Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips

Cell adhesion is required for essential biological functions such as migration, tissue formation and wound healing, and it is mediated by individual molecules that bind specifically to ligands on other cells or on the extracellular matrix. Atomic force microscopy (AFM) has been successfully used to measure cell adhesion at both single molecule and whole cell levels. However, the measurement of inherent cell adhesion properties requires a constant cell-probe contact area during indentation, a requirement which is not fulfilled in common pyramidal or spherical AFM tips. We developed a procedure using focused ion beam (FIB) technology by which we modified silicon pyramidal AFM cantilever tips to obtain flat-ended cylindrical tips with a constant and known area of contact. The tips were validated on elastic gels and living cells. Cylindrical tips showed a fairly linear force-indentation behaviour on both gels and cells for indentations >200 nm. Cylindrical tips coated with ligands were used to quantify inherent dynamic cell adhesion and elastic properties. Force, work of adhesion and elasticity showed a marked dynamic response. In contrast, the deformation applied to the cells before rupture was fairly constant within the probed dynamic range. Taken together, these results suggest that the dynamic adhesion strength is counterbalanced by the dynamic elastic response to keep a constant cell deformation regardless of the applied pulling rate.     

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.     

Changes in abscisic acid and flower pigments during floral senescence of petunia

The present work was focused on abscisic acid (ABA) changes in three differently coloured petunias during flower development and senescence. The ABA content was studied in correlation with changes of flower pigments and other phytohormones. The variations of anthocyanins and endogenous hormones were induced by treatments with 1 or 2 mM amino-oxyacetic acid (AOA), 50, 100 μM thidiazuron (TDZ) and 50 μM 6-benzyladenine (BA). ABA content decreased during bud development and increased during senescence. The AOA reduced the anthocyanins content and avoided ABA increase, while the cytokinins (BA and TDZ) did not significantly affected anthocyanin contents but increased ABA content. TDZ doubled the ABA content compared to the control. However, the treatments did not affected flower life, confirming the secondary role of ABA during flower senescence.     

Propulsion Phase of the Single Leg Triple Hop Test in Women with Patellofemoral Pain Syndrome: A Biomechanical Study

Asymmetry in the alignment of the lower limbs during weight-bearing activities is associated with patellofemoral pain syndrome (PFPS), caused by an increase in patellofemoral (PF) joint stress. High neuromuscular demands are placed on the lower limb during the propulsion phase of the single leg triple hop test (SLTHT), which may influence biomechanical behavior. The aim of the present cross-sectional study was to compare kinematic, kinetic and muscle activity in the trunk and lower limb during propulsion in the SLTHT using women with PFPS and pain free controls. The following measurements were made using 20 women with PFPS and 20 controls during propulsion in the SLTHT: kinematics of the trunk, pelvis, hip, and knee; kinetics of the hip, knee and ankle; and muscle activation of the gluteus maximus (GM), gluteus medius (GMed), biceps femoris (BF) and vastus lateralis (VL). Differences between groups were calculated using three separate sets of multivariate analysis of variance for kinematics, kinetics, and electromyographic data. Women with PFPS exhibited ipsilateral trunk lean; greater trunk flexion; greater contralateral pelvic drop; greater hip adduction and internal rotation; greater ankle pronation; greater internal hip abductor and ankle supinator moments; lower internal hip, knee and ankle extensor moments; and greater GM, GMed, BL, and VL muscle activity. The results of the present study are related to abnormal movement patterns in women with PFPS. We speculated that these findings constitute strategies to control a deficient dynamic alignment of the trunk and lower limb and to avoid PF pain. However, the greater BF and VL activity and the extensor pattern found for the hip, knee, and ankle of women with PFPS may contribute to increased PF stress.     

Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice

The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkermansia muciniphila. However, CLA supplementation was not able to override the negative effects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.     

Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum

The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1). Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate]. Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.     

Seagrass sediments reveal the long‐term deterioration of an estuarine ecosystem

The study of a Posidonia australis sediment archive has provided a record of ecosystem dynamics and processes over the last 600 years in Oyster Harbour (SW Australia). Ecosystem shifts are a widespread phenomenon in coastal areas, and this study identifies baseline conditions and the time‐course of ecological change (cycles, trends, resilience and thresholds of ecosystem change) under environmental stress in seagrass‐dominated ecosystem. The shifts in the concentrations of chemical elements, carbonates, sediments <0.125 mm and stable carbon isotope signatures (δ¹³C) of the organic matter were detected between 1850s and 1920s, whereas the shift detected in P concentration occurred several decades later (1960s). The first degradation phase (1850s–1950s) follows the onset of European settlement in Australia and was characterized by a strong increase in sediment accumulation rates and fine‐grained particles, driven primarily by enhanced run‐off due to land clearance and agriculture in the catchment. About 80% of total seagrass area at Oyster Harbour was lost during the second phase of environmental degradation (1960s until present). The sharp increase in P concentration and the increasing contribution of algae and terrestrial inputs into the sedimentary organic matter pool around 1960s provides compelling evidence of the documented eutrophication of the estuary and the subsequent loss of seagrass meadows. The results presented demonstrate the power of seagrass sedimentary archives to reconstruct the trajectories of anthropogenic pressures on estuarine ecosystem and the associated regime shifts, which can be used to improve the capacity of scientists and environmental managers to understand, predict and better manage ecological change in these ecosystems.     

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.     

Estimating and Mapping the Population at Risk of Sleeping Sickness

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, persists as a public health problem in several sub-Saharan countries. Evidence-based, spatially explicit estimates of population at risk are needed to inform planning and implementation of field interventions, monitor disease trends, raise awareness and support advocacy. Comprehensive, geo-referenced epidemiological records from HAT-affected countries were combined with human population layers to map five categories of risk, ranging from “very high” to “very low,” and to estimate the corresponding at-risk population.

Results

Approximately 70 million people distributed over a surface of 1.55 million km² are estimated to be at different levels of risk of contracting HAT. Trypanosoma brucei gambiense accounts for 82.2% of the population at risk, the remaining 17.8% being at risk of infection from T. b. rhodesiense. Twenty-one million people live in areas classified as moderate to very high risk, where more than 1 HAT case per 10,000 inhabitants per annum is reported.

Discussion

Updated estimates of the population at risk of sleeping sickness were made, based on quantitative information on the reported cases and the geographic distribution of human population. Due to substantial methodological differences, it is not possible to make direct comparisons with previous figures for at-risk population. By contrast, it will be possible to explore trends in the future. The presented maps of different HAT risk levels will help to develop site-specific strategies for control and surveillance, and to monitor progress achieved by ongoing efforts aimed at the elimination of sleeping sickness.