Phylogenetic and functional analysis of histidine residues essential for pH-dependent multimerization of von Willebrand factor.

von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D'D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ~ 6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pK(a) ~6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D'D3 domains, and His-to-Ala mutagenesis identified His³95 and His46° in the D2 domain as critical for VWF multimerization. Replacement of His³95 with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His46° with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His46° depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.

Coronavirus disease 2019 (COVID-19) pandemic, central-line-associated bloodstream infection (CLABSI), and catheter-associated urinary tract infection (CAUTI): The urgent need to refocus on hardwiring prevention efforts.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has had a considerable impact on US hospitalizations, affecting processes and patient population. OBJECTIVE: To evaluate the impact of COVID-19 pandemic on central-line-associated bloodstream infections (CLABSIs) and catheter associated urinary tract infections (CAUTIs) in hospitals. METHODS: We performed a retrospective study of CLABSIs and CAUTIs in 78 US 12 months before COVID-19 and 6 months during COVID-19 pandemic. RESULTS: During the 2 study periods, there were 795,022 central-line days and 817,267 urinary catheter days. Compared to the period before the COVID-19 pandemic, CLABSI rates increased by 51.0% during the pandemic period from 0.56 to 0.85 per 1,000 line days (P < .001) and by 62.9% from 1.00 to 1.64 per 10,000 patient days (P < .001). Hospitals with monthly COVID-19 patients representing >10% of admissions had a National Health Safety Network (NHSN) device standardized infection ratio for CLABSI that was 2.38 times higher than hospitals with <5% prevalence during the pandemic period (P = .004). Coagulase-negative Staphylococcus CLABSIs increased by 130% from 0.07 to 0.17 events per 1,000 line days (P < .001), and Candida spp by 56.9% from 0.14 to 0.21 per 1,000 line days (P = .01). In contrast, no significant changes were identified for CAUTI (0.86 vs 0.77 per 1,000 catheter days; P = .19). CONCLUSIONS: The COVID-19 pandemic was associated with substantial increases in CLABSIs but not CAUTIs. Our findings underscore the importance of hardwiring processes for optimal line care and regular feedback on performance to maintain a safe environment.

Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor.

Endothelial cells assemble von Willebrand factor (VWF) multimers into ordered tubules within storage organelles called Weibel-Palade bodies, and tubular packing is necessary for the secretion of VWF filaments that can bind connective tissue and recruit platelets to sites of vascular injury. We now have recreated VWF tubule assembly in vitro, starting with only pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D'D3 domains. Assembly requires low pH and calcium ions and is reversed at neutral pH. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D'D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging.

Association of Race With Mortality Among Patients Hospitalized With Coronavirus Disease 2019 (COVID-19) at 92 US Hospitals.

IMPORTANCE: While current reports suggest that a disproportionate share of US coronavirus disease 2019 (COVID-19) cases and deaths are among Black residents, little information is available regarding how race is associated with in-hospital mortality. OBJECTIVE: To evaluate the association of race, adjusting for sociodemographic and clinical factors, on all-cause, in-hospital mortality for patients with COVID-19. DESIGN, SETTING, AND PARTICIPANTS: This cohort study included 11 210 adult patients (age ≥18 years) hospitalized with confirmed severe acute respiratory coronavirus 2 (SARS-CoV-2) between February 19, 2020, and May 31, 2020, in 92 hospitals in 12 states: Alabama (6 hospitals), Maryland (1 hospital), Florida (5 hospitals), Illinois (8 hospitals), Indiana (14 hospitals), Kansas (4 hospitals), Michigan (13 hospitals), New York (2 hospitals), Oklahoma (6 hospitals), Tennessee (4 hospitals), Texas (11 hospitals), and Wisconsin (18 hospitals). EXPOSURES: Confirmed SARS-CoV-2 infection by positive result on polymerase chain reaction testing of a nasopharyngeal sample. MAIN OUTCOMES AND MEASURES: Death during hospitalization was examined overall and by race. Race was self-reported and categorized as Black, White, and other or missing. Cox proportional hazards regression with mixed effects was used to evaluate associations between all-cause in-hospital mortality and patient characteristics while accounting for the random effects of hospital on the outcome. RESULTS: Of 11 210 patients with confirmed COVID-19 presenting to hospitals, 4180 (37.3%) were Black patients and 5583 (49.8%) were men. The median (interquartile range) age was 61 (46 to 74) years. Compared with White patients, Black patients were younger (median [interquartile range] age, 66 [50 to 80] years vs 61 [46 to 72] years), were more likely to be women (2259 [49.0%] vs 2293 [54.9%]), were more likely to have Medicaid insurance (611 [13.3%] vs 1031 [24.7%]), and had higher median (interquartile range) scores on the Neighborhood Deprivation Index (-0.11 [-0.70 to 0.56] vs 0.82 [0.08 to 1.76]) and the Elixhauser Comorbidity Index (21 [0 to 44] vs 22 [0 to 46]). All-cause in-hospital mortality among hospitalized White and Black patients was 23.1% (724 of 3218) and 19.2% (540 of 2812), respectively. After adjustment for age, sex, insurance, comorbidities, neighborhood deprivation, and site of care, there was no statistically significant difference in risk of mortality between Black and White patients (hazard ratio, 0.93; 95% CI, 0.80 to 1.09). CONCLUSIONS AND RELEVANCE: Although current reports suggest that Black patients represent a disproportionate share of COVID-19 infections and death in the United States, in this study, mortality for those able to access hospital care did not differ between Black and White patients after adjusting for sociodemographic factors and comorbidities.

The case for a population standardized infection ratio (SIR): A metric that marries the device SIR to the standardized utilization ratio (SUR).

BACKGROUND: The device standardized infection ratio (SIR) is used to compare unit and hospital performance for different publicly reported infections. Interventions to reduce unnecessary device use may select a higher-risk population, leading to a paradoxical increase in SIR for some high-performing facilities. The standardized utilization ratio (SUR) adjusts for device use for different units and facilities. METHODS: We calculated the device SIR (calculated based on actual device days) and population SIR (defined as Σ observed events divided by Σ predicted events based on predicted device days), adjusting for the facility SUR for both central-line-associated bloodstream infections (CLABSIs) and catheter-associated urinary tract infections (CAUTIs) in 84 hospitals from a single system for calendar years 2016 and 2017. RESULTS: The central-line SUR was 1.02 for 801,172 central-line days, with a device SIR of 0.76 and a population SIR of 0.78, a 1.6% relative increase. On the other hand, the urinary catheter SUR was 0.90 for 757,504 urinary catheter days, with a device SIR of 0.84 and a population SIR of 0.76, a 10.0% relative decrease. The cumulative attributable difference for CAUTI to a target SIR of 1 was -135.4 for the device SIR compared to -203.66 for the population SIR, a 50.8% increase in prevented events. CONCLUSION: Population SIR accounts for predicted device utilization; thus, it is an attractive metric with which to address overall risk of infection or harm to a patient population. It also reduces the risk of selection bias that may impact the device SIR with interventions to reduce device use.

Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv.

Two antifungal peptides, named EAFP1 and EAFP2, have been purified from the bark of Eucommia ulmoides Oliv. Each of the sequences consists of 41 residues with a N-terminal blockage by pyroglutamic acid determined by automated Edman degradation in combination with the tandem mass spectroscopy and the C-terminal ladder sequencing analysis. The primary structurs all contain 10 cysteines, which are cross-linked to form five disulfide bridges with a pairing pattern (C1-C5, C2-C9, C3-C6, C4-C7, C8-C10). This is the first finding of a plant antifungal peptide with a five-disulfide motif. EAFP1 and EAFP2 show characteristics of hevein domain and exhibit chitin-binding properties similar to the previously identified hevein-like peptides. They exhibit relatively broad spectra of antifungal activities against eight pathogenic fungi from cotton, wheat, potato, tomato and tobacco. The inhibition activity of EAFP1 and EAFP2 can be effective on both chitin-containing and chitin-free fungi. The values of IC(50) range from 35 to 155 microg/ml for EAFP1 and 18 to 109 microg/ml for EAFP2. Their antifungal effects are strongly antagonized by calcium ions.

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides.

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.

Purification and characterization of a neurotoxic peptide huwentoxin-III and a natural inactive mutant from the venom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang).

A novel neurotoxic peptide, named huwentoxin-III, and a natural mutant have been isolated from the venom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang). The average molecular weights of the two peptides were determined as 3853.35 and 3667.40 by mass spectrometry, respectively. Huwentoxin-III has 33 amino acid residues containing 6 cysteine residues. Its natural mutant is only truncated a tryptophan residue from C-terminals of huwentoxin-III. The sequences of the two peptides show 70.5% sequence similarity with that of lectin-like peptide SHL-I previously isolated from the venom of the same spider, while they cannot agglutinate human erythrocytes. Huwentoxin-III can reversibly paralyze cockroaches for several hours with an ED(50) of (192.95 +/- 120.84) microg/g (P=0.95) (mean +/- SD) and can enhance the muscular contractions elicited by stimulating the nerve of the isolated rat vas deferens, however, the mutant of huwentoxin-III has no such effect, which suggests that Trp33 was an important residue related to the biological function of huwentoxin-III.

A structural explanation for the antithrombotic activity of ARC1172, a DNA aptamer that binds von Willebrand factor domain A1.

ARC1172 is a 41-mer DNA aptamer selected to bind the A1 domain of von Willebrand factor (VWF). A derivative of ARC1172 with modifications to increase intravascular survival inhibits carotid artery thrombosis in a Cynomolgus macaque model and inhibits VWF-dependent platelet aggregation in humans, suggesting that such aptamers may be useful to prevent or treat thrombosis. In the crystal structure of a VWF A1-ARC1172 complex, the aptamer adopts a three-stem structure of mainly B-form DNA with three noncanonical base pairs and 9 unpaired residues, 6 of which are stabilized by base-base or base-deoxyribose stacking interactions. The aptamer-protein interface is characterized by cation-pi interactions involving Arg, Lys, and Gln residues, often stabilized by H-bonds with adjacent bases. The ARC1172 binding site on the A1 domain overlaps with that of botrocetin and clashes with glycoprotein Ibalpha binding at an adjacent site, which accounts for the antithrombotic activity of ARC1172 and related aptamers.

Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi.

Von Willebrand factor (VWF) dimerizes through C-terminal CK domains, and VWF dimers assemble into multimers in the Golgi by forming intersubunit disulfide bonds between D3 domains. This unusual oxidoreductase reaction requires the VWF propeptide (domains D1D2), which acts as an endogenous pH-dependent chaperone. The cysteines involved in multimer assembly were characterized by using a VWF construct that encodes the N-terminal D1D2D'D3 domains. Modification with thiol-specific reagents demonstrated that secreted D'D3 monomer contained reduced Cys, whereas D'D3 dimer and propeptide did not. Reduced Cys in the D'D3 monomer were alkylated with N-ethylmaleimide and analyzed by mass spectrometry. All 52 Cys within the D'D3 region were observed, and only Cys(1099) and Cys(1142) were modified by N-ethylmaleimide. When introduced into the D1D2D'D3 construct, the mutation C1099A or C1142A markedly impaired the formation of D'D3 dimers, and the double mutation prevented dimerization. In full-length VWF, the mutations C1099A and C1099A/C1142A prevented multimer assembly; the mutation C1142A allowed the formation of almost exclusively dimers, with few tetramers and no multimers larger than hexamers. Therefore, Cys(1099) and Cys(1142) are essential for the oxidoreductase mechanism of VWF multimerization. Cys(1142) is reported to form a Cys(1142)-Cys(1142) intersubunit bond, suggesting that Cys(1099) also participates in a Cys(1099)-Cys(1099) disulfide bond between D3 domains. This arrangement of intersubunit disulfide bonds implies that the dimeric N-terminal D'D3 domains of VWF subunits align in a parallel orientation within VWF multimers.

Surface morphology and kinetic properties in rapid growth of EAFP protein crystals investigated by atomic force microscopy.

The whole process of EAFP protein monoclinic crystal growth with an extremely fast rate has been observed by atomic force microscopy. The results showed that the patterns of the growth images in rapidly growing crystals are complicated. The two-dimensional multi-layered stacks of growth steps are characteristic of higher supersaturation and the growth of steps proceeds in a manner of strong anisotropic spiral dislocations dominantly under lower supersaturation conditions. The complex dislocation sources, including multiple dislocation and multi-interacting single dislocation sources, the constant step-split and the propagation of trooped steps were observed on the {100} surfaces of growing EAFP crystals. The step height of each layer generated either by two-dimensional nucleation at higher supersaturation or by screw dislocation at lower supersaturation is about 2-3 nm, which corresponds to the length of the crystallographic unit cell. Although the rate of advancement for each growth step is similar to that of other protein crystal growth, the unique way of the propagation distinct with the trooped steps, by which a bundle of steps are strapped together, would be responsible for the rapid growth of EAFP crystals. All features show a possible mechanism by which the fast growth of EAFP crystals could be attained. The structural basis of the growth mechanism is also discussed.

Structural mechanism governing the quaternary organization of monocot mannose-binding lectin revealed by the novel monomeric structure of an orchid lectin.

Two isoforms of an antifungal protein, gastrodianin, were isolated from two subspecies of the orchid Gastrodia elata, belonging to the protein superfamily of monocot mannose-specific lectins. In the context that all available structures in this superfamily are oligomers so far, the crystal structures of the orchid lectins, both at 2.0 A, revealed a novel monomeric structure. It resulted from the rearrangement of the C-terminal peptide inclusive of the 12th beta-strand, which changes from the "C-terminal exchange" into a "C-terminal self-assembly" mode. Thus, the overall tertiary scaffold is stabilized with an intramolecular beta-sheet instead of the hybrid observed on subunit/subunit interface in all known homologous dimeric or tetrameric lectins. In contrast to the constrained extended conformation with a cis peptide bond between residues 98 and 99 commonly occurring in oligomers, a beta-hairpin forms from position 97 to 101 with a normal trans peptide bond at the corresponding site in gastrodianin, which determines the topology of the C-terminal peptide and thereby its unique fold pattern. Sequence and structure comparison shows that residue replacement and insertion at the position where the beta-hairpin occurs in association with cis-trans inter-conversion of the specific peptide bond (97-98) are possibly responsible for such a radical structure switch between monomers and oligomers. Moreover, this seems to be a common melody controlling the quaternary states among bulb lectins through studies on sequence alignment. The observations revealed a structural mechanism by which the quaternary organization of monocot mannose binding lectins could be governed. The mutation experiment performed on maltose-binding protein-gastrodianin fusion protein followed by a few biochemical detections provides direct evidence to support this conclusion. Potential carbohydrate recognition sites and biological implications of the orchid lectin based on its monomeric state are also discussed in this paper.

Primary structural determination of N-terminally blocked peptides from the bark of Eucommia ulmoides Oliv by mass spectrometric analysis.

Sequencing of N-terminally blocked proteins/peptides is a challenge since these molecules inhibit processing by Edman degradation. By using high accuracy Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), the primary structures of two novel N-terminally blocked antifungal peptides (EAFP1 and EAFP2), purified from the bark of Eucommia ulmoides Oliv, have been determined. The results show that the high mass accuracy provided by FTICR mass spectrometry is effective to determine the N-terminally blocking group, and can simplify the task of spectral interpretation and improve the precision of sequence determination. The combination of MALDI-TOFMS with carboxyl peptidase Y digestion was used to determine the C-terminal 36- and 27-residue sequences of EAFP1 and EAFP2, respectively, to provide the sequence linkage information for tryptic fragments. Compared with traditional peptide chemistry the advantage of mass spectrometric techniques is their simplicity, speed and sensitivity.

Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif.

The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.

Crystal structure of a novel antifungal protein distinct with five disulfide bridges from Eucommia ulmoides Oliver at an atomic resolution.

EAFP2 is a novel antifungal protein isolated from the bark of the tree Eucommia ulmoides Oliver. It consists of 41 residues and is characterized with a five-disulfide motif and the inhibitory effects on the growth of both cell wall chitin-containing and chitin-free fungi. The crystal structure of EAFP2 at an atomic resolution of 0.84 A has been determined by using Shake-and-Bake direct methods with the program SnB. The phases obtained were of sufficient quality to permit the initial model built automatically and the structural refinement carried out using anisotropic displacement parameters resulted in a final crystallographic R factor of 6.8%. In the resulting structural model, all non-hydrogen protein atoms including an unusual pyroglutamyl acid residue at the N-terminal can fit to the articulated electron densities with one centre and more than 65% of the hydrogen atoms in the protein can be observed as individual peaks in the difference map. The general fold of EAFP2 is composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Ala27-Cys31) and a three-stranded antiparallel beta-sheet (Cys16-Ser18, Cys23-Ser25, and Cys35-Cys37) and cross-linked by five disulfide bridges. The tertiary structure of EAFP2 can be divided into two structural sectors, A and B. Sector A composed of residues 11-30 adopts a conformation similar to the chitin-binding domain in the hevein-like proteins and features a hydrophobic surface embraced a chitin-binding site (Tyr20, 22, 29, and Ser18). The distinct disulfide bridge Cys7-Cys37 connects the N-terminal ten residues with the C-terminal segment 35-41 to form the sector B, which features a cationic surface distributing all four positively charged residues, Arg6, 9, 36, and 40. Based on these structural features, the possible structural basis of the functional properties of EAFP2 is discussed.

Crystallization and preliminary crystallographic studies of a novel antifungal protein with five disulfide bridges from Eucommia ulmoides Oliver.

Two antifungal proteins, named Eucommia antifungal peptides 1 and 2 (EAFP1 and EAFP2), have been purified from the bark of the tree E. ulmoides Oliver and show a relatively broad spectrum of antifungal activity against several agriculturally important plant pathogens. One of these small proteins (EAFP2) has been crystallized. The crystal belongs to space group P2(1), with unit-cell parameters a = 19.01, b = 23.16, c = 30.69 A, beta = 98.54 degrees. 1.0 A resolution data were collected from an EAFP2 crystal and have been used to obtain phase information directly by an ab initio method.