Plasmonic Assemblies for Real-Time Single-Molecule Biosensing.

Their tunable optical properties and versatile surface functionalization have sparked applications of plasmonic assemblies in the fields of biosensing, nonlinear optics, and photonics. Particularly, in the field of biosensing, rapid advances have occurred in the use of plasmonic assemblies for real-time single-molecule sensing. Compared to individual particles, the use of assemblies as sensors provides stronger signals, more control over the optical properties, and access to a broader range of timescales. In the past years, they have been used to directly reveal single-molecule interactions, mechanical properties, and conformational dynamics. This review summarizes the development of real-time single-molecule sensors built around plasmonic assemblies. First, a brief overview of their optical properties is given, and then recent applications are described. The current challenges in the field and suggestions to overcome those challenges are discussed in detail. Their stability, specificity, and sensitivity as sensors provide a complementary approach to other single-molecule techniques like force spectroscopy and single-molecule fluorescence. In future applications, the impact in real-time sensing on ultralong timescales (hours) and ultrashort timescales (sub-millisecond), time windows that are difficult to access using other techniques, is particularly foreseen.

Heterogeneous Kinetics in the Functionalization of Single Plasmonic Nanoparticles.

The functionalization of gold nanoparticles with DNA has been studied extensively in solution; however, these ensemble measurements do not reveal particle-to-particle differences. Here we study the functionalization of gold nanorods with thiolated single-stranded DNA (ssDNA) at the single-particle level. We exploit the sensitivity of the plasmon resonance to the local refractive index to study the functionalization in real time using single-particle spectroscopy. We find particle-to-particle variations of the plasmon shift that are attributed to the particle size distribution and variations in ssDNA coverage. We find that the ssDNA coverage varies by ∼10% from particle to particle, beyond the expected variation due to Poisson statistics. Surprisingly, we find binding rates that differ from particle to particle by an order of magnitude, even though the buffer conditions are identical. We ascribe this heterogeneity to a distribution of activation energies caused by particle-to-particle variations in effective surface charge. These results yield insight into the kinetics of biofunctionalization at the single particle level and highlight that significant kinetic heterogeneity has to be taken into account in applications of functional particles. The presented methodology is easily extended to any nanoparticle coating and can be used to optimize functionalization protocols.

Rationally manipulating aptamer binding affinities in a stem-loop molecular beacon.

Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM - 203 µM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd's for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.

Addressing Emergency Department Care for Patients Experiencing Incarceration: A Narrative Review.

Patients experiencing incarceration face a multitude of healthcare disparities. These patients are disproportionately affected by a variety of chronic medical conditions. Patients who are incarcerated often remain shackled throughout their hospital course, experience bias from members of the healthcare team, and have many barriers to privacy given the omnipresence of corrections officers. Despite this, many physicians report little formal training on caring for this unique patient population. In this narrative review, we examine the current literature on patients who are incarcerated, especially as it pertains to their care in the emergency department (ED).We also propose solutions to address these barriers to care in the ED setting.

A 30% Volumetric Muscle Loss Does Not Result in Sustained Functional Deficits after a 90-Day Recovery in Rats.

Volumetric muscle loss (VML) is defined as the loss of skeletal muscle tissue which exceeds the body's repair capabilities leading to sustained functional deficits over time. Some etiologies leading to VML include traumatic injuries, congenital diseases, and degenerative myopathies. Currently, the lack of standardized animal models prevents an appropriate estimation of the severity of injury capable of exceeding self-regeneration. Recent work in our laboratory has shown that a 30% VML does not create a sustained functional loss in rats after 3 months. Therefore, the purpose of this study was to evaluate the percentage threshold of muscle loss that results in permanent functional deficits. We surgically created models of 30, 40, and 50% VML injuries in the tibialis anterior (TA) of rats, and subsequently evaluated TA function and structure after a 90-day recovery period. TA muscle force production was measured in situ by stimulating the sciatic nerve to obtain a maximum tetanic force. Results revealed that the maximum force produced by rats with a 30% VML was not significantly different from the uninjured muscle, while the maximum force of the 40% and 50% VML groups was significantly lower in comparison to the uninjured muscle. Overall, this study further supports our observations, suggesting that a 30% VML rat model is not suitable for VML studies. Thus, increasing VML percentages might provide an improved standardized and clinically relevant model for VML that produces a long-term deficit in muscle self-regeneration, while providing a strong base for future tissue engineering techniques in medicine.

A Comparison of Ovine Facial and Limb Muscle as a Primary Cell Source for Engineered Skeletal Muscle.

Volumetric muscle loss (VML) contributes to the number of soft tissue injuries that necessitate reconstructive surgery, but treatment options are often limited by tissue availability and donor site morbidity. To combat these issues, our laboratory has developed scaffold-free tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML injuries. Recently, we have begun experiments addressing VML in facial muscle, and the optimal starting cell population for engineered skeletal muscle tissue for this application may not be cells derived from hindlimb muscles due to reported heterogeneity of cell populations. Thus, the purpose of this study was to compare SMUs fabricated from both craniofacial and hindlimb sources to determine which cell source is best suited for the engineering of skeletal muscle. Herein, we assessed the development, structure, and function of SMUs derived from four muscle sources, including two hindlimb muscles (i.e., soleus and semimembranosus [SM]) and two craniofacial muscles (i.e., zygomaticus major and masseter). Overall, the zygomaticus major exhibited the least efficient digestion, and SMUs fabricated from this muscle exhibited the least aligned myosin heavy chain staining and consequently, the lowest average force production. Conversely, the SM muscle exhibited the most efficient digestion and the highest number of myotubes/mm2; however, the SM, masseter, and soleus groups were roughly equivalent in terms of force production and histological structure. Impact Statement An empirical comparison of the development, structure, and function of engineered skeletal muscle tissue fabricated from different muscles, including both craniofacial and hindlimb sources, will not only provide insight into innate regenerative mechanisms of skeletal muscle but also will give our team and other researchers the information necessary to determine which cell sources are best suited for the skeletal muscle tissue engineering.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 3-Month Recovery.

Much effort has been made to fabricate engineered tissues on a scale that is clinically relevant to humans; however, scale-up remains one of the most significant technological challenges of tissue engineering to date. To address this limitation, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of volumetric muscle loss (VML). The goal of this study was to evaluate the SMUs and ENCs in vitro, and to test the efficacy of our SMUs and ENCs in restoring muscle function in a clinically relevant large animal (sheep) model. The animals received a 30% VML injury to the peroneus tertius muscle and were allowed to recover for 3 months. The animals were divided into three experimental groups: VML injury without a repair (VML only), repair with an SMU (VML+SMU), or repair with an SMU and ENC (VML+SMU+ENC). We evaluated the SMUs before implantation and found that our single scaled-up SMUs were characterized by the presence of contracting myotubes, linearly aligned extracellular matrix proteins, and Pax7+ satellite cells. Three months after implantation, we found that the repair groups (VML+SMU and VML+SMU+ENC) had restored muscle mass and tetanic force production to a level that was statistically indistinguishable from the uninjured contralateral muscle after 3 months in vivo. Furthermore, we demonstrated the ability of our ENCs to effectively bridge the gap between native nerve and the repair site by eliciting a muscle contraction through direct electrical stimulation of the re-routed nerve. Impact statement The fabrication of tissues of clinically relevant sizes is one of the largest obstacles preventing engineered tissues from achieving widespread use in the clinic. This study aimed to combat this limitation by developing a fabrication method to scale-up tissue-engineered skeletal muscle for the treatment of volumetric muscle loss in a large animal (sheep) model and evaluating the efficacy of the tissue-engineered constructs after a 3-month recovery.

Determinants of 30-day Morbidity in Adult Cranioplasty: An ACS-NSQIP Analysis of 697 Cases.

Cranioplasty is performed to restore the function and anatomy of the skull. Many techniques are used, including replacement of the bone flap and reconstruction with autologous or synthetic materials. This study describes the complication profile of adult cranioplasty using a prospective national sample and identifies risk factors for 30-day morbidity. METHODS: The American College of Surgeon's National Surgery Quality Improvement Project database for 2015-2016 was utilized. Cases were identified by current procedural terminology code, size, and type (autologous/alloplastic). χ2, Fisher exact, and ANOVA tests compared demographic differences. Univariate and multivariate logistic regressions were performed to identify risk factors for 30-day morbidity and mortality. RESULTS: Six hundred ninety-seven cranioplasty cases were identified. Two cases used 2 types of cranioplasties and were counted in both groups. Five hundred forty-three cranioplasties were alloplastic, 57 were autologous, and 99 were classified as "Other." Age, race, diabetes, ventilator dependency, congestive heart failure, hypertension, wound infection, sepsis, and bleeding disorders were identified on univariate analysis to increase complication risk. Multivariate analysis identified age of the patient, systemic sepsis, and bleeding disorders as significant risk factors for complications. There was no difference in complications between cranioplasty types. Overall and medical complications were greater in cranioplasties >5 cm (P < 0.001). CONCLUSIONS: Cranioplasty is a morbid procedure, with a complication rate of 27.4% and a mortality rate of 3.0% in this national sample. Factors such as age, sepsis, bleeding disorders, and size increase risk. Identification and modification of risk factors may guide operative timing and influence informed consent.

Triangulating Nucleic Acid Conformations Using Multicolor Surface Energy Transfer.

Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to interlabel distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multicolor surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short-range (10-50 Å), mid-range (50-150 Å), and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.

Nanometal surface energy transfer optical ruler for measuring a human telomere structure.

Nanometal surface energy transfer (NSET) techniques on gold nanoparticles (AuNPs) have become an essential tool in molecular biophysics to identify structural details at long-range donor-acceptor distances. The NSET mechanism is well described, but it has been suggested that the use of large AuNPs in NSET may manipulate natural biomolecular function. If, in fact, such nonspecific interactions with the AuNP surface can be quantified or contained, then NSET may offer more potential in tracking biomolecular folding than the most comprehensive methods in conformer determination (X-ray crystallography, NMR, EPR). Here, we describe an NSET ruler capable of tracking Hybrid-2 telomere quadruplex folding and we demonstrate that nucleic acid appendage to AuNPs up to 10 nm in diameter does not manipulate biomolecular function. The quadruplex folding of Hybrid-2 sequences was tracked by monitoring the emission of a DY680 dye on selected basepairs in the telomere sequence when appended to the surface of AuNPs (5-10 nm). Emission-derived distances extracted from NSET theory correlate well to reported NMR structures of the hybrid quadruplex. Moreover, NSET theory calculates identical donor-acceptor distal points between DY680 and all sizes of AuNPs, indicating that the AuNP tether is not dominant or disruptive towards nucleic acid folding.