Association between BCL11B gene polymorphisms and age-related hearing loss in the elderly: A case-control study in Qingdao, China.

Age-related hearing loss is a complex disease caused by a combination of genetic and environmental factors, and a study have conducted animal experiments to explore the association between BCL11B heterozygosity and age-related hearing loss. The present study used established genetic models to examine the association between BCL11B gene polymorphisms and age-related hearing loss. A total of 410 older adults from two communities in Qingdao, China, participated in this study. The case group comprised individuals aged ≥ 60 years with age-related hearing loss, and the control group comprised individuals without age-related hearing loss from the same communities. The groups were matched 1:1 for age and sex. The individual characteristics of the participants were analyzed descriptively using the Mann-Whitney U test and the chi-square test. To explore the association between BCL11B gene polymorphisms and age-related hearing loss, conditional logistic regression was performed to construct genetic models for two single-nucleotide-polymorphisms (SNPs) of BCL11B, and haplotype analysis was conducted to construct their haplotype domains. Two SNP sites of the BCL11B gene, four genetic models of rs1152781 (additive, dominant, recessive, and codominant), and five genetic models of rs1152783 (additive, dominant, recessive, codominant, and over dominant) were significantly associated with age-related hearing loss in the models both unadjusted and adjusted for all covariates (P < 0.05). Additionally, a linkage disequilibrium between rs1152781 and rs1152783 was revealed through haplotype analysis. Our study revealed that BCL11B gene polymorphisms were significantly associated with age-related hearing loss.

A reliable elasticity sensing method for analysis of cell entosis using microfluidic cytometer.

Cell entosis is a novel cell death process starting from cell-in-cell invasion. In general, cancer cells own higher incidence rate of cell entosis comparing to non-cancerous cells. Studies arguing whether cell entosis is a tumor suppressing process or a tumor accelerating process can deepen our understanding of tumor development. Cell elasticity is recognized as one of tumor malignant biomarkers. There have been some researchers studying cell elasticity in cell entosis. However, existing cell elasticity sensing technique (i.e. micropipette aspiration) can hardly be reliable neither high-throughput. In this work, we introduce an elasticity sensing method for quantifying both cell elasticity in cell-in-cell structures and single floating cells using a microfluidic cytometer. We not only argue our cell elasticity sensing method is reliable for already occurred entosis but also apply such method on predicting the "outer" cells in entosis of different cell types. The elasticity sensing method proposed in this manuscript is able to provide an effective and reliable way to further study deeper mechanism in cell entosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-023-00264-0.

Genome-wide DNA methylation analysis of middle-aged and elderly monozygotic twins with age-related hearing loss in Qingdao, China.

OBJECTIVE: To explore the differences in DNA methylation associated with age-related hearing loss in a study of 57 twin pairs from China. DESIGN: Monozygotic twins were identified through the Qingdao Twin Registration system. The median age of participants was > 50 years. Their hearing thresholds were measured using a multilevel pure-tone audiometry assessment. The pure-tone audiometry was calculated at low frequencies (0.5, 1.0, and 2.0 kHz), speech frequencies (0.5, 1.0, 2.0, and 4.0 kHz), and high frequencies (4.0 and 8 kHz). The CpG sites were tested using a linear mixed-effects model, and the function of the cis-regulatory regions and ontological enrichments were predicted using the online Genomic Regions Enrichment of Annotations Tool. The differentially methylated regions were identified using a comb-p python library approach. RESULTS: In each of the PTA categories (low-, speech-, high-frequency), age-related hearing loss was detected in 25.9%, 19.3%, and 52.8% of participants. In the low-, speech- and high-frequency categories we identified 18, 42, and 12 individual CpG sites and 6, 11, and 6 differentially methylated regions. The CpG site located near DUSP4 had the strongest association with low- and speech-frequency, while the strongest association with high-frequency was near C21orf58. We identified associations of ALG10 with high-frequency hearing, C3 and LCK with low- and speech-frequency hearing, and GBX2 with low-frequency hearing. Top pathways that may be related to hearing, such as the Notch signaling pathway, were also identified. CONCLUSION: Our study is the first of its kind to identify these genes and their associated with DNA methylation may play essential roles in the hearing process. The results of our epigenome-wide association study on twins clarify the complex mechanisms underlying age-related hearing loss.

Selective single-bacteria extraction based on capture and release of microemulsion droplets.

Human host-associated microbial communities in body sites can reflect health status based on the population distribution and specific microbial properties in the heterogeneous community. Bacteria identification at the single-cell level provides a reliable biomarker and pathological information for clinical diagnosis. Nevertheless, biosamples obtained from some body sites cannot offer sufficient sample volume and number of target cells as required by most of the existing single-cell isolation methods such as flow cytometry. Herein we report a novel integrated microfluidic system, which consists of a microemulsion module for single-bacteria encapsulation and a sequential microdroplet capture and release module for selectively extracting only the single-bacteria encapsulated in microdroplets. We optimize the system for a success rate of the single-cell extraction to be > 38%. We further verify applicability of the system with prepared cell mixtures (Methylorubrum extorquens AM1 and Methylomicrobium album BG8) and biosamples collected from human skin, to quantify the population distribution of multiple key species in a heterogeneous microbial community. Results indicate perfect viability of the single-cell extracts and compatibility with downstream analyses such as PCR. Together, this research demonstrates that the reported single-bacteria extraction system can be applied in microbiome and pathology research and clinical diagnosis as a clinical or point-of-care device.

Inhibition of lysyl oxidase-like 2 overcomes adhesion-dependent drug resistance in the collagen-enriched liver cancer microenvironment.

The tumor microenvironment (TME) is considered to be one of the vital mediators of tumor progression. Extracellular matrix (ECM), infiltrating immune cells, and stromal cells collectively constitute the complex ecosystem with varied biochemical and biophysical properties. The development of liver cancer is strongly tied with fibrosis and cirrhosis that alters the microenvironmental landscape, especially ECM composition. Enhanced deposition and cross-linking of type I collagen are frequently detected in patients with liver cancer and have been shown to facilitate tumor growth and metastasis by epithelial-to-mesenchymal transition. However, information on the effect of collagen enrichment on drug resistance is lacking. Thus, the present study has comprehensively illustrated phenotypical and mechanistic changes in an in vitro mimicry of collagen-enriched TME and revealed that collagen enrichment could induce 5-fluorouracil (5FU) and sorafenib resistance in liver cancer cells through hypoxia-induced up-regulation of lysyl oxidase-like 2 (LOXL2). LOXL2, an enzyme that facilitates collagen cross-linking, enhances cell adhesion-mediated drug resistance by activating the integrin alpha 5 (ITGA5)/focal adhesion kinase (FAK)/phosphoinositide 3-kinase (PI3K)/rho-associated kinase 1 (ROCK1) signaling axis. Conclusion: We demonstrated that inhibition of LOXL2 in a collagen-enriched microenvironment synergistically promotes the efficacy of sorafenib and 5FU through deterioration of focal adhesion signaling. These findings have clinical implications for developing LOXL2-targeted strategies in patients with chemoresistant liver cancer and especially for those patients with advanced fibrosis and cirrhosis.

Correction: Antibody-coated microstructures for selective isolation of immune cells in blood.

Correction for 'Antibody-coated microstructures for selective isolation of immune cells in blood' by Jiyu Li et al., Lab Chip, 2020, 20, 1072-1082, DOI: 10.1039/D0LC00078G.

A Narrow Straight Microchannel Array for Analysis of Transiting Speed of Floating Cancer Cells.

Investigating floating cells along a narrow microchannel (e.g., a blood vessel) for their transiting speeds and the corresponding roles of cell physical properties can deepen our understanding of circulating tumor cells (CTCs) metastasis via blood vessels. Many existing studies focus on the cell transiting process in blood vessel-like microchannels; further analytical studies are desired to summarize behaviors of the floating cell movement under different conditions. In this work, we perform a theoretical analysis to establish a relation between the transiting speed and key cell physical properties. We also conduct computational fluid dynamics simulation and microfluidic experiments to verify the theoretical model. This work reveals key cell physical properties and the channel configurations determining the transiting speed. The reported model can be applied to other works with various dimensions of microchannels as a more general way to evaluate the cancer cell metastasis ability with microfluidics.

High-throughput deterministic pairing and coculturing of single cells in a microwell array using combined hydrodynamic and recirculation flow captures.

Single-cell level coculture facilitates the study of cellular interactions for uncovering unknown physiological mechanisms, which are crucial for the development of new therapies for diseases. However, efficient approaches for high-throughput deterministic pairing of single cells and traceable coculture remain lacking. In this study, we report a new microfluidic device, which combines hydrodynamic and recirculation flow captures, to achieve high-throughput and deterministic pairing of single cells in a microwell array for traceable coculture. Compared with the existing techniques, the developed device exhibits advantages with regard to pairing efficiency, throughput, determinacy, and traceability. Through repeating a two-step method, which sequentially captures single cells in a meandering channel and a microwell array, cell number and type can be easily controlled. Double and triple single-cell pairings have been demonstrated with an efficiency of 72.2% and 38.0%, respectively. Cellular engulfment using two breast cell lines is investigated on a developed microfluidic chip as a biological case study, in which the morphological characteristics and the incidence rate are analyzed. This research provides an efficient and reliable alternative for the coculture of single cells on the microfluidic platform for various biomedical applications, such as studying cellular engulfment and tumor sphere formation under single-cell pairing condition.

Label-free biosensor of phagocytosis for diagnosing bacterial infections.

Phagocytic cells recognize and phagocytose invading microbes for destruction. However, bacterial pathogens can remain hidden at low levels from conventional detection or replicate intracellularly after being phagocytosed by immune cells. Current phagocytosis-detection approaches involve flow cytometry or microscopic search for rare bacteria-internalized phagocytes among large populations of uninfected cells, which poses significant challenges in research and clinical settings. Hence it is imperative to develop a rapid, non-disruptive, and label-free phagocytosis detection approach. Using deformability assays and microscopic imaging, we have demonstrated for the first time that the presence of intracellular bacteria in phagocytic blood cells led to aberrant physical properties. Specifically, human monocytes with internalized bacteria of various species were stiffer and larger compared with uninfected monocytes. Taking advantage of these physical differences, a novel microfluidics-based biosensor platform was developed to passively sort, concentrate and quantify rare monocytes with internalized pathogens (MIP) from uninfected monocyte populations for phagocytosis detection. The clinical utility of the MIP platform was demonstrated by enriching and detecting bacteria-internalized monocytes from spiked human blood samples within 1.5 h. Patient-derived clinical isolates were used to validate the utility of the MIP platform further. This proof-of-concept presents a phagocytosis detection platform that could be used to rapidly diagnose microbial infections, especially in bloodstream infections (BSIs), thereby improving the clinical outcomes for point-of-care management.

Spreading and Migration of Nasopharyngeal Normal and Cancer Cells on Microgratings.

Cell spreading and migration play a pivotal role in many diseases such as tumor metastasis. In particular, nasopharyngeal tumor cells have known of their tendency of migration to pterygoid muscles and further distant metastasis. Although existing studies revealed key characteristics of the nasopharyngeal tumor cells, their migration preference is yet to be thoroughly understood, especially in the physical aspects including the microtopographical factors. Researchers have developed techniques in recent years to study microtopography-related cell behaviors but they are not yet applied in investigating the nasopharyngeal tumor cells. In this work, we elaborate the spreading and migration characteristics of normal and cancerous nasopharyngeal cells on micrograting substrates mimicking the microtopography of myotubes of the pterygoid muscles. We further apply interference reflection microscopy (IRM) to visualize the cell-substrate adhesion dynamics. We are interested in examining the microtopography-related cell spreading and migration behaviors and their correlations, providing insights for deeper understanding and more promising prediction on the nasopharyngeal tumor metastasis.

Nondestructive quantification of single-cell nuclear and cytoplasmic mechanical properties based on large whole-cell deformation.

The mechanical properties of cell nuclei have been recognized to reflect and modulate important cell behaviors such as migration and cancer cell malignant tendency. However, these nuclear properties are difficult to characterize accurately using conventional measurement methods, which are often based on probing or deforming local sites over a nuclear region. The corresponding results are sensitive to the measurement position, and they are not decoupled from the cytoplasmic properties. Microfluidics is widely recognized as a promising technique for bioassay and phenotyping. In this report, we develop a simple and nondestructive approach for the single-cell quantification of nuclear elasticity based on microfluidics by considering different deformation levels of a live cell captured along a confining microchannel. We apply two inlet pressure levels to drive the flow of human nasopharyngeal epithelial cells (NP460) and human nasopharyngeal cancerous cells (NPC43) into the microchannels. A model considering the essential intracellular components (cytoplasm and nucleus) for describing the mechanics of a cell deforming along the confining microchannel is used to back-calculate the cytoplasmic and nuclear properties. On the other hand, we also apply a widely used chemical nucleus extraction technique to examine its possible effects (e.g., reduced nuclear modulus and reduced lamin A/C expression). To determine if the decoupled nuclear properties are representative of cancer-related attributes, we classify the NP460 and NPC43 cells using the decoupled physical properties as classification factors, resulting in an accuracy of 79.1% and a cell-type specificity exceeding 74%. It should be mentioned that the cells can be recollected at the device outlet after the nondestructive measurement. Hence, the reported cell elasticity measurement can be combined with downstream genetic and biochemical assays for general cell research and cancer diagnostic applications.

A two-chip acoustofluidic particle manipulation platform with a detachable and reusable surface acoustic wave device.

This work describes a two-chip acoustofluidic platform for two-dimensional (2D) manipulation of microparticles in a closed microchamber on a reusable surface acoustic wave (SAW) device. This platform comprises two microfabricated chips: (1) a detachable silicon superstrate enclosed by a PDMS microfluidic chamber and (2) a reusable SAW device for generating standing SAW (SSAW), which is typically an expensive component. Critical to such a two-chip acoustofluidic platform is the selection of a suitable coupling agent at the interface of the SAW device and superstrate. To this end, we applied a polymer thin film as a coupling agent that balances between acoustic coupling efficiency, stability over time, and reusability. Recycling of the SAW device lowers the cost-barrier for acoustofluidic particle manipulation. The SSAW is transmitted into the silicon superstrate via the coupling agent to form a standing Lamb wave (SLW) to trap and move microparticles. The reported two-chip strategy enables the single-use microfluidic superstrates to avoid chemical and biological contaminations, while maintaining the merits of acoustofluidic manipulation of being noncontact and label-free and applicable to a wide range of microparticles with different shapes, density, polarity, and electrical properties.

Antibody-coated microstructures for selective isolation of immune cells in blood.

Cell isolation from blood is an important process for diagnosing immune diseases. There are still demands for a user-friendly approach to achieve high cell extraction efficiency and purity of a target immune cell subtype for more promising diagnosis and monitoring. For selective immune cell isolation, we developed a microstructured device, which consists of antibody-coated micropillars and micro-sieve arrays, for isolating a target immune cell subtype from bovine blood samples. The focusing micropillars can guide immune cells flowing to the subsequent micro-sieves based on deterministic lateral shifts of the cells. The arrangement of these microstructures is characterized and configured for the maximal cell capture rate. Surface modification with a selected antibody offers selective cell capture in the micro-sieves based on the antigen-antibody reaction. We prepare a cell mixture of human CD14-expressing leukemia cells (THP-1) and epithelial cells (MDA-MB-231) in diluted blood to characterize the cell isolation operation, with a selective cell isolation yield of >80%, cell purity of ∼100% and cell viability of >93%. Together, this microstructured device strategy can achieve high-yield selective isolation of immune cells from blood samples and support downstream genetic and biochemical cell analyses, contributing to the medical diagnosis of a broad range of immune diseases.

Biofluidic Random Laser Cytometer for Biophysical Phenotyping of Cell Suspensions.

Phenotypic profiling of single floating cells in liquid biopsies is the key to the era of precision medicine. A random laser in biofluids is a promising tool for the label-free characterization of the biophysical properties as a result of the high brightness and sharp peaks of the lasing spectra, yet previous reports were limited to the random laser in solid tissues with dense scattering. In this report, a random laser cytometer is demonstrated in an optofluidic device filled with gain medium and human breast normal/cancerous cells. The multiple lightscattering event induced by the microscale human cells promotes random lasing and influences the lasing properties in term of laser modes, spectral wavelengths, and lasing thresholds. A sensing strategy based on analyzing the lasing properties is developed to determine both the whole cell and the subcellular biophysical properties, and the malignant alterations of the cell suspensions are successfully detected. Our results provide a new approach to designing a label-free biophysical cytometer based on optofluidic random laser devices, which is advantageous for further research in the field of random laser bioapplication.

Elasticity-Modulated Microbeads for Classification of Floating Normal and Cancer Cells Using Confining Microchannels.

Engineered microbeads have a wide range of applications in cancer research including identification, characterization, and sorting of cancer cells. In particular, the microbead-based cancer identification techniques are mainly based on the known genetic or biochemical biomarkers; and detection specificity is yet to be improved. On the other hand, it has been discovered that biomechanical properties of cancer cells such as cell-body elasticity can be considered as cancer biomarkers. Here, we report a straightforward microfluidic classification scheme for floating/dissociated normal and cancer epithelial cells using a confining microchannel device together with calcium-alginate hydrogel microbeads. The hydrogel microbeads are generated based on the microfluidic emulsion process, with characterization on the process parameters (e.g., liquid driving pressure and cross-linking duration) in order to specify the resultant bead diameter and elasticity. These engineered microbeads are first mixed with a cell mixture of dissociated human nasopharyngeal epithelial cells (NP460) and nasopharyngeal carcinoma cells (NPC43). The cell elasticity can then be reflected from the locations of captured cells in the device. Experiments further demonstrate that the cell classification has a success rate of >95%. Furthermore, we performed the microbead-based cell classification on a whole blood sample containing floating human breast epithelial cells (MCF-10A) and breast cancer epithelial cells (MDA-MB-231) with a success rate of >75%, revealing its directly applicability to identification of circulating tumor cells in human blood. Together, this research demonstrates a new application of engineered hydrogel microbeads for classification of cells based on their mechanical properties.