsIn adjusted model, each 20 points decrease in HEI-AP score was associated with a significant increase of 1.70 μm (p<0.05) in retinal venular calibre. Each 0.1 log increase in percentage of total fat intake was associated with a significant increment of 1.84 μm (p<0.05) in retinal venular caliber. Additionally, each 0.1 log increase in percentage of mono-unsaturated fat intake was associated with an increment of 1.84 μm (p<0.01) in retinal venular caliber.ConclusionsIn this cross-sectional study, we found that women with higher fat and lower protein intakes, and lower diet quality tended to have wider retinal venular caliber, which is suggestive of suboptimal microvasculature.     

Mid-trimester maternal plasma cytokines and CRP as predictors of spontaneous preterm birth

Most previous studies of maternal cytokines and preterm birth have analyzed immunologic biomarkers after the onset of labor or membrane rupture; fewer have examined the systemic (blood) immune response prior to labor onset. We carried out a case–control study nested in a large (n = 5337) prospective, multi-center cohort. Cohort women had an interview, examination, and venipuncture at 24–26 weeks. Frozen plasma samples in women with spontaneous preterm birth (n = 207) and approximately 2 term controls per case (n = 444) were analyzed using Luminex multianalyte profiling technology. Fresh placentas were fixed, stained, and blindly assessed for histologic evidence of infection/inflammation, decidual vasculopathy, and infarction, and vaginal swabs were analyzed for bacterial vaginosis and fetal fibronectin concentration. High maternal matrix metalloproteinase-9 (MMP-9) concentration, but none of the other cytokines or C-reactive protein (CRP), was significantly associated with spontaneous preterm birth [adjusted OR = 1.7 (1.1–2.4)] and showed a dose–response relation across quartiles. No association was observed, however, between maternal MMP-9 and placental infection/inflammation, bacterial vaginosis, or vaginal fetal fibronectin concentration. Our results require confirmation in future studies but suggest that a systemic immune response implicating MMP-9 may have an etiologic role in spontaneous preterm birth.