Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

FAM46C-mediated tumor heterogeneity predicts extramedullary metastasis and poorer survival in multiple myeloma.

Cancers originate from a single cell according to Nowell's theory of clonal evolution. The enrichment of the most aggressive clones has been developed and the heterogeneity arises for genomic instability and environmental selection. Multiple myeloma (MM) is a multiple relapse plasma cell cancer generated from bone marrow. Although there were accumulating researches in multiple myeloma pathogenesis, the heterogeneity remains poorly understood. The participants enrolled in this study were 4 EMP+ (EMP, Extramedullary plasmacytoma) and 2 EMP- primarily untreated MM patients. Single cell RNA sequencing and analysis were conducted for the single cell suspension, which was sorted by flow cytometry from peripheral blood mononuclear cells or bone marrow cells. In our research, the results of single cell RNA sequencing show that FAM46C determines MM tumor heterogeneity predicting extramedullary metastasis by influencing RNA stability. Further, we integrated and analyzed 2280 multiple myeloma samples from 7 independent datasets, which uncover that FAM46C mediated tumor heterogeneity predicts poorer survival in multiple myeloma.

Quantitative haplotype-resolved analysis of mitochondrial DNA heteroplasmy in Human single oocytes, blastoids, and pluripotent stem cells.

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life.

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions.

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.

Inflammation Markers Have Important Value in Predicting Relapse in Patients with papillary thyroid carcinoma: A Long-Term Follow-Up Retrospective Study.

PURPOSE: Many markers of inflammation are increasingly found to have prognostic significance in some cancers. This study investigated the prognostic value of albumin/globulin (AGR), lymphocyte/monocyte ratio (LMR), and other inflammatory markers, including neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR), in patients with papillary thyroid carcinoma (PTC). METHODS: We retrospectively analyzed the data of 764 patients newly diagnosed with PTC (608 women, 156 men) aged 10-83 years. Univariate and multivariate analyses were used to analyze recurrence rates and assess potential prognostic factors. Furthermore, we used random survival forests to construct a random survival forest score (RSFscore). The correlations between various inflammatory factors and traditional prognostic factors were analyzed. We also compared the areas under the curve (AUCs) of the RSFscore and 4 inflammation-based markers. RESULTS: AGR, NLR, PLR, and LMR were strongly associated with invasive clinicopathological features (tumor size, lesions, lymph node metastasis, and lymph node metastasis rate) and postoperative recurrence. In the multivariate analysis, AGR and LMR were independent prognostic markers for recurrent PTC. Higher NLR and PLR values indicated a higher risk of recurrence, while higher LMR and AGR values suggested a lower recurrence risk. The predictive power of the combined indicators was stronger than that of single indicators alone. CONCLUSION: Compared to the analysis of a single indicator, the combination of inflammatory markers was more helpful in determining the risk of PTC recurrence, which has an important impact on predicting patients' cancer-free survival and quality of life.

Histologically resolved multiomics enables precise molecular profiling of human intratumor heterogeneity.

Both the composition of cell types and their spatial distribution in a tissue play a critical role in cellular function, organ development, and disease progression. For example, intratumor heterogeneity and the distribution of transcriptional and genetic events in single cells drive the genesis and development of cancer. However, it can be challenging to fully characterize the molecular profile of cells in a tissue with high spatial resolution because microscopy has limited ability to extract comprehensive genomic information, and the spatial resolution of genomic techniques tends to be limited by dissection. There is a growing need for tools that can be used to explore the relationship between histological features, gene expression patterns, and spatially correlated genomic alterations in healthy and diseased tissue samples. Here, we present a technique that combines label-free histology with spatially resolved multiomics in unfixed and unstained tissue sections. This approach leverages stimulated Raman scattering microscopy to provide chemical contrast that reveals histological tissue architecture, allowing for high-resolution in situ laser microdissection of regions of interests. These microtissue samples are then processed for DNA and RNA sequencing to identify unique genetic profiles that correspond to distinct anatomical regions. We demonstrate the capabilities of this technique by mapping gene expression and copy number alterations to histologically defined regions in human oral squamous cell carcinoma (OSCC). Our approach provides complementary insights in tumorigenesis and offers an integrative tool for macroscale cancer tissues with spatial multiomics assessments.

Fasting serum glucose and lymph node metastasis in non-diabetic PTC patients: a 10-Year multicenter retrospective study.

Background: Mostly current studies are limited to the impact of lymph node metastasis(LNM) on the prognosis of papillary thyroid cancer(PTC) or the impact of glucose metabolism on the occurrence of PTC, but no one has paid attention to the connection between fasting serum glucose(FSG) and LNM. The purpose of our study was to explore the relationship between FSG and LNM in non-diabetic PTC patients. Methods: In this study, we performed a multicenter, retrospective study on 6034 non-diabetic patients with PTC. The associations of FSG with three types of LNM including central lymph node metastasis (CLNM), lateral cervical lymph node metastasis (LLNM) and both were estimated. Results: Compared with PTC patients without LNM, those with LNM had higher FSG. We also found that FSG was associated with tumor extension, maximum tumor diameter and TSH. In order to further explore the association between FSG and different types of LNM, we analyzed three groups of data separately. Our study reveals that by comparing FSG between patients without LNM and patients with three LNM types, it was statistically different in the PTC patients with CLNM and the PTC patients with CLNM combined with LLNM. Conclusion: Our study provides evidence for the association of FSG and LNM in non-diabetic PTC patients, with a gradual increase in FSG over the course of the PTC from no lymph node metastasis to CLNM combined with LLNM. Meanwhile, higher FSG is a risk factor for CLNM and CLNM combined with LLNM. In the future, FSG might be used as an indicator for lymph node dissection in PTC patients. However, larger relative studies are needed.

Combined prognostic value of preoperative serum thyrotrophin and thyroid hormone concentration in papillary thyroid cancer.

BACKGROUND: A growing number of studies have found a close association between thyroid hormones and thyrotrophin (TSH), and they also have prognostic significance in some cancer types; this study aimed to investigate the prognostic value of free triiodothyronine (fT3), free thyroxine (fT4), fT3/fT4, TSH, and their combination in patients with papillary thyroid carcinoma (PTC). METHODS: This study retrospectively analyzed the relevant data of 726 newly diagnosed PTC patients. Both univariate and multivariate analyses were used to predict the recurrence rate, and a risk score was established. In addition, with the use of a random survival forest, a random forest (RF) score was constructed. After calculating the area under the curve (AUC), the diagnostic efficacy of risk score, RF score, and four indicators was compared. RESULTS: fT3, fT4, fT3/fT4, and TSH were strongly associated with some invasive clinicopathological features and postoperative recurrence. Patients with high expression of fT4 and TSH have a high risk of recurrence. By contrast, patients with high expression of fT3 and fT3/fT4 have a low risk of recurrence. At the same time, the combined use of various indicators is more helpful for establishing an accurate diagnosis. By comparison, we found that the RF score was better than the risk score in terms of predicting the recurrence of PTC. CONCLUSION: The diagnostic accuracy of a combination of fT3, fT4, fT3/fT4, and TSH can help improve our clinical estimate of the risk of recurrent PTC, thus allowing the development of a more effective treatment plan for patients.

Computational Identification of Preneoplastic Cells Displaying High Stemness and Risk of Cancer Progression.

Evidence points toward the differentiation state of cells as a marker of cancer risk and progression. Measuring the differentiation state of single cells in a preneoplastic population could thus enable novel strategies for early detection and risk prediction. Recent maps of somatic mutagenesis in normal tissues from young healthy individuals have revealed cancer driver mutations, indicating that these do not correlate well with differentiation state and that other molecular events also contribute to cancer development. We hypothesized that the differentiation state of single cells can be measured by estimating the regulatory activity of the transcription factors (TF) that control differentiation within that cell lineage. To this end, we present a novel computational method called CancerStemID that estimates a stemness index of cells from single-cell RNA sequencing data. CancerStemID is validated in two human esophageal squamous cell carcinoma (ESCC) cohorts, demonstrating how it can identify undifferentiated preneoplastic cells whose transcriptomic state is overrepresented in invasive cancer. Spatial transcriptomics and whole-genome bisulfite sequencing demonstrated that differentiation activity of tissue-specific TFs was decreased in cancer cells compared with the basal cell-of-origin layer and established that differentiation state correlated with differential DNA methylation at the promoters of these TFs, independently of underlying NOTCH1 and TP53 mutations. The findings were replicated in a mouse model of ESCC development, and the broad applicability of CancerStemID to other cancer-types was demonstrated. In summary, these data support an epigenetic stem-cell model of oncogenesis and highlight a novel computational strategy to identify stem-like preneoplastic cells that undergo positive selection. SIGNIFICANCE: This study develops a computational strategy to dissect the heterogeneity of differentiation states within a preneoplastic cell population, allowing identification of stem-like cells that may drive cancer progression.

Optical Cell Tagging for Spatially Resolved Single-Cell RNA Sequencing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for profiling gene expression of distinct cell populations at the single-cell level. However, the information of the positions of cells within the multicellular samples is missing in scRNA-seq datasets. To overcome this limitation, we herein develop OpTAG (optical cell tagging) as a new chemical platform for attaching functional tags onto cell surfaces in a spatially resolved manner. With OpTAG, we establish OpTAG-seq, which enables spatially resolved scRNA-seq. We apply OpTAG-seq to investigate the spatially defined transcriptional program in migrating cancer cells and identified a list of genes that are potential regulators for cancer cell migration and invasion. OpTAG-seq provides a convenient method for mapping cellular heterogeneity with spatial information within multicellular biological systems.

Maturation Process, Nutritional Profile, Bioactivities and Utilisation in Food Products of Red Pitaya Fruits: A Review.

Red pitaya (Hylocereus polyrhizus, red pulp with pink peel), also known as dragon fruit, is a well-known species of pitaya fruit. Pitaya seeds and peels have been reported to exhibit higher concentrations of total polyphenols, beta-cyanins and amino acid than pulp, while anthocyanins (i.e., cyanidin 3-glucoside, delphinidin 3-glucoside and pelargonidin 3-glucoside) were only detected in the pulp extracts. Beta-cyanins, phenolics and flavonoids were found to increase gradually during fruit maturation and pigmentation appeared earlier in the pulp than peel. The phytochemicals were extracted and purified by various techniques and broadly used as natural, low-cost, and beneficial healthy compounds in foods, including bakery, wine, dairy, meat and confectionery products. These bioactive components also exhibit regulative influences on the human gut microbiota, glycaemic response, lipid accumulation, inflammation, growth of microbials and mutagenicity, but the mechanisms are yet to be understood. The objective of this study was to systematically summarise the effect of red pitaya's maturation process on the nutritional profile and techno-functionality in a variety of food products. The findings of this review provide valuable suggestions for the red pitaya fruit processing industry, leading to novel formulations supported by molecular research.

Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia (HOL, n=22) and dysplasia (DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate (86%) than oral leukoplakia with hyperplasia (46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation (MT, n=19) and leukoplakia recurrence (LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease (FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.

A body map of somatic mutagenesis in morphologically normal human tissues.

Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.

Copy number alteration profiling facilitates differential diagnosis between ossifying fibroma and fibrous dysplasia of the jaws.

Ossifying fibroma (OF) and fibrous dysplasia (FD) are two fibro-osseous lesions with overlapping clinicopathological features, making diagnosis challenging. In this study, we applied a whole-genome shallow sequencing approach to facilitate differential diagnosis via precise profiling of copy number alterations (CNAs) using minute amounts of DNA extracted from morphologically correlated microdissected tissue samples. Freshly frozen tissue specimens from OF (n = 29) and FD (n = 28) patients were obtained for analysis. Lesion fibrous tissues and surrounding normal tissues were obtained by laser capture microdissection (LCM), with ~30-50 cells (5 000-10 000 µm2) per sample. We found that the rate of recurrent CNAs in OF cases was much higher (44.8%, 13 of 29) than that in FD cases (3.6%, 1 of 28). Sixty-nine percent (9 of 13) of the CNA-containing OF cases involved segmental amplifications and deletions on Chrs 7 and 12. We also identified eight CNA-associated genes (HILPDA, CALD1, C1GALT1, MICALL2, PHF14, AIMP2, MDM2, and CDK4) with amplified expression, which was consistent with the copy number changes. We further confirmed a jaw lesion with a previous uncertain diagnosis due to its ambiguous morphological features and the absence of GNAS mutation as OF based on the typical Chr 12 amplification pattern in its CNA profile. Moreover, analysis of a set of longitudinal samples collected from an individual with a cellular lesion in suspicion of OF at the first surgery, recurrence and the latest malignant transformation revealed identical CNA patterns at the three time points, suggesting that copy number profiling can be used as an important tool to identify borderline lesions or lesions with malignant potential. Overall, CNA profiling of fibro-osseous lesions can greatly improve differential diagnosis between OF and FD and help predict disease progression.

Analysis of multi-omics differences in left-side and right-side colon cancer.

BACKGROUND: Colon cancer is one of the most common tumors in the digestive tract. Studies of left-side colon cancer (LCC) and right-side colon cancer (RCC) show that these two subtypes have different prognoses, outcomes, and clinical responses to chemotherapy. Therefore, a better understanding of the importance of the clinical classifications of the anatomic subtypes of colon cancer is needed. METHODS: We collected colon cancer patients' transcriptome data, clinical information, and somatic mutation data from the Cancer Genome Atlas (TCGA) database portal. The transcriptome data were taken from 390 colon cancer patients (172 LCC samples and 218 RCC samples); the somatic mutation data included 142 LCC samples and 187 RCC samples. We compared the expression and prognostic differences of LCC and RCC by conducting a multi-omics analysis of each using the clinical characteristics, immune microenvironment, transcriptomic differences, and mutation differences. The prognostic signatures was validated using the internal testing set, complete set, and external testing set (GSE39582). We also verified the independent prognostic value of the signature. RESULTS: The results of our clinical characteristic analysis showed that RCC had a significantly worse prognosis than LCC. The analysis of the immune microenvironment showed that immune infiltration was more common in RCC than LCC. The results of differential gene analysis showed that there were 360 differentially expressed genes, with 142 upregulated genes in LCC and 218 upregulated genes in RCC. The mutation frequency of RCC was generally higher than that of LCC. BRAF and KRAS gene mutations were the dominant genes mutations in RCC, and they had a strong mutual exclusion with APC, while APC gene mutation was the dominant gene mutation in LCC. This suggests that the molecular mechanisms of RCC and LCC differed. The 4-mRNA and 6-mRNA in the prognostic signatures of LCC and RCC, respectively, were highly predictive and may be used as independent prognostic factors. CONCLUSION: The clinical classification of the anatomic subtypes of colon cancer is of great significance for early diagnosis and prognostic risk assessment. Our study provides directions for individualized treatment of left and right colon cancer.

The landscape of tumors-infiltrate immune cells in papillary thyroid carcinoma and its prognostic value.

INTRODUCTION: Thyroid cancer is a very common malignant tumor in the endocrine system, while the incidence of papillary thyroid carcinoma (PTC) throughout the world also shows a trend of increase year by year. In this study, we constructed two models: ICIscore and Riskscore. Combined with these two models, we can make more accurate and reasonable inferences about the prognosis of PTC patients. METHODS: We selected 481 PTC samples from TCGA and 147 PTC samples from GEO (49 samples in GSE33630, 65 samples in GSE35570 and 33 samples in GSE60542). We performed consistent clustering for them and divided them into three subgroups and screened differentially expressed genes from these three subgroups. Then we divided the differential genes into three subtypes. We also distinguished the up-regulated and down-regulated genes and calculated ICIscore for each PTC sample. ICIscore consists of two parts: (1) the PCAu was calculated from up-regulated genes. (2) the PCAd was calculated from down-regulated genes. The PCAu and PCAd of each sample were the first principal component of the relevant gene. What's more, we divided the patients into two groups and constructed mRNA prognostic signatures. Additionally we also verified the independent prognostic value of the signature. RESULTS: Though ICIscore, we were able to observe the relationship between immune infiltration and prognosis. The result suggests that the activation of the immune system may have both positive and negative consequences. Though Riskscore, we could make more accurate predictions about the prognosis of patients with PTC. Meanwhile, we also generated and validated the ICIscore group and Riskscore group respectively. CONCLUSION: All the research results show that by combining the two models constructed, ICIscore and Riskscore, we can make a more accurate and reasonable inference about the prognosis of patients with clinical PTC patients. This suggests that we can provide more effective and reasonable treatment plan for clinical PTC patients.

Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion.

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

Enhancing KDM5A and TLR activity improves the response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) therapies are now established as first-line treatments for multiple cancers, but many patients do not derive long-term benefit from ICB. Here, we report that increased amounts of histone 3 lysine 4 demethylase KDM5A in tumors markedly improved response to the treatment with the programmed cell death protein 1 (PD-1) antibody in mouse cancer models. In a screen for molecules that increased KDM5A abundance, we identified one (D18) that increased the efficacy of various ICB agents in three murine cancer models when used as a combination therapy. D18 potentiated ICB efficacy through two orthogonal mechanisms: (i) increasing KDM5A abundance, which suppressed expression of the gene PTEN (encoding phosphatase and tensin homolog) and increased programmed cell death ligand 1 abundance through a pathway involving PI3K-AKT-S6K1, and (ii) activating Toll-like receptors 7 and 8 (TLR7/8) signaling pathways. Combination treatment increased T cell activation and expansion, CD103+ tumor-infiltrating dendritic cells, and tumor-associated M1 macrophages, ultimately enhancing the overall recruitment of activated CD8+ T cells to tumors. In patients with melanoma, a high KDM5A gene signature correlated with KDM5A expression and could potentially serve as a marker of response to anti-PD-1 immunotherapy. Furthermore, our results indicated that bifunctional agents that enhance both KDM5A and TLR activity warrant investigation as combination therapies with ICB agents.

Surfactant and oil formulations for monodisperse droplet emulsion PCR.

Emulsion PCR has become a popular and widely applied method in biological research and clinical diagnostics to provide evenly amplified products and perform highly quantitative counting of target sequences. However, there is still a lack of information to support further development of appropriate water-in-oil emulsion formulations, which need to be both thermally and mechanically stable for digital amplification reactions. Here, we present a systematic survey of the oil and surfactant components of stable monodisperse w/o emulsions suitable for use with our previously developed micro-capillary array (MiCA)-based centrifugal emulsion generation method. Our findings show that a binary formula consisting of isopropyl palmitate and a silicone copolymer demonstrated the best performance, and provided a general guideline for the development of emulsion systems for digital PCR and emulsion amplification applications.

Single-cell RNA sequencing reveals chemokine self-feeding of myeloma cells promotes extramedullary metastasis.

In this study, we aimed to determine the mechanisms underlying the initial extramedullary translocation of myeloma cells from bone marrow into peripheral blood. We found that clonal circulating plasma cells (cPCs) are more frequently detected by flow cytometry in extramedullary plasmacytoma (EMP) patients and worsen their prognosis. It is technically much easier to collect single cPCs using FACS than it is to perform EMP biopsy. Therefore, combining EMP imaging with cPC detection may be a promising strategy for prognostic stratification. Here, using single-cell transcriptome analysis, we found that the chemokine CXCL12, a key molecule involved in CXCR4-dependent cell retention in the bone marrow, is abnormally upregulated in cPCs and might initially enable cPCs to evade bone marrow retention and translocate into the bloodstream.