Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling.

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Peritumoral B cells drive proangiogenic responses in HMGB1-enriched esophageal squamous cell carcinoma.

Several B-cell subsets with distinct functions and polarized cytokine profiles that extend beyond antibody production have been reported in different cancers. Here we have demonstrated that proliferating B cells were predominantly found in the peritumoral region of esophageal squamous cell carcinoma (ESCC). These B cells were enriched in tumor nests with high expression of high-mobility group box 1 (HMGB1). High densities of peritumoral proliferating B cells and concomitantly high intratumoral HMGB1 expression showed improved prognostic significance, surpassing prognostic stratification of ESCC patients based on HMGB1 positivity alone. This striking association led us to set up models to test whether cancer-derived HMGB1 could shape tumor microenvironment via modulation on B cells. Overexpression of HMGB1 in ESCC cell lines (KYSE510 and EC18) enhanced proliferation and migration of B cells. Transcriptomic analysis showed that migratory B cells exhibited high enrichment of proangiogenic genes. VEGF expression in proliferating B cells was induced upon co-culture of HMGB1-overexpressing tumor cells and B cells. Secretome array profiling of conditioned media (CM) from the co-culture revealed rich expression of proangiogenic proteins. Consequently, incubation of human umbilical vein endothelial cells with CM promoted angiogenesis in tube formation and migration assays. HMGB1 inhibitor, glycyrrhizin, abolishes all the observed proangiogenic phenotypes. Finally, co-injection of B cells and CM with HMGB1-overexpressing tumor cells, but not with glycyrrhizin, significantly enhanced tumor growth associated with increased microvascular density in ESCC xenograft mice model. Our results indicate that cancer-derived HMGB1 elevates angiogenesis in ESCC by shifting the balance toward proangiogenic signals in proliferating B cells.

Single-Nucleotide Variations, Insertions/Deletions and Copy Number Variations in Myelodysplastic Syndrome during Disease Progression Revealed by a Single-Cell DNA Sequencing Platform.

Myelodysplastic syndrome (MDS) is a clonal myeloid neoplasm characterized by ineffective hematopoiesis, cytopenia, dysplasia, and clonal instability, leading to leukemic transformation. Hypomethylating agents are the mainstay of treatment in higher-risk MDS. However, treatment resistance and disease transformation into acute myeloid leukemia (AML) is observed in the majority of patients and is indicative of a dismal outcome. The residual cell clones resistant to therapy or cell clones acquiring new genetic aberrations are two of the key events responsible for drug resistance. Bulk tumor sequencing often fails to detect these rare subclones that confer resistance to therapy. In this study, we employed a single-cell DNA (sc-DNA) sequencing approach to study the clonal heterogeneity and clonal evolution in two MDS patients refractory to HMA. In both patients, different single nucleotide variations (SNVs) or insertions and deletions (INDELs) were detected with bulk tumor sequencing. Rare cell clones with mutations that are undetectable by bulk tumor sequencing were detected by sc-DNA sequencing. In addition to SNVs and short INDELs, this study also revealed the presence of a clonal copy number loss of DNMT3A, TET2, and GATA2 as standalone events or in association with the small SNVs or INDELs detected during HMA resistance and disease progression.

Remediation of a phonological representation deficit in Chinese children with dyslexia: A comparison between metalinguistic training and working memory training.

A form-preparation task in the language production field was adopted to examine output phonological representations in Chinese dyslexia and their susceptibility to training. Forty-one Chinese children with dyslexia (7-11 years old) and 36 chronological age controls completed this task. The controls demonstrated a marginally significant syllable facilitation effect (d = -0.13), indicating their use of syllable-sized phonological representations during speech production, while the group with dyslexia showed a significantly different pattern (d = 0.04), opposite to the direction of a facilitation effect. The children with dyslexia were then randomly assigned to either metalinguistic training (N = 22) or working memory training (N = 19). Only the metalinguistic training subgroup demonstrated a significant syllable facilitation effect afterward (metalinguistic: d = -0.13; working memory: d = -0.01). The results suggest the presence of a phonological representation deficit at the syllable level in Chinese dyslexia and its possible remediation by metalinguistic training. Such a phonological deficit in readers of a logographic script strongly supports the impaired phonological representation view of developmental dyslexia. A video abstract of this article can be viewed at https://youtu.be/zT2Be0xMkh0.

Differential interaction between iron and mutant alpha-synuclein causes distinctive Parkinsonian phenotypes in Drosophila.

Alpha-synuclein aggregation is the central hallmark of both sporadic and familial Parkinson's disease (PD). Patients with different PD-causing genetic defects of alpha-synuclein usually show distinctive clinical features that are atypical to sporadic PD. Iron accumulation is invariably found in PD. Recent studies showed that mutant and wild-type alpha-synuclein may have differential interaction with iron and mutant alpha-synuclein toxicity could be preferentially exacerbated by iron. We hence hypothesized that iron overload could selectively influence mutant alpha-synuclein toxicity and disease phenotypes. To test the hypothesis, we investigated if Drosophila melanogaster over-expressing A53T, A30P, and wild-type (WT) alpha-synuclein have different responses to iron treatment. We showed that iron treatment induced similar reduction of survival rate in all flies but induced a more severe motor decline in A53T and A30P mutant alpha-synuclein expressing flies, suggesting interaction between mutant alpha-synuclein and iron. Although no significant difference in total head iron content was found among these flies, we demonstrated that iron treatment induced selective DA neuron loss in motor-related PPM3 cluster only in the flies that express A53T and A30P mutant alpha-synuclein. We provided the first in vivo evidence that iron overload could induce distinctive neuropathology and disease phenotypes in mutant but not WT alpha-synuclein expressing flies, providing insights to the cause of clinical features selectively exhibited by mutant alpha-synuclein carriers.

Drosophila models for studying iron-related neurodegenerative diseases.

In recent years, iron has been regarded as a common pathological feature of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and Friedreich's ataxia (FRDA). A number of genes involved in iron transport, storage and regulation have been found associated with initiation and progression of neurodegeneration. However, whether iron abnormalities represent a primary or secondary event still remains unknown. Due to the limitation in transgenic rodent model construction and transfection systems, the progress in unraveling the pathogenic role of different iron-related proteins in neurodegenerative diseases has been slow. Drosophila melanogaster, a simple organism which has a shorter lifespan and smaller genome with many conserved genes, and captures many features of human nervous system and neurodegeneration, may help speed up the progress. The characteristics that spatial- and temporal-specific transgenic Drosophila can be easily constructed and raised in large quantity with phenotype easily determined turn Drosophila into an excellent in vivo genetic system for screening iron-related modifiers in different neurodegenerative conditions and hence provide a better picture about the pathogenic contribution of different iron-related protein abnormalities. It is believed that identification of important iron-related genes that can largely stop or even reverse degenerative process in Drosophila models may lead to development of novel therapeutic strategies against neurodegenerative diseases.

Development of EEG alpha and theta oscillations in the maintenance stage of working memory.

Several studies have shown developmental changes in EEG oscillations during working memory tasks. Although the load-modulated theta and alpha activities in adults are well-documented, the findings are inconsistent if children possess the adult-like brain oscillations that are similarly modulated by memory load. The present study compares children's and adults' true theta and alpha EEG oscillations, separated from aperiodic components, in the maintenance stage of working memory. The EEG was recorded in 25 Chinese-speaking children (14 male, Mage = 9.4 yrs) and 31 adults (19 male, Mage = 20.8 yrs) in Hong Kong while they performed an n-back task that included four conditions differing in load (1- vs. 2-back) and stimulus type (Chinese character vs. visual pattern). The results show that aperiodic activities (i.e., broadband power and slope) during the maintenance stage in the n-back task were significantly higher in children than adults. The periodic theta and alpha oscillations also changed with age. More importantly, adults showed significant periodic theta increase with memory load, whereas such an effect was absent in children. Regardless of age, there was a significant alpha power decrease with load increase, and a significant theta power enhancement when maintaining visual patterns than Chinese characters. In adults, load-modulated alpha peak shift (towards higher frequency) was linked to higher behavioral efficiency in the n-back task. In children, higher load-modulated theta enhancement was linked to better behavioral efficiency. The findings suggest that the load-modulated theta power during working memory maintenance matures from childhood to adulthood.

ENOX2 inhibition enhances infiltration of effector memory T-cell and mediates response to chemotherapy in immune-quiescent nasopharyngeal carcinoma.

INTRODUCTION: The immunosuppressive tumor microenvironment is a major barrier for chemotherapy. Different chemosensitization approaches to reinstate immunological surveillance for cancers that are immune quiescent at the outset, have thus been devised. Cancer-specific ENOX2 expression is correlated with abnormal cell growth and has been proposed as a cellular target for anti-cancer activity. However, the potential effects of ENOX2 on the interaction between immune system and tumor cells remain elusive. OBJECTIVES: To understand the mechanisms by which tumor-intrinsic ENOX2-mediated alterations in anti-tumor activity of T-cells and response to chemotherapy. METHODS: In situ multiplexed immunohistochemistry with single cell and bulk RNA sequencing data from nasopharyngeal carcinoma (NPC) human tissues were used to define tumor phenotypes. Two NPC cell lines, with distinct ENOX2 expression, were used in a co-culture platform to study tumor-immune interactions between cancer cells/spheroids and T-cells. The effect of cisplatin treatment with ENOX2 inhibition by idronoxil (IDX) were tested in vitro and in vivo. Multi-parametric flow cytometry was used to characterize T-cell infiltrates in an NPC tumor humanized mouse model treated with combined treatment. RESULTS: NPC predominantly displayed an immune-excluded profile. This "cold-phenotype" was shown to exhibit higher ENOX2 expression and was associate with poorer progression-free survival (PFS). The therapeutic combination of IDX with cisplatin was effective in promoting CD8+ effector memory T cell (Tem) differentiation and mobilization. This Tem signature was highly cytotoxic, with Tem-mediated preferential lysis of higher ENOX2-expressing NPC cells. A combination-treated humanized mouse model showing dramatic shrinkage in tumors, were intra-tumoral Tem-enriched. CONCLUSION: Tumor-intrinsic ENOX2 expression is associated with tumor phenotype and PFS in NPC. Targeting ENOX2 with IDX and cisplatin impose qualitative control of T-cell response by preferentially increasing immune cells infiltration, Tem differentiation and tumor suppression. We suggest that ENOX2 inhibition may be a promising therapeutic strategy to enhance the effects of chemotherapy.

Shift in Tissue-Specific Immune Niches and CD137 Expression in Tuberculoma of Pembrolizumab-Treated Nasopharyngeal Carcinoma Patients.

The use of immune checkpoint inhibitors (ICIs) in cancer treatment has shown promise but can also have unintended consequences, such as reactivating latent tuberculosis (TB). To develop treatments that address ICIs-related adverse events, it is essential to understand cellular heterogeneity across healthy and pathological tissues. We performed cross-tissue multiplexed staining analysis on samples from two patients with TB reactivation during pembrolizumab treatment for metastatic nasopharyngeal carcinoma. CD8+ T cells, rather than CD4+ T cells, accumulated preferentially in the tuberculoma and were associated with increased production of IFNγ and expression of CD137. Additionally, CD137 enrichment played a role in the spatial organization of the tuberculoma, with specific interaction limited to spatial proximal cells between IFNγ+ CD137+ CD8+ T cells and IL12+ CD137+ type-1 macrophages. This unique feature was not observed in non-tumoral or tumoral tissues. Our analysis of public transcriptomic datasets supported the notion that this cellular interaction was more prominent in patients with durable ICI responses compared to those with non-ICI-related TB. We suggest that shifts towards CD137-rich immune niches are correlated with both off-target immune-related adverse events and anti-tumor efficacy. Targeting the tumor microenvironment through conditional activation of anti-CD137 signaling in combination with ICIs can modulate the reactivity of T cells and macrophages for localized tumor killing without the potential off-target immune-related risks associated with ICIs alone.

Efficient strategies based on behavioral and electrophysiological methods for epilepsy-related gene screening in the Drosophila model.

Introduction: With the advent of trio-based whole-exome sequencing, the identification of epilepsy candidate genes has become easier, resulting in a large number of potential genes that need to be validated in a whole-organism context. However, conducting animal experiments systematically and efficiently remains a challenge due to their laborious and time-consuming nature. This study aims to develop optimized strategies for validating epilepsy candidate genes using the Drosophila model. Methods: This study incorporate behavior, morphology, and electrophysiology for genetic manipulation and phenotypic examination. We utilized the Gal4/UAS system in combination with RNAi techniques to generate loss-of-function models. We performed a range of behavioral tests, including two previously unreported seizure phenotypes, to evaluate the seizure behavior of mutant and wild-type flies. We used Gal4/UAS-mGFP flies to observe the morphological alterations in the brain under a confocal microscope. We also implemented patch-clamp recordings, including a novel electrophysiological method for studying synapse function and improved methods for recording action potential currents and spontaneous EPSCs on targeted neurons. Results: We applied different techniques or methods mentioned above to investigate four epilepsy-associated genes, namely Tango14, Klp3A, Cac, and Sbf, based on their genotype-phenotype correlation. Our findings showcase the feasibility and efficiency of our screening system for confirming epilepsy candidate genes in the Drosophila model. Discussion: This efficient screening system holds the potential to significantly accelerate and optimize the process of identifying epilepsy candidate genes, particularly in conjunction with trio-based whole-exome sequencing.

Children With Chinese Dyslexia Acquiring English Literacy: Interaction Between Cognitive Subtypes of Dyslexia and Orthographies.

This study investigated the impact of Chinese dyslexia subtypes on English literacy skills (i.e., reading fluency and dictation) in Hong Kong children. Eighty-four Cantonese-speaking children officially diagnosed with dyslexia (Mage = 103 months) and 48 age-matched typically developing (TD) children were tested. Cluster analysis with performances on Chinese syllable awareness (CSA), Chinese phonemic awareness (CPA), Chinese phonological memory (CPM), Chinese orthographic awareness (COA), and matrix reasoning (MR) yielded three cognitive subtypes: the phonological deficit (PD) subtype, the orthographic deficit (OD) subtype, and the global deficit (GD) subtype. After controlling for English language experience, age, and gender, all three dyslexia subtype groups performed significantly worse in English word reading fluency and dictation than the TD children. In addition, the PD group performed worse in English PA; the OD group performed worse in English OA; and the GD group performed worse in all English skills except English PM. We compared the level of impairment in literacy between languages and dyslexia subtypes. In word reading fluency, all subtype groups experienced less impairment in English than Chinese, while the OD group showed the largest English advantage. In dictation, only the OD group showed a significant language effect favoring English. The findings suggest that different subtypes of Chinese dyslexia bear different risks for difficulties in English literacy.

Behavioral and neurophysiological aspects of working memory impairment in children with dyslexia.

The present study aimed to identify behavioral and neurophysiological correlates of dyslexia which could potentially predict reading difficulty. One hundred and three Chinese children with and without dyslexia (Grade 2 or 3, 7- to 11-year-old) completed both verbal and visual working memory (n-back) tasks with concurrent EEG recording. Data of 74 children with sufficient usable EEG data are reported here. Overall, the typically developing control group (N = 28) responded significantly faster and more accurately than the group with dyslexia (N = 46), in both types of tasks. Group differences were also found in EEG band power in the retention phase of the tasks. Moreover, forward stepwise logistic regression demonstrated that both behavioral and neurophysiological measures predicted reading difficulty uniquely. Dyslexia was associated with higher frontal midline theta activity and reduced upper-alpha power in the posterior region. This finding is discussed in relation to the neural efficiency hypothesis. Whether these behavioral and neurophysiological patterns can longitudinally predict later reading development among preliterate children requires further investigation.

Vimentin supports mitochondrial morphology and organization.

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.

Expression of Iron Transporters and Pathological Hallmarks of Parkinson's and Alzheimer's Diseases in the Brain of Young, Adult, and Aged Rats.

Iron accumulates progressively in the brain with age; however, the cause is unknown. We hypothesized that iron accumulation may be associated with the age-induced changes in the expression of iron metabolism proteins in the brain. Here, we systematically investigated iron content and the expression of two major iron importers, transferrin receptor 1 (TfR1) and divalent metal transporter (DMT1), two iron exporters, ferroportin 1 (Fpn1) and ceruloplasmin (CP), and hepcidin, along with the pathological hallmarks of Parkinson's (PD) and Alzheimer's diseases (AD) in the brain of young (3 months), adult (12 months), and aged (24 months) rats. We demonstrated that age has a region-specific effect on iron transport proteins along with iron content in the cortex, striatum, hippocampus, and substantia nigra. We also found an age-dependent increase in hyperphosphorylated tau, total beta-amyloid, and neurotoxic oligomeric aggregates in the cortex and hippocampus as well as an increase in α-synuclein and a decrease in tyrosine hydroxylase positive neurons in the substantia nigra. Our findings suggest that the age-dependent increase in brain iron may be partly due to the age-induced increase in DMT1 expression, rather than TfR1 and Fpn1 expression, and also imply that the increased brain iron is associated with expression of the pathological hallmarks of AD and PD.

Reducing iron in the brain: a novel pharmacologic mechanism of huperzine A in the treatment of Alzheimer's disease.

Huperzine A (HupA), a natural inhibitor of acetylcholinesterase derived from a plant, is a licensed anti-Alzheimer's disease (AD) drug in China and a nutraceutical in the United States. In addition to acting as an acetylcholinesterase inhibitor, HupA possesses neuroprotective properties. However, the relevant mechanism is unknown. Here, we showed that the neuroprotective effect of HupA was derived from a novel action on brain iron regulation. HupA treatment reduced insoluble and soluble beta amyloid levels, ameliorated amyloid plaques formation, and hyperphosphorylated tau in the cortex and hippocampus of APPswe/PS1dE9 transgenic AD mice. Also, HupA decreased beta amyloid oligomers and amyloid precursor protein levels, and increased A Disintegrin And Metalloprotease Domain 10 (ADAM10) expression in these treated AD mice. However, these beneficial effects of HupA were largely abolished by feeding the animals with a high iron diet. In parallel, we found that HupA decreased iron content in the brain and demonstrated that HupA also has a role to reduce the expression of transferrin-receptor 1 as well as the transferrin-bound iron uptake in cultured neurons. The findings implied that reducing iron in the brain is a novel mechanism of HupA in the treatment of Alzheimer's disease.

Lipopolysaccharides upregulate hepcidin in neuron via microglia and the IL-6/STAT3 signaling pathway.

Neuroinflammation is closely related to brain iron homeostasis. Our previous study demonstrated that lipopolysaccharides (LPS) can regulate expression of iron-regulatory peptide hepcidin; however, the mechanism is undefined. Here, we demonstrated that intracerebroventricular injection of LPS in rat brain upregulated hepcidin and downregulated ferroportin 1 in the cortex and substantia nigra. LPS increased hepcidin expression in neurons only when they were co-cultured with BV-2 microglia, and the upregulation was suppressed by IL-6 neutralizing antibody in vitro. In addition, IL-6 but not IL-1α, IL-1ß, or tumor necrosis factor-alpha increased hepcidin expression and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cortical neurons and MES23.5 dopaminergic neurons. These effects were blocked by the STAT3 inhibitor, stattic. Our results show that neurons are the major source of increased hepcidin expression in response to LPS challenge but microglia play a key mediator role by releasing IL-6 and recruiting the STAT3 pathway. We conclude that LPS upregulates hepcidin expression in neurons via microglia and the IL-6/STAT3 signaling pathway.