Fluorescence Microscopy Images Research Articles

A Review for Artificial Intelligence Based Protein Subcellular Localization.

Proteins need to be located in appropriate spatiotemporal contexts to carry out their diverse biological functions. Mislocalized proteins may lead to a broad range of diseases, such as cancer and Alzheimer's disease. Knowing where a target protein resides within a cell will give insights into tailored drug design for a disease. As the gold validation standard, the conventional wet lab uses fluorescent microscopy imaging, immunoelectron microscopy, and fluorescent biomarker tags for protein subcellular location identification. However, the booming era of proteomics and high-throughput sequencing generates tons of newly discovered proteins, making protein subcellular localization by wet-lab experiments a mission impossible. To tackle this concern, in the past decades, artificial intelligence (AI) and machine learning (ML), especially deep learning methods, have made significant progress in this research area. In this article, we review the latest advances in AI-based method development in three typical types of approaches, including sequence-based, knowledge-based, and image-based methods. We also elaborately discuss existing challenges and future directions in AI-based method development in this research field.

SC-Track: a robust cell-tracking algorithm for generating accurate single-cell lineages from diverse cell segmentations.

Computational analysis of fluorescent timelapse microscopy images at the single-cell level is a powerful approach to study cellular changes that dictate important cell fate decisions. Core to this approach is the need to generate reliable cell segmentations and classifications necessary for accurate quantitative analysis. Deep learning-based convolutional neural networks (CNNs) have emerged as a promising solution to these challenges. However, current CNNs are prone to produce noisy cell segmentations and classifications, which is a significant barrier to constructing accurate single-cell lineages. To address this, we developed a novel algorithm called Single Cell Track (SC-Track), which employs a hierarchical probabilistic cache cascade model based on biological observations of cell division and movement dynamics. Our results show that SC-Track performs better than a panel of publicly available cell trackers on a diverse set of cell segmentation types. This cell-tracking performance was achieved without any parameter adjustments, making SC-Track an excellent generalized algorithm that can maintain robust cell-tracking performance in varying cell segmentation qualities, cell morphological appearances and imaging conditions. Furthermore, SC-Track is equipped with a cell class correction function to improve the accuracy of cell classifications in multiclass cell segmentation time series. These features together make SC-Track a robust cell-tracking algorithm that works well with noisy cell instance segmentation and classification predictions from CNNs to generate accurate single-cell lineages and classifications.

Hexagonal single crystals of perylene with patterned heterogeneous domains

Hexagonal single crystals of perylene were grown on a substrate by solution evaporation. Fluorescence microscopy images showed patterned heterogeneous domains with different fluorescence colors. Polarized ultra-low-frequency Raman microscopy revealed that the crystal structure is homogeneous throughout the entire crystal despite the domains observed in the fluorescence images. We concluded that hexagonal crystals were formed by the further growth of the rhombic β-crystal, which is one of the two polymorphs of the perylene crystal. This mechanism explains the formation of domains with different crystal qualities, resulting in different fluorescence colors.

Abstract 2018: Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces

Abstract Introduction: Tumor Treating Fields (TTFields) are an anti-cancer treatment that use low intensity, alternating electric fields. Anti-mitotic treatment effects of TTFields have been, to date, considered the primary anti-cancer effect. Recent studies have shown additional impacts on cancer cell invasion and migration, among other effects, resulting in a complex anti-tumor response derived from a combination of factors. By deploying computational imaging tools to assess experimental conditions that apply TTFields treatment to 3D cancer multicellular spheroid cultures, the goal of our study is to elucidate important mechanisms and effect sizes on anti-tumor effects relating to the modulation of cancer cell-extracellular matrix (ECM) biophysical interactions. Methods: 3D multicellular tumor spheroids (MTS) are cultured using GFP-tagged U-87 MG glioma cells. A single 3D MTS is placed in a cell culture insert and embedded in collagen I ECM at 2.25 mg/mL. Fluorescent polystyrene microspheres are dispersed in the ECM to facilitate subsequent tracking of displacement fields induced by the MTS-ECM construct. Fluorescence microscopy imaging is acquired for all spheroids prior to treatment initiation. Then, with 3 replicates in each treatment condition, TTFields are applied for a duration of 48 hours at 200 kHz frequency with a no treatment condition as a control. At 48 hours, we acquire fluorescence microscopy images to track spheroid growth and bead-field displacement. To determine deformation fields induced by the cancer spheroid, we compare each image at 48 hours to their respective baseline image. Deformation fields are analyzed using a custom-developed analysis code that performs an initial alignment of the baseline and specific time point images using a global rigid registration. Next, we perform a B-spline based nonrigid image registration to generate an estimate of the deformation field vector at each pixel location within the image. Deformation intensity is measured within a circular region of interest (r = 900 µm) centered at the spheroid core. Results: The average magnitude of displacement for treatment and control groups is 0.52±0.2 µm and 1.07±0.1 µm, respectively. Analysis of deformation fields reveals a significant difference (p < 0.1) in the magnitude of deformation present between treatment and control groups. Conclusions: Implementation of our computational imaging analysis methods using rigid and nonrigid registration, we observe a 69.6% decrease in the magnitude of deformation in TTFields treated spheroids compared to control spheroids. Due to the decrease in deformation induced by cancer spheroids treated with TTFields, we conclude that TTFields impact cancer cell traction forces applied to the ECM to disrupt cancer cell invasion. Citation Format: Samantha M. Short, Haley J. Bowers, Jared A. Weis. Tumor treating fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2018.

Targeted inhibition of colorectal cancer proliferation: Thedual-modulatory role of 2,4-DTBP on anti-apoptotic Bcl-2 and Survivin proteins.

The anti-apoptotic proteins, Bcl-2 and Survivin, are consistently overexpressed in numerous human malignancies, notably in colorectal cancer. 2,4-Di-tert-butylphenol (2,4-DTBP) is a naturally occurring phenolic compound known for its diverse biological activities, including anti-cancer properties. The mechanism behind 2,4-DTBP-induced inhibition of cell proliferation and apoptosis in human colorectal cancer cells, specifically regarding Bcl-2 and Survivin, remains to be elucidated. In this study, we employed both in silico and invitro methodologies to underpin this interaction at the molecular level. Molecular docking demonstrated a substantial binding affinity of 2,4-DTBP towards Bcl-2 (ΔG = -9.8 kcal/mol) and Survivin (ΔG = -5.6 kcal/mol), suggesting a potential inhibitory effect. Further, molecular dynamic simulations complemented by MM-GBSA calculations confirmed the significant binding of 2,4-DTBP with Bcl-2 (dGbind = -54.85 ± 6.79 kcal/mol) and Survivin (dGbind = -32.36 ± 1.29 kcal/mol). Invitro assays using HCT116 colorectal cancer cells revealed that 2,4-DTBP inhibited proliferation and promoted apoptosis in both a dose- and time-dependent manner. Fluorescence imaging and scanning electron microscopy illustrated the classical features associated with apoptosis upon 2,4-DTBP exposure. Cell cycle analysis through flow cytometry highlighted a G1 phase arrest and apoptosis assay demonstrated increased apoptotic cell population. Notably, western blotting results indicated a decreased expression of Bcl-2 and Survivin post-treatment. Considering the cytoprotective roles of Bcl-2 and Survivin through the inhibition of mitochondrial dysfunction, our findings of disrupted mitochondrial bioenergetics, characterized by reduced ATP production and oxygen consumption, further accentuate the functional impairment of these proteins. Overall, the integration of in silico and invitro data suggests that 2,4-DTBP holds promise as a therapeutic agent targeting Bcl-2 and Survivin in colorectal cancer.

A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

Effective alignment method using a diamond notch knife for correlative array tomography.

Correlative array tomography, combining light and electron microscopy via serial sections, plays a crucial role in the three-dimensional ultrastructural visualization and molecular distribution analysis in biological structures. To address the challenges of aligning fluorescence and electron microscopy images and aligning serial sections of irregularly shaped biological specimens, we developed a diamond notch knife, a new tool for puncturing holes using a diamond needle. The diamond needle featured a triangular and right-angled tip, enabling the drilling of deep holes upon insertion into the polished block face. This study describes the application of the diamond notch knife in correlative array tomography.

Blind multi-Poissonian image deconvolution with sparse log-step gradient prior.

Blind image deconvolution plays a very important role in the fields such as astronomical observation and fluorescence microscopy imaging, in which the noise follows Poisson distribution. However, due to the ill-posedness, it is a very challenging task to reach a satisfactory result from a single blurred image especially when the power of the Poisson noise is at a high level. Therefore, in this paper, we try to achieve high-quality restoration results with multi-blurred images which are contaminated by Poisson noise. Firstly, we design a novel sparse log-step gradient prior which adopts a mixture of logarithm and step functions to regularize the image gradients and combine it with the Poisson distribution to formulate the blind multi-image deconvolution problem. Secondly, we incorporate the methods of variable splitting and Lagrange multiplier to convert the original problem into sub-problems, then we alternately solve them to achieve the estimation of all the blur kernels of corresponding blurred images. Besides, we also design a non-blind multi-image deconvolution algorithm which is based on the log-step gradient prior to reach the final restored image. Experimental results on both synthetic and real-world blurred images show that the proposed prior is very capable of suppressing negative artifacts caused by ill-posedness. The algorithm can achieve restored image of very high quality which is competitive with some state-of-the-art methods.

Evaluation method of modification effect of direct-to-plant SBS modifier on asphalt

To accurately evaluate the modification effect of direct-to-plant styrene–butadiene–styrene block copolymer (SBS) modifier on asphalt, a collection method for direct-to-plant SBS-modified asphalt was proposed in this paper, the qualitative and quantitative evaluation index system was established, and the feasibility of quantitative evaluation based on fluorescence microscopy images and visual inspection of the mixing effect was evaluated, the feasibility of quantitative evaluation indicators was also verified based on the asphalt indicators, rheological properties, and asphalt mixtures's pavement performances. The research results indicate that based on fluorescence microscopy images, by observing the distribution and swelling state of direct-to-plant SBS modifiers can qualitatively evaluate the modification effect of direct-to-plant SBS modifiers on asphalt. Based on the visual inspection of the mixing effect, observe the size and quantity of unmelted particles, determine the melting state of the direct-to-plant SBS modifier, and its modification effect on asphalt can be qualitatively evaluated. For the same direct-to-plant SBS-modified asphalt, the percentage of rotation viscosity between direct-to-plant SBS-modified asphalt and traditional SBS-modified asphalt at 135 ℃ (DAη(135℃)) and discrete coefficient of 135 ℃ rotational viscosity of direct-to-plant SBS-modified asphalt (CVη(135℃)) exhibit a linear relationship with direct-to-plant SBS-modified asphalt's penetration, softening point, and ductility, the smaller the CVη(135℃), the greater the DAη(135℃). The smaller the CVη(135℃) (the larger the DAη(135℃)), the larger the dynamic shear modulus (G*), the smaller the phase angle (δ), and the larger the rutting factor (G*/sinδ) at the same scanning temperature. The relationship between CVη(135℃) (DAη(135℃)) and direct-to-plant SBS modifier on the main technical indicators and rheological performance indicators of asphalt is relatively stable. There is a clear relationship between CVη(135℃) (DAη(135℃)) and the high-temperature stability, low-temperature crack resistance, and water stability of direct-to-plant SBS-modified asphalt mixture, all of which show that as CVη(135℃) decreases (DAη(135℃) increases), the pavement performances improve.

Optimal technique for canine mesenchymal stem cells labeling with novel SPIO, MIRB™: for MRI detection of transplanted stem cells canine stroke model

ABSTRACT Background Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes.” Method Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 μg Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. Result Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 μg Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 μg Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). Conclusion This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 μg Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

Fluorescent Neuronal Cells v2: multi-task, multi-format annotations for deep learning in microscopy

Fluorescent Neuronal Cells v2 is a collection of fluorescence microscopy images and the corresponding ground-truth annotations, designed to foster innovative research in the domains of Life Sciences and Deep Learning. This dataset encompasses three image collections wherein rodent neuronal cell nuclei and cytoplasm are stained with diverse markers to highlight their anatomical or functional characteristics. Specifically, we release 1874 high-resolution images alongside 750 corresponding ground-truth annotations for several learning tasks, including semantic segmentation, object detection and counting. The contribution is two-fold. First, thanks to the variety of annotations and their accessible formats, we anticipate our work will facilitate methodological advancements in computer vision approaches for segmentation, detection, feature extraction, unsupervised and self-supervised learning, transfer learning, and related areas. Second, by enabling extensive exploration and benchmarking, we hope Fluorescent Neuronal Cells v2 will catalyze breakthroughs in fluorescence microscopy analysis and promote cutting-edge discoveries in life sciences.

Label-free visualization and quantification of the drug-type-dependent response of tumor spheroids by dynamic optical coherence tomography

We demonstrate label-free dynamic optical coherence tomography (D-OCT)-based visualization and quantitative assessment of patterns of tumor spheroid response to three anti-cancer drugs. The study involved treating human breast adenocarcinoma (MCF-7 cell-line) with paclitaxel (PTX), tamoxifen citrate (TAM), and doxorubicin (DOX) at concentrations of 0 (control), 0.1, 1, and 10 µM for 1, 3, and 6 days. In addition, fluorescence microscopy imaging was performed for reference. The D-OCT imaging was performed using a custom-built OCT device. Two algorithms, namely logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document}) were used to visualize the tissue dynamics. The spheroids treated with 0.1 and 1 µM TAM appeared similar to the control spheroid, whereas those treated with 10 µM TAM had significant structural corruption and decreasing LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} over treatment time. The spheroids treated with PTX had decreasing volumes and decrease of LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} signals over time at most PTX concentrations. The spheroids treated with DOX had decreasing and increasing volumes over time at DOX concentrations of 1 and 10 µM, respectively. Meanwhile, the LIV and OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document} signals decreased over treatment time at all DOX concentrations. The D-OCT, particularly OCDSl\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_l$$\\end{document}, patterns were consistent with the fluorescence microscopic patterns. The diversity in the structural and D-OCT results among the drug types and among the concentrations are explained by the mechanisms of the drugs. The presented results suggest that D-OCT is useful for evaluating the difference in the tumor spheroid response to different drugs and it can be a useful tool for anti-cancer drug testing.

Strategic Construction of meso-Aryl-Substituted N,N-Carbonyl-Bridged Dipyrrinones as Small, Bright, and Tunable Fluorophores.

A series of novel N,N-carbonyl-bridged dipyrrinone fluorophores have been directly constructed from α-halogenated dipyrrinones, which are conveniently obtained from the acid-catalyzed hydrolysis of readily available α,α'-dihalodipyrrins. This novel methodology affords efficient modulation of the functional groups at both the meso- and α-positions of this fluorophore. These resultant dyes show tunable absorption and emission wavelengths, good molar absorption coefficients, relatively large Stokes shifts, and excellent fluorescence quantum yields up to 0.99, and have been successfully applied in both one- and two-photon fluorescence microscopy imaging in living cells.

A 20 MHz Repetition Rate, Sub-Picosecond Ti-Sapphire Laser for Fiber Delivery in Nonlinear Microscopy of the Skin.

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.

Abstract A037: Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer

Abstract Metastatic breast cancer is currently the most deadly and incurable stage of breast cancer. A recent metabolic screen has identified sialic acid as a key player in progression of breast cancer metastasis. In the present study, we metabolically target the hypersialylation of metastatic breast cancer cells using appropriately designed nutritionary interventions. Our rationales are the following: (a) The aberrantly sialylated, hyperbranched glycocalyx promotes metastasis by facilitating cellular migration and immune evasion. (b) As hypersialylation relies heavily on enhanced sialic acid biosynthesis from the oncogenic glycolytic surge and preferential dietary uptake of glucose by cancer cells, it stands to reason that dietary changes can impact the extent of sialylation of the glycocalyx. Considering these points, we hypothesized that it was possible to control the degree of glycocalyx sialylation in breast cancer cells using an appropriate nutritionary regimen, which will in turn modulate the progression to invasive phenotype. The goal is to harness the metabolism of cancer cells themselves to curb their metastatic potential. Using a specific ketone-body forming metabolite called 3-hydroxybutyrate (HB) and lowering of glucose concentration, the cell culture medium was modified to mimic ketogenic diet. The response of metastatic breast cancer cells and normal breast epithelial cells was then monitored. The two main measures of cellular response were: cell surface sialylation via fluorescence microscopy and fluorescence lifetime imaging (FLIM) of intracellular NADH as a marker of metabolic index. We are currently investigating the consequences of nutritionary interventions on metastatic potential of breast cancer cells by measuring their impact on cellular proliferation, clonogenicity and migratory abilities. We have observed that the main metabolite of ketogenic diet, HB, tends to protect metastatic breast cancer cells from DOX treatment when supplemented in a low-glucose (LG) medium. However, the extent of increase in cell surface sialylation is very modest in comparison to that in normal breast epithelial cells. This differential increase in sialylation implies that a ketogenic diet might significantly promote the proliferation of healthy cells over the proliferation of metastatic breast cancer cells. FLIM measurements indicate that the chemotherapeutic, Doxorubicin tends to induce an initial preference for glycolysis in invasive breast cancer cells. While an HB rich LG media by itself promotes glycolysis in these cells, it suppresses any further Doxorubicin induced onset of glycolysis pointing to a protective mechanism against the chemotherapeutic. Through this ongoing study, we aim to decipher the right set of nutrients that would “deshield” the breast cancer cells and lower their metastatic disposition. The ultimate goal of our research is to determine a paradigm of drugs and diet that metastatic breast cancer cells are most responsive to, and that would translate into a viable remedy for metastatic breast cancer with minimal side-effects. Citation Format: Mohini Kamra, Yuan-I Chen, Paula C Delgado, Tim Yeh, Amy Brock, Sapun H Parekh. Metabolic maneuvering of glycocalyx hypersialylation is a smart and healthy way of curbing metastatic potential in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A037.

UV-C pretreatment of wastewater-grown algal biomass for recover of biofuel precursors

In the present study, ultraviolet radiation C (UV-C) was used as a pretreatment tool for the optimal recovery of biofuel precursors from algae Chlorella vulgaris. The pretreatment efficiency of UV-C with fixed light intensity (1.173 mWcm−2) on 1.5 gL-1 algal biomass was observed at 0, 0.5, 1, 2, 5, 10, and 20 h of irradiation by measuring the released sugar content and soluble chemical oxygen demand (sCOD). The UV-C pretreated algal cells were also analyzed through impedance spectroscopy, fluorescence microscope, and digital images to assess the degree of cell wall disintegration. The released sugar content reached a maximum of 73.61 mg L-1 at 10 h of exposure, whereas the maximum sCOD of 190 mg L-1 was achieved at 20 h exposure. Image analysis indicated that the chlorophyll content declined exponentially from 7.21 to 0.14 µgmL-1 with UV-C exposure. An increase in UV-C exposure time resulted in decreased impedance and increased conductance. The SYTOX green fluorescence also increased with 10 h of exposure, followed by a decline. Therefore, the cell wall stability gets compromised and the permeability increases with increment in UV-C exposure. This triggers extensive release in cell wall sugar content with exposure. Further exposure destabilizes the cell wall leading to discreet breakage. This breakage disperses internal content to bulk liquid, causing elevated sCOD with reduced sugar content. The present study showed that UV-C to be a potential pretreatment strategy to release precursors to derive biofuels such as biohydrogen, bioethanol and others.

Investigating the impact of silencing an RNA-binding protein gene SlRBP1 on tomato photosynthesis through RNA-sequencing analysis

Photosynthesis in plants directly affects the synthesis and accumulation of organic matter, which directly influences crop yield. RNA-binding proteins (RBPs) are involved in the regulation of a variety of physiological functions in plants, while the functions of RBPs in photosynthesis have not been clearly elucidated. To investigate the effect of a glycine-rich RNA-binding protein (SlRBP1) in tomato on plant photosynthesis, a stably inherited SlRBP1 silenced plant in Alisa Craig was obtained by plant tissue culture using artificial small RNA interference. It turns out that the size of the tomato fruit was reduced and leaves significantly turned yellow. Chlorophyll(Chl) content measurement, Chl fluorescence imaging and chloroplast transmission electron microscopy revealed that the chloroplast morphology and structure of the leaves of tomato amiR-SlRBP1 silenced plants were disrupted, and the chlorophyll content was significantly reduced. Measurement of photosynthesis rate of wild-type and amiR-SlRBP1 silenced plants in the same period demonstrated that the photosynthetic rate of these plants was significantly reduced, and analysis of RNA-seq data indicated that silencing of SlRBP1 significantly reduced the expression of photosynthesis-related genes, such as PsaE, PsaL, and PsbY, and affected the yield of tomato fruits through photosynthesis.

Therapy-induced modulation of tumor vasculature and oxygenation in a murine glioblastoma model quantified by deep learning-based feature extraction

Glioblastoma presents characteristically with an exuberant, poorly functional vasculature that causes malperfusion, hypoxia and necrosis. Despite limited clinical efficacy, anti-angiogenesis resulting in vascular normalization remains a promising therapeutic approach. Yet, fundamental questions concerning anti-angiogenic therapy remain unanswered, partly due to the scale and resolution gap between microscopy and clinical imaging and a lack of quantitative data readouts. To what extend does treatment lead to vessel regression or vessel normalization and does it ameliorate or aggravate hypoxia? Clearly, a better understanding of the underlying mechanisms would greatly benefit the development of desperately needed improved treatment regimens. Here, using orthotopic transplantation of Gli36 cells, a widely used murine glioma model, we present a mesoscopic approach based on light sheet fluorescence microscopic imaging of wholemount stained tumors. Deep learning-based segmentation followed by automated feature extraction allowed quantitative analyses of the entire tumor vasculature and oxygenation statuses. Unexpectedly in this model, the response to both cytotoxic and anti-angiogenic therapy was dominated by vessel normalization with little evidence for vessel regression. Equally surprising, only cytotoxic therapy resulted in a significant alleviation of hypoxia. Taken together, we provide and evaluate a quantitative workflow that addresses some of the most urgent mechanistic questions in anti-angiogenic therapy.

Live cell fluorescence microscopy-an end-to-end workflow for high-throughput image and data analysis.

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.