On the electrophysiological response of bone cells using a Stokesian fluid stimulus probe for delivery of quantifiable localized picoNewton level forces

A Stokesian fluid stimulus probe (SFSP), capable of delivering quantifiable pN level hydrodynamic forces, is developed to distinguish the electrophysiological response of the cell process and cell body of osteocyte-like MLO-Y4 cells without touching the cell or its substrate. The hydrodynamic disturbance is a short lived (100 ms), constant strength pressure pulse that propagates nearly instantaneously through the medium creating a nearly spherical expanding fluid bolus surrounding a 0.8 μm micropipette tip. Laboratory model experiments show that the growth of the bolus and the pressure field can be closely modeled by quasi-steady Stokes flow through a circular orifice provided the tip Reynolds number, Re(t)<0.03. By measuring the deflection of the dendritic processes between discrete attachment sites, and applying a detailed ultrastructural model for the central actin filament bundle within the process, one is able to calculate the forces produced by the probe using elastic beam theory. One finds that forces between 1 and 2.3 pN are sufficient to initiate electrical signaling when applied to the cell process, but not the much softer cell body. Even more significantly, cellular excitation by the process only occurs when the probe is directed at discrete focal attachment sites along the cell process. This suggests that electrical signaling is initiated at discrete focal attachments along the cell process and that these sites are likely integrin-mediated complexes associated with stretch-activated ion channels though their molecular structure is unknown.     

Positive and negative effects of a dominant competitor on the settlement, growth, and survival of competing species in an epibenthic community

Because dominant competitors can monopolize resources, any positive effects they have on other species can have large community impacts. The solitary tunicate Ascidia ceratodes is a dominant competitor in the fouling community in Bodega Harbor, CA. This tunicate preempts primary substratum from competitors, but its thick tunic also allows other species to grow on its surface. The net effect of Ascidia on the community as a whole therefore depends on the balance between competitive and facilitative effects. In this study we evaluate the facilitative effects of Ascidia on different life stages of common competing species. We quantified larval settlement onto Ascidia compared to unoccupied space; we quantified the growth rate on Ascidia of small colonies of two common species; and we measured whether established colonies could escape overgrowth by Ascidia by growing onto its tunic. We found evidence for high rates of settlement on Ascidia, with some species showing higher and others lower settlement relative to that observed on free space. The growth rate of settlers was generally lower on Ascidia compared to primary substratum. We also found that colonial species established on primary space commonly escape overgrowth from Ascidia by growing onto Ascidia, as this occurs in about half of all encounters. This study indicates that the total effect of Ascidia on the community will depend on species-specific and life stage-specific effects, with likely long-term consequences for the diversity and composition of the community.     

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.     

Myocardial dysfunction in an animal model of cancer cachexia

AimsFatigue is a common occurrence in cancer patients regardless of tumor type or anti-tumor therapies and is an especially problematic symptom in persons with incurable tumor disease. In rodents, tumor-induced fatigue is associated with a progressive loss of skeletal muscle mass and increased expression of biomarkers of muscle protein degradation. The purpose of the present study was to determine if muscle wasting and expression of biomarkers of muscle protein degradation occur in the hearts of tumor-bearing mice, and if these effects of tumor growth are associated with changes in cardiac function.Main methodsThe colon26 adenocarcinoma cell line was implanted into female CD2F1 mice and skeletal muscle wasting, in vivo heart function, in vitro cardiomyocyte function, and biomarkers of muscle protein degradation were determined.Key findingsExpression of biomarkers of protein degradation were increased in both the gastrocnemius and heart muscle of tumor-bearing mice and caused systolic dysfunction in vivo. Cardiomyocyte function was significantly depressed during both cellular contraction and relaxation.SignificanceThese results suggest that heart muscle is directly affected by tumor growth, with myocardial function more severely compromised at the cellular level than what is observed using echocardiography.     

Progress with schistosome transgenesis

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.     

Relationships between cytology, bacteriology and vaginal discharge scores and reproductive performance in dairy cattle

The objective was to compare three diagnostic approaches for intrauterine infection and inflammation: scoring of vaginal contents; quantification of percentage of nucleated cells that were polymorphonuclear leukocytes (PMN) following endometrial cytology; and intra-uterine bacteriology. Dairy cows (n = 303) were examined twice, Days 28 (D28) and 42 (D42), where Day 0 = day of calving. Associations between gross vaginal inflammation scores, uterine cytology, and bacteriology, and subsequent reproductive performance were examined using multivariable models. There was fair agreement at D28 (Kappa = 0.29), but only slight agreement at D42 (Kappa < 0.15), between PMN% and gross vaginal inflammation score. Cows were categorized as having PMN% in the highest quartile (H), or not (L), at both D28 and D42; therefore, cows were categorized as PMNLL, PMNLH, PMNHL, or PMNHH. Cows in the highest PMN% quartile at both time periods were slower to conceive (P < 0.001) than those in all other quartiles (mean ± SEM 32.2 ± 2.3, 37.0 ± 5.3, 40.8 ± 4.1, and 55.3 ± 7.3 d from start of breeding to conception for PMNLL, PMNLH, PMNHL, and PMNHH PMN% cows, respectively). Milk yield was greater (P = 0.001) in cows in the lower quartiles for PMN% at D28 and D42 (i.e., PMNLL) than those in the PMNHH and PMNHL categories, with PMNLH intermediate (P = 0.001). We concluded that PMN% was a better predictor of reproductive performance than either intra-uterine bacteriology or gross vaginal inflammation score. Cows in the highest quartile for PMN% at both D28 and D42 had lower pregnancy rates, took longer to conceive, and had a lower milk yield than those in the lower PMN% categories.     

Testing Moran's theorem in an agroecosystem

The abundance and reproductive effort of populations frequently fluctuate across space and time, a phenomenon known as spatial synchrony. Knowledge of the causes of this behavior underlies the ability to manage species, protect the health of humans and the environment, and increase agricultural sustainability. We used an agroecosystem to test Moran's theorem – spatial synchrony results from environmental entrainment. The controlled conditions of the agroecosystem allowed us to create a highly correlated environment while negating the effects of the alternative hypotheses: dispersal and trophic interactions. Under such conditions, synchrony of fruit production by 4288 trees was high over six years in a 32.5 ha pistachio orchard and occurred at similar temporal frequency as weather patterns demonstrating the Moran effect. The spatial synchrony of fruit production was less than the presumed synchrony of the environment supporting research from microcosms and observational studies showing the Moran effect is degraded by local mechanisms. Indeed even under the homogeneous environment of this system, synchrony declined significantly with distance among trees. We present evidence suggesting that the correlation of the local environment affects intrinsic dynamics to cause these patterns. Our findings demonstrate that the Moran effect is, at minimum, partially responsible for the synchronous fruit production in this system. Agroecosystems are often overlooked in basic ecological research; this experiment provides an example of their comparative advantages for the study of some ecological questions.     

Discovery of selective glucocorticoid receptor modulator MK-5932

A series of partial agonists of the Glucocorticoid Receptor were prepared targeting reduced transactivation activity, while maintaining significant transrepression activity. Incorporation of an ortho-aryl amide produced compounds with the desired in vitro profile. Bioreactors consisting of Suspension cultures of Sf21 cells co expressing a CYP3A4 and NADPH-cytochrome P450 oxireductase were used to prepare the major metabolites of these compounds and revealed that oxidative N-dealkylation provided a pathway for formation of metabolites that were more agonistic than the parent partial agonists. Oxidative N-dealkylation was blocked in a new series of compounds, however oxidation alone was capable of producing full agonist metabolites. Incorporation of an ortho-primary amide and utilization of fluorine to modulate agonism afforded partial agonist MK-5932. Synthesis of the major metabolites of MK-5932 using bioreactor technology revealed that no significant GR-active metabolites were formed. Orally administered MK-5932 displayed anti-inflammatory efficacy in a Rat Oxazolone-induced chronic dermatitis model, while sparing plasma insulin.