Narirutin-Rich Celluclast Extract from Mandarin (Citrus unshiu) Peel Alleviates High-Fat Diet-Induced Obesity and Promotes Energy Metabolism in C57BL/6 Mice.

Mandarin peel, a main by-product from the processing of citrus juice, has been highlighted for its various bioactivities and functional ingredients. Our previous study proved the inhibitory effects of Celluclast extract from mandarin peel (MPCE) on lipid accumulation and differentiation in 3T3-L1 adipocytes. Therefore, the current study aimed to evaluate the anti-obesity effect of MPCE in high-fat diet (HFD)-induced obese mice. The high-performance liquid chromatography (HPLC) analysis exhibited that narirutin and hesperidin are the main active components of MPCE. Our current results showed that MPCE supplementation decreased adiposity by reducing body and organ weights in HFD-induced obese mice. MPCE also reduced triglyceride (TG), alanine transaminase (ALT), aspartate transaminase (AST), and leptin contents in the serum of HFD-fed mice. Moreover, MPCE significantly inhibited hepatic lipid accumulation by regulating the expression levels of proteins associated with lipid metabolism, including sterol regulatory element-binding protein (SREBP1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Furthermore, MPCE administration significantly inhibited both adipogenesis and lipogenesis, with modulation of energy metabolism by activating 5' adenosine monophosphate-activated protein kinase (AMPK) and lipolytic enzymes such as hormone-sensitive lipase (HSL) in the white adipose tissue (WAT). Altogether, our findings indicate that MPCE improves HFD-induced obesity and can be used as a curative agent in pharmaceuticals and nutraceuticals to alleviate obesity and related disorders.

Role of gut-derived bacterial lipopolysaccharide and peripheral TLR4 in immobilization stress-induced itch aggravation in a mouse model of atopic dermatitis.

Psychological stress and intestinal leakage are key factors in atopic dermatitis (AD) recurrence and exacerbation. Here, we demonstrate the mechanism underlying bacterial translocation across intestinal epithelial barrier damaged due to stress and further aggravation of trimellitic anhydride (TMA)-induced itch, which remain unclear, in AD mice. Immobilization (IMO) stress exacerbated scratching bouts and colon histological damage, and increased serum corticosterone and lipopolysaccharide (LPS). Orally administered fluorescein isothiocyanate (FITC)-dextran and surgically injected (into the colon) Cy5.5-conjugated LPS were detected in the serum and skin after IMO stress, respectively. The relative abundance of aerobic or facultative anaerobic bacteria was increased in the colon mucus layer, and Lactobacillus murinus, E. coli, Staphylococcus nepalensis, and several strains of Bacillus sp. were isolated from the spleens and mesenteric lymph nodes. Oral antibiotics or intestinal permeability blockers, such as lubiprostone (Lu), 2,4,6-triaminopyrimidine (TAP) and ML-7, inhibited IMO stress-associated itch; however, it was reinduced through intradermal or i.p. injection of LPS without IMO stress. I.p. injection of TAK-242 (resatorvid), a TLR4 inhibitor, abrogated IMO stress-associated itch, which was also confirmed in TLR4-KO mice. IMO stress alone did not cause itch in naïve mice. IMO stress-induced itch aggravation in TMA-treated AD mice might be attributed to the translocation of gut-derived bacterial cells and LPS, which activates peripheral TLR4 signaling.

Reactive Oxygen Species (ROS)-Assisted Nano-Therapeutics Surface-Decorated with Epidermal Growth Factor Fragments for Enhanced Wound Healing.

In this study, stimuli-responsive liberation of an epidermal growth factor fragment (EGFfr) is accomplished using nanofibrous meshes to improve wound healing effects. Electrospun nanofibers are fragmented by mechanical milling, followed by aminolysis to fabricate powdered nanofibrils (NFs). EGFfrs are covalently immobilized on NFs via thioketal linkers (EGFfr@TK@NF) for reactive oxygen species (ROS)-dependent liberation. EGFfr@TK@NF exhibits ROS-responsive liberation of EGFfr from the matrix at hydrogen peroxide (H2 O2 ) concentrations of 0-250 mm. Released EGFfr is confirmed to enhance the migration of HaCaT cell monolayers, and keratinocytic gene expression levels are significantly enhanced when H2 O2 is added to obtain the released fraction of NFs. An in vivo study on the dorsal wounds of mice reveals that EGFfr-immobilized NFs improve the expression levels of keratin1, 5, and 14 for 2 weeks when H2 O2 is added to the wound sites, suggesting that the wounded skin is re-epithelized with the original epidermis. Thus, EGFfrs-immobilized NFs are anticipated to be potential nanotherapeutics for wound treatment in combination with the conventional disinfection process with H2 O2 .

Assessment of phenotypic heterogeneity in Salmonella Typhimurium preadapted to ciprofloxacin and tetracycline.

Bacterial population exposed to stressful antibiotic conditions consists of various subpopulations such as tolerant, persister, and resistant cells. The aim of this study was to evaluate the phenotypic heterogeneity of Salmonella Typhimurium preadapted to sublethal concentrations of antibiotics. Salmonella Typhimurium cells were treated with 1/2 × MIC of antibiotics for the first 48 h and successively 1 × MIC for the second 24 h at 37°C, including untreated control (CON), no antibiotic and 1 × MIC ciprofloxacin (NON-CIP), 1/2 × MIC ciprofloxacin and 1 × MIC ciprofloxacin (CIP-CIP), 1/2 × MIC tetracycline and 1 × MIC ciprofloxacin (TET-CIP), no antibiotic and 1 × MIC tetracycline (NON-TET), 1/2 × MIC ciprofloxacin and 1 × MIC tetracycline (CIP-TET), and 1/2 × MIC tetracycline and 1 × MIC tetracycline (TET-TET). All treatments were evaluated by antibiotic susceptibility, ATP level, relative fitness, cross-resistance, and persistence. S. Typhimurium cells were more susceptible to non-adapted NON-CIP and NON-TET (>3-log reduction) than pre-adapted CIP-CIP, TET-CIP, CIP-TET, and TET-TET. CON exhibited the highest ATP level, corresponding to the viable cell number. The relative fitness levels were more than 0.95 for all treatments, except for NON-CIP (0.78). The resistance to ciprofloxacin and tetracycline was increased at all treatments with the exception of NON-TET. The persister cells were noticeably induced at CIP-TET treatment, showing more than 5 log CFU mL-1. The results suggest that the antibiotic preadaptation led to heterogeneous populations including persisters that can develop to resistance. This study provides new insight in the bacterial persistence associated with their potential risk and paves the way to design antibiotic therapy targeting dormant bacteria.

Age-associated spinal stenosis in the turquoise killifish.

Aging triggers spinal degeneration, including common spinal stenosis, which causes back and leg pain in older individuals, significantly impacting their quality of life. Here, we explored aging traits in turquoise killifish spines, potentially offering a model for age-linked spinal stenosis in humans. Aged turquoise killifish exhibited body shape deformation and increased vertebral collapse, which was further accelerated by spawning. High-resolution CT scans revealed suppressed cortical bone thickness and hemal arch area in vertebrae due to spawning, and osteophyte formation was observed in both aged and breeding fish populations. Scale mineralization mirrored these changes, increasing with age but being suppressed by spawning. The expression of sp7, sox9b, axin1, and wnt4a/b genes can be utilized to monitor age- and reproduction-dependent spine deformation. This study demonstrates that turquoise killifish and humans share certain phenotypes of age-related vertebral abnormalities, suggesting that turquoise killifish could serve as a potential model for studying human spinal stenosis.

ANO1-downregulation induced by schisandrathera D: a novel therapeutic target for the treatment of prostate and oral cancers.

Anoctamin 1 (ANO1), a drug target for various cancers, including prostate and oral cancers, is an intracellular calcium-activated chloride ion channel that plays various physiopathological roles, especially in the induction of cancer growth and metastasis. In this study, we tested a novel compound isolated from Schisandra sphenanthera, known as schisandrathera D, for its inhibitory effect on ANO1. Schisandrathera D dose-dependently suppressed the ANO1 activation-mediated decrease in fluorescence of yellow fluorescent protein; however, it did not affect the adenosine triphosphate-induced increase in the intracellular calcium concentration or forskolin-induced cystic fibrosis transmembrane conductance regulator activity. Specifically, schisandrathera D gradually decreased the levels of ANO1 protein and significantly reduced the cell viability in ANO1-expressing cells when compared to those in ANO1-knockout cells. These effects could be attributed to the fact that schisandrathera D displayed better binding capacity to ANO1 protein than the previously known ANO1 inhibitor, Ani9. Finally, schisandrathera D increased the levels of caspase-3 and cleaved poly (ADP-ribose) polymerase 1, thereby indicating that its anticancer effect is mediated through apoptosis. Thus, this study highlights that schisandrathera D, which reduces ANO1 protein levels, has apoptosis-mediated anticancer effects in prostate and oral cancers, and thus, can be further developed into an anticancer agent.

Menaquinone-7 ameliorates cerebrovascular calcification-associated memory decline in aged mice.

Menaquinone (MK)-7 is a vitamin K2 analog that functions as a cofactor of γ-glutamyl carboxylase involved in the activation of vitamin K (VK)-dependent proteins. The present study aimed to evaluate the effect of MK-7 on memory and cognitive function in aged C57BL/6 mice. Eighteen-month-old mice were raised for a further 4 months, fed on a standard or calcium-rich diet (3 % [w/w]), and were orally given MK-7 (40 and 400 µg/day/mouse) five times per week during the same period. The Morris water maze (MWM) test was performed at 19 and 22 months. The aged mice showed noticeable memory declines in the MWM test at all time points compared with 6-week-old mice, and this memory loss was significantly restored by the daily administration of high-dose MK-7 for 4 months. MK-7 administration also improved micro-computed tomography-based cerebrovascular calcification and aging-associated declines in growth arrest-specific 6, total and carboxylated matrix Gla proteins, and ganglioside levels in the brain of aged mice. It serologically reduced phosphorous levels in the blood, but not the urea, cholesterol, and calcium. Taken together, the long-term administration of MK-7 significantly improved age-related memory and cognitive impairments, possibly through inhibition of cerebrovascular calcification in aged mice, indicating that it can be used to develop new drugs for improving memory and cognitive function in older adults.

Monoclonal Antibody Functionalized, and L-lysine α-Oxidase Loaded PEGylated-Chitosan Nanoparticle for HER2/Neu Targeted Breast Cancer Therapy.

Herein, we designed a nanocarrier to deliver the LO specifically to HER2+ breast cancer (BC) cells, where functionalization of mAb (anti-HER2+) with PEGylated chitosan enabled it to target the HER2+ BC cells. Taking advantage of overexpression of HER2+ in cancer cells, our nanocarrier (CS-LO-PEG-HER NPs) exhibited promising potency and selectivity against HER2+ BC cells (BT474). The CS-LO-PEG-HER NPs demonstrated the cytotoxicity in BT474 cells by promoting reactive oxygen species, mitochondrial membrane potential loss, and nucleus damage. The biocompatibility of CS-LO-PEG-HER NPs was evidenced by the hemolysis assay and H & E staining of major organs. The CS-LO-PEG-HER NPs showed anticancer potency against the BT474-xenograft tumor-bearing mice, as evident by the reduction of tumor size and cell density. These results indicate that CS-LO-PEG-HER NPs are biocompatible with mice while inhibiting tumor growth through alter the oxidative stress. Overall, this work provides a promising approach for the delivery of LO for good therapeutic effect in combination with mAb.

Real-Time Longitudinal Evaluation of Tumor Blood Vessels Using a Compact Preclinical Fluorescence Imaging System.

Tumor angiogenesis is enhanced in all types of tumors to supply oxygen and nutrients for their growth and metastasis. With the development of anti-angiogenic drugs, the importance of technology that closely monitors tumor angiogenesis has also been emerging. However, to date, the technology for observing blood vessels requires specialized skills with expensive equipment, thereby limiting its applicability only to the laboratory setting. Here, we used a preclinical optical imaging system for small animals and, for the first time, observed, in real time, the entire process of blood vessel development in tumor-bearing mice injected with indocyanine green. Time-lapse sequential imaging revealed blood vessel volume and blood flow dynamics on a microscopic scale. Upon analyzing fluorescence dynamics at each stage of tumor progression, vessel volume and blood flow were found to increase as the tumor developed. Conversely, these vascular parameters decreased when the mice were treated with angiogenesis inhibitors, which suggests that the effects of drugs targeting angiogenesis can be rapidly and easily screened. The results of this study may help evaluate the efficacy of angiogenesis-targeting drugs by facilitating the observation of tumor blood vessels easily in a laboratory unit without large and complex equipment.

Anti-Inflammatory Activity of 4-((1R,2R)-3-Hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol Isolated from Juglans mandshurica Maxim. in LPS-Stimulated RAW 264.7 Macrophages and Zebrafish Larvae Model.

Juglans mandshurica Maxim., a traditional folk medicinal plant, is widely distributed in Korea and China. In our previous study, we isolated a new phenylpropanoid compound, 4-((1R,2R)-3-hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol (HHMP), from J. mandshurica. In the present study, we evaluated the anti-inflammatory activity of HHMP on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and zebrafish larvae. HHMP significantly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 production in a dose-dependent manner. Moreover, HHMP treatment considerably suppressed LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. We also demonstrated the mechanisms of HHMP inhibition of inflammatory responses in LPS-stimulated RAW 264.7 cells via Western blot analysis and immunofluorescence staining. Furthermore, HHMP significantly inhibited NO production in LPS-stimulated zebrafish larvae. Consequently, we established that HHMP significantly inhibited the LPS-induced activation of NF-κB and MAPK and the nuclear translocation of p65 in RAW 264.7 cells. Taken together, our findings demonstrate the effect of HHMP on LPS-induced inflammatory responses in vitro and in vivo, suggesting its potential to be used as a natural anti-inflammatory agent.

Dissecting the impact of target-binding kinetics of protein binders on tumor localization.

Systematic control of in vivo behavior of protein-based therapeutics is considered highly desirable for improving their clinical outcomes. Modulation of biochemical properties including molecular weight, surface charge, and binding affinity has thus been suggested to enhance their therapeutic effects. However, establishing a relationship between the binding affinity and tumor localization remains a debated issue. Here we investigate the influence of the binding affinity of proteins on tumor localization by using four repebodies having different affinities to EGFR. Biochemical analysis and molecular imaging provided direct evidence that optimal affinity with balanced target binding and dissociation can facilitate deep penetration and accumulation of protein binders in tumors by overcoming the binding-site-barrier effect. Our findings suggest that binding kinetics-based protein design can be implicated in the development of fine-tuned protein therapeutics for cancers.

1-Cinnamoyltrichilinin from Melia azedarach Causes Apoptosis through the p38 MAPK Pathway in HL-60 Human Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive type of human leukemia with a low survival rate, and its complete remission remains challenging. Although chemotherapy is the first-line treatment of AML, it exerts toxicity in noncancerous cells when used in high doses, thus necessitating the development of novel compounds with a high therapeutic window. This study aimed to investigate the anticancer effects of several compounds derived from the fruits of Melia azedarach (a tree with medicinal properties). Among them, 1-cinnamoyltrichilinin (CT) was found to strongly suppress the viability of HL-60 human leukemia cells. CT treatment induced apoptosis and increased nuclear fragmentation and fractional DNA content in HL-60 cells in a dose-dependent manner. CT induced phosphorylation of p38 mitogen-activated protein kinases (p38), though not of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), and activated Bcl-2 family proteins towards the proapoptosis and cleavage of caspase-3 and poly (ADP-ribose) polymerase. Both CT-mediated apoptosis and apoptotic protein expression were reversed by treatment with the p38 inhibitor, thereby indicating the p38 pathway to be critical in CT-stimulated apoptosis. The results collectively indicated CT to suppress HL-60 survival by activating the p38 pathway and inducing apoptosis, hence being a novel potential therapeutic agent for AML.

Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery.

With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.

Survival, prophage induction, and invasive properties of lysogenic Salmonella Typhimurium exposed to simulated gastrointestinal conditions.

This study was designed to evaluate the viability, prophage induction, invasive ability, and relative gene expression in lysogenic Salmonella Typhimurium exposed to the simulated gastric juice (SGJ) at pH 2 (SGJ-2), 3 (SGJ-3), 4 (SGJ-4), and 5 (SGJ-5) for 30 min followed by 0.5 % bile salts for 2 h. The susceptibility of lysogenic S. Typhimurium increased with decreasing pH value and increasing bile salt concentration. The lysogenic S. Typhimurium cells were least susceptible to SGJ-4 and SGJ-5, showing <1 log reduction. The highest prophage induction was observed by 3.34 log PFU/ml in lysogenic S. Typhimurium at SGJ-3 in the presence of 0.5 % bile salts. The numbers of invading lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5 were 3.57, 3.73, and 4.15 log CFU/cm(2), respectively. Most genes (hilA, hilC, hilD, invA, invE, invF, and sirA) were down-regulated in lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5. This study provides useful information for understanding physiological changes of lysogenic S. Typhimurium in the simulated gastrointestinal conditions.

In vitro assessment of the susceptibility of planktonic and attached cells of foodborne pathogens to bacteriophage p22-mediated salmonella lysates.

This study was designed to evaluate the lytic activity of bacteriophage P22 against Salmonella Typhimurium ATCC 19585 (Salmonella Typhimurium P22(-)) at various multiplicities of infections (MOIs), the susceptibility of preattached Salmonella cells against bacteriophage P22, and the effect of P22-mediated bacterial lysates (extracellular DNA) on the attachment ability of Listeria monocytogenes ATCC 7644 and enterohemorrhagic Escherichia coli ATCC 700927 to surfaces. The numbers of attached Salmonella Typhimurium P22(-) cells were effectively reduced to below the detection limit (1 log CFU/ml) at the fixed inoculum levels of 3 × 10(-) CFU/ml (MOI = 3.12) and 3 × 10(3) CFU/ml (MOI = 4.12) by bacteriophage P22. The attached Salmonella Typhimurium P22(-) cells remained more than 2 log CFU/ml, with increasing inoculum levels from 3 × 10(4) to 3 × 10(7) CFU/ml infected with 4 × 10(8) PFU/ml of P22. The number of preattached Salmonella Typhimurium P22(-) cells was noticeably reduced by 2.72 log in the presence of P22. The highest specific attachment ability values for Salmonella Typhimurium P22(-), Salmonella Typhimurium ATCC 23555 carrying P22 prophage (Salmonella Typhimurium P22(+)), L. monocytogenes, and enterohemorrhagic E. coli were 2.09, 1.06, 1.86, and 1.08, respectively, in the bacteriophage-mediated cell-free supernatants (CFS) containing high amounts of extracellular DNA. These results suggest that bacteriophages could potentially be used to effectively eliminate planktonic and preattached Salmonella Typhimurium P22(-) cells with increasing MOI. However, further research is needed to understand the role of bacteriophage-induced lysates in bacterial attachment, which can provide useful information for the therapeutic use of bacteriophage in the food system.