Research on Hepatocyte Regulation of PCSK9-LDLR and Its Related Drug Targets.

The prevalence of hyperlipidemia has increased significantly due to genetic, dietary, nutritional and pharmacological factors, and has become one of the most common pathological conditions in humans. Hyperlipidemia can lead to a range of diseases such as atherosclerosis, stroke, coronary heart disease, myocardial infarction, diabetes, and kidney failure, etc. High circulating low-density lipoprotein cholesterol (LDL-C) is one of the causes of hyperlipidemia. LDL-C in the blood binds to LDL receptor (LDLR) and regulates cholesterol homeostasis through endocytosis. In contrast, proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates LDLR degradation via the intracellular and extracellular pathways, leading to hyperlipidemia. Targeting PCSK9-synthesizing transcription factors and downstream molecules are important for development of new lipid-lowering drugs. Clinical trials regarding PCSK9 inhibitors have demonstrated a reduction in atherosclerotic cardiovascular disease events. The purpose of this review was to explore the target and mechanism of intracellular and extracellular pathways in degradation of LDLR and related drugs by PCSK9 in order to open up a new pathway for the development of new lipid-lowering drugs.

A Telomerase-Assisted Strategy for Regeneration of DNA Nanomachines in Living Cells.

DNA nanomachines have been engineered into diverse personalized devices for diagnostic imaging of biomarkers; however, the regeneration of DNA nanomachines in living cells remains challenging. Here, we report an ingenious DNA nanomachine that can implement telomerase (TE)-activated regeneration in living cells. Upon apurinic/apyrimidinic endonuclease 1 (APE1)-responsive initiation of the nanomachine, the walker of the nanomachine moves along tracks regenerated by TE, generating multiply amplified signals through which APE1 can be imaged in situ. Additionally, augmentation of the signal due to the regeneration of the nanomachines could reveal differential expression of TE in different cell lines. To the best of our knowledge, this is the first proof-of-concept demonstration of the use of biomarkers to assist in the regeneration of nanomachines in living cells. This study offers a new paradigm for the development of more applicable and efficient DNA nanomachines.

Intracellular Delivery of Micron-Sized Magnetic Particles through a Virus Infection Pathway.

Micron-sized magnetic particles (M-MPs) have low toxicity, strong magnetic signals, and long-term retention capability, which are significant advantages for their application in biomedical imaging. Unfortunately, M-MPs are only internalized by few cell types, such as macrophages and phagocytes, and because of this lack of active intracellular delivery, their applications are restricted. The emergence of self-assembled virus-like particles (VLPs) offers a viable approach to drive M-MPs into cells, although the specific mechanism has not been revealed. In this study, we investigated in detail the intracellular pathway of M-MPs mediated by VLPs using a fluorescence co-localization method. The results indicated that the intracellular movement of M-MPs was consistent with the virus infection pathway, specifically caveolae-dependent endocytosis, transportation through microtubules, and accumulation in the endoplasmic reticulum. This study provides experimental support for the active transport of M-MPs into other cell types, thereby further extending their applications.

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

Intracellular pathways involved in cell survival are deregulated in mouse and human spinal muscular atrophy motoneurons.

Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function.

Future of Personalized Therapy Targeting Aberrant Signaling Pathways in Multiple Myeloma.

Multiple myeloma (MM) is a genetically complex disease. Identification of mutations and aberrant signaling pathways that contribute to the progression of MM and drug resistance has potential to lead to specific targets and personalized treatment. Aberrant signal pathways include RAS pathway activation due to RAS or BRAF mutations (targeted by vemurafenib alone or combined with cobimetinib), BCL-2 overexpression in t(11:14) (targeted by venetoclax), JAK2 pathway activation (targeted by ruxolitinib), NF-κB pathway activation (treated with DANFIN combined with bortezomib), MDM2 overexpression, and PI3K/mTOR pathway activation (targeted by BEZ235). Cyclin D1 (CCND1) and MYC are also emerging as key potential targets. In addition, histone deacetylase inhibitors are already in use for the treatment of MM in combination therapy, and targeted inhibition of FGFR3 (AZD4547) is effective in myeloma cells with t(4;14) translocation. Bromodomain and extra terminal (BET) protein antagonists decrease the expression of MYC and have displayed promising antimyeloma activity. A better understanding of the alterations in signaling pathways that promote MM progression will further inform the development of precision therapy for patients.

The Lactate Receptor HCAR1 Modulates Neuronal Network Activity through the Activation of Gα and Gßγ Subunits.

The discovery of a G-protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional nonmetabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch clamp, we found that the activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature EPSC frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists 3,5-dihydroxybenzoic acid, 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1, the basal activity was increased compared with WT. HCAR1 mediates its effect in neurons through a Giα-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 downmodulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and α2A-adrenergic receptors, through a mechanism involving both its Giα and Gißγ subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a downmodulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Giα and Gißγ intracellular pathways to functionally interact with other Gi-coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENT Expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) was recently described in neurons. Here, we describe the physiological role of this G-protein-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCAR1 activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross talk of HCAR1 with other GPCRs, such as GABAB, adenosine A1- and α2A-adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.

Delivery Pathway Regulation of 3',3″-Bis-Peptide-siRNA Conjugate via Nanocarrier Architecture Engineering.

Small interfering RNA (siRNA) has been continuously explored for clinical applications. However, neither nanocarriers nor conjugates have been able to remove the obstacles. In this study, we employed a combined nanochemistry strategy to optimize its delivery dilemma, where different interactions and assembly modes were cooperatively introduced into the forming process of siRNA/lipids nanoplexes. In the nanoplexes, the 3',3″-bis-peptide-siRNA conjugate (pp-siRNA) and gemini-like cationic lipids (CLDs) were employed as dual regulators to improve their bio-behavior. We demonstrated that the "cicada pupa"-shaped nanoplexes of MT-pp-siRNA/CLDs (MT represented the mixed two-phase method) exhibited more compact multi-sandwich structure (∼25 layers), controllable size (∼150 nm), and lower zeta potential (∼22 mV) than other comparable nanoplexes and presented an increased siRNA protection and stability. Significantly, the nanoplex was internalized into melanoma cells by almost caveolae-mediated endocytosis and macropinocytosis (∼99.46%), and later reduced/avoided lysosomal degradation. Finally, the nanoplex facilitated the silencing of mRNA of the mutant B-Raf protein (down by ∼60%). In addition, pp-siRNA had a high intracellular sustainability, a significantly prolonged circulating time, and accumulation in tumor tissues in vivo. Our results have demonstrated that the combined approach can improve the intracellular fate of siRNA, which opens up novel avenues for efficient siRNA delivery.

Routes and machinery of primary cilium biogenesis.

Primary cilia are solitary, microtubule-based protrusions of the cell surface that play fundamental roles as photosensors, mechanosensors and biochemical sensors. Primary cilia dysfunction results in a long list of developmental and degenerative disorders that combine to give rise to a large spectrum of human diseases affecting almost any major body organ. Depending on the cell type, primary ciliogenesis is initiated intracellularly, as in fibroblasts, or at the cell surface, as in renal polarized epithelial cells. In this review, we have focused on the routes of primary ciliogenesis placing particular emphasis on the recently described pathway in renal polarized epithelial cells by which the midbody remnant resulting from a previous cell division event enables the centrosome for initiation of primary cilium assembly. The protein machinery implicated in primary cilium formation in epithelial cells, including the machinery best known for its involvement in establishing cell polarity and polarized membrane trafficking, is also discussed.

Radioresistance in head and neck squamous cell carcinoma: Biological bases and therapeutic implications.

Head and neck squamous cell carcinoma (HNSCC) is strongly associated with alcohol and tobacco consumption. Lately, the incidence of human papillomavirus (HPV)-related tumors has shown a significant increase, and HPV-related tumors show distinctive features if compared with the HPV-negative counterpart. Locally advanced HNSCC can be treated with concomitant chemoradiotherapy, but early recurrences sometimes occur. Relapses are often related to an intrinsic radioresistance of the tumors. Alterations in intracellular pathways, primarily involved in cell proliferation, apoptosis, and DNA repair, can lead to radioresistance. Preclinical and clinical evidence highlighted that 3 main pathways, including the epidermal growth factor receptor (EGFR), the phosphotidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and the p53 signaling cascades, play a crucial role in radioresistance development. A future approach may consist in the association of radiotherapy (RT) and selective inhibition of the key pathways involved in radioresistance. Phase I, II, and III clinical trials are currently testing these novel treatment strategies.