Luciferase-Decorated Gold Nanorods as Dual-Modal Contrast Agents for Tumor-Targeted High-Performance Bioluminescence/Photoacoustic Imaging.

Gold nanorods (AuNRs) have been considered highly compelling materials for early cancer diagnosis and have aroused a burgeoning fascination among the biomedical sectors. By leveraging the versatile tunable optical properties of AuNRs, herein, we have developed a novel tumor-targeted dual-modal nanoprobe (FFA) that exhibits excellent bioluminescence and photoacoustic imaging performance for early tumor diagnosis. FFA has been synthesized by anchoring the recombinant bioluminescent firefly luciferase protein (Fluc) on the folate-conjugated AuNRs via the PEG linker. TEM images and UV-vis studies confirm the nanorod morphology and successful conjugation of the biomolecules to AuNRs. The nanoprobe FFA relies on the ability of the folate module to target the folate receptor-positive tumor cells actively, and simultaneously, the Fluc module facilitates excellent bioluminescent properties in physiological conditions. The success of chemical engineering in the present study enables stronger bioluminescent signals in the folate receptor-positive cells (Skov3, Hela, and MCF-7) than in folate receptor-negative cells (A549, 293T, MCF-10A, and HepG2). Additionally, the AuNRs induced strong photoacoustic conversion performance, enhancing the resolution of tumor imaging. No apparent toxicity was detected at the cellular and mouse tissue levels, manifesting the biocompatibility nature of the nanoprobe. Prompted by the positive merits of FFA, the in vivo animal studies were performed, and a notable enhancement was observed in the bioluminescent/photoacoustic intensity of the nanoprobe in the tumor region compared to that in the folate-blocking region. Therefore, this synergistic dual-modal bioluminescent and photoacoustic imaging platform holds great potential as a tumor-targeted contrast agent for early tumor diagnosis with high-performance imaging information.

Increased Expression of KNa1.2 Channel by MAPK Pathway Regulates Neuronal Activity Following Traumatic Brain Injury.

Recent studies have indicated that functional abnormalities in the KNa1.2 channel are linked to epileptic encephalopathies. However, the role of KNa1.2 channel in traumatic brain injury (TBI) remains limited. We collected brain tissue from the TBI mice and patients with post-traumatic epilepsy (PTE) to determine changes in KNa1.2 channel following TBI. We also investigated whether the MAPK pathway, which was activated by the released cytokines after injury, regulated KNa1.2 channel in in vitro. Finally, to elucidate the physiological significance of KNa1.2 channel in neuronal excitability, we utilized the null mutant-Kcnt2-/- mice and compared their behavior patterns, seizure susceptibility, and neuronal firing properties to wild type (WT) mice. TBI was induced in both Kcnt2-/- and WT mice to investigate any differences between the two groups under pathological condition. Our findings revealed that the expression of KNa1.2 channel was notably increased only during the acute phase following TBI, while no significant elevation was observed during the late phase. Furthermore, we identified the released cytokines and activated MAPK pathway in the neurons after TBI and confirmed that KNa1.2 channel was enhanced by the MAPK pathway via stimulation of TNF-α. Subsequently, compared to WT mice, neurons from Kcnt2-/- mice showed increased neuronal excitability and Kcnt2-/- mice displayed motor deficits and enhanced seizure susceptibility, which suggested that KNa1.2 channel may be neuroprotective. Therefore, this study suggests that enhanced KNa1.2 channel, facilitated by the inflammatory response, may exert a protective role in an acute phase of the TBI model.

RNA N6-methyladenosine modification in arthritis: New insights into pathogenesis.

The commonest type of eukaryotic RNA modification, N6-methyladenosine (m6A), has drawn increased scrutiny in the context of pathological functioning as well as relevance in determination of RNA stability, splicing, transportation, localization, and translation efficiency. The m6A modification plays an important role in several types of arthritis, especially osteoarthritis and rheumatoid arthritis. Recent studies have reported that m6A modification regulates arthritis pathology in cells, such as chondrocytes and synoviocytes via immune responses and inflammatory responses through functional proteins classified as writers, erasers, and readers. The aim of this review was to highlight recent advances relevant to m6A modification in the context of arthritis pathogenesis and detail underlying molecular mechanisms, regulatory functions, clinical applications, and future perspectives of m6A in arthritis with the aim of providing a foundation for future research directions.

Engineering Synthetic CopT/A-Based Genetic Biosensors for miRNA Imaging and Functional Gene Regulation.

Synthetic genetic biosensors that can operate at the transcriptional and translation levels have been widely applied in the control of cellular behaviors and functions. However, the regulation of genetic circuits is often accompanied by the introduction of exogenous substances or the endogenous generation of inhibitory products, which would bring uncontrollable hazards to biological safety and reduce the efficiency of the system. Here, we described a miRNA-responsive CopT-CopA (miCop) genetic biosensor system to realize real-time monitoring of the intracellular expression of miRNA-124a during neurogenesis or miRNA-122 under the stimulation of extracellular drugs in living cells and animals. Furthermore, to prove the modularity of the system, we engineered this miCop to tune the expression of the DTA (diphtheria toxin A) gene and showed its powerful capacity to kill cancer cells by inducing apoptosis and cell cycle arrest based on miRNA response. This study provides an effective means to couple miRNA sensing with miRNA-responsive gene modulation, which may open up new diagnostic or therapeutic applications.

Harnessing PUF-Based Reporters for Noninvasive Imaging of the MicroRNA Dynamics in Differentiation.

Precise characterization of miRNA expression patterns is critical to exploit the complexity of miRNA regulation in biology. Herein, we developed a Pumilio/FBF (PUF) protein-based engineering luciferase reporter system, PUF/miR, to quantitatively and non-invasively sense miRNA activity in living cells and animal models. We verified the feasibility of this reporter by monitoring the expression of several types of miRNAs (miRNA-9, 124a, 1, and 133a) in neural and muscle differentiated cells as well as subcutaneous or tibial anterior muscles in mice. The quantitative RT-PCR also validated the reliability and quantitative consistency of bioluminescence imaging in detecting miRNA expression. We further effectively employed this reporter system to visualize the expression of miRNA-1 and miRNA-133a in mouse models of skeletal muscle injury. As a non-invasive and convenient innovative approach, our results have realized the positive bioluminescence imaging of endogenous miRNAs in vitro and in vivo using the PUF/miR system. We believe that this approach would provide a potential means for noninvasive monitoring of disease-related miRNAs and could facilitate a deeper understanding of miRNA biology.

A ratiometric dual luciferase reporter for quantitative monitoring of pre-mRNA splicing efficiency in vivo.

Precursor messenger RNA (pre-mRNA) splicing is critical for cell growth and development, and errors in RNA splicing frequently cause cellular dysfunction, abnormal gene expression, and a variety of human diseases. However, there is currently a lack of reliable systems to noninvasively monitor the mRNA splicing efficiency in cells and animals. Here, we described the design of a genetically engineered ratiometric dual luciferase reporter to continuously quantify the changes in mRNA splice variants in vivo. This reporter system is encoded within a single polypeptide but on separate exons, thus generating two distinct luciferase signals derived from spliced and unspliced mRNAs. With this reporter, the two kinds of luciferase in the same individual can minimize the influence of indirect factors on splicing, and the ratio of these two luciferase intensities represents the dynamic splicing efficiency of pre-mRNA. Our study offers a convenient and robust tool for the screening and identification of small molecules or trans-acting factors that affect the efficiency of specific splicing reactions.

In Vivo Visualization of RNAi Efficiency Using a Pumilio/FBF Protein-Based Reporter.

As a strategy that induces gene silencing by the delivery of small interfering RNA (siRNA) targeting a specific gene locus into cells or tissues, RNA interference (RNAi) technology holds the potential to be a powerful tool in a range of intractable disorder therapeutics. However, reliable noninvasive probes for visualizing the siRNA delivery and silencing efficiency have become a major obstacle in siRNA-based treatment. Here, we describe the development of an RNA-binding protein Pumilio/FBF (PUF)-based reporter probe for the monitoring of siRNA delivery efficiency and functional screening of effective siRNA target sites in vivo. This reporter consisted of a Firefly luciferase (Fluc) gene whose expression is regulated by the unique interaction architecture of the PUF protein with its Nanos response element (NRE) target RNA. We showed that a robust and rapid increase in the luminescence signal was detected by the successful delivery of siRNA against the enhanced green fluorescent protein (EGFP) or p53 genes into mammalian cells or the livers of mice. The delivery efficiencies of various commercial transfection vehicles were quantitatively evaluated with this reporter. In addition, we also employed in vivo bioluminescence imaging to screen and identify the most potent siRNA targeting p53. Our study indicates that the positive-readout reporter represents a promising indicator for siRNA optimization and visualization, advancing the development of siRNA therapeutic products.

Maternal exposure to triclosan during lactation alters social behaviors and the hippocampal ultrastructure in adult mouse offspring.

We recently reported that exposure to triclosan (TCS), a broad-spectrum antibacterial agent, affects social behaviors in adult mice, however, the long-lasting effects of TCS exposure during early life on social behaviors are still elusive. The present study aimed to investigate the long-lasting impacts of adding TCS to the maternal drinking water during lactation on the social behaviors of adult mouse offspring and to explore the potential mechanism underlying these effects. The behavioral results showed that TCS exposure decreased body weight, increased depression-like behavior and decreased social dominance in both male and female offspring, as well as increased anxiety-like behavior and bedding preference in female offspring. In addition, enzyme-linked immunosorbent assay (ELISA) indicated that TCS exposure increased peripheral proinflammatory cytokine levels, altered serum oxytocin (OT) levels, and downregulated the expression of postsynaptic density protein 95 (PSD-95) in the hippocampus. Morphological analysis by transmission electron microscopy (TEM) demonstrated that exposure to TCS induced morphological changes to synapses and neurons in the hippocampus of offspring. These findings suggested that TCS exposure during lactation contributed to abnormal social behaviors accompanied by increased peripheral inflammation and altered hippocampal neuroplasticity, which provides a deeper understanding of the effects of TCS exposure during early life on brain function and behavioral phenotypes.

Acoustic Trauma Causes Cochlear Pericyte-to-Myofibroblast-Like Cell Transformation and Vascular Degeneration, and Transplantation of New Pericytes Prevents Vascular Atrophy.

Acoustic trauma disrupts cochlear blood flow and damages sensory hair cells. Damage and regression of capillaries after acoustic trauma have long been observed, but the underlying mechanism of pathology has not been understood. We show herein that loud sound causes change of phenotype from neural/glial antigen 2 positive/α-smooth muscle actin negative to neural/glial antigen 2 positive/α-smooth muscle actin positive in some pericytes (PCs) on strial capillaries that is strongly associated with up-regulation of transforming growth factor-ß1. The acoustic trauma also reduced capillary density and increased deposition of matrix proteins, particularly in the vicinity of transformed PCs. In a newly established in vitro three-dimensional endothelial cell (EC) and PC co-culture model, transformed PCs induced thicker capillary-like branches in ECs and increased collagen IV and laminin expression. Transplantation of exogenous PCs derived from neonatal day 10 mouse cochleae to acoustic traumatized cochleae, however, significantly attenuated the decreased vascular density in the stria. Transplantation of PCs pretransfected with adeno-associated virus 1-vascular endothelial growth factor-A165 under control of a hypoxia-response element markedly promotes vascular volume and blood flow, increased proliferation of PCs and ECs, and attenuated loud sound-caused loss in endocochlear potential and hearing. Our results indicate that loud sound-triggered PC transformation contributes to capillary wall thickening and regression, and young PC transplantation effectively rehabilitates the vascular regression and improves hearing.

A Secreted Reporter for Blood Monitoring of Pyroptotic Cell Death.

Pyroptotic cell death is a phenomenon that runs through all life activities and plays an important role in physiological and pathological processes of the body's metabolism. It is of big biological significance to understand the phenomenon and nature of cell pyroptosis. In the process of cell pyroptosis, the pore-forming effector gasdermin D (GSDMD) is cleaved to form oligomers, which are inserted into the cell membrane, causing rapid cell death. However, the effective cell death induced by GSDMD complicates our ability to understand the behavior of pyroptosis. In this work, we performed molecular mutagenesis to develop a genetically encoded pyroptotic reporter, where a secreted Gaussia luciferase (Gluc) was strategically placed in the p30-p20 tolerated junction of GSDMD to support natural pyrophosphorylation and promote live imaging of cell pyroptosis. In addition, we demonstrated that this fused Gluc-GSDMD reporter executed inflammatory body-dependent pyroptosis in response to extracellular stimuli, and that the lysed p30-GSDMD can be secreted out of the cell and can be detected in the culture medium and animal blood. Therefore, our study provides a valuable tool that not only noninvasive and real-time monitoring of cell pyroptosis, but also affords a high-throughput functional screening of pyroptosis-targeted compounds in cultured cells and animal models.

Quantifying the change in streamflow complexity in the Yangtze River.

Hydrological processes of the Yangtze River have changed over the past decades due to environmental change and human activity. This paper uses sample entropy to investigate the spatial distribution and dynamic change in streamflow series complexity in the Yangtze River, China. In this study, the complexity of the streamflow series is quantified by entropy analysis. Daily streamflow series for four stations located in the mainstem and two control stations of the two largest freshwater lakes were analysed for the past 60 years. The results showed that the complexity of the streamflow series showed an obvious spatial difference and an increasing trend from upstream to downstream in the Yangtze River. There was a negative relationship between the annual streamflow and the corresponding sample entropy, and their peak-to-valley values showed well-corresponding relationships. The complexity of the runoff series at the Cuntan, Yichang, and Datong stations showed a continuous increasing trend, while that of the Hankou station showed a decreasing trend. The Three Gorges Dam changed the streamflow series complexity in the middle reach of the Yangtze River during the initial impoundment stage, while it had only slight impacts during the fully operational stage. Compared to the mainstem reaches, the streamflow series complexity of the two lakes showed no obvious change. The complexity of the streamflow series in the mainstem of the Yangtze River has been influenced by dam construction. The study could provide a scientific reference for understanding the flow dynamic evolution in the Yangtze River.

ZNF830 mediates cancer chemoresistance through promoting homologous-recombination repair.

Homologous recombination (HR), which mediates the repair of DNA double-strand breaks (DSB), is crucial for maintaining genomic integrity and enhancing survival in response to chemotherapy and radiotherapy in human cancers. However, the mechanisms of HR repair in treatment resistance for the improvement of cancer therapy remains unclear. Here, we report that the zinc finger protein 830 (ZNF830) promotes HR repair and the survival of cancer cells in response to DNA damage. Mechanistically, ZNF830 directly participates in DNA end resection via interacting with CtIP and regulating CtIP recruitment to DNA damage sites. Moreover, the recruitment of ZNF830 at DNA damage sites is dependent on its phosphorylation at serine 362 by ATR. ZNF830 directly and preferentially binds to double-strand DNA with its 3' or 5' overhang through the Zinc finger (Znf) domain, facilitating HR repair and maintaining genome stability. Thus, our study identified a novel function of ZNF830 as a HR repair regulator in DNA end resection, conferring the chemoresistance to genotoxic therapy for cancers those that overexpress ZNF830.

Coarse-grained molecular dynamics study of wettability influence on protein translocation through solid nanopores.

Protein translocation through nanopores is widely involved in molecular sensing and analyzing devices, whereby nanopore surface properties are crucial. However, fundamental understanding of how these properties affect protein motion inside nanopores remains lacking. In this work, we study the influence of nanopore surface wettability on voltage-driven protein translocation through nanopores with coarse-grained molecular dynamics simulations. The results show that the electrophoretic mobility of protein translocation increases as the contact angle of nanopore surface increases from 0° to 90°, but becomes almost constant as the contact angle is above 90°. This observation can be attributed to the variation of the friction coefficient of protein translocation through the nanopores with different nanopore contact angles. We further show that the interaction between nanopore and water, rather than that between the nanopore and protein, dominates the protein transport in nanopores. These findings provide new insights into protein translocation dynamics across nanopores and will be beneficial to the design of high-efficiency nanopore devices for single molecule protein sensing.

Isoform-specific regulation of osteogenic factors by polypeptide N-Acetylgalactosaminyltransferases 1 and 4.

The family of UDP-GalNAc polypeptide: N-Acetylgalactosaminlytransfersases (ppGalNAcTs) catalyzes the initial step of O-linked protein glycosylation. Mucin-type O-glycoproteins are abundant in the bone and may play an important role in osteogenesis. Herein, we examined the effects of ppGalNAc-T isoforms on osteogenesis of MC3T3-E1 pre-osteoblasts. We found that ppGalNAc-T1 and -T4 isoforms were highly expressed during osteogenesis of MC3T3-E1 and their knockdown by short hairpin RNA (shRNA) decreased osteoblast formation and bone mineralization. Knockdown of ppGalNAc-T1 or -T4 decreased mRNA and protein levels of bone sialoprotein (BSP). Knockdown of ppGalNAc-T1decreased mRNA levels of osteocalcin (OC), osteoprotegerin (OPG). Knockdown ofppGalNAc-T4 isoform decreased mRNA levels of OC, OPG and vitamin D receptor (VDR). While knockdown of T1 or T4 isoforms did not change the expression of osteopontin (OPN), COLLI, receptor activator for nuclear factor-κB ligand (RANKL) and transforming growth factor-ß (TGF-ß). Our results demonstrated that the ppGalNAc-T4 was highly expressed in MC3T3-E1 cells during osteogenesis for the first time. We also found that ppGalNAc-T1 and -T4 affected the expression of different osteogenic factors, suggesting distinct roles ppGalNAc-T isoformsplay in regulating osteogenesis in vitro.

Structural changes in thestrial blood-labyrinth barrier of aged C57BL/6 mice.

Tight control over cochlear blood flow (CoBF) and the blood-labyrinth barrier (BLB) in the striavascularis is critical for maintaining the ionic, fluid and energy balance necessary for hearing function. Inefficient CoBF and disruption of BLB integrity have long been considered major etiologic factors in a variety of hearing disorders. In this study, we investigate structural changes in the BLB of the striavascularis in age-graded C57BL/6 mice (1 to 21 months) with a focus on changes in two blood barrier accessory cells, namely pericytes (PCs) and perivascular-resident macrophage-like melanocytes (PVM/Ms). Decreased capillary density was detectable at 6 months, with significant capillary degeneration seen in 9- to 21-month-old mice. Reduced capillary density was highly correlated with lower numbers of PCs and PVM/Ms. "Drop-out" of PCs and "activation" of PVM/Ms were seen at 6 months, with drastic changes being observed by 21 months. With newly established in vitro three-dimensional cell-based co-culture models, we demonstrate that PCs and PVM/Ms are essential for maintaining cochlear vascular architecture and stability.

Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the intrastrial fluid-blood barrier.

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid-blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid-blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid-blood barrier to both low- and high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid-blood barrier for establishing a normal endocochlear potential hearing threshold.

Perivascular macrophage-like melanocyte responsiveness to acoustic trauma--a salient feature of strial barrier associated hearing loss.

Tissue perivascular resident macrophages (PVM/Ms), a hybrid cell type with characteristics of both macrophages and melanocytes, are critical for establishing and maintaining the endocochlear potential (EP) required for hearing. The PVM/Ms modulate expression of tight- and adherens-junction proteins in the endothelial barrier of the stria vascularis (intrastrial fluid-blood barrier) through secretion of a signaling molecule, pigment epithelium growth factor (PEDF). Here, we identify a significant link between abnormalities in PVM/Ms and endothelial barrier breakdown from acoustic trauma to the mouse ear. We find that acoustic trauma causes activation of PVM/Ms and physical detachment from capillary walls. Concurrent with the detachment, we find loosened tight junctions between endothelial cells and decreased production of tight- and adherens-junction protein, resulting in leakage of serum proteins from the damaged barrier. A key factor in the intrastrial fluid-blood barrier hyperpermeability exhibited in the mice is down-regulation of PVM/M modulated PEDF production. We demonstrate that delivery of PEDF to the damaged ear ameliorates hearing loss by restoring intrastrial fluid-blood barrier integrity. PEDF up-regulates expression of tight junction-associated proteins (ZO-1 and VE-cadherin) and PVM/M stabilizing neural cell adhesion molecule (NCAM-120). These studies point to the critical role PVM/Ms play in regulating intrastrial fluid-blood barrier integrity in healthy and noise-damaged ears.

4-amino-2-trifluoromethyl-phenyl retinate alleviates lipopolysaccharide-induced acute myocardial injury through activation of the KLF4/p62 axis.

Ferroptosis plays a pivotal role in the pathological process of sepsis-induced cardiomyopathy (SIC). All-trans retinoic acid (ATRA) enhances the host immune response to lipopolysaccharides (LPS). This study investigated the role of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a derivative of ATRA, in myocardial injury caused by sepsis. Male C57BL/6 mice were intraperitoneally injected with LPS to establish a sepsis model. H9c2 cells were stimulated by LPS to establish an injury model. We observed that ATPR improved myocardial injury in mice, which was presented in terms of an increased glutathione (GSH) level and reduced production of malondialdehyde (MDA), as well as an increased number of mitochondrial cristae and maintenance of the mitochondrial membrane integrity. ATPR improved cardiac function in the LPS-injured mice. It inhibited the inflammatory response as evidenced by the decreasing mRNA levels of TNF-α and IL-6. The elevated protein expression levels of Nrf2, SLC7A11, GPX4, and FTH1 in mice and H9c2 cells showed that ATPR inhibited ferroptosis. Immunoprecipitation of LPS-stimulated H9c2 cells demonstrated that ATPR increased the interaction between p62 and Keap1. ATPR upregulated the KLF4 and p62 protein expression. However, the inhibition of Nrf2 by ML385 reduced the protective effect of ATPR in LPS-treated H9c2 cells. Furthermore, we used siRNA to knock down KLF4 in H9c2 cells and found that the KLF4 knockdown eliminated the inhibition of ferroptosis by ATPR in H9c2 cells. Therefore, ATPR alleviates LPS-induced myocardial injury by inhibiting ferroptosis via the KLF4/p62 axis.

A Ratiometric Gene-Switch System for miRNA Sensing and Gene Regulation.

microRNAs (miRNAs) are a class of non-coding, small RNAs that play an important role in diverse biological processes and diseases. By regulating the expression of eukaryotic genes post-transcriptionally in a sequence-specific manner, miRNAs are widely used to design synthetic RNA switches. However, most of the RNA switches are often dependent on the corresponding ligand molecules, whose specificity and concentration would affect the efficiency of synthetic RNA circuits. Here, a fused transcriptional repressor Gal4BD-Rluc based gene-switch system Gal-miR for miRNA visualization and gene regulation is described. By placing a luciferase downstream gene under the control of endogenous miRNA machinery, the Gal-miR system makes the conversion of miRNA-mediated gene silencing into a ratiometric bioluminescent signal, which quantitatively reflected miRNA-206 activity during myogenic differentiation. Moreover, it demonstrates that this gene-switch system can effectively inhibit breast cancer cell viability, migration and invasion under the control of specific miRNAs by replacing the downstream gene with melittin functional gene. The study proposes a powerful modular genetic design for achieving precise control of transgene expression in a miRNA responsive way, as well as visualizing the dynamics of miRNA activity.