Imaging Assays Research Articles

Dipsacus Asperoides-Derived Exosomes-Like Nanoparticles Inhibit the Progression of Osteosarcoma via Activating P38/JNK Signaling Pathway.

Osteosarcoma is a prevalent and highly malignant primary bone tumor. However, current clinical therapeutic drugs for osteosarcoma are not suitable for long-term use due to significant side effects. Therefore, there is an urgent need to develop new drugs with fewer side effects. Dipsacus asperoides C. Y. Cheng et T. M. Ai, a traditional Chinese medicine, is commonly used for its anti-inflammatory, anti-pain, bone fracture healing, and anti-tumor effects. In this study, we investigated the effects of exosome-like nanoparticles derived from Dipsacus asperoides (DAELNs) on osteosarcoma cells in vitro and in vivo. DAELNs were isolated and purified from Dipsacus asperoides and their physical and chemical properties were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The cellular uptake of DAELNs in osteosarcoma cells was analyzed by PKH26 staining. The proliferation, invasion, migration, and apoptosis of osteosarcoma cells were assessed using CCK8 assay, EdU assay, colony-formation assay, transwell assay, wound healing assay, and mitochondrial membrane potential measurement, respectively. The regulatory mechanism of DAELNs inhibiting the progression of osteosarcoma via activating P38/JNK signaling pathway was investigated using Western blotting and immunohistochemistry. Moreover, the therapeutic effects of DAELNs were evaluated using in vivo small animal imaging assay, HE staining, and immunohistochemistry. Our results showed that DAELNs inhibited the proliferation, invasion, migration, and fostered the apoptosis of osteosarcoma cells in vitro and suppressed the tumor growth of osteosarcoma cells in a xenograft nude mouse model. Furthermore, the bio-distribution of DiD-labeled DAELNs showed preferential targeting of osteosarcoma tumors and excellent biosafety in histological analysis of the liver and kidney. Mechanistically, DAELNs activated the P38/JNK signaling pathway-induced apoptosis. Taken together, DAELNs are novel, natural, and osteosarcoma-targeted agents that can serve as safe and effective therapeutic approaches for the treatment of osteosarcoma.

Active oxygen generation induced by the glucose sensor TaHXK7-1A decreased the drought resistance of transgenic Arabidopsis and wheat (Triticum aestivum L.)

Improving wheat drought resistance is of great significance for grain production and food security. Hexokinases (HXKs) play a role in sugar signal transduction and are involved in abiotic stress responses in wheat. To clarify the relationship between HXKs and drought stress in wheat, we used the rice active oxygen induction gene OsHXK1 as a reference sequence and the homologously cloned wheat TaHXK7-1A gene. TaHXK7-1A was localized in the nucleus and cell membrane. Under drought stress, over-expression of TaHXK7-1A increased the contents of O2·－ and malondialdehyde (MDA) and significantly up-regulated the respiratory burst oxidative homologue (RBOHs) genes in transgenic Arabidopsis. In addition, the over-expression of TaHXK7-1A inhibited the growth of Arabidopsis seedlings and increased ROS accumulation under 6 % exogenous glucose treatment. Gene silencing of TaHXK7-1 decreased the contents of O2·－ and MDA in wheat leaves under drought stress, and the RBOHs was significantly down-regulated, which improved the drought resistance of wheat. The results of yeast one-hybrid, EMSA, and dual-luciferase assays showed that TabHLH148-5A bound to the E-box motif of the TaHXK7-1A promoter and inhibited the expression of TaHXK7-1A. In addition, yeast two-hybrid and luciferase complementation imaging assays showed that TaHXK7-1A interacted with TaGRF3-4A. These results indicate that the glucose sensor TaHXK7-1A was negatively regulated by TabHLH148-5A, interacted with TaGRF3-4A, and negatively regulated wheat drought resistance by regulating RBOHs expression and inducing ROS production, thus providing a theoretical basis for revealing the molecular mechanism of wheat drought resistance.

P017 An activity-based chemiluminescence assay targeting granzyme A for the detection of Inflammatory Bowel Diseases

Abstract Background Inflammatory Bowel Diseases (IBD) represent a pressing global health concern lacking precise diagnostic tools for accurate patient stratification. Conventional methods, such as colonoscopy are extremely invasive whilst generic inflammatory markers (such as CRP or Calprotectin), are not specific enough and pose limitations in assessing disease activity, urging the need for novel, non-invasive diagnostic approaches which can rapidly inform clinicians on the inflammatory status of a patients bowels. Methods Our study focused on developing a non-invasive chemiluminescence assay targeting active granzyme A (GzmA) in stool supernatants. Employing a multifaceted approach encompassing functional, chemical, and imaging assays on human cells and gut tissue biopsies, we discerned elevated GzmA levels predominantly secreted by CD8+ T cells in inflamed gut tissues. Results The design of GzmA-INH, a specific inhibitor for GzmA, underscored the prevalence of active extracellular GzmA and it’s role in driving inflammation in the guts - revealing promising potential as a novel IBD biomarker. Further, we synthesized GzmA-CL, a highly reactive and selective chemiluminescent reporter for GzmA. Integrating GzmA-CL into a robust antibody-coated 96-well plate assay (Figure 1a), we analyzed 30 IBD biosamples along with healthy controls. Notably, substantially higher GzmA levels were observed in IBD patients, with discernible distinctions between ulcerative colitis (UC) and Crohn’s disease (CD) (Figure 1b). These levels correlated positively with established biomarkers, including faecal calprotectin and immune cell counts. Conclusion Our investigation highlighted active extracellular GzmA as a noteworthy biomarker in gut inflammation. The rapid, selective, and sensitive nature of our chemiluminescence assay presents promising prospects for precise IBD diagnosis and advancements in personalized medicine.

A Novel Multi-Functional Fluorescence Probe for the Detection of Al3+/Zn2+/Cd2+ and its Practical Applications.

A novel multi-functional fluorescence probe HMIC based on hydrazide Schiff base has been successfully synthesized and characterized. It can distinguish Al3+/Zn2+/Cd2+ in ethanol, in which fluorescence emission with different colors (blue for Al3+, orange for Zn2+, and green for Cd2+) were presented. The limits of detection of HMIC towards three ions were calculated from the titration curve as 7.70 × 10- 9 M, 4.64 × 10- 9 M, and 1.35 × 10- 8 M, respectively. The structures of HMIC and its complexes were investigated using UV-Vis spectra, Job's plot, infrared spectra, mass spectrometry, 1H-NMR and DFT calculations. Practical application studies have also demonstrated that HMIC can be applied to real samples with a low impact of potential interferents. Cytotoxicity and cellular imaging assays have shown that HMIC has good cellular permeability and potential antitumor effects. Interestingly, HMIC can image Al3+, Zn2+ and Cd2+ in the cells with different fluorescence signals.

Assessment of unmixing approaches for the quantitation of SERS nanoparticles in highly multiplexed spectral images

AbstractSurface‐enhanced Raman scattering nanoparticles (SERS NPs) offer powerful optical contrast features for imaging assays. Their gold core enhances the inelastic scattering cross section, allowing highly sensitive and rapid detection, and their characteristic sets of narrow spectral bands give them unsurpassed multiplexing capabilities. Multiplexed hyperspectral images are commonly unmixed using a compensation matrix of reference spectra to produce quantitative image channels illustrating the distribution of each material. It is these unmixed channels that are fit for interpretation from assays utilizing SERS NP contrast agents. Some factors that may impact SERS NP quantitative and dynamic range capabilities may include endogenous background heterogeneity, the ability of unmixing algorithms to account for signal variances, and linear system conditioning imposed by contrast agent signals. We report on hyperspectral Raman imaging of mixtures of SERS NPs from an expanded library of contrast agents. We study increasing plexity and varying degrees of system conditioning as inputs to a diverse set of classical, non‐negatively constrained, and regularized regression algorithms to investigate which signal features and unmixing methods deliver the most promising quantitation performance with the least error. Raman imaging of SERS NP mixtures is performed on controlled substrates and representative biological specimens, and experimental results are compared against ground truth data. We evaluate spectral fitting fidelity, quantitation, and specificity correlations with system conditioning. Spectral unmixing with a regularized hybrid of least squares regression with principal component analysis (HLP) algorithm approximated spectra with 3.5× better fitting fidelity and 3× better quantitation robustness with tissue background compared with simpler unmixing routines.

Dementia exacerbates periodontal bone loss in females.

Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and insitu imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aβ42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). Alveolar bone loss and insitu osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aβ42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.

Altered metabolism and DAM-signatures in female brains and microglia with aging

Despite Alzheimer’s disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo ‘metabolic reprogramming’, which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became ‘male-like.’ Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17β-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Molecular mechanisms of sensitivity and resistance to radiotherapy.

The molecular mechanisms underlying sensitivity and resistance to radiotherapy is an area of active investigation and discovery as its clinical applications have the potential to improve cancer patients' outcomes. In addition to the traditional pathways of radiation biology, our knowledge now includes molecular pathways of radiation sensitivity and resistance which have provided insights into potential targets for enhancing radiotherapy efficacy. Sensitivity to radiotherapy is influenced by the intricate interplay of various molecular mechanisms involved in DNA damage repair, apoptosis, cellular senescence, and epigenetics. Translationally, there have been several successful applications of this new knowledge into the clinic, such as biomarkers for improved response to chemo-radiation. New therapies to modify radiation response, such as the poly (ADP-ribose) polymerase (PARP) inhibitors, derived from research on DNA repair pathways leading to radiotherapy resistance, are being used clinically. In addition, p53-mediated pathways are critical for DNA damage related apoptosis, cellular senescence, and cell cycle arrest. As the understanding of genetic markers, molecular profiling, molecular imaging, and functional assays improve, these advances once translated clinically, will help propel modern radiation therapy towards more precise and individualized practices.

Abstract B085: K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC

Abstract Background: The 5-year survival rate of pancreatic cancer is extremely low compared with other cancers, although it has increased to 12% in 2023 in the United States. 80% of pancreatic ductal adenocarcinoma (PDAC) patients are not eligible for surgical procedure, and unfortunately, PDAC is refractory to standard-of-care chemotherapy. Thus, developing novel and more effective treatment strategies for PDAC is urgently needed. We previously reported keratin 17 (K17) as a novel negative prognostic and predictive biomarker, whose overexpression confers the resistance to chemotherapies. Here, we investigated the mechanisms of chemoresistance and tumor-specific vulnerabilities that can be exploited for K17-mediated targeted therapies. Methods: Survival analyses, immunohistochemistry (IHC), and matrix assisted laser desorption/ionization mass spectrometry (MALDI MSI) metabolite imaging were applied in patient derived samples. The K17 stable expression cell line models (KPC and PANC-1) and K17 loss-of-function cell line model (L3.6) were generated, where the analyses were applied including western blots, qRT-PCR, mass spectrometric metabolomics analysis, immunofluorescence imaging, cell viability and proliferation assays. KPC cells with or without K17 expression were orthotopically implanted into the head of the pancreas of c57B6J mice for tumor growth and treatment study. Results: Unbiased metabolomic studies in isogenic PDAC models identified several key metabolic pathways that are upregulated in the presence of K17. We demonstrate that K17 increases pyrimidine biosynthesis, a pathway that drives chemoresistance. Patient dataset analysis revealed that K17 expression and enzymes involved in pyrimidine, but not purine, de novo biosynthesis is associated with shorter patient survival. Furthermore, K17-expressing cells were more sensitive to Brequinar, a specific inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in de novo pyrimidine biosynthetic pathway. Targeting DHODH by small interfering RNA or by Brequinar with Gemcitabine synergistically inhibited the viability of K17- positive cells. Importantly, the combination of Gemcitabine and Brequinar significantly inhibited the growth of K17-expressing tumors and extended survival of mice bearing K17-positive tumors. Mechanistically, K17 increased mitochondrial DHODH translocation which made these cells more sensitive to Brequinar. We also found that the coil 2 domain of K17 was crucial for triggering metabolic reprogramming and promoting tumor aggression. Conclusions: Overall, we define a novel connection between K17, nucleotide metabolism, and chemoresistance in PDAC. We report for the first time that K17, a negative biomarker of the most aggressive PDAC subtype, drives chemoresistance by up-regulating de novo pyrimidine biosynthesis, which provides a tumor-specific vulnerability: DHODH. Further study could lead to the development of a DHODH targeted therapy for K17-expressing PDAC. Citation Format: Yinghuan Lyu, Kenneth Shroyer, Chun-Hao Pan, Robert Tseng, Md Afjalus Siraj, Girish Halemirle Rajacharya, Bo Chen, Katie L Donnelly, Michael Horowitz, Carlos Mauricio Mejia Arbelaez, Sumedha Chowdhury, Lyanne Oblein, Natalia Marchenko, Luisa Escobar-Hoyos, Pankaj K. Singh. K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B085.

AcMYB10 Involved in Anthocyanin Regulation of 'Hongyang' Kiwifruit Induced via Fruit Bagging and High-Postharvest-Temperature Treatments.

Light and temperature are key factors influencing the accumulation of anthocyanin in fruit crops. To assess the effects of fruit bagging during development and high post-ripening temperature on 'Hongyang' kiwifruit, we compared the pigmentation phenotypes and expression levels of anthocyanin-related genes between bagged and unbagged treatments, and between 25 °C and 37 °C postharvest storage temperatures. Both the bagging and 25 °C treatments showed better pigmentation phenotypes with higher anthocyanin concentrations. The results of the qRT-PCR analysis revealed that the gene expression levels of LDOX (leucoanthocyanidin dioxygenase), F3GT (UDP-flavonoid 3-O-glycosyltransferase ), AcMYB10, and AcbHLH42 were strongly correlated and upregulated by both the bagging treatment and 25 °C storage. The results of bimolecular fluorescence complementation and luciferase complementation imaging assays indicated an interaction between AcMYB10 and AcbHLH42 in plant cells, whereas the results of a yeast one-hybrid assay further demonstrated that AcMYB10 activated the promoters of AcLODX and AcF3GT. These results strongly suggest that enhanced anthocyanin synthesis is caused by the promoted expression of AcLODX and AcF3GT, regulated by the complex formed by AcMYB10-AcbHLH42.

Individual herpes simplex virus 1 (HSV-1) particles exit by exocytosis and accumulate at preferential egress sites.

The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.

Use of novel taurine-chitosan mediated liposomes for enhancing the oral absorption of doxorubicin via the TAUT transporter

Our research aimed to enhance the oral bioavailability of doxorubicin hydrochloride (DOX·HCl) while minimizing the potential for myocardial toxicity. To achieve this goal, we developed a new method that utilizes a coating material to encapsulate the drug in liposomes, which can specifically target intestinal taurine transporter proteins. This coating material, TAU-CS, was created by combining taurine with chitosan. We characterized TAU-CS using various methods, including 1H NMR, FT-IR, and scanning electron microscopy (SEM). The resulting liposomes exhibited a regular spherical morphology, with a particle size of 195.7 nm, an encapsulation efficiency of 91.23 %, and a zeta potential of +11.65 mV. Under simulated gastrointestinal conditions, TAU-CS/LIP@DOX·HCl exhibited good stability and slow release. Pharmacokinetic studies revealed that, compared with DOX·HCl, TAU-CS/LIP@DOX·HCl had a relative bioavailability of 342 %. Intracellular uptake, immunofluorescence imaging, and permeation assays confirmed that the taurine transporter protein mediates the intestinal uptake of these liposomes. Our study suggested that liposomes coated with TAU-CS could serve as an effective oral delivery system and that targeting the taurine transporter protein shows promise in enhancing drug absorption.

Simple models to understand complex disease: 10 years of progress from Caenorhabditis elegans models of amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

The nematode Caenorhabditis elegans are a powerful model system to study human disease, with numerous experimental advantages including significant genetic and cellular homology to vertebrate animals, a short lifespan, and tractable behavioral, molecular biology and imaging assays. Beginning with the identification of SOD1 as a genetic cause of amyotrophic lateral sclerosis (ALS), C. elegans have contributed to a deeper understanding of the mechanistic underpinnings of this devastating neurodegenerative disease. More recently this work has expanded to encompass models of other types of ALS and the related disease frontotemporal lobar degeneration (FTLD-TDP), including those characterized by mutation or accumulation of the proteins TDP-43, C9orf72, FUS, HnRNPA2B1, ALS2, DCTN1, CHCHD10, ELP3, TUBA4A, CAV1, UBQLN2, ATXN3, TIA1, KIF5A, VAPB, GRN, and RAB38. In this review we summarize these models and the progress and insights from the last ten years of using C. elegans to study the neurodegenerative diseases ALS and FTLD-TDP.

Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-d-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic β-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

A near-infrared emitting “off-on” fluorescent probe for bioimaging of Pd(Ⅱ) ions in living cells and mice

BackgroundThe surging consumption of palladium in modern industry has given rise to its accumulation in the ecosystem, posing conspicuous toxicity to aquatic organisms and human health. The investigation of palladium in biological systems is highly demanded for the in-depth understanding of its dynamics and behaviors. Fluorescence imaging serves as a powerful approach to assess palladium species in biological systems, and currently most of the sensing probes are applicable to living cells. Effective tracking of palladium species in living organisms is challenging, which requires sufficient hydrophilicity and imaging depth of the probes. ResultsBased on an intramolecular charge transfer (ICT) mechanism, a distyryl boron dipyrromethene (BODIPY) derivative (DISBDP-Pd) has been prepared for the near-infrared (NIR) fluorescence imaging of Pd2+ ions. Two additional methoxy triethylene glycol (TEG) chains could serve as flexible and hydrophilic moieties to enhance the aqueous solubility and cell permeability of the extended conjugate. Solution studies revealed that DISBDP-Pd exhibited a NIR fluorescence enhancement signal exclusively to Pd2+ ions (detection limit as low as 0.85 ppb) with negligible interference from Pd0 species and other closely related metal ions. Computational calculations have been performed to rationalize the binding mode and the mechanism of action. Fluorescence imaging assays have been conducted on A549 human non-small cell lung carcinoma cells and mouse models. Exhibiting negligible cytotoxicity, DISBDP-Pd demonstrated concentration-related fluorescence enhancement signals in response to Pd2+ ions in living cells and mice. SignificanceDISBDP-Pd exhibits advantages over many small molecule palladium probes in terms of satisfactory aqueous solubility, high sensitivity and selectivity, and biocompatible NIR emission property, which are particularly favorable for the sensing application in biological environments. The design strategy of this probe can potentially be adopted for the functionalization of other BODIPY probes implemented for NIR fluorescence bioimaging.

Qualification of a multiplexed tissue imaging assay and detection of novel patterns of HER2 heterogeneity in breast cancer.

Emerging data suggests that HER2 intratumoral heterogeneity (ITH) is associated with therapy resistance, highlighting the need for new strategies to assess HER2 ITH. A promising approach is leveraging multiplexed tissue analysis techniques such as cyclic immunofluorescence (CyCIF), which enable visualization and quantification of 10-60 antigens at single-cell resolution from individual tissue sections. In this study, we qualified a breast cancer-specific antibody panel, including HER2, ER, and PR, for multiplexed tissue imaging. We then compared the performance of these antibodies against established clinical standards using pixel-, cell- and tissue-level analyses, utilizing 866 tissue cores (representing 294 patients). To ensure reliability, the CyCIF antibodies were qualified against HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) data from the same samples. Our findings demonstrate the successful qualification of a breast cancer antibody panel for CyCIF, showing high concordance with established clinical antibodies. Subsequently, we employed the qualified antibodies, along with antibodies for CD45, CD68, PD-L1, p53, Ki67, pRB, and AR, to characterize 567 HER2+ invasive breast cancer samples from 189 patients. Through single-cell analysis, we identified four distinct cell clusters within HER2+ breast cancer exhibiting heterogeneous HER2 expression. Furthermore, these clusters displayed variations in ER, PR, p53, AR, and PD-L1 expression. To quantify the extent of heterogeneity, we calculated heterogeneity scores based on the diversity among these clusters. Our analysis revealed expression patterns that are relevant to breast cancer biology, with correlations to HER2 ITH and potential relevance to clinical outcomes.

Pipette-operable microfluidic devices with hydrophobic valves in sequential dispensing with various liquid samples: multiplex disease assay by RT-LAMP.

Microfluidic dispensing technologies often require additional equipment, posing challenges for their integration into point-of-care testing (POCT) applications. In response to this challenge, we have developed a pipette-operable microfluidic device fabricated using 3D printing technology for precise liquid dispensing. This device features three reaction chambers and three distinct hydrophobic valves to control the flow direction of liquids. Through these valves, the pipette-operable microfluidic device can sequentially dispense and isolate the liquid into the three reaction chambers, allowing for the individual conduction of three distinct reactions. These hydrophobic valves, with optimized flow resistance and burst pressure, can sustain a volumetric flow rate of up to 25 μL s-1, making them compatible with a standard pipette, a syringe, or a dropper operation. Furthermore, the device is successfully used to operate with various liquids, including BSA, DMEM, FBS, plasma, and blood, representing that the device has the potential to be used for various applications. Additionally, distinct RT-LAMP primer sets have been incorporated for diagnosing SARS-CoV-2, influenza A, and influenza B within each chamber through lyophilization. This pipette-operable microfluidic device serves as a versatile tool for diagnosing these three diseases using a single loading process, with results readable by the naked eye or image assay within 30 minutes of incubation. Finally, the design concepts are extended to engineer a microfluidic device with 20 reaction chambers, offering significant potential for multi-disease diagnostics.

High-contrast imaging of cellular non-repetitive drug-resistant genes via in situ dead Cas12a-labeled PCR.

In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms.

Predicting the phytotoxic mechanism of action of LiCoO2 nanomaterials using a novel multiplexed algal cytological imaging (MACI) assay and machine learning

This work uses novel high-throughput phenotypic profiling techniques to elucidate the most probable phytotoxic mechanism of action of LiCoO2 nanosheets.

Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-Tcells against glioblastoma.

CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function invitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.