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.

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.

Melatonin Potentiates Sensitivity to 5-Fluorouracil in Gastric Cancer Cells by Upregulating Autophagy and Downregulating Myosin Light-Chain Kinase.

5-Fluorouracil is an effective chemotherapeutic drug for gastric cancer. However, the acquisition of chemotherapeutic resistance remains a challenge in treatment. Melatonin can enhance the therapeutic effect of 5-fluorouracil; however, the underlying mechanisms are not well understood. We investigated the effects of combinations of melatonin and 5-fluorouracil on the proliferation, migration and invasion of gastric cancer cells. Melatonin significantly potentiated the 5-fluorouracil-mediated inhibition of proliferation, migration and invasion in gastric cancer cells, which potentiates sensitivity to 5-FU by promoting the activation of Beclin-1-dependent autophagy and targeting the myosin light-chain kinase (MLCK) signaling pathway. Previous studies have shown that autophagy might be associated with the MLCK signaling pathway. The autophagy inhibitor, 3-methyladenine, effectively rescued the migratory and invasive capabilities of gastric cancer cells, while also reducing expression level of MLCK and the phosphorylation level of MLC. This indicates that autophagy is involved in tumor metastasis, which may be related to inhibition of the MLCK signaling pathway. Our findings indicate that melatonin can improve the effectiveness of 5-fluorouracil in gastric cancer and could be used as a supplemental agent in the treatment of gastric cancer with 5-fluorouracil.

Cell-type-specific alternative polyadenylation promotes oncogenic gene expression in non-small cell lung cancer progression.

Disrupted alternative polyadenylation (APA) is frequently involved in tumorigenesis and cancer progression by regulating the gene expression of oncogenes and tumor suppressors. However, limited knowledge of tumor-type- and cell-type-specific APA events may lead to novel APA events and their functions being overlooked. Here, we compared APA events across different cell types in non-small cell lung cancer (NSCLC) and normal tissues and identified functionally related APA events in NSCLC. We found several cell-specific 3'-UTR alterations that regulate gene expression changes showed prognostic value in NSCLC. We further investigated the function of APA-mediated 3'-UTR shortening through loss of microRNA (miRNA)-binding sites, and we identified and experimentally validated several oncogene-miRNA-tumor suppressor axes. According to our analyses, we found SPARC as an APA-regulated oncogene in cancer-associated fibroblasts in NSCLC. Knockdown of SPARC attenuates lung cancer cell invasion and metastasis. Moreover, we found high SPARC expression associated with resistance to several drugs except cisplatin. NSCLC patients with high SPARC expression could benefit more compared to low-SPARC-expression patients with cisplatin treatment. Overall, our comprehensive analysis of cell-specific APA events shed light on the regulatory mechanism of cell-specific oncogenes and provided opportunities for combination of APA-regulated therapeutic target and cell-specific therapy development.

Chelate-functionalized magnetic micelles for sequestration of cisplatin.

Many cancer patients suffer permanent hearing loss due to accumulation of ototoxic cisplatin in the inner ear. In this study, two types of 100 nm magnetic micelles were developed to sequester cisplatin from aqueous solutions, with the goal of eliminating cochlear ototoxins via magnetic microsurgery. The micellar surface was quantitatively functionalized with anionic S-rich ligands and the micelle core encapsulated superparamagnetic iron oxide nanoparticles. Exceptionally effective sequestration is demonstrated, with removal of greater than 95 and 50% of solution Pt, by means of centrifugal filtration and magnetic extraction. Attraction between negatively charged micellar surfaces and cationic Pt-species played a critical role and was only partially screened by physiologic salt solution. Importantly, magnetic micelles introduce negligible impact on the integrity of inner ear hair cells, demonstrating excellent biocompatibility. This study showcases successful magnetic sequestration of Pt-based ototoxins using highly applicable nano-micellar materials. More generally, these examples highlight features of the micelle-water interfacial environment that are important in developing nanomaterials for metallo-medicinal applications.

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.

Pericytes control vascular stability and auditory spiral ganglion neuron survival.

The inner ear has a rich population of pericytes, a multi-functional mural cell essential for sensory hair cell heath and normal hearing. However, the mechanics of how pericytes contribute to the homeostasis of the auditory vascular-neuronal complex in the spiral ganglion are not yet known. In this study, using an inducible and conditional pericyte depletion mouse (PDGFRB-CreERT2; ROSA26iDTR) model, we demonstrate, for the first time, that pericyte depletion causes loss of vascular volume and spiral ganglion neurons (SGNs) and adversely affects hearing sensitivity. Using an in vitro trans-well co-culture system, we show pericytes markedly promote neurite and vascular branch growth in neonatal SGN explants and adult SGNs. The pericyte-controlled neural growth is strongly mediated by pericyte-released exosomes containing vascular endothelial growth factor-A (VEGF-A). Treatment of neonatal SGN explants or adult SGNs with pericyte-derived exosomes significantly enhances angiogenesis, SGN survival, and neurite growth, all of which were inhibited by a selective blocker of VEGF receptor 2 (Flk1). Our study demonstrates that pericytes in the adult ear are critical for vascular stability and SGN health. Cross-talk between pericytes and SGNs via exosomes is essential for neuronal and vascular health and normal hearing.

The Emerging Role of Pericyte-Derived Extracellular Vesicles in Vascular and Neurological Health.

Pericytes (PCs), as a central component of the neurovascular unit, contribute to the regenerative potential of the central nervous system (CNS) and peripheral nervous system (PNS) by virtue of their role in blood flow regulation, angiogenesis, maintenance of the BBB, neurogenesis, and neuroprotection. Emerging evidence indicates that PCs also have a role in mediating cell-to-cell communication through the secretion of extracellular vesicles (EVs). Extracellular vesicles are cell-derived, micro- to nano-sized vesicles that transport cell constituents such as proteins, nucleic acids, and lipids from a parent originating cell to a recipient cell. PC-derived EVs (PC-EVs) play a crucial homeostatic role in neurovascular disease, as they promote angiogenesis, maintain the integrity of the blood-tissue barrier, and provide neuroprotection. The cargo carried by PC-EVs includes growth factors such as endothelial growth factor (VEGF), connecting tissue growth factors (CTGFs), fibroblast growth factors, angiopoietin 1, and neurotrophic growth factors such as brain-derived neurotrophic growth factor (BDNF), neuron growth factor (NGF), and glial-derived neurotrophic factor (GDNF), as well as cytokines such as interleukin (IL)-6, IL-8, IL-10, and MCP-1. The PC-EVs also carry miRNA and circular RNA linked to neurovascular health and the progression of several vascular and neuronal diseases. Therapeutic strategies employing PC-EVs have potential in the treatment of vascular and neurodegenerative diseases. This review discusses current research on the characteristic features of EVs secreted by PCs and their role in neuronal and vascular health and disease.

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.

VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.

Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea. However, their role in different types of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model and noise-exposed mouse model, we show that loss of pericytes leads to marked changes in vascular structure, in turn leading to vascular degeneration and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models combined with exogenous donor pericytes, we show that pericytes, signaled by VEGF isoform A165 (VEGFA165), vigorously drive new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1-mediated (AAV1-mediated) VEGFA165 viral vector to pericyte-depleted or noise-exposed animals prevented and regenerated lost pericytes, improved blood supply, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for vascular regeneration, vascular stability, and hearing in adults. The restoration of vascular function in the damaged cochlea, including in noise-exposed animals, suggests that VEGFA165 gene therapy could be a new strategy for ameliorating vascular associated hearing disorders.

CircRNA ANXA2 Promotes Lung Cancer Proliferation and Metastasis by Upregulating PDPK1 Expression.

Lung cancer is a common malignant tumor that seriously threatens human health. It has become the top malignant tumor in terms of morbidity and mortality. In recent years, circRNA, a special noncoding RNA molecule, has attracted considerable interest. This study focused on the role of circRNA ANXA2 (circANXA2) in lung cancer and the molecular mechanism of cancer promotion. Real-time quantitative PCR (RT-PCR) was used in detecting the expression abundance of circANXA2 in different lung cancer cells and tissues. The subcellular localization of circANXA2 was detected through fluorescence in situ hybridization. circANXA2 expression was knocked down through siRNA. CCK-8, clone formation assay, and TUNEL assay were used in evaluating the effects of circANXA2 on cell proliferation, clone formation ability, and apoptosis. The role of circANXA2 in tumor proliferation was further verified in vivo using the tumor transplantation model in nude mice. The molecular mechanism of circANXA2 was investigated with luciferase activity assay and RT-PCR. The expression abundance of circANXA2 is high in lung cancer cell lines and tissues. Knocking down of circANXA2 inhibits the proliferation and clonogenesis of the lung cancer cells. Knocking down circANXA2 promotes apoptosis. circANXA2 further affects downstream PDPK1 expression by regulating miR-33a-5p and thereby affecting the malignancy of the lung cancer cells. circANXA2 inhibits miR-33a-5p activity by directly interacting with miR-33a-5p. circANXA2 regulates the transcription of the miR-33a-5p downstream target gene PDPK1 and affects the malignant progression of lung cancer.

Evaluation of a dynamic navigation system for training students in dental implant placement.

OBJECTIVE: Computer-guided simulation systems may offer a novel training approach in many surgical fields. This study aimed to compare dental students' learning progress in dental implants placement between a dynamic navigation system and a traditional training method using a simulation model. METHODS: Senior dental students with no implant placement experience were randomly assigned to implant placement training using a dynamic navigation system or a traditional freehand protocol. After training, 3-dimensional (3D) deviation at implant platform, 3D deviation at implant apex, and deviation of implant axis between the planned and placed implant positions were measured using superimposed cone beam computed tomography scans. RESULTS: Six students were trained in this study. Students showed significantly greater improvement in implant placement after training using the dynamic navigation system than after using the traditional freehand protocol. Overall deviation of implant axis (P < 0.001) and 3D apex deviation (P = 0.014) improved with training using the dynamic navigation system, but differences in 3D platform deviation (P = 0.513) were not statistically significant. CONCLUSIONS: A dynamic navigation system may be a useful teaching tool in the early development of clinical skills in implant placement for the novice practitioners. Novice practitioners exhibited significant improvement in angulation deviation across implant placement attempts with dynamic navigation system training.

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.

A Functionalized Polydopamine Theranostic Nanoprobe for Efficient Imaging of miRNA-21 and In Vivo Synergetic Cancer Therapy.

MicroRNAs (miRNAs) are emerging as vital biomarkers since their abnormal expression is associated with various disease types including cancer. Therefore, it is essential to develop a sensitive and specific platform to monitor the dynamic expression of miRNAs for early clinical diagnosis and treatment. In this study, we designed a functionalized polydopamine (PDA)-based theranostic nanoprobe for efficient detection of miRNA-21 and in vivo synergistic cancer therapy. PDA was modified with polyethylene glycol (PEG) and the obtained PDA-PEG nanoparticles showed good stability in different solutions. PDA-PEG nanoparticles were loaded with fluorescein isothiocyanate (FITC)-labeled hairpin DNA (hpDNA) and an anticancer drug doxorubicin (DOX). In the absence of miRNA-21, PDA effectively quenched the fluorescence of FITC-labeled hpDNA. The presence of miRNA-21 specifically recognized hpDNA and induced the dissociation of hpDNA from PDA-PEG and subsequently recovered the fluorescence signals. Upon cellular uptake of these nanoprobes, a dose-dependent fluorescence activation and synergetic cytotoxic effect were observed due to the release of DOX and inhibition of miRNA-21 function. Furthermore, PDA-PEG-DOX-hpDNA nanoparticles can afford long-term monitoring of miRNA-21 and combined therapeutic efficacy in the nude mice bearing 4T1 tumors. Our results demonstrate the capability of PDA-PEG-DOX-hpDNA as a theranostic nanoprobe for continuously tracking of miRNAs and synergetic cancer therapy.

Sulfur dioxide derivatives produce antidepressant- and anxiolytic-like effects in mice.

Sulfur dioxide (SO2) can be endogenously generated from sulfur-containing amino acids in animals and humans. Increasing evidence shows that endogenous SO2 may act as a gaseous molecule to participate in many physiological and pathological processes. However, the role of SO2 and its derivatives in the central nervous system remains poorly understood. The present study explored the protective effects of exogenous SO2 derivatives (Na2SO3:NaHSO3, 3:1 M/M) on cellular injury in vitro by using the cell proliferation assay (MTS), cell counting kit 8 assay (CCK-8), and cyto-flow assay in the corticosterone (CORT)-induced PC12 cell injury model. We also examined the antidepressant and anxiolytic effects of SO2 derivatives on the chronic mild stress (CMS)-induced depression mouse model by using the open field test, novelty suppressed feeding test, forced swimming test, tail suspension test, and sucrose preference test. In the MTS and CCK-8 assays, we found that preexposure of SO2 derivatives significantly blocked CORT-induced decrease of cellular survival without causing any negative effects. Results from the cyto-flow assay indicated that treatment with SO2 derivatives could reverse CORT-induced early and late apoptosis of PC12 cells. Systemic treatment with SO2 derivatives produced markedly antidepressant- and anxiolytic-like activities in mice under normal condition and rapidly reversed CMS-induced depressive- and anxiety-like behaviors. In conclusion, these findings indicate that exogenous SO2 derivatives show protective properties against the detrimental effects of stress and exert antidepressant- and anxiolytic-like actions. The present study suggests that exogenous SO2 derivatives are potential therapeutic agents for the treatment of depression, anxiety, and other stress-related diseases.

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.

Vascular regeneration in adult mouse cochlea stimulated by VEGF-A165 and driven by NG2-derived cells ex vivo.

Can damaged or degenerated vessels be regenerated in the ear? The question is clinically important, as disruption of cochlear blood flow is seen in a wide variety of hearing disorders, including in loud sound-induced hearing loss (endothelial injury), ageing-related hearing loss (lost vascular density), and genetic hearing loss (e.g., Norrie disease: strial avascularization). Progression in cochlear blood flow (CBF) pathology can parallel progression in hair cell and hearing loss. However, neither new vessel growth in the ear, nor the role of angiogenesis in hearing, have been investigated. In this study, we used an established ex vivo tissue explant model in conjunction with a matrigel matrix model to demonstrate for the first time that new vessels can be generated by activating a vascular endothelial growth factor (VEGF-A) signal. Most intriguingly, we found that the pattern of the newly formed vessels resembles the natural 'mesh pattern' of in situ strial vessels, with both lumen and expression of tight junctions. Sphigosine-1-phosphate (S1P) in synergy with VEGF-A control new vessel size and growth. Using transgenic neural/glial antigen 2 (NG2) fluorescent reporter mice, we have furthermore discovered that the progenitors of "de novo" strial vessels are NG2-derived cells. Taken together, our data demonstrates that damaged strial microvessels can be regenerated by reprogramming NG2-derived angiogenic cells. Restoration of the functional vasculature may be critical for recovery of vascular dysfunction related hearing loss.

Microvascular networks in the area of the auditory peripheral nervous system.

Using transgenic fluorescent reporter mice in combination with an established tissue clearing method, we detail heretofore optically opaque regions of the spiral lamina and spiral limbus where the auditory peripheral nervous system is located and provide insight into changes in cochlear vascular density with ageing. We found a relatively dense and branched vascular network in young adults, but a less dense and thinned network in aged adults. Significant reduction in vascular density starts early at the age of 180 days in the region of the spiral limbus (SL) and continues into old age at 540 days. Loss of vascular volume in the region of spiral ganglion neurons (SGN) is delayed until the age of 540 days. In addition, we observed that two vascular accessory cells are closely associated with the microvascular system: perivascular resident macrophages and pericytes. Morphologically, perivascular resident macrophages undergo drastic changes from postnatal P7 to young adult (P30). In postnatal animals, most perivascular resident macrophages exhibit a spherical or nodular shape. In young adult mice, the majority of perivascular resident macrophages are elongated and display an orientation parallel to the vessels. In our imaging, some of the perivascular resident macrophages are caught in the act of transmigrating from the blood circulation. Pericytes also display morphological heterogeneity. In the P7 mice, pericytes are prominent on the capillary walls, relatively large and punctate, and less uniform. In contrast, pericytes in the P30 mice are relatively flat and uniform, and less densely distributed on the vascular network. With triple fluorescence labeling, we did not find obvious physical connection between the two systems, unlike neuronal-vascular coupling found in brain. However, using a fluorescent (FITC-conjugated dextran) tracer and the enzymatic tracer horseradish peroxidase (HRP), we observed robust neurovascular exchange, likely through transcytotic transport, evidenced by multiple vesicles present in the endothelial cells. Taken together, our data demonstrate the effectiveness of tissue-clearing methods as an aid in imaging the vascular architecture of the SL and SGNs in whole mounted mouse cochlear preparations. Structure is indicative of function. The finding of differences in vascular structure in postnatal and young adult mice may correspond with variation in hearing refinement after birth and indicate the status of functional activity. The decrease in capillary network density in the older animals may reflect the decreased energy demand from peripheral neural activity. The finding of active transcytotic transport from blood to neurons opens a potential therapeutic avenue for delivery of various growth factors and gene vectors into the inner ear to target SGNs.

Vitamin D and DDX4 regulate the proliferation and invasion of ovarian cancer cells.

Ovarian cancer is one of the most commonly occurring types of cancer and one of the most common causes of cancer-associated mortality in women. Diagnosis of ovarian cancer at an early stage is difficult due to the lack of specific symptoms. In the present study, it is demonstrated that active vitamin D treatment prohibited the proliferation and invasion of ovarian cancer cells, and the expression level of a germ cell specific marker DEAD (Asp-Glu-Ala-Asp)-box helicase 4 (DDX4), which is overexpressed in ovarian cancer, was downregulated by active vitamin D treatment. Knockdown of DDX4 by siRNA could also suppress the invasive ability of ovarian cancer cells. Therefore, DDX4 may be considered as a diagnostic marker of ovarian cancer, and vitamin D may be a candidate drug for ovarian cancer therapy.