Ice cover extent drives phytoplankton and bacterial community structure in a large north-temperate lake: implications for a warming climate.

Mid-winter limnological surveys of Lake Erie captured extremes in ice extent ranging from expansive ice cover in 2010 and 2011 to nearly ice-free waters in 2012. Consistent with a warming climate, ice cover on the Great Lakes is in decline, thus the ice-free condition encountered may foreshadow the lakes future winter state. Here, we show that pronounced changes in annual ice cover are accompanied by equally important shifts in phytoplankton and bacterial community structure. Expansive ice cover supported phytoplankton blooms of filamentous diatoms. By comparison, ice free conditions promoted the growth of smaller sized cells that attained lower total biomass. We propose that isothermal mixing and elevated turbidity in the absence of ice cover resulted in light limitation of the phytoplankton during winter. Additional insights into microbial community dynamics were gleaned from short 16S rRNA tag (Itag) Illumina sequencing. UniFrac analysis of Itag sequences showed clear separation of microbial communities related to presence or absence of ice cover. Whereas the ecological implications of the changing bacterial community are unclear at this time, it is likely that the observed shift from a phytoplankton community dominated by filamentous diatoms to smaller cells will have far reaching ecosystem effects including food web disruptions.

Broad conditions favor the evolution of phase-variable loci.

UNLABELLED: Simple sequence repeat (SSR) tracts produce stochastic on-off switching, or phase variation, in the expression of a panoply of surface molecules in many bacterial commensals and pathogens. A change to the number of repeats in a tract may alter the phase of the translational reading frame, which toggles the on-off state of the switch. Here, we construct an in silico SSR locus with mutational dynamics calibrated to those of the Haemophilus influenzae mod locus. We simulate its evolution in a regimen of two alternating environments, simultaneously varying the selection coefficient, s, and the epoch length, T. Some recent work in a simpler (two-locus) model suggested that stochastic switching in a regimen of two alternating environments may be evolutionarily favored only if the selection coefficients in the two environments are nearly equal ("symmetric") or selection is very strong. This finding was puzzling, as it greatly restricted the conditions under which stochastic switching might evolve. Instead, we find agreement with other recent theoretical work, observing selective utility for stochastic switching if the product sT is large enough for the favored state to nearly fix in both environments. Symmetry is required neither in s nor in sT. Because we simulate finite populations and use a detailed model of the SSR locus, we are also able to examine the impact of population size and of several SSR locus parameters. Our results indicate that conditions favoring evolution and maintenance of SSR loci in bacteria are quite broad. IMPORTANCE: Bacteria experience frequent changes of environment during the infection cycle. One means to rapidly adapt is stochastic switching: a bacterial lineage will stochastically produce a variety of genotypes, so that some descendants will survive if the environment changes. Stochastic switching mediated by simple sequence repeat (SSR) loci is widespread among bacterial commensals and pathogens and influences critical interactions with host surfaces or immune effectors, thereby affecting host persistence, transmission, and virulence. Here, we use the most detailed in silico model of an SSR locus to date, with its phase variation calibrated to match the mod locus of Haemophilus influenzae. The type III restriction-modification system encoded by mod participates in the regulation of multiple other genes; thus, SSR-mediated phase variation of mod has far-reaching cis-regulatory effects. This coupling of phase-variable switching to complex phenotypic effects has been described as the "phasevarion" and is central to understanding the infection cycle of bacterial commensals and pathogens.

Response of female cuttlefish Sepia officinalis (Cephalopoda) to mirrors and conspecifics: evidence for signaling in female cuttlefish.

Cuttlefish have a large repertoire of body patterns that are used for camouflage and interspecific signaling. Intraspecific signaling by male cuttlefish has been well documented but studies on signaling by females are lacking. We found that females displayed a newly described body pattern termed Splotch toward their mirror image and female conspecifics, but not to males, prey or inanimate objects. Female cuttlefish may use the Splotch body pattern as an intraspecific signal, possibly to reduce agonistic interactions. The ability of females to produce a consistent body pattern in response to conspecifics and mirrors suggests that they can recognize same-sex conspecifics using visual cues, despite the lack of sexual dimorphism visible to human observers.

A parallel approach to post source decay MALDI-TOF analysis.

We present a novel enhancement to matrix-assisted laser desorption ionization (MALDI) post-source decay (PSD) analysis whereby fragment ions from multiple precursor ions are acquired into the same spectrum without employing a timed ion gate to preselect each parent ion. Fragment ions are matched to their corresponding precursor ions by comparing spectra acquired at slightly different reflectron electric fields. By measuring the difference in time-of-flight (TOF) between the two spectra for each fragment, it is possible to calculate the mass of the fragment ion and its parent. This new "parallel PSD" technique reduces analysis time and consumes less sample than conventional PSD, which requires an ion gate for serial preselection of precursor ions.

Separation of nicotine metabolites by capillary zone electrophoresis and capillary zone electrophoresis/mass spectrometry.

The use of capillary zone electrophoresis (CZE) and capillary zone electrophoresis/mass spectrometry (CZE/MS) has been demonstrated, in principle, for the separation of nicotine and nicotine metabolites. The buffer system developed for separation and detection by CZE/UV was modified for use in CZE/MS analysis. Several of the metabolites are isobaric and tandem mass spectrometric (MS/MS) techniques have been used to differentiate such analytes.

Hydrogen/deuterium exchange using a coaxial sheath-flow interface for capillary electrophoresis/mass spectrometry.

The interfacing of capillary electrophoresis (CE) with mass spectrometry (MS) is well established and may be accomplished by use of either a coaxial arrangement or by employing a liquid T-junction. In both these interfaces a make-up flow is introduced. This is required because of the mismatch in flow rates for capillary electrophoresis approximately nL/min and 'true' electrospray approximately 2-10 microL/min. Electrical connectivity may also be established where the liquid flows meet (the introduction of nanospray renders the use of make-up flow unnecessary). Hydrogen/deuterium (H/D) exchange occurs in solution when there are labile hydrogen atoms present in a molecule. The establishment of the presence and the number of such exchangeable hydrogen atoms may be of importance in the identification and differentiation of compounds. It may also be an aid in the structural elucidation of unknown materials. We have investigated the feasibility of carrying out H/D exchange via a CE/MS interface. This involved the addition of D2O to the sheath flow and our preliminary results showing the separations of drug substances, subsequently undergoing exchange, are presented.

Evaluation of antibacterial activity of belizean plants: an improved method.

The influences of medium type, inoculum density, and a cold incubation on antimicrobial assay sensitivity were tested. The largest and most distinct zones were produced using nutrient agar and the 1/10 4 inoculum density for Staphylococcus aureus and Proteus mirabilis but a 1/10 12 inoculum density for Pseudomonas aeruginosa and Escherichia coli . The greatest number of zones were detected without cold incubation. Using this method, eight plants from Belize were screened for antibacterial activity. Six plants showed activity against the four organisms tested. Both inoculum density and medium type played important roles in assay sensitivity; however, inoculum density was of more practical significance.

A capillary gas chromatographic assay with nitrogen phosphorus detection for the quantification of topiramate in human plasma, urine and whole blood.

An accurate and robust method involving liquid liquid extraction and capillary gas chromatographic (GC) assay with nitrogen phosphorus detection (NPD) was developed and validated for the quantitative determination of topiramate [2,3:4,5-bis-O-(-1-methylethylidene)-beta-D-fructopyranose sulfamate], Topamax, an anticonvulsant drug, in human plasma, urine, and whole blood. The galactopyranose analog of topiramate was used as the internal standard. A DB-5, fused silica capillary column (J&W Scientific, Folsom, CA) was used, yielding typical retention times of 4.95 min for topiramate and 5.32 min for the internal standard in human plasma. The assay involved organic extraction with methyl t-butyl ether (MTBE) from base, a back extraction into acid and a second extraction in MTBE. The organic solvent was evaporated, and the residue was redissolved and injected for analysis. The standard curve was validated from 0.5 to 50 microg/ml(-1) for human plasma and whole blood, and from 1.0 to 50 microg/ml(-1) for urine. Peak area ratios of drug to internal standard were determined and used to construct a standard curve. The resulting chromatograms showed no endogenous interfering peaks with the respective blank human fluids. Chromatograms corresponding to topiramate and the internal standard produced sharp peaks that were well resolved. This assay showed precision and accuracy of < or = 5%. Two minor human metabolites of topiramate did not interfere with the assay. This assay was successfully applied to determine the pharmacokinetics of topiramate during the development of this drug.

Variations in expression of copper/zinc superoxide dismutase in villous trophoblast of the human placenta with gestational age.

This study investigated expression of the key antioxidant enzyme copper/zinc superoxide dismutase in the villous trophoblast of the human placenta at different gestational ages from 8 weeks (last menstrual period) to term. Immunostaining for the enzyme was observed in the cytotrophoblast cells at all stages. Staining was generally absent from the syncytiotrophoblast at 8 weeks, except for small isolated areas close to the basal surface. The size and location of these areas suggested they were the result of recent cytotrophoblastic fusion. By 10 weeks, examples were more frequent and diffuse staining throughout most of the syncytiotrophoblast was observed at 12 weeks. The intensity of the immunostaining within the syncytiotrophoblast continued to increase until 14 weeks, by which time it matched generally that within the cytotrophoblast cells. A similar pattern of staining was observed within term material. These results are entirely consistent with the hypothesis that the oxygen tension within the intervillous space is low throughout the first trimester of pregnancy. They support the idea that an effective maternal circulation to the human placenta is only established at the start of the second trimester.

Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture.

We have recently shown using dansyl-L-lysine exclusion studies that the release of human chorionic gonadotrophin (HCG) in conjunction with L-lactate dehydrogenase (LDH) from first trimester villi during organ culture is symptomatic of syncytiotrophoblast degeneration. The purpose of this study was to examine chorionic villi at the ultrastructural level in order to determine events occurring during organ culture. The tissue was sampled after 0, 24, 48 and 120 h in culture and processed for electron microscopy. In addition to confirming the previously recorded syncytial degeneration, the electron micrographs showed clearly the generation of a new syncytiotrophoblast layer. The new layer, derived from differentiating cytotrophoblast cells, was largely formed by 48 h and was maintained for at least 120 h in culture. This study demonstrates a model which provides an opportunity to study the differentiation of cytotrophoblast cells whilst they retain their anatomical relationships within the villous structure.

An in vitro model for the study of wound healing in first trimester human placenta.

The placenta operates as a vital interface between the mother and fetus. In addition to facilitating fetal nourishment, it acts as a barrier both to potentially deleterious agents and to contact between their two immune systems. As a consequence, damage to the placenta, even on a relatively small scale, could be very dangerous to the fetus. Therefore, wound repair mechanisms are likely to be of great importance in ensuring that an intact placental barrier is re-established as soon as possible. By use of an in vitro method for injuring and subsequently culturing small pieces of first trimester villous tissue, we have observed a number of indications that a wound response is initiated. Pronounced expression of transforming growth factor-beta1, heavy infiltration of macrophages and late deposition of tenascin in the region of the wound all provide good evidence of some form of healing activity. Furthermore, we have noted that these indicators are suggestive of 'adult-type' rather than 'fetal-type' repair processes.

Human chorionic gonadotrophin release and tissue viability in placental organ culture.

The use of human chorionic gonadotrophin (HCG) secretion as a measure of viability during the organ culture of human first trimester placental tissue has become a popular practice. It has been suggested that if cultured tissue is releasing large amounts of this protein hormone, there is a high level of viability. We have found, however, that the cytosolic enzyme L-lactate dehydrogenase is released into the culture supernatant in a similar daily pattern as HCG, suggesting that tissue disruption may be occurring, resulting in some of the observed hormone release. In addition, we have shown that the uptake of the fluorescent dye dansyl-L-lysine into the syncytium increases significantly from day 0 to day 4, suggesting a loss of syncytial membrane integrity. Electron micrographs show further evidence of the syncytial degeneration at the ultrastructural level, displaying extensive vacuolation and poor microvillous cover. In contrast to the degenerated state of the syncytiotrophoblast, a high level of bromodeoxyuridine incorporation is observed for cytotrophoblasts and, in particular, stromal cells up to 5 days in culture. Overall, the results suggest that the use of HCG release as a determinant of tissue viability in placental organ culture should be treated with a degree of caution.

Development of the chick chorioallantoic capillary plexus under normoxic and normobaric hypoxic and hyperoxic conditions: a morphometric study.

Fertile eggs from the domestic fowl were incubated under normobaric normoxic (21% O2), hypoxic (14% O2), and hyperoxic (40% O2) conditions in order to examine the influence of the prevailing oxygen level on the growth and maturation of the chorioallantoic membrane. Eggs were sampled at regular stages throughout incubation for morphometric analysis. Under normoxic conditions, maturation of the capillary plexus occurred in two distinct stages, both of which contributed to a reduction in the thickness of the air-blood barrier. Between days 6 and 10, the capillaries sprouted and fused to form a dense plexus. Subsequently, between days 10 and 14, this plexus invaginated into the chorionic epithelium. Differentiation of the chorioallantoic membrane appeared maximal by the end of this period. Hypoxia resulted in diminished growth of the embryo and chorioallantoic membrane, but in accelerated maturation of the capillary plexus. Hyperoxia had a less marked effect but appeared to retard the final invagination of the plexus, resulting in a thicker air-blood barrier.

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

Morphometric differences between the placental vasculature of non-smokers, smokers and ex-smokers.

The aim of this study was to determine whether cigarette smoking during pregnancy has an adverse effect upon the placenta's capacity for gaseous exchange. Using recently developed stereological techniques, in conjunction with perfusion fixation, computer-assisted measurements were made on the placentas of 15 non-smokers, 15 moderate smokers, 15 heavy smokers and 13 ex-smokers, 7 of whom stopped smoking during the course of the pregnancy. Compared with the placentas of non-smokers and of those who stopped before pregnancy, it was found that the placentas of smokers and of those who stopped after conception exhibited a reduced capillary volume fraction, and an increased thickness of the villous membrane. Although they must impair gaseous exchange across the placenta, these changes were less severe than suggested by previously published reports. Nonetheless it is clear that in order to prevent these changes women should stop smoking before conception rather than during the course of a pregnancy.

PIP: To learn more about the mechanism of action whereby cigarette smoking leads to a significant reduction in birth weight, computer-assisted measurements were made of the affects of cigarette smoking on placental structure. The study sample included 15 non-smokers, 15 moderate (1-15 cigarettes/day) smokers), 15 heavy (over 15 cigarettes/day) smokers, and 13 ex-smokers, 7 of whom stopped smoking during the index pregnancy. Mean cigarette consumption was 9.3/day in the moderate smoker group and 22.2/day in the heavy smoker group. Pre-conceptional ex-smokers stopped, on average, 13 weeks before conception, while post-conception ex-smokers stopped, on average, 12 weeks after conception. There was no significant difference in birthweight between offspring of non-smokers, moderate smokers, and ex-smokers; however, the infants of heavy smokers were significantly smaller. The terminal villi from all subjects, even women who smoked 30 cigarettes/day, showed large dilated fetal capillaries, with smooth luminal outlines, and no evidence of widespread trophoblastic damage. In terms of placental vasculature, the only significant finding was a slight reduction in the capillary volume fraction in women who had smoked at any stage of pregnancy compared to non-smokers or women who quit smoking before conception. In addition, the villous membrane was significantly thicker among smokers and post- conception stoppers compared to women in the other 2 groups. These findings suggest a less severe effect on the placental vasculature than reported earlier; however, the increased thickness of the villous membrane of smokers observed in this study could compromise gas transfer to the fetus and thus cause growth retardation.

The chorioallantoic capillary plexus of the chicken egg: a microvascular corrosion casting study.

The chorioallantoic membrane of the avian egg serves as the principal organ of respiratory gaseous exchange for the embryo until close to hatching. It lies closely apposed to the inner shell membrane and contains an extremely dense capillary plexus supplied by the allantoic blood vessels. This study applied the microvascular corrosion casting technique to investigate the three-dimensional arrangement of the plexus at various stages of incubation. Casts were produced between days 6 and 14 of incubation, and their appearances were compared with those obtained from traditionally sectioned material and from freeze-cleaved specimens. By day 6 the capillary network was remarkably profuse but showed considerable regional variation in vessel density. In some areas there were only short capillary buds whereas in other areas fusion had taken place so that a true plexus was formed. By day 10 the capillaries had become confluent to such a degree that the cast consisted of a thin sheet of resin perforated only by an array of small irregularly shaped orifices. These corresponded closely in size to the intervening columns of chorionic epithelial cells seen in the sectioned material. It is clear from the appearances of the casts that the capillary surface density becomes maximal at approximately day 10 of incubation. From then on in incubation any increase in the diffusing capacity of the chorioallantoic membrane must be the result of either an increase in its overall surface area, or a decrease in the thickness of the air-blood barrier.

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.

Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells.

In cultured granule cells prelabeled with [3H]arachidonate the activation of excitatory amino acid receptors by various agonists results in a dose-dependent stimulation of [3H]arachidonic acid release. Glutamate and aspartate were the most potent agonists, whereas N-methyl-D-aspartate, kainate and quisqualate were less potent. Other neurotransmitter receptor agonists--GABA, baclofen and norepinephrine--were inactive, while carbachol induced only a slight effect. Since the transmitter-mediated release of [3H]arachidonate was blocked by phencyclidine, a selective inhibitor of NMDA-sensitive glutamate receptors, it can be inferred that the effects of all other receptor agonists were indirectly mediated via the release of glutamate from granule cells. Aspartate-evoked release was Ca2+-dependent and was abolished by the glutamate receptor inhibitors: Mg2+ ions and 2-amino-5-phosphonovalerate. The inhibitors of phospholipase A2, quinacrine and p-bromophenacyl bromide, decreased the release of [3H]arachidonate in a dose-related manner.