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1.
Int J Mol Sci ; 24(11)2023 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-37298286

RESUMO

T-cell recognition of antigens is complex, leading to biochemical and cellular events that impart both specific and targeted immune responses. The end result is an array of cytokines that facilitate the direction and intensity of the immune reaction-such as T-cell proliferation, differentiation, macrophage activation, and B-cell isotype switching-all of which may be necessary and appropriate to eliminate the antigen and induce adaptive immunity. Using in silico docking to identify small molecules that putatively bind to the T-cell Cß-FG loop, we have shown in vitro using an antigen presentation assay that T-cell signalling is altered. The idea of modulating T-cell signalling independently of antigens by directly targeting the FG loop is novel and warrants further study.


Assuntos
Transdução de Sinais , Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos/metabolismo , Citocinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
2.
J Biol Chem ; 297(2): 101011, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34324829

RESUMO

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.


Assuntos
Bacteroides thetaiotaomicron/enzimologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Simulação de Acoplamento Molecular/métodos , Polissacarídeos/metabolismo , Elementos Estruturais de Proteínas , Bacteroides thetaiotaomicron/química , Cristalografia por Raios X , Glicosilação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Especificidade por Substrato
3.
PLoS Comput Biol ; 17(7): e1009103, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34310592

RESUMO

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.


Assuntos
Proteínas de Bactérias , Glicosídeo Hidrolases , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biologia Computacional , Medição da Troca de Deutério , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Dinâmica Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , Streptococcus pyogenes/enzimologia , Especificidade por Substrato
4.
Phys Chem Chem Phys ; 24(21): 13015-13025, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35583143

RESUMO

This study investigated the effect of 2-methylimidazole (2-MIM) addition on the fluorescence of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate using low-cost density functional theory (DFT) and Time-Dependent DFT calculations on single crystal X-ray geometries of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate hydrate (1), 2-MIM (2), and the 1 : 1 co-crystal of (1) and (2), (3). At low concentrations (1 : 1-1 : 10) of 2-MIM, the fluorophore shows a decrease in the fluorescence intensity, but at higher concentrations (above 1 : 10) the fluorescence excitation maximum shifted from 354 nm to 405 nm, with a significant emission intensity increase. The changed excitation and emission profile at high concentrations is due to the deprotonation of the coumarin's phenolic group, which was confirmed by the increased shielding of the aromatic protons in the titration 1H NMR spectra. The experimental fluorescence data between the 1 : 1 and 1 : 10 ratios agreed with the theoretical fluorescence data, with a redshift and decreased intensity when comparing (1) and (3). The data indicated that combining the fluorophore with 2-MIM increased levels of vibronic coupling between 2-MIM and the fluorophore decreasing de-excitation efficiency. These increased vibronic changes were due to charge transfer between the fluorophore and 2-MIM in (3). The subtle movement of the proton, H(5) toward N(2') (0.07 Å) caused a significant decrease in fluorescence due to electron density distribution (EDD) changes. This was identified by comparison of the EDD in the excited (S1) and ground (S0) states plotted as an isosurface of EDD difference. For the higher concentrations, an alternative excitation pathway was explored by modifying the crystal geometry of (3) based on 1H NMR spectroscopy data to resemble excitoplexes. Theses excitoplex geometries reflected the fluorescence profile of the fluorophore with high concentrations of 2-MIM; there were dramatic changes in the theoretical fluorescence pathway, which was 100% vibronic coupling compared to 15.31% in the free fluorophore. At this concentration, the de-excitation pathway causes remodelling of the lactone ring via stretching/breaking the CO bond in the S1 causing increased fluorescence by movement of the transition dipole moment. These results reflect previous studies, but the methods used are less experimentally and computationally expensive. This study is among the first to explain charge transfer fluorescence using crystalline geometries. This study will be of interest to the fields of crystal engineering and fluorescence spectroscopy.


Assuntos
Prótons , Teoria Quântica , Corantes Fluorescentes , Imidazóis , Umbeliferonas , Difração de Raios X
5.
J Endovasc Ther ; 28(4): 614-622, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34018880

RESUMO

PURPOSE: Uncontrolled pelvic hemorrhage from trauma is associated with mortality rates above 30%. The ability of an intervention to reduce blood loss from pelvic trauma is paramount to its success. The objective of this study was to determine if computed tomography volumetric analysis could be used to quantify blood loss in a porcine endovascular pelvic hemorrhage model. MATERIALS AND METHODS: Yorkshire swine under general anesthesia underwent balloon dilation and rupture of the profunda femoris artery, which was confirmed by digital subtraction angiography. Computed tomography angiography and postprocessing segmentation were performed to quantify pelvic hemorrhage volume at 5 and 30 minutes after injury. Continuous hemodynamic and iliofemoral flow data were obtained. Baseline and postinjury hemoglobin, hematocrit and lactate were collected. RESULTS: Of 6 animals enrolled, 5 survived the 30-minute post-injury period. One animal died at 15 minutes. Median volume of pelvic hemorrhage was 141±106 cm3 at 5 minutes and 302±79 cm3 at 30 minutes with a 114% median increase in hematoma volume over 25 minutes (p=0.040). There was a significant decrease in mean arterial pressure (107 to 71 mm Hg, p=0.030) and iliofemoral flow (561 to 122 mL/min, p=0.014) at 30 minutes postinjury, but no significant changes in hemoglobin, hematocrit, or heart rate. CONCLUSION: Computed tomography volumetric analysis can be used to quantify rate and volume of blood loss in a porcine endovascular pelvic hemorrhage model. Future studies can incorporate this approach when evaluating the effect of hemorrhage control interventions associated with pelvic fractures.


Assuntos
Hemorragia , Ossos Pélvicos , Angiografia Digital , Animais , Tomografia Computadorizada de Feixe Cônico , Hemorragia/diagnóstico por imagem , Hemorragia/etiologia , Suínos , Resultado do Tratamento
6.
J Phys Chem A ; 125(45): 9736-9756, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34731566

RESUMO

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.


Assuntos
Teofilina , Cristalização , Ligação de Hidrogênio , Solubilidade , Molhabilidade
7.
Glycobiology ; 30(4): 268-279, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-31172182

RESUMO

The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan. EndoS and EndoS2, produced by Streptococcus pyogenes, are endoglycosidases with remarkable specificity for Asn297 glycosylation, making them ideal tools for chemoenzymatic synthesis. Although both enzymes are specific for IgG, EndoS2 recognizes a wider range of glycans than EndoS. Recent progress has been made in understanding the structural basis for their activities on antibodies. In this review, we examine the molecular mechanism of glycosidic bond cleavage by these enzymes and how specific point mutations convert them into glycosynthases. We also discuss the structural basis for differences in the glycan repertoire that IgG-active endoglycosidases recognize, which focuses on the structure of the loops within the glycoside hydrolase (GH) domain. Finally, we discuss the important contributions of carbohydrate binding modules (CBMs) to endoglycosidase activity, and how CBMs work in concert with GH domains to produce optimal activity on IgG.


Assuntos
Glicosídeo Hidrolases/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Polissacarídeos/metabolismo , Animais , Glicosilação , Humanos , Modelos Moleculares , Estrutura Molecular , Polissacarídeos/química , Streptococcus pyogenes/enzimologia
8.
J Phys Chem A ; 122(11): 3031-3044, 2018 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-29481082

RESUMO

Experimental charge density distribution studies, complemented by quantum mechanical theoretical calculations, of a host-guest system composed of a macrocycle (1) and barbital (2) in a 1:1 ratio (3) have been carried out via high-resolution single-crystal X-ray diffraction. The data were modeled using the conventional multipole model of electron density according to the Hansen-Coppens formalism. The asymmetric unit of macrocycle 1 contained an intraannular ethanol molecule and an extraannular acetonitrile molecule, and the asymmetric unit of 3 also contained an intraannular ethanol molecule. Visual comparison of the conformations of the macrocyclic ring shows the rotation by 180° of an amide bond attributed to competitive hydrogen bonding. It was found that the intraannular and extraannular molecules inside were orientated to maximize the number of hydrogen bonds present, with the presence of barbital in 3 resulting in the greatest stabilization. Hydrogen bonds ranging in strength from 4 to 70 kJ mol-1 were the main stabilizing force. Further analysis of the electrostatic potential among 1, 2, and 3 showed significant charge redistribution when cocrystallization occurred, which was further confirmed by a comparison of atomic charges. The findings presented herein introduce the possibility of high-resolution X-ray crystallography playing a more prominent role in the drug design process.


Assuntos
Barbital/química , Compostos Macrocíclicos/química , Teoria Quântica , Sítios de Ligação , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular
9.
Phys Chem Chem Phys ; 18(41): 28802-28818, 2016 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-27722530

RESUMO

Experimental charge density distribution studies of two polymorphic forms of piroxicam, ß-piroxicam (1) and piroxicam monohydrate (2), were carried out via high-resolution single crystal X-ray diffraction experiments and multipole refinement. The asymmetric unit of (2) consists of two discrete piroxicam molecules, (2a) and (2b), and two water molecules. Geometry differs between (1) and (2) due to the zwitterionic nature of (2) which results in the rotation of the pyridine ring around the C(10)-N(2) bond by approximately 180°. Consequently, the pyridine and amide are no longer co-planar and (2) forms two exclusive, strong hydrogen bonds, H(3)O(4) and H(2)O(3), with bond energies of 66.14 kJ mol-1 and 112.82 kJ mol-1 for (2a), and 58.35 kJ mol-1 and 159.51 kJ mol-1 for (2b), respectively. Proton transfer between O(3) and N(3) in (2) results in significant differences in surface electrostatic potentials. This is clarified by the calculation of atomic charges in the zwitterion that shows the formally positive charge of the pyridyl nitrogen which is redistributed over the whole of the pyridine ring instead of concentrating at N-H. Similarly, the negative charge of the oxygen is distributed across the benzothiazine carboxamide moiety. The multipole derived lattice energy for (1) is -304 kJ mol-1 and that for (2) is -571 kJ mol-1, which is in agreement with the experimentally determined observations of higher solubility and dissolution rates of (1) compared to (2).

10.
Phys Chem Chem Phys ; 17(9): 6667, 2015 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-25658441

RESUMO

Correction for 'Experimental and theoretical charge density distribution in Pigment Yellow 101' by Jonathan J. Du et al., Phys. Chem. Chem. Phys., 2015, DOI: 10.1039/c4cp04302b.

11.
Phys Chem Chem Phys ; 17(6): 4677-86, 2015 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-25588361

RESUMO

The charge density distribution in 2,2'-dihydroxy-1,1'-naphthalazine (Pigment Yellow 101; P.Y.101) has been determined using high-resolution X-ray diffraction and multipole refinement, along with density functional theory calculations. Topological analysis of the resulting densities highlights the localisation of single/double bonds in the central C=N-N=C moiety of the molecule in its ground state. The density in the N-N is examined in detail, where we show that very small differences between experiment and theory are amplified by use of the Laplacian of the density. Quantification of hydrogen bonds highlights the importance of the intramolecular N-H···O interaction, known to be vital for retention of fluorescence in the solid state, relative to the many but weak intermolecular contacts located. However, a popular method for deriving H-bond strengths from density data appears to struggle with the intramolecular N-H···O interaction. We also show that theoretical estimation of anisotropic displacements for hydrogen atoms brings little benefit overall, and degrades agreement with experiment for one intra-molecular contact.

12.
Sci Adv ; 10(15): eadk8157, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38598628

RESUMO

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.


Assuntos
Anticorpos Biespecíficos , Imunoglobulina G , Dimerização , Tirosina/metabolismo , Serina/metabolismo , Biologia Computacional
13.
Nat Commun ; 15(1): 5123, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38879612

RESUMO

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.


Assuntos
Proteínas de Bactérias , Bacteroides thetaiotaomicron , Microbioma Gastrointestinal , Polissacarídeos , Polissacarídeos/metabolismo , Humanos , Bacteroides thetaiotaomicron/metabolismo , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Especificidade por Substrato , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Manose/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Família Multigênica
14.
bioRxiv ; 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36747840

RESUMO

Immunoglobulin G (IgG) antibodies contain a single, complex N -glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

15.
Nat Commun ; 14(1): 2853, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37202422

RESUMO

Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.


Assuntos
Imunoglobulina G , Receptores de IgG , Animais , Camundongos , Glicosilação , Receptores de IgG/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Polissacarídeos/química
16.
CVIR Endovasc ; 6(1): 47, 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37843596

RESUMO

PURPOSE: To assess the effectiveness of trans-arterial vascular interventions in treatment of civilian gunshot wounds (GSW). MATERIALS AND METHODS: A retrospective review was performed at a level-1 trauma center to include 46 consecutive adults admitted due to GSW related hemorrhage and treated with endovascular interventions from July 2018 to July 2022. Patient demographics and procedural metrics were retrieved. Primary outcomes of interest include technical success and in-hospital mortality. Factors of mortality were assessed using a logistic regression model. RESULTS: Twenty-one patients were brought to the endovascular suite directly (endovascular group) from the trauma bay and 25 patients after treatment in the operating room (OR group). The OR group had higher hemodynamic instability (48.0% vs 19.0%, p = 0.040), lower hemoglobin (12.9 vs 10.1, p = 0.001) and platelet counts (235.2 vs 155.1, p = 0.003), and worse Acute Physiology and Chronic Health Evaluation (APACHE) score (4.1 vs 10.2, p < 0.0001) at the time of initial presentation. Technical success was achieved in all 40 cases in which targeted embolization was attempted (100%). Empiric embolization was performed in 6/46 (13.0%) patients based on computed tomographic angiogram (CTA) and operative findings. Stent-grafts were placed in 3 patients for subclavian artery injuries. Availability of pre-intervention CTA was associated with shorter fluoroscopy time (19.8 ± 12.1 vs 30.7 ± 18.6 min, p = 0.030). A total of 41 patients were discharged in stable condition (89.1%). Hollow organ injury was associated with mortality (p = 0.039). CONCLUSION: Endovascular embolization and stenting were effective in managing hemorrhage due to GSW in a carefully selected population. Hollow organ injury was a statistically significant predictor of mortality. Pre-intervention CTA enabled targeted, shorter and equally effective procedures.

17.
Biotechnol Adv ; 67: 108201, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37336296

RESUMO

Antibody based drugs, including IgG monoclonal antibodies, are an expanding class of therapeutics widely employed to treat cancer, autoimmune and infectious diseases. IgG antibodies have a conserved N-glycosylation site at Asn297 that bears complex type N-glycans which, along with other less conserved N- and O-glycosylation sites, fine-tune effector functions, complement activation, and half-life of antibodies. Fucosylation, galactosylation, sialylation, bisection and mannosylation all generate glycoforms that interact in a specific manner with different cellular antibody receptors and are linked to a distinct functional profile. Antibodies, including those employed in clinical settings, are generated with a mixture of glycoforms attached to them, which has an impact on their efficacy, stability and effector functions. It is therefore of great interest to produce antibodies containing only tailored glycoforms with specific effects associated with them. To this end, several antibody engineering strategies have been developed, including the usage of engineered mammalian cell lines, in vitro and in vivo glycoengineering.


Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Animais , Anticorpos Monoclonais/metabolismo , Imunoglobulina G/metabolismo , Glicosilação , Polissacarídeos , Linhagem Celular , Mamíferos
18.
Methods Mol Biol ; 2674: 147-167, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37258966

RESUMO

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.


Assuntos
Acetilglucosaminidase , Polissacarídeos , Humanos , Especificidade por Substrato , Acetilglucosaminidase/metabolismo , Hidrólise , Cinética , Espectrometria de Massas/métodos , Polissacarídeos/química
19.
Nat Commun ; 14(1): 1705, 2023 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-36973249

RESUMO

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-ß-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.


Assuntos
Glicosídeo Hidrolases , Evasão da Resposta Imune , Humanos , Glicosídeo Hidrolases/metabolismo , Streptococcus pyogenes , Imunoglobulina G , Polissacarídeos/metabolismo
20.
JACC Case Rep ; 4(1): 59-62, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-35036946

RESUMO

We present the case of a 25-year-old woman with desmoplakin cardiomyopathy-related myocarditis. Her high-sensitivity troponin and symptoms improved with pulse steroid therapy and mycophenolate mofetil. The literature lacks data to effectively guide the management of recurrent myocarditis in desmoplakin cardiomyopathy. (Level of Difficulty: Advanced.).

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