Tumor cell-derived LC3B+extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90α-IL-6/IL-8 signaling axis.

BACKGROUND: Inflammatory factors are being recognized as critical modulators of host antitumor immunity in liver cancer. We have previously shown that tumor cell-released LC3B positive extracellular vesicles (LC3B+ EVs) are responsible for malignant progression by dampening antitumor immunity. However, the relationship between LC3B+ EVs and inflammatory factors in the regulation of the liver cancer microenvironment remains unclear. METHODS: Flow cytometry analyses were performed to examine the panel of 12 cytokines, the main source of positive cytokines, and plasma LC3B+ EVs carrying HSP90α in peripheral blood of liver cancer patients. We correlated the levels of plasma IL-6, IL-8 with LC3B+ EVs carrying HSP90α and with prognosis. In vitro culture of healthy donor leukocytes with liver cancer-derived LC3B+ EVs was performed to evaluate the potential effect of blocking HSP90α, IL-6 or IL-8 alone or in combination with PD-1 inhibitor on CD8+ T cell function. We also investigated the potential associations of MAP1LC3B, HSP90AA1, IL6 or IL8 with immunotherapy efficacy using the TCGA databases. RESULTS: In liver cancer patients, plasma IL-6 and IL-8 levels were significantly higher than in healthy controls and associated with poor clinical outcome. In peripheral blood, levels of plasma LC3B+ EVs carrying HSP90α were significantly elevated in HCC patients and positively associated with IL-6 and IL-8 levels, which are predominantly secreted by monocytes and neutrophils. Moreover, LC3B+ EVs from human liver cancer cells promoted the secretion of IL-6 and IL-8 by leukocytes through HSP90α. Besides, we show that the cytokines IL-6 and IL-8 secreted by LC3B+ EVs-induced leukocytes were involved in the inhibition of CD8+ T-cell function, while blockade of the HSP90α on the LC3B+ EVs, IL-6, or IL-8 could enhance anti-PD-1-induced T cell reinvigoration. Finally, patients who received anti-PD-1/PD-L1 immunotherapy with high MAP1LC3B, HSP90AA1, IL6, or IL8 expression had a lower immunotherapy efficacy. CONCLUSIONS: Our data suggest that liver cancer-derived LC3B+ EVs promote a pro-oncogenic inflammatory microenvironment by carrying membrane-bound HSP90α. Targeting HSP90α on the LC3B+ EVs, IL-6, or IL-8 may synergize with anti-PD-1 treatment to enhance the CD8+ T-cell functions, which may provide novel combination strategies in the clinic for the treatment of liver cancer.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion.

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

Small extracellular vesicles' enrichment from biological fluids using an acoustic trap.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 µL min-1. The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

The influence of daily life events on learning crafting and the intervening roles of academic emotions and regulatory focus in high school students.

BACKGROUND: The adaptation of students to academic challenges in high school is crucial for academic performance. This study proposes the concept of "learning crafting," a previously under-researched area, and investigates its associated variables. METHODS: Using a diary method, we studied 187 Chinese high school students (64% female; Mage = 15.57) over a 9-day period. We examined the effect of daily life events on learning crafting, and considered academic emotions as mediators and regulatory focus as moderators. RESULTS: Hierarchical linear modeling revealed that daily positive events were positively correlated with learning crafting at both within-person and between-person levels. Positive academic emotions served as mediators of this relationship. Furthermore, promotion focus had a positive moderating effect on the relationship between daily positive events and positive academic emotions. Conversely, daily negative events were only negatively correlated with learning crafting at the between-person level, and no additional significant relationships were identified. CONCLUSION: This study elucidated the effect of daily life events on learning crafting, its mediating mechanisms, and conditional factors. These results not only contribute to crafting theory, but also provide theoretical underpinnings for future interventions targeting high school students' learning crafting.

Impact of IoT System Imperfections and Passenger Errors on Cruise Ship Evacuation Delay.

Cruise ships and other naval vessels include automated Internet of Things (IoT)-based evacuation systems for the passengers and crew to assist them in case of emergencies and accidents. The technical challenges of assisting passengers and crew to safety during emergencies include various aspects such as sensor failures, imperfections in the sound or display systems that are used to direct evacuees, the timely selection of optimum evacuation routes for the evacuees, as well as computation and communication delays that may occur in the IoT infrastructure due to intense activities during an emergency. In addition, during an emergency, the evacuees may be confused or in a panic, and may make mistakes in following the directions offered by the evacuation system. Therefore, the purpose of this work is to analyze the effect of two important aspects that can have an adverse effect on the passengers' evacuation time, namely (a) the computer processing and communication delays, and (b) the errors that may be made by the evacuees in following instructions. The approach we take uses simulation with a representative existing cruise ship model, which dynamically computes the best exit paths for each passenger, with a deadline-driven Adaptive Navigation Strategy (ANS). Our simulation results reveal that delays in the evacuees' reception of instructions can significantly increase the total time needed for passenger evacuation. In contrast, we observe that passenger behavior errors also affect the evacuation duration, but with less effect on the total time needed to evacuate passengers. These findings demonstrate the importance of the design of passenger evacuation systems in a way that takes into account all realistic features of the ship's indoor evacuation environment, including the importance of having high-performance data processing and communication systems that will not result in congestion and communication delays.

Long Non-Coding RNA-Cardiac-Inducing RNA 6 Mediates Repair of Infarcted Hearts by Inducing Mesenchymal Stem Cell Differentiation into Cardiogenic Cells through Cyclin-Dependent Kinase 1.

This study aims to investigate the induction effect of LncRNA-CIR6 on MSC differentiation into cardiogenic cells in vitro and in vivo. In addition to pretreatment with Ro-3306 (a CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was further transfected into the hearts of C57BL/6 mice via 100 µL of AAV9-cTnT-LncRNA-CIR6-ZsGreen intravenous injection. After three weeks of transfection followed by AMI surgery, hUCMSCs (5 × 105/100 µL) were injected intravenously one week later. Cardiac function was evaluated using VEVO 2100 and electric mapping nine days after cell injection. Immunofluorescence, Evans blue-TTC, Masson staining, FACS, and Western blotting were employed to determine relevant indicators. LncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00 ± 0.58)% and hUCMSCs (95.43 ± 2.13)% into cardiogenic cells, as determined by the expression of cTnT using immunofluorescence and FACS. High cTNT expression was observed in MSCs after transfection with LncRNA-CIR6 by Western blotting. Compared with the MI group, cardiac contraction and conduction function in MI hearts treated with LncRNA-CIR6 or combined with MSCs injection groups were significantly increased, and the areas of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was on Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0-50/190-255 in the sequence. CDK1, a protein found to be related to the proliferation and differentiation of cardiomyocytes, was located in the functional region of the LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed a high expression of CDK1. LncRNA-CIR6 mediates the repair of infarcted hearts by inducing MSC differentiation into cardiogenic cells through CDK1.

Biodegradable Hydrogenated Dimer Acid-Based Plasticizers for PLA with Excellent Plasticization, Thermal Stability and Gas Resistance.

The use of vegetable oil-dervied plasticizers to enhance the flexibility of polylactic acid (PLA) has received much attention due to their renewability, inexpensiveness and biodegradation. However, the double bonds in vegetable oil-based plasticizers limit their compatibility with PLA, resulting in PLA-derived products with reduced flexibility. Herein, we examined soybean oil-derived hydrogenated dimer acid-based polyethylene glycol methyl ether esters (HDA-2n, 2n = 2, 4, 6 or 8, referring to the ethoxy units) developed via the direct esterification of saturated hydrogenated dimer acid and polyethylene glycol monomethyl ethers. The resulting HDA-2n was first used as a plasticizer for PLA, and the effects of the ethoxy units in HDA-2n on the overall performance of the plasticized PLA were systematically investigated. The results showed that, compared with PLA blended with dioctyl terephthalate (DOTP), the PLA plasticized by HDA-8 with the maximum number of ethoxy units (PLA/HDA-8) exhibited better low-temperature resistance (40.1 °C vs. 15.3 °C), thermal stability (246.8 °C vs. 327.6 °C) and gas barrier properties. Additionally, the biodegradation results showed that HDA-8 could be biodegraded by directly burying it in soil. All results suggest that HDA-8 could be used as green alternative to the traditional petroleum-based plasticizer DOTP, which is applied in the PLA industry.

Chemical Recycling of Thiol Epoxy Thermosets via Light-Driven C-C Bond Cleavage.

Epoxy thermosets are high-volume materials that play a central role in a wide range of engineering applications; however, technologies to recycle these polymers remain rare. Here, we present a catalytic, light-driven method that enables chemical recycling of industrially relevant thiol epoxy thermosets to their original monomer at ambient temperature. This strategy relies on the proton-coupled electron transfer (PCET) activation of hydroxy groups within the polymer network to generate key alkoxy radicals that promote the fragmentation of the polymer through C-C bond ß-scission. The method fully depolymerizes insoluble thiol epoxy thermosets into well-defined mixtures of small-molecule products, which can collectively be converted into the original monomer via a one-step dealkylation process. Notably, this process is selective and efficient even in the presence of other commodity plastics and additives commonly found in commercial applications. These results constitute an important step toward making epoxy thermosets recyclable and more generally exemplify the potential of PCET to offer a more sustainable end-of-life for a diverse array of commercial plastics.

Identification of Potential Indicators for Survival in Patients with Thyroid Cancer Based on Expression of FAM3 Members.

Thyroid cancer (THCA) is a common head and neck malignancy. The family with sequence similarity 3 (FAM3) is a cytokine-like gene family with four members, which is presumed to participate in the development of many cancer types. However, the expression patterns of FAM3s in THCA and their prognostic values, have not yet been established. We investigated differential expressions of FAM3 mRNA and protein in THCA, then validated the findings for FAM3B by immunohistochemistry. We also investigated survival data with respect to FAM3 expression patterns in patients with THCA. FAM3s information regarding their relationships with clinical pathological parameters were obtained and FAM3 mutations were assessed. KEGG and GO pathway regarding FAM3C were obtained using online databases. To investigate potential correlations between FAM3s and immune cell infiltration, we investigated the roles of FAM3s in immune cells of patients with THCA. The mRNA expression of FAM3C were significantly elevated in THCA tissues; high expression levels of FAM3C protein were also observed in THCA tissues. A significant association between the pathological stage and the expression of FAM3C was found in patients with THCA. Patients with THCA who had high mRNA expression levels of FAM3C exhibited significantly more favorable prognosis, compared with patients who had low mRNA expression levels of FAM3C. Overall, FAM3C may play vital roles in the pathogenesis and development of THCA, and these findings constitute novel insights for biomarkers of immunotherapeutic targeted agents and may aid in the identification of prognostic biomarkers for THCA.

Photoredox Catalysis in Photocontrolled Cationic Polymerizations of Vinyl Ethers.

Advances in photocontrolled polymerizations have expanded the scope of polymer architectures and structures that can be synthesized for various applications. The majority of these polymerizations have been developed for radical processes, which limits the diversity of monomers that can be used in macromolecular design. More recent developments of photocontrolled cationic polymerizations have taken a step toward addressing this limitation and have expanded the palette of monomers that can be used in stimuli-regulated polymerizations, enabling the synthesis of previously inaccessible polymeric structures. This Account will detail our group's studies on cationic polymerization processes where chain growth is regulated by light and highlight how these methods can be combined with other stimuli-controlled polymerizations to precisely dictate macromolecular structure.Photoinitiated cationic polymerizations are well-studied and important processes that have control over initiation. However, we wanted to develop systems where we had spatiotemporal control over both polymer initiation and chain growth. This additional command over the reaction provides the ability to manipulate the growing polymer with an external stimulus during a polymerization, which can be used to control structure. To achieve this goal, we set out to develop a method to photoreversibly generate a cation at a growing chain end that could participate in a controlled polymerization process. We took inspiration from previous work on cationic degenerate chain transfer polymerizations of vinyl ethers that used thiocarbonylthio chain transfer agents. These polymerizations were initiated by a strong acid and gave well-defined poly(vinyl ether)s. We posited that we could remove the acid initiator in these systems and reversibly oxidize the thiocarbonylthio chain ends in these reactions with a photocatalyst to give a photocontrolled cationic polymerization of vinyl ethers. This Account will focus on our journey to discover cationic photocontrolled polymerizations. We will summarize our initial developments and detail our mechanistic understanding of these reactions using both organic and inorganic based photocatalysts, and we will outline more recent efforts to expand cationic degenerate chain transfer polymerizations to other thioacetal initiators. Finally, we will detail how these photocontrolled cationic polymerizations can be used to switch monomer selectivity in situ using light to control polymer structure. At the end of the Account, we will discuss our vision for future potential applications of these photocontrolled cationic polymerizations in the synthesis of novel block copolymers and next generation cross-linked networks.

Prostatitis No.1 traditional chinese medicine significantly exhibited anti-inflammation role on prostatitis through miR-205-5p/YES1.

Prostatitis is one common male disease with a high prevalence. Traditional Chinese medicine (TCM) has been used as an alternative method for the treatment. However, the molecular mechanism of Prostatitis No.1 Traditional Chinese Medicine (P1TCM) on prostatitis is still unclear. For this purpose, the rat models were constructed and treated with PITCM of control, model, low (10 g/kg/d), medium (20 g/kg/d), and high (40 g/kg/d), as well as the transfections of medium dosage+NC mimic, and medium dosage+miR-205-5p mimic, medium dosage+NC mimic+pc-NC, medium dosage+miR-205-5p mimic+pc-NC, and medium dosage+miR-205-5p mimic+pc-v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1). Real-time quantitative PCR (qPCR) and western blotting analyses were carried out to evaluate the expression of miR-205-5p and YES1, respectively. The levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α) were assessed by enzyme-linked immunosorbent assay (ELISA). The targeting role of miR-205-5p on YES1 was predicted by StarBase and verified by a dual-luciferase reporter gene assay. Results showed that the optimal treatment of P1TCM relieved the damage of prostate tissue, decreased the immunity and inflammation factors, and reduced the expression level of miR-205-5p in prostate tissue and serum. miR-205-5p mimics significantly relieved tissue damage and reduced immunity and inflammatory functions. miR-205-5p targeted YES1. YES1 was significantly upregulated in medium dosage treatment compared with Control, while downregulated compared with the Model. YES1 was also upregulated in prostatitis patients. The pc-YES1 reversed the function of the miR-205-5p mimic. In conclusion, P1TCM significantly relieved the tissue damage and reduced prostate patients' inflammatory functions through miR-205-5p/YES1, which might be essential for clinical studies.

The Resistance of Maize to Ustilago maydis Infection Is Correlated with the Degree of Methyl Esterification of Pectin in the Cell Wall.

Common smut caused by Ustilago maydis is one of the dominant fungal diseases in plants. The resistance mechanism to U. maydis infection involving alterations in the cell wall is poorly studied. In this study, the resistant single segment substitution line (SSSL) R445 and its susceptible recurrent parent line Ye478 of maize were infected with U. maydis, and the changes in cell wall components and structure were studied at 0, 2, 4, 8, and 12 days postinfection. In R445 and Ye478, the contents of cellulose, hemicellulose, pectin, and lignin increased by varying degrees, and pectin methylesterase (PME) activity increased. The changes in hemicellulose and pectin in the cell wall after U. maydis infection were analyzed via immunolabeling using monoclonal antibodies against hemicellulsic xylans and high/low-methylated pectin. U. maydis infection altered methyl esterification of pectin, and the degree of methyl esterification was correlated with the resistance of maize to U. maydis. Furthermore, the relationship between methyl esterification of pectin and host resistance was validated using 15 maize inbred lines with different resistance levels. The results revealed that cell wall components, particularly pectin, were important factors affecting the colonization and propagation of U. maydis in maize, and methyl esterification of pectin played a role in the resistance of maize to U. maydis infection.

BSA-Seq and Transcriptomic Analysis Provide Candidate Genes Associated with Inflorescence Architecture and Kernel Orientation by Phytohormone Homeostasis in Maize.

The developmental plasticity of the maize inflorescence depends on meristems, which directly affect reproductive potential and yield. However, the molecular roles of upper floral meristem (UFM) and lower floral meristem (LFM) in inflorescence and kernel development have not been fully elucidated. In this study, we characterized the reversed kernel1 (rk1) novel mutant, which contains kernels with giant embryos but shows normal vegetative growth like the wild type (WT). Total RNA was extracted from the inflorescence at three stages for transcriptomic analysis. A total of 250.16-Gb clean reads were generated, and 26,248 unigenes were assembled and annotated. Gene ontology analyses of differentially expressed genes (DEGs) detected in the sexual organ formation stage revealed that cell differentiation, organ development, phytohormonal responses and carbohydrate metabolism were enriched. The DEGs associated with the regulation of phytohormone levels and signaling were mainly expressed, including auxin (IAA), jasmonic acid (JA), gibberellins (GA), and abscisic acid (ABA). The transcriptome, hormone evaluation and immunohistochemistry observation revealed that phytohormone homeostasis were affected in rk1. BSA-Seq and transcriptomic analysis also provide candidate genes to regulate UFM and LFM development. These results provide novel insights for understanding the regulatory mechanism of UFM and LFM development in maize and other plants.

Dominant complementation of biological pathways in maize hybrid lines is associated with heterosis.

MAIN CONCLUSION: Allele-specific expressed genes (ASEGs) are widespread in maize hybrid lines and play important roles of complementation of biological pathways in heterosis. Heterosis (hybrid vigor) is an important phenomenon with both theoretical and practical value. However, our understanding of the genetic and molecular mechanisms behind heterosis is still limited. Here, we analyzed a comprehensive dataset of maize (Zea mays L.), including RNA-seq data from three hybrid-parent triplets (HPTs) and acetylated protein data from one HPT. The gene expression patterns exhibited extensive variation between the hybrids and their parents, and a substantial number of allele-specific expressed genes (ASEGs) were identified in the hybrids. Notably, ASEGs from different HPTs were significantly enriched in various conserved pathways. The parental alleles of ASEGs with fewer deleterious single-nucleotide polymorphisms were more likely to be expressed in hybrid lines than other parental alleles. ASEGs were mainly enriched in the functional gene ontology terms protein biosynthesis, photosynthesis, and metabolism. In addition, the ASEGs across the three HPTs were involved in key photosynthetic pathways and might enhance the photosynthetic efficiency of the hybrids. These findings suggest that ASEGs involved in complementary biological pathways in maize hybrids contribute to heterosis, shedding new light on the molecular mechanism of heterosis.

Sheathless acoustic based flow cell sorter for enrichment of rare cells.

Cell enrichment is a powerful tool in many kinds of cell research, especially in applications with low abundance cell types. In this work, we developed a microfluidic fluorescence activated cell sorting device that was able to perform on-demand, low loss cell detection, and sorting. The chip utilizes three-dimensional acoustic standing waves to position all cells in the same fluid velocity regime without sheath. When the cells pass through a laser interrogation region, the scattering and fluorescent signals are detected, translated and transported to software. The target cells are then identified by gating on the plots. Short bursts of standing acoustic waves are triggered by order from PC to sort target cells within predefined gating region. For very low abundance and rare labeled lymphocytes mixed with high concentration unlabeled white blood cells (WBCs), (1-100 labeled lymphocytes are diluted in 106 WBCs in 1 ml volume fluid), the device is able to remove more than 98% WBCs and recover labeled lymphocytes with efficiency of 80%. We further demonstrated that this device worked with real clinical samples by successfully isolating fetal nucleated red blood cells (FNRBCs) in the blood samples from pregnant women with male fetus. The obtained cells were sequenced and the expressions of (sex determining region Y) SRY genes were tested to determine fetal cell proportion. In genetic analysis, the proportion of fetal cells in the final picked sample is up to 40.64%. With this ability, the device proposed could be valuable for biomedical applications involving fetal cells, circulating tumor cells, and stem cells.

Autophagy and cellular immune responses.

Autophagy constitutes a mechanism for the sequestration and lysosomal degradation of various cytoplasmic structures, including damaged organelles and invading microorganisms. Autophagy not only represents an essential cell-intrinsic mechanism to protect against internal and external stress conditions but also shapes cellular immunity. Recent evidence indicates that autophagic responses in antigen-donor cells affect the release of several cytokines and "danger signals." Thus, especially when it precedes cell death, autophagy alerts innate immune effectors to elicit cognate immune responses. Autophagy is also important for the differentiation, survival, and activation of myeloid and lymphoid cells. Accordingly, inherited mutations in autophagy-relevant genes are associated with immune diseases, whereas oncogenesis-associated autophagic defects promote the escape of developing tumors from immunosurveillance. Here, we discuss the regulation of autophagy in the course of cellular immune responses and emphasize its impact on the immunogenicity of antigen-donor cells and on the activity of antigen-presenting cells and T lymphocytes.

Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.

Digenic heterozygous mutations of KCNH2 and SCN5A induced young and early-onset long QT syndrome and sinoatrial node dysfunction.

INTRODUCTION: Long QT syndrome (LQTS) is a life-threatening inherited channelopathy, and prolonged QT intervals easily trigger malignant arrhythmias, especially torsades de pointes and ventricular fibrillation. MATERIALS AND METHODS: The proband with overlapped phenotypes of LQTS and sinoatrial node dysfunction underwent some necessary examinations, including echocardiography, electrocardiogram (ECG), and Holter monitoring. Next, whole-exome sequencing was performed, and candidate genes were validated by Sanger sequencing. RNA secondary structure and protein physical-chemical parameter analyses were used to predict the possible structural change of the proteins induced by the mutations. RESULTS: We identified the digenic heterozygous mutations of KCNH2 p.307_308del (NM_001204798, c.921_923del) and SCN5A p.R1865H (NM_001160160, c.G5594A) in the female and young proband (II: 1) of LQTS and ventricular fibrillation with repeat syncope at rest. Subsequently, she occurred with obvious sinus arrest with persistent ventricular pacing of implantable cardioverter-defibrillator. The heterozygous SCN5Ap.R1865H was carried by her father and sister but not carried by I:2. II:1 carried with KCNH2 p.307_308del as a de novo mutation, but not existed in other family members. RNA secondary structure of KCNH2 p.307_308del showed a false regional double helix, and its amino acids' hydrophobicity was significantly weakened. For the Nav 1.5 protein property, SCN5A p.R1865H slightly increased the molecular weight and aliphatic index but reduced the instability index. CONCLUSIONS: The digenic heterozygous KCNH2 and SCN5A mutations were associated with young early-onset long QT syndrome and sinoatrial node dysfunction.