Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis.

Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow.

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF). With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Most High-Intensity Transient Signals Are Not Associated With Specific Surgical Maneuvers.

BACKGROUND: Mortality after congenital heart defect surgery has dropped dramatically in the last few decades. Current research on long-term outcomes has focused on preventing secondary neurological sequelae, for which embolic burden is suspected. In children, little is known of the correlation between specific surgical maneuvers and embolic burden. Transcranial Doppler ultrasound is highly useful for detecting emboli but has not been widely used with infants and children. METHODS: Bilateral middle cerebral artery blood flow was continuously monitored from sternal incision to chest closure in 20 infants undergoing congenital heart defect repair or palliative surgery. Embolus counts for specific maneuvers were recorded using widely accepted criteria for identifying emboli via high-intensity transient signals (HITS). RESULTS: An average of only 13% of all HITS detected during an operation were correlated with any of the surgical maneuvers of interest. The highest mean number of HITS associated with a specific maneuver occurred during cross-clamp removal. Cross-clamp placement also had elevated HITS counts that significantly differed from other maneuvers. CONCLUSIONS: In this study of infants undergoing cardiac surgery with cardiopulmonary bypass, the great majority of HITS detected are not definitively associated with a specific subset of surgical maneuvers. Among the measured maneuvers, removal of the aortic cross-clamp was associated with the greatest occurrence of HITS. Future recommended research efforts include identifying and confirming other sources for emboli and longitudinal outcome studies to determine if limiting embolic burden affects long-term neurological outcomes.

Real-Time Cerebral Hemodynamic Response to Tactile Somatosensory Stimulation.

BACKGROUND AND PURPOSE: Recent studies in rodents suggest that somatosensory stimulation could provide neuroprotection during ischemic stroke by inducing plasticity in the cortex-vasculature relationship. While functional magnetic resonance imaging (fMRI) has shown that somatosensory stimulation increases cerebral blood flow (CBF) over several seconds, subsecond changes in CBF in the basal cerebral arteries have rarely been studied due to temporal resolution limitations. This study characterized hemodynamic changes in the middle cerebral arteries (MCAs) during somatosensory stimulation with high temporal resolution (100 samples/s) using functional transcranial Doppler ultrasound (fTCD). METHODS: Pneumotactile somatosensory stimulation, consisting of punctate pressure pulses traversing the glabrous skin of the hand at 25 cm/s, was used to induce CBF velocity (CBFV) response curves. Changes in CBFV were measured in the bilateral MCAs using fTCD. All 12 subjects underwent three consecutive trials consisting of 20 seconds of stimulation followed by 5 minutes of rest. RESULTS: Sharp, bilateral increases in CBFV of about 20% (left MCA = 20.5%, right MCA = 18.8%) and sharp decreases in pulsatility index of about 8% were observed during stimulation. Left lateralization of up to 3.9% was also observed. The magnitude of the initial increase in CBFV showed significant adaptation between subsequent trials. CONCLUSIONS: Pneumotactile somatosensory stimulation is a potent stimulus that can evoke large, rapid hemodynamic changes, with adaptation between successive stimulus applications. Due to its high temporal resolution, fTCD is useful for identifying quickly evolving hemodynamic responses, and for correlating changes in hemodynamic parameters such as pulsatility index (PI) and CBFV.

Cerebral hemodynamics during scene viewing: Hemispheric lateralization predicts temporal gaze behavior associated with distinct modes of visual processing.

Systematic patterns of eye movements during scene perception suggest a functional distinction between 2 viewing modes: an ambient mode (characterized by short fixations and large saccades) thought to reflect dorsal activity involved with spatial analysis, and a focal mode (characterized by long fixations and small saccades) thought to reflect ventral activity involved with object analysis. Little neuroscientific evidence exists supporting this claim. Here, functional transcranial Doppler ultrasound (fTCD) was used to investigate whether these modes show hemispheric specialization. Participants viewed scenes for 20 s under instructions to search or memorize. Overall, early viewing was right lateralized, whereas later viewing was left lateralized. This right-to-left shift interacted with viewing task (more pronounced in the memory task). Importantly, changes in lateralization correlated with changes in eye movements. This is the first demonstration of right hemisphere bias for eye movements servicing spatial analysis and left hemisphere bias for eye movements servicing object analysis. (PsycINFO Database Record

Functional Transcranial Doppler Ultrasound for Measurement of Hemispheric Lateralization During Visual Memory and Visual Search Cognitive Tasks.

Functional transcranial Doppler ultrasound (fTCD) is a noninvasive sensing modality that measures cerebral blood flow velocity (CBFV) with high temporal resolution. CBFV change is correlated to changes in cerebral oxygen uptake, enabling fTCD to measure brain activity and lateralization with high accuracy. However, few studies have examined the relationship of CBFV change during visual search and visual memory tasks. Here a protocol to compare lateralization between these two similar cognitive tasks using fTCD is demonstrated. Ten healthy volunteers (age 21±2 years) were shown visual scenes on a computer and performed visual search and visual memory tasks while CBFV in the bilateral middle cerebral arteries was monitored with fTCD. Each subject completed 40 trials, consisting of baseline (25 s), calibration (variable), instruction (2.5 s), and task (20 s) epochs. Lateralization was computed for each task by calculating the bilateral CBFV envelope percent change from baseline and subtracting the right side from the left side. The results showed significant lateralization ( ) of the visual memory and visual search tasks, with memory reaching lateralization of 1.6% and search reaching lateralization of 0.5%, suggesting that search is more right lateralized (and therefore may be related to "holistic" or global perception) and memory is more left lateralized (and therefore may be related to local perception). This method could be used to compare cerebral activity for any related cognitive tasks as long as the same stimulus is used in all tasks. The protocol is straightforward and the equipment is inexpensive, introducing a low-cost high temporal resolution technique to further study lateralization of the brain.

Studies of metabolite-protein interactions: a review.

The study of metabolomics can provide valuable information about biochemical pathways and processes at the molecular level. There have been many reports that have examined the structure, identity and concentrations of metabolites in biological systems. However, the binding of metabolites with proteins is also of growing interest. This review examines past reports that have looked at the binding of various types of metabolites with proteins. An overview of the techniques that have been used to characterize and study metabolite-protein binding is first provided. This is followed by examples of studies that have investigated the binding of hormones, fatty acids, drugs or other xenobiotics, and their metabolites with transport proteins and receptors. These examples include reports that have considered the structure of the resulting solute-protein complexes, the nature of the binding sites, the strength of these interactions, the variations in these interactions with solute structure, and the kinetics of these reactions. The possible effects of metabolic diseases on these processes, including the impact of alterations in the structure and function of proteins, are also considered.