Exosomal LINC00853 promotes progression of gastric cancer via the MAP17/PDZK1/AKT signaling pathway.

Although rare, there is ongoing research into biomarkers that predict the onset and recurrence of gastric cancer, particularly focusing on substances found in exosomes. Long non-coding RNAs (lncRNAs) have garnered attention for their potential in diagnosing gastric cancer. This study investigates the role of lncRNAs in gastric cancer, focusing on their presence in exosomes as potential biomarkers for the disease's onset and recurrence. We utilized the ArrayStar Human LncRNA array 2.0 to analyze lncRNA expression in tissues from early-stage gastric cancer patients. Our analysis highlighted LINC00853, which was significantly upregulated in cancer tissues and implicated in promoting epithelial-mesenchymal transition via the MAP17/PDZK1/AKT pathway. Functional studies on AGS and MKN74 gastric cancer cell lines demonstrated that LINC00853 facilitates cell proliferation, invasion, and migration. Additionally, RNA immunoprecipitation and electrophoretic mobility shift assays confirmed LINC00853 interaction with MAP17. Importantly, LINC00853 was also detected in exosomes from both patient samples and cell lines, and its downregulation led to decreased tumorigenicity in AGS cells. These findings suggest that both cellular and exosomal LINC00853 contribute to gastric cancer pathogenesis and may serve as valuable biomarkers for the disease.

Impact of resident participation on surgical outcomes in laparoscopically assisted vaginal hysterectomy.

OBJECTIVE: To compare surgical outcomes in patients with benign diseases who underwent laparoscopically assisted vaginal hysterectomy (LAVH) to determine the association between surgical outcomes and resident participation in the gynecologic field. METHODS: A single-center retrospective study was conducted of patients diagnosed with benign gynecologic diseases who underwent LAVH between January 2010 and December 2015. Clinicopathologic characteristics and surgical outcomes were compared between the resident involvement and non-involvement groups. The primary endpoint was the 30-day postoperative morbidity. Observers were propensity matched for 17 covariates for resident involvement or non-involvement. RESULTS: Of the 683 patients involved in the study, 165 underwent LAVH with resident involvement and 518 underwent surgery without resident involvement. After propensity score matching (157 observations), 30-day postoperative morbidity occurred in 6 (3.8%) and 4 (2.5%) patients in the resident involvement and non-involvement groups, respectively (P = 0.501). The length of hospital stay differed significantly between the two groups: 5 days in the resident involvement group and 4 days in the non-involvement group (P < 0.001). On multivariate analysis, Charlson Comorbidity Index >2 (odds ratio [OR] 8.01, 95% confidence interval [CI] 2.68-23.96; P < 0.001), operative time (OR 1.02, 95% CI 1.01-1.03; P < 0.001), and estimated blood loss (OR 1.00, 95% CI 1.00-1.00; P < 0.001) were significantly associated with 30-day morbidity, but resident involvement was not statistically significant. CONCLUSION: There was no significant difference in the 30-day morbidity rate when residents participated in LAVH. These findings suggest that resident participation in LAVH may be a viable approach to ensure both residency education and patient safety.

No Interference of H9 Extract on Trastuzumab Pharmacokinetics in Their Combinations.

Trastuzumab is used to treat breast cancer patients overexpressing human epidermal growth factor receptor 2, but resistance and toxicity limit its uses, leading to attention to trastuzumab combinations. Recently, the synergistic effect of trastuzumab and H9 extract (H9) combination against breast cancer has been reported. Because drug exposure determines its efficacy and toxicity, the question of whether H9 changes trastuzumab exposure in the body has been raised. Therefore, this study aimed to characterize trastuzumab pharmacokinetics and elucidate the effect of H9 on trastuzumab pharmacokinetics at a combination dose that shows synergism in mice. As a result, trastuzumab showed linear pharmacokinetics after its intravenous administration from 1 to 10 mg/kg. In the combination of trastuzumab and H9, single and 2-week treatments of oral H9 (500 mg/kg) did not influence trastuzumab pharmacokinetics. In the multiple-combination treatments of trastuzumab and H9 showing their synergistic effect (3 weeks of trastuzumab with 2 weeks of H9), the pharmacokinetic profile of trastuzumab was comparable to that of 3 weeks of trastuzumab alone. In tissue distribution, the tissue to plasma ratios of trastuzumab below 1.0 indicated its limited distributions within the tissues, and these patterns were unaffected by H9. These results suggest that the systemic and local exposures of trastuzumab are unchanged by single and multiple-combination treatments of H9.

Adipose Tissue-Derived Mesenchymal Stem Cells Extend the Lifespan and Enhance Liver Function in Hepatocyte Organoids.

In this study, we generated hepatocyte organoids (HOs) using frozen-thawed primary hepatocytes (PHs) within a three-dimensional (3D) Matrigel dome culture in a porcine model. Previously studied hepatocyte organoid analogs, spheroids, or hepatocyte aggregates created using PHs in 3D culture systems have limitations in their in vitro lifespans. By co-culturing adipose tissue-derived mesenchymal stem cells (A-MSCs) with HOs within a 3D Matrigel dome culture, we achieved a 3.5-fold increase in the in vitro lifespan and enhanced liver function compared to a conventional two-dimensional (2D) monolayer culture, i.e., more than twice that of the HO group cultured alone, reaching up to 126 d. Although PHs were used to generate HOs, we identified markers associated with cholangiocyte organoids such as cytokeratin 19 and epithelial cellular adhesion molecule (EPCAM). Co-culturing A-MSCs with HOs increased the secretion of albumin and urea and glucose consumption compared to HOs cultured alone. After more than 100 d, we observed the upregulation of tumor protein P53 (TP53)-P21 and downregulation of EPCAM, albumin (ALB), and cytochrome P450 family 3 subfamily A member 29 (CYP3A29). Therefore, HOs with function and longevity improved through co-culturing with A-MSCs can be used to create large-scale human hepatotoxicity testing models and precise livestock nutrition assessment tools.

Gemcitabine and rapamycin-loaded mixed polymeric thermogel for metastatic pancreatic cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death and has a poor 5-year overall survival. The superior therapeutic benefits of combination or co-administration of drugs as intraperitoneal chemotherapy have increased interest in developing strategies to deliver chemotherapeutic agents to patients safely. In this study, we prepared a gel comprising the thermosensitive poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) polymer and gemcitabine (GEM), which is currently used as the primary chemotherapy for PDAC and rapamycin (RAPA), a mammalian TOR (mTOR) inhibitor, to deliver the drug through intraperitoneal injection. We performed in vitro cytotoxicity experiments to verify the synergistic effects of the two drugs at different molar ratios and characterized the physicochemical properties of the GEM, RAPA, and GEM/RAPA-loaded thermosensitive PLGA-PEG-PLGA gels, hereafter referred to as (g(G), g(R), and g(GR)), respectively. The g(GR) comprising PLGA-PEG-PLGA polymer (25% w/v) and GEM and RAPA at a molar ratio of 11:1 showed synergism and was optimized. An in vitro cytotoxicity assay was performed by treating Panc-1-luc2 tumor spheroids with g(G), g(R), or g(GR). The g(GR) treatment group showed a 2.75-fold higher inhibition rate than the non-treated (NT) and vehicle-treated groups. Furthermore, in vivo drug release assay in mice by intraperitoneal injection of g(G), g(R), or g(GR) showed a more rapid release rate of GEM than RAPA, similar to the in vitro release pattern. The drugs in the gel were released faster in vivo than in vitro and degraded in 48 h. In addition, g(GR) showed the highest anti-tumor efficacy with no toxicity to mice. These results provide evidence for the safety and efficacy of g(GR) for intraperitoneal drug delivery. This study will assist in developing and clinically administering topical anti-cancer formulations.

Synergistic Encapsulation of Paclitaxel and Sorafenib by Methoxy Poly(Ethylene Glycol)-b-Poly(Caprolactone) Polymeric Micelles for Ovarian Cancer Therapy.

Ovarian cancer has a high mortality rate due to difficult detection at an early stage. It is necessary to develop a novel anticancer treatment that demonstrates improved efficacy while reducing toxicity. Here, using the freeze-drying method, micelles encapsulating paclitaxel (PTX) and sorafenib (SRF) with various polymers were prepared, and the optimal polymer (mPEG-b-PCL) was selected by measuring drug loading (%), encapsulation efficiency (%), particle size, polydispersity index, and zeta potential. The final formulation was selected based on a molar ratio (PTX:SRF = 1:2.3) with synergistic effects on two ovarian cancer cell lines (SKOV3-red-fluc, HeyA8). In the in vitro release assay, PTX/SRF micelles showed a slower release than PTX and SRF single micelles. In pharmacokinetic evaluation, PTX/SRF micelles showed improved bioavailability compared to PTX/SRF solution. In in vivo toxicity assays, no significant differences were observed in body weight between the micellar formulation and the control group. The anticancer effect of PTX/SRF combination therapy was improved compared to the use of a single drug. In the xenografted BALB/c mouse model, the tumor growth inhibition rate of PTX/SRF micelles was 90.44%. Accordingly, PTX/SRF micelles showed improved anticancer effects compared to single-drug therapy in ovarian cancer (SKOV3-red-fluc).

Modularized dynamic cell culture platform for efficient production of extracellular vesicles and sequential analysis.

Extracellular vesicles (EVs) are nanometer-sized particles naturally secreted by cells for intercellular communication that encapsulate bioactive cargo, such as proteins and RNA, with a lipid bilayer. Tumor cell-derived EVs (tdEVs) are particularly promising biomarkers for cancer research because their contents reflect the cell of origin. In most studies, tdEVs have been obtained from cancer cells cultured under static conditions, thus lacking the ability to recapitulate the microenvironment of cells in vivo. Recent developments in perfusable cell culture systems have allowed oxygen and a nutrient gradient to mimic the physiological and cellular microenvironment. However, as these systems are perfused by circulating the culture medium within the unified structure, independently harvesting cells and EVs at each time point for analysis presents a limitation. In this study, a modularized cell culture system is designed for the perfusion and real-time collection of EVs. The system consists of three detachable chambers, one each for fresh medium, cell culture, and EV collection. The fresh medium flows from the medium chamber to the culture chamber at a flow rate controlled by the hydraulic pressure injected with a syringe pump. When the culture medium containing EVs exceeds a certain volume within the chamber, it overflows into the collection chamber to harvest EVs. The compact and modularized chambers are highly interoperable with conventional cell culture modalities used in the laboratory, thus enabling various EV-based assays.

Evaluation of pH-Sensitive Polymeric Micelles Using Citraconic Amide Bonds for the Co-Delivery of Paclitaxel, Etoposide, and Rapamycin.

Paclitaxel (PTX), etoposide (ETP), and rapamycin (RAPA) have different mechanisms, allowing multiple pathways to be targeted simultaneously, effectively treating various cancers. However, these drugs have a low hydrosolubility, limiting clinical applications. Therefore, we used pH-sensitive polymeric micelles to effectively control the drug release in cancer cells and to improve the water solubility of PTX, ETP, and RAPA. The synergistic effect of PTX, ETP, and RAPA was evaluated in gastric cancer, and the combination index values were evaluated. Thin-film hydration was used to prepare PTX/ETP/RAPA-loaded mPEG-pH-PCL micelles, and various physicochemical properties of these micelles were evaluated. In vitro cytotoxicity, pH-sensitivity, drug release profiles, in vivo pharmacokinetics, and biodistribution studies of PTX/ETP/RAPA-loaded mPEG-pH-PCL micelles were evaluated. In the pH-sensitivity evaluation, the size of the micelles increased more rapidly at a pH of 5.5 than at a pH of 7.4. The release rate of each drug increased with decreasing pH values in PTX/ETP/RAPA-loaded mPEG-pH-PCL micelles. In vitro and in vivo studies demonstrated that PTX/ETP/RAPA-loaded mPEG-pH-PCL micelles exhibit different drug release behaviors depending on the pH of the tumor and normal tissues and increased bioavailability and circulation time in the blood than solutions. Therefore, we propose that PTX/ETP/RAPA- loaded mPEG-pH-PCL micelles are advantageous for gastric cancer treatment in drug delivery systems.

Risk Assessment and Evaluation of Analytical Method of Polycyclic Aromatic Hydrocarbons (PAHs) for Deep-Fat Fried Pork Products in Korea.

Polycyclic aromatic hydrocarbons (PAHs) are produced during incomplete combustion of organic matter. Many of them are likely to be carcinogenic and cause mutations. In this study, the PAH4 (benzo[a]pyrene (BaP), benz[a]anthracene (BaA), chrysene (CHR), benzo[b]fluoranthene (BbF)) content in deep-fat fried pork was evaluated according to temperature and time, and a risk assessment was conducted. The high performance liquid chromatography-fluorescence detection (HPLC-FLD) method for PAH4 analysis was validated by determining linearity (R2), recovery, limit of detection (LOD), and limit of quantitation (LOQ). The linearity was R2 ≥ 0.99. The PAH4 level was dependent on the temperature, time, and nature of the edible oil. Before heat treatment, the PAH4 content of pork was 0.38 µg/kg. The PAH4 content of deep-fat fried pork ranged from 0.86 to 6.86 µg/kg according to temperature (160, 180, 200 °C) and time (3, 6, 9 min). Exposure to PAH4 via the consumption of deep-fat fried pork for different age groups among the Korean population was 0.01-0.89 µg-TEQBaP/kg/day, with the margin of exposure calculated as 7.88 × 104-5.22 × 106. The PAH4 content and risk of exposure increased proportionally with the heat treatment temperature and time. The survey provided important information in terms of evaluating the health risks that PAH compounds can cause in people's diets due to the heat treatment of pork.

Mosloflavone-Resveratrol Hybrid TMS-HDMF-5z Exhibits Potent In Vitro and In Vivo Anti-Inflammatory Effects Through NF-κB, AP-1, and JAK/STAT Inactivation.

TMS-HDMF-5z is a hybrid of the natural products mosloflavone and resveratrol. It was discovered to show potent inhibitory effects against lipopolysaccharide (LPS)-induced production of inflammatory mediators in RAW 264.7 macrophages. However, its mechanism of action is unknown. Hence this study aimed to demonstrate and explore in vitro and in vivo anti-inflammatory effects of TMS-HDMF-5z and its mechanism of action employing RAW 264.7 macrophages and carrageenan-induced hind paw edema. This work revealed that TMS-HDMF-5z suppressed the LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein, mRNA, and promoter binding levels and tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, and interferon-ß (IFN-ß) at the mRNA expression in RAW 264.7 macrophages. The results showed that TMS-HDMF-5z reduced the transcription and DNA binding activities of nuclear factor-κB (NF-κB) through inhibiting nuclear translocation of p65 and phosphorylation of κB inhibitor α (IκBα), IκB kinase (IKK), and TGF-ß activated kinase 1 (TAK1). Additionally, TMS-HDMF-5z attenuated the LPS-induced transcriptional and DNA binding activities of activator protein-1 (AP-1) by suppressing nuclear translocation of phosphorylated c-Fos, c-Jun, and activating transcription factor 2 (ATF2). TMS-HDMF-5z also reduced the LPS-induced phosphorylation of Janus kinase 1/2 (JAK1/2), signal transducers and activators of transcription 1/3 (STAT1/3), p38 mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 (MK2). In rats, TMS-HDMF-5z alleviated carrageenan-induced hind paw edema through the suppressing iNOS and COX-2 via NF-κB, AP-1, and STAT1/3 inactivation. Collectively, the TMS-HDMF-5z-mediated inhibition of NF-κB, AP-1, and STAT1/3 offer an opportunity for the development of a potential treatment for inflammatory diseases.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop.

Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.

Chaga mushroom extract induces autophagy via the AMPK-mTOR signaling pathway in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaga mushrooms (Inonotus obliquus) are commonly used in traditional treatments in Eastern Europe and Asia due to their diverse pharmacological effects, including anti-tumor and immunologic effects. Thus, many cancer patients take Chaga mushrooms as a complementary medicine, even during chemotherapy or radiotherapy. However, few studies have investigated the effects or molecular targets of Chaga mushrooms in breast cancer. AIM OF THE STUDY: Herein, we examined the anticancer effects of Chaga mushrooms in different types of breast cancer cell lines, and explored the underlying molecular mechanism to better understand their effects and benefits. MATERIALS AND METHODS: Chaga mushroom extract (CME) was prepared by extracting Chaga mushrooms with 70% ethanol. The cytotoxic effects of CME were assessed by MTT assay and protein expressions were evaluated by western blotting. To evaluate in vivo anti-tumor effects of CME, CME (2 g/kg) was orally administered to 4T1 tumor-bearing BALB/c mice every other day over 30 days (15 administrations), and tumor sizes were measured. Silica gel column chromatography was used to fractionate CME, and major constituents responsible for cytotoxic effects of CME were identified by 1H/13C-NMR and LC-MS. RESULTS: CME inhibited the proliferation of 4T1 mouse breast cancer cells in a dose and time-dependent manner. The expression of LC3 and phosphorylation of AMPK were increased by CME, while the phosphorylation of mTOR, S6, and S6K1 were suppressed, suggesting that CME induced autophagy by activating AMPK and inhibiting mTOR signaling pathways. Consistent with its observed cytotoxic effect in vitro, CME effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition, inotodiol and trametenolic acid were identified as the major constituents responsible for the cytotoxic effects of CME on breast cancer cells. Moreover, inotodiol and trametenolic acid-enriched fractions both exhibited cytotoxic effects regardless of breast cancer cell subtypes and did not interfere with the cytotoxic effects of conventional drugs. CONCLUSIONS: Taken together, Chaga mushroom extract induced autophagy by activating AMPK and inhibiting the mTOR signaling pathway. Our data suggest Chaga mushrooms may be a beneficial complementary medicine for breast cancer patients.

Umbilical cord arterial blood gas analysis in term singleton pregnancies: a retrospective analysis over 11 years.

OBJECTIVE: Given that the large volume of data on cord arterial blood gas analysis (ABGA) have been rarely addressed in Korean population, we aimed to examine the incidence, associated factors, and neonatal outcomes in cases of low cord pH, and investigate the incidence of cerebral palsy (CP). METHODS: From data of all consecutive term singleton pregnancies delivered in our institution from 2006 to 2016 (n=15,701), cases with cord ABGA (n=14,221) available were included. We collected information on maternal clinical characteristics and delivery outcomes and also examined neonatal and infant outcomes, including neonatal intensive care unit (NICU) admission and CP, in cases with low cord pH, defined as a pH <7.1. RESULTS: Rates of low Apgar scores at 1 minute (<4) and 5 minutes (<7) were 0.6% (n=79) and 0.4% (n=58), respectively. Rates of cord pH <7.2, <7.1, and <7.0 were 7.1% (n=1,011), 1.1% (n=163), and 0.3% (n=38), respectively. Among cases with low cord pH, 30.1% (n=49/163) were admitted to the NICU and 11.0% (n=18/163) required ventilator support. Ultrasonography of the brain was performed in 28.8% (n=47/163), with abnormal findings observed in 27.7% (n=13/47). Among cases with low cord pH, 1.8% (n=3/163) were subsequently diagnosed with CP, including 2 cases of spastic CP and 1 of ataxic CP. CONCLUSION: Although low cord pH was a relatively frequent finding observed in 1 out of every 87 cases, hypoxic-ischemic encephalopathy-related CP was found in only 1 out of 7,111 term singleton deliveries over 11 years in our institution.

Patriscabrin F from the roots of Patrinia scabra attenuates LPS-induced inflammation by downregulating NF-κB, AP-1, IRF3, and STAT1/3 activation in RAW 264.7 macrophages.

BACKGROUND: The roots of Partrinia scabra have been used as a medicinal herb in Asia. We previously reported that the inhibitory effect of patriscabrin F on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was the most potent than that of other isolated iridoids from the roots of P. scabra. PURPOSE: We investigated the anti-inflammatory activity of patriscabrin F as an active compound of P. scabra and related signaling cascade in LPS-activated macrophages. METHOD: The anti-inflammatory activities of patriscabrin F were determined according to its inhibitory effects on NO, prostaglandin E2 (PGE2), and pro-inflammatory cytokines. The molecular mechanisms were revealed by analyzing nuclear factor-κB (NF-κB), activator protein-1 (AP-1), interferon regulatory factor 3 (IRF3), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. RESULTS: Patriscabrin F inhibited the LPS-induced production of NO, PGE2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in both bone-marrow derived macrophages (BMDMs) and RAW 264.7 macrophages. Patriscabrin F downregulated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), TNF-α, IL-1ß, and IL-6 at the transcriptional level. Patriscabrin F suppressed LPS-induced NF-κB activation by decreasing p65 nuclear translocation, inhibitory κBα (IκBα) phosphorylation, and IκB kinase (IKK)α/ß phosphorylation. Patriscabrin F attenuated LPS-induced AP-1 activity by inhibiting c-Fos phosphorylation. Patriscabrin F suppressed the LPS-induced phosphorylation of IRF3, JAK1/JAK2, and STAT1/STAT3. CONCLUSION: Taken together, our findings suggest patriscabrin F may exhibit anti-inflammatory properties via the inhibition of NF-κB, AP-1, IRF3, and JAK-STAT activation in LPS-induced macrophages.

Shockwaves Suppress Adipocyte Differentiation via Decrease in PPARγ.

Adipogenesis is a crucial cellular process that contributes to the expansion of adipose tissue in obesity. Shockwaves are mechanical stimuli that transmit signals to cause biological responses. The purpose of this study is to evaluate the effects of shockwaves on adipogenesis. We treated 3T3L-1 cells and human primary preadipocytes for differentiation with or without shockwaves. Western blots and quantitative real-time reverse transcriptase PCR (qRT-PCR) for adipocyte markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPα) were performed. Extracellular adenosine triphosphate (ATP) and intracellular cyclic adenosine monophosphate (cAMP) levels, which are known to affect adipocyte differentiation, were measured. Shockwave treatment decreased intracellular lipid droplet accumulation in primary human preadipocytes and 3T3-L1 cells after 11-12 days of differentiation. Levels of key adipogenic transcriptional factors PPARγ and/or C/EBPα were lower in shockwave-treated human primary preadipocytes and 3T3L-1 cells after 12-13 days of differentiation than in shockwave-untreated cells. Shockwave treatment induced release of extracellular ATP from preadipocytes and decreased intracellular cAMP levels. Shockwave-treated preadipocytes showed a higher level of ß-catenin and less PPARγ expression than shockwave-untreated cells. Supplementation with 8-bromo-cAMP analog after shockwave treatment rescued adipocyte differentiation by preventing the effect of shockwaves on ß-catenin, Wnt10b mRNA, and PPARγ expression. Low-energy shockwaves suppressed adipocyte differentiation by decreasing PPARγ. Our study suggests an insight into potential uses of shockwave-treatment for obesity.

Characterization of the copper-sensing transcriptional regulator CopR from the hyperthermophilic archeaon Thermococcus onnurineus NA1.

A putative copper ion-sensing transcriptional regulator CopR (TON_0836) from Thermococcus onnurineus NA1 was characterized. The CopR protein consists of a winged helix-turn-helix DNA-binding domain in the amino-terminal region and a TRASH domain that is assumed to be involved in metal ion-sensing in the carboxyl-terminal region. The CopR protein was most strongly bound to a region between its own gene promoter and a counter directional promoter region for copper efflux system CopA. When the divalent metals such as nickel, cobalt, copper, and iron were present, the CopR protein was dissociated from the target promoters on electrophoretic mobility shift assay (EMSA). The highest sensible ion is copper which affected protein releasing under 10 µM concentrations. CopR recognizes a significant upstream region of TATA box on CopR own promoter and acts as a transcriptional repressor in an in vitro transcription assay. Through site-directed mutagenesis of the DNA-binding domain, R34M mutant protein completely lost the DNA-binding activity on target promoter. When the conserved cysteine residues in C144XXC147 motif 1 of the TRASH domain were mutated into glycine, the double cysteine residue mutant protein alone lost the copper-binding activity. Therefore, CopR is a copper-sensing transcriptional regulator and acts as a repressor for autoregulation and for a putative copper efflux system CopA of T. onnurineus NA1.

Repurposing mosloflavone/5,6,7-trimethoxyflavone-resveratrol hybrids: Discovery of novel p38-α MAPK inhibitors as potent interceptors of macrophage-dependent production of proinflammatory mediators.

Herein, we address repurposing hybrids of mosloflavone or 5,6,7-trimethoxyflavone with amide analogs of resveratrol from anticancer leads to novel potent anti-inflammatory chemical entities. To unveil the potent anti-inflammatory molecules, biological evaluations were initiated in LPS-induced RAW 264.7 macrophages at 1 µM concentration. Promising compounds were further evaluated at various concentrations. Multiple proinflammatory mediators were assessed including NO, PGE2, IL-6, TNF-α and IL-1ß. Compound 5z inhibited the induced production of NO, PGE2, IL-6, TNF-α and IL-1ß at the low 1 µM concentration by 44.76, 35.71, 53.48, 29.39 and 41.02%, respectively. Compound 5z elicited IC50 values as low as 2.11 and 0.98 µM against NO and PGE2 production respectively. Compounds 5q and 5g showed potent submicromolar IC50 values of 0.31 and 0.59 µM respectively against PGE2 production. Reverse docking of compound 5z suggested p38-α MAPK, which is a key signaling molecule within the pathways controlling the transcription of proinflammatory mediators, as the molecular target. Biochemical testing confirmed these compounds as p38-α MAPK inhibitors explaining its potent inhibition of proinflammatory mediators' production. Collectively, the results presented 5z as a promising compound for further development of anti-inflammatory agents for treatment of macrophages-and/or immune mediated inflammatory diseases.

Caffeoyloxy-5,6-dihydro-4-methyl-(2H)-pyran-2-one isolated from the leaves of Olinia usambarensis attenuates LPS-induced inflammatory mediators by inactivating AP-1 and NF-κB.

We have previously reported the isolation of four compounds, caffeoyloxy-5,6-dihydro-4-methyl-(2H)-pyran-2-one (CDMP), olinioside, caffeic acid and 3-hydroxylup-12-en-28-oic acid, from the leaves of Olinia usambarensis. Here, we evaluated the inhibitory effects of these compounds on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages, and found that CDMP is the most potent of these two pro-inflammatory mediators (IC50; 12.12 µM and 10.78 µM, respectively). Consistent with these results, CDMP also down-regulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 6 (IL-6) at the protein and mRNA levels in LPS-treated RAW 264.7 macrophages. Furthermore, CDMP suppressed LPS-induced nuclear factor κB (NF-κB) activation by decreasing p65 nuclear translocation through the phosphorylation and degradation of the inhibitory κBα (IκBα). CDMP also attenuated LPS-induced transcriptional and DNA-binding activities of activator protein 1 (AP-1) by suppressing the phosphorylation and expression of c-Fos and c-Jun. Finally, CDMP considerably suppressed the LPS-induced phosphorylation of c-Jun N-terminal kinase (JNK), but did not affect the phosphorylation of p38 or extracellular signal-regulated kinase (ERK). Taken together, our data suggest that CDMP down-regulates genes encoding pro-inflammatory mediators and cytokines, such as iNOS, COX-2, TNF-α, IL-1ß, and IL-6 via NF-κB and JNK/AP-1 inactivation in LPS-induced RAW 264.7 macrophages.

Incarceration of early gravid uterus with adenomyosis and myoma: report of two patients managed with uterine reduction.

Although gravid uterine incarceration is typically diagnosed during the early second trimester, we encountered two unusual cases in early pregnancy. A 34-year-old multiparous woman with adenomyosis presented at 7 + 2 weeks of gestation with increased urinary frequency and a sensation of incomplete bladder emptying. The uterine incarceration was successfully reduced by manual reduction and pessary insertion, and she delivered a normal infant at term. In the second case, a 31-year-old nulliparous woman with a large myoma complained of dysuria, acute urinary retention, and intense back pain at 6 weeks of gestation. Manual reduction was successful in the knee-chest position. Subsequent pessary insertion failed; however, a slight reduction in pain was achieved. After a week, the fetus spontaneously aborted. In summary, gravid uterine incarceration is a rare but potentially fatal condition for the fetus, and a suspicion of this condition in patients with urinary symptoms, especially urinary retention and pelvic pain, is important in the early gestation period.