Advance of microfluidic flow cytometry enabling high-throughput characterization of single-cell electrical and structural properties.

This paper reported a micro flow cytometer capable of high-throughput characterization of single-cell electrical and structural features based on constrictional microchannels and deep neural networks. When single cells traveled through microchannels with constricted cross-sectional areas, they effectively blocked concentrated electric field lines, producing large impedance variations. Meanwhile, the traveling cells were confined within the cross-sectional areas of the constrictional microchannels, enabling the capture of high-quality images without losing focuses. Then single-cell features from impedance profiles and optical images were extracted from customized recurrent and convolution networks (RNN and CNN), which were further fused for cell-type classification based on support vector machines (SVM). As a demonstration, two leukemia cell lines (e.g., HL60 vs. Jurkat) were analyzed, producing high-classification accuracies of 99.3% based on electrical features extracted from Long Short-Term Memory (LSTM) of RNN, 96.7% based on structural features extracted from Resnet18 of CNN and 100.0% based on combined features enabled by SVM. The microfluidic flow cytometry developed in this study may provide a new perspective for the field of single-cell analysis.

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-µFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-µFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-µFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-µFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-µFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-µFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

A Unified Therapeutic-Prophylactic Tissue-Engineering Scaffold Demonstrated to Prevent Tumor Recurrence and Overcoming Infection toward Bone Remodeling.

Osteosarcoma occurs in children and adolescents frequently and leads to a high fatality rate. Although surgical resection is the most common methods in clinic, patients always suffer from tumor metastasis and recurrence and it is difficult for them to self-repair large bone defects. Furthermore, the postoperative infection from bacteria triggers an inflammatory response and hinders the bone-repair process. This work demonstrates a gadolinium (Gd)-complex and molybdenum sulfide (MoS2 ) co-doped N-acryloyl glycinamide (NAGA)/gelatin methacrylate (Gel-MA) multifunctional hydrogel (GMNG). The combination between NAGA and Gel-MA endows the GMNG with attractive mechanical properties and controllable degradation ability. The MoS2 improves the hydrogel system, which has excellent photothermal ability to kill tumor cells and inhibit bacterial infection both in vitro and in vivo. Based on the Gd-complex, the magnetic resonance imaging (MRI) effect can be used to monitor the position and degradation situation of the hydrogel. Notably, accompanied by the degradation of GMNG hydrogel, the gradually released Gd3+ from the hydrogel exhibits osteogenic property and could promote new bone formation efficiently in vivo. Therefore, this strategy supplies a method to prepare multifunctional bone-defect-repair materials and is expected to represent a significant guidance and reference to the development of biomaterials for bone tissue engineering.

[Clinical and genetic analysis of three children with KBG syndrome due to novel variants of ANKRD11 gene].

OBJECTIVE: To explore the clinical and genetic characteristics of three children with KBG syndrome. METHODS: Clinical data of the three children from two families who have presented at the First Affiliated Hospital of Zhengzhou University between October 2019 and September 2020 and their family members were collected. Trio-whole exome sequencing (trio-WES) and Sanger sequencing were carried out. RESULTS: All children had feeding difficulties, congenital heart defects and facial dysmorphism. The sib- pair from family 1 was found to harbor a novel de novo heterozygous c.6270delT (p.Q2091Rfs*84) variant of the ANKRD11 gene, whilst the child from family 2 was found to harbor a novel heterozygous c.6858delC (p.D2286Efs*51) variant of the ANKRD11 gene, which was inherited from his mother who had a mild clinical phenotype. CONCLUSION: The heterozygous frameshift variants of the ANKRD11 gene probably underlay the disease in the three children. Above findings have enriched the spectrum of the ANKRD11 gene variants.

Genome Mining and Metabolic Profiling Reveal Cytotoxic Cyclodipeptides in Streptomyces hygrospinosus var. Beijingensis.

Two new cyclodipeptide (CDP) derivatives (1-2) and another seven known cyclodipeptides (3-9) were isolated from Streptomyces 26D9-414 by the genome mining approach combined with genetic dereplication and the "one strain many compounds" (OSMAC) strategy. The structures of the new CDPs were established on the basis of 1D- and 2D-NMR and comparative electronic circular dichroism (ECD) spectra analysis. The biosynthetic gene clusters (BGCs) for these CDPs were identified through antiSMASH analysis. The relevance between this cdp cluster and the identified nine CDPs was established by genetic interruption manipulation. The newly discovered natural compound 2 displayed comparable cytotoxicity against MDA-MB-231 and SW480 with that of cisplatin, a widely used chemotherapeutic agent for the treatment of various cancers.

Tanshinone IIa alleviates LPS-induced oxidative stress in dairy cow mammary epithelial cells by activating the Nrf2 signalling pathway.

OBJECTIVE: Mastitis is the most prevalent disease in dairy cows worldwide. Evidence has emerged that oxidative stress plays a crucial role in the development of mastitis. This study aimed to investigate the antioxidative effects of tanshinone IIa (Tan IIa) on LPS-induced oxidative stress in dairy cow mammary epithelial cells (CMECs). METHODS AND RESULTS: We examined the levels of ROS and MDA in LPS-treated CMECs after supplementation with Tan IIa using detection kits and found that Tan IIa significantly inhibited the upregulation of these factors. In addition, we also found that Tan IIa significantly reversed the decrease in mitochondrial membrane potential induced by LPS. Moreover, Tan IIa improved the activities of antioxidant enzymes, which were decreased by LPS. Finally, we examined the probable pathway in which Tan IIa exerted its antioxidant effects using qPCR and western blotting and found that Tan IIa significantly activated the Keap1/Nrf2 signalling pathway. CONCLUSION: These results suggest that Tan IIa might become a possible therapeutic agent for the treatment of dairy cow mastitis by weakening oxidative stress induced by LPS in CMECs.

Developments of Conventional and Microfluidic Flow Cytometry Enabling High-Throughput Characterization of Single Cells.

This article first reviews scientific meanings of single-cell analysis by highlighting two key scientific problems: landscape reconstruction of cellular identities during dynamic immune processes and mechanisms of tumor origin and evolution. Secondly, the article reviews clinical demands of single-cell analysis, which are complete blood counting enabled by optoelectronic flow cytometry and diagnosis of hematologic malignancies enabled by multicolor fluorescent flow cytometry. Then, this article focuses on the developments of optoelectronic flow cytometry for the complete blood counting by comparing conventional counterparts of hematology analyzers (e.g., DxH 900 of Beckman Coulter, XN-1000 of Sysmex, ADVIA 2120i of Siemens, and CELL-DYN Ruby of Abbott) and microfluidic counterparts (e.g., microfluidic impedance and imaging flow cytometry). Future directions of optoelectronic flow cytometry are indicated where intrinsic rather than dependent biophysical parameters of blood cells must be measured, and they can replace blood smears as the gold standard of blood analysis in the near future.

Assessment of attractancy and safeness of (E)-coniferyl alcohol for management of female adults of Oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: Bactrocera dorsalis is a devastating pest on fruits and vegetables because the adult female is the key factor that determines the population density of offspring and the degree of host damage. Unfortunately, there is still a lack of effective female attractants for behavioral control. Males of B. dorsalis fed on methyl eugenol (ME) were shown to be more sexually attracted to females and, therefore, were more successful in mating over ME-deprived males. RESULTS: In the current study, we demonstrated that (E)-coniferyl alcohol (E-CF), one of the ME metabolites in males, was highly attractive to sexually-mature females in laboratory bioassays. During the dusk courtship period, mature females showed the highest response to E-CF. However, there were no significant differences in olfactory responses to E-CF between virgin and mated mature females. Moreover, no obvious signs and symptoms of toxicity or death were observed in mice during a 14-day acute oral toxicity test. Toxicologically, no significant changes were observed in body weight, water intake, food consumption and absolute and relative organ weights between control and treated groups of healthy-looking mice, implying that E-CF could be regarded as non-toxic. Furthermore, cytotoxicity assessment revealed that E-CF was non-toxic against human fetal lung fibroblast 1 (HFL1), human breast cancer (MDA-MB-231), mouse embryonic hepatocytes (BNL-CL.2) and Spodoptera frugiperda ovary (SF-9) cell lines. CONCLUSIONS: E-CF proved to be an effective, promising and eco-friendly lure to B. dorsalis females. Therefore, this study may facilitate the development of novel control strategies against B. dorsalis in the field.

Classification of tumor subtypes leveraging constriction-channel based impedance flow cytometry and optical imaging.

As label-free biomarkers, electrical properties of single cells have been widely used for cell-type classification and cell-status evaluation. However, as intrinsic bioelectrical markers, previously reported membrane capacitance and cytoplasmic resistance (e.g., specific membrane capacitance Cspecific membrane and cytoplasmic conductivity σcytoplasm ) of tumor subtypes were derived from tens of single cells, lacking statistical significance due to low cell numbers. In this study, tumor subtypes were constructed based on phenotype (treatment with 4-methylumbelliferone) or genotype (knockdown of ROCK1) modifications and then aspirated through a constriction-channel based impedance flow cytometry to characterize single-cell Cspecific membrane and σcytoplasm . Thousands of single tumor cells with phenotype modifications were measured, resulting in significant differences in 1.64 ± 0.43 µF/cm2 vs. 1.55 ± 0.47 µF/cm2 of Cspecific membrane and 0.96 ± 0.37 S/m vs. 1.24 ± 0.47 S/m of σcytoplasm for 95C cells (792 cells of 95C-control vs. 1529 cells of 95C-pheno-mod); 2.56 ± 0.88 µF/cm2 vs. 2.33 ± 0.56 µF/cm2 of Cspecific membrane and 0.83 ± 0.18 S/m vs. 0.93 ± 0.25 S/m of σcytoplasm for H1299 cells (962 cells of H1299-control vs. 637 cells of H1299-pheno-mod). Furthermore, thousands of single tumor cells with genotype modifications were measured, resulting in significant differences in 3.82 ± 0.92 vs. 3.18 ± 0.47 µF/cm2 of Cspecific membrane and 0.47 ± 0.05 vs. 0.52 ± 0.05 S/m of σcytoplasm (1100 cells of A549-control vs. 1100 cells of A549-geno-mod). These results indicate that as intrinsic bioelectrical markers, specific membrane capacitance and cytoplasmic conductivity can be used to classify tumor subtypes.

Tanshinone IIA regulates microRNA­125b/foxp3/caspase­1 signaling and inhibits cell viability of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common disease with high prevalence worldwide, affecting hundreds of thousands of patients every year. Although its progress can be inhibited by concurrent chemoradiotherapy and platinum­based agents, there is also a need for novel drugs to treat NPC. The present study identified tanshinone IIA as a potent drug that could suppress the proliferation of HK1 cells by enhancing pyroptosis via regulation of the miR­125b/foxp3/caspase­1 signaling pathway. Firstly, the effects of tanshinone IIA on HK1 cells were assessed and it was confirmed that treatment with tanshinone IIA significantly decreased the proliferation of HK1 cells, with increased activity of caspase­3 and caspase­9. Then, the pyroptosis levels after tanshinone IIA administration were detected. The results showed that tanshinone IIA enhanced pyroptosis in a dose­dependent manner. Furthermore, the mechanism underlying the effects of tanshinone IIA on HK1 cells were explored. It was found that transfection with a microRNA (miR)­125b agomir and a small interfering RNA (si)­foxp3 plasmid reversed the inhibitory effect induced by tanshinone IIA, accompanied by an increase in reactive oxygen species levels and lactate dehydrogenase release, indicating a critical role of miR­125b/foxp3 signaling in pyroptosis in HK1 cells. In conclusion, the present study demonstrates that tanshinone IIA enhances pyroptosis and inhibits the proliferation of HK1 cells by modulating miR­125b/foxp3/caspase­1/GSDMD signaling. It is the first study to reveal the inhibitory effect of tanshinone IIA on HK1 cells and to demonstrate the critical role of miR­125b/foxp3 signaling in mediating these effects, providing robust evidence for the treatment of NPC.

Advances of Single-Cell Protein Analysis.

Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review summarizes recent advances of various approaches to single-cell protein analysis. We begin by discussing conventional characterization approaches, including fluorescence flow cytometry, mass cytometry, enzyme-linked immunospot assay, and capillary electrophoresis. We then detail the landmark advances of microfluidic approaches for analyzing single-cell protein expressions, including microfluidic fluorescent flow cytometry, droplet-based microfluidics, microwell-based assay (microengraving), microchamber-based assay (barcoding microchips), and single-cell Western blotting, among which the advantages and limitations are compared. Looking forward, we discuss future research opportunities and challenges for multiplexity, analyte, throughput, and sensitivity of the microfluidic approaches, which we believe will prompt the research of single-cell proteins such as the molecular mechanism of cell biology, as well as the clinical applications for tumor treatment and drug development.

Near-Infrared Tunable Laser Absorption Spectroscopic Acetylene Sensor System Using a Novel Three Mirror-Based, Dense Pattern Gas Cell.

By contrast with the widely reported traditional two mirror-based Herriott cell, a three mirror-based dense pattern gas cell was proposed, of which the modeling and design were proven to be effective through a comparison between the simulated spot pattern and effective path length and those of the experimental results. A mechanical structure was designed to adjust the position/angle of the three mirrors for aligning the optical path. The experimentally measured reflection number was 60, resulting in an optical path length of ~11 m, which agrees well with the theoretical value of 10.95 m. Combined with a near-infrared laser with a center wavenumber located at an acetylene (C2H2) absorption line of 6521.2 cm-1, a C2H2 sensor system was established to verify the feasibility of the three mirror-based gas cell. Assisted by a data acquisition (DAQ) card, a LabVIEW platform was developed to generate the drive signal of the laser and acquire the second harmonic (2f) signal from the output of the detector. Through Allan variance analysis, the limit of detection (LoD) of the sensor system is 4.36 ppm at an average time of 0.5 s; as the average time exceeds 10 s, the LoD is <1 ppm. The proposed model and design of the three mirror-based gas cell can be used to realize similar gas cells with different absorption path lengths for gas detection based on infrared absorption spectroscopy.

Development of microfluidic platform capable of high-throughput absolute quantification of single-cell multiple intracellular proteins from tumor cell lines and patient tumor samples.

Quantification of single-cell proteins plays key roles in cell heterogeneity while due to technical limitations absolute numbers of multiple intracellular proteins from large populations of single cells were still missing, leading to compromised results in cell-type classifications. This paper presents a microfluidic platform capable of high-throughput absolute quantification of single-cell multiple types of intracellular proteins where cells stained with fluorescent labelled antibodies are aspirated into the constriction microchannels with excited fluorescent signals detected and translated into numbers of binding sites of targeted proteins based on calibration curves formed by flushing gradient solutions of fluorescent labelled antibodies directly into constriction microchannels. Based on this approach, single-cell numbers of binding sites of ß-actin, α-tubulin and ß-tubulin from tens of thousands of five representative tumor cell lines were first quantified, reporting cell-type classification rates of 83.0 ± 7.1%. Then single-cell numbers of binding sites of ß-actin, biotin and RhoA from thousands of five tumor cell lines with varieties in malignant levels were quantified, reporting cell-type classification rates of 93.7 ± 2.8%. Furthermore, single-cell numbers of binding sites of Ras, c-Myc and p53 from thousands of cells derived from two oral tumor lines of CAL 27, WSU-HN6 and two oral tumor patient samples were quantified, contributing to high classifications of both tumor cell lines (98.6%) and tumor patient samples (83.4%). In conclusion, the developed microfluidic platform was capable of quantifying multiple intracellular proteins from large populations of single cells, and the collected data of protein expressions enabled effective cell-type classifications.

Anti-Inflammation Effect of Small Molecule Oligopeptides Prepared from Panax ginseng C. A. Meyer in Rats.

The present study was designed to investigate the anti-inflammatory effects of ginseng oligopeptides (GOPs). For the anti-inflammatory activity, dextran-induced paw edema and granuloma models were used in Sprague-Dawley rats (180⁻200 g, 12 weeks old, n = 10). Rats were treated orally with GOPs (0, 62.5, 125, 250 and 500 mg/kg) for prophylaxis. In the granuloma model, the levels of NO, Tumor necrosis factor-α (TNF-α), interleukin IL-ß, and interleukin IL-10 in serum were evaluated. In addition, in the edema model, the level of TNF-α, prostaglandin E2 (PGE2), Leukotriene D4 (LTD4), and the platelet activating factor (RAF) in paw tissue were detected. PCR assessed the effect of GOPs on the expression of MAPK and NF-κB. The results showed that oral administration of GOPs inhibited inflammation caused by cotton pellet and dextran. GOPs significantly inhibited the edema formation via MAPK and NF-κB. These findings suggested that GOPs have a beneficial effect on acute and chronic inflammation, and the mechanism possibly mediated by inhibiting gene expression involved in inflammation and downregulating inflammatory mediators.

Crossing constriction channel-based microfluidic cytometry capable of electrically phenotyping large populations of single cells.

This paper presents a crossing constriction channel-based microfluidic system for high-throughput characterization of specific membrane capacitance (Csm) and cytoplasm conductivity (σcy) of single cells. In operations, cells in suspension were forced through the major constriction channel and instead of invading the side constriction channel, they effectively sealed the side constriction channel, which led to variations in impedance data. Based on an equivalent circuit model, these raw impedance data were translated into Csm and σcy. As a demonstration, the developed microfluidic system quantified Csm (3.01 ± 0.92 µF cm-2) and σcy (0.36 ± 0.08 S m-1) of 100 000 A549 cells, which could generate reliable results by properly controlling cell positions during their traveling in the crossing constriction channels. Furthermore, the developed microfluidic impedance cytometry was used to distinguish paired low- and high-metastatic carcinoma cell types of SACC-83 (ncell = ∼100 000) and SACC-LM cells (ncell = ∼100 000), distinguishing significant differences in both Csm (3.16 ± 0.90 vs. 2.79 ± 0.67 µF cm-2) and σcy (0.36 ± 0.06 vs.0.41 ± 0.08 S m-1). As high-throughput microfluidic impedance cytometry, this technique may add a new marker-free dimension to flow cytometry in single-cell analysis.

Microfluidic Analyzer Enabling Quantitative Measurements of Specific Intracellular Proteins at the Single-Cell Level.

This paper presents a microfluidic instrument capable of quantifying single-cell specific intracellular proteins, which are composed of three functioning modules and two software platforms. Under the control of a LabVIEW platform, a pressure module flushed cells stained with fluorescent antibodies through a microfluidic module with fluorescent intensities quantified by a fluorescent module and translated into the numbers of specific intracellular proteins at the single-cell level using a MATLAB platform. Detection ranges and resolutions of the analyzer were characterized as 896.78⁻6.78 × 105 and 334.60 nM for Alexa 488, 314.60⁻2.11 × 105 and 153.98 nM for FITC, and 77.03⁻5.24 × 104 and 37.17 nM for FITC-labelled anti-beta-actin antibodies. As a demonstration, the numbers of single-cell beta-actins of two paired oral tumor cell types and two oral patient samples were quantified as: 1.12 ± 0.77 × 106/cell (salivary adenoid cystic carcinoma parental cell line (SACC-83), ncell = 13,689) vs. 0.90 ± 0.58 × 105/cell (salivary adenoid cystic carcinoma lung metastasis cell line (SACC-LM), ncell = 15,341); 0.89 ± 0.69 × 106/cell (oral carcinoma cell line (CAL 27), ncell = 7357) vs. 0.93 ± 0.69 × 106/cell (oral carcinoma lymphatic metastasis cell line (CAL 27-LN2), ncell = 6276); and 0.86 ± 0.52 × 106/cell (patient I) vs. 0.85 ± 0.58 × 106/cell (patient II). These results (1) validated the developed analyzer with a throughput of 10 cells/s and a processing capability of ~10,000 cells for each cell type, and (2) revealed that as an internal control in cell analysis, the expressions of beta-actins remained stable in oral tumors with different malignant levels.

Mesenchymal stem cells loaded with paclitaxel-poly(lactic-co-glycolic acid) nanoparticles for glioma-targeting therapy.

BACKGROUND: Mesenchymal stem cells (MSCs) possess inherent tropism towards tumor cells, and so have attracted increased attention as targeted-therapy vehicles for glioma treatment. PURPOSE: The objective of this study was to demonstrate the injection of MSCs loaded with paclitaxel (Ptx)-encapsulated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for orthotopic glioma therapy in rats. METHODS: Ptx-PLGA NP-loaded MSC was obtained by incubating MSCs with Ptx-PLGA NPs. The drug transfer and cytotoxicity of Ptx-PLGA NP-loaded MSC against tumor cells were investigated in the transwell system. Biodistribution and antitumor activity was evaluated in the orthotopic glioma rats after contralateral injection. RESULTS: The optimal dose of MSC-loaded Ptx-PLGA NPs (1 pg/cell Ptx) had little effect on MSC-migration capacity, cell cycle, or multilineage-differentiation potential. Compared with Ptx-primed MSCs, Ptx-PLGA NP-primed MSCs had enhanced sustained Ptx release in the form of free Ptx and Ptx NPs. Ptx transfer from MSCs to glioma cells could induce tumor cell death in vitro. As for distribution in vivo, NP-loaded fluorescent MSCs were tracked throughout the tumor mass for 2 days after therapeutic injection. Survival was significantly longer after contralateral implantation of Ptx-PLGA NP-loaded MSCs than those injected with Ptx-primed MSCs or Ptx-PLGA NPs alone. CONCLUSION: Based on timing and sufficient Ptx transfer from the MSCs to the tumor cells, Ptx-PLGA NP-loaded MSC is effective for glioma treatment. Incorporation of chemotherapeutic drug-loaded NPs into MSCs is a promising strategy for tumor-targeted therapy.

A Microfluidic Fluorescent Flow Cytometry Capable of Quantifying Cell Sizes and Numbers of Specific Cytosolic Proteins.

This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of ß-actins at the single-cell level were quantified as 14.2 ± 1.7 µm and 9.62 ± 4.29 × 105 (A549, ncell = 14 242), 13.0 ± 2.0 µm and 6.46 ± 3.34 × 105 (Hep G2, ncell = 35 932), 13.8 ± 1.9 µm and 1.58 ± 0.90 × 106 (MCF 10 A, ncell = 16 650), and 12.7 ± 1.5 µm and 1.09 ± 0.49 × 106 (HeLa, ncell = 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.

Ginseng oligopeptides protect against irradiation-induced immune dysfunction and intestinal injury.

Intestinal injury and immune dysfunction are commonly encountered after irradiation therapy. While the curative abilities of ginseng root have been reported in prior studies, there is little known regarding its role in immunoregulation of intestinal repairability in cancer patients treated with irradiation. Our current study aims to closely examine the protective effects of ginseng-derived small molecule oligopeptides (Panax ginseng C. A. Mey.) (GOP) against irradiation-induced immune dysfunction and subsequent intestinal injury, using in vitro and in vivo models. Expectedly, irradiation treatment resulted in increased intestinal permeability along with mucosal injury in both Caco-2 cells and mice, probably due to disruption of the intestinal epithelial barrier, leading to high plasma lipopolysaccharide (LPS) and pro-inflammatory cytokines levels. However, when the cells were treated with GOP, this led to diminished concentration of plasma LPS and cytokines (IL-1 and TNF-α), suggesting its dampening effect on inflammatory and oxidative stress, and potential role in restoring normal baseline intestinal permeability. Moreover, the Caco-2 cells treated with GOP showed high trans-epithelial electrical resistance (TEER) and low FITC-dextran paracellular permeability when compared to the control group. This could be explained by the higher levels of tight junction proteins (ZO-1 and Occludin) expression along with reduced expression of the apoptosis-related proteins (Bax and Caspase-3) noticed in the GOP-treated cells, highlighting its role in preserving intestinal permeability, through prevention of their degradation while maintaining normal levels of expression. Further confirmatory in vivo data showed that GOP-treated mice exhibited high concentrations of lymphocytes (CD3+, CD4+, CD8+) in the intestine, to rescue the irradiation-induced damage and restore baseline intestinal integrity. Therefore, we propose that GOP can be used as an adjuvant therapy to attenuate irradiation-induced immune dysfunction and intestinal injury in cancer patients.

Mechanical property characterization of hundreds of single nuclei based on microfluidic constriction channel.

As label-free biomarkers, the mechanical properties of nuclei are widely treated as promising biomechanical markers for cell type classification and cellular status evaluation. However, previously reported mechanical parameters were derived from only around 10 nuclei, lacking statistical significances due to low sample numbers. To address this issue, nuclei were first isolated from SW620 and A549 cells, respectively, using a chemical treatment method. This was followed by aspirating them through two types of microfluidic constriction channels for mechanical property characterization. In this study, hundreds of nuclei were characterized, producing passage times of 0.5 ± 1.2 s for SW620 nuclei in type I constriction channel (n = 153), 0.045 ± 0.047 s for SW620 nuclei in type II constriction channel (n = 215) and 0.50 ± 0.86 s for A549 nuclei in type II constriction channel. In addition, neural network based pattern recognition was used to classify the nuclei isolated from SW620 and A549 cells, producing successful classification rates of 87.2% for diameters of nuclei, 85.5% for passage times of nuclei and 89.3% for both passage times and diameters of nuclei. These results indicate that the characterization of the mechanical properties of nuclei may contribute to the classification of different tumor cells.