Development of a rinsing separation method for exosome isolation and comparison to conventional methods.

OBJECTIVE: Exosomes contain valuable biomarkers for many diseases. Tragically, standardized isolation methods and subsequent characterization criteria for exosomes remain limited. Therefore, we developed a new exosome isolation method, termed rinsing separation, and compared its advantages and weaknesses relative to the existing ultracentrifugation and ExoQuick precipitation methods. MATERIALS AND METHODS: Rinsing separation utilizes heparin and glutaraldehyde as a fixative to isolate exosomes, and was developed using the culture supernatant from mesenchymal stem cells (MSCs). The isolated exosomes were characterized and compared by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot. RESULTS: Consistent with known exosome parameters, exosomes isolated using each method ranged in size from 30 to 150 nm and demonstrated the characteristic cup-shaped morphology. Moreover, the exosome markers CD63 and TSG101 were observed in the lysate of all exosome samples that were isolated using each method. Several advantages and drawbacks were noted for each exosome isolation method. Most notably, ultracentrifugation resulted in fewer, but highly pure, exosomes, and samples generated using the ExoQuick precipitation method contained the most contaminating debris. Samples obtained using pour rinsing separation method represented an amalgam of these two fractions, but were isolated in significantly less time. CONCLUSIONS: In this study, we propose rinsing separation as a new method of isolating exosomes. This method is convenient, and the resulting exosomes are highly pure. Moreover, rinsing separation offers time- and cost-efficiency advantages, making it a promising approach for exosome isolation for clinical applications.

In situ multiproperty measurements of individual nanomaterials in SEM and correlation with their atomic structures.

The relationship between property and structure is one of the most important fundamental questions in the field of nanomaterials and nanodevices. Understanding the multiproperties of a given nano-object also aids in the development of novel nanomaterials and nanodevices. In this paper, we develop for the first time a comprehensive platform for in situ multiproperty measurements of individual nanomaterials using a scanning electron microscope (SEM). Mechanical, electrical, electromechanical, optical, and photoelectronic properties of individual nanomaterials, with lengths that range from less than 200 nm to 20 µm, can be measured in situ with an SEM on the platform under precisely controlled single-axial strain and environment. An individual single-walled carbon nanotube (SWCNT) was measured on the platform. Three-terminal electronic measurements in a field effect transistor structure showed that the SWCNT was semiconducting and agreed with the structure characterization by transmission electron microscopy after the in situ measurements. Importantly, we observed a bandgap increase of this SWCNT with increasing axial strain, and for the first time, the experimental results quantitatively agree with theoretical predictions calculated using the chirality of the SWCNT. The vibration performance of the SWCNT, a double-walled CNT, and a triple-walled CNT were also studied as a function of axial strain, and were proved to be in good agreement with classical beam theory, although the CNTs only have one, two, or three atomic layers, respectively. Our platform has wide applications in correlating multiproperties of the same individual nanostructures with their atomic structures.

A platform for in-situ multi-probe electronic measurements and modification of nanodevices inside a transmission electron microscope.

We developed a new platform that enables in-situ four-probe electronic measurements, in-situ three-probe field-effect measurements, nanomanipulation, and in-situ modification of nanodevices inside a transmission electron microscope (TEM). The platform includes a specially designed chip-holder and a silicon (Si) chip with suspended metal electrodes. The chip-holder can hold one Si chip with a size up to 3 mm × 3 mm and provides four electrical connections that can be connected to the micrometer-sized electrodes on the Si chip by wire-bonding. The other side of the electrical connections on the chip-holder is connected to the electronic instruments outside the TEM through a commercial Nanofactory SPM-TEM holder. The Si chip with suspended metal electrodes on one of its edges was fabricated by lithography and wet etching. Carbon nanotubes (CNTs), InAs nanowires, and tungsten disulfide nanowires were placed to stride over and connect to the suspended electrodes on the Si chip by nanomanipulations inside a scanning electron microscope (SEM). By using the platform, I-V curves of an individual single-walled CNT connecting to four electrodes were in-situ measured between any two of the four suspended electrodes, and a high-resolution TEM image of the same CNT was obtained. Furthermore, four-terminal I-V measurement on an InAs nanowire was achieved on this platform, and with a movable probe used as a gate electrode, field-effect measurement on the same InAs nanowire device was accomplished in SEM. In addition, by using the movable probe on the SPM-TEM holder, we could further in-situ modify nanomaterial and nanodevices. The present work demonstrates a method that allows a direct correlation between the atomic-level structure and the electronic property of nanomaterials or nanodevices whose structure can be further modified in-situ.

Transversally and axially tunable carbon nanotube resonators in situ fabricated and studied inside a scanning electron microscope.

We report a new design of carbon nanotube (CNT) resonator, whose resonance frequency can be tuned not only transversally by a gate voltage, but also by the axial strain applied through directly pulling the CNT. The resonators are fabricated from individual suspended single-walled CNT (SWCNT) in situ inside a scanning electron microscope. The resonance frequency of a SWCNT resonator can be tuned by more than 20 times with an increase of quality factor when the axial strain of the SWCNT is only increased from nearly zero to 2% at room temperature. The transversal gate-tuning ability is found to be weaker than the axial-tuning ability and decrease with increasing the axial strain. The gate voltage can hardly tune the resonance frequency when the initial axial strain is larger than 0.35% and the CNT acts like a tied string. The relationship among resonance frequency, gate voltage, and initial axial strain of the CNT obtained presently will allow for the designs of CNT resonators with high frequency and large tuning range. The present resonator also shows ultrahigh sensitivity in displacement and force detection, with a resolution being better than 2.4 pm and 0.55 pN, respectively.

AtMYB44 regulates resistance to the green peach aphid and diamondback moth by activating EIN2-affected defences in Arabidopsis.

Recently we showed that the transcription activator AtMYB44 regulates expression of EIN2, a gene essential for ethylene signalling and insect resistance, in Arabidopsis thaliana (Arabidopsis). To link the transactivation with insect resistance, we investigated the wild-type and atmyb44 mutant plants, genetically Complemented atmyb44 (Catmyb44) and AtMYB44-Overexpression Transgenic Arabidopsis (MYB44OTA). We found that AtMYB44 played a critical role in Arabidopsis resistance to the phloem-feeding generalist green peach aphid (Myzus persicae Sulzer) and leaf-chewing specialist caterpillar diamondback moth (Plutella xylostella L.). AtMYB44 was required not only for the development of constitutive resistance but also for the induction of resistance by both herbivorous insects. Levels of constitutive and herbivore-induced resistance were consistent with corresponding amounts of the AtMYB44 protein constitutively produced in MYB44OTA and induced by herbivory in Catmyb44. In both cases, AtMYB44 promoted EIN2 expression to a greater extent in MYB44OTA than in Catmyb44. However, AtMYB44-promoted EIN2 expression was arrested with reduced resistance levels in the EIN2-deficient Arabidopsis mutant ein2-1 and the MYB44OTA ein2-1 hybrid. In the different plant genotypes, only MYB44OTA constitutively displayed phloem-based defences, which are specific to phloem-feeding insects, and robust expression of genes involved in the biosynthesis of glucosinolates, which are the secondary plant metabolites known as deterrents to generalist herbivores. Phloem-based defences and glucosinolate-related gene expression were not detected in ein2-1 and MYB44OTA ein2-1. These results establish a genetic connection between the regulatory role of AtMYB44 in EIN2 expression and the development of Arabidopsis resistance to insects.

An efficacy analysis for nasopharyngeal carcinoma screening of different screening intervals.

OBJECTIVE: To evaluate the impact of different screening intervals on screening for nasopharyngeal carcinoma (NPC). METHODS: A Markov model was constructed, based on the natural history of NPC. The 5-year mortality rate of NPC was the major measurement to evaluate the efficacies of 16 screening strategies. Parameters for the model were derived from published literature. RESULTS: Screening reduced the 5-year mortality rate for NPC by 20.4 - 43.3%, compared with the equivalent rate without screening. The 5 year mortality rate and the NPC pick-up rate with strategy A1 (annual screening) were 23.6% and 83.9%, respectively. Compared with strategy A1, strategy B1 (annual screening for seropositive subjects; biennial screening for seronegative subjects) had a similar 5-year mortality rate (24.0%) and a slightly smaller NPC pick-up rate (81.7%), but led to a 39.3% reduction in total screenings. Compared with all other strategies excluding strategy A1, strategy B1 achieved the lowest 5-year mortality rate and the largest NPC pick-up rate. CONCLUSIONS: Strategy B1 had the highest efficacy for NPC screening.