Live-dead assay on unlabeled cells using phase imaging with computational specificity

Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, the step of exogenous staining makes these methods undesirable for rapid, nondestructive, and long-term investigation. Here, we present an instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity. This concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and nucleus area for the labeled and unlabeled populations reveal that the chemical reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals to assessing the effectiveness of cancer treatments

Artificial confocal microscopy for deep label-free imaging

Widefield microscopy methods applied to optically thick specimens are faced with reduced contrast due to spatial crosstalk, in which the signal at each point is the result of a superposition from neighboring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, which come at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We augmented a laser scanning confocal instrument with a quantitative phase imaging module, which provides optical pathlength maps of the specimen on the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered and the data acquisition is automated. Remarkably, the ACM images present significantly stronger depth sectioning than the input images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. Furthermore, taking the estimated fluorescence volumes, as annotation for the phase data, we extracted dry mass information for individual nuclei. Finally, our results indicate that the network learning can be transferred between spheroids suspended in different media.Comment: 35 pages, 6 figure

Electrothermal soft manipulator enabling safe transport and handling of thin cell/tissue sheets and bioelectronic devices

“Living” cell sheets or bioelectronic chips have great potentials to improve the quality of diagnostics and therapies. However, handling these thin and delicate materials remains a grand challenge because the external force applied for gripping and releasing can easily deform or damage the materials. This study presents a soft manipulator that can manipulate and transport cell/tissue sheets and ultrathin wearable biosensing devices seamlessly by recapitulating how a cephalopod’s suction cup works. The soft manipulator consists of an ultrafast thermo-responsive, microchanneled hydrogel layer with tissue-like softness and an electric heater layer. The electric current to the manipulator drives microchannels of the gel to shrink/expand and results in a pressure change through the microchannels. The manipulator can lift/detach an object within 10 s and can be used repeatedly over 50 times. This soft manipulator would be highly useful for safe and reliable assembly and implantation of therapeutic cell/tissue sheets and biosensing devices

NEDD9 Is a Positive Regulator of Epithelial-Mesenchymal Transition and Promotes Invasion in Aggressive Breast Cancer

Epithelial to mesenchymal transition (EMT) plays an important role in many biological processes. The latest studies revealed that aggressive breast cancer, especially the triple-negative breast cancer (TNBC) subtype was frequently associated with apparent EMT, but the mechanisms are still unclear. NEDD9/HEF1/Cas-L is a member of the Cas protein family and was identified as a metastasis marker in multiple cancer types. In this study, we wished to discern the role of NEDD9 in breast cancer progression and to investigate the molecular mechanism by which NEDD9 regulates EMT and promotes invasion in triple-negative breast cancer. We showed that expression of NEDD9 was frequently upregulated in TNBC cell lines, and in aggressive breast tumors, especially in TNBC subtype. Knockdown of endogenous NEDD9 reduced the migration, invasion and proliferation of TNBC cells. Moreover, ectopic overexpression of NEDD9 in mammary epithelial cells led to a string of events including the trigger of EMT, activation of ERK signaling, increase of several EMT-inducing transcription factors and promotion of their interactions with the E-cadherin promoter. Data presented in this report contribute to the understanding of the mechanisms by which NEDD9 promotes EMT, and provide useful clues to the evaluation of the potential of NEDD9 as a responsive molecular target for TNBC chemotherapy

Genome-Wide Analysis of Histone H3 Lysine9 Modifications in Human Mesenchymal Stem Cell Osteogenic Differentiation

Mesenchymal stem cells (MSCs) possess self-renewal and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms such as histone modifications could be critical for determining the fate of stem cells. In this study, full human genome promoter microarrays and expression microarrays were used to explore the roles of histone modifications (H3K9Ac and H3K9Me2) upon the induction of MSC osteogenic differentiation. Our results revealed that the enrichment of H3K9Ac was decreased globally at the gene promoters, whereas the number of promoters enriched with H3K9Me2 was increased evidently upon osteogenic induction. By a combined analysis of data from both ChIP-on-chip and expression microarrays, a number of differentially expressed genes regulated by H3K9Ac and/or H3K9Me2 were identified, implicating their roles in several biological events, such as cell cycle withdraw and cytoskeleton reconstruction that were essential to differentiation process. In addition, our results showed that the vitamin D receptor played a trans-repression role via alternations of H3K9Ac and H3K9Me2 upon MSC osteogenic differentiation. Data from this study suggested that gene activation and silencing controlled by changes of H3K9Ac and H3K9Me2, respectively, were crucial to MSC osteogenic differentiation

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Surgical treatment of distal radius fractures: impact on forearm rotation in non-elderly patients

Abstract Forearm rotation restriction (FRR) is common after surgery for distal radius fractures (DRFs). The aim of the current study was to investigate the effect of DRFs on forearm rotation. This retrospective study reviewed patients with DRFs who underwent surgical treatment from January 2019 to December 2021. The patients' basic data and radiographic parameters were analyzed. Forearm rotation, including pronation and supination, was assessed using a standard goniometer. The Patient-Rated Wrist Evaluation (PRWE) score was evaluated, and the incidence of FRR at the 6-month follow-up was recorded. Univariate and multivariate logistic regression analyses were performed to identify risk factors correlated with FRR. A total of 127 patients with DRFs were included in this study. After surgery, 46 cases were considered to have FRR, with a rate of 36.2%, while the remaining 81 cases (63.8%) did not have FRR. The PRWE scores were 22.8 ± 5.2 and 17.9 ± 4.2 in the FRR group and non-FRR group, respectively, and the difference was statistically significant (P  0 mm (OR, 1.37; 95% CI 1.06–1.78) were independently associated with the incidence of FRR. The FRR is associated with an increased PRWE score and may have had some impact on the patient's daily life. Fractures involving the sigmoid notch, dorsal angulation, and radial shortening deformity were found to be correlated with the incidence of FRR. Preoperative risk notification and intraoperative preventive measures are necessary for these patients

Protocol for photocatalytic upcycling of non-biodegradable plastics into platform chemicals at ambient conditions

Summary: Upcycling plastics presents an opportunity not only to reduce plastic waste, but also to provide an alternative carbon source to fossil fuels. Herein, we present a protocol to upcycle plastics with resin codes 2–7 using a commercially available base-metal photocatalyst. We first conducted batch reactions, followed by a continuous, segmented flow system for gram-scale upcycling into value-added platform chemicals. This protocol, employing tandem carbon-hydrogen bond oxidation/carbon-carbon bond cleavage reactions, can be useful for photocatalytically transforming plastics at ambient conditions.For complete details on the use and execution of this protocol, please refer to Li et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics