LIN28A loss of function is associated with Parkinson's disease pathogenesis.

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.

Copy number variation of ß-defensin genes in Behçet's disease.

OBJECTIVES: Behçet's disease (BD) may be triggered by infectious agents in genetically susceptible persons. Human ß-defensin 2 is an inducible antimicrobial peptide, the level of which can be influenced by copy number (CN) of the DEFB4. We investigated the relationship between copy number variation (CNV) of DEFB4 and BD. METHODS: One hundred and ninety-seven patients with BD and 197 healthy controls were enrolled. After measuring CN of DEFB4 with a paralogue ratio test, the CNV was compared between patients and controls. CNV was also analysed in comparison with the clinical manifestations of BD. RESULTS: The CN of DEFB4 was unimodally distributed among the study subjects with mean CN of 4.57 and standard deviation of 1.28. BD samples had numerically lower CN than controls, but the difference was not statistically significant (4.49 ± 1.21 vs. 4.65 ± 1.36, p=0.245). Regarding the relationship between CN of DEFB4 and clinical manifestations, there was no difference of CNV depending on the clinical manifestations. CONCLUSIONS: We found no significant difference in CNV of DEFB4 between patients with BD and controls. Our results suggest that CNV of DEFB4 may not contribute to the pathogenesis of BD.

Selecting coronavirus disease 2019 patients with negligible risk of progression: early experience from non-hospital isolation facility in Korea.

BACKGROUND/AIMS: As the novel coronavirus (coronavirus disease 2019 [COVID-19]) outbreak progresses rapidly, staying home is recommended for suspected patients; however, the safety of this recommendation is uncertain. In Korea, non-hospital facilities called "living and treatment centers (LTCs)" have been established since 5 March 2020. The LTCs provided a unique opportunity to evaluate the safety of selection criteria for low-risk groups. METHODS: Between 5 March and 9 April 2020, patients with COVID-19 who met the following criteria were admitted to the LTC; alert, age below 65 years old, no underlying disease or well-controlled underlying disease, body temperature below 38.0°C, whether taking antipyretics or not, and no dyspnea. Patients were closely observed by doctors or nurses' interviews twice a day and transferred to hospitals when symptoms worsened. RESULTS: A total of 113 patients were admitted to the LTC; 52.2% were female, with a median age of 25 years (interquartile range, 21.5 to 39.5). Of 113 patients, 54 (47.8%) were asymptomatic at diagnosis, and 15 (13.3%) had no symptoms until they were released from isolation. During the follow-up period, two (1.8%) patients were transferred to a hospital but did not progress to severe status during hospitalization. CONCLUSION: The risk of progression was negligible in COVID-19 patients who met the admission criteria for LTC at the time of diagnosis. LTCs could be a safe alternative considering shortage of hospital beds.

Fabry-Perot cavity resonance enabling highly polarization-sensitive double-layer gold grating.

We present experimental and theoretical investigations on the polarization properties of a single- and a double-layer gold (Au) grating, serving as a wire grid polarizer. Two layers of Au gratings form a cavity that effectively modulates the transmission and reflection of linearly polarized light. Theoretical calculations based on a transfer matrix method reveals that the double-layer Au grating structure creates an optical cavity exhibiting Fabry-Perot (FP) resonance modes. As compared to a single-layer grating, the FP cavity resonance modes of the double-layer grating significantly enhance the transmission of the transverse magnetic (TM) mode, while suppressing the transmission of the transverse electric (TE) mode. As a result, the extinction ratio of TM to TE transmission for the double-layer grating structure is improved by a factor of approximately 8 in the mid-wave infrared region of 3.4-6 µm. Furthermore, excellent infrared imagery is obtained with over a 600% increase in the ratio of the TM-output voltage (Vθ = 0°) to TE-output voltage (Vθ = 90°). This double-layer Au grating structure has great potential for use in polarimetric imaging applications due to its superior ability to resolve linear polarization signatures.

Biotunable Nanoplasmonic Filter on Few-Layer MoS2 for Rapid and Highly Sensitive Cytokine Optoelectronic Immunosensing.

Monitoring of the time-varying immune status of a diseased host often requires rapid and sensitive detection of cytokines. Metallic nanoparticle-based localized surface plasmon resonance (LSPR) biosensors hold promise to meet this clinical need by permitting label-free detection of target biomolecules. These biosensors, however, continue to suffer from relatively low sensitivity as compared to conventional immunoassay methods that involve labeling processes. Their response speeds also need to be further improved to enable rapid cytokine quantification for critical care in a timely manner. In this paper, we report an immunobiosensing device integrating a biotunable nanoplasmonic optical filter and a highly sensitive few-layer molybdenum disulfide (MoS2) photoconductive component, which can serve as a generic device platform to meet the need of rapid cytokine detection with high sensitivity. The nanoplasmonic filter consists of anticytokine antibody-conjugated gold nanoparticles on a SiO2 thin layer that is placed 170 µm above a few-layer MoS2 photoconductive flake device. The principle of the biosensor operation is based on tuning the delivery of incident light to the few-layer MoS2 photoconductive flake thorough the nanoplasmonic filter by means of biomolecular surface binding-induced LSPR shifts. The tuning is dependent on cytokine concentration on the nanoplasmonic filter and optoelectronically detected by the few-layer MoS2 device. Using the developed optoelectronic biosensor, we have demonstrated label-free detection of IL-1ß, a pro-inflammatory cytokine, with a detection limit as low as 250 fg/mL (14 fM), a large dynamic range of 106, and a short assay time of 10 min. The presented biosensing approach could be further developed and generalized for point-of-care diagnosis, wearable bio/chemical sensing, and environmental monitoring.

Cyclewise Operation of Printed MoS2 Transistor Biosensors for Rapid Biomolecule Quantification at Femtomolar Levels.

Field-effect transistors made from MoS2 and other emerging layered semiconductors have been demonstrated to be able to serve as ultrasensitive biosensors. However, such nanoelectronic sensors still suffer seriously from a series of challenges associated with the poor compatibility between electronic structures and liquid analytes. These challenges hinder the practical biosensing applications that demand rapid, low-noise, highly specific biomolecule quantification at femtomolar levels. To address such challenges, we study a cyclewise process for operating MoS2 transistor biosensors, in which a series of reagent fluids are delivered to the sensor in a time-sequenced manner and periodically set the sensor into four assay-cycle stages, including incubation, flushing, drying, and electrical measurement. Running multiple cycles of such an assay can acquire a time-dependent sensor response signal quantifying the reaction kinetics of analyte-receptor binding. This cyclewise detection approach can avoid the liquid-solution-induced electrochemical damage, screening, and nonspecific adsorption to the sensor and therefore improves the transistor sensor's durability, sensitivity, specificity, and signal-to-noise ratio. These advantages in combination with the inherent high sensitivity of MoS2 biosensors allow for rapid biomolecule quantification at femtomolar levels. We have demonstrated the cyclewise quantification of Interleukin-1ß in pure and complex solutions (e.g., serum and saliva) with a detection limit of ∼1 fM and a total detection time ∼23 min. This work leverages the superior properties of layered semiconductors for biosensing applications and advances the techniques toward realizing fast real-time immunoassay for low-abundance biomolecule detection.

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure.

Immunomodulatory drugs-agents regulating the immune response-are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 µL), short assay time (∼30 min), heightened sensitivity (∼20-30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment.

Doxycycline supplementation allows for the culture of human ESCs/iPSCs with media changes at 3-day intervals.

Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures, as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline, an anti-bacterial agent, had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study, we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions, hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change, while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus, hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties, indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs.

Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics.

Atomically layered transition metal dichalcogenides (TMDCs) exhibit a significant potential to enable next-generation low-cost transistor biosensors that permit single-molecule-level quantification of biomolecules. To realize such potential biosensing capability, device-oriented research is needed for calibrating the sensor responses to enable the quantification of the affinities/kinetics of biomolecule interactions. In this work, we demonstrated MoS2-based transistor biosensors capable of detecting tumor necrosis factor--alpha (TNF-α) with a detection limit as low as 60 fM. Such a detection limit was achieved in both linear and subthreshold regimes of MoS2 transistors. In both regimes, all sets of transistors exhibited consistent calibrated responses with respect to TNF-α concentration, and they resulted in a standard curve, from which the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM. Based on this calibrated sensor model, the time-dependent binding kinetics was also measured and the association/dissociation rates of the antibody-(TNF-α) pair were extracted to be (5.03 ± 0.16) × 10(8) M(-1) s(-1) and (1.97 ± 0.08) × 10(-4) s(-1), respectively. This work advanced the critical device physics for leveraging the excellent electronic/structural properties of TMDCs in biosensing applications as well as the research capability in analyzing the biomolecule interactions with fM-level sensitivities.

Integrated nanoplasmonic sensing for cellular functional immunoanalysis using human blood.

Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for a cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays, a technique to quantify intercellular-signaling proteins secreted by blood immune cells, allow determination of the functional response of the donor's immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on a cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key to rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45 cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-α secreted from cells as few as 1000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 µL) and a total assay time 3 times shorter than that of the conventional enzyme-linked immunosorbent assay (ELISA).

Surface-micromachined microfiltration membranes for efficient isolation and functional immunophenotyping of subpopulations of immune cells.

An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this "bulk" assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay ("AlphaLISA"), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples.

An integrated microfluidic platform for in situ cellular cytokine secretion immunophenotyping.

Rapid, quantitative detection of cell-secreted biomarker proteins with a low sample volume holds great promise to advance cellular immunophenotyping techniques for personalized diagnosis and treatment of infectious diseases. Here we achieved such an assay with the THP-1 human acute moncytic leukemia cell line (a model for human monocyte) using a highly integrated microfluidic platform incorporating a no-wash bead-based chemiluminescence immunodetection scheme. Our microfluidic device allowed us to stimulate cells with lipopolysaccharide (LPS), which is an endotoxin causing septic shock due to severely pronounced immune response of the human body, under a well-controlled on-chip environment. Tumor necrosis factor-alpha (TNF-α) secreted from stimulated THP-1 cells was subsequently measured within the device with no flushing process required. Our study achieved high-sensitivity cellular immunophenotyping with 20-fold fewer cells than current cell-stimulation assay. The total assay time was also 7 times shorter than that of a conventional enzyme-linked immunosorbent assay (ELISA). Our strategy of monitoring immune cell functions in situ using a microfluidic platform could impact future medical treatments of acute infectious diseases and immune disorders by enabling a rapid, sample-efficient cellular immunophenotyping analysis.

Optofluidic detection for cellular phenotyping.

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.