ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

Helicopter Scene Response for Stroke Patients: A 5-Year Experience.

OBJECTIVE: The purpose of this study was to examine the usefulness of an emergency medical service (EMS)-requested air medical helicopter response directly to the scene for a patient with clinical evidence of an ischemic cerebrovascular accident (CVA) and transport to a regional comprehensive CVA center. METHODS: CareFlight, an air medical critical care transportation service, is based in Dayton, OH. The 3 CareFlight helicopters are geographically located and provided transport to all CVA scene patients in this study. A retrospective chart review was completed for all CareFlight CVA scene flights for 5 years (2011-2015). A total of 136 adult patients were transported. EMS criteria included CVA symptom presence for less than 3 hours or awoke abnormal, nonhypoglycemia, and a significantly positive Cincinnati Prehospital Stroke Scale. RESULTS: The majority of patients (75%) met all 3 EMS CVA scene criteria; 27.5% of these patients received peripheral tissue plasminogen activator, and 9.8% underwent a neurointerventional procedure. CONCLUSION: Using a 3-step EMS triage for acute CVA, air medical transport from the scene to a comprehensive stroke center allowed for the timely administration of tissue plasminogen activator and/or a neurointerventional procedure in a substantive percentage of patients. Further investigation into air medical scene response for acute stroke is warranted.

Molecular Analysis of Lipid-Reactive Vδ1 γδ T Cells Identified by CD1c Tetramers.

CD1c is abundantly expressed on human dendritic cells (DC) and B cells, where it binds and displays lipid Ags to T cells. In this study, we report that CD1c tetramers carrying Mycobacterium tuberculosis phosphomycoketide bind γδ TCRs. An unbiased method of ligand-based TCR selection detects interactions only with Vδ1(+) TCRs, and mutational analyses demonstrate a role of the Vδ1 domain during recognition. These results strengthen evidence for a role of CD1c in the γδ T cell response, providing biophysical evidence for CD1c-γδ TCR interactions and a named foreign Ag. Surprisingly, TCRs also bind CD1c complexes formed with diverse lipids such as lysophosphatidylcholine, sulfatide, or mannosyl-phosophomycoketide, but not lipopeptide ligands. Dissection of TCR interactions with CD1c carrying foreign Ags, permissive ligands, and nonpermissive lipid ligands clarifies the molecular basis of the frequently observed but poorly understood phenomenon of mixed self- and foreign Ag reactivity in the CD1 system.

A 29-year-old with gunshot wound to the spine.

A mobile intensive care unit (MICU) was dispatched to transport a critically injured patient with a gunshot wound to the spine from a community hospital to a level I trauma center. The patient transported suffered from a gunshot wound to the left posterior midthoracic region. The patient experienced transient traumatic cardiac arrest before transfer. The MICU crew arrived at the emergency department and found the patient intubated and with a chest tube. Fluid resuscitation was continued, and the patient was transported. At the level I trauma center, the patient was admitted in critical condition. The patient was declared brain-dead on postinjury day 8. Spinal immobilization in penetrating trauma is a controversial topic. This patient met the historic clinical indication for spinal immobilization. The patient's injuries included multiple cervical vertebrae fractures and spinal cord disruption from the penetrating projectile, with the bullet remaining in the patient. Interfacility management by the MICU crew was focused on adequate ventilations and immobilization while continuing to address the patient's shock state. Penetrating injuries to the spinal cord can be devastating. Being aware of the pathophysiology of penetrating spinal injuries, along with current evidence-based practice, will assist providers in making sound clinical decisions for their patients.

The prevalence of positive drug and alcohol screens in elderly trauma patients.

BACKGROUND: Alcohol and drug abuse are recognized to be significantly prevalent in trauma patients, and are frequent harbingers of injury. The incidence of substance abuse in elderly trauma patients has, however, been limitedly examined. The authors sought to identify the spectrum of positive alcohol and drug toxicology screens in patients ≥65 years admitted to a Level I trauma center. METHODS: Patients ≥65 years old admitted to an American College of Surgeons (ACS) Level I trauma center over a 60--month period were identified from the trauma registry. Demographic data, blood alcohol content (BAC), and urine drug screen (UDS) results at admission were obtained and analyzed. The positive results were compared with individuals below 65 years in different substance categories using Fisher's exact test. RESULTS: In the 5-year period studied, of the 4139 patients ≥65 years, 1302 (31.5%) underwent toxicological substance screening. A positive BAC was present in 11.1% of these patients and a positive UDS in 48.3%. The mean BAC level in those tested was 163 mg/dL and 69% of patients had a level >80 mg/dL. CONCLUSIONS: These data show that alcohol and drug abuse are an issue in patients ≥65 years in our institution, though not as pervasive a problem as in younger populations. Admission toxicology screens, however, are important as an aid to identify geriatric individuals who may require intervention.

Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal.

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.

γδ T cell receptors recognize the non-classical major histocompatibility complex (MHC) molecule T22 via conserved anchor residues in a MHC peptide-like fashion.

The molecular mechanisms by which γδ T cells recognize ligand remain a mystery. The non-classical MHC molecule T22 represents the best characterized ligand for murine γδ T cells, with a motif (W … EGYEL) present in the γδ T cell receptor complementary-determining region 3δ (CDR3δ) loop mediating γδ T cell recognition of this molecule. Produced through V(D)J recombination, this loop is quite diverse, with different numbers and chemical types of amino acids between Trp and EGYEL, which have unknown functional consequences for T22 recognition. We have investigated the biophysical and structural effects of CDR3δ loop diversity, revealing a range of affinities for T22 but a common thermodynamic pattern. Mutagenesis of these CDR3δ loops defines the key anchor residues involved in T22 recognition as W … EGYEL, similar to those found for the G8 CDR3δ loop, and demonstrates that spacer residues modulate but are not required for T22 recognition. Comparison of the location of these residues in the T22 interface reveals a striking similarity to peptide anchor residues in classically presented MHC peptides, with the key Trp residue of the CDR3δ motif completing the deficient peptide-binding groove of T22. This suggests that γδ T cell recognition of T22 utilizes the conserved ligand-presenting nature of the MHC fold.

The 2.5 Å structure of CD1c in complex with a mycobacterial lipid reveals an open groove ideally suited for diverse antigen presentation.

CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-ß1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A' pocket, aided by a unique exit portal underneath the α1 helix. Most striking was an open F' pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

Chaperone-like N-methyl peptide inhibitors of polyglutamine aggregation.

Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of

Macrocyclic scaffold for the collagen triple helix.

[structure: see text] Three strands of natural collagen are linked by covalent bonds prior to their folding into a triple helix. We report on a synthetic collagen in which the strands are pendent on a rigid macrocyclic scaffold of C(3) symmetry. The scaffold confers substantial conformational stability upon the collagen triple helix and makes its folding independent of concentration, both desirable attributes for exploring and exploiting synthetic collagens.

Synthesis of a small library of 3-(carboranylalkyl)thymidines and their biological evaluation as substrates for human thymidine kinases 1 and 2.

A small library consisting of two series of thymidine derivatives containing o-carboranylalkyl groups at the N-3 position was prepared. In both series, alkyl spacers of 2-7 methylene units were placed between the o-carborane cage and the thymidine scaffold. In one series, an additional dihydroxypropyl substituent was introduced at the second carbon atom of the carborane cage. In the series of N-3-substituted carboranyl thymidines without additional dihydroxypropyl substituent, three steps were required to obtain the target compounds in overall yields as high as 75%, while in the series of N-3-substituted carboranyl thymidines with additional dihydroxypropyl substituent, 9-10 steps were necessary with significantly lower overall yield. All target compounds were good substrates of human cytosolic thymidine kinase 1 while they were, if at all, poor substrates of the mitochondrial thymidine kinase 2. There was only a minor difference in phosphorylation rates between N-3-substituted carboranyl thymidines with additional dihydroxypropyl substituents with thymidine kinase 1 (range: 13-49% relative to thymidine) and their counterparts lacking this group (range: 11-57% relative to thymidine). Tether lengths of two and five methylene groups in both series gave the highest enzyme activities in the present study. A hypothesis for this result is presented.