RESUMO
PURPOSE: This study evaluated the use of intraoperative local injection of liposomal bupivacaine to decrease opioid use in the early postoperative period for patients undergoing outpatient thumb carpometacarpal joint arthroplasty. METHODS: A prospective, randomized, controlled, single-blinded study was designed to compare 2 groups of patients for opioid use, pain scores, and nonopioid pill consumption within 5 days after surgery. The investigational group received an intraoperative injection of 10 ml (133 mg) liposomal bupivacaine. The control group received no local anesthetic. All patients were anesthetized with a standardized supraclavicular nerve block and were prescribed equal amounts of oral narcotic analgesic. Outcomes were assessed by collecting the data from postoperative patient-reported diaries. RESULTS: The experimental group reported a significantly lower total opioid consumption for the 5 days after surgery. Daily opioid use, as measured by both opioid pill equivalent count and morphine milligram equivalent in addition to postoperative pain scores and nonopioid pill consumption, was not different between groups. CONCLUSIONS: Intraoperative injection of liposomal bupivacaine was shown to decrease total opioid intake during the 5 days after thumb carpometacarpal arthroplasty. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic II.
Assuntos
Analgésicos Opioides , Bloqueio do Plexo Braquial , Analgésicos Opioides/uso terapêutico , Anestésicos Locais/uso terapêutico , Artroplastia , Bupivacaína , Humanos , Medição da Dor , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/prevenção & controle , Período Pós-Operatório , Estudos Prospectivos , PolegarRESUMO
Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (-0.23 cm(3) per mol = -0.38 Å(3) per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm(3)/mol (3.7 Å(3) per molecule) for adding and 2.4 cm(3)/mol (4.0 Å(3) per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.
Assuntos
Modelos Moleculares , Peptídeos/química , Pressão , Estabilidade Proteica , Estrutura Secundária de Proteína , Simulação por Computador , Método de Monte Carlo , Desdobramento de ProteínaRESUMO
The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7â 10(7) (amino acids)(2)/s or 6.1â 10(-9) cm(2)/s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a Ïf-value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (Ïf = 0) in agreement with a diffusing boundary mechanism.
Assuntos
Modelos Moleculares , Peptídeos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Algoritmos , Simulação por Computador , Cinética , Modelos Químicos , Peptídeos/genética , Estabilidade Proteica/efeitos dos fármacos , Termodinâmica , Ureia/química , Ureia/farmacologiaRESUMO
Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.
Assuntos
Dobramento de Proteína , Proteínas/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Proteínas/química , TermodinâmicaRESUMO
The intramolecular and intermolecular vibrational energy flow in a polyproline peptide with a total number of nine amino acids in the solvent dimethyl sulfoxide is investigated using time-resolved infrared (IR) spectroscopy. Azobenzene covalently bound to a proline sequence containing nitrophenylalanine as a local sensor for vibrational excess energy serves as a heat source. Information on through-space distances in the polyproline peptides is obtained by independent Förster resonance energy transfer measurements. Photoexcitation of the azobenzene and subsequent internal conversion yield strong vibrational excitation of the molecule acting as a local heat source. The relaxation of excess heat, its transfer along the peptide and to the solvent is monitored by the response of the nitro-group in nitrophenylalanine acting as internal thermometer. After optical excitation, vibrational excess energy is observed via changes in the IR absorption spectra of the peptide. The nitrophenylalanine bands reveal that the vibrational excess energy flows in the peptide over distances of more than 20 Å and arrives delayed by up to 7 ps at the outer positions of the peptide. The vibrational excess energy is transferred to the surrounding solvent on a time scale of 10-20 ps. The experimental observations are analyzed by different heat conduction models. Isotropic heat conduction in three dimensions away from the azobenzene heat source is not able to describe the observations. One-dimensional heat dissipation along the polyproline peptide combined with a slower transversal heat transfer to the solvent surrounding well reproduces the observations.
Assuntos
Transferência de Energia , Peptídeos , Temperatura Alta , Peptídeos/química , Solventes/química , VibraçãoRESUMO
A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (DeltaH(0) = 35 +/- 4 kJ/mol) which is nearly compensated in free energy by the entropy change (DeltaS(0) = 112 +/- 20 Jxmol(-1)xK(-1)). The unlocking reaction has a time constant of about 1 mus at 5 degrees C and is enthalpy-limited with an activation energy of 32 +/- 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 x 10(11) s(-1). In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 +/- 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.
Assuntos
Proteínas dos Microfilamentos/química , Sequência de Aminoácidos , Transferência de Energia/efeitos dos fármacos , Guanidina/farmacologia , Cinética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coloração e Rotulagem , TemperaturaRESUMO
PURPOSE: Proximal row carpectomy (PRC) is a motion-sparing procedure for degenerative disorders of the proximal carpal row. Reported results at a minimum 10-year follow-up consistently show maintenance of strength, motion, and satisfaction with an average conversion rate to radiocarpal arthrodesis of 12%. We hypothesized that PRC would continue to provide a high level of satisfaction and function at a minimum of 20 years. METHODS: Seventeen wrists in 16 patients, including 7 laborers, underwent PRC for symptomatic degenerative disorders of the proximal carpal row at an average age of 36 years. Patients returned for radiographic and clinical evaluation, and the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) questionnaire and Patient-Related Wrist Evaluation were used for subjective assessment. Follow-up was a minimum of 20 years (average, 24 y). RESULTS: Eleven wrists (65%) underwent no further surgery at a minimum 20-year follow-up. The average time to failure of PRC, defined as the time from PRC to radiocarpal arthrodesis, was 11 years (range, 8 mo to 20 y). Ten of 11 patients who did not undergo radiocarpal arthrodesis continued to be satisfied, with minimal decrease in motion and grip strength compared with the uninvolved side. Average score for QuickDASH was 16 and for Patient-Related Wrist Evaluation was 26. The flexion-extension arc was 68°, and grip strength was 72% of the contralateral side. All patients returned to their original employment. There was no correlation between degenerative radiographic changes and satisfaction level. The predicted probability of failure revealed a higher risk in patients who underwent PRC at a younger age, which leveled off at age 40 years. CONCLUSIONS: PRC provides satisfaction at a minimum of 20 years with a survival rate of 65%. Whereas we recommend a minimum age for PRC between 35 and 40 years, young patients should not be excluded as PRC candidates; these patients should undergo appropriate preoperative counseling of their increased failure risk secondary to their young age. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.
Assuntos
Artrodese/métodos , Ossos do Carpo/cirurgia , Rádio (Anatomia)/cirurgia , Amplitude de Movimento Articular/fisiologia , Articulação do Punho/cirurgia , Adulto , Fatores Etários , Artrodese/efeitos adversos , Ossos do Carpo/diagnóstico por imagem , Distribuição de Qui-Quadrado , Estudos de Coortes , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/diagnóstico por imagem , Osteoartrite/fisiopatologia , Osteoartrite/cirurgia , Osteotomia/efeitos adversos , Osteotomia/métodos , Medição da Dor , Dor Pós-Operatória/fisiopatologia , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/fisiopatologia , Radiografia , Rádio (Anatomia)/diagnóstico por imagem , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Articulação do Punho/diagnóstico por imagem , Articulação do Punho/fisiopatologiaRESUMO
Protein/solvent interactions largely influence protein dynamics, particularly motions in unfolded and intrinsically disordered proteins (IDPs). Here, we apply triplet-triplet energy transfer (TTET) to investigate the coupling of internal protein motions to solvent motions by determining the effect of solvent viscosity (η) and macromolecular crowding on the rate constants of loop formation (kc) in several unfolded polypeptide chains including IDPs. The results show that the viscosity dependence of loop formation depends on amino acid sequence, loop length, and co-solute size. Below a critical size (rc), co-solutes exert a maximum effect, indicating that under these conditions microviscosity experienced by chain motions matches macroviscosity of the solvent. rc depends on chain stiffness and reflects the length scale of the chain motions, i.e., it is related to the persistence length. Above rc, the effect of solvent viscosity decreases with increasing co-solute size. For co-solutes typically used to mimic cellular environments, a scaling of kc â η-0.1 is observed, suggesting that dynamics in unfolded proteins are only marginally modulated in cells. The effect of solvent viscosity on kc in the small co-solute limit (below rc) increases with increasing chain length and chain flexibility. Formation of long and very flexible loops exhibits a kc â η-1 viscosity dependence, indicating full solvent coupling. Shorter and less flexible loops show weaker solvent coupling with values as low as kc â η-0.75 ± 0.02. Coupling of formation of short loops to solvent motions is very little affected by amino acid sequence, but solvent coupling of long-range loop formation is decreased by side chain sterics.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Solventes/química , Dobramento de Proteína , Viscosidade , Peptídeos/químicaRESUMO
Internal dynamics of proteins are essential for protein folding and function. Dynamics in unfolded proteins are of particular interest since they are the basis for many cellular processes like folding, misfolding, aggregation, and amyloid formation and also determine the properties of intrinsically disordered proteins (IDPs). It is still an open question of what governs motions in unfolded proteins and whether they encounter major energy barriers. Here we use triplet-triplet energy transfer (TTET) in unfolded homopolypeptide chains and IDPs to characterize the barriers for local and long-range loop formation. The results show that the formation of short loops encounters major energy barriers with activation energies (Ea) up to 18 kJ/mol (corrected for effects of temperature on water viscosity) with very little dependence on amino acid sequence. For poly(Gly-Ser) and polySer chains the barrier decreases with increasing loop size and reaches a limiting value of 4.6 ± 0.4 kJ/mol for long and flexible chains. This observation is in accordance with the concept of internal friction encountered by chain motions due to steric effects, which is high for local motions and decreases with increasing loop size. Comparison with the results from the viscosity dependence of loop formation shows a negative correlation between Ea and the sensitivity of the reaction to solvent viscosity (α) in accordance with the Grote-Hynes theory of memory friction. The Arrhenius pre-exponential factor (A) also decreases with increasing loop size, indicating increased entropic costs for loop formation. Long-range loop formation in the investigated sequences derived from IDPs shows increased Ea and A compared with poly(Gly-Ser) and polySer chains. This increase is exclusively due to steric effects that cause additional internal friction, whereas intramolecular hydrogen bonds, dispersion forces, and charge interactions do not affect the activation parameters.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Temperatura , Fricção , Dobramento de Proteína , Sequência de AminoácidosRESUMO
Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in alpha-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 micros at 5 degrees C. Local helix formation occurs with a time constant of approximately 400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with position-independent elementary time constants of approximately 50 ns for the addition and of approximately 65 ns for the removal of an alpha-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.
Assuntos
Transferência de Energia , Peptídeos/química , Sequência de Aminoácidos , Simulação por Computador , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Temperatura , Termodinâmica , Ureia/farmacologiaRESUMO
PURPOSE: Two common techniques for fixation of extra-articular fractures at the proximal phalanx base are transarticular (across the metacarpophalangeal joint) and extra-articular cross-pinning. The aim of this study was to assess the complications and outcomes of these 2 techniques. Our hypothesis was that transarticular and extra-articular closed reduction and percutaneous pinning of base of proximal phalanx fractures have similar complication rates and outcomes. METHODS: A retrospective chart review identified 338 patients with base of proximal phalanx fractures. We treated 50 isolated fractures with closed reduction and percutaneous pinning using 1 of 2 techniques: transarticular (25 fractures through the metacarpal head) or extra-articular (25 fractures cross-pinned through the base of the proximal phalanx). Outcome measures included total active motion and complications. RESULTS: We found a substantial overall complication rate in both groups. The mean total active motion for the transarticular group and cross-pinning group was 201° and 198°, respectively. Proximal interphalangeal joint motion was notably affected; nearly half of the patients in each group had flexion loss greater than 20° (average, 27°) at the proximal interphalangeal joint. Nearly a third of patients in both groups had fixed flexion contracture greater than 15° at the proximal interphalangeal joint. There were more secondary procedures in the transarticular group (6) than in the cross-pinning group (2). There was no statistical significance between groups in any of the outcome parameters used. CONCLUSIONS: Closed pinning minimizes additional soft tissue injury and allows for early motion, but neither fixation method was superior in terms of the measured parameters. In addition, overall results were not as good as what has been reported in the literature.
Assuntos
Pinos Ortopédicos , Fios Ortopédicos , Traumatismos dos Dedos/cirurgia , Fixação Interna de Fraturas/métodos , Fraturas Ósseas/cirurgia , Complicações Pós-Operatórias/epidemiologia , Amplitude de Movimento Articular/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Feminino , Traumatismos dos Dedos/diagnóstico por imagem , Fraturas Ósseas/diagnóstico por imagem , Humanos , Luxações Articulares , Masculino , Pessoa de Meia-Idade , Radiografia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Obtaining patient-reported outcomes (PROs) is becoming a standard component of patient care. For nonacademic practices, this can be challenging. From this perspective, we designed a nearly autonomous patient outcomes reporting system. We then conducted a prospective, cohort pilot study to assess the efficacy of the system. METHODS: We created an automated system to gather PROs. All operative patients for 4 surgeons in an upper-extremity private practice were asked to participate. These patients completed the Quick Disabilities of the Arm, Shoulder, and Hand (QuickDASH) questionnaires preoperatively and received follow-up e-mails requesting patients to complete additional QuickDASH questionnaires at 3, 6, and 12 weeks postoperatively and to complete a 13-week postoperative satisfaction survey. Response rates and satisfaction levels are reported with descriptive statistics. RESULTS: Sixty-two percent of participants completed the 3-week assessment, 55% completed the 6-week assessment, and 43% completed the 12-week assessment. Overall, 35% of patients completed all questionnaires, and 73% completed at least 1 postoperative assessment. The collection of follow-up questionnaires required no additional time from the clinical staff, surgeon, or a research associate. CONCLUSIONS: Automated e-mail assessments can collect reliable clinical data, with minimal surgeon or staff intervention required to administer and collect data, minimizing the financial cost. For nonacademic practices, without access to additional research resources, such a system is feasible. Further improvements in communication with patients could increase response rates.
Assuntos
Avaliação da Deficiência , Mãos , Humanos , Projetos Piloto , Mãos/cirurgia , Estudos Prospectivos , Inquéritos e QuestionáriosRESUMO
PURPOSE: Sensibility testing plays a role in the diagnosis of carpal tunnel syndrome (CTS). No single physical examination test has proven to be of critical value in the diagnosis, especially when compared with electrodiagnostic testing (EDX). The purpose of this study was to define which digits are most affected by CTS, both subjectively and with objective sensibility testing. METHODS: A prospective series of 35 patients (40 hands) with EDX-positive, isolated CTS were evaluated preoperatively using 2 objective sensibility tests: static 2-point discrimination (2PD) and abbreviated Semmes-Weinstein monofilament (SWMF) testing. Detailed surveys of subjective symptoms were also collected. RESULTS: Patients identified the middle finger as the most symptomatic over all others (51%). Objective 2PD results of each digit mirrored the subjective data, with higher values for the middle finger (mean 6.07 mm, (p < .0001). Values for the index finger failed to show a significant difference from the ulnar-innervated small finger. The most symptomatic finger matched 2PD results in over two thirds of patients. The SWMF testing showed similar, statistically significant results (middle > thumb > index > small). Correlations failed between EDX, symptoms, and SWMF results or 2PD in the index finger. Positive but weak correlation (p = .002, r = .42) was found between EDX and 2PD only in the middle fingers. CONCLUSIONS: The middle finger is the most likely to show changes in 2PD in patients with positive EDX findings for CTS. Middle finger 2PD is best able to correlate with EDX when compared with 2PD of other digits. The SWMF testing also shows the middle digit testing as more sensitive, but this finding may be difficult to use clinically. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic I.
Assuntos
Síndrome do Túnel Carpal/diagnóstico , Eletromiografia/métodos , Dedos/inervação , Exame Físico/métodos , Adulto , Idoso , Síndrome do Túnel Carpal/cirurgia , Estudos de Coortes , Descompressão Cirúrgica/métodos , Técnicas de Diagnóstico Neurológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Limiar da Dor , Cuidados Pós-Operatórios/métodos , Cuidados Pré-Operatórios , Estudos Prospectivos , Recuperação de Função Fisiológica , Medição de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Thioxoamide (thioamide) bonds are nearly isosteric substitutions for amides but have altered hydrogen-bonding and photophysical properties. They are thus well-suited backbone modifications for physicochemical studies on peptides and proteins. The effect of thioxoamides on protein structure and stability has not been subject to detailed experimental investigations up to date. We used alanine-based model peptides to test the influence of single thioxoamide bonds on alpha-helix structure and stability. The results from circular dichroism measurements show that thioxoamides are strongly helix-destabilizing. The effect of an oxo-to-thioxoamide backbone substitution is of similar magnitude as an alanine-to-glycine substitution resulting in a helix destabilization of about 7 kJ/mol. NMR characterization of a helical peptide with a thioxopeptide bond near the N-terminus indicates that the thioxopeptide moiety is tolerated in helical structures. The thioxoamide group is engaged in an i, i+4 hydrogen bond, arguing against the formation of a 3(10)-helical structure as suggested for the N-termini of alpha-helices in general and for thioxopeptides in particular.
Assuntos
Peptídeos/química , Tioamidas/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Atypical mycobacterial infections of upper extremity synovial-lined structures are often misdiagnosed and unrecognized. Despite an increasing incidence, lack of physician awareness of these pathogens may result in considerable delay in diagnosis and management, potentially leading to permanent disability. The authors conducted a literature review and analyzed 31 cases of penetrating atypical mycobacterial infection to better understand the clinical characteristics and to evaluate their posttreatment complication rate compared with available literature. Medical records for culture-positive cases of tenosynovial or intra-articular atypical mycobacterial infections of the upper extremity that were treated were retrospectively reviewed. Treatment outcomes were analyzed against published case reviews and case series. Thirty-one cases of penetrating atypical mycobacterial infection were identified. Mycobacterium marinum (n=11) was the most common organism and was associated with aquatic exposure. Twenty-eight cases received empiric treatment, 17 of which received contraindicated treatment. Patients saw an average of 5 physicians prior to receiving an accurate diagnosis, and the mean time to diagnosis was 10 months. All cases received antibiotic treatment in addition to surgical management. Twenty cases (68%) failed treatment outcomes. Delay in diagnosis and inappropriate management of atypical mycobacterial infections may lead to a treatment failure rate that is higher than what has been reported in the literature. Mycobacterium avium and M fortuitum had significantly higher failure rates than other organisms. A high index of suspicion is required to make a diagnosis and prevent residual disability. [Orthopedics. 2018; 41(3):e383-e388.].
Assuntos
Artrite Infecciosa , Infecções por Mycobacterium não Tuberculosas , Tenossinovite , Antibacterianos/uso terapêutico , Artrite Infecciosa/complicações , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/microbiologia , Artrite Infecciosa/terapia , Terapia Combinada , Feminino , Seguimentos , Humanos , Masculino , Infecções por Mycobacterium não Tuberculosas/complicações , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/terapia , Procedimentos Ortopédicos , Estudos Retrospectivos , Tenossinovite/complicações , Tenossinovite/diagnóstico , Tenossinovite/microbiologia , Tenossinovite/terapia , Resultado do Tratamento , Extremidade SuperiorRESUMO
The characterization of the free energy barriers has been a major goal in studies on the mechanism of protein folding. Testing the effect of mutations or denaturants on protein folding reactions revealed that transition state movement is rare, suggesting that folding barriers are robust and narrow maxima on the free energy landscape. Here we demonstrate that the application of multiple perturbations allows the observation of small transition state movements that escape detection in single perturbation experiments. We used tendamistat as a model protein to test the broadness of the free energy barriers. Tendamistat folds over two consecutive transition states and through a high-energy intermediate. Measuring the combined effect of temperature and denaturant on the position of the transition state in the wild-type protein and in several mutants revealed that the early transition state shows significant transition state movement. Its accessible surface area state becomes more native-like with destabilization of the native state by temperature. To the same extent, the entropy of the early transition state becomes more native-like with increasing denaturant concentration, in accordance with Hammond behavior. The position of the late transition state, in contrast, is much less sensitive to the applied perturbations. These results suggest that the barriers in protein folding become increasingly narrow as the folding polypeptide chain approaches the native state.
Assuntos
Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Peptídeos/genética , Peptídeos/metabolismo , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
Intrachain diffusion processes play an important role in protein folding and function. In this chapter we discuss the application of triplet-triplet energy transfer to directly measure rate constants for intrachain contact formation in polypeptide chains. The photochemistry of triplet-triplet energy transfer is described, experimental prerequisites of the method are discussed, and a detailed description of the experimental protocols and data analysis is given.
Assuntos
Modelos Moleculares , Peptídeos/química , Fotoquímica/métodos , Conformação Proteica , Dobramento de Proteína , Aminoácidos/química , Cinética , Xantonas/químicaRESUMO
The structural characterization of transition states is essential for understanding the mechanism of protein folding. Analyzing the effect of mutations on protein stability and folding kinetics in phi-value analysis is commonly used to gain information about the presence of side-chain interactions in transition states. Recently, specific binding of ligands to engineered binding sites was applied to monitor the formation of local structures in transition states (psi analysis). A surprising result from psi analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between phi and psi values measured in the same protein. Here, we show that psi values cannot be analyzed in the same way as other rate-equilibrium free energy relationships due to the involvement of bimolecular reactions that may have different dissociation constants for the native, unfolded and transition state. As a consequence, psi values reflect the relative binding energy (kappa) of the transition state only for the extreme values of kappa=0 or kappa=1. In all other cases, non-linear rate-equilibrium free-energy relationships (Leffler plots) are observed. This apparently indicates the presence of parallel folding pathways even if folding occurs over a single homogeneous transition state. Consequently, the results from Leffler plots do not yield information about the structural properties of the transition state. This explains the lack of agreement between results from psi analysis and other methods used to characterize protein folding transition states. We further show that the same considerations apply for the analysis of the effect of pH on protein folding.
Assuntos
Dobramento de Proteína , Proteínas/química , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Químicos , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , TermodinâmicaRESUMO
Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.
Assuntos
Guanidina/química , Peptídeos/química , Conformação Proteica , Ureia/química , Estrutura Molecular , Peptídeos/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Viscosidade , Xantonas/química , Xantonas/metabolismoRESUMO
Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.