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The current methods for detecting myelin changes in ischemic stroke are indirect and cannot accurately reflect their status. This study aimed to develop a novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin. Compounds 7a and 7b were synthesized by linking the MeDAS group and Gadolinium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate. Compound 7a was selected for characterization and further study. Cell uptake, cytotoxicity, and magnetic resonance imaging scans were performed on cells. In vitro experiments on frozen brain sections from 7-day-old, 8-week-old, and ischemic stroke rats were compared with commercially available Luxol Fast Blue staining. After HPLC and MR scanning, brain tissue was soaked in 7a and scanned using T 1 WI and T 1 maps sequences. Spectrophotometer results showed that compounds 7a and 7b had fluorescent properties. MR scans indicated that the compounds had contrast agent properties. Cells could uptake 7a and exhibited high signals in imaging scans. Compound 7a brain tissue staining showed more fluorescence in myelin-rich regions and identified injury sites in ischemic stroke rats. MR scanning of brain sections provided clear myelin contrast. A novel fluorescent-magnetic resonance dual-modal molecular imaging probe for direct imaging of myelin was successfully developed and tested in rats with ischemic stroke. These findings provide new insights for the clinical diagnosis of demyelinating diseases. • Compounds 7a and 7b have fluorescent properties from spectrophotometer results. • Compounds 7a and 7b have MRI contrast agent properties. • Our novel dual-mode probes effectively visualized the rat infarction model. • Our study may provide new ideas for diagnosing demyelinating diseases. [ABSTRACT FROM AUTHOR]

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood–brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability. [ABSTRACT FROM AUTHOR]

Autologous cell-based therapy using dermal sheath cup (DSC) cells was reported as a new treatment for male and female pattern hair loss. However, the mechanisms underlying its action remain unclear. We investigated the mechanisms underlying the efficacy of DSC cells in cell-based therapy. We conducted multivariate analysis to categorize individuals based on treatment response as responders and non-responders. The differentially expressed genes in DSC cells from the two groups were evaluated using bulk transcriptome, quantitative polymerase chain reaction, and single-cell transcriptome analyses. We performed live cell imaging combined with immunostaining to characterize the DSC subpopulation associated with responders. We identified nine and three genes as high efficacy (HE) and low efficacy (LE) marker genes, respectively. The HE subpopulations were enriched for cell migration-related genes in single-cell analysis. In contrast, the LE subpopulation was enriched for basement membrane and vasculature-related genes. Moreover, DSC cells in culture were immunocytochemically and morphologically heterogeneous, expressing characteristic factors. Furthermore, live cell imaging showed that DSC cells expressing integrin subunit alpha 6 (ITGA6), an HE subpopulation gene, had markedly higher mobility than those expressing the LE subpopulation genes collagen type IV or CD36. ITGA6-positive DSC cells, with superior migratory activity, may contribute to cell-based therapy by promoting cell migration into nearby hair follicles. • Analyzed clinical results of cell therapy for PHL with multivariate analysis. • Identified differentially expressed genes between responders and non-responders. • Identified clinical efficacy-associated subpopulation by single cell analysis. • This subpopulation expressed integrin subunit alpha 6 (ITGA6). • ITGA6 + subpopulation had high migratory activity contributing to PHL cell therapy. [ABSTRACT FROM AUTHOR]

As an important posttranslational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or KO of SUMOconjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 KO cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes. [ABSTRACT FROM AUTHOR]

Cell therapy is a promising treatment method that uses living cells to address a variety of diseases and conditions, including cardiovascular diseases, neurologic disorders and certain cancers. As interest in cell therapy grows, there is a need to shift to a more efficient, scalable and automated manufacturing process that can produce high-quality products at a lower cost. One way to achieve this is using non-invasive imaging and real-time image analysis techniques to monitor and control the manufacturing process. This work presents a machine learning-based image analysis pipeline that includes semantic segmentation and anomaly detection capabilities. This method can be easily implemented even when given a limited dataset of annotated images, is able to segment cells and debris and can identify anomalies such as contamination or hardware failure. [ABSTRACT FROM AUTHOR]

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics--that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFN) stimulation. We found that IRF1 responds to low concentration or short duration IFN stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFN stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression. [ABSTRACT FROM AUTHOR]

The innate immune system provides the first line of defense against pathogens and cellular insults and is activated by pattern recognition receptors sensing pathogen- or damageassociated molecular patterns. This activation can result in inflammation via cytokine release as well as the induction of lytic regulated cell death (RCD). Innate immune signaling can also induce the expression of interferon regulatory factor 1 (IRF1), an important molecule in regulating downstream inflammation and cell death. While IRF1 has been shown to modulate some RCD pathways, a comprehensive evaluation of its role in inflammatory cell death pathways is lacking. Here, we examined the role of IRF1 in cell death during inflammasome and PANoptosome activation using live cell imaging, Western blotting, and ELISA in primary murine macrophages. IRF1 contributed to the induction of ZBP1- (Z-DNA binding protein 1), AIM2- (absent in melanoma-2), RIPK1- (receptor interacting protein kinase 1), and NLRP12 (NOD-like receptor family, pyrin domain–containing 12)-PANoptosome activation and PANoptosis. Furthermore, IRF1 regulated the cell death under conditions where inflammasomes, along with caspase-8 and RIPK3, act as integral components of PANoptosomes to drive PANoptosis. However, it was dispensable for other inflammasomes that form independent of the PANoptosome to drive pyroptosis. Overall, these findings define IRF1 as an upstream regulator of PANoptosis and suggest that modulating the activation of molecules in the IRF1 pathway could be used as a strategy to treat inflammatory and infectious diseases associated with aberrant inflammatory cell death. [ABSTRACT FROM AUTHOR]

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells. [ABSTRACT FROM AUTHOR]

Mechanical stretching is experienced ubiquitously by human tissues and their constituent cells and impacts biochemical and (mechano)biological processes relevant to health and disease. Numerous cell-stretching devices (CSDs) have been developed and used to apply mechanical strain to cells and tissues in in vitro settings, however, few have shown compatibility with live-cell microscopy. In most microscopy-compatible CSDs, imaging is performed post-mechanical stretching while live-cell imaging during cyclic stretching is very rarely shown, especially at high stretching frequencies and for long experiments (i.e., hours long). Live-cell imaging during cell stretching has revealed distinct time-dependent biomechanical features of cells, such as cell fluidization at high amplitudes or frequencies, or cracking followed by recovery of cell monolayers. Basic human functions such as breathing and digestion require mechanical stretching of cells and tissues. However, when it comes to laboratory experiments, the mechanical stretching that cells experience in the body is not often replicated, limiting the biomimetic nature of the studies and the relevance of results. Herein, we establish the importance of mechanical stretching during in vitro investigations by reviewing seminal works performed using cell-stretching platforms, highlighting important outcomes of these works as well as the engineering characteristics of the platforms used. Emphasis is placed on the compatibility of cell-stretching devices (CSDs) with live-cell imaging as well as their limitations and on the research advancements that could arise from live-cell imaging performed during cell stretching. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients. In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients. CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases. Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment. [Display omitted] • CTCs were characterized using a biomarker panel (+EpCAM, +CK, +AFP, −CD45, +DRAQ) in LC and HCC patients using IFC. • Surface markers, EpCAM + CK, were the most expressed biomarkers on CTCs in LC vs. HCC followed by AFP, EpCAM and CK. • The cell area of AFP was significantly higher in the LC group when compared with the HCC group (P value = 0.035). • CTCs cell area (EpCAM+CK+ AFP) performed well with an AUC of 0.92, in detecting early-stage and AFP-negative HCC and LC cases. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique. Adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging requires consideration of (amplified) signal generation complexity, an appropriate cell delivery mechanism, and the capacity for single-copy sequence imaging. Single-copy sequence imaging is achieved both with and without signal amplification, whereas SNP or single-nucleotide variant (SNV) detection requires signal amplification. CRISPR-based imaging with specificity at the SNV and SNP levels was achieved in both live and fixed cells. The versatility of paired RNA aptamer-based signal generation enables highly multiplexed live-cell imaging of six repetitive sequences. CRISPR-based imaging was used in a clinical setting, allowing the detection of Patau syndrome (in the form of Chr13 trisomy) in patient-derived cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel ultrasmall organosilica nanoparticles (ONs) were synthesized by a one-step hydrothermal method. • Ultrasmall ONs with avoiding aggregation-induced quenching effect exhibit strong solid-state orange fluorescence. • Ultrasmall ONs can be used in fingerprints detection, white light-emitting diodes, and fluorescence imaging of lysosomes. Organosilica nanoparticles (ONs), which are a new type of photoluminescent nanomaterial (PM) with excellent biocompatibility, have caught more attention in recent years. However, their applications are significantly impeded by the complicated preparation process, poor photostability, and especially aggregation-induced quenching. The present study was aimed to design and prepare solid-state fluorescent ONs to avoid aggregation-induced quenching effect. In addition, the uses of ONs for fingerprint detection, white light-emitting diodes (WLEDs) and lysosome-targetable cellular imaging were demonstrated. Here, for the first time, we designed and prepared novel solid-state fluorescent ultrasmall ONs with orange-emitting photoluminescence via a one-step hydrothermal method. The prepared solid-state fluorescent ONs could be successfully employed in fingerprint detection, WLEDs fabrication and cellular imaging. Intriguingly, the ultrasmall ONs specifically localized to lysosomes rather than other subcellular organelles across distinct cell lines, including cancer cells and noncancerous cells. Collectively, these data showed that the new ONs presented in this study could be ideal candidates for PMs in biological and photoelectric applications. [ABSTRACT FROM AUTHOR]

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the β-adrenergic agonist isoproterenol (1 μ M) in the presence of the CB1R agonist arachidonyl-2′-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage. [ABSTRACT FROM AUTHOR]

Synthesis of monoclinic Y 2 O 3 remains a challenging task due to complex formation conditions such as high pressure or high temperature environments. Monoclinic yttria exhibits better photoluminescence characteristics compared to the cubic phase and hence can be a better host for nanophosphors. Reports are available predicting the stability of monoclinic phase at low temperature in extremely fine particles. But experimental preparation of this phase at low temperature is not yet reported. Here we report the first-time preparation of monoclinic Y 2 O 3 at 90 °C using simple laboratory hot air oven. The synthesized nanoparticles exhibit intense PL emission in the blue region and hence can find applications in display industry. The band structure calculations were carried out using density functional theory and the excitonic Bohr radius was estimated using the electron as well as hole effective masses determined from band structure. The cytotoxicity studies reveal non-toxic nature the particles. The fluorescence imaging experiments show that the material is capable of emitting blue fluorescence in L6 skeletal cell lines and it ensures the applicability of this material in the biomedical field. [ABSTRACT FROM AUTHOR]

Water-soluble Mn2+ doped CsPbCl 3 nanocrystals coating by 6-aminocaproic acid (ACA) ligand were prepared by a simple ligand assisted reprecipitation method at room temperature. The CsPbCl 3 :Mn2+/ACA nanocrystals were almost cubic phase and had mean size of about 10 nm. Due to the coating and passivation of ACA, the CsPbCl 3 :Mn2+/ACA nanocrystals retained the initial fluorescence intensity of 20% after being dispersed in deionized water for 20 days. The orange LED device based on CsPbCl 3 :Mn2+/ACA nanocrystals were prepared with excellent luminescence properties. In the cytotoxicity test, even if the MCF-7 cells were cultured for 24 h at the concentration of CsPbCl 3 :Mn2+/ACA nanocrystals as high as 160 μg·ml−1, the cell survival rate was still 70%. Confocal fluorescent cell imaging results showed that the nanocrystals had remarkable fluorescent cell imaging effect even after 24 h. The synthesized CsPbCl 3 :Mn2+/ACA nanocrystals have nice water solubility, good water stability, low cytotoxicity and highly luminescence properties, and have great application potential in the field of orange LED and fluorescent probes. [ABSTRACT FROM AUTHOR]

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success. [ABSTRACT FROM AUTHOR]

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6 h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at -70 kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division. [ABSTRACT FROM AUTHOR]

Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane. Mechanotransduction is involved in a variety of cell and tissue processes. For this, mechanosensitive structures, such as integrin adhesions, the cytoskeleton, or the plasma membrane, are key to sense, integrate, and transmit mechanical forces. Novel imaging methods and molecular sensors have unveiled how molecular mechanosensing occurs in these structures within the context of live cells. These include super-resolution imaging, single particle tracking, and molecular force sensors. Super-resolution microscopy and single protein tracking have revealed the 3D nanoscale organization and dynamics of integrin adhesion structures in various cell types, which are divided into functional nanolayers with specific paths for protein diffusion. Forces on actin regulators and F-actin trigger conformational changes controlling actin regulator function and binding, and therefore F-actin assembly and architecture. In migrating cells, mechanical plasticity emerges from global geometrical reorganization of actin networks under loads, or forces from elongating actin filaments controlling locally actin regulators dynamics and functions. Cell surface mechanics are emerging as a key parameter controlling cell motility, division, and differentiation. Variations in cortical tension and membrane-to-cortex attachment regulate the timing of differentiation while the nanoscale architecture of the actin cortex is linked to cortical tension. [ABSTRACT FROM AUTHOR]

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Due to the damage during production, transportation and installation, some defects inevitably occur in the solar cells, which will reduce the power generation efficiency. Benefiting from the development of deep learning, the performance of solar cell defect detection has been improved by a considerable margin. However, a problem persists that a model trained on one batch of data may not work on another batch following different production schemes. Moreover, compared with monocrystalline cells, more impurities on the surface of polycrystalline cells make it more difficult to inspect defects and obtain labels. To address this problem, we formulate it as a monocrystalline to polycrystalline task based on an unsupervised domain adaptation task in transfer learning. A standard network with CNN architecture is adopted as our classifier, which can tell whether the solar cell is cracked. Based on that, we embed adversarial learning in the network, where the domain discriminator is added to distinguish data domains, so that the feature extractor can learn domain-invariant features. Considering the scenario of defect detection in Electroluminescence (EL) images, we further propose attention-based transfer learning and class-aware domain discriminator to enhance the effect of knowledge transfer. When detecting a batch of new polycrystalline products, our method can improve F1-score by 0.2631, and the recall and precision can reach 84.70% and 90.15%, respectively. • Using transfer learning to adapt defect detection model to various production lines. • Attention mechanism is introduced in knowledge transfer to optimize features. • Align distribution of channel attention to reduce feature distribution gap. • Proposing class-aware domain classification to combat the impact of class imbalance. [ABSTRACT FROM AUTHOR]

Compared with visible fluorescence carbon dots (CDs), the multi-emission (visible and near infrared) fluorescence CDs based platform (V-N Platform) with blood-brain barrier penetration, real-time brain imaging and drug loading capacities, is of great significance. Herein, a novel type of multi-emission fluorescence CDs (GT) was created to penetrate BBB for brain imaging and local drug delivery, which was synthesized by hydrothermal method, using endogenous l -tryptophan and d -glucose. The capability for in vitro imaging was verified through the experiments on cell imaging. Moreover, the ability of in vivo imaging, especially for brain imaging, was confirmed by experiments in mice and zebrafish. In addition, the in vitro and in vivo experiments preliminarily confirmed its potential to act as a drug delivery platform. This V-N Platform (GT) will be expected to have a significant impact on the researches for CDs and the theranostics of brain diseases. A novel kind of multi-emission fluorescence carbon dots (GT) was created with NIR fluorescence to act as the "V-N Platform", using endogenous nutrients. With the BBB-penetrating ability and drug loading potential, GT could be expected to with the potential of theranostics for brain diseases. [Display omitted] • GT CDs was synthesized using two endogenous nutrients (d -glucose and l -tryptophan) as the precursor by hydrothermal method. • The retained bio-activity of d -glucose and l -tryptophan enabled GT to cross the BBB effectively. • Carbonization endowed GT with multi-emission fluorescence, especially the NIR fluorescence. • GT could be used as fluorescent probe at multi-level,especially the brain imaging in real-time. • Nanoscale ensured GT as a drug carrier for loading drugs with various polarity. [ABSTRACT FROM AUTHOR]

Fluorescence lifetimes and quantum yields featuring polycyclic aromatic hydrocarbons (PAHs) and other organics constituting pyrogenic carbon particulate matter (PM) are seldom measured. In this work, PM sampled in a fuel-rich ethylene flame was firstly separated in organic carbon (OC), soluble in dichloromethane, and refractory organic carbon (ROC), soluble in N -methyl pyrrolidinone, and then analyzed by size exclusion chromatography (SEC) coupled with online UV and fluorescence detection, and by offline fluorescence spectroscopy and mass spectrometry. It was found that three classes of differently light emitting carbon dots (CDs) could be bottom-up synthesized in the same flame system by selecting appropriately the residence time. Actually, OC presented blue fluorescence regardless the residence time, whereas ROC sampled at low and high residence time emitted fluorescence in the green (green CDs) and in the yellow (yellow CDs) region, respectively. The SEC molecular weight of all CDs presented similar trimodal distributions, centered around 300, 1000 and 10,000 u. For the first time fluorescence lifetimes and quantum yields of pyrogenic CD fractions were measured as additional parameters useful for discriminating the fluorescent components and inferring their structural properties, with the support of mass spectrometry. The different spectroscopic features of CDs could be associated to different compositional characteristics as the polydispersity of molecular components featuring blue CDs, opposed to the oligomer-like nature of green and yellow CDs. Pyrogenic CDs showed different fluorescence emission ranges, quantum yield and lifetimes, appealing for their possible applications in the fields of imaging, electronics and sensors. [Display omitted] [ABSTRACT FROM AUTHOR]

To investigate correlations between clinical and histopathologic characteristics of idiopathic epiretinal membrane (ERM). Retrospective interventional case series. In total, 87 eyes from 87 patients with idiopathic ERM who underwent pars plana vitrectomy with peeling of the ERM from 2019 to 2020 were included. The outcomes of clinical ophthalmic examination, including measurement of best-corrected visual acuity (BCVA) and spectral-domain OCT (SD-OCT), before and after surgery were reviewed. Surgical specimens were fixed in formalin and embedded in paraffin for histologic and immunohistochemical analysis. The association between morphological characteristics revealed on SD-OCT images and the cellular composition of the surgically excised ERM demonstrated with immunohistochemical staining were the main outcome measures. Changes in the BCVA and central macular thickness (CMT) were assessed through a comparison of preoperative and postoperative measurements. Based on SD-OCT morphological characteristics in the foveal area, 15 cases were classified into group 1A (mainly outer retinal thickening), 39 into group 1B (more tenting of the outer retina and distorted inner retina), and 33 into group 1C (prominent inner retina thickening). Overall, postoperative final BCVA and CMT at 1 year improved in all groups. Patients who presented with a better initial BCVA exhibited a more favorable final BCVA. Epiretinal membranes in group 1C demonstrated the greatest decrease in CMT compared with those in groups 1B and 1A, but the final CMT did not differ among the groups. A negative correlation between the density of hyalocytes (P = 0.003) and myofibroblasts (P = 0.047) was noted between the 3 groups. Total cell density and glial cell density of the ERMs were strongly associated with poor final BCVA and BCVA improvement. The present study provides new histopathologic information regarding the formation and progression of idiopathic ERM. Glial cell proliferation plays a predominant role in these processes. Epiretinal membranes with high cellularity and glial cell density may cause damage to the retina structure, resulting in poor postoperative visual outcomes. These findings provide additional evidence supporting early surgical intervention in patients with idiopathic ERM reported with visual disturbance. [ABSTRACT FROM AUTHOR]

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement. • Equine and swine antral follicles were injected with liposome nanoparticles. • Follicular fluid, granulosa cells and follicle wall biopsy (FWB) were collected. • Fluorescence imaging revealed successful intrafollicular binding of liposomes to mural granulosa cells and progressive migration to other follicle cell layers. • Intrafollicular injection of nanomolecules in combination with the FWB technique is a promising approach for future diagnostic and treatment. [ABSTRACT FROM AUTHOR]

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase. [ABSTRACT FROM AUTHOR]

[Display omitted] Fluorescent red-emissive carbon dots (RCDs) were synthesized using the solvothermal method with citric acid as a carbon source, N , N -dimethylformamide as a nitrogen source, and formamide as a solvent. The as-synthesized RCDs show red fluorescence in an aqueous solution and have an excellent stability towards photobleaching as well as extremely low cytotoxicity and are successfully used for cell and zebrafish imaging. The results indicate that RCDs have potential applications in both in vitro and in vivo bioimaging. [ABSTRACT FROM AUTHOR]

In order to further visualize intracellular dynamics, precise imaging of endogenous proteins in live cells was performed using an antigen-binding fragment (Fab)-based Quenchbody (Q-body). The transfected Q-body probe showed an antigen-dependent fluorescence response, enabling the clear visualization and sorting of cells expressing p53, a tumor suppressor biomarker. [ABSTRACT FROM AUTHOR]

Native broken-rice starch was used to create starch nanoparticles (StNPs) with particle sizes ranging from 100 nm to 800 nm. The fluorescent isothiocyanate poly- l -lysine StNPs (FITC-PLL-StNPs) were created in two steps. First, the StNPs were electrostatically modified by poly- l -lysine (PLL) molecules rich in amino acids. Second, fluorescein isothiocyanate reacted with some amino groups on PLL molecules (FITC). Fluorescence spectrophotometry was used to determine the degree of substitution (DS) and fluorescent properties of fluorescent starches. The study found that FITC-PLL-StNP-200 has higher fluorescence stability, more phagocytic cells, and a better and clearer fluorescence detecting effect than FITC-PLL-St, FITC-PLL-StNP-100, FITC-PLL-StNP-400, and FITC-PLL-StNP-800. The biological evaluation results showed that FITC-PLL-StNP-200 did not affect the viability of HeLa cells at the lysosome labeling concentration. These findings suggest that FITC-PLL-StNP-200 has strong and stable fluorescence, indicating that FITC-PLL-StNP-200 can be used as a fluorescent probe and lysosome marker in a variety of applications, particularly in biomedicine. [Display omitted] • Fluorecent starch nanoparticles (StNP) were synthesized. • FITC-PLL-StNP-200 has clearer fluorescence detecting effect. • FITC-PLL-StNP-200 has higher phagocytic amount of Hela cells. • FITC-PLL-StNP-200 is safe and possesses excellent biocompatibility. [ABSTRACT FROM AUTHOR]

Imaging flow cytometry (IFC), a technique originally designed for cellular imaging, is featured by the parallel acquisition in brightfield (BF), fluorescence (FL), and side scattering channels. Introduced to the field of subvisible particle analysis in biopharmaceuticals roughly ten years ago, it has the potential to yield additional information, e.g., on particle origin. Here, we present an extensive, systematic development of masks for IFC image analysis to optimize the accuracy of size determination of polystyrene beads and pharmaceutically relevant particles (protein, silicone oil) in BF and FL channels. Based on the developed masks, particle sizing and counting by IFC are compared to flow imaging microscopy (FIM). Mask verification based on fluorescent polystyrene particles revealed good agreement between sizes obtained from IFC and FIM. In the evaluation of counting accuracy, IFC reported lower concentrations for polystyrene particle standards than FIM. For the analysis of fluorescently stained silicone oil and protein particles however, IFC FL imaging reported higher particle concentrations in the low micrometer size range. Overall, we identified IFC as suitable tool to generate supportive data for particle characterization purposes or trouble shooting activities, but not as routine quantitative technique, e.g., for subvisible particle analysis during drug product development or quality control. [ABSTRACT FROM AUTHOR]

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes. [ABSTRACT FROM AUTHOR]

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein stromal interaction molecule 1 (STIM1) physically interacts with plasma membrane protein Orai1 to induce calcium release--activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long-chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here using the acyl resin--assisted capture assay, we show that STIM1 is also rapidly S-acylated at cysteine 437 upon ER calcium store depletion. Using a combination of live cell imaging and electrophysiology approaches with a mutant STIM1 protein, which could not be S-acylated, we determined that the S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating the CRAC channel assembly and function. [ABSTRACT FROM AUTHOR]

Mitochondrial viscosity and mitochondrial membrane potential (MMP) are vital parameters for reflecting mitochondrial conditions and they are closely correlative in various biological events. Accordingly, simultaneous detection of mitochondrial viscosity and MMP is imperative for elucidating their interactions, further exploring the mechanism of diseases, and investigating numerous pathological procedures. However, such profound research remains a big challenge due to the lack of high-performing single fluorescent probes capable of simultaneously visualizing of these two parameters. In this study, we developed a new kind of fluorescent carbon dots (CDs) that can successfully realize simultaneous monitoring of mitochondrial viscosity and MMP in living cells. The fluorescence of CDs can be specifically turned on as the viscosity of solvents increases from 0.89 to 945 cP. Meanwhile, the CDs accumulate in mitochondria under normal MMP conditions, switch to exist in both mitochondria and nucleoli under reduced MMP, and present only in nucleoli with the vanishment of MMP. Thus, various MMP situations can be easily differentiated through the changed spatial distributions of CDs. More importantly, based on the developed CDs, an increased mitochondrial viscosity accompanying a decreased MMP was simultaneously tracked in drug-stimulated cells. We envision that the developed CDs has a great potential to be utilized as a promising fluorescent probe for mitochondrion-related biology studies. The carbon dots were elaborately designed for simultaneous determination of mitochondrial viscosity and membrane potential through their fluorescence variation and location migration from mitochondria to nucleoli. [Display omitted] • The fluorescence of Carbon Dots (CDs) was specifically turned on as the viscosity of solvents increased. • Fluctuation of mitochondrial membrane potential was visualized through migration of CDs from mitochondria to nucleoli. • An increased mitochondrial viscosity accompanying a decreased membrane potential was simultaneously monitored in cells. [ABSTRACT FROM AUTHOR]

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma. [ABSTRACT FROM AUTHOR]

[Display omitted] In the biomedical field, stimuli-responsive systems comprising smart nanoplatforms based on metal–organic frameworks (MOFs) have garnered immense attention in view of their high surface area, biocompatibility, facile synthesis, tunable structural features, adjustable pore size, and highly versatile composition. Interestingly, stimuli-responsive MOFs are excellent nanoplatforms for biomedical applications as they are designed to specifically change their properties upon interior stimuli in target tissues (e.g. , pH and redox reaction triggering agents) or exposure to exterior stimuli like temperature, various wavelengths of light and magnetic fields. Compared to other single-imaging techniques either alone or in combination with each other, synergistic therapy based on multimodal imaging-guided strategies deliver high diagnostic accuracy, as well as superior therapeutic efficiency. In order to fully comprehend the field of stimuli-responsive MOFs, herein, the basic mechanistic chemistry of stimuli-responsive MOFs along with the latest progress in the development of cell imaging and theranostic nanoplatforms for biomedical imaging, phototherapy and cancer chemotherapy is elaborated. [ABSTRACT FROM AUTHOR]

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSa, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSa or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSa and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSa to ATM. Consistently, MutSa and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSa, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage. [ABSTRACT FROM AUTHOR]

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization. [ABSTRACT FROM AUTHOR]

• Promising G-quadruplex binders: chroman derivatives ligands & their BF2-complexes. • These compounds stabilize parallel and mixed hybrid G-quadruplex structures. • In vitro and cellular studies: biophysical analyses demonstrate binding affinity in both contexts. • Molecular docking: computational study supports binding of chroman complexes to G-quadruplex. • Potential fluorescent ligands: these compounds offer new options for G-quadruplex targeting ligands. Currently, G-quadruplex structure targeting strategies are considered as a promising anticancer approach. The purpose of this research is to expand the options for G-quadruplex targeting ligands, particularly emphasizing fluorescent ligands. Our study contributes to the field by providing additional choices for G-quadruplex binders with a notable focus on selectivity towards G-quadruplex topology over duplex DNA. In the search of selective and potent G-quadruplex binders, here we discuss an analysis of a few chroman derivatives ligands named A and C and their respective borondifluoride complexes B and D as a quadruplex targeting compounds which found to stabilize G-quadruplex structure. To investigate the binding characteristics of these molecules with G-quadruplex vs. duplex selectivity, In vitro biophysical studies were performed by steady-state fluorescence, UV–visible titration, fluorescent TO displacement assay, CD thermal melting, circular dichroism spectroscopy, and cellular imaging by employing both telomeric and PRCC G-quadruplex forming sequences. Our investigation shows that these chromam ligands and their complexes are able to selectively bind and stabilize parallel and mixed hybrid topology of G-quadruplex both In vitro and in cellular conditions. A molecular docking and MD simulation study also suggests the binding of these compounds with G-quadruplex conformation. Collectively our study suggests these chroman complexes for the first time as a potentially useful fluorescent chemical product for G-quadruplex specific ligands and expands an option for G-quadruplex targeting ligands. [Display omitted] [ABSTRACT FROM AUTHOR]

A fluorescent probe (probe 1) with selectivity and sensitivity for H 2 S and N 2 H 4 has been developed. Probe 1 exhibits dual-excitation and dual-emission fluorescence response to H 2 S and fluorescence enhancement toward N 2 H 4 with distinct naked-eye color changes. Moreover, probe 1 displayed low detection limits for H 2 S and N 2 H 4 of 0.90 nM and 23.6 nM, respectively. The reaction mechanism of probe 1 toward H 2 S and N 2 H 4 was established using 1H NMR and HR-MS spectroscopy. Additionally, probe 1 was used for monitoring and differentiating H 2 S and N 2 H 4 in HeLa cells and displays good biocompatibility and low cytotoxicity. • Probe 1 exhibits dual-emission fluorescence-enhanced detection of H 2 S in mitochondria. • Probe 1 exhibits a turn-on response for the detection of N 2 H 4 in mitochondria. • Probe 1 exhibits rapid response, good optical performance, high selectivity, and high sensitivity in solution. • Probe 1 displays multi species detection in the mitochondria of HeLa cells and zebrafish. • Detection using Probe 1 can be achieved using a smartphone with a chromaticity APP. [ABSTRACT FROM AUTHOR]

[Display omitted] • Summarization of recent progress in the biomedical applications of water-soluble polythiophenes, covering biosensing, cell imaging, gene delivery, drug delivery, cancer therapy, and tissue engineering. • Emphasis on polymerization methods of water-soluble polythiophenes. • Description of the mechanisms underlying the biomedical applications of water-soluble polythiophenes. • Outlining of challenges and future developments in the use of water-soluble polythiophenes for biomedical applications. Water-soluble polythiophenes (PTs) have gained attention for their potential in medical uses. PTs have strong electrical and optical properties and are sensitive to outside influences. Recently, they have been widely applied for detecting nucleic acid, adenosine triphosphate, antibiotics, cancer biomarkers, and metal ions. They are also used for imaging cells, delivering genes and drugs, photodynamic, photothermal, sonodynamic treatments, and tissue engineering. However, there is still no comprehensive review covering these applications. This review briefly outlines the medical uses of water-soluble PTs, including biosensing, cell imaging, gene and drug delivery, cancer treatment, and tissue engineering. It also discusses the significant challenges and future directions for water-soluble PTs. [ABSTRACT FROM AUTHOR]

[Display omitted] • A dual-locked fluorescence probe (TPA-DNP) for LDs and HClO was designed. • TPA-DNP could detect LDs and HClO simultaneously by "turn-on" fluorescence singal. • TPA-DNP had successfully been applied in imaging HClO within LDs accumulation environments. • The real-time dynamic imaging of LDs and HClO by TPA-DNP under the LPS-induced inflammatory system. Inflammation is a key factor leading to the occurrence and development of many diseases, both lipid droplets (LDs) and hypochlorous acid (HClO/ClO-) are regarded as the important biomarkers of inflammation. Therefore, it is of great significance to develop an efficient single chemical sensor that can simultaneously detect these two biomarkers. To achieve the goal, we developed a dual-locked fluorescence probe (TPA-DNP) by fusing two targets activated reporting system, its implementation was achieved by turning-on the fluorescence of TPA-DNP through LDs and HClO/ClO- simultaneously. In simulated LDs environment, TPA-DNP displayed excellent selectivity to HClO/ClO-, high sensitivity (LOD = 0.527 μM) and strong anti-interference ability. In addition, cell and zebrafish imaging experiments showed that TPA-DNP could be utilized to visualize exogenous/endogenous HClO/ClO- in LDs environment, and could also be used to observe the impact of LDs changes on the HClO/ClO- detection. On the basis, TPA-DNP served as a favorable tool to achieve visualization of inflammatory dynamic changes. [ABSTRACT FROM AUTHOR]

Micro- and nanoplastics (MNPs) are ubiquitous in the environment, resulting in the uptake of MNPs by a variety of organisms, including humans, leading to particle-cell interaction. Human macrophages derived from THP-1 cell lines take up Polystyrene (PS), a widespread plastic. The question therefore arises whether primary human macrophages also take up PS micro- and nanobeads (MNBs) and how they react to this stimulation. Major aim of this study is to visualize this uptake and to validate the isolation of macrophages from peripheral blood mononuclear cells (PBMCs) to assess the impact of MNPs on human macrophages. Uptake of macrophages from THP-1 cell lines and PBMCs was examined by transmission electron microscopy (TEM), scanning electron microscopy and live cell imaging. In addition, the reaction of the macrophages was analyzed in terms of metabolic activity, cytotoxicity, production of reactive oxygen species (ROS) and macrophage polarization. This study is the first to visualize PS MNBs in primary human cells using TEM and live cell imaging. Metabolic activity was size- and concentration-dependent, necrosis and ROS were increased. The methods demonstrated in this study outline an approach to assess the influence of MNP exposure on human macrophages and help investigating the consequences of worldwide plastic pollution. [Display omitted] • Human monocyte-derived macrophages as novel in vitro model to study effects of MNP. • Concentration-dependent cytoplasmic uptake of PS MNBs visualized by TEM and SEM. • Uptake of PS MNBs by primary human macrophages is visible in live cell imaging. • Effects of different sized, non-fluorescent PS-MNBs in physiological concentration. • PS MNBs induce nitric oxide formation and cytotoxic effects in human macrophages. [ABSTRACT FROM AUTHOR]

• Constructing a complex (1) with precise structures by introducing luminescent groups triphenylphosphine (TPA), is used for treatment and imaging. • Within 0.5 h, 1 can be distributed into cells, performing effective cell imaging. • 1 can be used for CDT, promoting oxidative stress and causing cell apoptosis. The combination of imaging and treatment is the fundamental strategy for achieving precise anticancer treatment. This article constructs a copper(I) complex (1) by aminourea derivative containing triphenylamine (TPA). 1 with precise structure, can be used for cell imaging and chemodynamic therapy (CDT). Within 0.5 h, 1 can be distributed into the mitochondria of T24 cells, performing effective cell imaging and then promoting oxidative stress and causing cell apoptosis. A precise complex with luminescent triphenylphosphine (TPA) group, is used for the effective cell imaging, promoting oxidative stress and causing cell apoptosis. [Display omitted] [ABSTRACT FROM AUTHOR]

• Novel indium(III) phthalocyanines baring mercaptopyridine groups and their quaternized derivatives were synthesized. • Singlet oxygen generation and photostability of these phthalocyanines were investigated. • Electrochemical biosensing assay conducted with disposable pencil graphite electrodes. • Interaction of phthalocyanines with AS1411 examined by electrochemical impedance spectroscopy as well as spectroscopic techniques. • The developed targeted PDT agent has the potential for breast cancer treatment in the future. In the study, the novel indium(III) phthalocyanines containing 4-mercaptopyridine groups as substituents on the peripheral (Pc3) or non-peripheral (Pc8) tetra-and peripheral octa (Pc13) positions were synthesized. After then, the synthesized phthalocyanines were converted to their water-soluble derivatives that are Pc5, Pc10, and Pc15 by the quaternization of the nitrogen atoms on the substituents. These new phthalocyanines have been characterized by various spectroscopic methods such as FT-IR, 1H NMR, elemental analysis, UV–vis, MALDI-TOF mass spectra. Photochemical properties were also investigated for the synthesized phthalocyanines. In this study; quaternized derivatives were studied in DMSO and aqueous solutions, while non-quaternized derivatives were studied only in DMSO. The interaction between AS1411 and three phthalocyanines (Pc5, Pc10 , and Pc15) was investigated using spectroscopic methods and electrochemical impedance spectroscopy technique combined with pencil graphite electrodes (PGEs). The cytotoxic activity of AS1411 and each phthalocyanine was investigated in the MCF-7 and doxorubicin (Dox) resistant MCF-7/DOX-R cell lines in the presence and absence of Dox. The photodynamic therapy (PDT) activity of Pc5, Pc10 , and Pc15 and their AS1411 conjugates was investigated on MCF-7, MCF-7/DOX-R and MCF-10A cells. The IC 50 values for MCF-7 cells were found as 1.1, 0.46, and 0.46 mM for Pc5 and 0.58, 0.34, and 0.32 mM for Pc5 -AS1411 conjugate in the absence of irradiation and radiation with 7 J and 20 J. The values for MCF-7/DOX-R cells were found 0.81, 0.30, and 0.11 mM for Pc5 and 0.39, 0.12, and 0.15 mM for Pc5 -AS1411 conjugate without irradiation and radiation with 7 J and 20 J, respectively. Apoptosis and cell imaging studies showed that conjugation of the phthalocyanines with AS1411 induced apoptosis in both cell lines and increases the intracellular entry of phthalocyanine Pc5. [ABSTRACT FROM AUTHOR]

Cryopreserved cell banks are fundamental for off-the-shelf mesenchymal stromal cell (MSC) therapies. However, cryopreserved MSCs are functionally impaired after thaw. Since metabolic fitness is linked to the functional potency of MSCs, we tested whether cultured (fresh) and thawed MSCs exhibit metabolic differences. Donor- and age-matched cultured and cryopreserved human umbilical cord or bone marrow MSCs were lifted or thawed respectively, suspended in xeno-free media, and cultured at 37C, 5% CO2. Mitochondrial morphology was assessed by live cell imaging of Mitotracker-stained cultures. Cellular energetics were quantified using a Seahorse metabolic analyzer after 6-, 24-, and 48-hours post-thaw. Thawed cells exhibited mitochondrial fusion and reduced networking, accompanied by reduced metabolic flux. These effects lasted 24-48 hours, depending on the donor. MSCs exposed to IFN-γ and TNF-α have increased potency, so we tested whether this strategy enhances MSC metabolism. Indeed, cytokine preconditioning shifted MSCs into glycolysis, independent of dose or time, and this effect was retained during cryopreservation and thaw. We next tested whether the glycolytic state of preconditioned MSCs improves their function. Fresh and thawed MSCs were challenged with IFN-γ and TNF-α and changes in functional biomarkers including IDO-1 quantified using RT-qPCR. Six and 24 hours after thaw, the preconditioned MSCs produced equivalent IDO-1 to fresh cells, suggesting that cytokine preconditioning may be a useful strategy to accelerate metabolic and functional recovery of cryopreserved MSCs. [ABSTRACT FROM AUTHOR]

The biomechanical regulation of the cytoskeleton and cell adhesions underlies various essential cellular functions. Studying them requires visualizing their nanostructure and molecular dynamics with evermore precise spatio-temporal resolution. In this review we will focus on the recent advances in single molecule fluorescence imaging techniques and discuss how they improve our understanding of mechanically sensitive cellular structures such as adhesions and the cytoskeleton. We will also discuss future directions for research, emphasizing on the 3D nature of cellular structures and tissues, their mechanical regulation at the molecule level, as well as how super-resolution microscopy will enhance our knowledge on protein structure and conformational changes in the cellular context. [ABSTRACT FROM AUTHOR]

The Tubulin Code revolutionizes our understanding of microtubule dynamics and functions, proposing a nuanced system governed by tubulin isotypes, posttranslational modifications (PTMs) and microtubule-associated proteins (MAPs). Tubulin isotypes, diverse across species, contribute structural complexity, and are thought to influence microtubule functions. PTMs encode dynamic information on microtubules, which are read by several microtubule interacting proteins and impact on cellular processes. Here we discuss recent technological and methodological advances, such as in genome engineering, live cell imaging, expansion microscopy, and cryo-electron microscopy that reveal new elements and levels of complexity of the tubulin code, including new modifying enzymes and nanopatterns of PTMs on individual microtubules. The Tubulin Code's exploration holds transformative potential, guiding therapeutic strategies and illuminating connections to diseases like cancer and neurodegenerative disorders, underscoring its relevance in decoding fundamental cellular language. [ABSTRACT FROM AUTHOR]

Fluorescent probes have revolutionized biological imaging by enabling the real-time visualization of cellular processes under physiological conditions. However, their size and potential perturbative nature can pose challenges in retaining the integrity of biological functions. This manuscript highlights recent advancements in the development of small fluorescent probes for optical imaging studies. Single benzene-based fluorophores offer versatility with minimal disruption, exhibiting diverse properties like aggregation-induced emission and pH responsiveness. Fluorescent nucleobases enable precise labeling of nucleic acids without compromising function, offering high sensitivity and compatibility with biochemistry studies. Bright yet small fluorescent amino acids provide an interesting alternative to bulky fusion proteins, facilitating non-invasive imaging of cellular events with high precision. These miniaturized fluorophores promise enhanced capabilities for studying biological systems in a non-invasive manner, fostering further innovations in molecular imaging. [ABSTRACT FROM AUTHOR]

Perennial ryegrass (Lolium perenne) is a fructan-accumulating plant constituting one of the most important grassland species with high herbage production, nutritive value and digestibility for grazing cattle. Although fructans were reported to be involved in plant defense acting as antioxidants or stress signals, their contribution in root protection is still to be explored. In roots, atypical defense is provided by the "Root Extracellular Trap" or "RET" at the root-soil interface. The molecular composition and structural organization of the RET are essential to provide root defense against pathogen attacks and abiotic stresses. The RET was reported to be mainly composed of polysaccharides (homogalacturonan, xylogalacturonan, xyloglucan) and proteoglycans such as arabinogalactan proteins (AGPs). Our aim is to characterize the RET composition of L. perenne using cell imaging techniques and a wide range of monoclonal antibodies directed against epitopes from cell wall glycomolecules and to investigate the potential presence of fructans. Interestingly, we found that both mucilage and cell wall surface of border cells were enriched in AGP epitopes. An increased amount of the AGP-containing mucilage was produced by L. perenne root tip in response to both elicitor and osmotic stress. Fructan epitopes were also detected in root cap cells and appeared to be released in the RET under stress conditions. Taken together our findings suggest that AGPs together with fructans are involved in root response of L. perenne to environmental stresses. • The root tip of perennial ryegrass produced border cells of different morphologies. • The root extracellular trap (RET) of perennial ryegrass contained abundant mucilage rich in arabinogalactan protein (AGP). • Fructan epitopes are released within the RET in response to PEG-induced osmotic stress and flagellin 22 elicitation. • Fructans, together with AGPs are promising candidates in root response to environmental stress in perennial ryegrass. [ABSTRACT FROM AUTHOR]