Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19.

We present a supercomputer-driven pipeline for in silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. Ensemble docking makes use of MD results by docking compound databases into representative protein binding-site conformations, thus taking into account the dynamic properties of the binding sites. We also describe preliminary results obtained for 24 systems involving eight proteins of the proteome of SARS-CoV-2. The MD involves temperature replica exchange enhanced sampling, making use of massively parallel supercomputing to quickly sample the configurational space of protein drug targets. Using the Summit supercomputer at the Oak Ridge National Laboratory, more than 1 ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to 10 configurations of each of the 24 SARS-CoV-2 systems using AutoDock Vina. Comparison to experiment demonstrates remarkably high hit rates for the top scoring tranches of compounds identified by our ensemble approach. We also demonstrate that, using Autodock-GPU on Summit, it is possible to perform exhaustive docking of one billion compounds in under 24 h. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and artificial intelligence (AI) methods to cluster MD trajectories and rescore docking poses.

Public engagement with information on renewable energy developments: The case of single, semi-urban wind turbines.

This paper explores perceptions of public engagement with information on renewable energy developments. It draws on a case study of proposals by a major supermarket chain to construct single wind turbines in two semi-urban locations in the UK, analysing data from interviews with key actors in the planning process and focus groups with local residents. The paper concludes that key actors often had high expectations of how local people should engage with information, and sometimes implied that members of the public who were incapable of filtering or processing information in an organised or targeted fashion had no productive role to play in the planning process. It shows how the specific nature of the proposals (single wind turbines in semi-urban locations proposed by a commercial private sector developer) shaped local residents' information needs and concerns in a way that challenged key actors' expectations of how the public should engage with information.

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19.

We present a supercomputer-driven pipeline for in-silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. We also describe preliminary results obtained for 23 systems involving eight protein targets of the proteome of SARS CoV-2. THe MD performed is temperature replica-exchange enhanced sampling, making use of the massively parallel supercomputing on the SUMMIT supercomputer at Oak Ridge National Laboratory, with which more than 1ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to ten configurations of each of the 23 SARS CoV-2 systems using AutoDock Vina. We also demonstrate that using Autodock-GPU on SUMMIT, it is possible to perform exhaustive docking of one billion compounds in under 24 hours. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and AI methods to cluster MD trajectories and rescore docking poses.

Modeling Mercury in Proteins.

Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling.

Cell volume and permeability of oxygen-and glucose-deprived retina in vitro.

Rabbit retina was deprived of O(2) and glucose in vitro for up to four hours at 37 C. Intracellular volume was measured, using inulin as an extracellular marker. After a 30-minute latency, cells swelled rapidly to more than twice normal volume while extracellular volume was unchanged. Intracellular accumulation of water was not reversed by resupply of oxygen and glucose. Permeability to small molecules was assessed with mannitol. The ratio of mannitol space to inulin space averaged 1.0 in controls. This ratio remained 1.0 up to 30 minutes of deprivation, but increased to 1.2 by 60 minutes. Permeability to large molecules was assessed from the rate of loss of isotopically labeled cell protein into the medium. There was no difference between control and deprived retinas up to three hours.

Comparison of aspirin alone versus aspirin plus ticlopidine after coronary artery stenting.

This prospective nonrandomized study was performed comparing aspirin alone (n = 46) versus aspirin and ticlopidine (p = 338) following native coronary artery stenting. There were significantly more stent thrombosis events in the aspirin-only group than in the aspirin and ticlopidine group (6.5% vs 0.9%, p = 0.02) and significantly more Q-wave myocardial infarctions and cardiac-related deaths in the aspirin-only group than in the aspirin and ticlopidine group (6.5% vs 0%, p = 0.002 and 4.4% vs 0.3% p = 0.02, respectively).

Angiographic and procedural outcome after coronary angioplasty in high-risk subsets using a decremental diameter (tapered) balloon catheter. Tapered Balloon Registry Investigators.

The angiographic and clinical outcomes of 115 patients (129 lesions) treated at 11 clinical centers using a decremental diameter (tapered) balloon catheter were evaluated. The presence of marked tapering of the reference vessel, lesion location involving a bifurcation or anastomosis of a saphenous vein graft, or total coronary occlusion where estimation of the distal vessel size was difficult were indications for this device. The tapered balloon was used as the initial dilatation device in 62 patients (73 narrowings), and as a secondary device in 53 patients (56 narrowings). Lesions selected for tapered balloon angioplasty were generally complex (95% had > or = 1 and 60% had > or = 2 adverse morphologic features). Vessel diameters were larger in the proximal reference segments (3.07 +/- 0.52 mm) than in distal ones (2.48 +/- 0.45 mm) (p<0.001). After tapered balloon angioplasty, the minimal lumen diameter increased from 0.85 +/- 0.34 mm to 2.13 +/- 0.50 mm (p<0.001), and the percent diameter stenosis decreased from 69 +/- 12% to 24 +/- 12% (p<0.001). Coronary dissections occurred in 20% of lesions; they were severe in 4% (National Heart, Lung, and Blood Institute grade C to F). Abrupt closure occurred in 4.3% of patients (2.6% immediate; 1.7% delayed). Procedural success was obtained in 110 patients (96%); major complications (in-hospital death, myocardial infarction, or emergency coronary bypass surgery) occurred in 3 patients (2.7%). Coronary angioplasty using the tapered balloon catheter appears to be a safe and effective technique for the treatment of complex lesion subsets, particularly those involving coronary arteries with marked segmental tapering.

Efficacy of coronary stenting in the management of cardiac allograft vasculopathy.

We undertook a study to determine the efficacy of stents in reducing restenosis in cardiac allograft vasculopathy. The result shows that coronary stenting significantly reduces restenosis in cardiac allograft vasculopathy compared with balloon angioplasty alone.

Comparison of balloon angioplasty versus debulking devices versus stenting in right coronary ostial lesions.

Angioplasty of aorto-ostial stenosis is associated with lower procedural success and a higher complication rate. The aim of the present study was to compare the acute and long-term results of balloon and new device angioplasty in 110 consecutive patients with right coronary ostial lesions. Patients were divided into 3 groups according to the angioplasty device used: group I (balloon only, n = 26), group II (debulking devices including excimer laser, directional and rotational atherectomy, n = 26), group III (stent, n = 58). Procedural success was highest in group III (96%) followed by group I (88%), and group II (77%). In-hospital complications were similar among the groups (p = NS). Patients in group III achieved the highest acute gain (2.61 mm) followed by groups II (1.92 mm), and I (1.39 mm, p <0.05). During follow up, target lesion revascularization and/or bypass surgery was required in 24% of patients in group III compared with 47% and 40% in groups I and II, respectively (p <0.05). Cardiac-event free survival was highest in the stent group (74%, p <0.005) and was similar between the balloon (39%) and debulking device groups (45%). Thus, among the currently available technologies, stenting of right coronary ostial lesions appears to provide excellent angiographic and long-term results.

Attenuation of interleukin-8 production by inhibiting nuclear factor-kappaB translocation using decoy oligonucleotides.

Interleukin-8 (IL-8), a monocyte-derived neutrophil chemoattractant factor, is a polymorphonuclear neutrophil chemotaxin that is involved in a number of inflammatory disorders. Transcription of the IL-8 gene is controlled by regulatory proteins, including nuclear factor-kappaB (NF-kappaB), a family of proteins that is important in the transcriptional control of a number of genes. When cells are activated, NF-kappaB translocates from the cytoplasm to the nucleus, where it activates transcription by binding to a specific sequence within the 5' untranslated region of the gene. During translocation, NF-kappaB is potentially susceptible to diversion by oligonucleotides that contain the binding sequence for this protein. In the current study, we produced phosphorothioate-modified oligonucleotides containing the specific DNA sequence that NF-kappaB binds within the IL-8 gene. We then investigated the effects of transfection of monocytes with these oligonucleotides on interleukin-1beta (IL-1beta)-stimulated IL-8 production, IL-8 mRNA expression, and NF-kappaB binding activity. We found that transfection with these oligonucleotides significantly inhibited monocyte IL-8 production. A single-stranded oligonucleotide with two copies of the NF-kappaB-binding sequence was the most potent of those tested. This single-stranded oligonucleotide also inhibited IL-1beta-induced translocation of NF-kappaB to the nucleus and reduced IL-8 mRNA expression. These studies demonstrated that monocyte production of IL-8 can be attenuated using a single-stranded oligonucleotide that binds a transcriptional activating protein before it translocates to the cell nucleus. This approach ultimately may be useful in the control of inflammation involved in a number of diseases.

Synthesis and breakdown of different sized retinal proteins in darkness and during photic stimulation.

Rabbit retinas were maintained in vitro in medium resembling cerebrospinal fluid and were exposed to (3)H-leucine or (14)C-leucine in double-labeling experiments designed to measure rates of protein turnover and to determine the effects of photic stimulation on protein synthesis and degradation. The retinas were solubilized, and the proteins separated according to size by polyacrylamide gel electrophoresis. The gels were cut into 95 slices and each slice differentially counted. Protein content of the slices was estimated from Coomassie Blue staining, and molecular weight (MW) from distribution along the gel of MW standards. The retinas appeared to function nearly normally in vitro for many hours. Protein renewal was rapid and reproducible at 0.55 +/- 0.01 (S.E.M.) %/h and remained quite constant for at least 7 h. Synthesis and degradation were approximately equal. Two retinas were maintained in vitro at 37 degrees C for 52 h, and showed good preservation of morphology, electrophysiological response to light, and protein synthesis. Total synthesis of new polypeptides was at the rate of 103 nmole per g of protein, per h; there was a sharp peak in the 33,000 to 43,000 MW range. Proteins in every size group were very heterogeneous with respect to breakdown coefficients (i.e. longevity) which were the prime determinants of the amount of each protein present. Fractional renewal showed a highly significance (p<0.0001) correlation with MW, due apparently to a reduction in maximal longevity as size increased. Neither synthesis nor degradation was significantly affected by intense continuous light or flashing light, though the latter increased uptake of 2-deoxyglucose by 38%.

Platelet activation and restenosis after coronary stenting: flow cytometric detection of wound-induced platelet activation.

BACKGROUND: Platelet activation has been implicated in restenosis after percutaneous transluminal coronary angioplasty (PTCA), but previous studies may have been confounded by factors such as elastic recoil and arterial remodelling. Restenosis after coronary stenting is unlikely to be affected by these factors. METHODS: Forty-nine patients who had stenting for acute or impending closure after PTCA were included in the study. Patients with restenosis (> or = 50% stenosis by angiography) and without restenosis were selected using a case-control design. Restenosis was determined by the caliper method. Patients were tested for platelet activation 1-4 years after their procedure while taking their usual medications (including aspirin). Reliability testing was conducted with 11 healthy subjects. Platelet activation was measured in blood leaving a bleeding-time wound (wound-induced platelet activation), using flow cytometry. Blood was collected from the wound site 1 and 2 min after the incision. Monoclonal antibodies were used to test for activation of glycoprotein (GP) IIb/IIIa (PAC-1), GPIIb/IIIa ligand binding (anti-ligand-induced binding site 1: anti-LIBS-1), and P-selectin expression (AC1.2). RESULTS: Short-term intersample reliability was very good to excellent for anti-LIBS-1 and AC1.2 (intraclass correlation coefficients 0.79-0.96), but only fair for PAC-1. Patients with restenosis (n = 25) had greater activation in all measures than patients without restenosis (n = 24); the difference was significant for GPIIb/IIIa ligand binding at 1 min (P = 0.03). The correlation between GPIIb/IIIa ligand binding at 1 min and percent stenosis at follow-up was also significant (P = 0.03). Patients taking nitrates had lower activation; after eliminating these patients, GPIIb/IIIa ligand binding was greater among patients with restenosis at both 1 and 2 min (P = 0.04 for both). CONCLUSIONS: The results suggest that increased GPIIb/IIIa ligand binding may be associated with restenosis after coronary stenting. The results also suggest that the wound-induced platelet activation method is a reliable and valid measure of platelet activity.

Chemical release from topical formulations across synthetic membranes: infinite dose.

Drug release rates from topical preparations are sometimes measured by monitoring the cumulative mass of drug appearing in a receptor solution (MR). If the topical formulation and receptor solution are in direct contact, then MR increases linearly with square root of t. When a synthetic membrane is placed between the topical formulation and receptor solution, drug appearance in the receptor solution is delayed and MR is not immediately linear in square root of t. As a result, linear regressions of MR with square root of t produce positive values for the square root of t-intercept. Here, we mathematically model chemical release from an infinite-dose, topical formulation across synthetic membrane to quanitiatively determine the physical meaning of the square root of t-intercept. To correctly determine drug diffusivity in the topical formulation, the experiment must be conducted long enough that MR is linear in square root of t. Theoretically based procedures are presented for testing which data should not be used in linear regression of MR with square root of t. Theoretical predictions are compared with previously published experimental results for ethyl salicylate across a poly(dimethylsiloxane) (Silastic) membrane and for hydrocortisone across several different synthetic membranes.

Cocaine-associated rhabdomyolysis.

Three male patients developed a total of four episodes of acute rhabdomyolysis associated with documented cocaine intoxication (two caused "crack" and two caused by intravenous cocaine). Included is one patient who developed rhabdomyolysis after injecting cocaine and then redeveloped it 6 months later on "rechallenge." One of the four cases resulted in death related to severe hyperkalemia and ischemic bowel. The remaining three episodes followed a course of nonoliguric renal failure.

Functional homogeneity of leucine pool in retina cells.

Data on leucine metabolism in isolated rabbit retina are examined for evidence, for or against, a common intracellular pool of free leucine. Data include values for: concentrations, transport rates, degradative metabolism and protein incorporation of labelled leucine measured over a wide range of concentrations; protein incorporation of labelled threonine, measured simultaneously; and an indirect measurement of protein breakdown. The fall in labelled leucine incorporation into protein, when medium leucine was reduced below 100 microM, corresponded closely with the fall in intracellular specific activity predicted from rate of influx of labelled leucine from medium and rate of release of unlabelled leucine from protein breakdown. Protein incorporation of labelled leucine competed with decarboxylation and outward transport and reduced the free intracellular leucine in about the amounts predicted for a common pool. Implications for measurements using labelled amino acid are discussed.

Mycobacterium kansasii infections in patients positive for human immunodeficiency virus.

The experience with Mycobacterium kansasii infections in patients who are infected with human immunodeficiency virus (HIV) at Parkland Memorial Hospital in Dallas is presented, and the literature on such infections is reviewed. The absolute and relative paucity of reports of M. kansasii infections in HIV-positive patients is emphasized. M. kansasii infections in HIV-positive patients are classified as either pulmonary or disseminated. Evidence of the lack of therapeutic response in patients with disseminated infections and of the potential for therapeutic response in patients with infections limited to the lung is reviewed and documented. Other unresolved diagnostic and therapeutic issues concerning M. kansasii infections in HIV-positive patients are reviewed.

Evidence from phospholipid metabolism changes for muscarinic cholinergic receptors on rat anterior pituitary cells.

In incubations of dissociated adult rat anterior pituitary cells with [32P]orthophosphate, carbamylcholine causes an increase in phosphatidic acid labeling accompanied by a small reduction in phosphatidylinositol labeling. Carbamylcholine and oxotremorine produce about the same maximum change while that caused by pilocarpine is smaller. Low concentrations of atropine, quinuclidinyl benzilate, and scopolamine completely inhibit the effect caused by carbamylcholine, whereas d-tubocurarine does not decrease the stimulation, even at higher concentrations. The muscarinic antagonists alone produce a rise in phosphatidylinositol labeling and a drop in phosphatidic acid labeling, an effect opposite from that produced by the agonists, but d-tubocurarine alone has no effect. Thus changes in phospholipid metabolism are mediated through muscarinic cholinergic receptors in dissociated rat anterior pituitary cells, confirming the presence of functional binding sites. The present studies also demonstrate the utility of experiments on precursor incorporation into phospholipids in identifying the existence on cells or tissues of certain receptor classes.

Protein synthesis in central nervous tissue: studies on retina in vitro.

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.