Extremely Severe Case of COVID-19 Pneumonia Recovered Despite Bad Prognostic Indicators: a Didactic Report.

COVID-19 is a highly infectious respiratory disease caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Starting from Wuhan (China) where it was firstly reported, it rapidly spread to the rest of the world, causing a pandemic with more than 300,000 deaths to date. We report an extremely severe case of coronavirus pneumonia in an over 80-year-old patient with hypertension, coronary heart disease, chronic heart failure, and chronic obstructive pulmonary disease. Despite a clearly poor anamnestic and clinical prognostic forecast, she was successfully discharged thanks to a careful evaluation of the case and of the complications that have arisen. Although a higher vulnerability of geriatric patients has been observed, the literature on elderly COVID-19 patients has remained very scarce, especially in those over 80. The article aims to explore factors that may allow the successful outcome and provides important elements to better understand this disease.

Correction to: A PRISMA-compliant systematic review of the endpoints employed to evaluate symptomatic treatments for primary headaches.

Following publication of the original article [1], we have been notified that the name of author five was spelled incorrectly as M. Ferrili, when the correct spelling is MAN Ferilli.

A PRISMA-compliant systematic review of the endpoints employed to evaluate symptomatic treatments for primary headaches.

BACKGROUND: Primary headache are prevalent and debilitating disorders. Acute pain cessation is one of the key points in their treatment. Many drugs have been studied but the design of the trials is not usually homogeneous. Efficacy of the trial is determined depending on the selected primary endpoint and usually other different outcomes are measured. We aim to critically appraise which were the employed outcomes through a systematic review. METHODS: We conducted a systematic review of literature focusing on studies on primary headache evaluating acute relief of pain, following the PRISMA guideline. The study population included patients participating in a controlled study about symptomatic treatment. The comparator could be placebo or the standard of care. The collected information was the primary outcome of the study and all secondary outcomes. We evaluated the studied drug, the year of publication and the type of journal. We performed a search and we screened all the potential papers and reviewed them considering inclusion/exclusion criteria. RESULTS: The search showed 4288 clinical trials that were screened and 794 full articles were assessed for eligibility for a final inclusion of 495 papers. The studies were published in headache specific journals (58%), general journals (21.6%) and neuroscience journals (20.4%). Migraine was the most studied headache, in 87.8% studies, followed by tension type headache in 4.7%. Regarding the most evaluated drug, triptans represented 68.6% of all studies, followed by non-steroidal anti-inflammatories (25.1%). Only 4.6% of the papers evaluated ergots and 1.6% analyzed opioids. The most frequent primary endpoint was the relief of the headache at a determinate moment, in 54.1%. Primary endpoint was evaluated at 2-h in 69.9% of the studies. Concerning other endpoints, tolerance was the most frequently addressed (83%), followed by headache relief (71.1%), improvement of other symptoms (62.5%) and presence of relapse (54%). The number of secondary endpoints increased from 4.2 (SD = 2.0) before 1991 to 6.39 after 2013 (p = 0.001). CONCLUSION: Headache relief has been the most employed primary endpoint but headache disappearance starts to be firmly considered. The number of secondary endpoints increases over time and other outcomes such as disability, quality of life and patients' preference are receiving attention.

The allosteric regulation of pyruvate kinase.

Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.

Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide.

The gene encoding pyruvate kinase type I (PKI) of Escherichia coli was amplified by PCR, cloned and sequenced. The gene product was overexpressed in E. coli, using an inducible T7 RNA polymerase-based expression system. The transformed cells contained sixtyfold the enzyme activity of the reference cells and enabled the purification of 30 mg of highly active PKI from 1 liter of culture. The gene sequence was determined and found to be different from the one previously reported, i.e., the T nucleotide at position 1351 was missing. This resulted in a downstream shift of the stop codon, thus the deduced polypeptide was 470 amino acids long instead of 462. In addition the twelve C-terminal amino acids of the former sequence were changed.

Crystallization and preliminary X-ray analysis of pyruvate kinase type I from Escherichia coli.

Crystals of the fructose-l,6-bisphosphate-dependent pyruvate kinase from Escherichia coli have been grown using the hanging-drop vapour-diffusion technique. The space group was found to be C222(1) with cell dimensions a = 76.8, b = 247.5, c = 132.6 A. Diffraction data to 3.0 A resolution have been recorded and the enzyme molecular symmetry analysed through inspection of the self-rotation function. The crystallized protein is in the allosterically inactive T state.

Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.

BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation. RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme. CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.

Affinity labelling of the catalytic and allosteric ATP binding sites on pyruvate kinase type I from Escherichia coli.

The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.

The outB gene of Bacillus subtilis codes for NAD synthetase.

The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH3-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH3-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 degrees C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+. The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.

Divergent binding sites in pyruvate kinases I and II from Escherichia coli.

Pyridoxal 5'-phosphate incorporation into pyruvate kinase II from E. coli was decreased by the substrate phosphoenolpyruvate and increased by the allosteric activator ribose 5-phosphate, the total incorporation being linearly related to inactivation. Four lysyl residues were substantially modified, whatever the incubation conditions were while two additional residues became reactive only in the presence of the allosteric activator. Six tryptic peptides containing modified lysines were purified and sequenced. They defined five regions of pyruvate kinase II, since one of them contained two labelled lysines and included a peptide which also appeared independently. Sequence comparison with E. coli type I, yeast and cat muscle pyruvate kinases shows that the binding regions of pyruvate kinase II are clearly divergent from those of pyruvate kinase I and of the eukaryotic enzymes.

Fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli: ligand promoted conformational changes.

The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands. With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands. The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.

Bacterial pyruvate kinases have a shorter N-terminal domain.

The N-terminal portions of the two forms of pyruvate kinase (EC2.7.1.40) from Escherichia coli have been sequenced up the 48th and 43rd residue, respectively. Comparison with the known primary structures shows that bacterial enzymes lack a substantial portion of the N-terminal sequence with respect to pyruvate kinases from vertebrates. This makes the suggested functional role of the N-terminal domain unlikely [Muirhead, H. (1990) Biochem. Soc. Trans. 18, 193-196] although an elongation of this domain with evolution is apparent.

Fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli. Nature of bonds involved in the allosteric mechanism.

The allosteric properties of the fructose-1,6-bis-phosphate-activated pyruvate kinase from Escherichia coli were examined in the presence of a number of fructose bisphosphate analogues, as well as of increased ionic strength (NaCl) and of the hydrogen-bond-breaking agent, formamide. Fructose 2,6-bisphosphate, ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate gave allosteric activation (additive to that of fructose 1,6-bisphosphate). Formamide always decreased Vmax, but left unchanged the Km for phosphoenolpyruvate, while it decreased the concentration of fructose bisphosphate required to give half-maximal activity (K0.5). NaCl increased the K0.5 for both phosphoenolpyruvate and fructose bisphosphate, leaving Vmax unchanged. These results are consistent with ionic binding of fructose bisphosphate through phosphates and with a critical role of hydrogen bonds in stabilizing both the inactive and the active enzyme conformers.

Primary structure of three peptides at the catalytic and allosteric sites of the fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli.

Three peptides containing 6-pyridoxyllysine have been isolated from the tryptic digest of the allosteric fructose-1,6-bisphosphate-dependent pyruvate kinase from Escherichia coli, which had been almost completely inactivated with pyridoxal 5'-phosphate. The labelled peptides have been sequenced. The comparison of their sequences with the primary structure of the cat muscle pyruvate kinase allowed to state that peptide I fits the region spanning residues 423-438 (53% identity), peptide II corresponds to residues 442-457 (44% identity) and peptide III encompasses residues 342-368 (70% identity). These findings are discussed in connection with our previous results on the involvement of the three peptides in the catalytic and regulatory properties of the enzyme (Valentini, G., Speranza, M.L., Iadarola, P., Ferri, G. & Malcovati, M. (1988) Biol. Chem. Hoppe-Seyler 369, 1219-1226) and in connection with their location in the three-dimensional structure of the cat muscle pyruvate kinase (Muirhead, H., Clayden, D.A., Lorimer, C.G., Fothergill-Gilmore, L.A., Schiltz, E. & Schmitt, W. (1986) EMBO J. 5, 475-481).

Reactivity of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli with pyridoxal 5'-phosphate.

The allosteric fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli was modified with pyridoxal 5'-phosphate in the presence and in the absence of phosphoenolpyruvate, fructose 1,6-bisphosphate, MgADP and MgATP. In all cases a time-dependent inactivation was observed, but the rate and the extent of inactivation varied according to the conditions used. The kinetic properties of the partially inactivated enzyme were differently modified by addition of substrates and effectors to the modification mixture, the parameters mostly affected being those concerning fructose 1,6-bisphosphate. Tryptic peptides obtained from fully inactivated pyruvate kinase in the different conditions have been separated. In all conditions three main 6-pyridoxyllysine-containing peptides were present, the amounts of which showed significant differences in the presence of fructose 1,6-bisphosphate and MgADP. The function of the labelled peptides and the evidence supporting the physical existence of different conformational states are discussed. The main conclusion concerns the involvement of one of the above peptides in the binding of the allosteric effector fructose 1,6-bisphosphate.

Influence of fibronectin on the fibrillogenesis of type I and type III collagen.

The in vitro self-assembly of type I and type III calf skin collagen in the presence of fibronectin was studied turbidimetrically. Fibronectin delayed the fibrillogenesis of type III collagen but accelerated that of type I collagen. The effect of fibronectin was concentration-dependent. The lag phase was more altered than the growth phase in the presence of fibronectin while the final turbidity and the amount of fibrils formed were unmodified. Fibrils obtained in the presence of fibronectin all showed native banding pattern. Fibronectin bound partly to collagen fibrils; the amount of bound fibronectin was similar for the two types of collagen and tended to be constant at increasing fibronectin concentrations. It is suggested that the antithetic effect of fibronectin on type I and type III collagen fibrillogenesis may arise both from the different affinity of fibronectin to the two types of collagen and the different aggregation properties of each collagen type.

A simple procedure for the purification of neutral salt soluble type I collagen from skin.

Intact monomeric type I collagen was purified from fetal bovine skin by a simple and time saving procedure. Saline precipitates of mixed skin collagens, in 4 M NaCl, were extracted with a limited volume of dilute acetic acid, taken in the proportion of 1 ml per g of original wet skin; NaCl in the precipitate was not removed by dialysis. The salt concentration in the extraction medium in the above conditions, selectively solubilized type I collagen.

Conformation and kinetic properties of photosynthetic glyceraldehyde-3-phosphate dehydrogenase "in vivo".

Photosynthetic GAPDH has been studied in chloroplast extracts, obtained in presence of physiological concentrations of NADP and NAD. The enzyme is shown to have a molecular weight of 600,000, on the basis of zymograms obtained after electrophoresis on polyacrylamide gradient gels. Km values of 0.08 and 0.16 mM, respectively, were found for NADP and NAD. The same V is reached with both NADP and NAD. The two coenzymes bind to the enzyme at the same catalytic site.

Apparent subunit heterogeneity of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

NAD dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from spinach leaves has been previously investigated: the enzyme, homogeneous on conventional chromatography and analytical disc electrophoresis, shows on denaturing condition, non identical subunits of 37,000 and 14,000 daltons which have been separated in equimolar amounts after sodium dodecyl sulphate treatment (Speranza and Gozzer, 1978). Affinity chromatography carried out on these enyzme preparations allowed to separate a fully active enzyme of 150,000 daltons M.W. from a protein of 70,000 daltons M.W. which is devoid of glyceraldehyde-3-phosphate dehydrogenase activity: the purified enzyme shows 37,000 daltons subunits, while the second protein is made up of 14,000 daltons subunits. It is also reported that the 70,000 daltons protein could be separated from the enzyme by selective crystallization in the presence of organic solvents. It is concluded that the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves, as the homologous enzyme from other sources, is a tetramer of 37,000 daltons subunits.

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.