Expert-Level Immunofixation Electrophoresis Image Recognition based on Explainable and Generalizable Deep Learning.

BACKGROUND: Immunofixation electrophoresis (IFE) is important for diagnosis of plasma cell disorders (PCDs). Manual analysis of IFE images is time-consuming and potentially subjective. An artificial intelligence (AI) system for automatic and accurate IFE image recognition is desirable. METHODS: In total, 12 703 expert-annotated IFE images (9182 from a new IFE imaging system and 3521 from an old one) were used to develop and test an AI system that was an ensemble of 3 deep neural networks. The model takes an IFE image as input and predicts the presence of 8 basic patterns (IgA-, IgA-, IgG-, IgG-, IgM-, IgM-, light chain and ) and their combinations. Score-based class activation maps (Score-CAMs) were used for visual explanation of the models prediction. RESULTS: The AI model achieved an average accuracy, sensitivity, and specificity of 99.82, 93.17, and 99.93, respectively, for detection of the 8 basic patterns, which outperformed 4 junior experts with 1 years experience and was comparable to a senior expert with 5 years experience. The Score-CAMs gave a reasonable visual explanation of the prediction by highlighting the target aligned regions in the bands and indicating potentially unreliable predictions. When trained with only the new system images, the models performance was still higher than junior experts on both the new and old IFE systems, with average accuracy of 99.91 and 99.81, respectively. CONCLUSIONS: Our AI system achieved human-level performance in automatic recognition of IFE images, with high explainability and generalizability. It has the potential to improve the efficiency and reliability of diagnosis of PCDs.

Construction and characterization of microsatellite markers for the Schistosoma japonicum isolate from a hilly area of China based on whole genome sequencing.

Schistosoma japonicum had once caused the greatest disease burden in China and has still been transmitted in some hilly areas, for example, in Shitai of Anhui province, where rodents are projected to be the main reservoir. This may lead to a critical need of molecular tools with high efficiency in monitoring the dynamic of the rodent-associated S. japonicum, as an appropriate amount of schistosome input can re-establish its life cycle in a place with snails and then result in the re-emergence of schistosomiasis. Therefore, the goal of this study was to develop high polymorphic microsatellites from the whole genome of rodent-associated S. japonicum strain to monitor its transmission dynamic. We sampled the hilly schistosome isolate from Shitai of Anhui in China and sequenced the parasite with the next-generation sequencing technology. The whole genome was assembled with four different approaches. We then developed 71 microsatellite markers at a genome-wide scale throughout two best assembled genomes. Based on their chromosome mapping and the expected length of targeted sequences, we selected 24 markers for the development of multiplex reactions. Two multiplexes composed of 10 loci were finally developed, and their potential was revealed by their successful application on and capturing the genetic diversity of three schistosome populations. The selected 10 markers, each with clear chromosome location and characteristics, will be greatly useful in tracing the dispersal pathways or/and dynamics of the rodent-associated S. japonicum or others in the hilly area of China or elsewhere.

Breaking the Bottleneck in Anticancer Drug Development: Efficient Utilization of Synthetic Biology.

Natural products have multifarious bioactivities against bacteria, fungi, viruses, cancers and other diseases due to their diverse structures. Nearly 65% of anticancer drugs are natural products or their derivatives. Thus, natural products play significant roles in clinical cancer therapy. With the development of biosynthetic technologies, an increasing number of natural products have been discovered and developed as candidates for clinical cancer therapy. Here, we aim to summarize the anticancer natural products approved from 1950 to 2021 and discuss their molecular mechanisms. We also describe the available synthetic biology tools and highlight their applications in the development of natural products.

Ethnic, geographic, and seasonal differences of vitamin D status among adults in south-west China.

BACKGROUND: There are limited data on vitamin D status of Sichuan province, and no investigation has been carried out on the correlations of 25(OH)D and BTMs between healthy Hans and Tibetans of Sichuan province. This study aimed to examine 25(OH)D levels around Sichuan province and to assess differences by ethnicity, age, gender, sunlight exposure, geographic location, and seasons. METHODS: Blood samples from 2317 healthy adults aged of 18 to 75 years and of Han and Tibetan ethnicities were collected in six regions and during four seasons. Serum 25(OH)D2 and 25(OH)D3 levels were measured by LC-MS/MS method. Serum total P1NP and ß-CTX were measured by immunoassay. RESULTS: Participants aged 18-40 years showed significantly lower 25(OH)D levels than participants aged 41-75 years old (P < .0001). The median serum 25(OH)D level for males was significantly higher than that of females (P < .0001). Serum 25(OH)D levels among four seasons and different districts varied significantly (P < .0001). In addition, the 25(OH)D level of Tibetans was significantly lower than that of Hans, while the serum total P1NP and ß-CTX levels of Tibetans were significantly higher than those of Hans (P < .0001). CONCLUSION: Adult population was more common to have vitamin D deficiency/insufficiency among Tibetans, females, north regions and in spring and winter.

Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis.

Considerable evidence suggests breast cancer metastasis arises from cells undergoing epithelial-to-mesenchymal-transition (EMT) and cancer stem-like cells (CSCs). Using a microfluidic device that enriches migratory breast cancer cells with enhanced capacity for tumor formation and metastasis, we identified genes differentially expressed in migratory cells by high-throughput single-cell RNA-sequencing. Migratory cells exhibited overall signatures of EMT and CSCs with variable expression of marker genes, and they retained expression profiles of EMT over time. With single-cell resolution, we discovered intermediate EMT states and distinct epithelial and mesenchymal sub-populations of migratory cells, indicating breast cancer cells can migrate rapidly while retaining an epithelial state. Migratory cells showed differential profiles for regulators of oxidative stress, mitochondrial morphology, and the proteasome, revealing potential vulnerabilities and unexpected consequences of drugs. We also identified novel genes correlated with cell migration and outcomes in breast cancer as potential prognostic biomarkers and therapeutic targets to block migratory cells in metastasis.

Phase transition and electronic properties of skutterudite-type IrP3 under high pressure.

Binary skutterudite-type IrP3 possesses a unique structural configuration that exhibits unusual electronic, thermoelectric, and dynamical properties and can be applied in thermoelectric generators; IrP3 has unique square (P4) rings stacked with a relatively loose arrangement and thus has been expected to exhibit fascinating evolution in the bonding patterns and electronic properties under high pressure. Herein, we systematically investigated the global energetically stable structures of IrP3 under ambient- and high-pressure conditions using the swarm intelligence-based structure searching technique in combination with first-principles calculations. Our theoretical prediction shows that the skutterudite-type structure with the Im3[combining macron] symmetry is most stable under ambient conditions. An orthorhombic structure with the Pmma space group was predicted to be energetically superior to the Im3[combining macron] phase above 47.60 GPa. The abrupt volume collapse at the corresponding phase boundaries even reached 14.67%, stemming from the abrupt collapse of large voids in the Im3[combining macron] phase. To explore the possibility of the occurrence of pressure-induced metallization and superconducting states under compressive conditions, the electronic band structures were investigated. Our results showed that the Im3[combining macron] phase was a narrow-gap semiconductor with the band gap of 1.04 eV, whereas the high-pressure Pmma IrP3 was a metallic phase with the superconducting transition temperature of 2.40 K. The current results are beneficial for the further understanding of other skutterudite-type compounds under high pressure.

Higher stochasticity in comammox Nitrospira community assembly in upland soils than the adjacent paddy soils at a regional scale.

Understanding the assembly mechanisms of microbial communities, particularly comammox Nitrospira, in agroecosystems is crucial for sustainable agriculture. However, the large-scale distribution and assembly processes of comammox Nitrospira in agricultural soils remain largely elusive. We investigated comammox Nitrospira abundance, community structure, and assembly processes in 16 paired upland peanuts and water-logged paddy soils in south China. Higher abundance, richness, and network complexity of comammox Nitrospira were observed in upland soils than in paddy soils, indicating a preference for upland soils over paddy soils among comammox Nitrospira taxa in agricultural environments. Clade A.2.1 and clade A.1 were the predominant comammox Nitrospira taxa in upland and paddy soils, respectively. Soil pH was the most crucial factor shaping comammox Nitrospira community structure. Stochastic processes were found to predominantly drive comammox Nitrospira community assembly in both upland and paddy soils, with deterministic processes playing a more important role in paddy soils than in upland soils. Overall, our findings demonstrate the higher stochasticity of comammox Nitrospira in upland soils than in the adjacent paddy soils, which may have implications for autotrophic nitrification in acidic agricultural soils.

Population Genetics of Oncomelania hupensis Snails from New-Emerging Snail Habitats in a Currently Schistosoma japonicum Non-Endemic Area.

Schistosomiasis is still one of the most significant neglected tropical diseases worldwide, and China is endemic for Schistosoma japonicum. With its great achievement in schistosomiasis control, the government of China has set the goal to eliminate the parasitic disease at the country level by 2030. However, one major challenge is the remaining huge areas of habitats for the intermediate host Oncomelania hupensis. This is further exacerbated by an increasing number of new emerging snail habitats reported each year. Therefore, population genetics on snails in such areas will be useful in evaluation of snail control effect and/or dispersal. We then sampled snails from new emerging habitats in Taicang of Jiangsu, China, a currently S. japonicum non-endemic area from 2014 to 2017, and performed population genetic analyses based on nine microsatellites. Results showed that all snail populations had low genetic diversity, and most genetic variations originated from within snail populations. The estimated effective population size for the 2015 population was infinitive. All snails could be separated into two clusters, and further DIYABC analysis revealed that both the 2016 and the 2017 populations may derive from the 2015, indicating that the 2017 population must have been missed in the field survey performed in 2016. These findings may have implications in development of more practical guidelines for snail monitoring and control.

Surface Modification of Ultrafiltration Membranes with 1,4-Benzoquinone and Polyetheramines to Improve Fouling Resistance.

Membrane fouling remains a key challenge for membrane separations. Hydrophilic membrane surface modification can mitigate irreversible foulant deposition, thereby improving fouling resistance. We report new hydrophilic membrane coatings based on 1,4-benzoquinone and various commercially available polyetheramines. These coatings, prepared from 1,4-benzoquinone and Jeffamine EDR 148, poly(benzoquinone-Jeffamine EDR 148) (p(BQ-EDR 148)), were used to modify polysulfone (PS) ultrafiltration membranes. In fouling experiments using an oil/water emulsion, membranes exhibited comparable fouling resistance to that of polydopamine (pDA)-modified membranes. Based on contact angle measurements, p(BQ-EDR 148) and pDA-modified membranes have similar levels of hydrophilicity, and both exhibited higher threshold flux values than those of their unmodified analogues. Based on their similar threshold flux values, p(BQ-EDR 148)-modified (76 LMH) and pDA-modified membranes (74 LMH) should have similar fouling resistance. Moreover, the mean pore size of p(BQ-EDR 148)-modified membranes can be tuned, while keeping the pure water permeance constant, by changing the deposition time and molar ratio of benzoquinone to EDR 148 in the modification solution.

In vivo efficiency of praziquantel treatment of single-sex Schistosoma japonicum aged three months old in mice.

Schistosomiasis is a major neglected tropical disease mainly caused by Schistosoma haematobium, S. japonicum and S. mansoni, and results in the greatest disease burden. Mass drug administration (MDA) with praziquantel (PZQ), a single drug only available for the disease, has played a vital role in schistosomiasis control. Therefore, any possibility of selection of the parasites for PZQ resistance or low sensitivity may hamper the 2030's target of global disease elimination. We had experimentally demonstrated the long-term survival and reproductive potential of single-sex (of either sex) S. japonicum infections in definitive hosts mice. What has not yet been adequately addressed is whether the long live single-sex schistosomes remain sensitive to PZQ, and what reproduction potential for those schistosomes surviving treatment may have. We therefore performed experimental mice studies to explore the treatment effectiveness of PZQ (at total doses of 200 or 400 mg/kg, corresponding to the sub-standard or standard treatment doses in humans) for single-sex S. japonicum aged three months old. The results showed that no treatment efficiency was observed on female schistosomes, whereas on male schistosomes only at PZQ 400 mg/kg a significant higher efficiency in reducing worm burdens was observed. Moreover, either schistosome males or females surviving PZQ treatment remained their reproduction potential as normal. The results indicate that long (i.e., three months) live single-sex S. japonicum can easily survive the current treatment strategy, and moreover, any schistosomes, if with PZQ resistance or low sensitivity, could be easily transmitted in nature. Therefore, in order to realize the target for the national and the global schistosomiasis elimination, there is undoubtedly a great need for refining PZQ administration and dosage, looking for alternative therapies, and/or developing vaccines against schistosome.

Modifying structural polymorphs and tuning electronic properties in pressure-stabilized binary Ir-Sb phases.

The search for novel structures and chemical stoichiometry of binary Ir-Sb compounds is of great importance in view of their catalytic applications. Based on the results of swarm structure searching technique combined with density functional theory, we proposed the hitherto unknown Ir-Sb phase diagram in a wide pressure range with various chemical compositions. Besides two ambient pressure phases of IrSb3-Im3̄ and IrSb2-P21/c, five novel phases of IrSb-C2/c, IrSb-P1̄, IrSb2-P4̄21m, IrSb2-I4/mmm and Ir2Sb-Pmmn were identified at high pressures. The phonon dispersion curves reveal that these phases are all dynamically stable. The calculated electronic results show that a mixed behavior of covalent, ionic and metallic bonds simultaneously exits in these novel phases. A pressure-induced electronic topological transition in Ir2Sb-Pmmn phase occurs according to the theoretical electronic band structures, while is not shown in other stoichiometries of the Ir-Sb system. Our work provides a potential opportunity for experimental synthesis of crystal structures with different chemical stoichiometries of the binary Ir-Sb system.

Mesenchymal Stem/Stromal Cell Engulfment Reveals Metastatic Advantage in Breast Cancer.

Twenty percent of breast cancer (BC) patients develop distant metastasis for which there is no cure. Mesenchymal stem/stromal cells (MSCs) in the tumor microenvironment were shown to stimulate metastasis, but the mechanisms are unclear. Here, we identified and quantified cancer cells engulfing stromal cells in clinical samples of BC metastasis by dual immunostaining for EZH2 and ALDH1 expression. Using flow cytometry and a microfluidic single-cell paring and retrieval platform, we show that MSC engulfment capacity is associated with BC cell metastatic potential and generates cells with mesenchymal-like, invasion, and stem cell traits. Whole-transcriptome analyses of selectively retrieved engulfing BC cells identify a gene signature of MSC engulfment consisting of WNT5A, MSR1, ELMO1, IL1RL2, ZPLD1, and SIRPB1. These results delineate a mechanism by which MSCs in the tumor microenvironment promote metastasis and provide a microfluidic platform with the potential to predict BC metastasis in clinical samples.

Plasminogen Activator Inhibitor 1 (PAI1) Promotes Actin Cytoskeleton Reorganization and Glycolytic Metabolism in Triple-Negative Breast Cancer.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression. IMPLICATIONS: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

Hydro-Seq enables contamination-free high-throughput single-cell RNA-sequencing for circulating tumor cells.

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.

The effects of salt concentration and foulant surface charge on hydrocarbon fouling of a poly(vinylidene fluoride) microfiltration membrane.

The effects of inorganic salts and organic hydrocarbons on membrane fouling are often investigated independently. However, in many cases, these foulants are commonly found together, and such mixtures are rarely the subject of fouling studies. In this study, crude oil-in-water emulsions were formulated at three different added NaCl concentrations, 0, 10-3 and 10-1 M. Surface properties, such as surface tension and surface charge, of these emulsions and a poly(vinylidene fluoride) (PVDF) microfiltration (MF) membrane were characterized. The Derjaguin-Landau-Verwey-Overbeek (DLVO) model was utilized to simulate membrane-oil droplet and oil layer-oil droplet surface interactions. The DLVO model qualitatively predicted increasing fouling propensity with increasing emulsion salt concentration. The PVDF MF membrane was challenged with crude oil-in-water emulsions in constant permeate flux crossflow fouling tests to characterize the fouling propensity of the various emulsions, and the results were consistent with the model predictions.

Selective Photomechanical Detachment and Retrieval of Divided Sister Cells from Enclosed Microfluidics for Downstream Analyses.

Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells, a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photomechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube-polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using the presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.

Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme.

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10-100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study.

3D cell culture in the extracellular matrix (ECM), which not only provides structural support to cellular constituents, but also initiates regulatory biochemical cues for a variety of important cell functions in tissue, has become more and more important in understanding cancer pathology and drug testing. Although the ECM-gel has been used in cell culture both in bulk and on-chip, previous studies focused on collective cell behavior rather than single-cell heterogeneity. To track the behavior of each individual cell, we have developed a gel-island chip, which can form thousands of islands containing single cells encapsulated by the desired ECM. Optimized by Poisson's distribution, the device can attain 34% single cell capture efficiency of the exact number of single cells per island. A good culture media exchange rate and high cell viability can be achieved in the gel-islands. The cells in the islands can be automatically counted for high-throughput analysis. As a proof of concept, we monitored the proliferation and differentiation of single Notch+ (stem-like) T47D breast cancer cells. The 3D collagen gel environment was found to be favorable for the stem-like phenotype through better self-renewal and de-differentiation (Notch- to Notch+ transition). More interestingly, we found that the Notch- de-differentiated cells were more resistant to doxorubicin and cisplatin than the Notch+ cells. Combining the 3D ECM culture and single cell resolution, the presented platform can automatically analyze the individual cell behaviors of hundreds of cells using a small amount of drug and reagents.

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.