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Abstract Previous studies arguing for parental care in dinosaurs have been primarily based on fossil accumulations of adults and hatchlings, perinatal and post-hatchlings in nests and nest areas, and evidence of brooding, the majority of which date to the Late Cretaceous. Similarly, the general body proportions of preserved embryonic skeletons of the much older Early Jurassic Massospondylus have been used to suggest that hatchlings were unable to forage for themselves. Here, we approach the question of parental care in dinosaurs by using a combined morphological, chemical, and biomechanical approach to compare early embryonic and hatchling bones of the Early Jurassic sauropodomorph Lufengosaurus with those of extant avian taxa with known levels of parental care. We compare femora, the main weight-bearing limb bone, at various embryonic and post-embryonic stages in a precocious and an altricial extant avian dinosaur with those of embryonic and hatchling Lufengosaurus, and find that the rate and degree of bone development in Lufengosaurus is closer to that of the highly altricial Columba (pigeon) than the precocious Gallus (chicken), providing strong support for the hypothesis that Lufengosaurus was fully altricial. We suggest that the limb bones of Lufengosaurus hatchlings were not strong enough to forage for themselves and would likely need parental feeding.

Aim: Preeclampsia is a complex and serious pregnancy disorder that leads to maternal and neonatal mortality worldwide. Kefir peptides (KPs), derived from various prebiotic fermentations in whole milk by kefir grains, were investigated for their potential therapeutic effects. In this study, we used the L-NAME in drinking water to induce a preeclampsia-like condition in spontaneous hypertension stroke-prone (SHRSP) pregnant rats. Main methods: The rats were assigned to five groups: the normal group (WKY rats), the untreated group (SHRSP rat control pregnant), the L-NAME/Mock group (SHRSP rats fed with L-NAME water), the L-NAME/KPs-LD group (SHRSP rats fed with L-NAME water and low-dose KPs diets), and the L-NAME/KPs-HD group (SHRSP rats fed with L-NAME water and high-dose KPs diets) for a 20-day experiment. Chorioallantois membrane (CAM) assay was applied for ex vivo angiogenesis study of KPs treatment. Key findings: Data showed that rats in the L-NAME group developed severe hypertension, proteinuria, placental damage, and embryo resorption. Pre-administration of KPs significantly reduced hypertension, proteinuria, improved generalized endothelial dysfunction, and decreased levels of anti-HIF-1α, sFLT1, anti-TNF-α, and IL-6 in the placenta of SHRSP rats. In ex vivo CAM study, L-NAME administration in chicken embryos resulted in lower vessel density and hemorrhage; however, angiogenesis was observed after KPs-HD treatment. Significance: The results indicate that kefir peptides improve renal lesions, prevent renal parenchyma damage, and balance endothelial and angiogenic dysfunction in both maternal and fetal sites in L-NAME-induced SHRSP pregnant rats.

Background: Pulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. Methods: The TGF-β1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. Results: The A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-β1-induced MRC-5 cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. Conclusions: The Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.

Abstract Background The development of alcohol-associated liver disease (ALD) is influenced by the amount and duration of alcohol consumption. The resulting liver damage can range from reversible stages, such as steatosis, steatohepatitis and alcoholic fibrosis, to the advanced and irreversible stage of cirrhosis. Aldo-keto reductase family 1 member A1 (AKR1A1) is a member of the aldo-keto reductase family that catalyzes the reduction of aldehyde groups to their corresponding alcohols in an NADPH-dependent manner. AKR1A1 was found to be downregulated in patients diagnosed with ALD. This study aims to interpret the protective effects of AKR1A1 on the development of ALD. Methods A 5% alcohol-fed (AF) Akr1a1 knockout (Akr1a1 −/−) mouse model and an AML12 hepatocyte model were used. The effects of AKR1A1 on liver function, inflammation, oxidative stress, lipid accumulation, and fibrosis were assessed by ELISA, western blotting, RT‒PCR, and a variety of histological staining methods in AF-induced wild-type (WT) and Akr1a1 −/− mice compared to control liquid diet-fed (PF) WT and Akr1a1 −/− mice. Results The results demonstrated that AF-WT mice expressed higher levels of AKR1A1 than WT mice fed a control diet, and they did not show any noticeable liver steatosis. However, AF-Akr1a1 −/− mice displayed a lower survival rate and more severe liver injury than AF-WT mice, as demonstrated by increased proinflammatory cytokines, oxidative stress, lipid accumulation, fibrosis, and reduced antioxidant enzymes in their livers. Additionally, elevated levels of 4-HNE and p53 phosphorylation were observed in AF-Akr1a1 −/− mice, suggesting that the loss of AKR1A1 led to increased 4-HNE accumulation and subsequent activation of p53, which contributed to the progression of ALD. Furthermore, in AML12 hepatocytes, Akr1a1 knockdown aggravated oxidative stress and steatosis induced by palmitic acid/oleic acid (P/O) inflammation induced by lipopolysaccharide (LPS), and fibrosis induced by TGF-β1. Conclusions This loss-of-function study suggests that AKR1A1 plays a liver-protective role during chronic alcohol consumption by reducing the accumulation of 4-HNE and inhibiting 4-HNE-mediated p53 activation.

Treating diabetic wounds effectively remains a significant clinical challenge. Emerging studies suggest that microRNAs (miRNAs) play crucial roles in various physiological and pathological processes and hold promise as therapeutic tools. This study investigates the miRNA expression profile in keratinocytes using a cell model of diabetic wounds. Microarray analysis revealed that 43 miRNAs from wounded keratinocytes incubated under diabetic conditions (high glucose/hypoxia) exhibited a two-fold change in expression compared to those incubated under normal conditions (low glucose/normoxia). Quantitative RT-PCR confirmed significant differences in the expression of eight miRNAs, with miR-3138 and miR-3679-5p being further analyzed for their roles in keratinocyte migration. Transfection with a miR-3138 mimic and a miR-3679-5p inhibitor indicated that upregulation of miR-3138 and downregulation of miR-3679-5p enhance keratinocyte migration in both normal and diabetic wounds. Pathway and gene ontology (GO) analyses identified potential pathways and functional annotations associated with miR-3138 and miR-3679-5p in diabetic wound healing. Potential human gene targets of miR-3138 and miR-3679-5p were predicted using a three-way comparison of the TargetScan, miRDB, and DIANA databases. This study elucidates the miRNA expression signature of human keratinocytes in a diabetes-like environment, providing deeper insights into the pathogenesis of diabetic wounds.

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF’s protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF’s mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-β1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF’s ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1β and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-β1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening lung disease with high mortality rates. The limited availability of effective drugs for IPF treatment, coupled with concerns regarding adverse effects and restricted responsiveness, underscores the need for alternative approaches. Kefir peptides (KPs) have demonstrated antioxidative, anti-inflammatory, and antifibrotic properties, along with the capability to modulate gut microbiota. This study aims to investigate the impact of KPs on bleomycin-induced pulmonary fibrosis. Methods: Mice were treated with KPs for four days, followed by intratracheal injection of bleomycin for 21 days. Comprehensive assessments included pulmonary functional tests, micro-computed tomography (µ-CT), in vivo image analysis using MMPsense750, evaluation of inflammation- and fibrosis-related gene expression in lung tissue, and histopathological examinations. Furthermore, a detailed investigation of the gut microbiota community was performed using full-length 16 S rRNA sequencing in control mice, bleomycin-induced fibrotic mice, and KPs-pretreated fibrotic mice. Results: In KPs-pretreated bleomycin-induced lung fibrotic mice, notable outcomes included the absence of significant bodyweight loss, enhanced pulmonary functions, restored lung tissue architecture, and diminished thickening of inter-alveolar septa, as elucidated by morphological and histopathological analyses. Concurrently, a reduction in the expression levels of oxidative biomarkers, inflammatory factors, and fibrotic indicators was observed. Moreover, 16 S rRNA sequencing demonstrated that KPs pretreatment induced alterations in the relative abundances of gut microbiota, notably affecting Barnesiella_intestinihominis, Kineothrix_alysoides, and Clostridium_viride. Conclusions: Kefir peptides exerted preventive effects, protecting mice against bleomycin-induced lung oxidative stress, inflammation, and fibrosis. These effects are likely linked to modifications in the gut microbiota community. The findings highlight the therapeutic potential of KPs in mitigating pulmonary fibrosis and advocate for additional exploration in clinical settings.

Huntington’s disease (HD) is one of the inheritable neurodegenerative diseases, and these diseases share several similar pathological characteristics, such as abnormal neuronal morphology. miR-196a is a potential target to provide neuroprotective functions, and has been reported to enhance polymerization of neuronal microtubules in HD. While microtubules and microfilaments are two important components of the neuronal cytoskeleton, whether miR-196a improves neuronal microfilaments is still unknown. Here, we identify insulin-like growth factor 2 mRNA binding protein 3 (IMP3), and show that miR-196a directly suppresses IMP3 to increase neurite outgrowth in neurons. In addition, IMP3 disturbs neurite outgrowth in vitro and in vivo, and worsens the microfilament polymerization. Moreover, insulin-like growth factor-II (IGF2) is identified as the downstream target of IMP3, and miR-196a downregulates IMP3 to upregulate IGF2, which increases microfilamental filopodia numbers and activates Cdc42 to increase neurite outgrowth. Besides, miR-196a increases neurite outgrowth through IGF2 in different HD models. Finally, higher expression of IMP3 and lower expression IGF2 are observed in HD transgenic mice and patients, and increase the formation of aggregates in the HD cell model. Taken together, miR-196a enhances polymerization of neuronal microfilaments through suppressing IMP3 and upregulating IGF2 in HD, supporting the neuroprotective functions of miR-196a through neuronal cytoskeleton in HD.

Leptospirosis, caused by pathogenic spirochetes of the Leptospira genus, is a common zoonosis in tropical and subtropical regions and can lead to an epidemic following heavy rainfall or flooding. The primary reservoirs of Leptospira include rodents, wild animals, dogs, cats, amphibians, and others, but the brown rat (Rattus norvegicus) remains the main source of human Leptospirosis. Humans are often accidental hosts and they can be infected through cuts, abrasions, mucosa, conjunctiva, or by ingesting contaminated water. The clinical manifestation of leptospirosis can vary from mild, nonspecific symptoms to a fatal outcome involving liver and renal failure, pulmonary hemorrhage, meningitis, and septic shock. The severity of fatal outcomes is likely to be due to virulence factors, host susceptibility, and epidemiological conditions. L. interrogans are associated with high-risk individuals, particularly patients older than 60 years of age in clinical settings. The current case study showed a foreign worker who presented with rapidly deteriorating clinical signs of fever, jaundice, impaired consciousness, and oliguric acute renal failure. Drawing from our experience, it is advisable to consider the possibility of leptospirosis diagnosis in patients who show clinical symptoms such as fever, hepatic failure with jaundice, and acute renal failure. This is particularly important for those individuals with a prior history of pathogen exposure. This case study had a strong suspicion of leptospirosis, which was confirmed by the microscopic agglutination test (MAT) and, later, the patient’s recovery following treatment.

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy, is a rare and drug-resistant form of developmental and epileptic encephalopathies, which is both debilitating and challenging to manage, typically arising during the first year of life, with seizures often triggered by fever, infections, or vaccinations. It is characterized by frequent and prolonged seizures, developmental delays, and various other neurological and behavioral impairments. Most cases result from pathogenic mutations in the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene, which encodes a critical voltage-gated sodium channel subunit involved in neuronal excitability. Precision medicine offers significant potential for improving DS diagnosis and treatment. Early genetic testing enables timely and accurate diagnosis. Advances in our understanding of DS’s underlying genetic mechanisms and neurobiology have enabled the development of targeted therapies, such as gene therapy, offering more effective and less invasive treatment options for patients with DS. Targeted and gene therapies provide hope for more effective and personalized treatments. However, research into novel approaches remains in its early stages, and their clinical application remains to be seen. This review addresses the current understanding of clinical DS features, genetic involvement in DS development, and outcomes of novel DS therapies.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC. Methods To evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft. Results An in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice. Conclusions Our data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.

Aims: Vascular calcification (VC) and osteoporosis were previously considered two distinct diseases. However, current understanding indicates that they share common pathogenetic mechanisms. The available medicines for treating VC and osteoporosis are limited. We previously demonstrated that kefir peptides (KPs) alleviated atherosclerosis in high-fat diet (HFD)-induced apolipoprotein E knockout (ApoE−/−) mice. The present study further addressed the preventive effects of KPs on VC and osteoporosis in ApoE−/− mice fed a high-cholesterol atherogenic diet (AD).Main methods: Seven-week-old ApoE−/− and wild-type C57BL/6 mice were randomly divided into five groups (n = 6). The development of VC and osteoporosis was evaluated after AD feeding for 13 weeks in KP-treated ApoE−/− mice and compared to C57BL/6 and ApoE−/− mice fed a standard chow diet (CD).Key findings: The results indicated that KP-treated ApoE−/− mice exhibited lower serum total cholesterol, oxidized low-density lipoprotein (ox-LDL), malondialdehyde (MDA) levels, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) activities, which suggested that KPs prevented hyperlipidemia and possible damages to the liver and muscle in ApoE−/− mice. KPs reduced serum tumor necrosis factor-α (TNF-α) and the local expression of TNF-α, IL-1β, and macrophage-specific CD68 markers in aortic tissues, which suggested that KPs inhibited inflammatory responses in AD-fed ApoE−/− mice. KPs reduced the deposition of lipid, collagen, and calcium minerals in the aortic roots of AD-fed ApoE−/− mice, which suggested that KPs inhibited the calcific progression of atherosclerotic plaques. KPs exerted osteoprotective effects in AD-fed ApoE−/− mice, which was evidenced by lower levels of the bone resorption marker CTX-1 and higher levels of the bone formation marker P1NP. KPs improved cortical bone mineral density and bone volume and reduced trabecular bone loss in femurs.Significance: The present data suggested that KPs attenuated VC and osteoporosis by reducing oxidative stress and inflammatory responses in AD-fed ApoE−/− mice. Our findings contribute to the application of KPs as preventive medicines for the treatment of hyperlipidemia-induced vascular and bone degeneration.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

A novel kefir exopolysaccharides (KEPS) derived from kefir grain fermentation were found to have a small molecular weight (12 kDa) compared to the traditionally high molecular weight (12,000 kDa) of kefiran (KE). KE has been shown to possess antioxidant, blood pressure-lowering, and immune-modulating effects. In this study, we characterized KEPS and KE and evaluated their anti-inflammatory properties in vitro using RAW264.7 macrophages. The main monosaccharide components were identified as glucose (98.1 ± 0.06%) in KEPS and galactose (45.36 ± 0.16%) and glucose (47.13 ± 0.06%) in KE, respectively. Both KEPS and KE significantly reduced IL-6 secretion in lipopolysaccharide (LPS)-stimulated macrophages. We further investigated their effects in LPS-induced systemic injury in male and female NF-κB-luciferase+/+ transgenic mice. Mice received oral KEPS (100 mg/kg) or KE (100 mg/kg) for seven days, followed by LPS or saline injection. KEPS and KE inhibited NF-κB signaling, as indicated by reduced luciferase expression and phosphorylated NF-κB levels. LPS-induced systemic injury increased luciferase signals, especially in the kidney, spleen, pancreas, lung, and gut tissues of female mice compared to male mice. Additionally, it upregulated inflammatory mediators in these organs. However, KEPS and KE effectively suppressed the expression of inflammatory mediators, including p-MAPK and IL-6. These findings demonstrate that KEPS can alleviate LPS-induced systemic damage by inhibiting NF-κB/MAPK signaling, suggesting their potential as a treatment for inflammatory disorders.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1β, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). μ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, β-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.

2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) is the main active ingredient extracted from Polygonum multiflorum Thunb. (PMT), which has been reported to possess extensive pharmacological properties. Nevertheless, the exact role of THSG in pulmonary fibrosis has not been demonstrated yet. The main purpose of this study was to investigate the protective effect of THSG against bleomycin (BLM)-induced lung fibrosis in a murine model, and explore the underlying mechanisms of THSG in transforming growth factor-beta 1 (TGF-β1)-induced fibrogenesis using MRC-5 human lung fibroblast cells. We found that THSG significantly attenuated lung injury by reducing fibrosis and extracellular matrix deposition. THSG treatment significantly downregulated the expression levels of TGF-β1, fibronectin, α-SMA, CTGF, and TGFBR2, however, upregulated the expression levels of antioxidants (SOD-1 and catalase) and LC3B in the lungs of BLM-treated mice. THSG treatment decreased the expression levels of fibronectin, α-SMA, and CTGF in TGF-β1-stimulated MRC-5 cells. Conversely, THSG increased the expression levels of SOD-1 and catalase. Furthermore, treatment of THSG profoundly reduced the TGF-β1-induced generation of reactive oxygen species (ROS). In addition, THSG restored TGF-β1-induced impaired autophagy, accompany by increasing the protein levels of LC3B-II and Beclin 1. Mechanism study indicated that THSG significantly reduced TGF-β1-induced increase of TGFBR2 expression and phosphorylation of Smad2/3, Akt, mTOR, and ERK1/2 in MRC-5 cells. These findings suggest that THSG may be considered as an anti-fibrotic drug for the treatment of pulmonary fibrosis.

E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.

Comorbidities are common in children with epilepsy, with nearly half of the patients having at least one comorbidity. Attention deficit hyperactivity disorder (ADHD) is a psychiatric disorder characterized by hyperactivity and inattentiveness level disproportional to the child’s developmental stage. The burden of ADHD in children with epilepsy is high and can adversely affect the patients’ clinical outcomes, psychosocial aspects, and quality of life. Several hypotheses were proposed to explain the high burden of ADHD in childhood epilepsy; the well-established bidirectional connection and shared genetic/non-genetic factors between epilepsy and comorbid ADHD largely rule out the possibility of a chance in this association. Stimulants are effective in children with comorbid ADHD, and the current body of evidence supports their safety within the approved dose. Nonetheless, safety data should be further studied in randomized, double-blinded, placebo-controlled trials. Comorbid ADHD is still under-recognized in clinical practice. Early identification and management of comorbid ADHD are crucial to optimize the prognosis and reduce the risk of adverse long-term neurodevelopmental outcomes. The identification of the shared genetic background of epilepsy and ADHD can open the gate for tailoring treatment options for these patients through precision medicine.

Abstract Increased heart rate (HR) is a reaction to head-to-toe gravito-inertial (G) force. The anti-G straining manoeuvre (AGSM) is the crucial technique for withstanding a high-G load. Previous studies reported the main effects of HR only or AGSM only on G tolerance. We assessed the combined effect of HR and AGSM on the outcome of 9G profile exposure. A total of 530 attempts for the 9G profile were extracted to clarify the association of interest. Subjects with an AGSM effectiveness of less than 2.5G had a 2.14-fold higher likelihood of failing in the 9G profile. Trainees with HR increases of less than 20% in the first five seconds also had higher odds of 9G profile intolerance (adjusted OR 1.83, 95% CI 1.09–3.07). The adjusted OR of 9G profile disqualification was 2.93 (95% CI 1.19–7.20) for participants with smaller HR increases and lower AGSM effectiveness. The negative effect of a smaller HR increase on the outcome was likely to be affected by improved AGSM effectiveness (adjusted OR 1.26, 95% CI 0.65–2.42). We speculate that low AGSM effectiveness and a small HR increase were separately associated with failure of high-G challenge. Nonetheless, good AGSM performance seemed to reduce the negative effect of weak HR responses on the dependent variable.

Dinosaurs had some of the most complex dentitions known. Here, Reisz et al. characterize dental development across embryonic, hatchling and adult Lufengosaurus, an Early Jurassic sauropodomorph dinosaur, and suggest that derived sauropod dentition evolved by paedomorphosis (juvenilization).

Osteoporosis is a clinically prevalent comorbidity in patients with hemophilia. A preventive effect of kefir peptides (KPs) on postmenopausal osteoporosis has been proved. The aim of this study was to evaluate the therapeutic effect of KPs for the treatment of osteoporosis in coagulation factor VIII (FVIII) gene knockout mice (F8KO), a model of hemophilia A. In this study, male F8KO mice at 20 weeks of age were orally administered different doses of KPs for 8 weeks. The therapeutic effects of KPs were shown in the femoral trabeculae and the 4th lumbar vertebrae, which increased the trabecular bone mineral density (BMD), bone volume (Tb.BV/TV), and trabecular number (Tb.N) and decreased the trabecular separation (Tb.Sp), and they were also observed in the femoral cortical bones, in which the mechanical properties were enhanced in a dose-dependent manner. Characterization of receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and interleukin 6 (IL-6) demonstrated that the serum RANKL/OPG ratio and IL-6 levels were significantly decreased in the F8KO mice after the KP treatment. Tartrate-resistant acid phosphatase (TRAP) staining of mature osteoclasts indicated that the therapeutic effect of KPs in F8KO mice was associated with the functions of KPs to inhibit RANKL-induced osteoclastogenesis by reducing serum RANKL/OPG ratio and IL-6 secretion. The present study is the first to address the potentials of KPs for the treatment of hemophilia-induced osteoporosis in mice and it also provides useful information for the application of KPs as a complementary therapy for the treatment of osteoporosis in hemophilic patients.

Acute respiratory distress syndrome (ARDS) is a common destructive syndrome with high morbidity and mortality rates. Currently, few effective therapeutic interventions for ARDS are available. Clinical trials have shown that the effectiveness of aspirin is inconsistent. The contribution of platelets to the inflammatory response leading to the development of ARDS is increasingly recognized. The antiplatelet agent aspirin reportedly exerts a protective effect on acid- and hyperoxia-induced lung injury in murine models. Our previous study showed that pretreatment with aspirin exerts protective effects on hyperoxia-induced lung injury in mice. However, the mechanisms and therapeutic efficacy of aspirin in the posttreatment of hyperoxia-induced acute lung injury (ALI) remain unclear. In this study, we used a homozygous NF-κB-luciferase+/+ transgenic mouse model and treated mice with low-dose (25 μg/g) or high-dose (50 μg/g) aspirin at 0, 24, and 48 h after exposure to hyperoxia (inspired oxygen fraction (FiO2) > 95%). Hyperoxia-induced lung injury significantly increased the activation of NF-κB in the lung and increased the levels of macrophages infiltrating the lung and reactive oxygen species (ROS), increased the HO-1, NF-κB, TNF-α, IL-1β, and IL-4 protein levels, and reduced the CC10, SPC, eNOS, Nrp-1, and IκBα protein levels in the lung tissue. Pulmonary edema and alveolar infiltration of neutrophils were also observed in the lung tissue of mice exposed to hyperoxia. However, in vivo imaging revealed that posttreatment with aspirin reduced luciferase expression, suggesting that aspirin might reduce NF-κB activation. Posttreatment with aspirin also reduced hyperoxia-induced increases in the numbers of lung macrophages, intracellular ROS levels, and the expression of TNF-α, IL-1β, and IL-4; it also increased CC10, SPC and Nrp-1 levels compared with hyperoxia exposure alone. Lung histopathology also indicated that the aspirin posttreatment significantly reduced neutrophil infiltration and lung edema compared with hyperoxia exposure alone. Aspirin effectively induces an anti-inflammatory response in a model of hyperoxia-induced lung injury. Thus, aspirin may have potential as a novel treatment for hyperoxia-induced ALI.

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.

E7050 is a potent inhibitor of c-Met receptor tyrosine kinase and has potential for cancer therapy. However, the underlying molecular mechanism involved in the anti-cancer property of E7050 has not been fully elucidated. The main objective of this study was to investigate the anti-tumor activity of E7050 in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells in vitro and in vivo, and to define its mechanisms. Our results revealed that E7050 reduced cell viability of MES-SA/Dx5 cells, which was associated with the induction of apoptosis and S phase cell cycle arrest. Additionally, E7050 treatment significantly upregulated the expression of Bax, cleaved PARP, cleaved caspase-3, p21, p53 and cyclin D1, while it downregulated the expression of survivin and cyclin A. On the other hand, the mechanistic study demonstrated that E7050 inhibited the phosphorylation of c-Met, Src, Akt and p38 in HGF-stimulated MES-SA/Dx5 cells. Further in vivo experiments showed that treatment of athymic nude mice carrying MES-SA/Dx5 xenograft tumors with E7050 remarkably suppressed tumor growth. E7050 treatment also decreased the expression of Ki-67 and p-Met, and increased the expression of cleaved caspase-3 in MES-SA/Dx5 tumor sections. Therefore, E7050 is a promising drug that can be developed for the treatment of multidrug-resistant uterine sarcoma.

Asthma is a chronic respiratory disease with symptoms such as expiratory airflow narrowing and airway hyperresponsiveness (AHR). Millions of people suffer from asthma and are at risk of life-threatening conditions. Lactoferrin (LF) is a glycoprotein with multiple physiological functions, including antioxidant, anti-inflammatory, antimicrobial, and antitumoral activities. LF has been shown to function in immunoregulatory activities in ovalbumin (OVA)-induced delayed type hypersensitivity (DTH) in mice. Hence, the purpose of this study was to investigate the roles of LF in AHR and the functions of dendritic cells (DCs) and Th2-related responses in asthma. Twenty 8-week-old male BALB/c mice were divided into normal control (NC), ovalbumin (OVA)-sensitized, and OVA-sensitized with low dose of LF (100 mg/kg) or high dose of LF (300 mg/kg) treatment groups. The mice were challenged by intranasal instillation with 5% OVA on the 21st to 27th day after the start of the sensitization period. The AHR, cytokines in bronchoalveolar lavage fluid, and pulmonary histology of each mouse were measured. Serum OVA-specific IgE and IgG1 and OVA-specific splenocyte responses were further detected. The results showed that LF exhibited protective effects in ameliorating AHR, as well as lung inflammation and damage, in reducing the expression of Th2 cytokines and the secretion of allergen-specific antibodies, in influencing the functions of DCs, and in decreasing the level of Th2 immune responses in a BALB/c mouse model of OVA-induced allergic asthma. Importantly, we demonstrated that LF has practical application in reducing DC-induced Th2 cell responses in asthma. In conclusion, LF exhibits anti-inflammation and immunoregulation activities in OVA-induced allergic asthma. These results suggest that LF may act as a supplement to prevent asthma-induced lung injury and provide an additional agent for reducing asthma severity.

Lung cancer is heterogeneous and challenging to cope with once it has progressed. Chemotherapy is the first step once no active driver mutation has been discovered. Non-antitumor drugs have been found to be beneficial when used as adjuvants to chemotherapy. In this study, the additive effect and mechanism of metformin combined with pemetrexed in non-small-cell lung cancer (NSCLC) cells were elucidated. Three NSCLC cell lines, A549, H1975, and HCC827, were used to analyze tumor cell proliferation, colony formation and the cell cycle in vitro when exposed to metformin alone, pemetrexed alone or their combination. We found that combination treatment in three cell lines exerted antiproliferative effects through cell cycle arrest in the S phase. An ex vivo chicken chorioallantoic membrane (CAM) assay was used to examine the antiangiogenic effect of metformin combined with pemetrexed on vascular structure formation. We further created an A549 orthotopic xenograft model with an in vivo imaging system (IVIS) and explored the associated indicators involved in the tumorigenic process. The in vitro results showed that the combination of metformin and pemetrexed exhibited an antiproliferative effect in reducing cell viability and colony formation, the downregulation of cyclin D1 and A2 and the upregulation of CDKN1B, which are involved in the G1/S phase. For antiangiogenic effects, the combination therapy inhibited the vascular structure, as proven by the CAM assay. We elucidated that combination therapy could target VEGFA and Endoglin by RT-qPCR, ELISA and histopathological findings in an A549 orthotopic NSCLC xenograft model. Our research demonstrated the additive antiproliferative and antiangiogenic effects of the combination of metformin with pemetrexed in NSCLC and could be applied to clinical lung cancer therapy.

Abstract Background Metformin is proven to improve the prognosis of various cancers, but it is unknown if metformin could ameliorate hypopharyngeal cancer in diabetes mellitus patients. This was a retrospective cohort study, and the effect and survival outcome of metformin on hypopharyngeal cancer with diabetes mellitus was investigated. Methods There were 141 hypopharyngeal cancer patients collected in a tertiary referral center from December 1st, 2011 to December 31st, 2013. There were 49 patients without diabetes mellitus (DM) and 92 patients with DM. In the 92 DM patients, there were 43 patients with metformin used and 49 patients without metformin used. All received patients followed up until September 1st, 2015. Results There was no significant difference in patients’ characteristics between the non-DM and DM groups, and also no significant difference in clinical T stage, N stage, metastatic condition, and disease stage between the non-DM and DM groups. DM with metformin patients had lower metastasis rates and better overall survival (OS) (p = 0.011) and disease-free survival (DFS) (p = 0.004) compared to non-DM and DM without metformin. Multivariate analysis also showed a better OS and DFS in DM-Met (+) with advanced hypopharyngeal cancer but not in early stage. Conclusion There was less distant metastasis and better survival outcomes in hypopharyngeal cancer DM patients who use metformin.

Abstract Background Autologous platelet concentrates are currently widely used across different areas of regenerative medicine in order to enhance the wound healing process. Although several protocols for platelet concentrates are available, their application remains difficult due to different protocols leading to distinct products with vary potential biological uses. In this study, we attempted to make a platelet patch (PP) using mixtures of platelet rich plasma (PRP) injection and platelet rich fibrin (PRF) to promote wound repair and regeneration. Results Experiments were performed using a full-thickness wound model in mini-pigs. Autologous PRP, PRF and PP were prepared immediately before creating four full-thickness skin wounds in pigs. We quantified concentrations of platelets, thrombin and various growth factors to ensure that the desired effect can be produced. After surgery, hydrocolloid dressing, PRP injection, PRF and PP was applied to experimentally induced wounds. Application efficacy was evaluated by measurement of wound sizes and histological examination. The results indicated that all wounds showed a significant size reduction. Wound repair efficacy in response to PP treatment exhibited enhanced re-epithelialization compared to PRP and PRF (P < 0.05) and higher wound contraction than did PRF application (P < 0.05). Another aspect, experiment using DsRed transgenic pigs as blood donors demonstrated that leucocytes in PP were incorporated into the wound bed at the end of the study, suggesting that leucocytes activity is stimulated in response to PP application. Safety of the experimental processes was also confirmed by examination of organ biopsies. Conclusions We used a mini-pig model to evaluate the efficacy of lab-made PP on induced full-thickness wound healing. Results demonstrated that application of one piece of PP was enough to obtain comparable efficacy versus general utilization of PRP or PRF for wound care. We also demonstrated that leucocytes in PP were incorporated into the wound bed and no safety concerns have been found in the whole experiment. This study provides a novel and feasible method for veterinary or clinical wound care.

Abstract Introduction Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs). Methods Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration. Results An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups. Conclusion Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.

Abstract Objective Influenza is an acute respiratory disease caused by the influenza virus which circulates annually in populations of different species. Madin-Darby Canine Kidney (MDCK) is the most widely utilized cell-line for conducting influenza research. However, the infectivity of various influenza strains in MDCK cells is not equivalent and the productivity of viral propagation is also limited. Results We tested the functional adequacy of two MDCK-lineage cell lines, conventional MDCK and MDCK/London, were evaluated by assessing their infectivity of different influenza viral strains with focus forming assays and the cellular toxicity caused by influenza infections by lactate dehydrogenase assay. Moreover, the sensitivity of cells in the presence of the antiviral agent ribavirin was assessed by MTT assay. Our results showed that MDCK/London cells efficiently propagate virus across all influenza viruses tested, are comparable to the utility of Mv1Lu cells, and are superior to conventional MDCK cells in replicating virus as indicated by an increase in virus of three to four logs, particularly in H3N2 infection. Also, the MDCK/London cells were more sensitive to the presence of antiviral drug than conventional MDCK cells. In conclusion, MDCK/London cell line could be a better platform for influenza studies and vaccine development.

Background: Acute traumatic wounds are a common and distressing event for patients presenting to plastic surgery clinics. There are no definitive conclusions regarding the benefits of collagen-based wound care products toward the application on full-thickness wounds compared to traditional ointment or gauze care. In this clinical trial, we aimed to define the potential benefits of collagen-based dressings on healing through the comparison of collagen-based composite dressings with antibiotic ointment dressings. Methods: This was a randomized two-tailed controlled clinical trial. The acute full-thickness wounds underwent wound cleansing, debridement, and were secured with different dressings in both experimental and control groups. The wounds were subsequently photographed weekly, and the wound areas were calculated using ImageJ software to evaluate wound healing rate. Results: The results demonstrate that the collagen-based triple layer composite dressings which consisted of silver sheet, collagen sponge, and foam films promoted better epithelialization and healing than antibiotic ointment treatment. Collagen supplementation improved wound healing and epithelialization by providing a dry environment during the late stage of wound healing. Conclusion: We demonstrated that the collagen-based triple layer wound dressing could potentially increase the healing rate when used for traumatic full-thickness wound care compared to the conventional antibiotic ointment dressing, providing a new prospective in the future applications of wound care.

End-stage renal disease (ESRD) patients experience oxidative stress due to excess exogenous or endogenous oxidants and insufficient antioxidants. Hence, oxidative stress and inflammation cause endothelial damage, contributing to vascular dysfunction and atherosclerosis. Therefore, ESRD patients suffer more cardiovascular and hospitalization events than healthy people. This study aims to test the correlations between ROS, SOD3, IL-2, IL-6, and IL-18 and the first kidney disease-related hospitalization or death events in ESRD patients undergoing regular hemodialysis. A total of 212 participants was enrolled, including 45 normal healthy adults and 167 ESRD patients on regular dialysis. Blood samples from all participants were collected for ROS, SOD3, IL-2, IL-6, and IL-18 measurement at the beginning of the study, and every kidney disease-related admission or death was recorded for the next year. Multivariate analysis was conducted by fitting a linear regression model, logistic regression model, and Cox proportional hazards model to estimate the adjusted effects of risk factors, prognostic factors, or predictors on continuous, binary, and survival outcome data. The results showed that plasma SOD3 and serum IL-18 were two strong predictors of the first kidney disease-related hospitalization or death. In the Cox proportional hazards models (run in R), higher IL-18 concentration (>69.054 pg/mL) was associated with a hazard ratio of 3.376 for the first kidney disease-related hospitalization or death (95% CI: 1.2644 to 9.012), while log(SOD3) < 4.723 and dialysis clearance (Kt/V; 1.11 < value < 1.869) had a hazard ratio = 0.2730 (95% CI: 0.1133 to 0.6576) for reducing future kidney disease-related hospitalization or death. Other markers, including body mass index (BMI), transferrin saturation, total iron binding capacity, and sodium and alkaline phosphate, were also found to be significant in our study. These results reveal the new predictors SOD3 and IL-18 for the medical care of end-stage renal disease patients.

Air pollution triggers a tissue-specific inflammatory response. However, studies on the association between exposure to air pollutants and chronic rhinosinusitis (CRS) risk remain limited. Thus, we conducted this nationwide study to define the association between air pollution and CRS. We used the Longitudinal Health Insurance Database (LHID) and Taiwan Air Quality-Monitoring Database (TAQMD) to conduct a population-based cohort study. Study participants were recruited from the LHID, a data subset of the National Health Insurance Research Database that randomly sampled one million individuals. TAQMD has been an air pollutant database since 1998. In univariate and multivariate Cox proportional hazards regression models, adjusted hazard ratios (aHRs) and 95% CIs of CRS in five air pollutants were accounted. We adjusted for age, sex, urbanization level, insurance fee, comorbidities, and pollutant levels in the multivariate model. The total number of participants enrolled in this study was 160,504. The average age was 40.46 ± 14.62 years; males constituted 43.8% of the total participants. The percentages of alcoholism, tobacco dependence, and COPD were 1.5%, 2.8%, and 28.3%, respectively. After adjustment for age, sex, urbanization level, insurance fee, and comorbidities, the highest levels of air pollutants, including PM2.5 (aHR = 1.14, 95% CI = 1.06–1.22), NO2 (aHR = 1.07, 95% CI = 1.00–1.15), and PM10 (aHR = 1.13, 95% CI = 1.05–1.21) had a significantly greater CRS risk; we selected the lower concentration as the reference but did not correlate with CO. We found a significantly increased risk of CRS in residents with air pollutant exposure.

Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery.

Abstract Background Allergen-specific immunotherapy (ASIT) has the potential to modify allergic diseases, and it is also considered a potential therapy for allergic asthma. House dust mite (HDM) allergens, a common source of airborne allergen in human diseases, have been developed as an immunotherapy for patients with allergic asthma via the subcutaneous and sublingual routes. Oral immunotherapy with repeated allergen ingestion is emerging as another potential modality of ASIT. The aim of this study was to evaluate the therapeutic efficacy of the oral ingestion of HDM extracts in a murine model of allergic asthma. Methods BABL/c mice were sensitized twice by intraperitoneal injection of HDM extracts and Al(OH)3 on day 1 and day 8. Then, the mice received challenge to induce airway inflammation by intratracheal instillation of HDM extracts on days 29–31. The treatment group received immunotherapy with oral HDM extracts ingestion before the challenge. All the mice were sacrificed on day 32 for bronchoalveolar inflammatory cytokines, mediastinal lymph node T cells, lung histology, and serum HDM-specific immunoglobulins analyses. Results Upon HDM sensitization and following challenge, a robust Th2 cell response and eosinophilic airway inflammation were observed in mice of the positive control group. The mice treated with HDM extracts ingestion had decreased eosinophilic airway inflammation, suppressed HDM-specific Th2 cell responses in the mediastinal lymph nodes, and attenuated serum HDM-specific IgE levels. Conclusions Oral immunotherapy with HDM extracts ingestion was demonstrated to have a partial therapeutic effect in the murine model of allergic asthma. This study may serve as the basis for the further development of oral immunotherapy with HDM extracts in allergic asthma.

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non‐small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke‐derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine‐derived nitrosamine ketone (NNK), reduced the sensitivity of wild‐type EGFR‐expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P‐treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c‐MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c‐MET reduces B[α]P‐induced Akt activation. The CSE‐treated NSCLC cells are sensitive to the c‐MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c‐MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history.

Telomeres cap the ends of eukaryotic chromosomes and are indispensable chromatin structures for genome protection and replication. Telomere length maintenance has been attributed to several functional modulators, including telomerase, the shelterin complex, and the CST complex, synergizing with DNA replication, repair, and the RNA metabolism pathway components. As dysfunctional telomere maintenance and telomerase activation are associated with several human diseases, including cancer, the molecular mechanisms behind telomere length regulation and protection need particular emphasis. Cancer cells exhibit telomerase activation, enabling replicative immortality. Telomerase reverse transcriptase (TERT) activation is involved in cancer development through diverse activities other than mediating telomere elongation. This review describes the telomere functions, the role of functional modulators, the implications in cancer development, and the future therapeutic opportunities.

Whether meconium-stained amniotic fluid (MSAF) serves as an indicator of fetal distress is under debate; however, the presence of MSAF concerns both obstetricians and pediatricians because meconium aspiration is a major contributor to neonatal morbidity and mortality, even with appropriate treatment. The present study suggested that thick meconium in infants might be associated with poor outcomes compared with thin meconium based on chart reviews. In addition, cell survival assays following the incubation of various meconium concentrations with monolayers of human epithelial and embryonic lung fibroblast cell lines were consistent with the results obtained from chart reviews. Exposure to meconium resulted in the significant release of nitrite from A549 and HEL299 cells. Medicinal agents, including dexamethasone, L-Nω-nitro-arginine methylester (L-NAME), and NS-398 significantly reduced the meconium-induced release of nitrite. These results support the hypothesis that thick meconium is a risk factor for neonates who require resuscitation, and inflammation appears to serve as the primary mechanism for meconium-associated lung injury. A better understanding of the relationship between nitrite and inflammation could result in the development of promising treatments for meconium aspiration syndrome (MAS).

Background and Objectives: In the intensive care unit (ICU), renal failure and respiratory failure are two of the most common organ failures in patients with systemic inflammatory response syndrome (SIRS). These clinical symptoms usually result from sepsis, trauma, hypermetabolism or shock. If this syndrome is caused by septic shock, the Surviving Sepsis Campaign Bundle suggests that vasopressin be given to maintain mean arterial pressure (MAP) > 65 mmHg if the patient is hypotensive after fluid resuscitation. Nevertheless, it is important to note that some studies found an effect of various mean arterial pressures on organ function; for example, a MAP of less than 75 mmHg was associated with the risk of acute kidney injury (AKI). However, no published study has evaluated the risk factors of mortality in the subgroup of acute kidney injury with respiratory failure, and little is known of the impact of general risk factors that may increase the mortality rate. Materials and Methods: The objective of this study was to determine the risk factors that might directly affect survival in critically ill patients with multiple organ failure in this subgroup. We retrospectively constructed a cohort study of patients who were admitted to the ICUs, including medical, surgical, and neurological, over 24 months (2015.1 to 2016.12) at Chiayi Chang Gung Memorial Hospital. We only considered patients who met the criteria of acute renal injury according to the Acute Kidney Injury Network (AKIN) and were undergoing mechanical ventilator support due to acute respiratory failure at admission. Results: Data showed that the overall ICU and hospital mortality rate was 63.5%. The most common cause of ICU admission in this cohort study was cardiovascular disease (31.7%) followed by respiratory disease (28.6%). Most patients (73%) suffered sepsis during their ICU admission and the mean length of hospital stay was 24.32 ± 25.73 days. In general, the factors independently associated with in-hospital mortality were lactate > 51.8 mg/dL, MAP ≤ 77.16 mmHg, and pH ≤ 7.22. The risk of in-patient mortality was analyzed using a multivariable Cox regression survival model. Adjusting for other covariates, MAP ≤ 77.16 mmHg was associated with higher probability of in-hospital death [OR = 3.06 (1.374–6.853), p = 0.006]. The other independent outcome predictor of mortality was pH ≤ 7.22 [OR = 2.40 (1.122–5.147), p = 0.024]. Kaplan-Meier survival curves were calculated and the log rank statistic was highly significant. Conclusions: Acute kidney injury combined with respiratory failure is associated with high mortality. High mean arterial pressure and normal blood pH might improve these outcomes. Therefore, the acid–base status and MAP should be considered when attempting to predict outcome. Moreover, the blood pressure targets for acute kidney injury in critical care should not be similar to those recommended for the general population and might prevent mortality.

Lung cancer remains a challenge in daily practice. Chemotherapy is first considered for advanced lung adenocarcinoma bearing no active driver mutations. Maintaining drug efficacy and overcoming drug resistance are essential. This study aimed to explore the real-world use of anti-diabetic agent metformin in combination with pemetrexed-based platinum doublets in a first-line setting. We retrospectively collected data during 2004~2013 from TaiwaN′s National Health Insurance Research Database to access the survival benefit of metformin combined with pemetrexed-based platinum doublets as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. Demographic data and information regarding platinum reagents, diabetes medications, and metformin doses were gathered, and overall survival status regarding metformin use was analyzed. Overall survival status based on the daily dose and the calculated cumulative defined daily dose (DDD) of metformin prescribed during the first 3 months after lung cancer was diagnosed was also assessed. A total of 495 patients were enrolled with a mean age of 67 years old, and the majority of the patients were male. After adjusting for age, sex, diabetes medication, and platinum reagents used, the adjusted hazard ratio (HR) for the metformin-user group was 0.61 (95% confidence interval (CI); 0.46~0.79; p < 0.001). The metformin-user group had a survival benefit (log-rank p < 0.001). We analyzed metformin dosing during the first 3 months after lung cancer diagnosis, and for a daily dose ≥ 1500 mg, the adjusted hazard ratio (aHR) was 0.42 (95% CI; 0.27~0.65; p < 0.001). Regarding the cumulative DDD of metformin, a DDD equal to or exceeding 21 resulted in aHR of 0.48 (95% CI; 0.34~0.69; p < 0.001). In this study, we found that the combination of metformin and pemetrexed-based platinum doublets provides a robust survival benefit as a first-line therapy for diabetic patients with advanced lung adenocarcinoma. It is worth conducting a large and randomized clinical trial to further investigate the antitumor effects of metformin on advanced lung adenocarcinoma when used as a first-ling therapy, including in non-diabetic patients.

Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat FVIII knockout (FVIII KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-Fe3O4) to carry the B-domain-deleted (BDD)-FVIII plasmid, which was delivered into the FVIII KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD-FVIII plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-Fe3O4 nanoparticles. The FVIII KO mice that received the DPPC-Fe3O4 plasmid complex were assessed by staining the ferric ion of DPPC-Fe3O4 nanoparticles with Prussian blue in liver tissue. The bleeding of the FVIII KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in FVIII KO mice and might be a promising approach to treating hemophilia A patients in clinical settings.

The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow's colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals either deceased or euthanized were necropsied and intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.

Hypopharyngeal squamous cell carcinoma (HSCC) is usually diagnosed at an advanced stage, leading to a poor prognosis. Even after improvement of surgical techniques, chemotherapy, and radiation technology, the survival rate of HSCC remains poor. Metformin, which is commonly used for type 2 diabetes mellitus (DM), has been suggested to reduce the risk of various cancer types. However, only a few clinical studies mentioned the relationship between metformin use and HSCC. Hence, the aim of this study was to elucidate the specific effect and mechanism of action of metformin in hypopharyngeal cancer. We first assessed whether metformin use has an effect on hypopharyngeal cancer patients with DM by conducting a retrospective cohort study. Our results showed that DM hypopharyngeal cancer patients who used metformin exhibited significantly better overall survival rates than that without metformin treatment. The cell-based analysis further indicated that metformin treatment regulated p38/JNK pathway to reduce Cyclin D1 and Bcl-2 expressions. In addition, metformin activated the pathways of AMPKα and MEK/ERK to phosphorylate p27(Thr198) and reduce mTOR phosphorylation in cells. These actions direct cells toward G1 cell cycle arrest, apoptosis, and autophagy. Our results, through combining a clinical cohort analysis with an in vitro study, demonstrate that metformin can be used for drug repositioning in the treatment of DM patients with hypopharyngeal cancer.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Osteoporosis is a major skeletal disease associated with estrogen deficiency in postmenopausal women. Kefir-fermented peptides (KPs) are bioactive peptides with health-promoting benefits that are produced from the degradation of dairy milk proteins by the probiotic microflora in kefir grains. This study aimed to evaluate the effects of KPs on osteoporosis prevention and the modulation of the composition of the gut microbiota in ovariectomized (OVX) mice. OVX mice receiving an 8-week oral gavage of 100 mg of KPs and 100 mg of KPs + 10 mg Ca exhibited lower trabecular separation (Tb. Sp), and higher bone mineral density (BMD), trabecular number (Tb. N) and bone volume (BV/TV), than OVX groups receiving Ca alone and untreated mice, and these effects were also reflected in bones with better mechanical properties of strength and fracture toughness. The gut microbiota of the cecal contents was examined by 16S rDNA amplicon sequencing. α-Diversity analysis indicated that the gut microbiota of OVX mice was enriched more than that of sham mice, but the diversity was not changed significantly. Treatment with KPs caused increased microbiota richness and diversity in OVX mice compared with those in sham mice. The microbiota composition changed markedly in OVX mice compared with that in sham mice. Following the oral administration of KPs for 8 weeks, the abundances of Alloprevotella, Anaerostipes, Parasutterella, Romboutsia, Ruminococcus_1 and Streptococcus genera were restored to levels close to those in the sham group. However, the correlation of these bacterial populations with bone metabolism needs further investigation. Taken together, KPs prevent menopausal osteoporosis and mildly modulate the structure of the gut microbiota in OVX mice.

The increased prevalence of renal dysfunction and chronic kidney disease (CKD) and the high costs and poor outcomes of treatment are a significant health issue. The consequence of chronic high blood pressure is the increased prevalence of target organ end-stage renal disease, which has been proven to be a strong independent risk factor for adverse cardiovascular disease. A previous study showed that kefir products have anti-inflammatory and anti-hypertensive activities and immunological modulation functions. However, no data regarding the beneficial effects of kefir peptides (KPs) on salt-induced renal damage or related kidney diseases are available. In this study, KPs were orally administered to aged salt-induced stroke-prone spontaneously hypertensive (SHRSP) rats, and the effects of KPs against inflammation and oxidative stress and their ability to protect against renal dysfunction were evaluated. Fifty-five-week-old SHRSP rats under induction with 1% NaCl in drinking water for 4 weeks showed multiple renal injuries with increased renal inflammation, fibrosis, oxidative stress, tubular atrophy, and glomerulosclerosis. In contrast, oral gavage with KPs reduced the urine protein to creatinine (UPC) ratio, the fractional excretion of electrolytes (FeNa and FeCl), extracellular matrix deposition, and the interstitial fibrotic α-smooth muscle actin (α-SMA) levels in salt-induced SHRSP rats. The renal infiltration of inflammatory cells; the release of monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), and the cytokine nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) and transforming growth factor-β (TGF-β); the reactive oxygen species (ROS) levels; and histopathological lesions were also decreased in salt-induced SHRSP rats. Furthermore, KP treatment significantly increased the renal superoxide dismutase (SOD) activity and the glomerular filtration rate (GFR), which exerted potent protection against salt-induced chronic kidney disease in SHRSP rats. The results of this study suggest that KPs ameliorate salt-induced renal damage, tubular atrophy, and glomerular dysfunction through anti-inflammatory, antioxidative stress, and antifibrotic activities, and might be a promising protective agent against high salt-induced renovascular-related diseases.

Acute respiratory distress syndrome (ARDS) is a common and devastating syndrome that contributes to serious morbidities and mortality in critically ill patients. No known pharmacologic therapy is beneficial in the treatment of ARDS, and the only effective management is through a protective lung strategy. Platelets play a crucial role in the pathogenesis of ARDS, and antiplatelet therapy may be a potential medication for ARDS. In this review, we introduce the overall pathogenesis of ARDS, and then focus on platelet-related mechanisms underlying the development of ARDS, including platelet adhesion to the injured vessel wall, platelet-leukocyte-endothelium interactions, platelet-related lipid mediators, and neutrophil extracellular traps. We further summarize antiplatelet therapy, including aspirin, glycoprotein IIb/IIIa receptor antagonists, and P2Y12 inhibitors for ARDS in experimental and clinical studies and a meta-analysis. Novel aspirin-derived agents, aspirin-triggered lipoxin, and aspirin-triggered resolvin D1 are also described here. In this narrative review, we summarize the current knowledge of the role of platelets in the pathogenesis of ARDS, and the potential benefits of antiplatelet therapy for the prevention and treatment of ARDS.

Acute lung injury (ALI), a common cause of morbidity and mortality in intensive care units, results from either direct intra-alveolar injury or indirect injury following systemic inflammation and oxidative stress. Adequate tissue oxygenation often requires additional supplemental oxygen. However, hyperoxia causes lung injury and pathological changes. Notably, preclinical data suggest that aspirin modulates numerous platelet-mediated processes involved in ALI development and resolution. Our previous study suggested that prehospital aspirin use reduced the risk of ALI in critically ill patients. This research uses an in vivo imaging system (IVIS) to investigate the mechanisms of aspirin’s anti-inflammatory and antioxidant effects on hyperoxia-induced ALI in nuclear factor κB (NF-κB)–luciferase transgenic mice. To define mechanisms through which NF-κB causes disease, we developed transgenic mice that express luciferase under the control of NF-κB, enabling real-time in vivo imaging of NF-κB activity in intact animals. An NF-κB-dependent bioluminescent signal was used in transgenic mice carrying the luciferase genes to monitor the anti-inflammatory effects of aspirin. These results demonstrated that pretreatment with aspirin reduced luciferase expression, indicating that aspirin reduces NF-κB activation. In addition, aspirin reduced reactive oxygen species expression, the number of macrophages, neutrophil infiltration and lung edema compared with treatment with only hyperoxia treatment. In addition, we demonstrated that pretreatment with aspirin significantly reduced the protein levels of phosphorylated protein kinase B, NF-κB and tumor necrosis factor α in NF-κB–luciferase+/+ transgenic mice. Thus, the effects of aspirin on the anti-inflammatory response and reactive oxygen species suppressive are hypothesized to occur through the NF-κB signaling pathway. This study demonstrated that aspirin exerts a protective effect for hyperoxia-induced lung injury and thus is currently the drug conventionally used for hyperoxia-induced lung injury.

The collection of exhaled breath condensate (EBC) is a noninvasive method that can be used to monitor the inflammatory status of patients with chronic airway diseases. We aimed to study differences in cytokine expression between patients with exacerbations of chronic obstructive pulmonary disease (COPD) and patients with asthma attacks. Methods: Using a custom-made device and methods based on American Thoracic Society (ATS)/European Respiratory Society (ERS) recommendations, EBC samples were collected from nine COPD patients, 12 asthma patients and 10 healthy individuals. Cytokine concentrations in serum and EBC were measured via commercial ELISA kits. Results: Of four cytokines measured in EBC [interleukin-8 (IL-8), IL-17, IL-4 and tumor necrosis factor-α (TNF-α)], only IL-8 was significantly higher in COPD than in asthma patients (5.27 ± 0.18 vs. 4.36 ± 0.34 pg/mL, p = 0.001). Moreover, COPD patients had higher serum IL-8 than asthma patients (10.57 ± 0.55 vs. 5.15 ± 0.24 pg/mL, p