A pre-admission triaging tool to predict severe COVID-19 cases: ABCD score.

INTRODUCTION: Isolation of SARS-CoV-2-infected individuals is an important COVID-19 pandemic control measure. While most cases have uncomplicated infection, a small proportion of them has developed life-threatening disease. We set up a retrospective study to determine preadmission triaging tool to predict the development of severe COVID-19. MATERIALS AND METHODS: A retrospective study was conducted from 1 October 2020 to 31 January 2021 with enrolment of all SARS-CoV-2 PCR-confirmed persons aged ≥13 years. The disease severity was assessed on admission and daily throughout the hospitalisation. Test-positive individuals were considered as having "severe COVID-19" if they had ≥1 of the following: room air oxygen saturation 30 breaths/minute, signs of severe respiratory distress, or received mechanical ventilation and/or vasopressor therapy. Uni- and multi-variate analyses using SPSS Statistics Ver. 26 were performed. RESULTS: We showed that age ≥ 60 years, BMI ≥ 30.0, presentation on days 7-12 of illness, and ≥1 comorbidity were associated with development of severe COVID-19. A scoring system based on the four variables is a useful COVID-19 risk assessment tool. A total score ≥2 had a sensitivity of 60.9%, specificity of 88.2%, positive predictive value of 37.8% and negative predictive value of 95.0%. CONCLUSION: Development of preadmission triaging tool can help health care providers (HCPs) decide on the placement of test-positive individuals to appropriate isolation facilities according to the risk of developing severe COVID-19.

CLOCK is a substrate of SUMO and sumoylation of CLOCK upregulates the transcriptional activity of estrogen receptor-α.

Disruption of the circadian rhythm is now believed to associate with a number of hormone-related cancers, such as breast cancer, in which aberrant estrogen receptor-α (ERα) signaling is a major contributor. However, the molecular mechanisms underlying the function of core clock proteins in cancer are still largely undefined. In this study, we showed that circadian locomotor output cycles kaput (CLOCK), a key circadian protein, can interact with ERα. Furthermore, this interaction was enhanced by estrogen. We also showed that CLOCK can be sumoylated and sumoylation of CLOCK, which is also stimulated by estrogen, had two consequences: (1) it increased the transcriptional activity of CLOCK; and (2) it increased the CLOCK-modulated transcriptional activity of ERα, as shown by increased transcription of cyclin D1. Sumoylation of CLOCK occurred at two lysine residues, K67 and K851. The enhancement of ERα transcriptional activity exerted by wild-type but not mutant (2K/2R) CLOCK in response to estrogen indicated that sumoylation of CLOCK may have an important role in estrogen-dependent signaling. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay conducted with breast cancer cell lines (MCF-7 and T47D) demonstrated that sumoylation of CLOCK stimulated cell growth and increased the proportion of S phase cells in the cell cycle. The results of this study uncovered new insight into the connection between a major circadian protein and a major estrogen-dependent transcription factor, providing the basis for further research into the involvement of circadian proteins in breast cancer.

Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.

Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.

Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity.

The three-dimensional structure of Zymomonas mobilis pyruvate decarboxylase shows that the carboxyl-terminal region of the protein occludes the active site. This observation is consistent with earlier suggestions that the active site is inaccessible to solvent during catalysis. However, the carboxyl-terminal region must move aside to allow entry of the substrate, and again to permit the products to leave. Here we have examined the role of the carboxyl terminus by making 15 variants of the enzyme with serial deletions. The activity is largely unaffected by removal of up to seven residues but deletion of the next two, R561 and S560, results in a drastic loss of activity. Five of these deletion mutants were purified and fully characterized and showed progressive decreases in activity, in the ability to discriminate between pyruvate and larger substrates, and in cofactor affinity. Several substitution mutants at residues R561 and S560 were prepared, purified, and fully characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-terminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrimination between substrates and for decarboxylation to occur.

Effect of mutagenesis at serine 653 of Arabidopsis thaliana acetohydroxyacid synthase on the sensitivity to imidazolinone and sulfonylurea herbicides.

Resistance to sulfonylurea and imidazolinone herbicides can occur by mutations in acetohydroxyacid synthase (EC 4.1.3.18). Changing serine 653 to asparagine is known to cause insensitivity to imidazolinones but not to sulfonylureas. Here, S-653 of the Arabidopsis thaliana enzyme was mutated to alanine, threonine and phenylalanine. The purified mutated enzymes resemble wild-type in their enzymatic properties. The threonine and phenylalanine mutants are imidazolinone-resistant and the latter is also slightly sulfonylurea-resistant. The alanine mutant remains sensitive to both herbicides. The results suggest that the beta-hydroxyl group is not required for imidazolinone binding and that the size of the side-chain determines resistance.

Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase.

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.

Implant reconstruction following removal of tumors of the head and neck.

Functional maxillofacial rehabilitation following tumor ablation depends on many variables. These include the magnitude of hard or soft tissue defects, the use of adjunctive chemotherapy or radiation therapy, and the presence of underlying systemic disease. The physiologic effects of tumor ablation and radiation therapy on local tissues and grafted bone are discussed in this article in relation to the ultimate use of implant supported prostheses. Cases are presented to illustrate a variety of clinical situations.

Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.

Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold.

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase.

Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene from Arabidopsis thaliana with part of the chloroplast transit sequence removed was cloned into the bacterial expression vector pT7-7 and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by an extensive procedure involving (NH4)2SO4 fractionation followed by hydrophobic and anion-exchange chromatography. The purified enzyme appears as a single band on SDS/PAGE with a molecular mass of about 61 kDa. On gel filtration the enzyme is a dimer, migrating as a single peak with molecular masses of 109 and 113 kDa in the absence and presence of FAD respectively. Ion spray MS analysis yielded a mass of 63864 Da. The enzyme has optimum activity in the pH range 6.5-8.5 and exhibits absolute dependence on the three cofactors FAD, Mg2+ and thiamine diphosphate for activity. It displays negatively co-operative kinetics with respect to pyruvate concentration. A model was derived to explain the non-hyperbolic substrate-saturation curve, involving interaction between the active sites of the dimer. The Km for the first active site was found to be 8.01 +/- 0.66 mM; the Km for the second active site could not be accurately determined but was estimated to be approx. 100 mM. The enzyme is insensitive to valine, leucine and isoleucine but is strongly inhibited by the sulphonylurea herbicide, chlorsulphuron, and the imidazolinone herbicide, imazapyr. Inhibition by both herbicides exhibits slow-binding kinetics, whereas chlorsulphuron also shows tight-binding inhibition.

MAST 96.

The Military Anti-Shock Trouser, or MAST suit, is a controversial device that has been used to support blood pressure in hypotensive trauma patients. Most studies on humans have shown that the device has limited clinical utility. In this study, a telephone survey of all 50 State Emergency Medical Services was conducted to determine the nature and extent of MAST suit usage in the United States. The trend in MAST suit usage in San Diego County over the last 7 years was also analyzed. Thirty (60%) states still require MAST suits to be carried on ambulances. In San Diego County, MAST suit inflations for adult, hypotensive (systolic blood pressure < 90 mmHg,) blunt trauma patients has declined from 37% in 1987, to 2% in 1993. Despite a lack of data supporting efficacy in areas of severe hypotensive shock, blunt trauma, long transport times, and pelvic fractures, states continue to expend resources on the MAST suit. It is for this reason that we believe that the clinical use of the MAST suit should be based upon medical control philosophy rather than legislation.

Low plasma cholesterol predicts an increased risk of lung cancer in elderly women.

BACKGROUND: There have been significant efforts in the United States to lower high cholesterol levels. Studies of men, however, have found a higher total cancer mortality rate at lower levels of plasma cholesterol. Many of these studies have found that lung cancer is more closely associated than other cancers with low cholesterol. Of the studies that include women, none has demonstrated a statistically significant inverse association between low cholesterol and lung cancer. METHODS: We examined the relation between very low plasma cholesterol levels ( < 160 mg/dl) and lung cancer death in an 18-year prospective study of 2,011 men and 2,327 women. RESULTS: After adjusting for age, body mass index, smoking, and education, the relative hazard of lung cancer mortality for those with low cholesterol ( < 160 mg/dl) compared with all other cholesterol levels ( > or = 160 mg/dl) was 1.75 among men (P = 0.28) and 3.29 among women (P = 0.02). Excluding those who died within 5 years of baseline did not change the results. CONCLUSIONS: Both men and women with baseline plasma cholesterol levels < 160 mg/dl were more likely to die of lung cancer. This difference was statistically significant in women. The association could not be explained by occult malignancy, smoking, or socioeconomic status.

Functional assay of plasma antithrombin using polyethylene glycol (PEG) defibrinated plasma.

Polyethylene glycol(PEG) was used to precipitate fibrinogen to prepare defibrinated plasma in the two stage clotting assay of antithrombin activity. Five percent PEG-8000 precipitated fibrinogen from plasma without loss of antithrombin activity in the defibrinated plasma. Fibrin degradation products(FDP) as high as 640 ug/ml did not interfere the two stage clotting assay using PEG defibrinated plasma possibly because part of FDP was precipitated by PEG in the process of plasma defibrination. The two stage clotting assay was very sensitive to the changes of antithrombin activity in the range of 60%-100% of normal level. The assay was reproducible and correlated with chromogenic assay. The decrease of plasma antithrombin activity in a baboon septic shock model was demonstrated with this assay.

Retrospective study comparing the pathophysiology of antibiotic-treated and untreated Escherichia coli- and Staphylococcus aureus-infused baboons.

Escherichia coli and Staphylococcus aureus are the most common pathogens encountered in septic shock. This is a descriptive study in which the pathophysiologic response to infusions of LD100 concentrations of E. coli and S. aureus are staged and compared. Equivalent concentrations of both organisms were infused over a 2 hr period into antibiotic-treated and untreated animals with the following results: 1) The apparent clearance of E. coli was less than that of S. aureus over the 2-hr infusion period, but far greater during the next 8 hr in both antibiotic-treated and untreated animals. Thus the clearance of E. coli fits a one-compartment (intravascular), and that of S. aureus fits a two-compartment (intra- and extravascular) model. 2) The intensity of the cardiovascular, temperature, and metabolic response to E. coli was greater, whereas that of the disseminated intravascular coagulant (DIC) response to S. aureus was greater. We conclude, therefore, that the response to E. coli consists of four stages with no invasion and colonization of tissues, whereas the response to S. aureus consists of two stages with invasion and colonization of tissues.

Free radicals and septic shock in primates: the role of tumor necrosis factor.

The role of free radicals in septic-shock-associated tissue injury and the mechanisms underlying the generation of free radicals in sepsis was investigated in a primate model using electron spin resonance (ESR) spectroscopy and spin-trapping techniques paired with physiological measurements. Baboons were administered the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) during infusions of live Escherichia coli (E. coli) with or without challenge with tumor necrosis factor (TNF). ESR spectra suggesting the trapping of carbon-centered and oxygen-centered radicals were detected in liver lipid extracts of E. coli infused animals which exhibited pathophysiological changes indicative of sepsis. In animals demonstrating a toxic response to E. coli. TNF challenge appeared to intensify the ESR signal observed. These data provide evidence of free radical production during sepsis and suggest a role for TNF in the production of these radicals.

Antithrombin-III prevents the lethal effects of Escherichia coli infusion in baboons.

Infusion of Escherichia coli (LD100) was followed by coagulopathic and cell injury responses, cardiovascular collapse, and death in 18 to 32 hr in four out of four baboons. Infusion of AT-III in sufficient amounts to achieve AT-III levels of more than 4 units/ml of plasma before and during the infusion of E. coli reduced the intensity of the coagulopathic and cell injury response and prevented vascular collapse and death in four out of four baboons. Failure to achieve AT-III levels of more than six units/ml at T +60 min during the infusion of E. coli resulted in failure to prevent its lethal effects in three out of three baboons even though levels as high as 10 units/ml were achieved later in the course of the experiment. These studies suggest that thrombin and/or its products can contribute to the inflammatory response to E. coli and that AT-III is of potential value as a prophylactic but not as a therapeutic agent in the treatment of patients at high risk of developing gram negative sepsis.