Imaging Techniques in Pharmacological Precision Medicine.

Biomedical imaging is a powerful tool for medical diagnostics and personalized medicines. Examples of commonly used imaging modalities include Positron Emission Tomography (PET), Ultrasound (US), Single Photon Emission Computed Tomography (SPECT), and hybrid imaging. By combining these modalities, scientists can gain a comprehensive view and better understand physiology and pathology at the preclinical, clinical, and multiscale levels. This can aid in the accuracy of medical diagnoses and treatment decisions. Moreover, biomedical imaging allows for evaluating the metabolic, functional, and structural details of living tissues. This can be particularly useful for the early diagnosis of diseases such as cancer and for the application of personalized medicines. In the case of hybrid imaging, two or more modalities are combined to produce a high-resolution image with enhanced sensitivity and specificity. This can significantly improve the accuracy of diagnosis and offer more detailed treatment plans. In this book chapter, we showcase how continued advancements in biomedical imaging technology can potentially revolutionize medical diagnostics and personalized medicine.

Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning.

Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

Identification of a t(X;17)(q28;q21) generating a KANSL1-MTCP1 gene fusion leading to dysregulated expression of MTCP1 in acute myeloid leukemia.

Chromosomal translocations and generating fusion genes are closely associated with disease initiation and progression in acute myeloid leukemia (AML). In this study, we identified a novel t(X;17)(q28;q21) chromosomal rearrangement in a patient with acute monocytic leukemia. Using RNA-sequencing, we identified a KANSL1-MTCP1 and a KANSL1-CMC4 fusion gene. 5'-UTR sequences of the KANSL1 gene were found to become fused upstream of the coding sequence region of the MTCP1 and CMC4 genes, respectively, resulting in an aberrantly high expression of these genes. Functional studies revealed that overexpression of the MTCP1 gene induced an increased cell proliferation and partial blockage of cell differentiation, suggesting that the aberrant expression of MTCP1 is of critical importance in leukemogenesis.

Controlling cell-cell interactions using surface acoustic waves.

The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.

Acoustofluidic bacteria separation.

Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate E. coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells (RBCs) shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care (POC) sepsis diagnostic device.

Cell separation using tilted-angle standing surface acoustic waves.

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.

Acoustofluidic Transfer of Inflammatory Cells from Human Sputum Samples.

For sputum analysis, the transfer of inflammatory cells from liquefied sputum samples to a culture medium or buffer solution is a critical step because it removes the inflammatory cells from the presence of residual dithiothreitol (DTT), a reagent that reduces cell viability and interferes with further sputum analyses. In this work, we report an acoustofluidic platform for transferring inflammatory cells using standing surface acoustic waves (SSAW). In particular, we exploit the acoustic radiation force generated from a SSAW field to actively transfer inflammatory cells from a solution containing residual DTT to a buffer solution. The viability and integrity of the inflammatory cells are maintained during the acoustofluidic-based cell transfer process. Our acoustofluidic technique removes residual DTT generated in sputum liquefaction and facilitates immunophenotyping of major inflammatory cells from sputum samples. It enables cell transfer in a continuous flow, which aids the development of an automated, integrated system for on-chip sputum processing and analysis.

On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves.

Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based "acoustic tweezers" that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve real-time control of a standing surface acoustic wave field, which enables flexible manipulation of most known microparticles. The power density required by our acoustic device is significantly lower than its optical counterparts (10,000,000 times less than optical tweezers and 100 times less than optoelectronic tweezers), which renders the technique more biocompatible and amenable to miniaturization. Cell-viability tests were conducted to verify the tweezers' compatibility with biological objects. With its advantages in biocompatibility, miniaturization, and versatility, the acoustic tweezers presented here will become a powerful tool for many disciplines of science and engineering.

Standing surface acoustic wave based cell coculture.

Precise reconstruction of heterotypic cell-cell interactions in vitro requires the coculture of different cell types in a highly controlled manner. In this article, we report a standing surface acoustic wave (SSAW)-based cell coculture platform. In our approach, different types of cells are patterned sequentially in the SSAW field to form an organized cell coculture. To validate our platform, we demonstrate a coculture of epithelial cancer cells and endothelial cells. Real-time monitoring of cell migration dynamics reveals increased cancer cell mobility when cancer cells are cocultured with endothelial cells. Our SSAW-based cell coculture platform has the advantages of contactless cell manipulation, high biocompatibility, high controllability, simplicity, and minimal interference of the cellular microenvironment. The SSAW technique demonstrated here can be a valuable analytical tool for various biological studies involving heterotypic cell-cell interactions.

Acoustic propulsion of nanorod motors inside living cells.

The ultrasonic propulsion of rod-shaped nanomotors inside living HeLa cells is demonstrated. These nanomotors (gold rods about 300 nm in diameter and about 3 mm long) attach strongly to the external surface of the cells, and are readily internalized by incubation with the cells for periods longer than 24 h. Once inside the cells, the nanorod motors can be activated by resonant ultrasound operating at 4 MHz, and show axial propulsion as well as spinning. The intracellular propulsion does not involve chemical fuels or high-power ultrasound and the HeLa cells remain viable. Ultrasonic propulsion of nanomotors may thus provide a new tool for probing the response of living cells to internal mechanical excitation, for controllably manipulating intracellular organelles, and for biomedical applications.

An on-chip, multichannel droplet sorter using standing surface acoustic waves.

The emerging field of droplet microfluidics requires effective on-chip handling and sorting of droplets. In this work, we demonstrate a microfluidic device that is capable of sorting picoliter water-in-oil droplets into multiple outputs using standing surface acoustic waves (SSAW). This device integrates a single-layer microfluidic channel with interdigital transducers (IDTs) to achieve on-chip droplet generation and sorting. Within the SSAW field, water-in-oil droplets experience an acoustic radiation force and are pushed toward the acoustic pressure node. As a result, by tuning the frequency of the SSAW excitation, the position of the pressure nodes can be changed and droplets can be sorted to different outlets at rates up to 222 droplets s(-1). With its advantages in simplicity, controllability, versatility, noninvasiveness, and capability to be integrated with other on-chip components such as droplet manipulation and optical detection units, the technique presented here could be valuable for the development of droplet-based micro total analysis systems (µTAS).

Steering acoustically propelled nanowire motors toward cells in a biologically compatible environment using magnetic fields.

The recent discovery of fuel-free propulsion of nanomotors using acoustic energy has provided a new avenue for using nanomotors in biocompatible media. Crucial to the application of nanomotors in biosensing and biomedical applications is the ability to remotely control and steer them toward targets of interest, such as specific cells and tissues. We demonstrate in vitro magnetic steering of acoustically powered nanorod motors in a biologically compatible environment. Steering was accomplished by incorporating (40 ± 5) nm thick nickel stripes into the electrochemically grown nanowires. An external magnetic field of 40-45 mT was used to orient the motors, which were acoustically propelled along their long axes. In the absence of a magnetic field, (300 ± 30) nm diameter, (4.3 ± 0.2) µm long nanowires with (40 ± 5) nm thick magnetic stripes exhibit the same self-acoustophoretic behavior, including pattern formation into concentric nanowire circles, aligned spinning chains, and autonomous axial motion, as their non-magnetic counterparts. In a magnetic field, these wires and their paths are oriented as evidenced by their relatively linear trajectories. Coordinated motion of multiple motors and targeting of individual motors toward HeLa cells with micrometer-level precision was demonstrated.

Association between BUN/creatinine ratio and the risk of in-hospital mortality in patients with trauma-related acute respiratory distress syndrome: a single-centre retrospective cohort from the MIMIC database.

OBJECTIVE: Recent studies have shown that blood urea nitrogen to creatinine (BUN/Cr) ratio might be an effective marker for the prognosis of patients with respiratory diseases. Herein, we aimed to assess the association between BUN/Cr ratio and the risk of in-hospital mortality in patients with trauma-related acute respiratory distress syndrome (ARDS). DESIGN: A retrospective cohort study. SETTING AND PARTICIPANTS: 1034 patients were extracted from the Medical Information Mart for Intensive Care-III (MIMIC-III) database. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome of the study was in-hospital mortality, defined by the vital status at the time of hospital discharge (ie, survivors and non-survivors). RESULTS: Of the total patients, 191 (18.5%) died in hospital. The median follow-up duration was 16.0 (8.3-26.6) days. The results showed that high level of BUN/Cr ratio was significantly associated with an increased risk of in-hospital mortality (15.54-21.43: HR=2.00, 95% CI: (1.18 to 3.38); >21.43: HR=1.76, 95% CI: (1.04 to 2.99)) of patients with trauma-related ARDS. In patients with trauma-related ARDS that aged ≥65 years old, male and female, Onychomycosis Severity Index (OSI)>98, Revised Trauma Score (RTS)>11, Simplified Acute Physiology Score II (SAPS-II)>37 and sequential organ failure assessment (SOFA) scores≤7, BUN/Cr ratio was also related to the increased risk of in-hospital mortality (all p<0.05). The predictive performance of BUN/Cr ratio for in-hospital mortality was superior to BUN or Cr, respectively, with the area under the curve of receiver operator characteristic curve at 0.6, and that association was observed in age, gender, OSI, RTS, SAPS-II and SOFA score subgroups. CONCLUSION: BUN/Cr ratio may be a potential biomarker for the risk of in-hospital mortality of trauma-related ARDS, which may help the clinicians to identify high-risk individuals and to implement clinical interventions.

New realm of precision multiplexing enabled by massively-parallel single molecule UltraPCR.

PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.

Self-Healing Silver Nanowires and Reduced Graphene Oxide/Polyurethane Composite Film Based on the Diels-Alder Reaction under Infrared Radiation.

The hybrid composite of silver nanowires (AgNWs) and reduced graphene oxide (RGO) was synthesized in situ by an improved polyol-thermal method. The AgNWs-RGO with mass contents of 5-37 wt% was added into the thermo-reversible Diels-Alder reaction polyurethane (DA-PU) matrix with the AgNWs as the main conductor and the RGO as the auxiliary conductor to prepare self-healing composite conductive films. Further, the electrical conductivity, thermal conductivity, mechanical properties, infrared thermal response, and self-healing property of the composite film under infrared light irradiation were studied. The experimental results demonstrate that the AgNWs-RGO endows the composite film with good electrical and thermal conductivity and infrared thermal response ability, while the mechanical properties of the composite film decrease as the AgNWs-RGO mass content increases. The self-healing efficiency of the composite film is higher than that of the pure DA-PU under infrared light irradiation due to the good infrared photothermal response ability of the AgNWs-RGO. When the mass content of AgNWs-RGO in the composite film was 25 wt%, the AgNWs-RGO showed good dispersion in composite films, and the resistivity, thermal conductivity, and tensile strength of the composite film were 0.544 Ω·m, 0.3039 W·m-1·K-1, and 9.05 MPa, respectively. The infrared photothermal conversion temperature of the composite film is 158.5 °C (3450 lux for 1 min), and the infrared photothermal self-healing efficiency is 118% (3450 lux for 600 s). The AgNWs-RGO also improves the multiple self-healing ability of the composite film. The use of a high mass content of AgNWs-RGO in the composite film is beneficial in obtaining high multiple self-healing efficiencies. The first and the fifth infrared thermal self-healing efficiencies of the composite film with AgNWs-RGO of 35 wt% are 105% and 86%, respectively, and the resistivity of the composite film changes little and still maintains good conductivity.

Near-Infrared-Light-Assisted Self-Healing Graphene-Thermopolyurethane Composite Films.

Graphene-thermopolyurethane (G-TPU) composite films were fabricated and the effects of the TPU initial concentration, characteristics of TPU, and graphene loading on the electrical, mechanical, thermal, infrared thermal response and near-infrared-light-assisted self-healing properties of the composite films were investigated in detail. The experimental results demonstrate that the comprehensive performances of the composite film are related to the initial concentration of the TPU solution and the characteristics of the TPU and the graphene loading. The composite film prepared from TPU solution with low initial concentration can have conductivity under the condition of low graphene content. However, the composite film prepared with appropriate initial concentration of TPU solution and high graphene loading is conducive to obtain high conductivity. After 60 s of near-infrared illumination, the temperature of the composite film first increases and then decreases with the increase in graphene loading until it reaches saturation. The near-infrared light thermal response of the composite film with high graphene loading is related to the initial concentration of TPU solution, while the near-IR thermal response of the composite film with low graphene loading is independent of the initial concentration of TPU. The surface micro-cracks of the composite film almost disappeared after 10 min of near-infrared illumination. The resistance of the conductive composite film increases after healed. The composite film prepared with low melting point TPU is more favorable to obtain high near-IR thermal self-healing efficiency.

Luminescence properties of Ba4Yb3F17:Er3+ nanocrystals embedded in glass ceramics for optical thermometry.

Transparent glass ceramics (GCs) containing Ba4Yb3F17:Er3+ nanocrystals were successfully fabricated by a traditional melt-quenching method. The formation of Ba4Yb3F17 nanocrystals was confirmed by X-ray diffraction, transmission electron microscopy, and selected area electron diffraction. Compared with the precursor glass, the enhanced emission intensity and lifetime of GCs indicate that the Er3+ ions incorporate into the Ba4Yb3F17 nanocrystals after crystallization. The color tuning properties with doping under 980 nm excitation have been systematically discussed. It was found that the red/green ratio increased with Er3+ ion doping and the corresponding color changed from greenish-yellow to yellow-green. Furthermore, the temperature-dependent luminescence properties were studied in detail by the fluorescence intensity ratio (FIR) technique. The monotonic change of FIR with temperature indicates that this material is suitable for temperature sensing. At a temperature of 450 K, the relative sensitivity of the prepared sample reached its maximal value of 0.20% K-1. The results show that the GCs containing Ba4Yb3F17:Er3+ nanocrystals are candidate materials for temperature sensing.

Massive PD-L1 and CD8 double positive TILs characterize an immunosuppressive microenvironment with high mutational burden in lung cancer.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) expressed on tumor and immune cells are both associated with the response to programmed cell death 1 (PD-1) pathway blockade therapy. Here, we examine the role of CD8+PD-L1+ tumor-infiltrating lymphocyte (Tils) in the tumor microenvironment of non-small cell lung cancer (NSCLC). METHODS: Tumor tissue samples of a total of 378 patients from two NSCLC cohorts were collected retrospectively. Tumor genetic variations were measured by targeted next-generation sequencing of 543 oncogenes. Tils were assessed by multiplex immunohistochemistry assay. Correlations among Tils, tumor genetic variations, and clinicopathological characteristics were analyzed. RESULTS: The levels of CD8+PD-L1+ Tils varied in NSCLC tumor tissues. Tumor samples with high CD8+PD-L1+ Tils had higher levels of CD8+ Tils, CD68+ macrophages, PD-L1+ tumor cells, PD-1+ Tils, and CD163+ M2-type macrophages, and also had a higher tumor mutation burden, all of which collectively constituted a typically hot but immunosuppressive tumor microenvironment. Therefore, in a non-immunotherapy cohort, we observed that the higher the CD8+PD-L1+ Tils level in the tumor tissue, the worse the prognosis (progression-free survival; cohort A, stage I-II tumor; p=0.005). Contrarily, in an immunotherapy cohort, where the immune suppression was blocked by anti-PD-1 treatment, the higher the CD8+PD-L1+ Tils level, the better the response to the anti-PD-1 treatment (complete response/partial response vs stable disease/progressive disease; cohort B; p=0.0337). CONCLUSIONS: CD8+PD-L1+ Tils may be an indicator of the hot but immunosuppressive tumor microenvironment which is related to a high tumor mutation burden. PD-1 pathway blockade therapy can help to mitigate this immunosuppression and obtain better curative effects.

[Prognosis of patients with intracerebral hemorrhage].

OBJECTIVE: To study the related factors of patients with intracerebral hemorrhage (ICH) so as to guide treatment and predict prognosis. METHODS: The prognostic factors of 463 cases with intracerebral hemorrhage were analyzed with single factor and Logistic regression analyses. RESULTS: Age, Glasgow coma scale, amount of hemorrhage, NIHSS score, mean arterial blood pressure, with or without ventricular breakage, with or without midline shift and the incidence of complications at random blood glucose levels were analyzed for the correlation with the prognosis of patients. The poor prognosis group had significant differences with the good prognosis group with regards to these factors. The average age of patients with a poor prognosis was 71 years old, the average hematoma volume 29 ml and the average GCS score 11.2 versus 65 years old, 15 ml, 15.1 for those with a good prognosis (P < 0.05). Logistic regression analysis showed that age, amount of hemorrhage and disturbance of consciousness was an independent adverse prognostic factor for cerebral hemorrhage at three months. The OR values were 1.32, 8.66 and 1.08 respectively. CONCLUSION: The etiologies of ICH are diverse. Maintaining normal blood pressure is an important preventive measure for ICH. Hematoma volume, disturbance of consciousness and age may be used to predict the prognosis of cerebral hemorrhage.