Binding Kinetics Toolkit for Analyzing Transient Molecular Conformations and Computing Free Energy Landscapes.

Understanding ligand binding kinetics and thermodynamics, which involves investigating the free, transient, and final complex conformations, is important in fundamental studies and applications for chemical and biomedical systems. Examining the important but transient ligand-protein-bound conformations, in addition to experimentally determined structures, also provides a more accurate estimation for drug efficacy and selectivity. Moreover, obtaining the entire picture of the free energy landscape during ligand binding/unbinding processes is critical in understanding binding mechanisms. Here, we present a Binding Kinetics Toolkit (BKiT) that includes several utilities to analyze trajectories and compute a free energy and kinetics profile. BKiT uses principal component space to generate approximated unbinding or conformational transition coordinates for accurately describing and easily visualizing the molecular motions. We implemented a new partitioning approach to assign indexes along the approximated coordinates that can be used as milestones or microstates. The program can generate input files to run many short classical molecular dynamics simulations and uses milestoning theory to construct the free energy profile and estimate binding residence time. We first validated the method with a host-guest system, aspirin unbinding from ß-cyclodextrin, and then applied the protocol to pyrazolourea compounds and cyclin-dependent kinase 8 and cyclin C complexes, a kinase system of pharmacological interest. Overall, our approaches yielded good agreement with published results and suggest ligand design strategies. The computed unbinding free energy landscape also provides a more complete picture of ligand-receptor binding barriers and stable local minima for deepening our understanding of molecular recognition. BKiT is easy to use and has extensible features for future expansion of utilities for postanalysis and calculations.

GeomBD3: Brownian Dynamics Simulation Software for Biological and Engineered Systems.

GeomBD3 is a robust Brownian dynamics simulation package designed to easily handle natural or engineered systems in diverse environments and arrangements. The software package described herein allows users to design, execute, and analyze BD simulations. The simulations use all-atom, rigid molecular models that diffuse according to overdamped Langevin dynamics and interact through electrostatic, Lennard-Jones, and ligand desolvation potentials. The program automatically calculates molecular association rates, surface residence times, and association statistics for any number of user-defined association criteria. Users can also extract molecular association pathways, diffusion coefficients, intermolecular interaction energies, intermolecular contact probability maps, and more using the provided supplementary analysis scripts. We detail the use of the package from start to finish and apply it to a protein-ligand system and a large nucleic acid biosensor. GeomBD3 provides a versatile tool for researchers from various disciplines that can aid in rational design of engineered systems or play an explanatory role as a complement to experiments. GeomBD version 3 is available on our website at http://chemcha-gpu0.ucr.edu/geombd3/ and KBbox at https://kbbox.h-its.org/toolbox/methods/molecular-simulation/geombd/.

The hallucal interphalangeal ossicle: anatomy and basis for ultrasound-guided surgical shaving.

Painful lesions on the plantar aspect of the first interphalangeal joint (IPJ) of the hallux can be attributed to structures called ossicles, nodules, or sesamoids. The aims of the present study were first to verify that ultrasonography (US) is a high-sensitivity tool for diagnosing an interphalangeal ossicle (IO), and second to prove that US-guided-shaving surgery ("milling") is a safe and feasible technique for remodeling the IO. The study is divided into three parts. In the first part, the prevalence of IOs was estimated in 12 cadaver feet using US, anatomical dissection, and fluoroscopy. In the second, a detailed US and morphological description of the IO was obtained. In the third, six cadaver feet were subjected to surgical milling. IO prevalence was 41.6% in gross anatomy, 41.6% in US examination and just 16.6% in fluoroscopy. The ossicles had a mean length of 4 mm (± 2 mm) and a width of 7 mm (± 2 mm). The ossicles could be completely shaved in all specimens without injuring important anatomical structures. Our results indicate that US is a more precise tool for diagnosing an IO than X-ray. Moreover, our US-guided mini-invasive surgical technique appears feasible and safe.

Anatomical basis of a safe mini-invasive technique for lengthening of the anterior gastrocnemius aponeurosis.

BACKGROUND: The surgical procedure itself of lengthening the gastrocnemius muscle aponeurosis is performed to treat multiple musculoskeletal, neurological and metabolical pathologies related to a gastro-soleus unit contracture such as plantar fasciitis, Achilles tendinopathy, metatarsalgia, cerebral palsy, or diabetic foot ulcerations. Therefore, the aim of our research was to prove the effectiveness and safety of a new ultrasound-guided surgery-technique for the lengthening of the anterior gastrocnemius muscle aponeurosis, the "GIAR"- technique: the gastrocnemius-intramuscular aponeurosis release. METHODS AND RESULTS: An ultrasound-guided surgical GIAR on ten fresh-frozen specimens (10 donors, 8 male, 2 females, 5 left and 5 right) was performed. Exclusion criteria of the donated bodies to science were BMI above 35 (impaired ultrasound echogenicity), signs of traumas in the ankle and crural region, a history of ankle or foot ischemic vascular disorder, surgery or space-occupying mass lesions. The surgical procedures were performed by two podiatric surgeons with more than 6 years of experience in ultrasound-guided procedures. The anterior gastrocnemius muscle aponeurosis was entirely transected in 10 over 10 specimens, with a mean portal length of 2 mm (± 1 mm). The mean gain at the ankle joint ROM after the GIAR was 7.9° (± 1.1°). No damages of important anatomical structures could be found. CONCLUSION: Results of this study indicate that our novel ultrasound-guided surgery for the lengthening of the anterior gastrocnemius muscle aponeurosis (GIAR) might be an effective and safe procedure.

Anatomic basis for a new ultrasound-guided, mini-invasive technique for release of the deep transverse metatarsal ligament.

INTRODUCTION: Morton's neuroma is an entrapment neuropathy of the third common plantar digital nerve, caused by the deep transverse metatarsal ligament (DTML). Minimally invasive or percutaneous surgery is a very common procedure, but surgical effectivity of this technique remains controversial. The goal of our study was to prove the effectiveness and safety of a new ultrasound-guided technique for DTML-release in a cadaver model. MATERIALS, METHODS, AND RESULTS: The DTML was visualized in 10 fresh frozen donated body to science-feet (eight male and two females, five left and five right) using an US device (GE Logic R7; 13 MHz linear probe, Madrid, Spain). Consecutively, minimally invasive ultrasound-guided surgery was performed. Exclusion criteria of the donated bodies to science were previous history of forefoot surgery and space occupying mass lesions. The complete release of the ligament was achieved in all specimens without damage of any important anatomical structures as proven by anatomical dissection. CONCLUSIONS: The results of this study indicate that our novel approach of an ultrasound-guided release of the DTML is safer and more effective compared to blind techniques. The DTML could reliably be visualized and securely cut through a dorsal, minimally invasive surgical incision of only 2 mm.

Ultrasound-guided decompression surgery of the distal tarsal tunnel: a novel technique for the distal tarsal tunnel syndrome-part III.

BACKGROUND: The aim of this study was to provide a safe ultrasound-guided minimally invasive surgical approach for a distal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them have been examined by high-resolution ultrasound at the distal tarsal tunnel. The surgical approach has been marked throughout the course of the medial intermuscular septum (MIS, the lateral fascia of the abductor hallucis muscle). After the previous steps, nerve decompression was carried out through a MIS release through a 2.5 mm (± 0.5 mm) surgical portal. As a result, an effective release of the MIS has been obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that this novel ultrasound-guided minimally invasive surgical approach for the release of the MIS might be an effective, safe and quick decompression technique treating selected patients with a distal tarsal tunnel syndrome.

Ultrasound-guided decompression surgery of the tarsal tunnel: a novel technique for the proximal tarsal tunnel syndrome-Part II.

BACKGROUND: The aim of this study is to provide a safe ultrasound-guided minimally invasive surgical approach for a proximal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them were examined by high resolution ultrasound at the medial ankle region. The surgical approach was marked throughout the course of the flexor retinaculum (laciniate ligament). Once the previous steps were done, the flexor retinaculum release technique was carried out with a 2-mm entry only. As a result, an effective and safe release of the flexor retinaculum was obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that our novel ultrasound-guided minimally invasive surgical approach for the release of the flexor retinaculum might be an effective, safe and quick decompression technique treating selected patients with a proximal tarsal tunnel syndrome.

Integration of GIS, Electromagnetic and Electrical Methods in the Delimitation of Groundwater Polluted by Effluent Discharge (Salamanca, Spain): A Case Study.

The present work envisages the possible geometry of a contaminated plume of groundwater near hospital facilities by combining GIS (Geographic Information System) and geophysical methods. The rock underlying the soil and thin sedimentary cover of the study area is moderately fractured quartzite, which makes aquifers vulnerable to pollution. The GIS methodology is used to calculate the area that would be affected by the effluent source of residual water, based on algorithms that consider ground surface mapping (slopes, orientations, accumulated costs and cost per distance). Geophysical methods (electromagnetic induction and electric resistivity tomography) use changes in the electrical conductivity or resistivity of the subsurface to determine the geometry of the discharge and the degree of contamination. The model presented would allow a preliminary investigation regarding potential corrective measures.

Savanna vegetation-fire-climate relationships differ among continents.

Ecologists have long sought to understand the factors controlling the structure of savanna vegetation. Using data from 2154 sites in savannas across Africa, Australia, and South America, we found that increasing moisture availability drives increases in fire and tree basal area, whereas fire reduces tree basal area. However, among continents, the magnitude of these effects varied substantially, so that a single model cannot adequately represent savanna woody biomass across these regions. Historical and environmental differences drive the regional variation in the functional relationships between woody vegetation, fire, and climate. These same differences will determine the regional responses of vegetation to future climates, with implications for global carbon stocks.

Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentameric ion channels that account for the effects of nicotine. Recent genetic studies have highlighted the importance of variants of the CHRNA5/A3/B4 genomic cluster in human nicotine dependence. Among these genetic variants those found in non-coding segments of the cluster may contribute to the pathophysiology of tobacco use through alterations in the expression of these genes. To discern the in vivo effects of the cluster, we generated a transgenic mouse overexpressing the human CHRNA5/A3/B4 cluster using a bacterial artificial chromosome. Transgenic mice showed increased functional α3ß4-nAChRs in brain regions where these subunits are highly expressed under normal physiological conditions. Moreover, they exhibited increased sensitivity to the pharmacological effects of nicotine along with higher activation of the medial habenula and reduced activation of dopaminergic neurons in the ventral tegmental area after acute nicotine administration. Importantly, transgenic mice showed increased acquisition of nicotine self-administration (0.015 mg/kg per infusion) and a differential response in the progressive ratio test. Our study provides the first in vivo evidence of the involvement of the CHRNA5/A3/B4 genomic cluster in nicotine addiction through modifying the activity of brain regions responsible for the balance between the rewarding and the aversive properties of this drug.

Overexpression of α3/α5/ß4 nicotinic receptor subunits modifies impulsive-like behavior.

Recent studies have revealed that sequence variants in genes encoding the α3/α5/ß4 nicotinic acetylcholine receptor subunits are associated with nicotine dependence. In this study, we evaluated two specific aspects of executive functioning related to drug addiction (impulsivity and working memory) in transgenic mice over expressing α3/α5/ß4 nicotinic receptor subunits. Impulsivity and working memory were evaluated in an operant delayed alternation task, where mice must inhibit responding between 2 and 8s in order to receive food reinforcement. Working memory was also evaluated in a spontaneous alternation task in an open field. Transgenic mice showed less impulsive-like behavior than wild-type controls, and this behavioral phenotype was related to the number of copies of the transgene. Thus, transgenic Line 22 (16-28 copies) showed a more pronounced phenotype than Line 30 (4-5 copies). Overexpression of these subunits in Line 22 reduced spontaneous alternation behavior suggesting deficits in working memory processing in this particular paradigm. These results reveal the involvement of α3/α5/ß4 nicotinic receptor subunits in working memory and impulsivity, two behavioral traits directly related to the vulnerability to develop nicotine dependence.

Land-use changes alter radiative energy and water vapor fluxes of a tall-grass Andropogon field and a savanna-woodland continuum in the Orinoco lowlands.

Changes in land use in the Orinoco lowlands affect the daily trends of energy and water vapor fluxes. We analyzed these fluxes along a disturbance gradient beginning from a cultivated tall-grass Andropogon field (S1) and extending over three savanna sites with increasing woody cover over herbaceous vegetation. The savanna sites encompass a herbaceous savanna (S2), a tree savanna (S3) and a woodland savanna (S4). In the wet season, there were differences in the radiation budget: seasonally averaged albedo for S1 (0.17) exceeded that of S2-S4 (0.13-0.14). Eddy covariance fluxes indicate that the partitioning of the daily net radiation (Rn) into sensible and latent heat (lambda E) fluxes depends on land use. During the wet season, evapotranspiration (i.e., lambda E) over the S1-S4 sites accounted for a variable fraction of Rn (i.e., 0.75, 0.52, 0.67 and 0.68, respectively). Therefore, the Bowen ratio was typically below 1. As the dry season progressed, the lambda E/Rn ratio decreased markedly with increasing air and canopy temperatures and air humidity mole fraction deficit. The maximum evaporation rate over the S1-S4 sites was 3.2, 2.5, 3.5 and 4.1 mm day(-1), respectively, and the annual values were 721, 538, 771 and 732 mm year(-1), respectively, equivalent to 49, 65, 52 and 88% of the rainfall. Soil water content fell from a maximum above 0.28 in the wet season to 0.030, 0.026, 0.030 and 0.028 m(3) m(-3) at sites S1-S4, respectively, in the dry season. Leaf area index was greatly reduced as herbaceous vegetation dried out.

Land-use changes alter CO2 flux patterns of a tall-grass Andropogon field and a savanna-woodland continuum in the Orinoco lowlands.

Land use changes in the savannas of the Orinoco lowlands have resulted in a mosaic of vegetation. To elucidate how these changes have affected carbon exchanges with the atmosphere, we measured CO2 fluxes by eddy covariance and soil CO2 efflux systems along a disturbance gradient beginning with a cultivated tall-grass Andropogon field (S1) and extending over three savanna sites with increasing woody cover growing above native herbaceous vegetation. The savanna sites included a herbaceous savanna (S2), a tree savanna (S3) and a woodland savanna (S4). During the wet season, maximum diurnal net ecosystem exchange (NEE) over the S1-S4 sites was 6.6-9.3, 6.6-7.9, 10.6-11.3 and 9.3-10.6 micromol m(-2) s(-1), respectively. The rate of CO2 uptake over S1 was lower than that for C4 grasses elsewhere because of pasture degradation. Soil respiration and temperature were exponentially related when soil water content (theta) was above 0.083 m(3) m(-3); however, soil respiration declined markedly as theta decreased from 0.083-0.090 to 0.033-0.056 m(3) m(-3). There were bursts of CO2 emission when dry soils were rewetted by rainfall. During the wet season, all sites constituted carbon sinks with maximum net daily ecosystem production (NEP) of 2.1, 1.7, 2.1 and 2.1 g C m(-2) day(-1), respectively. During the dry season, the savanna sites (S2-S4) became carbon sources with maximum emission fluxes of -0.5, -1.4 and -1.6 g C m(-2) day(-1), respectively, whereas the tall-grass field (S1) remained a carbon sink with a maximum NEP of 0.3 g C m(-2) day(-1) at the end of the season. For all measurement periods, annual NEP of sites S1-S4 was 366, 6, 116 and 139 g C m(-2), respectively. Comparisons of carbon source/sink dynamics across a wide range of savannas indicate that savanna carbon budgets can change in sign and magnitude. On an annual basis, gross primary production over the S1-S4 stands was 797, 803, 136 and 1230 g C m(-2), respectively. Net primary productivity (NPP) of the S1-S4 stands, calculated from eddy covariance measurements as the daily sum of NEE and day and night heterotrophic respiration was 498, 169, 181 and 402 g C m-2 year-1, respectively. These values were slightly higher than NPP based on harvest measurements (432, 162, 176 and 386 g C m(-2) year(-1), respectively), presumably because fine roots were incompletely harvested. Soil water content limited carbon uptake at all sites, and water-use efficiency (WUE) was related to rainfall dynamics. During the dry season, all sites except the cultivated tall-grass Andropogon field (S1) had a negative WUE. Although our results are specific to the Orinoco vegetational mosaic, the effects of land-use practices on the controls and physiological functions of the studied ecosystems may be generalized to other savannas.

Water flux through a semi-deciduous forest grove of the Orinoco savannas.

Water relations were analysed in a semi-deciduous forest grove occurring in the oxisols of the Orinoco savannas. This grove has a shallow unconsolidated ironstone cuirass, which is overlaid by a sandy loam layer (0.0-0.5 m) that contains more than 90% of the grove forest root phytomass. Evapotranspiration and through drainage were calculated by using data from the soil profile as related to physical characteristics of the site root zone, hydraulic conductivity, volumetric water content and potential hydraulic gradient. Mean annual evapotranspiration was 783 mm year-1 and annual through drainage below the root zone was 14% (162 mm year-1) of the gross rainfall. This drainage recharged the 42% of the annual saturation deficit of the water table. Similar mean annual evapotranspiration (770 mm year-1) was also calculated by using the water balance components. The mean daily coupling omega factor (Ω) between the grove canopy and the surrounding atmosphere indicated that a high degree of coupling (Ω=0.14±0.16) occurs in the grove and evapotranspiration was mainly controlled by surface conductance. As the dry season proceeded, the soil saturation deficit (δθ) increased rapidly resulting in a threshold surface conductance (0.030-0.005 m s-1) for δθ ranging from 0.05 to 0.10. Hypotheses to explain the omnipresence of perennial species in the wide range of physical conditions in neotropical savannas are discussed.