Clinical and radiographic phenotypes of patients with multifocal subcortical versus cortical cerebral infarcts.

BACKGROUND AND PURPOSE: Infarct topology is a key determinant in classification of a stroke as potentially embolic, with cortical and multifocal lesions being presumed embolic. Whether isolated subcortical multifocal infarcts are likely embolic has not been well studied. METHODS: A prospective, single-center cohort study of consecutive patients with acute multifocal strokes confirmed on diffusion-weighting imaging (DWI) was queried, and patients compared according to the presence of isolated subcortical infarct topology versus cortical ± subcortical topology. Descriptive statistics and multivariable logistic regression were used to determine independent predictors of cryptogenic, subcortical infarcts. RESULTS: Of 1739 patients screened, 743 had complete diagnostic testing with DWI evidence of acute infarction, 183 (24.6%) of whom had a multifocal stroke pattern. Isolated subcortical involvement was disproportionate among patients with ESUS (64.9%) when compared to patients with cardioembolic (24.3%) or large vessel disease (10.8%, p<0.01). Following multivariable adjustment, independent predictors of isolated subcortical multifocal infarction were milder strokes (OR 0.94, 95%CI 0.89-0.98) and higher grade Fazekas score (OR 2.32, 95%CI 1.02-5.29), while cardioembolism (OR 0.30, 95%CI 0.08-1.13) and large vessel disease (OR 0.27, 95%CI 0.08-0.91) remained inversely associated (as compared to ESUS). CONCLUSIONS: These data suggest that multifocal subcortical infarctions are less likely to have an associated proximal embolic source than multifocal infarctions with cortical involvement. The strong association with chronic microvascular disease suggests this topology is more consistent with acute-on-chronic microvascular injury rather than an occult embolic source.

Unique Clinicopathologic Subclassifiers of Cryptogenic Cerebral Emboli.

INTRODUCTION: Ipsilateral nonstenotic (<50%) internal carotid artery (ICA) plaque, cardiac atriopathy, and patent foramen ovale (PFO) may account for a substantial proportion of embolic stroke of undetermined source (ESUS). METHODS: Consecutive stroke patients at our center (2019-2021) with unilateral, anterior circulation ESUS were categorized into the following mutually exclusive etiologies: (1) nonstenotic ipsilateral ICA plaque (NSP, ≥3mm in maximal axial diameter), (2) sex-adjusted mod-to-severe left atrial enlargement (LAE), (3) PFO, and (4) "occult ESUS" (patients who failed to meet criteria for these 3 groups). Descriptive statistics and multivariable logistic regression were used to model group characteristics. RESULTS: Of 132 included patients, the median age was 65 (IQR 56-73), 74 (56%) of whom were White, and 54 (41%) were female. Twenty-one patients (16%) had NSP proximal to the infarct territory, 17 (13%) had LAE, 9 (7%) had a PFO, and 85 (64%) had no other mechanism. Patients with LAE were older (p=0.004), and had more frequent intracranial occlusions of the internal carotid and proximal middle cerebral artery (p=0.048), while tobacco use was most commonly found among patients with NSP (75%) when compared to other ESUS groups (p=0.02). Five of 9 patients with LAE who underwent outpatient telemetry had paroxysmal atrial fibrillation (56%), while zero patients with PFO or NSP had paroxysmal atrial fibrillation (p=0.005). Older age (adjusted OR [aOR] 1.05, 95%CI 1.03-1.07), coronary artery disease (aOR 3.22, 95%CI 1.61-6.44) and hypertension (aOR 2.16, 95%CI 1.14-4.06) were independently associated with LAE, while only tobacco use was associated with NSP when compared to other ESUS subclassifiers (OR 3.18, 95%CI 1.08-0.42). Age and tobacco use were both inversely associated with PFO (aOR 0.93, 95%CI 0.88-0.98, and aOR 0.10, 95%CI 0.02-0.90, respectively). CONCLUSIONS: Certain clinical and radiographic features may be useful in predicting the proximal source of occult cerebral emboli, and can be used for cost-effective outpatient diagnostic testing.

New-onset Bell's palsy after neuroinvasive West Nile virus.

In this case report, a patient was diagnosed with new-onset Bell's palsy 3 weeks after the onset of neuroinvasive West Nile virus. This was the second case report of West Nile virus-associated Bell's palsy, highlighting the need to monitor these patients for peripheral neuropathies. This case report is also intended to raise awareness about the prevalence of West Nile virus in the USA.

Unexpected dependence on pH of NO release from Paracoccus pantotrophus cytochrome cd1.

A previous study of nitrite reduction by Paracoccus pantotrophus cytochrome cd(1) at pH 7.0 identified early reaction intermediates. The c-heme rapidly oxidised and nitrite was reduced to NO at the d(1)-heme. A slower equilibration of electrons followed, forming a stable complex assigned as 55% cFe(III)d(1)Fe(II)-NO and 45% cFe(II)d(1)Fe(II)-NO(+). No catalytically competent NO release was observed. Here we show that at pH 6.0, a significant proportion of the enzyme undergoes turnover and releases NO. An early intermediate, which was previously overlooked, is also identified; enzyme immediately following product release is a candidate. However, even at pH 6.0 a considerable fraction of the enzyme remains bound to NO so another component is required for full product release. The kinetically stable product formed at the end of the reaction differs significantly at pH 6.0 and 7.0, as does its rate of formation; thus the reaction is critically dependent on pH.

The CcmE protein of the c-type cytochrome biogenesis system: unusual in vitro heme incorporation into apo-CcmE and transfer from holo-CcmE to apocytochrome.

Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro. apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (K(d), 200 nM), rather than ferrous heme. The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE. Reduction under certain conditions of the ferric heme-CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein. The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE. Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE. Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced. The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products. In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE. These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.

Interaction of heme with variants of the heme chaperone CcmE carrying active site mutations and a cleavable N-terminal His tag.

Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form.

A novel, kinetically stable, catalytically active, all-ferric, nitrite-bound complex of Paracoccus pantotrophus cytochrome cd1.

The oxidized form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase, as isolated, has bis-histidinyl co-ordination of the c haem and His/Tyr co-ordination of the d(1) haem. On reduction, the haem co-ordinations change to His/Met and His/vacant respectively. If the latter form of the enzyme is reoxidized, a conformer is generated in which the ferric c haem is His/Met co-ordinated; this can revert to the 'as isolated' state of the enzyme over approx. 20 min at room temperature. However, addition of nitrite to the enzyme after a cycle of reduction and reoxidation produces a kinetically stable, all-ferric complex with nitrite bound to the d(1) haem and His/Met co-ordination of the c haem. This complex is catalytically active with the physiological electron donor protein pseudoazurin. The effective dissociation constant for nitrite is 2 mM. Evidence is presented that d(1) haem is optimized to bind nitrite, as opposed to other anions that are commonly good ligands to ferric haem. The all-ferric nitrite bound state of the enzyme could not be generated stoichiometrically by mixing nitrite with the 'as isolated' conformer of cytochrome cd(1) without redox cycling.

Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.

Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine.

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.