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To explore the effects of high cervical spinal cord electrical stimulation (cSCS) on the recovery of neurological function and its possible mechanism in rats with traumatic brain injury (TBI). 72 rats were randomly divided into: (1) a sham group; (2) a traumatic brain injury (TBI) group; (3) a TBI+cSCS group; (4) a LY294002+TBI+cSCS group. The degree of neurological dysfunction was evaluated by modified Neurological severity score (mNSS). The pathological changes of the brain tissue in the injured area were observed by HE staining, and the apoptosis of neuron cells were observed by TUNEL staining. The expressions of BDNF and VEGFmRNA were detected by polymerase chain reaction (PCR), and the expressions of p-AKT, AKT, Bcl-2, Bax and caspase-3 proteins were detected by western blot. Compared with that of the TBI and LY294002+TBI+cSCS groups, the mNSS of the TBI+cSCS group were significantly lower on day 3 and 7 ( P0.05). Compared with that in the TBI and LY294002+TBI+cSCS groups, the apoptosis of neuron cells in the TBI+cSCS group decreased significantly ( P0.05). Compared with the TBI and LY294002+TBI+cSCS group, the expression of Bcl-2 protein increased and the expressions of Bax and Caspase-3 proteins decreased in the TBI+cSCS group ( P0.05). Compared with that in the TBI and LY294002+TBI+cSCS groups, the intensity of p-Akt/Akt in the TBI+cSCS group increased ( P0.05). We found that cSCS had a protective effect on neuron cells after craniocerebral injury and could improve neurological dysfunction in rats, the mechanism of which might be that cSCS made the PI3K/Akt pathway more active after TBI.

Individuals with autosomal dominant polycystic kidney disease have a higher incidence of stone formation than the general population. However, there are no cystic animal models known to develop stones. Cystic mice compound heterozygous for hypomorphic Pkd1(V) and Pkd1(RC) alleles develop cystic kidneys within a few weeks of birth but live beyond 20 wk of age, allowing for the study of cystic comorbidities including stone formation. Cystic Pkd1(V)(/)(RC) mice were euthanized at 3, 13, or 26 wk of age, and their kidneys were analyzed by microcomputed tomography (µCT) for stone formation. Mice had occasional mineral aggregates that could be detected by µCT analysis at 3 wk of age. At 13 or 26 wk of age, numerous white masses were visible beneath the kidney surface. µCT analysis confirmed the masses to be large mineral stone deposits throughout the renal cortex, with mineral content increasing with age. Staining of histological sections with alizarin red and von Kossa suggested that the stone deposits were composed primarily of calcium and phosphate. Microdissection confirmed stones localized within cyst lumens. Analysis of individual stones by µCT and infrared spectroscopy confirmed apatite mineral composition. Urinalysis revealed elevated levels of phosphate and citrate at 3 wk of age and lower pH and elevated levels of calcium and citrate at 13 wk of age, suggesting altered phosphate and calcium homeostasis as a potential cause of mineralization and renal stone formation. This is the first animal model exhibiting overt kidney stone formation in the context of cystic kidney disease. NEW & NOTEWORTHY Compound heterozygous Pkd1(V/RC) mice were found to form calcium phosphate-containing stones within cysts of the renal cortex by 13 wk of age. This is the first polycystic kidney disease animal model exhibiting spontaneous stone formation. A growing body of evidence suggests a link between renal stone formation and cystic kidney disease. This mouse model may be useful for studying the interplay between stone and cyst formation and the functional role of polycystins in mineral homeostasis.

Glucocorticoid-induced TNF receptor-related (GITR) can act as a co-stimulatory receptor, representing a potential target for safely enhancing immunotherapy efficacy. GITR is triggered by a GITR ligand or an agonist antibody and activates CD8

Ethyl ferulate (EF) is abundant in Rhizoma Chuanxiong and grains (e.g., rice and maize) and possesses antioxidative, antiapoptotic, antirheumatic, and anti-inflammatory properties. However, its effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) is still unknown. In the present study, we found that EF significantly alleviated LPS-induced pathological damage and neutrophil infiltration and inhibited the gene expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in murine lung tissues. Moreover, EF reduced the gene expression of TNF-α, IL-1β, IL-6, and iNOS and decreased the production of NO in LPS-stimulated RAW264.7 cells and BMDMs. Mechanistic experiments revealed that EF prominently activated the AMPK/Nrf2 pathway and promoted Nrf2 nuclear translocation. AMPK inhibition (Compound C) and Nrf2 inhibition (ML385) abolished the beneficial effect of EF on the inflammatory response. Furthermore, the protective effect of EF on LPS-induced ALI was not observed in Nrf2 knockout mice. Taken together, the results of our study suggest that EF ameliorates LPS-induced ALI in an AMPK/Nrf2-dependent manner. These findings provide a foundation for developing EF as a new anti-inflammatory agent for LPS-induced ALI/ARDS therapy. [Image: see text]

Primary cilia protrude from the apical surface of many cell types and act as a sensory organelle that regulates diverse biological processes ranging from chemo- and mechanosensation to signaling. Ciliary dysfunction is associated with a wide array of genetic disorders, known as ciliopathies. Polycystic lesions are commonly found in the kidney, liver, and pancreas of ciliopathy patients and mouse models. However, the pathogenesis of the pancreatic phenotype remains poorly understood. Chibby1 (Cby1), a small conserved coiled-coil protein, localizes to the ciliary base and plays a crucial role in ciliogenesis. Here, we report that Cby1-knockout (KO) mice develop severe exocrine pancreatic atrophy with dilated ducts during early postnatal development. A significant reduction in the number and length of cilia was observed in Cby1-KO pancreta. In the adult Cby1-KO pancreas, inflammatory cell infiltration and fibrosis were noticeable. Intriguingly, Cby1-KO acinar cells showed an accumulation of zymogen granules (ZGs) with altered polarity. Moreover, isolated acini from Cby1-KO pancreas exhibited defective ZG secretion in vitro. Collectively, our results suggest that, upon loss of Cby1, concomitant with ciliary defects, acinar cells accumulate ZGs due to defective exocytosis, leading to cell death and progressive exocrine pancreatic degeneration after birth.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

Objectives Acute lung injury (ALI) is a pulmonary manifestation of an acute systemic inflammatory response, which is associated with high morbidity and mortality. Accordingly, from the perspective of treating ALI, it is important to identify effective agents and elucidate the underlying modulatory mechanisms. β-Caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene that has anti-cancer and anti-inflammatory activities. However, the effects of BCP on ALI have yet to be ascertained. Methods ALI was induced intratracheally, injected with 5 mg/kg LPS and treated with BCP. The bone marrow-derived macrophages (BMDMs) were obtained and cultured then challenged with 100 ng/ml LPS for 4 h, with or without BCP pre-treatment for 30 min. Key findings BCP significantly ameliorates LPS-induced mouse ALI, which is related to an alleviation of neutrophil infiltration and reduction in cytokine production. In vitro, BCP was found to reduce the expression of interleukin-6, interleukin-1β and tumour necrosis factor-α, and suppresses the MAPK signalling pathway in BMDMs, which is associated with the inhibition of TAK1 phosphorylation and an enhancement of MKP-1 expression. Conclusions Our data indicate that BCP protects against inflammatory responses and is a potential therapeutic agent for the treatment of LPS-induced acute lung injury.

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15−/− zebrafish using transcription activator‐like effector nuclease technology. Whole‐mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15−/− zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15−/−; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression., Dhx15 is essential for hematopoietic stem/progenitor cell (HSPC) development in zebrafish; HSPC failure is mediated by the unfolded protein response (UPR) pathway in dhx15−/− zebrafish.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD + ), which reduced T cell proliferation and function. Because NAD + metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD + on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD + -mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

The astroglial network connected through gap junctions assembling from connexins physiologically balances the concentrations of ions and neurotransmitters around neurons. Astrocytic dysfunction has been associated with many neurological disorders including epilepsy. Dissociated gap junctions result in the increased activity of connexin hemichannels which triggers brain pathophysiological changes. Previous studies in patients and animal models of epilepsy indicate that the reduced gap junction coupling from assembled connexin hemichannels in the astrocytes may play an important role in epileptogenesis. This abnormal cell-to-cell communication is now emerging as an important feature of brain pathologies and being considered as a novel therapeutic target for controlling epileptogenesis. In particular, candidate drugs with ability of inhibition of connexin hemichannel activity and enhancement of gap junction formation in astrocytes should be explored to prevent epileptogenesis and control epilepsy.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Temozolomide (TMZ) is a conventional chemotherapeutic drug for glioma, however, its clinical application and efficacy is severely restricted by its drug resistance properties. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme, which can repair the DNA damage caused by TMZ. A large number of clinical data show that reducing the expression of MGMT can enhance the chemotherapeutic efficacy of TMZ. Therefore, in order to improve the resistance of glioma to TMZ, an angiopep-2 (A2) modified nanoprodrug of polytemozolomide (P(TMZ)n) that combines with MGMT siRNA (siMGMT) targeting MGMT was developed (A2/T/D/siMGMT). It not only increased the amount of TMZ within tumor lesion site, but also reduced MGMT expression in glioma. The in vitro experiments indicated that the A2/T/D/siMGMT effectively enhanced the cellular uptake of TMZ and siMGMT, and resulted in a significant cell apoptosis and cytotoxicity in the glioma cells. The in vivo experiments showed that glioma growth was inhibited and the survival time of animals were prolonged remarkably after A2/T/D/siMGMT was injected via tail vein. The results showed that the therapeutic effect of A2/T/D/siMGMT in the treatment of glioma was significantly improved.

Pathologic features of Alzheimer's disease (AD) include accumulation of amyloid β (Aβ) and hyperphosphorylated tau protein. We have shown previously that the chemokine-like receptor 1 (CMKLR1) is a functional receptor for Aβ, and CMKLR1 contributes to the uptake of Aβ. However, it is unclear whether CMKLR1 ameliorates or aggravates the process of AD. Here, we show that deletion of the gene coding for CMKLR1 significantly increased Aβ deposits in brains of both male and female amyloid β precursor protein/presenilin-1 mice. However, it markedly decreased the mortality of these mice. Behavioral studies found that CMKLR1 deficiency improved cognitive impairment of male and female amyloid β precursor protein/presenilin-1 mice and intracerebroventricular-streptozotocin injection AD mice. We further explored the effect of CMKLR1 on tau pathology. We found that CMKLR1 deficiency or inhibition attenuated the hyperphosphorylation of tau in brains of AD mice in vivo and in the neuronal cells in vitro. The expression of CMKLR1 on the neurons affected tau phosphorylation by participating in tau seeding. Together, these results uncover a novel mechanism of CMKLR1 in the pathologic process of AD and suggest that inhibiting the promotion effect of CMKLR1 on tau seeding may provide a new strategy for the treatment of AD. SIGNIFICANCE STATEMENT Evidence suggests that inflammation is involved in the pathologic progression of AD. The chemokine-like receptor 1 (CMKLR1), belonging to the family of GPCRs, is able to bind and uptake amyloid β. We show here, for the first time, that, although CMKLR1 deficiency increased amyloid β deposits in AD mice, it reduced the mortality and improved the cognitive deficits of AD mice. We furthermore show that CMKLR1 deficiency or inhibition attenuated tau hyperphosphorylation in brains of AD model mice in vivo and in neuronal cells in vitro. Finally, we first discovered that the expression of CMKLR1 on neurons affected tau phosphorylation by participating in tau seeding. These findings suggest that inhibition of CMKLR1 may provide a new strategy for the treatment of AD.

The proliferation of vascular smooth muscle cells (VSMCs) induced by oxidative injury is one of the main features in diabetes-accelerated atherosclerosis. Geranylgeranyl transferase-I (GGTase-I) is an essential enzyme mediating posttranslational modification, especially the geranylgeranylation of small GTPase, Rac1. Our previous studies found that GGTase-I played an important role in diabetes-accelerated atherosclerosis. However, its exact role is largely unclear. In this study, mouse conditional knockout of VSMC GGTase-I (Pggt1bΔ/Δ mice) was generated using the CRISPR/Cas9 system. The mouse model of diabetes-accelerated atherosclerosis was induced by streptozotocin injections and an atherogenic diet. We found that GGTase-I knockout attenuated diabetes-accelerated atherosclerosis in vivo and suppressed high-glucose-induced VSMC proliferation in vitro. Moreover, after a 16-week duration of diabetes, Pggt1bΔ/Δ mice exhibited lower α-smooth muscle actin (α-SMA) and nitrotyrosine level, Rac1 activity, p47phox and NOXO1 expression, and phospho-ERK1/2 and phosphor-JNK content than wild-type mice. Meanwhile, the same changes were found in Pggt1bΔ/Δ VSMCs cultured with high glucose (22.2 mM) in vitro. In conclusion, GGTase-I knockout efficiently blocked diabetes-accelerated atherosclerosis, and this protective effect must be related to the inhibition of VSMC proliferation. The potential mechanisms probably involved interfering Rac1 geranylgeranylation, inhibiting the assembly of NADPH oxidase cytosolic regulatory subunits, reducing oxidative injury, and decreasing ERK1/2 and JNK phosphorylation.

Sepsis-induced multiple organ dysfunction and inflammatory response are life-threatening symptoms without effective treatment. Fisetin, a dietary flavonoid extracted from berries and family Fabaceae, has displayed neuroprotective and anti-oxidant activities. In this study we investigated whether fisetin exerted a protective effect against sepsis-induced multiple organ dysfunction in mouse cecum ligation and puncture (CLP) model. The mice were injected with fisetin (10 mg/kg, ip) 0.5 h prior to CLP, and sacrificed 18 h after CLP. We found that fisetin administration significantly alleviated CLP-induced lung, liver and kidney injury, as well as the expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β in bronchoalveolar lavage fluid (BALF). In lipopolysaccharide (LPS)-treated mouse bone marrow-derived macrophages (BMDMs), application of fisetin (3–10 μM) dose- dependently inhibited the expression levels of IL-6, TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS). Furthermore, fisetin dose-dependently inhibited the phosphorylation of p38 MAPK, MK2, and transforming growth factor-β-activated kinase (TAK) 1 via attenuating the interaction between TAK1 and TAK-binding proteins (TAB) 1. These results demonstrate that fisetin is a promising agent for protecting against sepsis-induced inflammatory response and organ injury via inhibiting macrophage activation.

Cardiovascular manifestations account for marked morbi-mortality in autosomal dominant polycystic kidney disease (ADPKD). Pkd1- and Pkd2-deficient mice develop cardiac dysfunction, however the underlying mechanisms remain largely unclear. It is unknown whether impairment of polycystin-1 cleavage at the G-protein-coupled receptor proteolysis site, a significant ADPKD mutational mechanism, is involved in this process. We analyzed the impact of polycystin-1 cleavage on heart metabolism using Pkd1

Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI), which is widely used for anti-HIV-1. Evidences revealed that several central nervous system side effects could be observed in mice and patients with administration of EFV. However, the detailed mechanisms are still unknown. In this study, we investigated the effects of long-term EFV treatment on cognitive functions and the potential underlying mechanisms in mice. We maintained C57BL/6 mice aged 2 months with treatment containing 40 or 80 mg/kg/day EFV for 5 months, while control group treated with saline. The cognitive functions were evaluated by novel object recognition test, Barnes maze test and Morris water maze. The results showed significant short-term memory impairment in 40 and 80 mg/kg groups, and notable spatial learning and memory impairments in 80 mg/kg group, without any spontaneous activity alteration. Moreover, EFV induced impairments in dendritic integrity and synaptic plasticity in hippocampus. Furthermore, Significant increases were observed in the expression levels of pro-IL-1β, a similar tendency of TNF-α and phosphorylation of p65 of the 80 mg/kg group compared with control group. These results imply that long-term EFV treatment causes synaptic dysfunction resulting in cognitive deficits, which might be induced by the enhanced pro-inflammatory cytokines IL-1β and TNF-α via activating NF-κB pathway.

In order to search for novel checkpoint kinase 1/2 (Chk1) inhibitors, we have designed and synthesized a series of new compounds incorporating thienopyridazine core. Bioevaluation showed that compounds 10j, 10i, 13e and 10o exhibited relatively good inhibitory activity. Notably, compound 10o displayed high selectivity against a panel of kinases and inhibited Chk1/2 signaling pathway stimulated by DNA damage drugs in cellular level. Molecular docking of 10o to the ATP-binding site of Chk1 kinase domain indicated the existence of polar interactions between 10o and the ATP-ribose-binding residues of Chk1. In mouse HT-29 xenografts, a synergistic effect was observed. Co-treatment by CPT-11 and 10o significantly diminished the tumor volume, indicating the great potential of 10o as a candidate of Chk1/2 inhibitor.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic tumor microenvironment (TME) consisting of abundant activated pancreatic stellate cells (PSCs). PSCs play a key role in the refractory responses of PDAC to immunotherapy and chemotherapy and deactivating PSCs into quiescence through vitamin D receptor (VDR) signaling activation is a promising strategy for PDAC treatment. We observed p62 loss in PSCs hindered the deactivation efficacy of VDR ligands, and hypothesized that reversing p62 levels by inhibiting autophagy processing, which is responsible for p62 loss, could sensitize PSCs toward VDR ligands. Herein, we constructed a PSC deactivator with dual functions of VDR activation and autophagy inhibition, utilizing a pH-buffering micelle (LBM) with an inherent ability to block autophagic flux to encapsulate calcipotriol (Cal), a VDR ligand. This Cal-loaded LBM (C-LBM) could efficiently reprogram PSCs, modulate the fibrotic TME, and alter immunosuppression. In combination with PD-1 antagonists and chemotherapy, C-LBM showed superior antitumor efficacy and significantly prolonged the survival of PDAC mice. These findings suggest that synergistic autophagy blockade and VDR signaling activation are promising therapeutic approaches to reprogram PSCs and improve the PDAC response to immunotherapy.

The immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO1) and the PD-1/PD-L1 axis are potent mechanisms that impede effective anti-tumor immunity in ovarian cancer. However, whether the IDO pathway regulates PD-1 expression in T cells is currently unknown. Here we show that tumoral IDO1 expression led to profound changes in tryptophan, nicotinate/nicotinamide, and purine metabolic pathways in the ovarian tumor microenvironment, and to an increased frequency of PD-1+CD8+ tumor infiltrating T cells. We determined that activation of the aryl hydrocarbon receptor (AHR) by kynurenine induced PD-1 expression, and this effect was significantly abrogated by the AHR antagonist CH223191. Mechanistically, kynurenine alters chromatin accessibility in regulatory regions of T cell inhibitory receptors, allowing AHR to bind to consensus XRE motifs in the promoter region of PD-1. These results enable the design of strategies to target the IDO1 and AHR pathways for enhancing anti-tumor immunity in ovarian cancer.

Idiopathic pulmonary fibrosis is a progressive interstitial pneumonia characterised by fibroblast accumulation, collagen deposition and extracellular matrix (ECM) remodelling. It was reported that Akt1 mediated idiopathic pulmonary fibrosis progression through regulating the apoptosis of alveolar macrophage, while its effect on macrophage-produced cytokines remains largely unknown. In the present study, we first examined the phosphorylation of Akt1 in lung sections from idiopathic pulmonary fibrosis patients by immunohistochemistry before applying a bleomycin-induced idiopathic pulmonary fibrosis model using Akt1−/− mice and Akt1+/+ littermates. The results showed that Akt1 was remarkably up-regulated in idiopathic pulmonary fibrosis patients, while in vivo studies revealed that Akt1-deficient mice had well-preserved alveolar structure and fewer collagens, secreted fewer matrix components, including alpha smooth-muscle actin and fibronectin and survived significantly longer than Akt1+/+ littermates. Additionally, the pro-fibrogenic cytokine IL-13 was down-regulated at least twofold in Akt1−/−mice compared to the Akt1+/+group on d 3 and 7 after bleomycin treatment. Furthermore, it was found that Akt1–/– macrophages displayed down-regulation of IL-13 compared to Akt1+/+ macrophages in which Akt1 was phosphorylated in response to IL-33 stimulation. These findings indicate that Akt1 modulates pulmonary fibrosis through inducing IL-13 production by macrophages, suggesting that targeting Akt1 may simultaneously block the fibrogenic processes of idiopathic pulmonary fibrosis.

An excessive inflammatory response in terminal airways, alveoli, and the lung interstitium eventually leads to pulmonary hypertension and chronic obstructive pulmonary disease. Proinflammatory cytokine interleukin-17A (IL-17A) has been implicated in the pathogenesis of pulmonary inflammatory diseases. MLN4924, an inhibitor of NEDD8-activating enzyme (NAE), is associated with the treatment of various types of cancers, but its role in the IL-17A-mediated inflammatory response has not been identified. Here, we report that MLN4924 can markedly reduce the expression of proinflammatory cytokines and chemokines such as IL-1β, IL-6, and CXCL-1 and neutrophilia in a mouse model of IL-17A adenovirus-induced pulmonary inflammation. MLN4924 significantly inhibited IL-17A-induced stabilization of mRNA of proinflammatory cytokines and chemokines in vitro. Mechanistically, MLN4924 significantly blocked the activation of MAPK and NF-κB pathways and interfered with the interaction between ACT1 and tumor necrosis factor receptor-associated factor proteins (TRAFs), thereby inhibiting TRAF6 ubiquitination. Taken together, our data uncover a previously uncharacterized inhibitory effect of MLN4924 on the IL-17A-mediated inflammatory response; this phenomenon may facilitate the development of MLN4924 into an effective small-molecule drug for the treatment of pulmonary inflammatory diseases.

Pancreatic ductal adenocarcinoma (PDAC) is a dominantly (~95%) KRAS-mutant cancer that has extremely poor prognosis, in part this is due to its strong intrinsic resistance towards almost all therapeutic agents. PDAC relies heavily on KRAS-transformed metabolism, including enhanced macropinocytosis and catabolism of extracellular albumin, to maintain its proliferation and progression. However, it has yet to be validated that whether such transformed metabolism could be exploited for the drug delivery to open therapeutic windows of cytotoxic agents in KRAS-mutant PDAC. In this study, we attempt to answer this question by focusing on the impact of two critical regulators of albumin catabolism, KRAS and the neonatal Fc receptor (FcRn), on the sensitivity of PDAC to doxorubicin (DOX, a model cytotoxic agent) and albumin-conjugated doxorubicin (DOX-ALB). Using cell lines and cell-derived xenografts with different KRAS genotypes and FcRn levels, we demonstrated that KRAS-enhanced macropinocytosis and reduced FcRn expression sensitize PDAC to DOX-ALB but not free DOX. In both in vitro and in vivo comparsion, the DOX-ALB demonstrated ~10 times enlarged therapeutic window compared with free DOX, in PDAC with KRAS mutation and reduced FcRn level, two events appear to occur simultaneously in the investigated PDAC. In summary, we conclude that albumin conjugation is an exploitable drug delivery strategy that significantly opens the therapeutic windows of otherwise undevelopable anti-cancer agents for KRAS-mutant PDAC therapy, and creates a new landscape for clinical evaluation and future translation of such compounds.

Staphylococcus aureus (S. aureus) infection leads to a severe inflammatory response and causes acute lung injury (ALI), eventually threatening human life. Therefore, it is of importance to find an agent to inhibit inflammation and reduce ALI. Here, we found that costunolide, a sesquiterpene lactone, displays anti-inflammatory effects and ameliorates heat-killed S. aureus (HKSA)-induced pneumonia. Costunolide treatment attenuated HKSA-induced murine ALI in which pulmonary neutrophil infiltration was inhibited, lung edema was decreased, and the production of pro-inflammatory cytokines was significantly reduced. In addition, costunolide dose-dependently inhibited the generation of IL-6, TNF-α, IL-1β, and keratinocyte-derived cytokine (KC), as well as the expression of iNOS, in HKSA-induced macrophages. Furthermore, costunolide attenuated the phosphorylation of p38 MAPK and cAMP response element-binding protein (CREB). Collectively, our findings suggested that costunolide is a promising agent for alleviating bacterial-induced ALI via the inhibition of the MAPK signaling pathways.

Schisandra chinensis decoction derived from the book of Waitai Miyao (Tao Wang, Tang dynasty) is often used in the treatment of idiopathic pulmonary fibrosis (IPF), which is included in the Grand Ceremony of Chinese formulae (Huairen Peng, 1994). Schisandrae Chinensis Fructus (Sch) is one of the most important herbs in this formula. According to the "Shennong's Herbal Classicherbal" of the Han Dynasty, Sch has sour taste, warm nature, which has the effect of tonifying qi and curing cough. In addition, according to the "Compendium of Materia Medica" of the Ming Dynasty, Sch is used to treat cough and asthma, which has the effect of moistening the lung and tonifying the kidney. However, the active ingredients of Sch absorption into the plasma and its pharmacological mechanism of treatment for IPF still remained unclear.Our research aimed at identifying the absorbed active ingredients and metabolized of Sch in rat plasma and the mechanism of anti-IPF based on serum pharmacochemistry.First, the rats were divided into control group and Sch group. Sch sample was orally administrated to the rats for seven days. The blood samples were drawn into an Eppendorf tube after the last dosing. The ultrahigh performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the absorption components and metabolites of Sch in rat plasma. Second, the network pharmacology combined with molecular docking analysis was further investigated to illuminate its potential mechanism of treatment for IPF by the biological targets regulating related pathways. Finally, the mechanism of action was verified by experimental in vitro and in vivo.A total of 78 compounds, consist of 13 prototype lignans and 65 metabolites (including isomers) were identified. Network pharmacology study and molecular docking analysis indicated that schisandrol A (L1) play an anti-fibrosis role by regulating the TGF-β signaling pathway. Experimental in vitro and in vivo verified that the schisandrol A could inhibiting pulmonary fibrosis through TGF-β signaling pathway. The effect and mechanism of schisandrol A inhibiting pulmonary fibrosis were reported for the first time.In this study, the absorption active ingredients of Sch in rat plasma were combined with the network pharmacology investigation and experimental in vitro and in vivo to elucidate its biological mechanism of treatment for IPF. The results provided a theoretical support for understanding the bioactive compounds and the pharmacological mechanism of Sch.

Aconitine (AC) is the primary bioactive and secondary metabolite alkaloidin of Aconitum species which is accounted for more than 60% of the total diester-diterpenoid alkaloids in Aconite. To evaluate the analgesic effects of AC, 4 different pain models including hot plate assay, acetic acid writhing assay, formalin and CFA induced pain models were adopted in this study. In hot plate experiment, AC treatment at concentration of 0.3 mg/kg and 0.9 mg/kg improved the pain thresholds of mice similar to the positive drug aspirin at the concentration of 200 mg/kg (17.12% and 20.27% VS 19.21%). In acetic acid writhing experiment, AC significantly reduced the number of mice writhing events caused by acetic acid, and the inhibition rates were 68% and 76%. These results demonstrated that AC treatment revealed significant analgesic effects in both acute thermal stimulus pain model and chemically-induced visceral pain model. The biphasic nociceptive responses induced by formalin were significantly inhibited after AC treatment for 1h or 2h. The inhibition rates were 33.23% and 20.25% of AC treatment for 1h at 0.3 mg/kg and 0.9 mg/kg in phase I. In phase II, the inhibition rates of AC and aspirin were 36.08%, 32.48% and 48.82% respectively, which means AC showed similar analgesic effect to non-steroidal anti-inflammatory compounds. In the chronic CFA-induced nociception model, AC treatment also improved mice pain threshold to 131.33% at 0.3 mg/kg, which was similar to aspirin group (152.03%). Above all, our results verified that AC had obviously analgesic effects in different mice pain models.

Patients receiving brain radiotherapy may suffer acute or chronic side effects. Ionizing radiation induces the production of intracellular reactive oxygen species and pro-inflammatory cytokines in the central nervous system, leading to brain damage. Complementary Chinese herbal medicine therapy may reduce radiotherapy-induced side effects. Flavonoids are a class of natural products which can be extracted from Chinese herbal medicine and have been shown to have neuroprotective and radioprotective properties. Flavonoids are effective antioxidants and can also inhibit regulatory enzymes or transcription factors important for controlling inflammatory mediators, affect oxidative stress through interaction with DNA and enhance genomic stability. In this paper, radiation-induced brain damage and the relevant molecular mechanism were summarized. The radio-neuro-protective effect of flavonoids, i.e., antioxidant, anti-inflammatory and maintaining genomic stability, were then reviewed. We concluded that flavonoids treatment may be a promising complementary therapy to prevent radiotherapy-induced brain pathophysiological changes and cognitive impairment.

A dense desmoplastic stroma formed by abundant extracellular matrix and stromal cells, including cancer-associated fibroblasts (CAFs) and immune cells, is a feature of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancer types. As the dominant cellular component of the PDAC stroma, CAFs orchestrate intensive and biologically diverse crosstalk with pancreatic cancer cells and immune cells and contribute to a unique PDAC tumor microenvironment promoting cancer proliferation, metastasis, and resistance against both chemo- and immunotherapies. Therefore, CAFs and CAF-related mechanisms have emerged as promising targets for PDAC therapy. However, several clinical setbacks and accumulating knowledge of the PDAC stroma have revealed the heterogeneity and multifaceted biological roles of CAFs, and concerns regarding "what to deliver" and "how to deliver" have arisen when designing CAF-targeted drug delivery systems to specifically inhibit tumor-supporting CAFs without impairing tumor-restricting CAFs. In this review, we will discuss the complexity of CAFs in the PDAC stroma as well as the potential opportunities and common misconceptions regarding drug delivery efforts targeting PDAC CAFs.

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease., eLife digest On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2. Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia – which are less than a thousandth of a millimeter thick – little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques. Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels. Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD.

Idiopathic pulmonary fibrosis (IPF) induces significant morbidity and mortality, for which there are limited therapeutic options available. Here, we found that tetraethylthiuram disulphide (disulfiram, DSF), a derivative of thiuram, used in the treatment of alcohol abuse, has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis via the attenuation of the fibroblast-to-myofibroblast transition, migration, and proliferation of fibroblasts. Furthermore, DSF inhibited the activation of primary pulmonary fibroblasts and fibroblast cell line under transforming growth factor-β 1 (TGF-β1) challenge. Mechanistically, the anti-fibrotic effect of DSF on fibroblasts depends on the inhibition of TGF-β signalling. We further determined that DSF interrupts the interaction between SMAD3 and TGF-β receptor Ι (TBR Ι), and identified that DSF directly binds with SMAD3, in which Trp326, Thr330, and Cys332 of SMAD3 are critical binding sites for DSF. Collectively, our results reveal a powerful anti-fibrotic function of DSF in pulmonary fibrosis through the inhibition of TGF-β/SMAD signalling in pulmonary fibroblasts, indicating that DSF is a promising therapeutic candidate for IPF.

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain (RBD, residues 319-541) and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice. The antibodies also effectively suppressed SARS-CoV-2 RBD binding to soluble ACE2, indicating that RBD-mFc may be further developed as a safe and effective SARS-CoV-2 vaccine.

IL-17A combined with TNF-α plays a vital role in inflammatory response and interference of the synergistic effect is an effective strategy for treating inflammatory diseases. Ellipticine, a natural alkaloid, has biological activities on anti-tumor and anti-HIV. However, it is still unknown whether ellipticine can inhibit IL-17A and TNF-α-mediated signaling and has treatment effect on PALI. Here, we reported that ellipticine significantly inhibited the production of pro-inflammatory cytokines and chemokines in pulmonary epithelial cell BEAS-2B treated with IL-17A and TNF-α, but not IL-17A or TNF-α alone. Meanwhile, ellipticine attenuated NF-κB and MAPKs activation in response to IL-17A and TNF-α treatment, inhibited Act1 and TRAF6-mediated NF-κB activation, and blocked the interaction of Act1 with TRAF6. Furthermore, we found that ellipticine significantly alleviated CAE and LPS-induced SAP/PALI. Ellipticine treatment dramatically reduced inflammatory cells infiltration, MPO activity, serum amylase and lipase activity and the protein concentration of BALF. Collectively, our findings indicate that ellipticine inhibits the synergistic effect of IL-17A and TNF-α by targeting on Act1 and TRAF6 interaction and is a potential therapeutic agent for the treatment of SAP/PALI.

Shikonin is the major active component in Lithospermum erythrorhizon and has pharmacological effects including reducing inflammation, aiding resistance to bacteria and promoting wound healing. However, the effect of shikonin on lipoteichoic acid (LTA)‑induced acute lung injury (ALI) remains to be elucidated. ALI is a serious illness resulting from significant pulmonary inflammation caused by various diseases, such as sepsis, acid aspiration and trauma. The present study found that shikonin significantly attenuated LTA‑induced ALI. Following shikonin treatment, the accumulation of pulmonary neutrophils and expression of TNFα, IL‑1β and IL‑6 were decreased in mice with LTA‑induced ALI. Furthermore, Shikonin promoted neutrophil apoptosis by increasing the activation of caspase‑3 and reducing the expression of the antiapoptotic myeloid cell leukemia‑1 (Mcl‑1) protein. However, shikonin treatment did not influence the expression of B‑cell lymphoma‑2. The findings of the present study demonstrated that shikonin protected against LTA‑induced ALI by promoting caspase-3 and Mcl‑1‑related neutrophil apoptosis, suggesting that shikonin is a potential agent that can be used in the treatment of sepsis‑mediated lung injury.

CREB-binding protein (CBP) participates in numerous transcription events. However, cell-intrinsic inhibitors of CBP are poorly defined. Here, we found that cellular USP12 interacts with the HAT domain of CBP and inhibits CBP’s acetyltransferase activity. Interestingly, USP12 positively regulates interferon (IFN) antiviral signaling independently of its deubiquitinase activity. Furthermore, we found that in IFN signaling USP12 translocates from the cytoplasm to the nucleus. The decrease in cytoplasmic USP12 facilitates CBP-induced acetylation and activation of IFN signaling proteins in the cytoplasm. Moreover, USP12 accumulation in the nucleus blocks CBP-induced acetylation of phosphorylated STAT1 (p-STAT1) and therefore inhibits the dephosphorylation effects of TCPTP on p-STAT1, which finally maintains nuclear p-STAT1 levels and IFN antiviral efficacy. USP12 nuclear translocation extends our understanding of the regulation of the strength of IFN antiviral signaling. Our study uncovers a cell-intrinsic regulation of CBP acetyltransferase activity and may provide potential strategies for IFN-based antiviral therapy., Author summary Activated p-STAT1 is a determinant for the strength of IFN antiviral signaling. We and other groups have demonstrated that activated p-STAT1 is regulated by multiple protein post-translational modifications, including phosphorylation, acetylation and ubiquitination. In this study, we revealed that CBP-mediated acetylation regulation of p-STAT1 is modulated by the deubiquitinase USP12 in a deubiquitinase activity-independent manner. USP12 translocates into the nucleus in IFN signaling, which critically regulates nuclear p-STAT1 levels and IFN antiviral activity by inhibiting CBP’s acetyltransferase activity. Importantly, we demonstrated that USP12 is a cell-intrinsic inhibitor of the acetyltransferase CBP. These findings promote the understanding of delicate regulation of both CBP-mediated acetylation and IFN antiviral signaling.

Serum amyloid A (SAA) is an early and sensitive biomarker of inflammatory diseases, but its role in acute pancreatitis (AP) is still unclear. Here, we used a caerulein-induced mouse model to investigate the role of SAA in AP and other related inflammatory responses. In our study, we found that the expression of a specific SAA isoform, SAA3, was significantly elevated in a caerulein-induced AP animal model. In addition, SAA3-knockout (Saa3-/- ) mice showed lower serum levels of amylase and lipase, tissue damage and proinflammatory cytokine production in the pancreas compared with those of wild-type mice in response to caerulein administration. AP-associated acute lung injury was also significantly attenuated in Saa3-/- mice. In our in vitro experiments, treatment with cholecystokinin and recombinant SAA3 significantly induced necroptosis and cytokine production. Moreover, we found that the regulatory effect of SAA3 on acinar cell necroptosis was through a receptor-interacting protein 3 (RIP3)-dependent manner. Collectively, our findings indicate that SAA3 is required for AP by inducing an RIP3-dependent necroptosis pathway in acinar cells and is a potential drug target for AP.

Isoalantolactone (IAL) is a sesquiterpene lactone extracted from roots of Inula helenium L and has shown anti-inflammatory effects. In this study we investigated the therapeutic effects of IAL on acute lung injury (ALI) and elucidated the mechanisms underlying its anti-inflammation potential in vitro and in vivo. Treatment with lipopolysaccharide (LPS, 100 ng/mL) drastically stimulated production of inflammatory mediators such as NO, TNF-α, IL-1β, and IL-6 in mouse bone marrow-derived macrophages (BMDMs), which was dose-dependently suppressed by pretreatment with IAL (2.5, 5, 10, 20 μM). We further revealed that IAL suppressed LPS-induced NF-κB, ERK, and Akt activation. Moreover, the downregulation of non-degradable K63-linked polyubiquitination of TRAF6, an upstream transcription factor of NF-κB, contributed to the anti-inflammatory effects of IAL. ALI was induced in mice by intratracheal injection of LPS (5 mg/kg). Administration of IAL (20 mg/kg, i.p.) significantly suppressed pulmonary pathological changes, neutrophil infiltration, pulmonary permeability, and pro-inflammatory cytokine expression. Our results demonstrate that IAL is a potential therapeutic reagent against inflammation and ALI.

Maximizing the pharmacological efficacy of albendazole (ABZ), an anti-echinococcosis drug, is essential in the long-term treatment of patients with echinococcosis. As a weakly alkaline drug, ABZ has a pH-dependent solubility that decreases dramatically from gastric fluid (pH 1.4) to intestinal fluid (pH 6.5), where it is absorbed. In this study, we endeavored to develop an optimized tablet formulation of ABZ to improve its dissolution and oral bioavailability from two aspects: a faster initial dissolution in the gastric pH condition (i.e., the "spring") and a more prolonged drug supersaturation in the intestinal pH condition (i.e., the "parachute"). To achieve this goal, ABZ-HCl salt was selected first, which demonstrated a higher intrinsic dissolution rate under pH 1.4 compared with the ABZ free base that is used in the commercial product Albenda. Second, by comparing the ABZ supersaturation kinetics under pH 6.5 in the presence of various polymers including poly(vinylpyrrolidone) (PVP), PVP/VA, hydroxypropyl methylcellulose (HPMC), and HPMC acetate succinate (HPMC-AS), HPMC-AS was found to be the most effective crystallization inhibitor for ABZ, likely due to the hydrophobic interaction between ABZ and HPMC-AS in an aqueous environment. The newly designed tablet formulation containing ABZ-HCl and HPMC-AS showed ∼3 times higher oral bioavailability compared with that of Albenda in Beagle dogs. More significantly, the anti-echinococcosis efficacy of the improved formulation was 2.4 times higher than that of Albenda in a secondary hepatic alveolar echinococcosis Sprague-Dawley rat model. The strategy of simultaneously improving the spring and parachute of an oral formulation of ABZ, by using a highly soluble salt and an effective polymeric crystallization inhibitor, was once again proven to be a viable and readily translatable approach to optimize the unsatisfactory oral medicines due to solubility and bioavailability limitations.

Alveolar echinococcosis (AE) is a chronic infectious parasitic disease that is fatal and still being neglected. Currently, the AE treatment recommended by the WHO is complete excision of the lesions, followed by the oral administration of albendazole (ABZ), the only effective first-line anti-AE drug, for two years. Unfortunately, complete excision of AE lesions is impossible in most cases, leaving the long-term use of ABZ as the only alternative. However, only about one-third of patients experience complete remission or cure with such treatments, largely because of the low oral bioavailability of ABZ caused by its very low solubility. To improve the oral bioavailability of ABZ, a novel nanocrystalline (NC) formulation of ABZ was obtained by spray-drying ABZ with a triblock copolymer poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Poloxamer 188), and its physical structure was confirmed by scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), wide-angle X-ray diffraction (WAXRD), and polarized optical microscopy (POM). The significantly reduced ABZ crystallite size coupled with prolonged ABZ supersaturation significantly improved the drug dissolution performance compared with that of the commercial ABZ oral product (Albenda), and the NC formulation showed an approximately 4.2-fold higher AUC than Albenda in a pharmacokinetic comparison in Beagle dogs as measured by the plasma concentration of albendazole sulfoxide, the active antiparasitic metabolite. Even more encouragingly, after 30 days of once-daily oral administration of the NC and Albenda formulations to SD rats with hepatic alveolar echinococcosis, the NC formulation demonstrated a cyst inhibition effect 3.7-fold greater than that of Albenda. We therefore conclude that the NC formulation could potentially be developed into an improved anti-AE drug therapy.

A growing body of evidence shows that the development and progression of gastric cancer (GC) is mainly associated to the presence of gastric cancer stem-like cells (GCSLCs). However, it is unclear how GCSLC population is maintained. This study aimed to explore the role of capillary morphogenesis gene 2 (CMG2) in GCSLC maintenance and the relevance to GC progression. We found that CMG2 was highly expressed in GC tissues and the expression levels were associated with the invasion depth and lymph node metastasis of GC, and inversely correlated with the survival of GC patients. Sorted CMG2High GC cells preferentially clustered in CD44High stem-like cell population, which expressed high levels of stemness-related genes with increased capabilities of self-renewal and tumorigenicity. Depletion of CMG2 gene resulted in reduction of GCSLC population with attenuated stemness and decrease of invasive and metastatic capabilities with subdued epithelial–mesenchymal transition phenotype in GC cells. Mechanistically, CMG2 interacted with LRP6 in GCSLCs to activate a Wnt/β-catenin pathway. Thus, our results demonstrate that CMG2 promotes GC progression by maintaining GCSLCs and can serve as a new prognostic indicator and a target for human GC therapy.

The aim of this paper was to compare the in vitro dissolution and in vivo bioavailability of three solubility enhancement technologies for β-lapachone (LPC), a poorly water soluble compound with extremely high crystallization propensity. LPC cocrystal was prepared by co-grinding LPC with resorcinol. LPC crystalline and amorphous solid dispersions (CSD and ASD) were obtained by spray drying with Poloxamer 188 and HPMC-AS, respectively. The cocrystal structure was solved by single crystal x-ray diffraction. All formulations were characterized by WAXRD, DSC, POM and SEM. USP II and intrinsic dissolution studies were used to compare the in vitro dissolution of these formulations, and a crossover dog pharmacokinetic study was used to compare their in vivo bioavailability. An 1:1 LPC-resorcinol cocrystal with higher solubility and faster dissolution rate was obtained, yet it converted to LPC crystal rapidly in solution. LPC/HPMC-AS ASD was confirmed to be amorphous and uniform, while the crystal and crystallite sizes of LPC in CSD were found to be ∼1–3 μm and around 40 nm, respectively. These formulations performed similarly during USP II dissolution, while demonstrated dramatically different oral bioavailability of ∼32%, ∼5%, and ∼1% in dogs, for CSD, co-crystal, and ASD, respectively. CSD showed the fastest intrinsic dissolution rate among the three. The three formulations showed poor IVIVC which could be due to rapid and unpredictable crystallization kinetics. Considering all the reasons, we conclude that for molecules with extremely high crystallization tendency that cannot be inhibited by any pharmaceutical excipients, size-reduction technologies such as CSD could be advantageous for oral bioavailability enhancement in vivo than technologies only generating transient but not sustained supersaturation.

Increasing studies have demonstrated that most tumors consisted a subpopulation of cells with stem cell properties, known as cancer stem cells (CSCs). Accumulating evidence indicated that CSCs may be critical driving force for several types of cancer. Hence, it was necessary to develop therapeutic approaches specifically targeting CSCs. In this review, first, the biological properties of CSCs were introduced, including the self-renewal and differentiation, high tumorigenesis and invasiveness, resistance to chemotherapy and radiotherapy, genetic and epigenetic variations. Meanwhile, CSCs-targeted therapeutic strategies were summarized, including targeting cell surface markers, signaling pathways, CSC niches, differentiation therapy, and drug resistance for CSCs. Furthermore, clinical trials on anti-CSCs therapies supported the efficacy of these therapies, as well as their combination with conventional chemotherapy and radiotherapy. CSCs could be significantly eradicated, eventually resulting in inhibited tumor growth, metastasis, and recurrence. Thus, selectively targeting CSCs with various agents may be a novel and promising therapeutic strategy against cancer.

Chemically incorporating Schiff base interactions into silicate frameworks endows the obtained mesoporous organosilica based drug carriers with ultrafast pH-responsive degradability and efficient anti-cancer drug release kinetics, resulting in enhanced cancer therapy.

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

Itraconazole (ITA), an old and widely prescribed antifungal drug with excellent safety profile, has more recently been demonstrated to be a multitarget antiangiogenesis agent affecting multiple angiogenic stimulatory signals and pathways, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, and mammalian target of rapamycin (mTOR). In this study, we developed two nanoparticle formulations, i.e., polymer micelles (IP2K) and albumin nanoparticles (IBSA), to solubilize the extremely hydrophobic and insoluble ITA to allow intravenous administration and pharmacokinetics (PK)/pharmacodynamics (PD) comparisons. Although none of the formulations showed strong antiproliferation potency against non-small cell lung cancer (NSCLC) cells in vitro, when administrated at the equivalent ITA dose to a NSCLC patient-derived xenograft (PDX) model, IBSA retarded while IP2K accelerated the tumor growth. We attributed the cause of this paradox to formulation-dependent PK and vascular manipulation: IBSA demonstrated a more sustained PK with a C

Cardiac tissue ischemia/hypoxia increases glycolysis and lactic acid accumulation in cardiomyocytes, leading to intracellular metabolic acidosis. Sodium bicarbonate cotransporters (NBCs) play a vital role in modulating intracellular pH and maintaining sodium ion concentrations in cardiomyocytes. Cardiomyocytes mainly express electrogenic sodium bicarbonate cotransporter (NBCe1), which has been demonstrated to participate in myocardial ischemia/reperfusion (I/R) injury. This review outlines the structural and functional properties of NBCe1, summarizes the signaling pathways and factors that may regulate the activity of NBCe1, and reviews the roles of NBCe1 in the pathogenesis of I/R-induced cardiac diseases. Further studies revealing the regulatory mechanisms of NBCe1 activity should provide novel therapeutic targets for preventing I/R-induced cardiac diseases.

Formyl peptide receptors (FPRs) are G protein-coupled chemoattractant receptors expressed mainly in phagocytic leukocytes. High expression of FPRs has also been detected in several cancers but the functions of FPR1 in tumor invasion and metastasis is poorly understood. In this study, we investigated the expression of FPRs in primary human colorectal cancer (CRC) and analyzed the association of FPRs expression with clinicopathological parameters. The levels of FPRs mRNA, especially those of FPR1, were significantly higher in colorectal tumors than in distant normal tissues and adjacent non-tumor tissues. FPR1 mRNA expression was also associated with tumor serosal infiltration. FPR1 protein expression was both in the colorectal epitheliums and tumor infiltrating neutrophils/macrophages. Furthermore, the functions of FPR1 in tumor invasion and tissue repair were investigated using the CRC cell lines SW480 and HT29. Higher cell surface expression of FPR1 is associated with significantly increased migration in SW480 cells compared with HT29 cells that have less FPR1 membrane expression. Finally, genetic deletion of fpr1 increased the survival rate of the resulting knockout mice compared with wild type littermates in a mouse model of colitis-associated colorectal cancer. Our data demonstrate that FPR1 may play an important role in tumor cell invasion in CRC patients.