Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles.

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the distinct optical properties of a heterogeneous nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed "hyperspectral counting", which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of fluorescent nanomaterials to estimate particle number counts in live cells and subcellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique with single-walled carbon nanotubes and identified an upper limit of the rate of uptake into cells─approximately 3,000 nanotubes endocytosed within 30 min. In contrast, conventional region-of-interest counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogeneous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify the cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for the nanotoxicology, drug/gene delivery, and nanosensor fields.

The silent pandemic: Emergent antibiotic resistances following the global response to SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic has highlighted the importance of the rapid development of vaccines and antivirals. However, the potential for the emergence of antibiotic resistances due to the increased use of antibacterial cleaning products and therapeutics presents an additional, underreported threat. Most antibacterial cleaners contain simple quaternary ammonium compounds (QACs); however, these compounds are steadily becoming less effective as antibacterial agents. QACs are extensively used in SARS-CoV-2-related sanitization in clinical and household settings. Similarly, due to the danger of secondary infections, antibiotic therapeutics are increasingly used as a component of COVID-19 treatment regimens, even in the absence of a bacterial infection diagnosis. The increased use of antibacterial agents as cleaners and therapeutics is anticipated to lead to novel resistances in the coming years.

Biomolecular Functionalization of a Nanomaterial To Control Stability and Retention within Live Cells.

Noncovalent hybrids of single-stranded DNA and single-walled carbon nanotubes (SWCNTs) have demonstrated applications in biomedical imaging and sensing due to their enhanced biocompatibility and photostable, environmentally responsive near-infrared (NIR) fluorescence. The fundamental properties of such DNA-SWCNTs have been studied to determine the correlative relationships between oligonucleotide sequence and length, SWCNT species, and the physical attributes of the resultant hybrids. However, intracellular environments introduce harsh conditions that can change the physical identities of the hybrid nanomaterials, thus altering their intrinsic optical properties. Here, through visible and NIR fluorescence imaging in addition to confocal Raman microscopy, we show that the oligonucleotide length controls the relative uptake, intracellular optical stability, and retention of DNA-SWCNTs in mammalian cells. Although the absolute NIR fluorescence intensity of DNA-SWCNTs in murine macrophages increases with increasing oligonucleotide length (from 12 to 60 nucleotides), we found that shorter oligonucleotide DNA-SWCNTs undergo a greater magnitude of spectral shift and are more rapidly internalized and expelled from the cell after 24 h. Furthermore, by labeling the DNA with a fluorophore that dequenches upon removal from the SWCNT surface, we found that shorter oligonucleotide strands are displaced from the SWCNT within the cell, altering the physical identity and changing the fate of the internalized nanomaterial. Finally, through a pharmacological inhibition study, we identified the mechanism of SWCNT expulsion from the cells as lysosomal exocytosis. These findings provide a fundamental understanding of the interactions between SWCNTs and live cells as well as evidence suggesting the ability to control the biological fate of the nanomaterials merely by varying the type of DNA wrapping.

Enhancing the Thermal Stability of Carbon Nanomaterials with DNA.

Single-walled carbon nanotubes (SWCNTs) have recently been utilized as fillers that reduce the flammability and enhance the strength and thermal conductivity of material composites. Enhancing the thermal stability of SWCNTs is crucial when these materials are applied to high temperature applications. In many instances, SWCNTs are applied to composites with surface coatings that are toxic to living organisms. Alternatively, single-stranded DNA, a naturally occurring biological polymer, has recently been utilized to form singly-dispersed hybrids with SWCNTs as well as suppress their known toxicological effects. These hybrids have shown unrivaled stabilities in both aqueous suspension or as a dried material. Furthermore, DNA has certain documented flame-retardant effects due to the creation of a protective char upon heating in the presence of oxygen. Herein, using various thermogravimetric analytical techniques, we find that single-stranded DNA has a significant flame-retardant effect on the SWCNTs, and effectively enhances their thermal stability. Hybridization with DNA results in the elevation of the thermal decomposition temperature of purified SWCNTs in excess of 200 °C. We translate this finding to other carbon nanomaterials including multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (RGO) and fullerene (C60), and show similar effects upon complexation with DNA. The rate of thermal decomposition of the SWCNTs was also explored and found to significantly depend upon the sequence of DNA that was used.

DNA Sequence Mediates Apparent Length Distribution in Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) functionalized with short single-stranded DNA have been extensively studied within the last decade for biomedical applications due to the high dispersion efficiency and intrinsic biocompatibility of DNA as well as the photostable and tunable fluorescence of SWCNTs. Characterization of their physical properties, particularly their length distribution, is of great importance regarding their application as a bioengineered research tool and clinical diagnostic agent. Conventionally, atomic force microscopy (AFM) has been used to quantify the length of DNA-SWCNTs by depositing the hybrids onto an electrostatically charged flat surface. Here, we demonstrate that hybrids of DNA-SWCNTs with different oligomeric DNA sequences ((GT)6 and (GT)30) differentially deposit on the AFM substrate, resulting in significant inaccuracies in the reported length distributions of the parent solutions. Using a solution-based surfactant exchange technique, we placed both samples into a common surfactant wrapping and found identical SWCNT length distributions upon surface deposition. Additionally, by spin-coating the surfactant-wrapped SWCNTs onto a substrate, thus mitigating effects of electrostatic interactions, we found length distributions that did not depend on DNA sequence but were significantly longer than electrostatic deposition methods, illuminating the inherent bias of the surface deposition method. Quantifying the coverage of DNA molecules on each SWCNT through both absorbance spectroscopy and direct observation, we found that the density of DNA per SWCNT was significantly higher in short (GT)6-SWCNTs (length < 100 nm) compared to long (GT)6-SWCNTs (length > 100 nm). In contrast, we found no dependence of the DNA density on SWCNT length in (GT)30-SWCNT hybrids. Thus, we attribute differences in the observed length distributions of DNA-SWCNTs to variations in electrostatic repulsion induced by sequence-dependent DNA density.

DNA-Carbon Nanotube Complexation Affinity and Photoluminescence Modulation Are Independent.

Short single-stranded DNA (ssDNA) has emerged as the natural polymer of choice for noncovalently functionalizing photoluminescent single-walled carbon nanotubes. In addition, specific empirically identified DNA sequences can be used to separate single species (chiralities) of nanotubes, with an exceptionally high purity. Currently, only limited general principles exist for designing DNA-nanotube hybrids amenable to separation processes, due in part to an incomplete understanding of the fundamental interactions between a DNA sequence and a specific nanotube structure, whereas even less is known in the design of nanotube-based sensors with determined optical properties. We therefore developed a combined experimental and analysis platform on the basis of time-resolved near-infrared fluorescence spectroscopy to extract the complete set of photoluminescence parameters that characterizes DNA-nanotube hybrids. Here, we systematically investigated the affinity of the d(GT)n oligonucleotide family for structurally defined carbon nanotubes by measuring photoluminescence response of the nanotube upon oligonucleotide displacement. We found, surprisingly, that the rate of displacement of the oligonucleotides is independent of the coverage on the nanotube, as inferred through the intrinsic optical properties of the hybrid. The kinetics of intensity modulation is essentially a single-exponential, and the time constants, which quantify the stability of DNA binding, span an order of magnitude. Surprisingly, these time constants do not depend on the intrinsic optical parameters within the hybrids, suggesting that the DNA-nanotube stability is not due to increased nanotube surface coverage by DNA. Further, a principal component analysis of the excitation and emission shifts along with intensity enhancement at equilibrium accurately identified the (8,6) nanotube as the partner chirality to (GT)6 ssDNA. When combined, the chirality-resolved equilibrium and kinetics data can guide the development of the DNA-nanotube pairs, with tunable stability and optical modulation. Additionally, this high-throughput optical platform could function as a primary screen for mapping the DNA-chirality recognition phase space.

Epidemiology of traumatic spinal injury: a descriptive study.

Acute injuries of the spine and spinal cord are among the most causes of severe disability and death after trauma. Data about spine fracture with or without cord damage are different. The aim of this study was to determine epidemiology and demographics of spinal injury in main trauma center, Guilan, an Iranian province. The present study was a descriptive study of all cases of traumatic spine injury. Who were admitted to Poursina Hospital, main trauma center of Gilan. The scoring Systems used to evaluate severity of injury were American Spinal Injury association (ASIA) and The Injury Severity Score. Among a total of 245 cases, 71.8%were male and 28.2% were female. Male/Female ratio was 2.55:1. The most common age group at which spinal injury occurred in males was 25-44year-olds and in females was 45-64 year olds. The most common causes were motorcycle vehicle accidents and falls. The most common fracture in spine was thoracolumbar (T10-L2). Among forty four of patient with abnormal findings on neurological examination, fifteen of them had complete spinal cord injury (class A of ASIA) and twenty nine of them had incomplete spinal cord injury (class B, C, D, of ASIA). Our focus on the spinal injury and its major etiology revealed that efforts should be made to prevention. More detailed information about the causes of spinal injury should be sought as it might lead to more targeted intervention.

Hemodynamic variation following induction and tracheal intubation--thiopental vs propofol.

BACKGROUND/AIM: Hemodynamic variations are inevitable during induction of anesthetic drugs. The present study, investigates the hemodynamic variations of two different drugs used for induction; Thiopental vs. Propofol. MATERIALS AND METHODS: In a prospective randomized double-blind study, from June 2003 to November 2004, 120 (ASA I and II) patients scheduled for elective surgery, were randomly divided into two equal groups. Patients were premedicated with midazolam (0.05 mg/kg) and fentanyl (1 microg/kg). Anesthesia was induced with either thiopental 5 mg/kg (group T) or propofol 2 mg/kg (group P). Neuromuscular blockade was achieved with atracurium (0.5 mg/kg) and anesthesia was maintained with halothane 1%, nitrous oxide (67%) in O2. Hemodynamic variable (systolic and diastolic blood pressure, mean arterial pressure and heart rate) were measured non-invasively in three periods: before drug administration, immediately after injection, prior to intubation, and finally immediately after intubation. RESULTS: the incidence of hemodynamic changes in systolic, diastolic, mean arterial blood pressures and heart rate were significantly higher in group T compared to group P. CONCLUSION: We conclude that Propofol causes less hemodynamic changes compared to Thiopental. Therefore, we recommend Propofol especially when dealing with hemodynamically compromised patients.