Measure of simultaneous tree sways and estimation of crown interactions among a group of trees

We present a technique to measure the simultaneous sway of a group of trees and reconstruct the frequency of crown collisions and sway dynamics of individual or groups of trees. We placed a biaxial clinometer (tiltmeter) at the live crown base in each of ten neighboring 15-m-tall lodgepole pine trees in Alberta, Canada. Tree bole rotation at tiltmeter mount height was recorded during windy conditions at a rate of 10 times/s for the cluster of trees. Rotation angles were used in a bole curve calculation to estimate tree displacement in 2-dimensional (x, y) space. Collision reconstruction was done in Arc/Info by assigning asymmetrical crown area dimensions (polygons) to calculated bole displacement for each tree. Reconstruction of each time step measured any overlaps between crown polygon areas. Crown polygon overlaps estimated in this manner allowed assessment of collision frequencies, area of crown overlap during collisions, and identification of the tree(s) that a subject tree contacted. Collision statistics are only given for trees interior to the sensored cluster (n=3). For 15.0 min of data with an average wind speed of 4.5 m/s and a maximum of 10.0 m/s there was an average of 65 collisions/min for each tree, and an average collision overlap area of 24%. This frequency and depth of collisions supports the notion that wind-induced crown interaction inhibits lateral shoot extension and is an important mechanism for the development of crown asymmetry and crown shyness. Insight into dynamic tree sway behavior and crown interactions will allow estimation and cultivation of a forest stand structure that is more resistant to damage from wind. The techniques of recording multiple simultaneous bole sway and their reconstruction are applicable to a broad range of wind-forest interaction research.     

Evolutionary aspects of intestinal bicarbonate secretion in fish

Experiments compared intestinal HCO3- secretion in the intestine of marine teleost Gulf toadfish, Opsanus beta, to representatives of early chondrostean and chondrichthyan fishes, the Siberian sturgeon, Acipenser baerii, and white-spotted bamboo shark, Chiloscyllium plagiosum, respectively. As seen in marine teleosts, luminal HCO3- concentrations were 10-fold plasma levels in all species when exposed to hyperosmotic conditions. While intestinal water absorption left Mg2+ and SO4(2-) concentrated in intestinal fluids up to four-fold ambient seawater concentrations, HCO3- was concentrated up to 50 times ambient levels as a result of intestinal HCO3- secretion. Reduced luminal Cl- concentrations in the intestine of all species suggest that HCO3- secretion also occurs via Cl-/HCO3- exchange in chondrostean and chondrichthyan fishes. Sturgeon began precipitating carbonates from the gut after only 3 days at 14 per thousand, a mechanism utilized by marine teleosts to reduce intestinal fluid osmolality and maintain calcium homeostasis. Analysis of published intestinal fluid composition in the cyclostome Lampetra fluviatilis reveals that this species likely also utilize intestinal HCO3- secretion for osmoregulation. Analysis of existing cyclostome data and our results indicate that intestinal Cl-/HCO3- exchange plays an integral role in maintaining hydromineral balance not only in teleosts, but in all fish (and perhaps other animals) with a need to drink seawater.     

Acid–base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid–base regulation during respiratory acidosis either by facilitating or constraining HCO₃ ⁻ accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid–base status during exposure to increasing levels of hypercapnia (nominally 1–5% CO₂). Fish exhibited a well-developed ability to increase plasma HCO₃ ⁻ levels with values of 39.8 ± 2.8 mmol l⁻¹ being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO₂ = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO₃ ⁻ during hypercapnia was associated with marked increases in branchial net acid excretion (J _NETH⁺) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J _NETH⁺ was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na⁺/K⁺-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO₃ ⁻ concentrations of 92.2 ± 4.8 mmol l⁻¹. At the highest level of hypercapnia, rectal fluid HCO₃ ⁻ levels were increased significantly to 141.8 ± 7.4 mmol l⁻¹ but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l⁻¹). Thus, the impressive accumulation of plasma HCO₃ ⁻ to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO₃ ⁻ excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO₃ ⁻ concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO₃ ⁻. The apparent inability of P. notatus to effectively excrete HCO₃ ⁻ following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.     

Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton

Retinoic acid (RA) plays important roles in diverse biological processes ranging from germ cell specification to limb patterning. RA ultimately exerts its effect in the nucleus, but how RA levels are being generated and maintained locally is less clear. Here, we have analyzed the zebrafish stocksteif mutant, which exhibits severe over-ossification of the entire vertebral column. stocksteif encodes cyp26b1, a cytochrome P450 member that metabolizes RA. The mutant is completely phenocopied by treating 4 dpf wild-type embryos with either RA or the pharmacological Cyp26 blocker R115866, thus identifying a previously unappreciated role for RA and cyp26b1 in osteogenesis of the vertebral column. Cyp26b1 is expressed within osteoblast cells, demonstrating that RA levels within these cells need to be tightly controlled. Furthermore, we have examined the effect of RA on osteoblasts in vivo. As numbers of osteoblasts do not change upon RA treatment, we suggest that RA causes increased activity of axial osteoblasts, ultimately resulting in defective skeletogenesis.     

Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.     

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.     

Sources of Variability in a Synthetic Gene Oscillator

Synthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits.