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Purpose: This study explores the sociodemographic, insurance coverage, and substance use differences among transgender and gender diverse (TGD) individuals currently using hormone therapy (HT) and those who have an interest in future HT use. Methods: We surveyed TGD individuals in Michigan in 2018 to examine sociodemographic, health insurance, and substance use differences between those who had used HT and those who were interested but had never accessed HT using logistic regression models. Results: Respondents (N = 536) were 80.1% White and 18.0% nonbinary. About two-thirds of the participants had ever used HT (65.7%). In multivariate analyses, nonbinary participants were much more likely to be interested in future HT use than transmasculine individuals (odds ratio [OR] = 6.91), yet no significant difference between transmasculine and transfeminine individuals was found. Black participants also had higher odds of interest in future HT use (OR = 8.79). Those who did not know if they had trans-specific insurance coverage (OR = 42.39) and those who had no trans-specific insurance coverage (OR = 4.50) were more likely to be in the future interest group compared with those who reported full trans care coverage. Those with a bachelor's degree were less likely to be in the future interest group than those with some college education or an associate's degree, as were heavy marijuana users. Conclusion: Nonbinary individuals may be interested in HT but lack access, and known health care disparities around race and socioeconomic status may also impact HT access. Standard and transparent insurance coverage for gender-affirming care is sorely needed.
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Purpose: Little is known about the prevalence and risks associated with transgender and gender diverse (TGD) persons' misuse of prescription pain relievers (PPRs). This study explores the relationship between PPR misuse and various sociodemographic identities and experiences of discrimination in health care among TGD adults. Methods: TGD participants (n=595) were recruited in 2018 to participate in a cross-sectional statewide trans health survey through convenience sampling. Chi-square tests of independence and logistic regressions were conducted to explore associations between sociodemographics and experiences of discrimination among persons who had ever misused PPRs, or who had misused PPRs in the past year. Results: Sociodemographics such as gender identity (odds ratio [OR]=0.44, p=0.01), race/ethnicity (OR=0.14, p<0.001), and sexual orientation influence TGD individuals likeliness of misusing PPRs (OR=0.40, p<0.001). Notably, those who were ever diagnosed with anxiety had a higher likeliness of having lifetime PPR misuse compared with those who were never diagnosed (OR=2.05, p=0.05), and those reporting past-year discrimination within the mental health care setting because of their gender identity were more than twice as likely to report past-year misuse than those who reported not experiencing it (OR=2.43, p=0.004). Conclusion: Certain subpopulations of TGD individuals may be at elevated risk of PPR misuse. It is imperative to acknowledge the impact of multimarginalized identities as well as differences across various identities and experiences within the TGD community while working to address non-PPR misuse.
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OBJECTIVE: Neuropathic pain is undertreated in children. Neurosurgical treatments of pediatric chronic pain are limited by the absence of both US Food and Drug Administration approval and pediatric-specific hardware, as well as weak referral patterns due to a lack of physician education. This study presents a single-institution retrospective case series of spinal cord stimulation (SCS) in children ≤ 19 years of age and a systematic review of SCS in children. The authors' findings may further validate the role of SCS as an effective treatment modality for varied neuropathic pain syndromes found in pediatric patients. METHODS: The study was a single-center, single-surgeon, retrospective case series of individuals treated between July 2017 and May 2022. The outcomes for pediatric patients with chronic neuropathic pain syndromes indicated by the multidisciplinary pain clinic for evaluation for SCS were cataloged. A systematic review and individual participant data (IPD) meta-analysis was performed for cases treated until May 2022, using PubMed, EMBASE, and Scopus to characterize outcomes of children with neuropathic pain treated with SCS. RESULTS: Twelve patients were evaluated and 9 were indicated for percutaneous or buried lead trials. Seven female and 2 male patients between the ages of 13 and 19 years were implanted with trial leads. Eight of 9 (89%) patients went on to receive permanent systems. The average trial length was 6 days, and the length of stay for both trial and implant was less than 1 day. Complication rates due to CSF leaks were 22% and 0% for trial and implant, respectively. Visual analog scale pain scores decreased from 9.2 to 2.9 (p = 0.0002) and the number of medications decreased from 4.9 to 2.1 (p = 0.0005). Functional status also improved for each patient. A systematic review identified 13 studies describing pediatric patients with SCS, including 12 providing IPD on 30 patients. In the IPD meta-analysis, pain was reduced in 16/16 (100%) of patients following surgery and in 25/26 (96.2%) at last follow-up. Medication use was decreased in 16/21 (76.2%), and functional outcomes were improved in 29/29 (100%). The complication rate was 5/30 (16.7%). CONCLUSIONS: SCS effectively decreases pain and medication use for pediatric neuropathic pain syndromes. Patients also report improved functional status, including improved matriculation, gainful employment, and physical activity. There is minimal high-quality literature describing neuromodulation for pain in children. Neuromodulation should be considered earlier as a viable alternative to escalating use of multiple drugs and as a potential mechanism to address tolerance, dependence, and addiction in pediatric patients.
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Dolor Crónico , Neuralgia , Estimulación de la Médula Espinal , Adolescente , Adulto , Niño , Dolor Crónico/terapia , Femenino , Humanos , Masculino , Neuralgia/terapia , Estudios Retrospectivos , Médula Espinal , Síndrome , Resultado del Tratamiento , Adulto JovenRESUMEN
Selective dorsal rhizotomy (SDR) has been a well-established neurosurgical treatment option for ambulatory children with spastic diplegic cerebral palsy to reduce spasticity. Outcomes for SDR for spastic lower extremity hemiparesis has been less well described. In our experience, hemi-SDR has been an excellent intervention for children with suboptimal spasticity control despite maximizing pharmacologic and chemodenervation treatments. In Video 1, we demonstrate a focal segmental hemi-SDR at the L5-S1 level in a 7-year-old male patient with spastic hemiparesis secondary to a dysembryoplastic neuroepithelial tumor in the right inferior frontoparietal area. Rhizotomy was performed with identification and selective sectioning of dorsal nerve roots with abnormal stimulation patterns as determined by electrophysiology and clinical correlation. Dorsal nerve root fibers with unsustained discharges were spared. Postoperatively, the patient participated well in inpatient and outpatient therapies with significant progress in his mobility and activities of daily living. The patient showed improvement in gait velocity (51%), internal pressure ratio (+0.05), and step length (41% on the left and 27% on the right) 20 months after hemi-SDR. He also demonstrated a step length ratio closer to 1 (0.89) showing a more equal step length bilaterally and improved weight acceptance on the affected side. There were no changes observed on the left upper extremity. This positive outcome on spasticity control and function supports the need for further prospective studies for hemi-SDR as a treatment option for children with spastic hemiparesis.
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Espasticidad Muscular , Rizotomía , Actividades Cotidianas , Niño , Análisis de la Marcha , Hemiplejía/etiología , Hemiplejía/cirugía , Humanos , Masculino , Espasticidad Muscular/etiología , Espasticidad Muscular/cirugía , Paresia/etiología , Paresia/cirugía , Estudios ProspectivosRESUMEN
PURPOSE: The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1ß, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients. PATIENTS AND METHODS: We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies. RESULTS: PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance. CONCLUSIONS: These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Carcinoma de Células Renales/tratamiento farmacológico , Indanos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Sulfonas/uso terapéutico , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Ensayos Clínicos Fase I como Asunto , Resistencia a Antineoplásicos , Femenino , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Imágenes de Resonancia Magnética Multiparamétrica/métodos , Estudios ProspectivosRESUMEN
Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Indanos/farmacología , Indanos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Transformación Celular Neoplásica , Resistencia a Antineoplásicos/efectos de los fármacos , Eritropoyetina/antagonistas & inhibidores , Eritropoyetina/sangre , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Indanos/administración & dosificación , Indoles/farmacología , Indoles/uso terapéutico , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Mutación , Pirroles/farmacología , Pirroles/uso terapéutico , Reproducibilidad de los Resultados , Sulfonas/administración & dosificación , Sunitinib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.
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Carcinoma de Células Renales/clasificación , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Mutación , Adenoma Oxifílico/clasificación , Adenoma Oxifílico/genética , Adenoma Oxifílico/patología , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , ADN de Neoplasias , Dosificación de Gen , Inestabilidad Genómica , Humanos , Neoplasias Renales/clasificación , Neoplasias Renales/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Polimorfismo de Nucleótido Simple , Conformación Proteica , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/genética , Translocación GenéticaRESUMEN
Women are more susceptible to multiple sclerosis (MS) and have more robust immune responses than men. However, men with MS tend to demonstrate a more progressive disease course than women, suggesting a disconnect between the severity of an immune attack and the CNS response to a given immune attack. We have previously shown in an MS model, experimental autoimmune encephalomyelitis, that autoantigen-sensitized XX lymph node cells, compared with XY, are more encephalitogenic. These studies demonstrated an effect of sex chromosomes in the induction of immune responses, but did not address a potential role of sex chromosomes in the CNS response to immune-mediated injury. Here, we examined this possibility using XX versus XY bone marrow chimeras reconstituted with a common immune system of one sex chromosomal type. We found that experimental autoimmune encephalomyelitis mice with an XY sex chromosome complement in the CNS, compared with XX, demonstrated greater clinical disease severity with more neuropathology in the spinal cord, cerebellum, and cerebral cortex. A candidate gene on the X chromosome, toll-like receptor 7, was then examined. Toll-like receptor 7 expression in cortical neurons was higher in mice with XY compared with mice with XX CNS, consistent with the known neurodegenerative role for toll-like receptor 7 in neurons. These results suggest that sex chromosome effects on neurodegeneration in the CNS run counter to effects on immune responses, and may bear relevance to the clinical enigma of greater MS susceptibility in women but faster disability progression in men. This is a demonstration of a direct effect of sex chromosome complement on neurodegeneration in a neurological disease.
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Sistema Nervioso Central/fisiopatología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica/genética , Degeneración Nerviosa/genética , Cromosomas Sexuales/genética , Análisis de Varianza , Animales , Trasplante de Médula Ósea , Femenino , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Masculino , Ratones , Degeneración Nerviosa/patología , Receptor Toll-Like 7/metabolismo , Quimera por TrasplanteRESUMEN
Estrogens can signal through either estrogen receptor α (ERα) or ß (ERß) to ameliorate experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of multiple sclerosis (MS). Cellular targets of estrogen-mediated neuroprotection are still being elucidated. Previously, we demonstrated that ERα on astrocytes, but not neurons, was critical for ERα ligand-mediated neuroprotection in EAE, including decreased T-cell and macrophage inflammation and decreased axonal loss. Here, we determined whether ERß on astrocytes or neurons could mediate neuroprotection in EAE, by selectively removing ERß from either of these cell types using Cre-loxP gene deletion. Our results demonstrated that, even though ERß ligand treatment was neuroprotective in EAE, this neuroprotection was not mediated through ERß on either astrocytes or neurons and did not involve a reduction in levels of CNS inflammation. Given the differential neuroprotective and anti-inflammatory effects mediated via ERα versus ERß on astrocytes, we looked for molecules within astrocytes that were affected by signaling through ERα, but not ERß. We found that ERα ligand treatment, but not ERß ligand treatment, decreased expression of the chemokines CCL2 and CCL7 by astrocytes in EAE. Together, our data show that neuroprotection in EAE mediated via ERß signaling does not require ERß on either astrocytes or neurons, whereas neuroprotection in EAE mediated via ERα signaling requires ERα on astrocytes and reduces astrocyte expression of proinflammatory chemokines. These findings reveal important cellular differences in the neuroprotective mechanisms of estrogen signaling through ERα and ERß in EAE.
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Antiinflamatorios no Esteroideos , Astrocitos/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/prevención & control , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor beta de Estrógeno/efectos de los fármacos , Estrógenos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Transducción de Señal/efectos de los fármacos , Animales , Acuaporina 4/fisiología , Axones/fisiología , Recuento de Células , Quimiocina CCL2/genética , Quimiocina CCL2/fisiología , Quimiocina CCL7/genética , Quimiocina CCL7/fisiología , Enfermedades Desmielinizantes/patología , Gliosis/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Médula Espinal/patologíaRESUMEN
We present an analysis of the key steps involved in the DNA-directed assembly of nanoparticles into crystallites and polycrystalline aggregates. Additionally, the rate of crystal growth as a function of increased DNA linker length, solution temperature, and self-complementary versus non-self-complementary DNA linker strands (1- versus 2-component systems) has been studied. The data show that the crystals grow via a 3-step process: an initial "random binding" phase resulting in disordered DNA-AuNP aggregates, followed by localized reorganization and subsequent growth of crystalline domain size, where the resulting crystals are well-ordered at all subsequent stages of growth.
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ADN/química , Oro/química , Nanopartículas del Metal/química , Secuencia de Bases , Coloides/química , Cristalización , Cinética , Modelos Químicos , Oligonucleótidos/química , Tamaño de la Partícula , Difracción de Rayos XRESUMEN
We chemically immobilized live, motile Escherichia coli on micrometer-scale, photocatalytically patterned silicon surfaces via amine- and carboxylic acid-based chemistries. Immobilization facilitated (i) controlled positioning; (ii) high resolution cell wall imaging via atomic force microscopy (AFM); and (iii) chemical analysis with time-of-flight-secondary ion mass spectrometry (ToF-SIMS). Spinning motion of tethered bacteria, captured with fast-acquisition video, proved microbe viability. We expect our protocols to open new experimental doors for basic and applied studies of microorganisms, from host-pathogen relationships, to microbial forensics and drug discovery, to biosensors and biofuel cell optimization.
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Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Escherichia coli/fisiología , Escherichia coli/ultraestructura , Silicio/químicaRESUMEN
We show that by correlating the radius of curvature of spherical gold nanoparticles of varying sizes with their respective thiol-terminated oligonucleotide loading densities, a mathematical relationship can be derived for predicting the loading of oligonucleotides on anisotropic gold nanomaterials. This mathematical relationship was tested with gold nanorods (radius 17.5 nm, length 475 nm) where the measured number of oligonucleotides per rod (3330 +/- 110) was within experimental error of the predicted loading of 3244 oligonucleotides from the derivation. Additionally, we show that once gold nanoparticles reach a diameter of approximately 60 nm the local surface experienced by the oligonucleotide is highly similar to that of a planar surface.
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Oro/química , Nanopartículas del Metal/química , Oligonucleótidos/química , Compuestos de Sulfhidrilo/química , Anisotropía , Modelos Moleculares , Conformación Molecular , Nanotubos/química , Tamaño de la Partícula , Sales (Química)/química , Propiedades de SuperficieRESUMEN
Experiments are presented that suggest DNA strands chemically immobilized on gold nanoparticle surfaces can engage in two types of hybridization: one that involves complementary strands and normal base pairing interactions and a second one assigned as a "slipping" interaction, which can additionally stabilize the aggregate structures through non-Watson-Crick type base pairing or interactions less complementary than the primary interaction. The curvature of the particles appears to be a major factor that contributes to the formation of these slipping interactions as evidenced by the observation that flat gold triangular nanoprism conjugates of the same sequence do not support them. Finally, these slipping interactions significantly stabilize nanoparticle aggregate structures, leading to large increases in T(m)'s and effective association constants as compared with free DNA and particles that do not have the appropriate sequence to maximize their contribution.
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Emparejamiento Base , ADN/química , ADN/ultraestructura , Hibridación in Situ/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Cristalización/métodos , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
We have determined the minimum number of base pairings necessary to stabilize DNA-Au NP aggregates as a function of salt concentration for particles between 15 and 150 nm in diameter. Significantly, we find that sequences containing a single base pair interaction are capable of effecting hybridization between 150 nm DNA-Au NPs. While traditional DNA hybridization involves two strands interacting in one dimension (1D, Z), we propose that hybridization in the context of an aggregate of polyvalent DNA-Au NP conjugates occurs in three dimensions (many oligonucleotides oriented perpendicular to the X, Y plane engage in base pairing), making nanoparticle assembly possible with three or fewer base pairings per DNA strand. These studies enabled us to compare the stability of duplex DNA free in solution and bound to the nanoparticle surface. We estimate that 4-8, 6-19, or 8-33 additional DNA bases must be added to free duplex DNA to achieve melting temperatures equivalent to hybridized systems formed from 15, 60, or 150 nm DNA-Au NPs, respectively. In addition, we estimate that the equilibrium binding constant (K(eq)) for 15 nm DNA-Au NPs (3 base pairs) is approximately 3 orders of magnitude higher than the K(eq) for the corresponding nanoparticle free system.
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ADN/química , Oro/química , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico/métodos , Emparejamiento Base , Temperatura de TransiciónRESUMEN
DNA-functionalized gold nanoparticles can be used to induce the formation and control the unit cell parameters of highly ordered face-centered cubic crystal lattices. Nanoparticle spacing increases linearly with longer DNA interconnect length, yielding maximum unit cell parameters of 77 nm and 0.52% inorganic-filled space for the DNA constructs studied. In general, we show that longer DNA connections result in a decrease in the overall crystallinity and order of the lattice due to greater conformational flexibility.
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Reactivos de Enlaces Cruzados/química , ADN/química , Oro/química , Nanopartículas del Metal/química , Secuencia de Bases , CristalizaciónRESUMEN
The detection of bacterial genomic DNA through a nonenzymatic nanomaterials-based amplification method, the bio bar code assay, is reported. The assay utilizes oligonucleotide-functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double-stranded DNA. These blockers bind to specific regions of the target DNA upon cooling and prevent the duplex DNA from rehybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide-functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (bar codes) act as amplification surrogates. The bar codes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 fM, and this is the first demonstration of a bar code-type assay for the detection of double-stranded, genomic DNA.
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ADN Bacteriano/análisis , Procesamiento Automatizado de Datos , Genoma Bacteriano , Bacillus subtilis/genética , Secuencia de Bases , Sondas de ADN , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.
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Luz , Nanopartículas del Metal/química , Ácidos Nucleicos/análisis , Plata/química , ADN/análisis , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here, we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by NMR. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.