COVID-19 Cliff Notes: A COVID-19 Multidisciplinary Care Compendium.

As we pass the nearly 9 month mark of the coronavirus virus disease 2019 (COVID-19) pandemic in the United States, we sought to compile a brief multi-disciplinary compendium of COVID-19 information learned to date. COVID-19 is an active viral pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that confers high morbidity and mortality. COVID-19 has been associated with: pulmonary compromise and acute respiratory distress syndrome, thrombotic events, inflammation and cytokine, and post-infectious syndromes. Mitigation of these complications and expeditious therapy are a global urgency; this is brief summary of current data and management approaches synthesized from publications, experience, cross-disciplinary expertise (Figure 1).

Frequency-domain interferometric second-harmonic spectroscopy.

We report a new spectroscopic technique to measure simultaneously the intensity and the phase of second-harmonic (SH) radiation over a broad spectral range without laser tuning. Temporally separated SH pulses from two sources, excited by the same broad-bandwidth 15-fs Ti:sapphire fundamental pulse, interfere in a spectrometer to yield frequency-domain interference fringes. We demonstrate the technique by measuring the strongly bias-dependent phase of SH radiation from a Si/SiO(2)/Cr metal-oxide-semiconductor capacitor in the spectral range of the SiE(1) critical point.

Receptor-mediated activation of Gsalpha: evidence for intramolecular signal transduction.

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of alphas (residues 236-394) to identify residues that play a role in receptor-mediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc- S49 lymphoma cells, which lack endogenous alphas. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an alpha helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding, are positioned to relay the receptor-initiated signal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of alphas with that of alphai2 in an alphas/alphai2/alphas chimera corrects the defect in receptor-mediated activation caused by alphai2 substitutions on the GTPase side of the interface. Thus, complementary interactions between residues across the domain interface seem to play a role in receptor-catalyzed activation.

Plasma membrane localization of G alpha z requires two signals.

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the alpha subunits are myristoylated and/or palmitoylated, whereas the gamma chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled alpha subunit, alpha z. Wild-type alpha z (alpha z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does alpha z-WT. Removal of the myristoylation site produced a mutant alpha z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of beta gamma with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on alpha z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on alpha z-WT. Together these results suggest that myristate and beta gamma promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on alpha z specific localization at the plasma membrane.

Femtosecond carrier-induced screening of dc electric-field-induced second-harmonic generation at the Si(001) SiO(2) interface.

Carrier-induced screening of the dc electric field at the Si(001)-SiO(2) interface is observed by intensity-dependent and femtosecond-time-resolved second-harmonic spectroscopy. The screening occurs on a time scale of ~?(p)(-1) , the reciprocal plasma frequency of the generated carriers.

Fatty acylation of alpha z. Effects of palmitoylation and myristoylation on alpha z signaling.

As the first step in an investigation of roles played by fatty acylation of G protein alpha chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three alpha subunits. At site I, only HA-tagged alpha q and alpha z functioned normally. alpha s, alpha q, and alpha z subunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged alpha z, we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly2 (G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys3 (C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Gly2 and Cys3 (G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of alpha z with the particulate fraction. Gz-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by alpha z was unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated alpha z mutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated alpha z. From these findings we conclude: (a) fatty acylations of alpha z increase its association with membranes; (b) myristoylation is not required for palmitoylation of alpha z or for its productive interactions with adenylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by alpha z.

Lipid modifications of trimeric G proteins.

G protein alpha subunits and beta gamma dimers are covalently modified by lipids. The emerging picture is one in which attached lipids provide more than just a nonspecific "glue" for sticking G proteins to membranes. We are only beginning to understand how different lipid modifications of different G protein subunits affect specific protein-protein interactions and localization to specific cellular sites. In addition, regulation of these modifications, particularly palmitoylation, can provide new ways to regulate signals transmitted by G proteins.

Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.

We have identified the palmitoylated cysteine residues of alpha q and alpha s, alpha subunits of two heterotrimeric G proteins. Mutational substitutions of serines for cysteines 9 and 10 in alpha q and cysteine 3 in alpha s profoundly alter behavior of the subunits expressed in HEK293 cells. Neither mutant alpha subunit incorporates palmitate; both mutant proteins are found in the soluble rather than the particulate fraction; mutant alpha q or alpha s cannot couple a co-expressed receptor to stimulation of phospholipase C or adenylylcyclase, respectively; cysteine substitution prevents a mutationally activated alpha q (R183C) from stimulating phospholipase C directly, and reduces but does not abolish the ability of a similarly activated alpha s (R201C) to stimulate cAMP synthesis. Substitution of a myristoylation sequence for the palmitoylation sites leads to labeling of alpha q and alpha s by myristate, rather than by palmitate. Myristoylation restores the abilities of both nonpalmitoylated alpha q and alpha s to attach to membranes and, in the case of alpha q, restores its ability to stimulate phospholipase C, whether triggered by the R183C mutation or by receptor activation. These findings identify palmitoylation as a critical determinant of membrane attachment for alpha q and alpha s and show that this modification is required for normal signaling by these proteins.

An audit of extradural infusion analgesia in children using bupivacaine and diamorphine.

One hundred and fifty extradural infusions of diamorphine and bupivacaine after major surgery in children were audited over a 15 month period. The majority of the children (69%) were less than 5 years of age. Analgesia was assessed or self-rated as 'very good' in over 75% of patients. Urinary retention was seen in 11% of patients and pruritus in 10%. Respiratory depression requiring intervention was only seen in one patient--a premature infant of 39 weeks post-conceptual age. Technical complications resulted in the early loss of 16.7% of the infusions. Although analgesia was good the complexity of the extradural infusion technique demanded significant medical and nursing time especially to overcome technical problems.

Monoclonal antibodies directed against a synthetic peptide corresponding to the alpha-bungarotoxin binding region of the acetylcholine receptor.

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.

Distribution of alpha-bungarotoxin binding sites over residues 173-204 of the alpha subunit of the acetylcholine receptor.

The binding of alpha-bungarotoxin to several synthetic peptides comprising different segments of the region 173-204 of the alpha subunit of the Torpedo acetylcholine receptor was investigated to further localize the neurotoxin-binding site on the primary sequence. When tested in a solid phase microwell assay system, a 32-amino acid peptide corresponding to residues 173-204 (32-mer) bound 125I-alpha-bungarotoxin with the same affinity (4.2 x 10(-8) M as determined from IC50 values) as the isolated alpha subunit (4.6 x 10(-8) M). The relative affinities of other antagonists (alpha-cobratoxin, d-tubocurarine) maintained the same rank order in this assay system as has been demonstrated with the intact receptor. Agonists competed with binding of toxin at millimolar concentrations but lost all rank order of potency. These findings demonstrate that peptide 173-204 contains many of the antagonist-binding determinants present on denatured alpha subunit but has lost specificity of agonist binding. To further localize the toxin-binding site, alpha-bungarotoxin binding to seven shorter peptides corresponding to portions of the 32-mer was investigated. 125I-alpha-Bungarotoxin bound to alpha subunit peptides 179-192, 181-198, 185-196, 186-196, and 193-204, but not to alpha subunit peptides 173-180 and 194-204. In a second assay, all of the peptides competed with binding of 125I-acetylcholine receptor to immobilized alpha-bungarotoxin. The apparent affinity was highest for the 173-204 32-mer (1.4 x 10(-7) M) and lowest for peptides 173-180 and 194-204 (greater than 10(-4) M). The affinity of the other peptides was intermediate (approximately 10(-5) M) and about 100-fold less than that of the 32-mer. The affinity of alpha-bungarotoxin was 3.5 x 10(-10) M, of isolated, native acetylcholine receptor, 3.2 x 10(-9) M, and of isolated denatured subunit, 1.2 x 10(-8) M, with this assay. The retention of some toxin-binding capacity by the shorter peptides indicates toxin-binding determinants are distributed over the entire length of the 32-mer. The determinants with higher affinity are located in the central region of the 32-mer between residues 179 and 196.

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate.

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic peptides in the study of the interaction of rabies virus and the acetylcholine receptor.

The neurotropism of some viruses may be explained in part by the attachment of these viruses to host cell receptors that are present on or even largely restricted to neurons. Rabies virus is an RNA virus that, after a period of replication in muscle, gains access to the central nervous system, where it selectively infects certain neuronal populations. The nicotinic acetylcholine receptor occurs in high density at the neuromuscular junction and is present in the central nervous system. Although several different cell surface constituents may act as attachment determinants for rabies, direct binding of radioactively labeled virus to affinity-purified acetylcholine receptor has been demonstrated. Binding of virus to the receptor was saturable and inhibited by up to 50% by alpha-bungarotoxin, a snake venom neurotoxin that binds at or near the acetylcholine binding site on the receptor. The molecular basis for the virus-receptor interaction may lie in an amino acid sequence similarity between the snake venom neurotoxins and a segment of the rabies virus glycoprotein. Two peptides (10 and 13 residues) of the rabies virus glycoprotein and homologous bungarotoxin peptides were synthesized and tested for ability to compete with labeled alpha-bungarotoxin for binding to the acetylcholine receptor. The peptides were found to compete with toxin binding with affinities comparable to those of the cholinergic ligands d-tubocurarine and nicotine. These findings indicate that a segment of the rabies virus glycoprotein interacts with the acetylcholine receptor at or near the acetylcholine binding site of the receptor. The similarity between the virus glycoprotein and the neurotoxin was further evidenced by the cross reaction of antibody raised against the virus 10-mer with the bungarotoxin 10-mer. Binding of rabies virus to the acetylcholine receptor or to other neuronal bungarotoxin-binding proteins may be related to the neurotropism of this virus. In addition, knowledge of both the region of the virus involved in binding and the binding domain on the receptor may be helpful in developing new strategies for treatment, especially for viruses that infect the central nervous system or evade the immune response through genetic drift. These strategies include development of antiviral agents that cross the blood-brain barrier and inhibit viral binding and the utilization as immunogens the regions of viruses, such as their binding domains, that are highly conserved among different strains.

Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor.

Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.

Binding of rabies virus to purified Torpedo acetylcholine receptor.

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Alpha-bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots.

The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.

Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.

A region of the alpha subunit of the nicotinic acetylcholine receptor containing the alpha-bungarotoxin-binding domain was mapped on the primary amino acid sequence in relation to asparagine-141, the presumed site of N-linked glycosylation. Proteolytic fragments of the alpha subunit, immobilized onto positively charged membrane filters, that bind 125I-labeled bungarotoxin were further analyzed on the basis of the size of the fragments and the presence of asparagine-141 as determined by susceptibility to digestion with endoglycosidase H. The bungarotoxin-binding site was found not to reside between amino acid residues 1 and 140 since bungarotoxin-binding fragments that are considerably larger than 140 amino acids and lack N-linked oligosaccharide chains were detected. The size of the smallest bungarotoxin-binding fragment containing asparagine-141 and the size of fragments produced by digestion with V8 protease further indicated that the bungarotoxin-binding site is contained within amino acid residues 153-241. A 32-amino acid synthetic peptide comprising a portion of this region (residues 173-204) was tested for its ability to bind 125I-labeled bungarotoxin. 125I-labeled bungarotoxin bound to the peptide and was competed by unlabeled bungarotoxin and d-tubocurarine with IC50 values of 0.5 microM and 2 mM, respectively. We conclude that a major determinant of the bungarotoxin-binding site on the alpha subunit resides between residues 173 and 204.