RESUMEN
Purpose: The American Society for Surgery of the Hand developed the Surgery Training and Educational Platform (STEP) in order to assess essential skills in hand surgery. The American Society for Surgery of the Hand designed modules spanning both osseous and soft tissue skills aimed to be cost effective for the purpose of orthopedic surgical education. The STEP curriculum was adapted and implemented at a single military orthopedic residency program. Methods: The following six modules were implemented: (1) depth of plunge, (2) scaphoid pinning, (3) phalangeal fracture pinning, (4) microsurgery, (5) full-thickness skin graft harvest, and (6) wrist arthroscopy. Both first- (PGY1) and second-year (PGY2) residents participated. Scores were calculated according to the original STEP curriculum criteria and were compared with historic data from the previous year. All residents responded to an evaluation questionnaire following the performance of the tasks. Results: The PGY2 cohort outperformed PGY1 cohorts across all modules except for the depth of plunge and scaphoid fixation modules. In the phalangeal pinning module, PGY2s did significantly better when compared with pooled PGY1 performance and their own PGY1 performance (P < .05). In the microsurgery module, PGY2s scored better than pooled PGY1s. In the full-thickness skin grafting module, PGY2s outperformed PGY1s (P < .05). On the post-task evaluation, residents unanimously responded that this was a valuable exercise, but the time required to complete all the modules was significant, similar to that of the previous year survey. Conclusions: The STEP simulation is a cost effective and reliable program to engage residents in hand surgery-related skills. However, adaptations should be encouraged according to institutional resources to provide the most inclusive training platform possible per institutional constraints. The STEP simulation is interpreted by residents as a valuable exercise but requires a significant time commitment that could be a barrier to implementation and regular use. Type of study/level of evidence: Therapeutic IV.
RESUMEN
OBJECTIVES: Tibiotalocalcaneal (TTC) nailing in the setting of acute ankle trauma has become increasingly popular. No consensus exists as to whether formal joint preparation is necessary, although there is some concern that residual motion at unprepared joints may lead to implant loosening and/or breakage. The objective of this study was to quantify the proportion of tibiotalar and subtalar articular surface destruction that occurs during reaming for TTC nail fixation. METHODS: Twelve cadaver lower extremities were procured. The specimens were pinned into neutral ankle and hindfoot alignment. A guidewire was inserted under fluoroscopy, followed by a 12-mm opening reamer. The specimens were then dissected, exposing the tibial plafond, talar dome, posterior facet of the talus, and posterior facet of the calcaneus. Images of each joint were obtained, and ImageJ software was used to calculate the total joint surface area and the area of articular destruction. RESULTS: The mean proportion of articular cartilage destruction was 9.3%, 10.3%, 8.9%, and 10.3% for the tibial plafond, talar dome, posterior facets of the talus, and posterior facets of the calcaneus, respectively. No joint destruction was observed in the middle facets of the subtalar joint. CONCLUSIONS: Reaming for TTC nail placement violates approximately 10% of each articular surface of the tibiotalar and subtalar joints. Retention of 90% of the articular surface may allow for residual motion at the joints and therefore potentially substantial stress on the implant. Formal joint preparation for the purposes of achieving fusion during TTC nail placement may be beneficial to prevent implant loosening or breakage.
Asunto(s)
Calcáneo , Fijación Intramedular de Fracturas , Artropatías , Articulación Talocalcánea , Humanos , Tobillo , Articulación Talocalcánea/cirugía , Artrodesis/métodos , Articulación del Tobillo/diagnóstico por imagen , Articulación del Tobillo/cirugía , Extremidad Inferior , Clavos Ortopédicos , Calcáneo/cirugíaRESUMEN
OBJECTIVE: Herniated nucleus pulposus (HNP) is one of the most common lumbar spine conditions treated surgically, often through a minimally invasive surgery (MIS) microdiscectomy approach. This technique attempts to reduce damage to the paraspinal muscular-ligamentous envelope. However, there are currently limited data regarding comparative outcomes using patient-reported outcome measures (PROMs) for one- and two-level MIS discectomies. The aim of this study was to quantify comparative clinical outcomes in patients undergoing one-level and two-level MIS lumbar microdiscectomy for HNP using PROMs. METHODS: The authors performed a retrospective review of patients undergoing MIS lumbar microdiscectomy between 2004 and 2019 for the primary diagnosis of HNP at a single academic institution. All patients had a minimum 1-year follow-up. Patient demographics and comorbidities were collected to establish baselines between cohorts. PROMs and minimal clinically important differences (MCIDs) were used to examine the patient's perception of operative success. Bivariate and multivariate linear/logistic regression analyses were used to compare one- and two-level discectomies. The bivariate analysis included the t-test and chi-square test, which were used to assess continuous and categorical variables, respectively. Statistical significance was established at p < 0.05. RESULTS: A total of 293 patients underwent one-level (n = 250) or two-level (n = 43) MIS discectomies. The mean follow-ups for the one- and two-level cohorts were 50.4 (SD 35.5) months and 61.6 (SD 39.8) months, respectively. Fewer female patients underwent two-level discectomies, and BMI and operative duration were higher in the two-level group (p < 0.001). Recurrent herniation requiring reoperation was recorded at rates of 6.80% and 11.6% in the one- and two-level groups, respectively (p = 0.270). Pre- and postoperative PROMs were largely similar between the cohorts; however, patients undergoing one-level discectomy had greater improvement in leg pain, and a significantly greater proportion of these patients achieved MCID for the leg pain visual analog scale score (p < 0.001). CONCLUSIONS: At the 1-year clinical follow-up, patients who underwent two-level discectomy had significantly less improvement in leg pain scores with lower achievement of MCID for leg pain improvement than patients undergoing one-level procedures. At the 1-year follow-up, there were no other significant differences in PROMs between the two cohorts. Given these findings, patients should be counseled regarding the anticipated outcomes to better manage expectations. Further studies are warranted to examine the long-term clinical outcomes associated with single- and multilevel MIS discectomy.
Asunto(s)
Discectomía , Desplazamiento del Disco Intervertebral , Humanos , Femenino , Resultado del Tratamiento , Discectomía/métodos , Desplazamiento del Disco Intervertebral/cirugía , Desplazamiento del Disco Intervertebral/etiología , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Dolor/cirugía , Vértebras Lumbares/cirugía , Estudios RetrospectivosRESUMEN
INTRODUCTION: Resorption after lumbar disk herniation is a common yet unpredictable finding. It is hypothesized that nearly 70% of lumbar herniated nucleus pulposus (HNP) undergo the resorption to a significant degree after acute herniation, which has led to nonoperative management before surgical planning. METHODS: This narrative review on the literature from 4 databases (MEDLINE, Cumulative Index to Nursing and Allied Health Literature, Scopus, and Cochrane) examines historical and recent advancements related to disk resorption. Studies were appraised for their description of the predictive factor (e.g., imaging or morphologic factors), pathophysiology, and treatment recommendations. OBSERVATIONS: We reviewed 68 articles considering the possibility of resorption of lumbar HNP. Recent literature has proposed various mechanisms (inflammation and neovascularization, dehydration, and mechanical traction) of lumbar disk resorption; however, consensus has yet to be established. Current factors that increase the likelihood of resorption include the initial size of the herniation, sequestration, percentage of rim enhancement on initial gadolinium-based magnetic resonance imaging (MRI), composition of inflammatory mediators, and involvement of the posterior longitudinal ligament. CONCLUSION: Heterogeneity in imaging and morphologic factors has led to uncertainty in the identification of which lumbar herniations will resorb. Current factors that increase the likelihood of disk resorption include the initial size of the herniation, sequestration, percentage of rim enhancement on initial MRI, composition of cellular and inflammatory mediators present, and involvement of the posterior longitudinal ligament. This review article highlights the role of disk resorption after herniation without surgical intervention and questions the role of traditional noninflammatory medications after acute herniation. Further research is warranted to refine the ideal patient profile for disk resorption to ultimately avoid unnecessary treatment, thus individualizing patient care.
Asunto(s)
Desplazamiento del Disco Intervertebral , Humanos , Desplazamiento del Disco Intervertebral/diagnóstico por imagen , Desplazamiento del Disco Intervertebral/terapia , Desplazamiento del Disco Intervertebral/patología , Imagen por Resonancia Magnética/métodos , Región LumbosacraRESUMEN
Modic changes (MC) and endplate abnormalities (EA) have been shown to impact preoperative symptoms and outcomes following spinal surgery. However, little is known about how these phenotypes impact cervical alignment. This study aimed to evaluate the impact that these phenotypes have on preoperative, postoperative, and changes in cervical alignment in patients undergoing anterior cervical discectomy and fusion (ACDF). We performed a retrospective study of prospectively collected data of ACDF patients at a single institution. Preoperative magnetic resonance imagings (MRIs) were used to assess for the MC and EA. Patients were subdivided into four groups: MC-only, EA-only, the combined Modic-Endplate-Complex (MEC), and patients without either phenotype. Pre and postoperative MRIs were used to assess alignment parameters. Associations with imaging phenotypes and alignment parameters were assessed, and statistical significance was set at p < 0.5. A total of 512 patients were included, with 84 MC-only patients, 166 EA-only patients, and 71 patients with MEC. Preoperative MC (p = 0.031) and the MEC (p = 0.039) had significantly lower preoperative T1 slope compared to controls. Lower preoperative T1 slope was a risk factor for MC (p = 0.020) and MEC (p = 0.029) and presence of MC (Type II) and the MEC (Type III) was predictive of lower preoperative T1 slope. There were no differences in postoperative alignment measures or patient reported outcome measures. MC and endplate pathologies such as the MEC appear to be associated with worse cervical alignment at baseline relative to patients without these phenotypes. Poor alignment may be an adaptive response to these degenerative findings or may be a risk factor for their development.
Asunto(s)
Vértebras Cervicales , Vértebras Cervicales/diagnóstico por imagen , Vértebras Cervicales/cirugía , Estudios Retrospectivos , Resultado del Tratamiento , Humanos , Imagen por Resonancia MagnéticaRESUMEN
A water-soluble thermochromic molecular switch with spectrally resolved fluorescence in its two interconvertible states can be assembled in three synthetic steps by integrating a fluorescent coumarin chromophore, a hydrophilic oligo(ethylene glycol) chain, and a switchable oxazole heterocycle in the same covalent skeleton. Measurements of its two emissions in separate detection channels of a fluorescence microscope permit the noninvasive and ratiometric sensing of temperature at the micrometer level with millisecond response in aqueous solutions and within hydrogel matrices. The ratiometric optical output of this fluorescent molecular switch overcomes the limitations of single-wavelength fluorescent probes and enables noninvasive temperature mapping at length scales that are not accessible to conventional thermometers based on physical contact.
Asunto(s)
Colorantes Fluorescentes , Agua , Fluorescencia , Espectrometría de Fluorescencia , Temperatura , TermómetrosRESUMEN
This study describes a novel, combined Modic changes (MC) and structural endplate abnormality phenotype of the cervical spine, which we have termed the Modic-Endplate-Complex (MEC), and its association with preoperative symptoms and outcomes in anterior cervical discectomy and fusion (ACDF) patients. This was a retrospective study of prospectively collected data at a single institution. Preoperative cervical magnetic resonance imagings were used to assess the presence of MC and endplate abnormalities. Patients were divided into four groups: MC-only, endplate abnormality-only, the MEC and controls. The MEC was defined as the presence of both a MC and endplate abnormality in the cervical spine. Phenotypes were further stratified by location and compared to controls. Associations with patient-reported outcome measures were assessed using regression controlling for baseline characteristics. A total of 628 patients were included, with 84 MC-only, 166 endplate abnormality-only, and 187 MEC patients. Both MC (p < 0.001) and endplate abnormalities (p < 0.001) were independently associated with one another. MC at the adjacent level (p = 0.018), endplate abnormalities (regardless of location) (p = 0.001), and the MEC within the fusion segment (p = 0.027) were all associated with higher Neck Disability Index scores. Both MC within the fusion segment (p = 0.008) and endplate abnormalities within the fusion segment (p = 0.017) associated with lower Veteran's Rand 12-item scores. MC and structural endplate abnormalities commonly manifest concomitantly in patients indicated for ACDF for degenerative pathology. Patients with the endplate pathology, including the MEC phenotype, reported significantly higher levels of postoperative disability following ACDF. These findings add valuable data to the prognostic assessment of degenerative cervical spine patients.
Asunto(s)
Degeneración del Disco Intervertebral , Fusión Vertebral , Vértebras Cervicales/diagnóstico por imagen , Vértebras Cervicales/cirugía , Discectomía , Humanos , Degeneración del Disco Intervertebral/diagnóstico por imagen , Degeneración del Disco Intervertebral/cirugía , Fenotipo , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: YouTube has become a popular source for patient education, though there are concerns regarding the quality and reliability of videos related to orthopaedic and neurosurgical procedures. This study aims to evaluate the credibility and educational content of videos on YouTube related to cervical fusion. Secondarily, the study aims to identify factors predictive of higher or lower quality videos. METHODS: A YouTube query using the search terms "cervical fusion" was performed, and the first 50 videos were included for analysis. Reliability was assessed using the Journal of the American Medical Association (JAMA) criteria. Educational quality was assessed using the Global Quality Score (GQS) and the Cervical Fusion Content Score (CFCS). Videos were stratified by content and source, and differences in JAMA, GQS, and CFCS scores were assessed. Multivariable linear regression was used to identify predictors of higher or lower JAMA, GQS, and CFCS scores. Statistical significance was established at P < 0.05. RESULTS: Total number of views was 6â221 816 with a mean of 124â436.32 ± 412â883.32 views per video. Physicians, academic, and medical sources had significantly higher mean JAMA scores (P = 0.042). Exercise training and nonsurgical management videos had significantly higher mean CFCS scores (P = 0.018). Videos by physicians (ß = 0.616; P = 0.025) were independently associated with higher JAMA scores. Advertisements were significant predictors of worse CFCS (ß = -3.978; P = 0.030), and videos by commercial sources predicted significantly lower JAMA scores (ß = -1.326; P = 0.006). CONCLUSIONS: While videos related to cervical fusion amassed a large viewership, they were poor in both quality and reliability. Videos by physicians were associated with higher reliability scores relative to other sources, whereas commercial sources and advertisements had significantly lower reliability and educational content scores. Currently, YouTube seems to be an unreliable source of information on cervical fusion for patients. LEVEL OF EVIDENCE: 4. CLINICAL RELEVANCE: The results of this study aid surgeons in counseling patients interested in cervical fusion, and suggest that publicly available videos regarding cervical fusion may not be an adequate tool for patient education at this time.
RESUMEN
We detail the preparation of highly fluorescent quantum dots (QDs), surface-engineered with multifunctional polymer ligands that are compact and readily compatible with strain-promoted click conjugation, and the use of these nanocrystals in immunofluorescence and in vivo imaging. The ligand design combines the benefits of mixed coordination (i.e., thiol and imidazole) with zwitterion motifs, yielding sterically-stabilized QDs that present a controllable number of azide groups, for easy conjugation to biomolecules via the selective click chemistry. The polymer coating was characterized using NMR spectroscopy to extract estimates of the diffusion coefficient, hydrodynamic size, and ligand density. The azide-functionalized QDs were conjugated to anti-tropomyosin receptor kinase B antibody (α-TrkB) or to the brain-derived neurotrophic factor (BDNF). These conjugates were highly effective for labeling the tropomyosin receptor kinase B (TrkB) in pyramidal neurons within cortical tissue and for monitoring the BDNF induced activation of TrkB signaling in live neuronal cells. Finally, the polymer-coated QDs were applied for in vivo imaging of Drosophila melanogaster embryos, where the QDs remained highly fluorescent and colloidally stable, with no measurable cytotoxicity. These materials would be of great use in various imaging applications, where a small size, ease of conjugation, and great colloidal stability for in vivo studies are needed.
Asunto(s)
Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Polímeros/química , Puntos Cuánticos/química , Animales , Azidas/química , Línea Celular , Química Clic , Drosophila melanogaster/embriología , Imidazoles/química , Ligandos , Neuronas/citología , Tamaño de la Partícula , Transducción de Señal , Compuestos de Sulfhidrilo/químicaRESUMEN
OBJECTIVE: To assess the impact of Modic changes (MC) on preoperative symptoms, and postoperative outcomes in anterior cervical discectomy and fusion (ACDF) patients. METHODS: We performed a retrospective study of prospectively collected data of ACDF patients at a single institution. Preoperative magnetic resonance imagings were used to assess the presence of MC. MC were stratified by type and location, and compared to patients without MC. Associations with symptoms, patient-reported measures, and surgical outcomes were assessed. RESULTS: A total of 861 patients were included, with 356 patients with MC (41.3%). MC more frequently occurred at C5-6 (15.1%), and type II was the most common type (61.2%). MC were associated with advanced age (p < 0.001), more levels fused (p < 0.001), a longer duration of symptoms, but not with specific symptoms. MC at C7-T1 resulted in higher postoperative disability (p < 0.001), but did not increase risk of adjacent segment degeneration or reoperation. CONCLUSION: This study is the first to systematically examine the impact of cervical MC, stratified by type and location, on outcomes in ACDF patients. Patients with MC were generally older, required larger fusions, and had longer duration of preoperative symptoms. While MC may not affect specific outcomes following ACDF, they may indicate a more debilitating preoperative state for patients.
RESUMEN
Objectives: 1) To evaluate return to play (RTP) timing in National Basketball Association (NBA) athletes following adductor injuries, and 2) to evaluate the effect of adductor injuries on player performance, game availability, and career longevity following RTP. Methods: Adductor injuries in NBA athletes from the 2009-2010 to 2018-2019 seasons were identified utilizing publicly available records via previously validated methodology. RTP time was calculated, and player performance and game availability were compared pre- vs. post-injury. Additionally, an injury-free control group matched for age, BMI, position, and experience was assembled to allow for comparisons in performance, availability, and career length. Results: In total, 79 adductor injuries across 65 NBA athletes were identified. The average injured player was 28.3 ± 4.0 years of age, and had 6.5 ± 4.2 seasons of NBA experience. Guards were injured more frequently than forwards or centers (49% vs 25% vs 25%, respectively). All players were able to RTP following first-time adductor injury after missing an average of 7.7 ± 9.8 games (median [IQR]: 4 [1-9]) and 16.9 ± 20.4 days (median [IQR]: 9 [3.5-20]). Twelve players (18.5%) suffered an adductor re-injury at a mean latency of 509.5 ± 503.9 days. Adductor injuries did not result in significant changes in any major statistical category (points, assists, rebounds, steals, blocks, turnovers, field goal percentage), player efficiency rating (PER), minutes/game, games/season, or a number of all-star selections (all P > 0.05) following RTP. Additionally, when compared to matched controls, no difference was found in pre- to post-injury change of PER, games/season, or minutes/game (all P > 0.05). Career longevity was not significantly different between groups (P = 0.44). Conclusion: Following adductor injury, NBA players returned to gameplay after missing an average of 16 to 17 days, or 7 to 8 games. Adductor injury did not affect player performance, nor game availability or career longevity.
Asunto(s)
Rendimiento Atlético , Baloncesto/lesiones , Músculo Esquelético/lesiones , Volver al Deporte , Muslo/lesiones , Adulto , Humanos , Masculino , Lesiones de Repetición , Factores de Tiempo , Adulto JovenRESUMEN
HYPOTHESIS: The purpose of this study was to correlate the Patient-Reported Outcomes Measurement Information System (PROMIS) computer adaptive testing (CAT) domains with commonly used legacy patient-reported outcome measures (PROMs) preoperatively in a population of patients presenting to a tertiary care orthopedic center for biceps and labral pathology. METHODS: Prospective data were collected on 175 patients scheduled to undergo isolated biceps tenodesis at a tertiary center. Enrollees completed legacy scores (Veterans RAND 12-Item Health Survey, Short Form 12, American Shoulder and Elbow Surgeons [ASES] Assessment Form, Single Assessment Numeric Evaluation, and Constant-Murley score) and PROMIS CAT questionnaires (Upper Extremity [UE], Pain Interference [PIF], and Depression). In addition, patients were asked to provide an assessment of the strength, function, and pain of the affected shoulder using a custom visual analog scale (VAS) questionnaire. Spearman rank correlations of the PROMIS CAT with legacy measures and the custom VAS were calculated. Floor and ceiling effects were assessed. RESULTS: The UE CAT yielded moderate correlations with the ASES score (r = 0.57) and the custom VAS strength score (r = 0.50-0.57). The PIF CAT demonstrated moderate correlations with the VAS pain score (r = 0.45) and high-moderate correlations with the ASES score, VR6D score, and UE CAT (r = 0.61-0.66). The Depression CAT demonstrated high-moderate correlations with the mental health legacy measures (r = -0.64 to -0.61). There was a significant relative floor effect for the Depression CAT at a score of 34.2 (19%). CONCLUSION: The PIF CAT was comparable to the ASES score, the current gold standard. Compared with legacy measures, both the UE CAT and PIF CAT are less burdensome and have few floor or ceiling effects. The PIF CAT may be a viable alternative to describe the physical and psychosocial impact of pain in biceps tenodesis patients.
Asunto(s)
Diagnóstico por Computador , Medición de Resultados Informados por el Paciente , Tendinopatía/cirugía , Tenodesis , Extremidad Superior , Adulto , Artroscopía , Evaluación de la Discapacidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético , Dimensión del Dolor , Estudios Prospectivos , Reproducibilidad de los Resultados , Encuestas y Cuestionarios , Tendinopatía/etiología , Resultado del TratamientoRESUMEN
HYPOTHESIS: We hypothesized that players in the National Basketball Association (NBA) who sustained a shoulder destabilizing injury could return to play (RTP) successfully at a high rate regardless of treatment type. METHODS: We used publicly available data to identify and evaluate 50 players who sustained an in-season shoulder instability event (subluxation/dislocation) while playing in the NBA. Demographic variables, return to NBA gameplay, incidence of surgery, time to RTP, recurrent instability events, and player efficiency rating (PER) were collected. Overall RTP was determined, and players were compared by type of injury and mode of treatment. RESULTS: All players (50/50) returned to game play after sustaining a shoulder instability event. In those treated nonoperatively, athletes who sustained shoulder subluxations returned after an average of 3.6 weeks, compared with 7.6 weeks in those who sustained a shoulder dislocation (P = .037). Players who underwent operative management returned after an average of 19 weeks. Athletes treated operatively were found to have a longer time interval between a recurrent instability event (70 weeks vs. 28.5 weeks, P = .001). CONCLUSION: We found 100% rate of RTP after a shoulder instability event in an NBA athlete. Players who experience shoulder dislocations were found to miss more time before RTP and were more likely to undergo surgical intervention compared with those who experienced a subluxation. Surgical repair maintained a longer interval between recurrent instability. Future investigations should aim to evaluate outcomes based on surgical procedures and identify possible risk factors predictive of recurrent instability or failure to RTP.
Asunto(s)
Baloncesto/lesiones , Tratamiento Conservador , Inestabilidad de la Articulación/cirugía , Volver al Deporte , Luxación del Hombro/cirugía , Lesiones del Hombro/cirugía , Adulto , Rendimiento Atlético , Humanos , Inestabilidad de la Articulación/terapia , Masculino , Recurrencia , Luxación del Hombro/terapia , Lesiones del Hombro/terapia , Factores de Tiempo , Adulto JovenRESUMEN
Light discrimination according to colour can confer survival advantages by guiding animals towards food and shelter and away from potentially harmful situations1,2. Such colour-dependent behaviour can be learned or innate. Data on innate colour preference in mammals remain controversial3 and there are limited data for simpler organisms4-7. Here we show that, when given a choice among blue, green and dim light, fruit flies exhibit an unexpectedly complex pattern of colour preference that changes according to the time of day. Flies show a strong preference for green in the early morning and late afternoon, a reduced green preference at midday and a robust avoidance of blue throughout the day. Genetic manipulations reveal that the peaks in green preference require rhodopsin-based visual photoreceptors and are controlled by the circadian clock. The midday reduction in green preference in favour of dim light depends on the transient receptor potential (TRP) channels dTRPA1 and Pyrexia, and is also timed by the clock. By contrast, avoidance of blue light is primarily mediated by multidendritic neurons, requires rhodopsin 7 and the TRP channel Painless, and is independent of the clock. Our findings show that several TRP channels are involved in colour-driven behaviour in Drosophila, and reveal distinct pathways of innate colour preference that coordinate the behavioural dynamics of flies in ambient light.
Asunto(s)
Relojes Circadianos/fisiología , Relojes Circadianos/efectos de la radiación , Color , Drosophila melanogaster/fisiología , Drosophila melanogaster/efectos de la radiación , Luz , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Antenas de Artrópodos/fisiología , Antenas de Artrópodos/efectos de la radiación , Dendritas/fisiología , Dendritas/efectos de la radiación , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Larva/fisiología , Larva/efectos de la radiación , Luz/efectos adversos , Masculino , Neuronas/fisiología , Neuronas/efectos de la radiación , Rodopsinas Sensoriales/metabolismo , Factores de Tiempo , Visión Ocular/efectos de la radiaciónRESUMEN
The connection of fluorescent chromophores to switchable heterocycles translates into molecular probes with ratiometric response to temperature. The opening and closing of their heterocyclic component equilibrates two emissive species with resolved fluorescence. Their relative emission intensities change monotonically with temperature to enable the visualization of thermal distributions at the microscale.
RESUMEN
A mechanism to photoactivate far-red/near-infrared fluorescence with infinite contrast and under mild visible illumination was designed around the photophysical properties of borondipyrromethene (BODIPY) dyes and the photochemical behavior of oxazine heterocycles. Specifically, the photoinduced and irreversible cleavage of an oxazine ring with a laser line at 405 nm extends the electronic conjugation of a BODIPY chromophore over a 3 H-indole auxochrome with a 2-(4-methoxyphenyl)ethenyl substituent in position 5. This structural transformation shifts bathochromically the main absorption band of the BODIPY component to allow the selective excitation of the photochemical product with a laser line of 633 nm and produce fluorescence between 600 and 850 nm. This combination of activation, excitation, and emission wavelengths permits the visualization of the cellular blastoderm of developing Drosophila melanogaster embryos with optimal contrast and essentially no autofluorescence from the biological specimen. Furthermore, the sequential acquisition of images, after the photoactivation event, enables the tracking of individual cells within the embryos in real time. Thus, our structural design and operating principles for the photoactivation of far-red/near-infrared fluorescence can evolve into invaluable probes to monitor cellular dynamics in vivo.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Pirroles/química , Animales , Drosophila melanogaster/embriología , Rayos Infrarrojos , Modelos Moleculares , Oxazinas/química , FotólisisRESUMEN
Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Trastornos del Crecimiento/genética , Animales , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Modelos Animales , Pez Cebra/genéticaRESUMEN
Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. IMPORTANCE: Bacteriophages (phages), viruses that infect bacteria, are the planet's most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed "superspreaders," that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments.
Asunto(s)
Bacteriólisis , Colifagos/crecimiento & desarrollo , ADN Bacteriano/genética , Escherichia coli/virología , Transferencia de Gen Horizontal , Transformación Bacteriana , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Maryland , Plásmidos , WyomingRESUMEN
This study reports the synthesis of a photoactivatable fluorophore with optimal photochemical and photophysical properties for the real-time tracking of motion in vivo. The photoactivation mechanism designed into this particular compound permits the conversion of an emissive reactant into an emissive product with resolved fluorescence, under mild illumination conditions that are impossible to replicate with conventional switching schemes based on bleaching. Indeed, the supramolecular delivery of these photoswitchable probes into the cellular blastoderm of Drosophila melanogaster embryos allows the real-time visualization of translocating molecules with no detrimental effects on the developing organisms. Thus, this innovative mechanism for fluorescence photoactivation can evolve into a general chemical tool to monitor dynamic processes in living biological specimens.
Asunto(s)
Colorantes Fluorescentes/química , Animales , Drosophila melanogaster , Procesos FotoquímicosRESUMEN
Several new DNA-targeting probes that exhibit binding-induced 'turn on' fluorescence are presented. Two of the dyes, orange emissive 1, (E)-4-(4(-4-methylpiperazin-1-yl)phenyl)6-(4-(4-methylpi-perazin-1-yl)styryl)pyrimidin-2-ol), and red emissive 2, (E)-4-(4(-4-methyl-piperazin-1-yl)-phenyl)6-(4-(4-methylpiperazin-1-yl)styryl)-1,3-propanedionato-κO,κO']difluoroborane), are brightly fluorescent when bound to DNA, but are virtually non-fluorescent in aqueous solutions. Confocal fluorescence microscopy of live BT474, MCF7 and HEK293 cells demonstrates that both probes are cell permeable and rapidly accumulated intracellularly into cell nuclei and the cytosol. Taking advantage of their environmental sensitivity, these two pools of fluorophores are readily resolved into separate channels, and thus, a single dye allows two-color imaging of the nuclear and cytosolic compartments.