Ecological and Phenotypic Diversification after a Continental Invasion in Neotropical Freshwater Stingrays.

Habitat transitions are key potential explanations for why some lineages have diversified and others have not-from Anolis lizards to Darwin's finches. The ecological ramifications of marine-to-freshwater transitions for fishes suggest evolutionary contingency: some lineages maintain their ancestral niches in novel habitats (niche conservatism), whereas others alter their ecological role. However, few studies have considered phenotypic, ecological, and lineage diversification concurrently to explore this issue. Here, we investigated the macroevolutionary history of the taxonomically and ecologically diverse Neotropical freshwater river rays (subfamily Potamotrygoninae), which invaded and diversified in the Amazon and other South American rivers during the late Oligocene to early Miocene. We generated a time-calibrated, multi-gene phylogeny for Potamotrygoninae and reconstructed evolutionary patterns of diet specialization. We measured functional morphological traits relevant for feeding and used comparative phylogenetic methods to examine how feeding morphology diversified over time. Potamotrygonine trophic and phenotypic diversity are evenly partitioned (non-overlapping) among internal clades for most of their history, until 20-16 mya, when more recent diversification suggests increasing overlap among phenotypes. Specialized piscivores (Heliotrygon and Paratrygon) evolved early in the history of freshwater stingrays, while later trophic specialization (molluscivory, insectivory, and crustacivory) evolved in the genus Potamotrygon. Potamotrygonins demonstrate ecological niche lability in diets and feeding apparatus; however, diversification has mostly been a gradual process through time. We suggest that competition is unlikely to have limited the potamotrygonine invasion and diversification in South America.

Geometric localization in supported elastic struts.

Localized deformation patterns are a common motif in morphogenesis and are increasingly finding applications in materials science and engineering, in such instances as mechanical memories. Here, we describe the emergence of spatially localized deformations in a minimal mechanical system by exploring the impact of growth and shear on the conformation of a semi-flexible filament connected to a pliable shearable substrate. We combine numerical simulations of a discrete rod model with theoretical analysis of the differential equations recovered in the continuum limit to quantify (in the form of scaling laws) how geometry, mechanics and growth act together to give rise to such localized structures in this system. We find that spatially localized deformations along the filament emerge for intermediate shear modulus and increasing growth. Finally, we use experiments on a 3D-printed multi-material model system to demonstrate that external control of the amount of shear and growth may be used to regulate the spatial extent of the localized strain texture.

Determining the lipid specificity of insoluble protein transmembrane domains.

While the specificity of protein-lipid interactions is a key feature in the function of biological membranes, studying the specifics of these interactions is challenging because most membrane proteins are insoluble in water due to the hydrophobic nature of their transmembrane domains (TMDs). Here, we introduce a method that overcomes this solubility limitation and identifies the affinity profile of protein TMDs to specific lipid formulations. Using 5 human TMDs as a sample group, our results demonstrate that TMDs are highly selective and that these specific lipid-TMD interactions can involve either a single lipid, or the combination of multiple lipid species.

Mechanical behavior of idealized, stingray-skeleton-inspired tiled composites as a function of geometry and material properties.

Tilings are constructs of repeated shapes covering a surface, common in both manmade and natural structures, but in particular are a defining characteristic of shark and ray skeletons. In these fishes, cartilaginous skeletal elements are wrapped in a surface tessellation, comprised of polygonal mineralized tiles linked by flexible joints, an arrangement believed to provide both stiffness and flexibility. The aim of this research is to use two-dimensional analytical models to evaluate the mechanical performance of stingray skeleton-inspired tessellations, as a function of their material and structural parameters. To calculate the effective modulus of modeled composites, we subdivided tiles and their surrounding joint material into simple shapes, for which mechanical properties (i.e. effective modulus) could be estimated using a modification of traditional Rule of Mixtures equations, that either assume uniform strain (Voigt) or uniform stress (Reuss) across a loaded composite material. The properties of joints (thickness, Young's modulus) and tiles (shape, area and Young's modulus) were then altered, and the effects of these tessellation parameters on the effective modulus of whole tessellations were observed. We show that for all examined tile shapes (triangle, square and hexagon) composite stiffness increased as the width of the joints was decreased and/or the stiffness of the tiles was increased; this supports hypotheses that the narrow joints and high tile to joint stiffness ratio in shark and ray cartilage optimize composite tissue stiffness. Our models also indicate that, for simple, uniaxial loading, square tessellations are least sensitive and hexagon tessellations most sensitive to changes in model parameters, indicating that hexagon tessellations are the most "tunable" to specific mechanical properties. Our models provide useful estimates for the tensile and compressive properties of 2d tiled composites under uniaxial loading. These results lay groundwork for future studies into more complex (e.g. biological) loading scenarios and three dimensional structural parameters of biological tilings, while also providing insight into the mechanical roles of tessellations in general and improving the design of bioinspired materials.

Multi-lineage MSC differentiation via engineered morphogen fields.

Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-ß1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-ß1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.

Intracellular electroporation site distributions: modeling examples for nsPEF and IRE pulse waveforms.

We illustrate expected electroporation (EP) responses to two classes of large electric field pulses by employing systems models, one of a cell in vitro and the other of multiple cells in vivo. The first pulse class involves "nsPEF" (nanosecond pulsed electric fields). The durations are less than a microsecond, but the magnitudes are extremely large, often 10 kV/cm or more, and all of the pores remain small. The second class involves "IRE" (irreversible electroporation). Durations are many microseconds to several milliseconds, but with magnitudes smaller than 10 kV/cm, and a wide range of pore sizes evolves. A key feature of both pulse classes is non-thermal cell killing by multiple pulses without delivering external drugs or genes. For small pulses the models respond passively (no pore creation) providing negative controls. For larger pulses transient aqueous pore populations evolve. These greatly increase local membrane conductance temporarily, causing rapid redistribution of fields near and within cells. This complex electrical behavior is generally not revealed by experiments reporting biological end points resulting from cumulative ionic and molecular transport through cell membranes. The underlying, heterogeneous pore population distributions are also not obtained from typical experiments. Further, traditional EP applications involving molecular delivery are usually assumed to create pores solely in the outer, plasma membrane (PM). In contrast, our examples support the occurrence of intracellular EP by both nsPEF and IRE, but with different intracellular spatial distributions of EP sites.

In silico estimates of cell electroporation by electrical incapacitation waveforms.

We use a system model of a cell and approximate magnitudes of electrical incapacitation (EI) device waveforms to estimate conditions that lead to responses with or without electroporation (EP) of cell membranes near electrodes. Single pulse waveforms of Taser X26 and Aegis MK63 devices were measured using a resistive load. For the present estimates the digitized waveforms were scaled in magnitude according to the inverse square radial distance from two tissue-penetrating electrodes, approximated as hemispheres. The corresponding tissue level electric fields were then used as inputs to the cell system model. A dynamic pore model for membrane electroporation (EP) was assigned to many different sites on the cell plasma membrane (PM). EI devices generate sufficiently large transmembrane voltage, U(m)(t), such that pores were created, evolving into a heterogeneous and time-dependent pore population. These approximate responses suggest that both waveforms can cause PM EP. Peripheral nerve damage by EP is a candidate side effect. More extensive EP is expected from the Taser X26 than the Aegis MK63, mainly due to the approximately eight-fold difference in the peak magnitudes. In silico examination of EI waveforms by multiscale modeling is warranted, and can involve whole body, tissue and cell level models that now exist and are rapidly being improved.

In situ observation of fluoride-ion-induced hydroxyapatite-collagen detachment on bone fracture surfaces by atomic force microscopy.

The topography of freshly fractured bovine and human bone surfaces was determined by the use of atomic force microscopy (AFM). Fracture surfaces from both kinds of samples exhibited complex landscapes formed by hydroxyapatite mineral platelets with lateral dimensions ranging from ∼90 nm × 60 nm to ∼20 nm × 20 nm. Novel AFM techniques were used to study these fracture surfaces during various chemical treatments. Significant topographical changes were observed following exposure to aqueous solutions of ethylenediaminetetraacetic acid (EDTA) or highly concentrated sodium fluoride (NaF). Both treatments resulted in the apparent loss of the hydroxyapatite mineral platelets on a timescale of a few seconds. Collagen fibrils situated beneath the overlying mineral platelets were clearly exposed and could be resolved with high spatial resolution in the acquired AFM images. Time-dependent mass loss experiments revealed that the applied agents (NaF or EDTA) had very different resulting effects. Despite the fact that the two treatments exhibited nearly identical results following examination by AFM, bulk bone samples treated with EDTA exhibited a ∼70% mass loss after 72 h, whereas for the NaF-treated samples, the mass loss was only of the order of ∼10%. These results support those obtained from previous mechanical testing experiments, suggesting that enhanced formation of superficial fluoroapatite dramatically weakens the protein-hydroxyapatite interfaces. Additionally, we discovered that treatment with aqueous solutions of NaF resulted in the effective extraction of noncollagenous proteins from bone powder.

Emerging linkages between substance abuse and HIV infection in St. Croix, US Virgin Islands.

In the US Virgin Islands 575 cases of AIDS had been reported to the Centers for Disease Control and Prevention through mid-2003. Although males continue to be most affected by HIV/AIDS, the feminization of the epidemic is evidenced by recent data showing rates of infection increasing rapidly among women. This paper focuses on the role of substance abuse and the socially and culturally based gender issues that influence risk and vulnerability to HIV in this setting. 254 chronically drug- or alcohol-involved men and women were recruited and interviewed using targeted sampling strategies. Crack use was overwhelmingly reported by females when compared to males (84.7% vs. 48.8%). Women also reported a significantly higher number of sexual partners in the past month (5.6 vs. 2.3) and significantly more occasions of unprotected vaginal sexual contact (11.2 vs. 6.5). Rates of self-reported HIV infection were elevated among women as well (8.8% vs. 1.4%). Women's precarious economic position and lack of access to legitimate income-generating activities tended to drive them into 'survival sex' to support their subsistence and drug needs. As such, it would appear that substance abuse has an emerging role in the spread of the epidemic in St. Croix, particularly among women.

The effect of serostatus on HIV risk behaviour change among women sex workers in Miami, Florida.

HIV prevention and risk reduction are especially salient and timely issues for women, particularly among those who are drug-involved or who exchange sex for drugs or money. Studies suggest that HIV-prevention measures can be effective with highly vulnerable women, and have the potential to produce significant reductions in risk behaviours among both HIV-negative and HIV-positive women. Within this context, this paper examines risk behaviours and HIV serostatus among 407 drug-involved women sex workers in Miami, Florida, and investigates the effects of participation in HIV testing, counselling, and a risk-reduction intervention on subsequent behavioural change among this population. Overall, at follow-up, the HIV-positive women were 2.4 times more likely than the HIV-negative women to have entered residential treatment for drug abuse, 2.2 times more likely to have decreased the number of their sex partners, 1.9 times more likely to have decreased the frequency of unprotected sex, 1.9 times more likely to have reduced their levels of alcohol use, and 2.3 times more likely to have decreased their crack use. These data support the importance of HIV testing and risk-reduction programmes for drug-involved women sex workers.

Microfabrication of individual 200 microm diameter transdermal microconduits using high voltage pulsing in salicylic acid and benzoic acid.

We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids.

Non-linearity of molecular transport through human skin due to electric stimulus.

The multilamellar bilayer system of the skin's stratum corneum (SC) provides the main barrier to transdermal transport of ions and charged molecules. Electrically driven transport of charged species at low trans-SC voltages (U(SC)<5 V) occurs predominantly via pre-existing aqueous pathways. In contrast, high voltage, (HV; U(SC)>50 V) has been hypothesized to involve electroporation within the SC's multilamellar bilayer membranes, creating new aqueous pathways that contribute to a rapid, large increase in transport. Thus, it might be expected that HV-pulses would always increase subsequent iontophoresis. Here we show, however, that for some charged molecules the opposite occurs, because the low skin resistance due to new aqueous pathways leads to an actual decrease in U(SC) for the same applied current, and the transport of some, highly charged molecules has a highly nonlinear dependence on U(SC).

Biological sensing of small field differences by magnetically sensitive chemical reactions.

There is evidence that animals can detect small changes in the Earth's magnetic field by two distinct mechanisms, one using the mineral magnetite as the primary sensor and one using magnetically sensitive chemical reactions. Magnetite responds by physically twisting, or even reorienting the whole organism in the case of some bacteria, but the magnetic dipoles of individual molecules are too small to respond in the same way. Here we assess whether reactions whose rates are affected by the orientation of reactants in magnetic fields could form the basis of a biological compass. We use a general model, incorporating biological components and design criteria, to calculate realistic constraints for such a compass. This model compares a chemical signal produced owing to magnetic field effects with stochastic noise and with changes due to physiological temperature variation. Our analysis shows that a chemically based biological compass is feasible with its size, for any given detection limit, being dependent on the magnetic sensitivity of the rate constant of the chemical reaction.

Simultaneous quantitative determination of electroporative molecular uptake and subsequent cell survival using gel microdrops and flow cytometry.

BACKGROUND: Electroporation is widely used to introduce molecules into cells, but conditions yielding maximal molecular uptake often result in low cell survival. We describe a high throughput method for analyzing populations of culturable cells simultaneously for molecular uptake and cell growth. METHODS: Cells are microencapsulated within agarose gel microdrops (GMDs), exposed to a polar tracer fluorescent molecule, electrically pulsed at various field strengths, and cultured. The GMDs are then analyzed at about 100,000 occupied GMDs per hour by flow cytometry for both uptake and microcolony formation. RESULTS: We demonstrate how the method can be used to optimize a parameter of interest (e.g., the applied field strength) with respect to both uptake and cell survival. Here, the optimal field strength is determined to be 1.7 kV/cm. Below this, there is lower molecular uptake. As the field strength is increased, the cell survival rate goes down. CONCLUSIONS: This method may be applicable to optimization of other electroporation parameters and alternative physical and chemical methods for cell loading.

Mechanism of transdermal drug delivery by electroporation.

Human skin is a complex system, providing a formidable obstacle to drug delivery Fig. 1 (1-3). In particular, the stratum corneum (SC) is the primary barrier to transdermal drug delivery. The stratum corneum is made up of corneocytes, which are flattened remnants of cells, surrounded by lipid bilayer membranes (2,3). Because lipid-based structures tend to exclude charged species, the multilamellar arrangement acts as a "brick wall" (4) to prevent ionic and molecular transport. Fig. 1. 1. Key features of skin, skin barriers (104) and hypothetical aqueous pathways. The stratum corneum (SC) is the dead outermost layer (≈ 20 µm hydrated thickness) that is the main barrier to transport, particularly for charged molecules (1-3,5). Appendages (sweat ducts and hair follicles) penetrate the SC, but are lined with a double cell layer with tight junctions (105), which prevents significant transport. Hypothetical transport sites are labeled: LV (low voltage): Iontophoresis involves transport through preexisting aqueous pathways associated with appendages or around corneocytes within the SC (7,8,53,55,106). MV (moderate voltage) pulses: A relatively fast (>10 ms) process of "macropore activation" is followed by cell lining electroporation, which is hypothesized to create new aqueous pathways at appendages ("appendageal electroporation") (40,107). HV (high voltage) pulses: A very fast (<10 µs) process is hypothesized to involve a primary event of aqueous pathway creation based on electroporation of multilamellar lipid bilayers separating corneocytes (27,61,63,91,108), and secondary processes such as localized heating, or pathwayenlarging chemical introduction. Lower right: The original "brick wall model" of the SC (the largest area skin feature) intended to treat permeation of lipophilic molecules through the continuous "mortar" (4).

An in vitro system for measuring the transdermal voltage and molecular flux across the skin in real time.

In many in vitro transdermal drug delivery experiments, the skin is placed within a permeation chamber, and measurements are taken every hour or so. However, during skin electroporation, significant molecular transport can occur within the first few minutes (1).

Spatially constrained skin electroporation with sodium thiosulfate and urea creates transdermal microconduits.

Controlled transport of molecules through the skin's main barrier, the stratum corneum (SC), is a long standing goal of transdermal drug delivery. Traditional, needle-based injection provides delivery of almost any water soluble compound, by creating a single large aqueous pathway in the form of the hollow core of a needle, through which drug is delivered by pressure-driven flow. We extend previous work to show that SC-spanning microconduits (here with diameters of about 200 microm) can be created in vivo by skin electroporation and low-toxicity, keratolytic molecules (here sodium thiosulfate and urea). A single microconduit in isolated SC can support volumetric flow of the order of 0.01 ml s(-1) by a pressure difference of only 0.01 atm (about 10(2) Pa), demonstrating that the SC barrier has been essentially eliminated within this microscopic area.

Biological effects due to weak electric and magnetic fields: the temperature variation threshold.

A large number of epidemiological and experimental studies suggest that prolonged (>100 s) weak 50-60-Hz electric and magnetic field (EMF) exposures may cause biological effects(NIEHS Working Group, NIH, 1998; Bersani, 1999). We show, however, that for typical temperature sensitivities of biochemical processes, realistic temperature variations during long exposures raise the threshold exposure by two to three orders of magnitude over a fundamental value, independent of the biophysical coupling mechanism. Temperature variations have been omitted in previous theoretical analyses of possible weak field effects, particularly stochastic resonance (Bezrukov and Vodyanoy 1997a. Nature. 385:319-321; Astumian et al., 1997 Nature. 338:632-633; Bezrukov and Vodyanoy, 1997b. Nature. 338:663; Dykman and McClintock, 1998. Nature. 391:344; McClintock, 1998;. Gammaitoni et al., 1998. Rev. Mod. Phys. 70:223-287). Although sensory systems usually respond to much shorter (approximately 1 s) exposures and can approach fundamental limits (Bialek, 1987 Annu. Rev. Biophys. Biophys. Chem. 16:455-468; Adair et al, 1998. Chaos. 8:576-587), our results significantly decrease the plausibility of effects for nonsensory biological systems due to prolonged, weak-field exposures.

Comparison of the effects of short, high-voltage and long, medium-voltage pulses on skin electrical and transport properties.

High-voltage pulses have been shown to increase rates of transport across skin by several orders of magnitude on a time scale of minutes to seconds. Two main pulse protocols have been employed to promote transport: the intermittent application of short ( approximately 1 ms) high-voltage (approximately 100 V across skin) pulses and a few applications of long (=100 ms) medium-voltage (>30 V across skin) pulses. In order to better evaluate the benefits of each protocol for transdermal drug delivery, we compared these two protocols in vitro in terms of changes in skin electrical properties and transport of sulforhodamine, a fluorescent polar molecule of 607 g/mol and a charge of -1. Whereas both protocols induced similar alterations and recovery processes of skin electrical resistance, long pulses of medium-voltage appeared to be more efficient in transporting molecules across skin. Skin resistance decreased by three (short pulses) and two (long pulses) orders of magnitude, followed by incomplete recovery in both cases. For the same total transported charge, long pulses induced faster and greater molecular transport across skin than short pulses. In addition, a greater fraction of the aqueous pathways created by the electric field was involved in molecular transport when using long pulse protocols. Transport was concentrated in localized transport regions (LTRs) for both protocols but LTRs created by long pulses were an order of magnitude larger than those formed by short pulses and the short pulses created an order of magnitude more LTRs. Overall, this study is consistent with the creation of fewer, but larger aqueous pathways by long, medium-voltage pulses in comparison to short, high-voltage pulses.

Spatially constrained localized transport regions due to skin electroporation.

Rapid, controlled molecular transport across human skin is of great interest for transdermal drug delivery and minimally invasive chemical sensing. Short, high-voltage pulses have been shown previously to create localized transport regions in the skin. Here, we show that these regions can be constrained to occur at specific sites using electrically insulating masks that restrict the field lines. The increase in total ionic and molecular transport per area was comparable to the levels observed in unconstrained electroporation of human skin. Constraining the area of intervention to encompass small areas of interest, a primary feature in the design of microdevices for transdermal drug delivery, can provide the same levels of flux as the unconstrained case.