Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

NIR-I emissive cyanine derived molecular probe for selective monitoring of hepatic albumin levels during hyperglycemia.

In this study, we report a small molecule optical marker BI-CyG derived from the structural engineering of a cyanine scaffold. The developed probe offers suitable advantages over existing cyanine-based albumin specific probes in terms of its excitation and emission wavelengths, which are 760 and 830-832 nm, respectively. Structural tuning of the cyanine architecture leading to extended π-conjugation and resulting in a suitable bathochromic shift in the emission wavelength of the probe is represented in this study. The probe besides emitting in the NIR region, also possesses the desirable characteristics of being a potential target selective optical marker, as established from various biophysical studies. Molecular modelling and simulation studies provided critical insights into the binding of the probe in the protein microenvironment, which was further supported by experimental studies. The probe displayed intracellular albumin selectivity and was utilized for demonstrating alteration in albumin levels in pathological states such as hyperglycemia in hepatic cells. The present study also sheds some light on using BI-CyG as an imaging probe and on the role of metformin as a suitable drug for balancing hyperglycemia-induced reduced intra-hepatic albumin levels. The study, thus, attempts to highlight the structural derivatization of cyanine to afford a potential probe for serum albumin and its deployment to image altering albumin levels in an induced pathological condition, hyperglycemia.

Preclinical evaluation of AGTR1-Targeting molecular probe for colorectal cancer imaging in orthotopic and liver metastasis mouse models.

Despite advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable for patients with distant liver metastasis. Fluorescence molecular imaging with specific probes is increasingly used to guide CRC surgical resection in real-time and treatment planning. Here, we demonstrate the targeted imaging capacity of an MPA-PEG4-N3-Ang II probe labeled with near-infrared (NIR) fluorescent dye targeting the angiotensin II (Ang II) type 1 receptor (AGTR1) that is significantly upregulated in CRC. MPA-PEG4-N3-Ang II was highly selective and specific to in vitro tumor cells and in vivo tumors in a mouse CRC xenograft model. The favorable ex vivo imaging and in vivo biodistribution of MPA-PEG4-N3-Ang II afforded tumor-specific accumulation with low background and >10 contrast tumor-to-colorectal values in multiple subcutaneous CRC models at 8 h following injection. Biodistribution analysis confirmed the probe's high uptake in HT29 and HCT116 orthotopic and liver metastatic models of CRC with signal-to-noise ratio (SNR) values of tumor-to-colorectal and -liver fluorescence of 5.8 ± 0.6, 5.3 ± 0.7, and 2.7 ± 0.5, 2.6 ± 0.5, respectively, enabling high-contrast intraoperative tumor visualization for surgical navigation. Given its rapid tumor targeting, precise tumor boundary delineation, durable tumor retention and docking study, MPA-PEG4-N3-Ang II is a promising high-contrast imaging agent for the clinical detection of CRC.

Covalent activity-based probes for imaging of serine proteases.

Serine proteases are one of the largest mechanistic classes of proteases. They regulate a plethora of biochemical pathways inside and outside the cell. Aberrant serine protease activity leads to a wide variety of human diseases. Reagents to visualize these activities can be used to gain insight into the biological roles of serine proteases. Moreover, they may find future use for the detection of serine proteases as biomarkers. In this review, we discuss small molecule tools to image serine protease activity. Specifically, we outline different covalent activity-based probes and their selectivity against various serine protease targets. We also describe their application in several imaging methods.

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4.

Chemokines and chemokine receptors are indispensable to play a key role in the development of malignant tumors. As one of the most widely expressed chemokine receptors, chemokine (C-X-C motif) receptor 4 (CXCR4) has been a popular research focus. In most tumors, CXCR4 expression is significantly upregulated. Moreover, integrated nuclide diagnosis and therapy targeting CXCR4 show great potential. [68Ga]Ga-pentixafor, a radioligand targeting CXCR4, exhibits a strong affinity for CXCR4 both in vivo and in vitro. However, [177Lu]Lu-pentixather, the therapeutic companion of [68Ga]Ga-pentixafor, requires significant refinement to mitigate its pronounced hepatic biodistribution. The objective of this study was to synthesize theranostic molecular tracers with superior CXCR4 targeting functions. The Daudi cell line, which highly expressed CXCR4, and the MM.1S cell line, which weakly expressed CXCR4, were used in this study. Based on the pharmacophore cyclo (-d-Tyr-n-me-d-Orn-l-Arg-L-2-NAL-Gly-) (CPCR4) of pentixafor, six tracers were synthesized: [124I]I-1 ([124I]I-CPCR4), [99mTc]Tc-2 ([99mTc]Tc-HYNIC-CPCR4), [124I]I-3 ([124I]I-pentixafor), [18F]AlF-4 ([18F]AlF-NETA-CPCR4), [99mTc]Tc-5 ([99mTc]Tc-MAG3-CPCR4) and [124I]I-6 ([124I]I-pentixafor-Ga) and their radiochemical purities were all higher than 95%. After positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, the [124I]I-6 group exhibited the best target-nontarget ratio. At the same time, comparing the [68Ga]Ga-pentixafor group with the [124I]I-6 group, we found that the [124I]I-6 group had a better target-nontarget ratio and lower uptake in nontarget organs. Therefore, compound 6 was selected for therapeutic radionuclide (131I) labeling, and the tumor-bearing animal models were treated with [131I]I-6. The volume of the tumor site was significantly reduced in the treatment group compared with the control group, and no significant side effects were found. [124I]I-6 and [131I]I-6 showed excellent affinity for targeting CXCR4, and they showed great potential for the integrated diagnosis and treatment of tumors with high CXCR4 expression.

Bifunctional molecular probe targeting tumor PD-L1 enhances anti-tumor efficacy by promoting ferroptosis in lung cancer mouse model.

PURPOSE: Immune checkpoint inhibitors (ICIs) targeting tumor-specific PD-1/PD-L1 significantly improve the overall survival rate of patients with advanced cancer by reactivating the immune system to attack cancer cells. To explore their tumor killing effect, we used the radionuclide iodine-131 (131I) to label the anti-PD-L1 antibody Atezolizumab (131I-PD-L1 mAb). METHOD: We prepared the radioimmunoassay molecular probe 131I-PD-L1 mAb by the chloramine-T method and evaluated its affinity using Lewis lung cancer (LLC) cells. The uptake of 131I-PD-L1 mAb by transplanted tumors was examined through SPECT and its in vivo distribution. We then compared the in vitro and in vivo anti-tumor efficacy of groups treated with control, PD-L1 mAb, 131I-PD-L1 mAb, and 131I-PD-L1 mAb + PD-L1 mAb combined treatment. We performed H&E staining to examine the changes in tumor, as well as the damage in major tissues and organs caused by potential side effects. The anti-tumor mechanism of 131I-PD-L1 mAb was analyzed by Western blot, RT-qPCR and immunohistochemistry (IHC). RESULT: 131I-PD-L1 mAb was highly stable and specific, and easily penetrated into tumor. 131I-PD-L1 mAb suppressed cancer cell proliferation in vitro, and inhibited tumor growth in vivo by inducing ferroptosis, thus prolonging the survival of experimental animals while demonstrating biological safety. CONCLUSION: Therefore, our study suggested that 131I-PD-L1 mAb affected the expression of tumor-related factors through ß-rays and thus promoted ferroptosis in tumor. Combined treatment showed better anti-tumor effect compared to single ICI treatment.

Synthesis and preclinical evaluation of a novel molecular probe [18F]AlF-NOTA-PEG2-Asp2-PDL1P for PET imaging of PD-L1 positive tumor.

Immunotherapy has brought great benefits to cancer patients, but only some patients benefit from it. Noninvasive, real-time and dynamic monitoring of the effectiveness of immunotherapy through PET imaging may provide assistance for the treatment plan of immunotherapy. In this study, we designed and synthesized a new targeted PD-L1 peptide NOTA-PEG2-Asp2-PDL1P, which was labeled with nuclide 18F to obtain a new imaging agent [18F]AlF-NOTA-PEG2-Asp2-PDL1P. The total radiochemical yield of [18F]AlF-NOTA-PEG2-Asp2-PDL1P was 13.7 % (Uncorrected radiochemical yield, n > 5). [18F]AlF-NOTA-PEG2-Asp2-PDL1P achieved high radiochemical purity (>95 %) with a molar activity more than 51.2 GBq/µmol. [18F]AlF-NOTA-PEG2-Asp2-PDL1P exhibited good hydrophilicity and had good stability both in vivo and in vitro, it can specifically targets B16F10 tumor with PD-L1 expression, and had a relatively high retention in tumor, a relatively fast clearance in vivo and a higher tumor-to-non-target ratio, all of which could make [18F]AlF-NOTA-PEG2-Asp2-PDL1P a potential tracer for PD-L1 prediction before clinical immunotherapy.

A rationally designed nuclei-targeting FAPI 04-based molecular probe with enhanced tumor uptake for PET/CT and fluorescence imaging.

PURPOSE: Fibroblast activation protein inhibitor (FAPI) -based probes have been widely studied in the diagnosis of various malignant tumors with positron emission tomography/computed tomography (PET/CT). However, current imaging studies of FAPI-based probes face challenges in rapid clearance rate and potential false-negative results. Furthermore, FAPI has been rarely explored in optical imaging. Considering this, further modifications are imperative to improve the properties of FAPI-based probes to address existing limitations and broaden their application scenarios. In this study, we rationally introduced methylene blue (MB) to FAPIs, thereby imparting nuclei-targeting and fluorescence imaging capabilities to the probes. Furthermore, we evaluated the added value of FAPI-based fluorescence imaging to traditional PET/CT, exploring the potential application of FAPI-based probes in intraoperative fluorescence imaging. METHODS: A new FAPI-based probe, namely NOTA-FAPI-MB, was designed for both PET/CT and fluorescence imaging by conjugation of MB. The targeting efficacy of the probe was evaluated on fibroblast activation protein (FAP)-transfected cell line and human primary cancer-associated fibroblasts (CAFs). Subsequently, PET/CT and fluorescence imaging were conducted on tumor-bearing mice. The tumor detection and boundary delineation were assessed by fluorescence imaging of tissues from hepatocellular carcinoma (HCC) patients. RESULTS: NOTA-FAPI-MB demonstrated exceptional targeting ability towards FAP-transfected cells and CAFs in comparison to NOTA-FAPI. This benefit arises from the cationic methylene blue (MB) affinity for anionic nucleic acids. PET/CT imaging of tumor-bearing mice revealed significantly higher tumor uptake of [18F]F-NOTA-FAPI-MB (standard uptake value of 2.20 ± 0.31) compared to [18F]F-FDG (standard uptake value of 1.66 ± 0.14). In vivo fluorescence imaging indicated prolonged retention at the tumor site, with retention lasting up to 24 h. In addition, the fluorescent probes enabled more precise lesion detection and tumor margin delineation than clinically used indocyanine green (ICG), achieving a 100.0% (6/6) tumor-positive rate for NOTA-FAPI-MB while 33.3% (2/6) for ICG. These findings highlighted the potential of NOTA-FAPI-MB in guiding intraoperative surgical procedures. CONCLUSIONS: The NOTA-FAPI-MB was successfully synthesized, in which FAPI and MB simultaneously contributed to the targeting effect. Notably, the nuclear delivery mechanism of the probes improved intracellular retention time and targeting efficacy, broadening the imaging time window for fluorescence imaging. In vivo PET/CT demonstrated favorable performance of NOTA-FAPI-MB compared to [18F]F-FDG. This study highlights the significance of fluorescence imaging as an adjunct technique to PET/CT. Furthermore, the encouraging results obtained from the imaging of human HCC tissues hold promise for the potential application of NOTA-FAPI-MB in intraoperative fluorescent surgery guidance within clinical settings.

A NIR-II Photoacoustic Probe for High Spatial Quantitative Imaging of Tumor Nitric Oxide in Vivo.

Nitric oxide (NO) exhibits both pro- and anti-tumor effects. Therefore, real-time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high-resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor-targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron-resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double-edged-sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO-mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti-cancer drug screening.

Activatable Dual-Optical Molecular Probe for Bioimaging Superoxide Anion in Epilepsy.

Superoxide anion (O2•-) plays a pivotal role in the generation of other reactive oxygen species within the body and is closely linked to epilepsy. Despite this connection, achieving precise imaging of O2•- during epilepsy pathology remains a formidable challenge. Herein, we develop an activatable molecular probe, CL-SA, to track the fluctuation of the level of O2•- in epilepsy through simultaneous fluorescence imaging and chemiluminescence sensing. The developed probe CL-SA demonstrated its efficacy in imaging of O2•- in neuronal cells, showcasing its dual optical imaging capability for O2•- in vitro. Furthermore, CL-SA was successfully used to observe aberrantly expressed O2•- in a mouse model of epilepsy. Overall, CL-SA provides us with a valuable tool for chemical and biomedical studies of O2•-, promoting the investigation of O2•- fluctuations in epilepsy, as well as providing a reliable means to explore the diagnosis and therapy of epilepsy.

Colabind: A Cloud-Based Approach for Prediction of Binding Sites Using Coarse-Grained Simulations with Molecular Probes.

Binding site prediction is a crucial step in understanding protein-ligand and protein-protein interactions (PPIs) with broad implications in drug discovery and bioinformatics. This study introduces Colabind, a robust, versatile, and user-friendly cloud-based approach that employs coarse-grained molecular dynamics simulations in the presence of molecular probes, mimicking fragments of drug-like compounds. Our method has demonstrated high effectiveness when validated across a diverse range of biological targets spanning various protein classes, successfully identifying orthosteric binding sites, as well as known druggable allosteric or PPI sites, in both experimentally determined and AI-predicted protein structures, consistently placing them among the top-ranked sites. Furthermore, we suggest that careful inspection of the identified regions with a high affinity for specific probes can provide valuable insights for the development of pharmacophore hypotheses. The approach is available at https://github.com/porekhov/CG_probeMD.

Molecular Imaging of Diffuse Cardiac Fibrosis with a Radiotracer That Targets Proteolyzed Collagen IV.

Purpose To develop an approach for in vivo detection of interstitial cardiac fibrosis using PET with a peptide tracer targeting proteolyzed collagen IV (T-peptide). Materials and Methods T-peptide was conjugated to the copper chelator MeCOSar (chemical name, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid) and radiolabeled with copper 64 (64Cu). PET/CT scans were acquired following intravenous delivery of 64Cu-T-peptide-MeCOSar (0.25 mg/kg; 18 MBq ± 2.7 [SD]) to male transgenic mice overexpressing ß2-adrenergic receptors with intermediate (7 months of age; n = 4 per group) to severe (10 months of age; n = 11 per group) cardiac fibrosis and their wild-type controls. PET scans were also performed following coadministration of the radiolabeled probe with nonlabeled T-peptide in excess to confirm binding specificity. PET data were analyzed by t tests for static scans and analysis of variance tests (one- or two-way) for dynamic scans. Results PET/CT scans revealed significantly elevated (2.24-4.26-fold; P < .05) 64Cu-T-peptide-MeCOSar binding in the fibrotic hearts of aged transgenic ß2-adrenergic receptor mice across the entire 45-minute acquisition period compared with healthy controls. The cardiac tracer accumulation and presence of diffuse cardiac fibrosis in older animals were confirmed by gamma counting (P < .05) and histologic evaluation, respectively. Coadministration of a nonradiolabeled probe in excess abolished the elevated radiotracer binding in the aged transgenic hearts. Importantly, PET tracer accumulation was also detected in younger (7 months of age) transgenic mice with intermediate cardiac fibrosis, although this was only apparent from 20 minutes following injection (1.6-2.2-fold binding increase; P < .05). Conclusion The T-peptide PET tracer targeting proteolyzed collagen IV provided a sensitive and specific approach of detecting diffuse cardiac fibrosis at varying degrees of severity in a transgenic mouse model. Keywords: Diffuse Cardiac Fibrosis, Molecular Peptide Probe, Molecular Imaging, PET/CT © RSNA, 2024.

[Imaging of In Vivo Oxygen Tension Based on Phosphorescence Lifetime Microscopy].

Molecular oxygen plays essential roles in aerobic organisms as a terminal electron acceptor in the electron transport chain in mitochondria. The intracellular oxygen concentration of the entire body is strictly regulated by a balance between the supply of oxygen from blood vessels and the consumption of oxygen in mitochondria. The disruption of oxygen homeostasis in the body often results in serious pathologies such as cancer, cerebral infarction, and chronic kidney disease, and thus considerable effort has been devoted to the development of suitable techniques allowing the qualitative and quantitative detection of tissue oxygen levels. This review focuses on recent advances in the visualization of oxygen levels in tissue based on phosphorescence lifetime measurements using exogenously small molecular oxygen probes. Specially, I introduce the principle of oxygen sensing by means of phosphorescence quenching, recent advances in intracellular and intravascular oxygen probes based on iridium(III) complexes, a system for measuring phosphorescence lifetime combined with confocal scanning microscopy, and the applications of these technologies to in vivo oxygen measurements, emphasizing the usefulness of iridium(III) complexes as biological oxygen probes.

Activity-Based Fluorescence Diagnostics for Cancer.

Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.

Recent Progress in Peptide-Based Molecular Probes for Disease Bioimaging.

Recent strides in molecular pathology have unveiled distinctive alterations at the molecular level throughout the onset and progression of diseases. Enhancing the in vivo visualization of these biomarkers is crucial for advancing disease classification, staging, and treatment strategies. Peptide-based molecular probes (PMPs) have emerged as versatile tools due to their exceptional ability to discern these molecular changes with unparalleled specificity and precision. In this Perspective, we first summarize the methodologies for crafting innovative functional peptides, emphasizing recent advancements in both peptide library technologies and computer-assisted peptide design approaches. Furthermore, we offer an overview of the latest advances in PMPs within the realm of biological imaging, showcasing their varied applications in diagnostic and therapeutic modalities. We also briefly address current challenges and potential future directions in this dynamic field.

Recent Advances of Aminopeptidases-Responsive Small-Molecular Probes for Bioimaging.

Aminopeptidases, enzymes with critical roles in human body, are emerging as vital biomarkers for metabolic processes and diseases. Aberrant aminopeptidase levels are often associated with diseases, particularly cancer. Small-molecule probes, such as fluorescent, fluorescent/photoacoustics, bioluminescent, and chemiluminescent probes, are essential tools in the study of aminopeptidases-related diseases. The fluorescent probes provide real-time insights into protein activities, offering high sensitivity in specific locations, and precise spatiotemporal results. Additionally, photoacoustic probes offer signals that are able to penetrate deeper tissues. Bioluminescent and chemiluminescent probes can enhance in vivo imaging abilities by reducing the background. This comprehensive review is focused on small-molecule probes that respond to four key aminopeptidases: aminopeptidase N, leucine aminopeptidase, Pyroglutamate aminopeptidase 1, and Prolyl Aminopeptidase, and their utilization in imaging tumors and afflicted regions. In this review, the design strategy of small-molecule probes, the variety of designs from previous studies, and the opportunities of future bioimaging applications are discussed, serving as a roadmap for future research, sparking innovations in aminopeptidase-responsive probe development, and enhancing our understanding of these enzymes in disease diagnostics and treatment.

Molecular Compressive Force Sensor for Mapping Forces at the Cell-Substrate Interface.

Mechanical forces are crucial for biological processes such as T cell antigen recognition. A suite of molecular tension probes to measure pulling forces have been reported over the past decade; however, there are no reports of molecular probes for measuring compressive forces, representing a gap in the current mechanobiology toolbox. To address this gap, we report a molecular compression reporter using pseudostable hairpins (M-CRUSH). The design principle was based on a pseudostable DNA structure that folds in response to an external compressive force. We created a library of DNA stem-loop hairpins with varying thermodynamic stability, and then used Förster Resonance Energy Transfer (FRET) to quantify hairpin folding stability as a function of temperature and crowding. We identified an optimal pseudostable DNA hairpin highly sensitive to molecular crowding that displayed a shift in melting temperature (Tm) of 7 °C in response to a PEG crowding agent. When immobilized on surfaces, this optimized DNA hairpin showed a 29 ± 6% increase in FRET index in response to 25% w/w PEG 8K. As a proof-of-concept demonstration, we employed M-CRUSH to map the compressive forces generated by primary naïve T cells. We noted dynamic compressive forces that were highly sensitive to antigen presentation and coreceptor engagement. Importantly, mechanical forces are generated by cytoskeletal protrusions caused by acto-myosin activity. This was confirmed by treating cells with cytoskeletal inhibitors, which resulted in a lower FRET response when compared to untreated cells. Furthermore, we showed that M-CRUSH signal is dependent on probe density with greater density probes showing enhanced signal. Finally, we demonstrated that M-CRUSH probes are modular and can be applied to different cell types by displaying a compressive signal observed under human platelets. M-CRUSH offers a powerful tool to complement tension sensors and map out compressive forces in living systems.

A novel pH-activated AIEgen probe for dynamic lysosome tracking and high-efficiency photodynamic therapy.

A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function.

For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800-2800 cm-1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.