Recent advances in scaffolding biomaterials for cultivated meat.

The emergence of cultivated meat provides a sustainable and ethical alternative to traditional animal agriculture, highlighting its increasing importance in the food industry. Biomaterial scaffolds are critical components in cultivated meat production for enabling cell adhesion, proliferation, differentiation, and orientation. While there's extensive research on scaffolding biomaterials, applying them to cultivated meat production poses distinct challenges, with each material offering its own set of advantages and disadvantages. This review summarizes the most recent scaffolding biomaterials used in the last five years for cell-cultured meat, detailing their respective advantages and disadvantages. We suggest future research directions and provide recommendations for scaffolds that support scalable, cost-effective, and safe high-quality meat production. Additionally, we highlight commercial challenges cultivated meat faces, encompassing bioreactor design, cell culture mediums, and regulatory and food safety issues. In summary, this review provides a comprehensive guide and valuable insights for researchers and companies in the field of cultivated meat production.

Role of Staphylococcus aureus's Buoyant Density in the Development of Biofilm Associated Antibiotic Susceptibility.

Biofilms are clusters of microorganisms that form at various interfaces, including those between air and liquid or liquid and solid. Due to their roles in enhancing wastewater treatment processes, and their unfortunate propensity to cause persistent human infections through lowering antibiotic susceptibility, understanding and managing bacterial biofilms is of paramount importance. A pivotal stage in biofilm development is the initial bacterial attachment to these interfaces. However, the determinants of bacterial cell choice in colonizing an interface first and heterogeneity in bacterial adhesion remain elusive. Our research has unveiled variations in the buoyant density of free-swimming Staphylococcus aureus cells, irrespective of their growth phase. Cells with a low cell buoyant density, characterized by fewer cell contents, exhibited lower susceptibility to antibiotic treatments (100 µg/mL vancomycin) and favored biofilm formation at air-liquid interfaces. In contrast, cells with higher cell buoyant density, which have richer cell contents, were more vulnerable to antibiotics and predominantly formed biofilms on liquid-solid interfaces when contained upright. Cells with low cell buoyant density were not able to revert to a more antibiotic sensitive and high cell buoyant density phenotype. In essence, S. aureus cells with higher cell buoyant density may be more inclined to adhere to upright substrates.

Detection of MicroRNAs Using Synthetic Toehold Switch in Mammalian Cells.

Engineering synthetic gene circuits to control cellular functions has a broad application in the field of synthetic biology. Synthetic RNA-based switches that can operate at the transcriptional and posttranscriptional level have also drawn significant interest for the application of next-generation therapeutics and diagnostics. Thus, RNA-based switchable platforms are needed to report dynamic cellular mechanisms which play an important role in cell development and diseases. Recently, several RNA-based switches have been designed and utilized for biosensing and molecular diagnostics. However, miRNA-based switches have not been well established or characterized, especially for eukaryotic translational control. Here, we designed a novel synthetic toehold switch for detection of exogenously and endogenously expressed miRNAs in CHO, HeLa, HEK 293, and MDA-MB-231 breast cancer cells. Multiplex detection of miR-155 and miR-21 was tested using two toehold switches to evaluate the orthogonality and programmability of this synthetic platform.

An M cell-targeting recombinant L. lactis vaccine against four H. pylori adhesins.

The acidic environment and enzyme degradation lead to oral vaccines often having little immune effect. Therefore, it is an attractive strategy to study an effective and safe oral vaccine delivery system that can promote gastrointestinal mucosal immune responses and inhibit antigen degradation. Moreover, the antigens uptake by microfold cells (M cells) is the determining step in initiating efficient immune responses. Therefore, M cell-targeting is one promising approach for enhancing oral vaccine potency. In the present study, an M cell-targeting L. lactis surface display system (plSAM) was built to favor the multivalent epitope vaccine antigen (FAdE) to achieve effective gastrointestinal mucosal immunity against Helicobacter pylori. Therefore, a recombinant Lactococcus lactic acid vaccine (LL-plSAM-FAdE) was successfully prepared, and its immunological properties and protective efficacy were analyzed. The results showed that LL-plSAM-FAdE can secretively express the recombinant proteins SAM-FAdE and display the SAM-FAdE on the bacterial cell surface. More importantly, LL-plSAM-FAdE effectively promoted the phagocytosis and transport of vaccine antigen by M cells in the gastrointestinal tract of mice, and simulated high levels of cellular and humoral immune responses against four key H. pylori adhesins (Urease, CagL, HpaA, and Lpp20) in the gastrointestinal tract, thus enabling effective prevention of H. pylori infection and to some extent eliminating H. pylori already present in the gastrointestinal tract. KEY POINTS: • M-cell-targeting L. lactis surface display system LL- plSAM was designed • This system displays H. pylori vaccine-promoted phagocytosis and transport of M cell • A promising vaccine candidate for controlling H. pylori infection was verified.

Recent advances of droplet-based microfluidics for engineering artificial cells.

Artificial cells, synthetic cells, or minimal cells are microengineered cell-like structures that mimic the biological functions of cells. Artificial cells are typically biological or polymeric membranes where biologically active components, including proteins, genes, and enzymes, are encapsulated. The goal of engineering artificial cells is to build a living cell with the least amount of parts and complexity. Artificial cells hold great potential for several applications, including membrane protein interactions, gene expression, biomaterials, and drug development. It is critical to generate robust, stable artificial cells using high throughput, easy-to-control, and flexible techniques. Recently, droplet-based microfluidic techniques have shown great potential for the synthesis of vesicles and artificial cells. Here, we summarized the recent advances in droplet-based microfluidic techniques for the fabrication of vesicles and artificial cells. We first reviewed the different types of droplet-based microfluidic devices, including flow-focusing, T-junction, and coflowing. Next, we discussed the formation of multi-compartmental vesicles and artificial cells based on droplet-based microfluidics. The applications of artificial cells for studying gene expression dynamics, artificial cell-cell communications, and mechanobiology are highlighted and discussed. Finally, the current challenges and future outlook of droplet-based microfluidic methods for engineering artificial cells are discussed. This review will provide insights into scientific research in synthetic biology, microfluidic devices, membrane interactions, and mechanobiology.

Morphology-based deep learning approach for predicting adipogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs).

Human mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes. These cells have been extensively employed in the field of cell-based therapies and regenerative medicine due to their inherent attributes of self-renewal and multipotency. Traditional approaches for assessing hMSCs differentiation capacity have relied heavily on labor-intensive techniques, such as RT-PCR, immunostaining, and Western blot, to identify specific biomarkers. However, these methods are not only time-consuming and economically demanding, but also require the fixation of cells, resulting in the loss of temporal data. Consequently, there is an emerging need for a more efficient and precise approach to predict hMSCs differentiation in live cells, particularly for osteogenic and adipogenic differentiation. In response to this need, we developed innovative approaches that combine live-cell imaging with cutting-edge deep learning techniques, specifically employing a convolutional neural network (CNN) to meticulously classify osteogenic and adipogenic differentiation. Specifically, four notable pre-trained CNN models, VGG 19, Inception V3, ResNet 18, and ResNet 50, were developed and tested for identifying adipogenic and osteogenic differentiated cells based on cell morphology changes. We rigorously evaluated the performance of these four models concerning binary and multi-class classification of differentiated cells at various time intervals, focusing on pivotal metrics such as accuracy, the area under the receiver operating characteristic curve (AUC), sensitivity, precision, and F1-score. Among these four different models, ResNet 50 has proven to be the most effective choice with the highest accuracy (0.9572 for binary, 0.9474 for multi-class) and AUC (0.9958 for binary, 0.9836 for multi-class) in both multi-class and binary classification tasks. Although VGG 19 matched the accuracy of ResNet 50 in both tasks, ResNet 50 consistently outperformed it in terms of AUC, underscoring its superior effectiveness in identifying differentiated cells. Overall, our study demonstrated the capability to use a CNN approach to predict stem cell fate based on morphology changes, which will potentially provide insights for the application of cell-based therapy and advance our understanding of regenerative medicine.

Long non-coding RNA (lncRNA) MALAT1 in regulating osteogenic and adipogenic differentiation using a double-stranded gapmer locked nucleic acid nanobiosensor.

Long non-coding RNAs (lncRNA) are non-protein coding RNA molecules that are longer than 200 nucleotides. The lncRNA molecule plays diverse roles in gene regulation, chromatin remodeling, and cellular processes, influencing various biological pathways. However, probing the complex dynamics of lncRNA in live cells is a challenging task. In this study, a double-stranded gapmer locked nucleic acid (ds-GapM-LNA) nanobiosensor is designed for visualizing the abundance and expression of lncRNA in live human bone-marrow-derived mesenchymal stem cells (hMSCs). The sensitivity, specificity, and stability were characterized. The results showed that this ds-GapM-LNA nanobiosensor has very good sensitivity, specificity, and stability, which allows for dissecting the regulatory roles of cellular processes during dynamic physiological events. By incorporating this nanobiosensor in living hMSC imaging, we elucidated lncRNA MALAT1 expression dynamics during osteogenic and adipogenic differentiation. The data reveal that lncRNA MALAT1 expression is correlated with distinct sub-stages of osteogenic and adipogenic differentiation.

Mitophagy suppresses motor neuron necroptotic mitochondrial damage and alleviates necroptosis that converges to SARM1 in acrylamide-induced dying-back neuropathy.

Acrylamide (ACR), a common industrial ingredient that is also found in many foodstuffs, induces dying-back neuropathy in humans and animals. However, the mechanisms remain poorly understood. Sterile alpha and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the central determinant of axonal degeneration and has crosstalk with different cell death programs to determine neuronal survival. Herein, we illustrated the role of SARM1 in ACR-induced dying-back neuropathy. We further demonstrated the upstream programmed cell death mechanism of this SARM1-dependent process. Spinal cord motor neurons that were induced to overexpress SARM1 underwent necroptosis rather than apoptosis in ACR neuropathy. Mechanically, non-canonical necroptotic pathways mediated mitochondrial permeability transition pore (mPTP) opening, reactive oxygen species (ROS) production, and mitochondrial fission. What's more, the final executioner of necroptosis, phosphorylation-activated mixed lineage kinase domain-like protein (MLKL), aggregated in mitochondrial fractions. Rapamycin intervention removed the impaired mitochondria, inhibited necroptosis for axon maintenance and neuronal survival, and alleviated ACR neuropathy. Our work clarified the functional links among mitophagy, necroptosis, and SARM1-dependent axonal destruction during ACR intoxication, providing novel therapeutic targets for dying-back neuropathies.

Synergistic anti-cancer and attenuation effects of thymosin on chemotherapeutic drug vinorelbine in tumor-bearing zebrafish.

Vinorelbine, the standard chemotherapy drug on advanced lung cancer, causes adverse events such as immunosuppression and bone marrow suppression. Thus, it is necessary to find drugs that could improve immune function and synergistically enhance the anti-tumor effect of vinorelbine. Thymosin is reported to inhibit tumor growth as an immunomodulator. Herein, to study the synergistic anti-cancer and attenuation effects of thymosin on vinorelbine, human lung cancer A549 cells that were labeled with CM-DiI were transplanted into zebrafish to establish the lung cancer xenotransplanted model. After treatment of vinorelbine and different concentrations of thymosin, the fluorescence intensity of CM-DiI-labeled A549 cells and the number of apoptotic muscle cells in the tumor-bearing zebrafish were detected. Besides, effects of thymosin on vinorelbine-reduced macrophages and T cells were identified in the transgenic zebrafish (Tg:zlyz-EGFP and Tg:rag2-DsRed). Then, the qRT-PCR was used to determine the alterations of the immune-related factors at the transcription level. Thymosin showed a marked synergistic anti-cancer effect with vinorelbine for the xenograft human lung cancer A549 cells, and the synergistic effect enhanced in a dose-dependent manner. Moreover, thymosin alleviated vinorelbine-induced muscle cell apoptosis, macrophage reduction, and T cell suppression. Compared with the vinorelbine group, co-administration with thymosin raised the mRNA levels of TNF-α, TNF-ß, INF-γ, and GM-CSF. Thus, thymosin possesses synergistic anti-cancer effect on vinorelbine, and has protective effect on vinorelbine-induced immunosuppression. Thymosin, as an adjuvant immunomodulatory therapy, has great potential in enhancing the clinical application of vinorelbine.

Detection of MicroRNA Expression Dynamics Using LNA/DNA Nanobiosensor.

The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2):81-5, 1989; Stahlberg and Bengtsson, Methods 50(4):282-288, 2010; Sanchez-Freire et al., Nat Protoc 7(5):829-838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible.

Atorvastatin Inhibits High-Fat Diet-Induced Lipid Metabolism Disorders in Rats by Inhibiting Bacteroides Reduction and Improving Metabolism.

Purpose: The prevalence of hyperlipidemia and related illnesses is on its rise, and atorvastatin is the frequently used hypolipidemic agent. However, there is still uncertainty about the mechanisms, especially the relationship between the lipid-lowering effect, intestinal microbiome, and metabolic profiles. We aim to intensively explain the mechanism of the hypolipidemic effect of atorvastatin through multi-omics perspective of intestinal microbiome and metabolomics. Methods: Multi-omics methods play an increasingly important role in the analysis of intestinal triggers and evaluation of metabolic disorders such as obesity, hyperlipidemia, and diabetes. Therefore, we were prompted to explore intestinal triggers, underlying biomarkers, and potential intervention targets of atorvastatin in the treatment of dyslipidemia through multi-omics. To achieve this, SPF Wistar rats were fed a high-fat diet or normal diet for 8 weeks. Atorvastatin was then administered to high-fat diet-fed rats. Results: By altering intestinal microbiome, a high-fat diet can affect feces and plasma metabolic profiles. Treatment with atorvastatin possibly increases the abundance of Bacteroides, thereby improving "propanoate metabolism" and "glycine, serine and threonine metabolism" in feces and plasma, and contributing to blood lipid reduction. Conclusion: Our study elucidated the intestinal triggers and metabolites of high-fat diet-induced dyslipidemia from the perspective of intestinal microbiome and metabolomics. It equally identified potential intervention targets of atorvastatin. This further explains the mechanism of the hypolipidemic effect of atorvastatin from a multi-omics perspective.

Notch signaling and fluid shear stress in regulating osteogenic differentiation.

Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells (hMSCs) are multipotent progenitor cells with the potential to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes, which have been utilized extensively in the field of bone tissue engineering and cell-based therapy. Although fluid shear stress plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood. Here, a locked nucleic acid (LNA)/DNA nanobiosensor was exploited to monitor mRNA gene expression of hMSCs that were exposed to physiologically relevant fluid shear stress to examine the regulatory role of Notch signaling during osteogenic differentiation. First, the effects of fluid shear stress on cell viability, proliferation, morphology, and osteogenic differentiation were investigated and compared. Our results showed shear stress modulates hMSCs morphology and osteogenic differentiation depending on the applied shear and duration. By incorporating this LNA/DNA nanobiosensor and alkaline phosphatase (ALP) staining, we further investigated the role of Notch signaling in regulating osteogenic differentiation. Pharmacological treatment is applied to disrupt Notch signaling to investigate the mechanisms that govern shear stress induced osteogenic differentiation. Our experimental results provide convincing evidence supporting that physiologically relevant shear stress regulates osteogenic differentiation through Notch signaling. Inhibition of Notch signaling mediates the effects of shear stress on osteogenic differentiation, with reduced ALP enzyme activity and decreased Dll4 mRNA expression. In conclusion, our results will add new information concerning osteogenic differentiation of hMSCs under shear stress and the regulatory role of Notch signaling. Further studies may elucidate the mechanisms underlying the mechanosensitive role of Notch signaling in stem cell differentiation.

Mitochondrial Localization of SARM1 in Acrylamide Intoxication Induces Mitophagy and Limits Neuropathy.

Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the defining molecule and central executioner of programmed axon death, also known as Wallerian degeneration. SARM1 has a mitochondrial targeting sequence, and it can bind to and stabilize PTEN-induced putative kinase 1 (PINK1) for mitophagy induction, but the deletion of the mitochondrial localization sequence is found to disrupt the mitochondrial localization of SARM1 in neurons without altering its ability to promote axon degeneration after axotomy. The biological significance of SARM1 mitochondrial localization remains elusive. In this study, we observed that the pro-degeneration factor, SARM1, was upregulated in acrylamide (ACR) neuropathy, a slow, Wallerian-like, programmed axonal death process. The upregulated SARM1 accumulated on mitochondria, interfered with mitochondrial dynamics, and activated PINK1-mediated mitophagy. Importantly, rapamycin (RAPA) intervention eliminated mitochondrial accumulation of SARM1 and partly attenuated ACR neuropathy. Thus, mitochondrial localization of SARM1 may contribute to its clearance through the SARM1-PINK1 mitophagy pathway, which inhibits axonal degeneration through a negative feedback loop. The mitochondrial localization of SARM1 complements the coordinated activity of the pro-survival factor, nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and SARM1 and is part of the self-limiting molecular mechanisms underpinning programmed axon death in ACR neuropathy. Mitophagy clearance of SARM1 is complementary to the coordinated activity of NMNAT2 and SARM1 in ACR neuropathy.

Oral Immunization With a M Cell-Targeting Recombinant L. Lactis Vaccine LL-plSAM-FVpE Stimulate Protective Immunity Against H. Pylori in Mice.

There are many virulence factors of H. pylori that contribute in diverse ways to gastric disease. Therefore, designing multivalent epitope vaccines against many key virulence factors virulence factors of H. pylori is a promising strategy to control H. pylori infection. In previous studies, we constructed a multivalent epitope vaccine FVpE against four key virulence factors of H. pylori (Urease, CagA, VacA, and NAP), and oral immunization with the FVpE vaccine plus a polysaccharide adjuvant (PA) containing lycium barbarum polysaccharide and chitosan could provide protection against H. pylori infection in the Mongolian gerbil model. Oral vaccines have many advantages over injected vaccines, such as improved safety and compliance, and easier manufacturing and administration. However, the harsh gastrointestinal (GI) environment, such as gastric acid and proteolytic enzymes, limits the development of oral vaccines to some extent. Oral vaccines need a gastrointestinal delivery system with high safety, low price and promoting vaccine antigen to stimulate immune response in the gastrointestinal mucosa. Lactic acid bacteria are gastrointestinal probiotics that have unique advantages as a delivery system for oral vaccines. In this study, a M cell-targeting surface display system for L. lactis named plSAM was designed to help vaccine antigens to stimulate effective immune responses in the gastrointestinal tract, and a M cell-targeting recombinant L. lactis vaccine LL-plSAM-FVpE was constructed by using the surface display system plSAM. recombinant L. lactis vaccine LL-plSAM-FVpE could secretively express the SAM-FVpE protein and display it on the bacterial surface. Moreover, experimental results confirmed that LL-plSAM-FVpE had an enhanced M cell-targeting property. In addition, LL-plSAM-FVpE had excellent M cell-targeting property to promote the phagocytosis and transport of the antigen SAM-FVpE by gastrointestinal M cells. More importantly, oral immunization of LL-plSAM-FVpE or SAM-FVpE plus PA can stimulate IgG and sIgA antibodies and CD4+ T cell immune responses against four virulence factors of H. pylori (Urease, CagA, VacA, and NAP), thus providing protective immunity against H. pylori infection in mice. The M cell-targeting recombinant L. lactis vaccine against various key H. pylori virulence factors could be a promising vaccine candidate for controlling H. pylori infection.

Pea Peptide Supplementation in Conjunction With Resistance Exercise Promotes Gains in Muscle Mass and Strength.

It is generally considered that protein supplementation and resistance exercise significantly increase muscle mass and muscle growth. As the hydrolysis products of proteins, peptides may play the crucial role on muscle growth. In this study, male rats were orally administrated 0.4 g/kg body weight of pea peptide combined with 8 weeks of moderate intensity resistance exercise training. After treatment, the body gains, upper limb grip, muscle thickness, and wet weight of biceps brachii were tested, and the cross-sectional area of biceps brachii muscle fiber and the types of muscle fibers were determined by HE staining, immunofluorescence staining, and lactate dehydrogenase activity, respectively. Western blot analysis was used to investigate the level of growth-signaling pathway-related proteins. The results showed that pea peptide supplementation combined with resistance exercise training significantly increased body weight, upper limb grip, muscle thickness, wet weight of biceps brachii, and cross-sectional area of muscle fiber. Meanwhile, pea peptide supplementation obviously elevated the ratio of fast-twitch fiber (type II) and the expression of muscle growth-signaling pathway-related proteins. In addition, the PP2 oligopeptide in pea peptide with the amino acid sequence of LDLPVL induced a more significant promotion on C2C12 cell growth than other oligopeptides.

Katsuwonus pelamis Peptide and its Complexes Protect Zebrafish and Mice From Hyperuricemia Through Promoting Kidney Excretion of Uric Acid and Inhibiting Liver Xanthine Oxidase Activity.

Katsuwonus pelamis peptide and its complexes have the effect of lowering uric acid (UA)-levels. To identify the effect and possible mechanisms, different concentrations of Katsuwonus pelamis peptide and its complexes were administered to the zebrafish and mice hyperuricemia models, and the UA level was measured. Meanwhile, the hyperuricemic mice were treated orally at 0.83, 1.67, and 5.00 mg/g body weight for 7 days with Katsuwonus pelamis peptide and the complexes groups, separately. The levels of serum UA (SUA), urinary UA (UUA), serum creatinine (SCR), blood urine nitrogen (BUN), and xanthine oxidase (XOD) activities were detected in each group. The results showed that the Katsuwonus pelamis peptide (125 µg/ml) and its complexes (83.3 and 250 µg/ml) effectively reduced UA level in zebrafish with hyperuricemia (p < 0.05). The Katsuwonus pelamis peptide at high concentration (5.00 mg/g) decreased the SUA level, SCR level, BUN level, and hepatic XOD activity, and the complexes (1.67 and 5.00 mg/g) significantly reduced the SUA level and hepatic XOD activity (p < 0.05) in the hyperuricemic mice. In addition, in a hyperuricemic mouse model, the UUA level was increased after treatment with Katsuwonus pelamis peptide and its complexes at high concentrations (p < 0.05). The total therapeutic effects in the Katsuwonus pelamis peptide complex group were better than those in the Katsuwonus pelamis peptide group. Thus, Katsuwonus pelamis peptide and its complexes may possibly be used to prevent hyperuricemia via promoting urate secretion and inhibiting XOD activity production.

Probing Notch1-Dll4 signaling in regulating osteogenic differentiation of human mesenchymal stem cells using single cell nanobiosensor.

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

Experimental and biophysical modeling of transcription and translation dynamics in bacterial- and mammalian-based cell-free expression systems.

Cell-free expression (CFE) systems have been used extensively in systems and synthetic biology as a promising platform for manufacturing proteins and chemicals. Currently, the most widely used CFE system is in vitro protein transcription and translation platform. As the rapidly increased applications and uses, it is crucial to have a standard biophysical model for quantitative studies of gene circuits, which will provide a fundamental understanding of basic working mechanisms of CFE systems. Current modeling approaches mainly focus on the characterization of E. coli-based CFE systems, a computational model that can be utilized for both bacterial- and mammalian-based CFE has not been investigated. Here, we developed a simple ODE (ordinary differential equation)-based biophysical model to simulate transcription and translation dynamics for both bacterial- and mammalian- based CFE systems. The key parameters were estimated and adjusted based on experimental results. We next tested four gene circuits to characterize kinetic dynamics of transcription and translation in E. coli- and HeLa-based CFE systems. The real-time transcription and translation were monitored using Broccoli aptamer, double stranded locked nucleic acid (dsLNA) probe and fluorescent protein. We demonstrated the difference of kinetic dynamics for transcription and translation in both systems, which will provide valuable information for quantitative genomic and proteomic studies. This simple biophysical model and the experimental data for both E. coli- and HeLa-based CFE will be useful for researchers that are interested in genetic engineering and CFE bio-manufacturing.

Single-Cell Sequencing to Unveil the Mystery of Embryonic Development.

Embryonic development is a fundamental physiological process that can provide tremendous insights into stem cell biology and regenerative medicine. In this process, cell fate decision is highly heterogeneous and dynamic, and investigations at the single-cell level can greatly facilitate the understanding of the molecular roadmap of embryonic development. Rapid advances in the technology of single-cell sequencing offer a perfectly useful tool to fulfill this purpose. Despite its great promise, single-cell sequencing is highly interdisciplinary, and successful applications in specific biological contexts require a general understanding of its diversity as well as the advantage versus limitations for each of its variants. Here, the technological principles of single-cell sequencing are consolidated and its applications in the study of embryonic development are summarized. First, the technology basics are presented and the available tools for each step including cell isolation, library construction, sequencing, and data analysis are discussed. Then, the works that employed single-cell sequencing are reviewed to investigate the specific processes of embryonic development, including preimplantation, peri-implantation, gastrulation, and organogenesis. Further, insights are provided on existing challenges and future research directions.

Tributyltin perturbs femoral cortical architecture and polar moment of inertia in rat.

BACKGROUND: Tributyltin, a well-known endocrine disruptor, is widely used in agriculture and industry. Previous studies have shown that tributyltin could cause deleterious effects on bone health by impairing the adipo-osteogenic balance in bone marrow. METHODS: To investigate further the effects of tributyltin on bone, weaned male SD rats were treated with tributyltin (0.5, 5 or 50 µg·kg- 1) or corn oil by gavage once every 3 days for 60 days in this study. Then, we analyzed the effects of tributyltin on geometry, the polar moment of inertia, mineral content, relative abundances of mRNA from representative genes related to adipogenesis and osteogenesis, serum calcium ion and inorganic phosphate levels. RESULTS: Micro-computed tomography analysis revealed that treatment with 50 µg·kg- 1 tributyltin caused an obvious decrease in femoral cortical cross sectional area, marrow area, periosteal circumference and derived polar moment of inertia in rats. However, other test results showed that exposure to tributyltin resulted in no significant changes in the expression of genes detected, femoral cancellous architecture, ash content, as well as serum calcium ion and inorganic phosphate levels. CONCLUSIONS: Exposure to a low dose of tributyltin from the prepubertal to adult stage produced adverse effects on skeletal architecture and strength.