Digital PCR for Single-Cell Analysis.

Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.

Spatial Omics Sequencing Based on Microfluidic Array Chips.

Spatial profiling technologies fill the gap left by the loss of spatial information in traditional single-cell sequencing, showing great application prospects. After just a few years of quick development, spatial profiling technologies have made great progress in resolution and simplicity. This review introduces the development of spatial omics sequencing based on microfluidic array chips and describes barcoding strategies using various microfluidic designs with simplicity and efficiency. At the same time, the pros and cons of each strategy are compared. Moreover, commercialized solutions for spatial profiling are also introduced. In the end, the future perspective of spatial omics sequencing and research directions are discussed.

Droplet-based transcriptome profiling of individual synapses.

Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.

Risk Factors and Spatial-Temporal Analysis of Porcine Reproductive and Respiratory Syndrome Seroprevalence in China Before and After African Swine Fever Outbreak.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious viral disease that causes great harm to the pig industry. PRRS virus (PRRSV), the causative agent of PRRS, is characterized by severe reproductive failure and respiratory confusion. This study performed a cross-sectional investigation of PRRSV seroprevalence and collected 14,134 serum samples in pig farms without PRRSV vaccination from 12 provinces and two cities in China from 2017 to 2021 to detect PRRSV antibodies by enzyme-linked immunosorbent assay (ELISA). The apparent and true PRRSV antibody prevalence was estimated and compared based on the Clopper-Pearson method and Pearson chi-square test, respectively. Risk factors associated with the PRRSV serological status of pig farms were analyzed through univariate and multivariable logistic regression analysis. An automatic autoregressive integrated moving average (ARIMA) model procedure was used for time-series analysis for PRRSV seroprevalence. Spatial clusters of high PRRSV seroprevalence were detected by SaTScan software. The total true PRRSV seroprevalence of the animal level was 62.56% (95% confidence interval [CI]: 61.74-63.37%). Additionally, 286 out of 316 pig farms were positive for PRRSV antibodies at the herd level. Pig farms without pseudorabies virus (PRV) infection were 5.413 (95% CI: 1.977-17.435) times more likely to be PRRSV antibody positive than those with PRV. Identically, the possibility of pig farms being PRRSV antibody positive before an African swine fever (ASF) outbreak was 3.104 (95% CI: 1.122-10.326) times more than after ASF. The odd ratio values of medium and large pig farms with PRRSV infection are 3.076 (95% CI: 1.005-9.498) and 6.098 (95% CI: 1.814-21.290). A fluctuant decline pattern for PRRSV prevalence was observed in the temporal analysis. Three significant clusters of high PRRSV seroprevalence were first detected in China, covering a time frame from January 2018 to September 2018, which reveals high PRRSV prevalence before the outbreak of ASF. These findings show the epidemic situation and spatial-temporal distribution of PRRSV infection in China in recent years and could help develop reasonable measures to prevent PRRSV infection.

Microfluidics Facilitates the Development of Single-Cell RNA Sequencing.

Single-cell RNA sequencing (scRNA-seq) technology provides a powerful tool for understanding complex biosystems at the single-cell and single-molecule level. The past decade has been a golden period for the development of single-cell sequencing, with scRNA-seq undergoing a tremendous leap in sensitivity and throughput. The application of droplet- and microwell-based microfluidics in scRNA-seq has contributed greatly to improving sequencing throughput. This review introduces the history of development and important technical factors of scRNA-seq. We mainly focus on the role of microfluidics in facilitating the development of scRNA-seq technology. To end, we discuss the future directions for scRNA-seq.

A Specific and Sensitive Aptamer-Based Digital PCR Chip for Salmonella typhimurium Detection.

Food poisoning and infectious diseases caused by Salmonella typhimurium (S. typhimurium) are serious public health concerns for human health and food safety. The diversity and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of S. typhimurium in food samples. A method capable of identification, detection, and quantification of S. typhimurium is essential for addressing these issues. In this study, aptamer-coated magnetic beads (Apt-MBs) are employed as capture bio-probes to specifically and selectively concentrate S. typhimurium in food samples. A self-priming chip-based digital PCR was then presented as another biosensor for on-site detection and quantification of S. typhimurium cells. The chip we developed was robust and did not require any external power for sample loading. The combination of Apt-MBs with an on-chip digital detection realized the integration into lab-on-a-chip-based biosensors for on-site monitoring of foodborne pathogens. It was possible to capture and detect S. typhimurium cells as low as 90 CFU/reaction with a capture efficiency of 94.5%. Additionally, the whole process only took about 2 h. This unique platform could also be used to monitor other target bacteria with high specificity and sensitivity by utilizing different aptamers. Furthermore, the platform has potential applications in point-of-care testing in the future.

A large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking based on a thermosetting oil.

The rapid and accurate detection of viable bacteria is of great importance in food quality monitoring and clinical diagnosis. Escherichia coli (E. coli) is a major pathogenic bacterium, which causes potential threats to food safety and human health. Therefore, rapid and portable methods for preventing E. coli outbreaks are needed. Single cell analysis can be performed at the single-cell level, which has great advantages for analysis and diagnosis. Herein, we employed a thermosetting oil to generate a large-scale pico-droplet array for viable bacteria digital counting and dynamic tracking. In this array, the droplets can be solidified without any inducers due to the cross-linking reaction of the hydrosilation of vinyl silicone oil and hydrosilicone oil. Single E. coli cells were encapsulated in solidified droplets to form a microcolony. Resazurin was used as a fluorescent indicator to achieve amplification of bacterial growth signals. This method can achieve digital counting of viable E. coli cells in 4 h. We achieved real-time monitoring of E. coli cell growth and division in droplets. It is rapid, simple, and does not require a pre-enrichment process when compared to the traditional plate counting method. We successfully applied the method for the enumeration of E. coli in milk. In conclusion, the thermosetting oil enables the immobilization of droplets to achieve real-time monitoring and digital counting of bacterial growth without impairing the flexibility of droplet microfluidics, and it has the potential to provide dynamic information at high resolution in this process.

Single-PanIN-seq unveils that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence.

ARID1A is one of the most frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and promoters. These broad and complex effects make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Here, we identified the anti-senescence effect of Arid1a deficiency in the progression of pancreatic intraepithelial neoplasia (PanIN) by profiling the transcriptome of individual PanINs in a mouse model. In a human cell line model, we found that ARID1A deficiency upregulates the expression of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which plays an essential role in attenuating the senescence induced by oncogenic KRAS through scavenging reactive oxygen species. As a subunit of the SWI/SNF chromatin remodeling complex, our ATAC sequencing data showed that ARID1A deficiency increases the accessibility of the enhancer region of ALDH1A1. This study provides the first evidence that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence through the upregulation of ALDH1A1 expression.

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding.

Droplet-based single cell sequencing technologies, such as inDrop, Drop-seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.

Transcriptomic and epigenetic regulation of hair cell regeneration in the mouse utricle and its potentiation by Atoh1.

The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.

Phenotypic heterogeneity of intellectual disability in patients with congenital insensitivity to pain with anhidrosis: A case report and literature review.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive heterogeneous disorder mainly caused by mutations in the neurotrophic tyrosine receptor kinase 1 gene ( NTRK1) and characterized by insensitivity to noxious stimuli, anhidrosis, and intellectual disability. We herein report the first north Han Chinese patient with CIPA who exhibited classic phenotypic features and severe intellectual disability caused by a homozygous c.851-33T>A mutation of NTRK1, resulting in aberrant splicing and an open reading frame shift. We reviewed the literature and performed in silico analysis to determine the association between mutations and intellectual disability in patients with CIPA. We found that intellectual disability was correlated with the specific Ntrk1 protein domain that a mutation jeopardized. Mutations located peripheral to the Ntrk1 protein do not influence important functional domains and tend to cause milder symptoms without intellectual disability. Mutations that involve critical amino acids in the protein are prone to cause severe symptoms, including intellectual disability.

Effective detection of variation in single-cell transcriptomes using MATQ-seq.

The quantification of transcriptional variation in single cells, particularly within the same cell population, is currently limited by the low sensitivity and high technical noise of single-cell RNA-seq assays. We report multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), a highly sensitive and quantitative method for single-cell sequencing of total RNA. By systematically determining technical noise, we show that MATQ-seq captures genuine biological variation between whole transcriptomes of single cells.

SIX2 haploinsufficiency causes conductive hearing loss with ptosis in humans.

The ossicles represent one of the most fundamental morphological features in evolutionary biology of the mammalians. The mobile ossicular morphology abnormalities result in the severe conductive hearing loss. Development and patterning of the middle ear malformation depend on genetic and environmental causes. However, the genetic basis for the risk of congenital ossicle malformation is poorly understood. We show here nine affected individuals in a Chinese pedigree who had bilateral conductive hearing loss with ptosis. We performed whole-genome sequencing and array comparative genomic hybridization (CGH) analysis on DNA samples from the Chinese pedigree. We confirmed the presence of a novel 60 kb heterozygous deletion in size, encompassing SIX2 in our family. Mutation screening in 169 sporadic cases with external ear and middle ear malformations identified no pathogenic variant or polymorphism. We suggest SIX2 haploinsufficiency as a potential congenital factor could be attributed to developmental malformation of the middle ear ossicles and upper eyelid. To the best of our knowledge, this is the first report to provide a description of copy number variation in the SIX2 gene resulting in syndromic conductive hearing loss.

Molecular and clinical analyses of 16q24.1 duplications involving FOXF1 identify an evolutionarily unstable large minisatellite.

BACKGROUND: Point mutations or genomic deletions of FOXF1 result in a lethal developmental lung disease Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins. However, the clinical consequences of the constitutively increased dosage of FOXF1 are unknown. METHODS: Copy-number variations and their parental origin were identified using a combination of array CGH, long-range PCR, DNA sequencing, and microsatellite analyses. Minisatellite sequences across different species were compared using a gready clustering algorithm and genome-wide analysis of the distribution of minisatellite sequences was performed using R statistical software. RESULTS: We report four unrelated families with 16q24.1 duplications encompassing entire FOXF1. In a 4-year-old boy with speech delay and a café-au-lait macule, we identified an ~15 kb 16q24.1 duplication inherited from the reportedly healthy father, in addition to a de novo ~1.09 Mb mosaic 17q11.2 NF1 deletion. In a 13-year-old patient with autism and mood disorder, we found an ~0.3 Mb duplication harboring FOXF1 and an ~0.5 Mb 16q23.3 duplication, both inherited from the father with bipolar disorder. In a 47-year old patient with pyloric stenosis, mesenterium commune, and aplasia of the appendix, we identified an ~0.4 Mb duplication in 16q24.1 encompassing 16 genes including FOXF1. The patient transmitted the duplication to her daughter, who presented with similar symptoms. In a fourth patient with speech and motor delay, and borderline intellectual disability, we identified an ~1.7 Mb FOXF1 duplication adjacent to a large minisatellite. This duplication has a complex structure and arose de novo on the maternal chromosome, likely as a result of a DNA replication error initiated by the adjacent large tandem repeat. Using bioinformatic and array CGH analyses of the minisatellite, we found a large variation of its size in several different species and individuals, demonstrating both its evolutionarily instability and population polymorphism. CONCLUSIONS: Our data indicate that constitutional duplication of FOXF1 in humans is not associated with any pediatric lung abnormalities. We propose that patients with gut malrotation, pyloric or duodenal stenosis, and gall bladder agenesis should be tested for FOXF1 alterations. We suggest that instability of minisatellites greater than 1 kb can lead to structural variation due to DNA replication errors.