Mapping a multiplexed zoo of mRNA expression.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

Stimulating macropinocytosis of peptide-drug conjugates through DNA-dependent protein kinase inhibition for treating KRAS-mutant cancer.

KRAS-mutant cancers, due to their protein targeting complexity, present significant therapeutic hurdles. The identification of the macropinocytic phenotype in these cancers has emerged as a promising alternative therapeutic target. Our study introduces MPD1, an macropinocytosis-targeting peptide-drug conjugates (PDC), which is developed to treat KRAS mutant cancers. This PDC is specifically designed to trigger a positive feedback loop through its caspase-3 cleavable characteristic. However, we observe that this loop is hindered by DNA-PK mediated DNA damage repair processes in cancer cells. To counter this impediment, we employ AZD7648, a DNA-PK inhibitor. Interestingly, the combined treatment of MPD1 and AZD7648 resulted in a 100% complete response rate in KRAS-mutant xenograft model. We focus on the synergic mechanism of it. We discover that AZD7648 specifically enhances macropinocytosis in KRAS-mutant cancer cells. Further analysis uncovers a significant correlation between the increase in macropinocytosis and PI3K signaling, driven by AMPK pathways. Also, AZD7648 reinforces the positive feedback loop, leading to escalated apoptosis and enhanced payload accumulation within tumors. AZD7648 possesses broad applications in augmenting nano-sized drug delivery and preventing DNA repair resistance. The promising efficacy and evident synergy underscore the potential of combining MPD1 with AZD7648 as a strategy for treating KRAS-mutant cancers.

Nanozyme-Initiated In Situ Cascade Reactions for Self-Amplified Biocatalytic Immunotherapy.

Biocatalytic nanomaterials have been verified to modulate the immunosuppressive state of an extensive range of solid tumors and directly induce antitumor immune response, which effectively combats the holdbacks in cancer immunotherapy. Herein, biomimetic cascade enzyme-initiated toxic-radical-generating devices (GHZD NCs) are fabricated by enveloping glucose oxidase (GOx), artificial nanozyme hemin, and sesquiterpene lactone endoperoxide derived dihydroartemisinin (DHA) into zeolitic imidazolate framework (ZIF-8) for enhanced biocatalytic immunotherapy. The GHZD NCs exhibit amplified multienzyme-mimic (glucose oxidase, peroxidase, and glutathione peroxidase) cascade reactions in artificial nanoscale proximity. Concurrently, a glutathione (GSH)-stimulated labile iron-current amplifier boosts C-centered free radicals, which endows the GHZD NCs with tumor-specific and self-circulating generation ability of vicious C-centered free radicals. Irreversible free radicals (·C and ·OH) and sustainable H2 O2 from sequential catalytic processes logically and selectively elevate the oxidative stress in the tumor, which further triggers an efficient immunogenic cell death (ICD) progress. In addition, the in situ nanozyme-based immunotherapy employed for tumor suppression successfully elicits the long-lasting immunological memory effect, which hinders the growth of distant tumors and lung metastasis.

High-resolution MRI of kidney microstructures at 7.05 T with an endo-colonic Wireless Amplified NMR detector.

To map the hemodynamic responses of kidney microstructures at 7.05 T with improved sensitivity, a Wireless Amplified NMR Detector (WAND) with cylindrical symmetry was fabricated as an endoluminal detector that can convert externally provided wireless signal at 600.71 MHz into amplified MR signals at 300.33 MHz. When this detector was inserted inside colonic lumens to sensitively observe adjacent kidneys, it could clearly identify kidney microstructures in the renal cortex and renal medullary. Owing to the higher achievable spatial resolution, differential hemodynamic responses of kidney microstructures under different breathing conditions could be individually quantified to estimate the underlying correlation between oxygen bearing capability and local levels of oxygen unsaturation. The WAND's ability to map Blood Oxygen Level Dependent (BOLD) signal responses in heterogeneous microstructures will pave way for early-stage diagnosis of kidney diseases, without the use of contrast agents for reduced tissue retention and toxicity.

Paperfluidic Chip Device for Small RNA Extraction, Amplification, and Multiplexed Analysis.

Small RNAs have been considered as potential biomarkers of various human diseases. Sensitive and multiplexed determination of small RNAs with point-of-care (POC) assay would be of great significance. Herein, an integrated paperfluidic chip device for multiplexed small RNA analysis was developed for the first time. In this system, the extraction and purification of small RNA was completed through a poly(ether sulfone) (PES) paper chip without the need for centrifugation. Subsequently, a newly designed hairpin probe-exponential amplification reaction (HP-EXPAR) was directly performed within the extraction paper chip. For the simultaneous realization of multiple detection, a multilayer paper chip was designed in a foldable manner with more portability and usability. Quantum dots (QDs) were employed as signal labels, which endowed this assay with high optical detection efficiency. Moreover, magnetic sheets were introduced as an alternative method for layer stacking, not only guaranteeing adjacent layers are in contact but also facilitating the sample dispersion. With these outstanding characteristics, our platform obtained a satisfactory sensitivity range from 3 × 105 to 3 × 108 copies with a limit of 3 × 106 copies. Additionally, the multiplex small RNA analyses from various cancer cells were in good agreement with the results of the real-time polymerase chain reaction (qRT-PCR). More importantly, simultaneous analysis of two types of miRNAs from clinical tumor samples demonstrated the clinical applicability of the system. Therefore, the proposed paper-based device shows great promise for POC applications in the future.