Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing.

By directly converting solar energy and carbon dioxide into biobased products, cyanobacteria are promising chassis for photosynthetic biosynthesis. To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales, exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance. To simplify and accelerate the screening for fast-growing cyanobacteria strains, a method called Individual Cyanobacteria Vitality Tests and Screening (iCyanVS) was established. We show that the 13C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations, thus greatly accelerating the process screening for fastest-growing cells. The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing. Therefore, this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.

Artificial intelligence-assisted automatic and index-based microbial single-cell sorting system for One-Cell-One-Tube.

Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in-depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)-assisted object detection model for cell phenotype screening and a cross-interface contact method for single-cell exporting, we developed an automatic and index-based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a microdroplet and automatically exported in a precisely indexed, "One-Cell-One-Tube" manner. The target cell is automatically identified based on an AI-assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross-interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single-cell culture or sequencing. The efficiency of the system for single-cell printing is >93%. The throughput of the system for single-cell printing is ~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI-assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single-cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single-cell sorting.

Therapeutic potential of stem cells from human exfoliated deciduous teeth infusion into patients with type 2 diabetes depends on basal lipid levels and islet function.

Mesenchymal stem cells (MSCs) hold great potential in treating patients with diabetes, but the therapeutic effects are not always achieved. Particularly, the clinical factors regulating MSC therapy in this setting are largely unknown. In this study, 24 patients with type 2 diabetes mellitus (T2DM) treated with insulin were selected to receive three intravenous infusions of stem cells from human exfoliated deciduous teeth (SHED) over the course of 6 weeks and were followed up for 12 months. We observed a significant reduction of glycosylated serum albumin level (P < .05) and glycosylated hemoglobin level (P < .05) after SHED transplantation. The total effective rate was 86.36% and 68.18%, respectively, at the end of treatment and follow-up periods. Three patients ceased insulin injections after SHED transplantation. A steamed bread meal test showed that the serum levels of postprandial C-peptide at 2 hours were significantly higher than those at the baseline (P < .05). Further analysis showed that patients with a high level of blood cholesterol and a low baseline level of C-peptide had poor response to SHED transplantation. Some patients experienced a transient fever (11.11%), fatigue (4.17%), or rash (1.39%) after SHED transplantation, which were easily resolved. In summary, SHED infusion is a safe and effective therapy to improve glucose metabolism and islet function in patients with T2DM. Blood lipid levels and baseline islet function may serve as key factors contributing to the therapeutic outcome of MSC transplantation in patients with T2DM.

Effects and mechanism of stem cells from human exfoliated deciduous teeth combined with hyperbaric oxygen therapy in type 2 diabetic rats.

OBJECTIVES: Mesenchymal stem cells (MSCs) are potentially ideal for type 2 diabetes treatment, owing to their multidirectional differentiation ability and immunomodulatory properties. Here we investigated whether the stem cells from human exfoliated deciduous teeth (SHED) in combination with hyperbaric oxygen (HBO) could treat type 2 diabetic rats, and explored the underlying mechanism. METHODS: SD rats were used to generate a type 2 diabetes model, which received stem cell therapy, HBO therapy, or both together. Before and after treatment, body weight, blood glucose, and serum insulin, blood lipid, pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6), and urinary proteins were measured and compared. After 6 weeks, rats were sacrificed and their organs were subjected to hematoxylin and eosin staining and immunofluorescence staining for insulin and glucagon; apoptosis and proliferation were analyzed in islet cells. Structural changes in islets were observed under an electron microscope. Expression levels of Pdx1, Ngn3, and Pax4 mRNAs in the pancreas were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: In comparison with diabetic mice, those treated with the combination or SHE therapy showed decreased blood glucose, insulin resistance, serum lipids, and pro-inflammatory cytokines and increased body weight and serum insulin. The morphology and structure of pancreatic islets improved, as evident from an increase in insulin-positive cells and a decrease in glucagon-positive cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of islet cells revealed the decreased apoptosis index, while Ki67 and proliferating cell nuclear antigen staining showed increased proliferation index. Pancreatic expression of Pdx1, Ngn3, and Pax4 was upregulated. CONCLUSION: SHED combined with HBO therapy was effective for treating type 2 diabetic rats. The underlying mechanism may involve SHED-mediated increase in the proliferation and trans-differentiation of islet ß-cells and decrease in pro-inflammatory cytokines and apoptosis of islets.

Pioglitazone Metformin Complex Improves Polycystic Ovary Syndrome Comorbid Psychological Distress via Inhibiting NLRP3 Inflammasome Activation: A Prospective Clinical Study.

OBJECTIVE: This study aimed at investigating the therapeutic effect and mechanism of pioglitazone metformin complex preparation (PM) in polycystic ovary syndrome (PCOS) comorbid psychological distress. METHODS: Seventy-five patients with PCOS comorbid psychological distress were randomly allocated into the PM, metformin, and placebo groups. The primary efficacy measure was the change from baseline to week 12 on the Symptom Checklist 90-R (SCL-90-R) scores. NLRP3 inflammasome, IL-1ß, IL-6, TNF-α, and biochemical parameters were determined at baseline and at week 12. The participants were required to meet the criteria for PCOS (Rotterdam, NIH) and psychological distress (any factor scores of SCL - 90 - R > 2). RESULTS: The participants had significantly high scores on the SCL-90-R scales of anxiety and depression. PM significantly decreased anxiety and depression symptom severity (from 2.31 ± 0.75 to 1.65 ± 0.38, p < 0.001, and from 2.08 ± 0.74 to 1.61 ± 0.46, p = 0.010, at week 12, respectively). PM significantly decreased the expression of NRPL3 and caspase-1. Patients in the PM group experienced a significant reduction in IL-1ß (from 98.42 ± 14.38 to 71.76 ± 13.66, p = 0.02), IL-6 (from 87.51 ± 8.74 to 71.98 ± 15.87, p = 0.02), and TNF-α (from 395.33 ± 88.55 to 281.98 ± 85.69, p = 0.04). PM was superior to metformin in reducing total testosterone (2.24 ± 0.74 versus 3.06 ± 0.83, p = 0.024, at week 12). CONCLUSIONS: This study is the first to reveal that PM alleviates psychological distress via inhibiting NLRP3 inflammasome and improves several markers, including total testosterone.

Effects and mechanism of stem cells from human exfoliated deciduous teeth combined with hyperbaric oxygen therapy in type 2 diabetic rats

OBJECTIVES: Mesenchymal stem cells (MSCs) are potentially ideal for type 2 diabetes treatment, owing to their multidirectional differentiation ability and immunomodulatory properties. Here we investigated whether the stem cells from human exfoliated deciduous teeth (SHED) in combination with hyperbaric oxygen (HBO) could treat type 2 diabetic rats, and explored the underlying mechanism. METHODS: SD rats were used to generate a type 2 diabetes model, which received stem cell therapy, HBO therapy, or both together. Before and after treatment, body weight, blood glucose, and serum insulin, blood lipid, pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6), and urinary proteins were measured and compared. After 6 weeks, rats were sacrificed and their organs were subjected to hematoxylin and eosin staining and immunofluorescence staining for insulin and glucagon; apoptosis and proliferation were analyzed in islet cells. Structural changes in islets were observed under an electron microscope. Expression levels of Pdx1, Ngn3, and Pax4 mRNAs in the pancreas were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: In comparison with diabetic mice, those treated with the combination or SHE therapy showed decreased blood glucose, insulin resistance, serum lipids, and pro-inflammatory cytokines and increased body weight and serum insulin. The morphology and structure of pancreatic islets improved, as evident from an increase in insulin-positive cells and a decrease in glucagon-positive cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of islet cells revealed the decreased apoptosis index, while Ki67 and proliferating cell nuclear antigen staining showed increased proliferation index. Pancreatic expression of Pdx1, Ngn3, and Pax4 was upregulated. CONCLUSION: SHED combined with HBO therapy was effective for treating type 2 diabetic rats. The underlying mechanism may involve SHED-mediated increase in the proliferation and trans-differentiation of islet β-cells and decrease in pro-inflammatory cytokines and apoptosis of islets.