Using Fluorescence Anisotropy for Ligand Binding Kinetics of Membrane Proteins.

Determining ligand binding kinetics provides an indirect route to probe the functional capabilities of the binding pocket of a membrane protein receptor. Presented in this unit are four ligand-binding protocols that provide data useful for characterizing membrane proteins, including equilibrium binding, thermostability, competitive ligand binding, and kinetic ligand binding. These techniques use fluorescence anisotropy, which is safer, less costly, and simpler to execute than radioactive ligand binding. Each protocol may be used on its own or in combination with others to quantify a number of ligand binding constants. © 2018 by John Wiley & Sons, Inc.

Cutaneous microvascular perfusion responses to insulin iontophoresis are differentially affected by insulin resistance after spinal cord injury.

NEW FINDINGS: What is the central question of this study? What impact does insulin resistance have on cutaneous perfusion responses to insulin iontophoresis in vascular beds with markedly reduced or functionally ablated sympathetic nervous system vasomotor function resulting from spinal cord injury? What is the main finding and its importance? Persons with spinal cord injury have sublesional microvascular endothelial dysfunction, as indicated by a blunted cutaneous perfusion response to acetylcholine iontophoresis, and the presence of insulin resistance has a further confounding effect on endothelium-mediated changes to cutaneous perfusion in the lower extremities. Endothelium-mediated mechanisms that regulate skin blood flow might play an integral role in optimizing skin perfusion in vascular beds with sympathetic nervous system vasomotor impairment, such as in spinal cord injury (SCI). Insulin is a vasoactive hormone and second messenger of nitric oxide that facilitates endothelium-mediated dilatation. The effects of insulin resistance (IR) on sublesional cutaneous perfusion responses to insulin provocation have yet to be described in persons with SCI. Persons with SCI and an able-bodied (AB) cohort were divided into subgroups based upon fasting plasma insulin concentration cut-offs for IR (≥13.13 mIU ml-1 ) or insulin sensitivity (IS; <13.13 mIU ml-1 ), as follows: AB, IS (ABIS, n = 21); SCI, IS (SCIS, n = 21); AB, IR (ABIR, n = 9); and SCI, IR (SCIR, n = 11). Laser Doppler flowmetry characterized peak blood perfusion unit (BPU) responses (percentage change from baseline) to insulin, acetylcholine or placebo iontophoresis in the lower extremities; BPU responses were log10 transformed to facilitate comparisons, and the net insulin response (NetIns) BPU response was calculated (insulin minus placebo BPU response). The NetIns was significantly greater in both IS groups compared with their corresponding IR group. The acetylcholine-mediated BPU responses in the SCI subgroups were significantly lower than those in the ABIS group. The proportional BPU responses of NetIns to acetylcholine in the IS cohorts (i.e. ABIS and SCIS) were significantly greater (P < 0.05) than that of each IR subgroup. The presence of IR has a confounding effect on sublesional microvascular endothelium-mediated cutaneous perfusion responses to provocation. Preservation of endothelial sensitivity to its agonists appears to be an important modifiable risk factor to optimize cutaneous perfusion in the lower extremities of persons with SCI.

Self-reported effects of cold temperature exposure in persons with tetraplegia.

OBJECTIVE: Cervical spinal cord injury (tetraplegia) is known to interrupt sympathetic vasculature control, thereby preventing shunting of blood from the periphery to central organs when exposed to cold temperatures. As a result, persons with tetraplegia are at risk to develop hypothermia. However, information regarding the discomfort experienced during the cooler months (late fall, winter, early spring) is overwhelmingly anecdotal. It is not known, with any certainty, how those with tetraplegia perceive cold and if discomfort in colder environments restricts them from performing activities that they routinely would perform. DESIGN: Prospective, two-group, self-report surveys. SETTING: VA Medical Center and Kessler Institute for Rehabilitation. PARTICIPANTS: Forty-four subjects with tetraplegia; 41 matched non-SCI controls. OUTCOME MEASURES: Tetraplegic and control groups responded "yes" or "no" when asked whether cold seasonal temperatures allowed comfort or negatively affected participation in routine activities. RESULTS: Percentage of responses of tetraplegia compared to controls was different as to whether they felt cold when others in the same room were comfortable (82 vs. 24%; χ2 = 28.2, P < 0.0001), felt comfortable outdoors (17 vs. 43%; χ2 = 6.8, P = 0.009), or whether cold negatively affected bathing routines (55 vs. 15%; χ2 = 14.8, P = 0.0001), keeping physician appointments (46 vs. 12%; χ2 = 11.3, P = 0.0008), thinking clearly (41 vs. 7%; χ2 = 12.9, P = 0.0003), and completing usual work duties (46 vs. 10%; χ2 = 13.3, P = 0.0003). CONCLUSION: Cold seasonal temperatures have a reported greater negative impact on personal comfort and ability to perform vital activities in persons with tetraplegia than that of non-SCI controls. These findings highlight the need to address thermoregulatory impairment in persons with tetraplegia.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 µg mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ∼1300 µg mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

Effect of Mild Cold Exposure on Cognition in Persons with Tetraplegia.

UNLABELLED: Persons with a cervical spinal cord injury (SCI) have impaired thermoregulatory mechanisms secondary to interrupted of motor, sensory, and sympathetic pathways. In this study, our primary aim was to determine the effect of cool temperature exposure on core body temperature (Tcore) and cognitive performance in persons with tetraplegia. Seven men with chronic tetraplegia (C3-C7, American Spinal Injury Association Impairment Scale [AIS] A-C) and seven able-bodied controls were exposed to 27°C temperature at baseline (BL) before being exposed to 18°C for ≤120 min (Cool Challenge). Rectal temperature (Tcore), distal skin temperatures (Tskavg), microvascular skin perfusion (LDFavg), and systolic blood pressure (SBP) were measured. Cognitive performance was assessed using Delayed Recall, Stroop Interference tests at the end of BL and Cool Challenge. After Cool Challenge, Tcore decreased -1.2±0.12°C (p<0.0001) in tetraplegics after an average of 109±15.9 min with no change in controls after 120 min. Tskavg declined in both groups, but decline was less in tetraplegics than in controls (-8.6±5.8% vs. -31.6±7.9%, respectively; p<0.0001). LDFavg declined only in controls (-72±17.9%; p<0.001). Plasma norepinephrine levels differed after Cool Challenge (tetraplegics vs. CONTROLS: 86±62 pg/mL vs. 832±431 pg/mL, respectively; p<0.01). SBP increased from BL to Cool Challenge only in controls (123±16 mm Hg to 149±17 mm Hg, respectively; p<0.01). Delayed Recall and Stroop Interference scores both declined in tetraplegics (-55±47.4%; p<0.05 and -3.9±3.8%; p<0.05, respectively), but not in controls. We conclude that persons with tetraplegia lack adequate thermoregulatory mechanisms to prevent downward drift in Tcore on exposure to cool temperatures. This decline in Tcore was associated with deterioration of working memory and executive function.