Targeting Labile Iron-Mediated Ferroptosis Provides a Potential Therapeutic Strategy for Rhabdomyolysis-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a global health problem that occurs in a variety of clinical settings. Despite some advances in supportive clinical care, no medicinal intervention has been demonstrated to reliably prevent AKI thus far. Therefore, it is highly necessary to investigate the pathophysiology and mechanisms involved in AKI for the discovery of therapeutics. In the current study, a robust change in the level of renal malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and elevated renal iron levels were observed in murine rhabdomyolysis-induced AKI (RM-AKI), which supports a pathogenic role of labile iron-mediated ferroptosis and provides a chance to utilize iron chelation for RM-AKI prevention. Given that the existing small molecule-based iron chelators did not show promising preventative effects against RM-AKI, we further designed and synthesized a new hydroxypyridinone-based iron chelator to potently inhibit labile iron-mediated ferroptosis. Lead compound AKI-02 was identified, which remarkably protected renal proximal tubular epithelial cells from ferroptosis as well as showed excellent iron chelation ability. Moreover, administration of AKI-02 led to renal function recovery, a result that was substantiated by the decreased contents of BUN and creatinine, as well as the reduced labile iron level and improved histopathology. Thus, our studies highlighted that targeting labile iron-mediated ferroptosis could provide therapeutic benefits against RM-AKI.

[Design, simulation and application of multichannel microfluidic chip for cell migration].

Cell migration is defined as the directional movement of cells toward a specific chemical concentration gradient, which plays a crucial role in embryo development, wound healing and tumor metastasis. However, current research methods showed low flux and are only suitable for single-factor assessment, and it was difficult to comprehensively consider the effects of other parameters such as different concentration gradients on cell migration behavior. In this paper, a four-channel microfluidic chip was designed. Its characteristics were as follows: it relied on laminar flow and diffusion mechanisms to establish and maintain a concentration gradient; it was suitable for observation of cell migration in different concentration gradient environment under a single microscope field; four cell isolation zones (20 µm width) were integrated into the microfluidic device to calibrate the initial cell position, which ensured the accuracy of the experimental results. In particular, we used COMSOL Multiphysics software to simulate the structure of the chip, which demonstrated the necessity of designing S-shaped microchannel and horizontal pressure balance channel to maintain concentration gradient. Finally, neutrophils were incubated with advanced glycation end products (AGEs, 0, 0.2, 0.5, 1.0 µmol·L -1), which were closely related to diabetes mellitus and its complications. The migration behavior of incubated neutrophils was studied in the 100 nmol·L -1 of chemokine (N-formylmethionyl-leucyl-phenyl-alanine) concentration gradient. The results prove the reliability and practicability of the microfluidic chip.

A Novel Microfluidic Device for the Neutrophil Functional Phenotype Analysis: Effects of Glucose and Its Derivatives AGEs.

Neutrophil dysfunction is closely related to the pathophysiology of patients with diabetes mellitus, but existing immunoassays are difficult to implement in clinical applications, and neutrophil's chemotaxis as a functional biomarker for diabetes mellitus prognostic remains largely unexplored. Herein, a novel microfluidic device consisted of four independent test units with four cell docking structures was developed to study the neutrophil chemotaxis, which allowed multiple cell migration observations under a single field of view (FOV) and guaranteed more reliable results. In vitro studies, the chemotaxis of healthy neutrophils to N-Formyl-Met-Leu-Phe (fMLP) gradient (0, 10, 100, and 1000 nM) was concentration-dependent. The distinct promotion or suppression in the chemotaxis of metformin or pravastatin pretreated cells were observed after exposure to 100 nM fMLP gradient, indicating the feasibility and efficiency of this novel microfluidic device for clinically relevant evaluation of neutrophil functional phenotype. Further, the chemotaxis of neutrophils pretreated with 25, 50, or 70 mM of glucose was quantitatively lower than that of the control groups (i.e., 5 mM normal serum level). Neutrophils exposed to highly concentrated advanced glycation end products (AGEs) (0.2, 0.5, or 1.0 µM; 0.13 µM normal serum AGEs level), a product of prolonged hyperglycemia, showed that the higher the AGEs concentration was, the weaker the migration speed became. Specifically, neutrophils exposed to high concentrations of glucose or AGEs also showed a stronger drifting along with the flow, further demonstrating the change of neutrophil chemotaxis. Interestingly, adding the N-benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1) (i.e., high-affinity RAGE inhibitor) into the migration medium with AGEs could hinder the binding between AGEs and AGE receptor (RAGE) located on the neutrophil, thereby keeping the normal chemotaxis of neutrophils than the ones incubated with AGEs alone. These results revealed the negative effects of high concentrations of glucose and AGEs on the neutrophil chemotaxis, suggesting that patients with diabetes should manage serum AGEs and also pay attention to blood glucose indexes. Overall, this novel microfluidic device could significantly characterize the chemotaxis of neutrophils and have the potential to be further improved into a tool for risk stratification of diabetes mellitus.

One therapeutic approach for triple-negative breast cancer: Checkpoint kinase 1 inhibitor AZD7762 combination with neoadjuvant carboplatin.

Carboplatin treatment is associated with potential benefits in practice in the neoadjuvant chemotherapy for Triple-negative breast cancer (TNBC) patients. In order to enhance its anti-tumor effects, new concepts for successful combination therapy are needed. Here, we interestingly found that the combination treatment of carboplatin with the Chk1 inhibitor AZD7762 synergistically inhibits TNBC cell growth in multiple TNBC cell lines in vitro. Mechanistically, we proved that prolonged carboplatin-treated induce cell mitotic arrest, and cells would fail to initiate the G2-M transition following the inhibition of the Chk1 pathway, leading to accumulation of DNA lesions. With this drug-in-combination treatment, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure is remarkably enhanced, which subsequently drives tumor cells multinucleation, polyploidization and apoptosis. Thus, our findings not only propose Chk1 as a therapeutic target for combination therapy with DNA-damaging agents such as carboplatin in TNBC, but also highlight that the induction of mitotic catastrophe could be considered as an alternative strategy for TNBC therapy.

MS2 device: smartphone-facilitated mobile nucleic acid analysis on microfluidic device.

Mobile sensing based on the integration of microfluidic devices and smartphones, so-called MS2 technology, has enabled many applications over recent years and continues to stimulate growing interest in both research communities and industries. In particular, MS2 technology has been proven to be able to be applied to molecular diagnostic analysis and can be implemented for basic research and clinical testing. However, the currently reported MS2-based nucleic acid analysis system has limited use in practical applications, because it is not integrated with quantitative PCR, multiplex PCR, and isothermal amplification functions, and lacks temperature control, image acquisition and real-time processing units with excellent performance. To provide a more universal and powerful platform, we here developed a novel MS2 device by integrating a thermocycler, a multi fluorescence detection unit, a PCR chip, an isothermal chip, and a smartphone. The MS2 device was approximately 325 mm (L) × 200 mm (W) × 200 mm (H) in volume and only 5 kg in weight, and showed an average power consumption of about 38.4 W. The entire nucleic acid amplification and analysis could be controlled through a self-made smartphone App. The maximum heating and cooling rates were 5 °C s-1 and 4 °C s-1, respectively. The entire PCR could be completed within 65 min. The temperature uniformity was less than 0.1 °C. Besides, the temperature stability over time (30 min) was within ±0.04 °C. Four optical channels were integrated (FAM, HEX, TAMRA, and ROX) on the MS2 device. In particular, the PCR-based detection sensitivity reached 1 copy per µL, and the amplification efficiency was calculated to be 106.8%. Besides, the MS2 device also was compatible with multiplex PCR and isothermal amplification. In short, the MS2 device showed performance consistent with that of traditional commercial equipment. Thus, the MS2 device provides an easy and integrated experimental platform for molecular diagnostic-related research and potential medical diagnostic applications.