Chemical Shifts of Artificial Monomers Used to Construct Heterogeneous-Backbone Protein Mimetics in Random Coil and Folded States.

The construction of protein-sized synthetic chains that blend natural amino acids with artificial monomers to create so-called heterogeneous-backbones is a powerful approach to generate complex folds and functions from bio-inspired agents. A variety of techniques from structural biology commonly used to study natural proteins have been adapted to investigate folding in these entities. In NMR characterization of proteins, proton chemical shift is a straightforward to acquire, information-rich metric that bears directly on a variety of properties related to folding. Leveraging chemical shift to gain insight into folding requires a set of reference chemical shift values corresponding to each building block type (i.e., the 20 canonical amino acids in the case of natural proteins) in a random coil state and knowledge of systematic changes in chemical shift associated with particular folded conformations. Although well documented for natural proteins, these issues remain unexplored in the context of protein mimetics. Here, we report random coil chemical shift values for a library of artificial amino acid monomers frequently used to construct heterogeneous-backbone protein analogues as well as a spectroscopic signature associated with one monomer class, ß3-residues bearing proteinogenic side chains, adopting a helical folded conformation. Collectively, these results will facilitate the continued utilization of NMR for the study of structure and dynamics in protein-like artificial backbones.

A high-yielding solid-phase total synthesis of daptomycin using a Fmoc SPPS stable kynurenine synthon.

A high-yielding total synthesis of daptomycin, an important clinical antibiotic, is described. Key to the development of this synthesis was the elucidation of a Camps cyclization reaction that occurs in the solid-phase when conventionally used kynurenine (Kyn) synthons, such as Fmoc-l-Kyn(Boc,CHO)-OH and Fmoc-l-Kyn(CHO,CHO)-OH, are exposed to 20% 2-methylpiperidine (2MP)/DMF. During the synthesis of daptomycin, this side reaction was accompanied by intractable peptide decomposition, which resulted in a low yield of Dap and a 4-quinolone containing peptide. The Camps cyclization was found to occur in solution when Boc-l-Kyn(Boc,CHO)-Ot-Bu and Boc-l-Kyn(CHO,CHO)-OMe were exposed to 20% 2MP/DMF giving the corresponding 4-quinolone amino acid. In contrast, Boc-l-Kyn(CHO)-OMe was stable under these conditions, demonstrating that removing one of the electron withdrawing groups from the aforementioned building blocks prevents enolization in 2MP/DMF. Hence, a new synthesis of daptomycin was developed using Fmoc-l-Kyn(Boc)-OH, which is prepared in two steps from Fmoc-l-Trp(Boc)-OH, that proceeded with an unprecedented 22% overall yield. The simplicity and efficiency of this synthesis will facilitate the preparation of analogs of daptomycin. In addition, the elucidation of this side reaction will simplify preparation of other Kyn-containing natural products via Fmoc SPPS.

Thermal stability and structure of glyceraldehyde-3-phosphate dehydrogenase from the coral Acropora millepora.

Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD+. Determination of the structure of the cofactor-enzyme complex (PDB ID 6PX2) revealed variable NAD+ occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.

Revisiting Perioperative Hair Removal Practices.

The standard of practice for perioperative hair removal is largely based on research that is outdated and underpowered. Although there is evidence to support the practice of clipping instead of shaving, current recommendations are to remove hair only when absolutely necessary. Human hair is bacteria-laden and challenging to disinfect, and clipping can be a safe method of hair removal that does not damage the skin. This article considers the removal of hair at the incision site with clippers, either before the patient enters the OR or in a manner that completely contains clipped hair, for every procedure, not just when absolutely necessary. There have been only two studies to date comparing clipping with no hair removal; more research is needed on this subject.

Classics in Chemical Neuroscience: Chlorpromazine.

The discovery of chlorpromazine in the early 1950s revolutionized the clinical treatment of schizophrenia, galvanized the development of psychopharmacology, and standardized protocols used for testing the clinical efficacy of antipsychotics. Furthermore, chlorpromazine expanded our understanding of the role of chemical messaging in neurotransmission and reduced the stigma associated with mental illness, facilitating deinstitutionalization in the 1960s and 1970s. In this review, we will discuss the synthesis, manufacturing, metabolism and pharmacokinetics, pharmacology, structure-activity relationship, and adverse effects of chlorpromazine. In conclusion, we summarize the history and significant contributions of chlorpromazine that have resulted in this potent first-generation antipsychotic maintaining its clinical relevance for nearly 70 years.

Neurophysiologic responses of peripheral nerve to anoxia: effects of hypothermia and age.

OBJECTIVE: Quantify how temperature and age modulate the effects of multiple periods of anoxia on peripheral nerve. METHODS: Nerve action potentials (NAP) recorded from rat sciatic nerve in vitro. Effects of multiple cycles of anoxia on the NAP are evaluated as functions of temperature and age. In some experiments, the nerve was held at constant temperature and others the nerve was hypothermic only during anoxia and returned to 36°C with restoration of oxygen. RESULTS: Hypothermia mitigates the effect of anoxia on the NAP, an effect that increases with multiple cycles of anoxia. Preservation of the NAP waveform after 5 cycles of anoxia is best when hypothermia is delivered only during anoxia. Hypothermia reduces the rate at which the NAP disappears during anoxia but has a limited effect on the rate of recovery. With intermittent hypothermia the amplitude of the NAP is best preserved with temperatures of 15-22°C. Velocity and duration are best preserved below 25°C. The loss of the NAP during anoxia is slower in the older nerves. CONCLUSIONS: Lower temperatures improve the recovery of the NAP from anoxia and increase the time it takes for the NAP to disappear during anoxia as does increasing age. SIGNIFICANCE: Document the effects of hypothermia on the anoxia response as a step toward understanding its nerve protectant effect.

Neurophysiologic responses of peripheral nerve to repeated episodes of anoxia.

OBJECTIVE: Determine the effects of serial episodes of anoxia in an in vitro peripheral nerve preparation. METHODS: The nerve action potential (NAP) from rat sciatic nerve was recorded during 5 cycles of anoxia and reperfusion. Multiple NAP parameters were analyzed as well as stimulus response curves. RESULTS: The amplitude of the NAP declined to half baseline in 865 s on the first cycle of anoxia and recovered to half baseline during recovery in 470 s. These times increased with successive cycles of anoxia. The current required to produce a half maximal NAP showed a variable initial decrease before increasing with anoxia. The paired-pulse response showed a decline at 2-3 ms interstimulus interval during anoxia but was less dependent of interstimulus interval during recovery. NAP amplitude and velocity decrease over successive cycles of anoxia at a rate greater than in the absence of anoxia. CONCLUSIONS: The NAP declines slowly when peripheral nerve is exposed to anoxia but returns at least twice as quickly when re-exposed to oxygen. Short periods of anoxia produce long lasting changes in the nerve suggesting greater resistance to anoxia. With serial episodes of anoxia there is gradual NAP amplitude reduction and increase in duration and latency. SIGNIFICANCE: Anoxic-preconditioning appears in isolated peripheral nerve.

Acute nerve stretch and the compound motor action potential.

In this paper, the acute changes in the compound motor action potential (CMAP) during mechanical stretch were studied in hamster sciatic nerve and compared to the changes that occur during compression.In response to stretch, the nerve physically broke when a mean force of 331 gm (3.3 N) was applied while the CMAP disappeared at an average stretch force of 73 gm (0.73 N). There were 5 primary measures of the CMAP used to describe the changes during the experiment: the normalized peak to peak amplitude, the normalized area under the curve (AUC), the normalized duration, the normalized velocity and the normalized velocity corrected for the additional path length the impulses travel when the nerve is stretched. Each of these measures was shown to contain information not available in the others.During stretch, the earliest change is a reduction in conduction velocity followed at higher stretch forces by declines in the amplitude of the CMAP. This is associated with the appearance of spontaneous EMG activity. With stretch forces < 40 gm (0.40 N), there is evidence of increased excitability since the corrected velocities increase above baseline values. In addition, there is a remarkable increase in the peak to peak amplitude of the CMAP after recovery from stretch < 40 gm, often to 20% above baseline.Multiple means of predicting when a change in the CMAP suggests a significant stretch are discussed and it is clear that a multifactorial approach using both velocity and amplitude parameters is important. In the case of pure compression, it is only the amplitude of the CMAP that is critical in predicting which changes in the CMAP are associated with significant compression.