Angio-Net: deep learning-based label-free detection and morphometric analysis of in vitro angiogenesis.

Despite significant advancements in three-dimensional (3D) cell culture technology and the acquisition of extensive data, there is an ongoing need for more effective and dependable data analysis methods. These concerns arise from the continued reliance on manual quantification techniques. In this study, we introduce a microphysiological system (MPS) that seamlessly integrates 3D cell culture to acquire large-scale imaging data and employs deep learning-based virtual staining for quantitative angiogenesis analysis. We utilize a standardized microfluidic device to obtain comprehensive angiogenesis data. Introducing Angio-Net, a novel solution that replaces conventional immunocytochemistry, we convert brightfield images into label-free virtual fluorescence images through the fusion of SegNet and cGAN. Moreover, we develop a tool capable of extracting morphological blood vessel features and automating their measurement, facilitating precise quantitative analysis. This integrated system proves to be invaluable for evaluating drug efficacy, including the assessment of anticancer drugs on targets such as the tumor microenvironment. Additionally, its unique ability to enable live cell imaging without the need for cell fixation promises to broaden the horizons of pharmaceutical and biological research. Our study pioneers a powerful approach to high-throughput angiogenesis analysis, marking a significant advancement in MPS.

Pleurospermum kamtschaticum extract induces apoptosis via mitochondrial pathway and NAG-1 expression in colon cancer cells.

To evaluate the anticarcinogenic activity of methanol extract of Pleurospermum kamtschaticum (PKE), we assessed its apoptosis-inducing capability in HT-29 colon carcinoma cells. PKE treatment for 2 h reduced cell viability in a dose-related manner, and induced apoptotic morphological changes. Flow cytometric analysis indicated that PKE treatment at 0.05 mg/ml induced early apoptosis in 66.2% of HT-29 cells. Additionally, Bcl-2 expression was substantially reduced in PKE-treated HT-29 cells, increasing the Bax/Bcl-2 ratio. The protein levels of procaspase-9 and procaspase-3 were decreased markedly, reflecting caspase-9 and caspase-3 activation, and resulting PARP cleavage was noted in the PKE-treated HT-29 cells. Furthermore, we detected increased NAG-1 expression in the PKE-treated HT-29 cells. In an in vivo study, intraperitoneal PKE administration suppressed the formation of tumor nodules in the lungs of mice. These results indicate that PKE can serve as a beneficial supplement in the treatment and the prevention of colon cancer.

Anti-ulcerogenic effect and HPLC analysis of the caffeoylquinic acid-rich extract from Ligularia stenocephala.

The leaves of three Ligularia species belonging to the family Compositae, Ligularia stenocephala, L. fischeri, and L. fischeri var. spiciformis, were qualitatively and quantitatively analyzed on the caffeoylquinic acids by HPLC and subjected to peroxynitrite-scavenging assay. The IC(50) of the MeOH extract of L. stenocephala was 1.62+/-0.03 mug/ml and the major caffeoylquinic acids of L. stenocephala were 5-O-caffeoylquinic acid, 3,5-di-O-caffeoyl-muco-quinic acid, and 3,5-di-O-caffeoylquinic acid. The compositions of caffeoylquinic acids were different for the three plants. Since percentage of total caffeoylquinic acids of the extract was highest (42.20% of the MeOH extract and 94.52% of the BuOH extract) in L. stenocephala and potent in peroxynitrite-scavenging assay, the extracts of L. stenocephala were chosen to perform in vivo anti-ulcerogenic activity. Treatment of mice with the MeOH- and BuOH extracts decreased the diameter of gastric lesions caused by HCl/ethanol- and indomethacin/bethanechol and decreased the volume of gastric juice, suggesting that caffeoylquinic acids have anti-ulcerogenic activity. These results suggest that the leaves of Ligularia species may help prevent or treat gastric ulcers.

Qualitative and quantitative determination of the caffeoylquinic acids on the Korean mountainous vegetables used for chwinamul and their peroxynitrite-scavenging effect.

Mountainous vegetables called chwinamul are used in Korea to promote health. Chwinamul was obtained from several plants belonging to the Compositae - e.g., Kalimeris yomena, Aster scaber, Solidago virga var. gigantea, Solidago viragaurea var. asiatica, Saussurea grandifolia, Ainsliaea acerifolia - were used for our experiments. Analytical methods for simultaneous determination of the caffeoylquinic acids (3,4-di-O-caffeoylquinic acid, 3,5-di-O-dicaffeoyl-epi-quinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3-O-p-coumaroyl-caffeoylquinic acids) were established for chwinamul. The kinds of constituents were identified from HPLC chromatograms and it was possible to calculate the percentage (w/w) of seven of these compounds in the dried plants and in the extracts. The proportion of caffeoylquinic acids in the extracts ranged from 20.25 to 38.35%. Since it is known that peroxynitrite (ONOO(-))-scavenging is beneficial for amelioration of obesity, diabetes mellitus, atherosclerosis and even Alzheimer's disease, assays for peroxynitrite-scavenging activity were performed on the seven chwinamul plants. Of the tested extracts, the MeOH extract of A. acerifolia had the most potent effect (IC(50) 1.49 +/- 0.68 microg/mL). These results suggest that chwinamul vegetables can be used for treatment or prevention of peroxynitrite-related diseases.

The MeOH extract of Pleurospermum kamtschaticum and its active component buddlejasaponin (IV) inhibits intrinsic and extrinsic hyperlipidemia and hypercholesterolemia in the rat.

The inhibitory effect of the MeOH extract of Pleurospermum kamtschaticum (Umbelliferase) and its fractions were tested in hyperlipidemic and hypercholesterolemic rats using four animal models induced using poloxamer-407 or using Triton WR-1339 as intrinsic inducers and by 30% corn oil or high cholesterol diet as extrinsic inducers. We measured serum triglyceride, total cholesterol, HDL-cholesterol and LDL-cholesterol levels as measures of its hypocholesterolemic or hypolipidemic effects. Since the MeOH extract and the BuOH fraction of Pleurospermum kamtschaticum were found to be active using these four hypolipidemic assays, its major saponin buddlejasaponin IV {BS(IV)} isolated from the BuOH fraction were also tested to demonstrate the active components. BS(IV) was found to significantly inhibit hypercholesterolemia and hyperlipidemia by extrinsic and intrinsic inducers. In particular, BS(IV) reduced the blood thiobarbituric acid reactive substance (TBARS) and hydroxy radical levels, and increased superoxide dismutase activity in high cholesterol diet-induced rats, thus suggesting that BS(IV) reduces oxidative stress caused by a high cholesterol diet. Moreover, these effects of BS(IV) were comparable to probucol, which was used as a positive control. These results suggested that Pleurospermum kamtschaticum which is traditionally used to treat atherosclerosis and its active major saponin BS(IV) could be used to treat hypercholesterolemia or hyperlipidemia.

Enhanced immunogenicity and protective efficacy with the use of interleukin-12-encapsulated microspheres plus AS01B in tuberculosis subunit vaccination.

Tuberculosis subunit vaccines codelivered with interleukin-12 (IL-12)-encapsulated microspheres (IL-12EM) are designed for a sustained release of IL-12 and could induce strong Th1 immune responses specific to Ag85A and ESAT-6. The adjuvant combination of IL-12EM plus AS01B was a more efficient way to induce a sustained Th1 immunity and protection against Mycobacterium tuberculosis.

Isolation of saponins with the inhibitory effect on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha production from Pleurospermum kamtschaticum.

As an attempt to search for bioactive natural products exerting antiinflammatory activity, we have isolated two saponins were isolated from the aerial portion of Pleurospermum kamtschaticum (Umbelliferae) by nitrite assay activity-directed chromatographic fractionation. They were identified as saikogenin F 3-O-{beta-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->3)]-beta-D-fucopyranoside} (buddlejasaponin IV, 1) and 3beta,16beta,23,28-tetrahydroxy-11alpha-methoxyolean-12-ene 3-O-{beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-beta-D-fucopyranoside} (buddlejasaponin IVa, 2). Compound 1 significantly inhibited nitric oxide (NO) production, and it also significantly decreased prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) release in the lipopolysaccharide (LPS)-activated macrophage Raw 264.7 cells whereas compound 2 was much less active. Saikogenin A (3) and -H (4) were obtained by hydrolyzing 1 and 2. Although these sapogenin showed strong NO inhibition, these effects were caused by the cytotoxic effect on Raw 264.7 cells. These results supported the notion that buddlejasaponin IV is a major inhibitors of NO, PGE2 and TNF-alpha production in P. kamtschaticum.

In vivo antihepatotoxic effects of Ligularia fischeri var. spiciformis and the identification of the active component, 3,4-dicaffeoylquinic acid.

Pretreatment with a methanolic extract of Ligularia fischeri var. spiciformis (Compositae) herb inhibited hepatotoxicities caused by CCl4, D-galactosamine (GalN), alpha-naphthylisothiocyanate (ANIT), and DL-ethionine in rats. An ethyl acetate (EtOAc) extract fractionated from the methanolic extract showed a strong inhibitory effect. A major component, 3,4-dicaffeoylquinic acid (DCQA), isolated from the methanolic extract was examined for antihepatotoxicity. Pretreatment with DCQA (5 and 10 mg/kg, p.o.) significantly reduced serum aminotransferases (alanine and aspartate), sorbitol dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, and lactate dehydrogenase activities during CCl4- or GalN-induced hepatotoxicity, suggesting that DCQA is a major principle for the antihepatotoxic activity of L. fischeri var. spiciformis. DCQA also partially restored bile flow and reduced total bilirubin and cholic acid concentrations in rats with ANIT-induced cholestasis. Treatment with DCQA inhibited the increase in triglyceride, cholesterol, and total lipids in DL-ethionine-induced fatty liver. These results support the traditionally held belief that this plant can be used for the treatment of jaundice and hepatic failure.

Primary carcinoid tumor of the bilateral testis associated with carcinoid syndrome.

Carcinoid tumors derived from neuroendocrine cells can release serotonin and other vasoactive substances into the systemic circulation, resulting in carcinoid syndrome. Testicular carcinoid, a rare disease accounting for less than 1% of all testicular neoplasms, rarely manifests symptoms of carcinoid syndrome. We describe a case of carcinoid syndrome arising from a primary testicular carcinoid tumor. A 21-year-old male patient presented with facial flushing and diarrhea for 5 years. He had an enlarged left testis and a 1-cm, ill-defined, hard, non-tender mass in his right testis. His 24 h urinary excretion of 5-hydroxyindoleacetic acid was elevated (16.1 mg/day). Somatostatin receptor scintigraphy correlated with carcinoid tumor in both testes. Following bilateral orchiectomy, the patient's facial flushing and diarrhea disappeared.

Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease.

This phase 1 clinical trial tested the safety of intramuscular gene transfer by using naked plasmid DNA encoding the gene for VEGF, and analyzed the potential therapeutic benefits in patients with severe peripheral arterial disease (PAD). This study was an open-labeled, dose- escalating, single-center trial on nine male patients with severe debilitating PAD who had not responded to conventional therapy. Seven had Buerger's disease and two had arteriosclerosis obliterans. Plasmid DNA (pCK) containing human VEGF165 was given by eight intramuscular injections in and around the area in need of new blood vessels. The study evaluated three escalating total doses (2, 4, and 8 mug of pCK- VEGF165), with half of each total dose given four weeks apart. The follow-up duration was nine months. The gene injections were well tolerated without significant side effects or laboratory abnormalities related to gene transfer. Three patients showed transient edema in their extremities. Ischemic pain of the affected limb was relieved or improved markedly in six of seven patients. Ischemic ulcers healed or improved in four of six patients. The mean ankle-brachial index (ABI) improved significantly. Six of nine patients showed an increase in collateral vessels around the injection sites demonstrated by digital subtraction angiography. However, there was no relationship between the degree of ABI improvement and the dose given. Mean plasma levels of VEGF did not increase significantly. In conclusion, intramuscular injections of pCK- VEGF165 can be performed safely to induce therapeutic angiogenesis in patients with severe PAD.

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, induces cell cycle arrest in ras-transformed human mammary epithelial cells.

Extracts of Artemisia asiatica Nakai (Asteraceae) possess anti-inflammatory and anti-oxidative activities. Eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone), one of the pharmacologically active ingredients derived from A. asiatica, was shown to induce apoptosis in human promyelocytic leukemia (HL-60) cells [Mutat Res 496 (2001) 191]. In the present study, we examined the cytostatic effects of eupatilin in H-ras-transformed human breast epithelial (MCF10A-ras) cells. Eupatilin inhibited the growth of MCF10A-ras cells in a concentration-dependent and time-related manner. To explore whether the anti-proliferative effects of eupatilin could be mediated through modulation of the cell cycle in MCF10A-ras, DNA contents were analyzed by the flow cytometry. Eupatilin inhibited the expression of cyclin D1, cyclin B1, Cdk2 and Cdc2 that are key regulators of the cell cycle. In addition, eupatilin treatment led to elevated expression of p53 and p27Kip1 that act as Cdk inhibitors. It has been known that the Ras-signaling pathway plays integral roles in the induction of cyclin D1. Eupatilin inhibited the activation of ERK1/2 as well as expression of Raf-1 and Ras in MCF10A-ras cells. Thus, the inhibitory effect of eupatilin on cyclin D1 expression appears to be mediated by targeting the Raf/MEK/ERK signaling cascades. Eupatilin did not change activation of Akt, an important component of cell-survival pathways. In conclusion, the anti-proliferative effect of eupatilin in MCF10A-ras cells is associated with its blockade of cell cycle progression which appears to be attributable in part to inhibition of ERK1/2 activation.

Sodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alpha.

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.

Monitoring of peptide acylation inside degrading PLGA microspheres by capillary electrophoresis and MALDI-TOF mass spectrometry.

The purpose of this research was to assess the acylation reactions of peptides, salmon calcitonin (sCT), human parathyroid hormone 1-34 (hPTH1-34) and leuprolide, in poly(lactic-co-glycolic acid) (PLGA) microspheres. Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for determining and monitoring peptide acylation and quantitating acylation products in the degrading PLGA microspheres. In the degrading PLGA microspheres of sCT and hPTH1-34, the acylation products were observed and determined to be adducts with glycolic acid units from degradable PLGA polymer by MALDI-TOF MS. In the microsphere of leuprolide, however, the acylation product was not observed even after 28 days of incubation at the release medium, which represents the different stabilities among peptides according to the primary structure. As the leuprolide contains tyrosine and serine having hydroxyl group of nucleophilic amino acids, the acylation reaction of peptide is shown to be mainly due to the primary amino groups of N-terminus or lysine residue. The complementary use of CE and MALDI-TOF MS will be useful for searching the counter measures as well as determining the peptide acylation in the manufactured formulations on the market.

In vivo kinetics and biodistribution of a HIV-1 DNA vaccine after administration in mice.

In this study we have investigated the pharmacokinetics and tissue distribution of GX-12, a multiple plasmid DNA vaccine for the treatment of HIV-1 infection. Plasmid DNA was rapidly degraded in blood with a half-life of 1.34 min and was no longer detectable at 90 min after intravenous injection in mice. After intramuscular injection, plasmid DNA concentration in the injection site rapidly declined to less than 1% of the initial concentration by 90 min post-injection. However, sub-picogram levels (per mg tissue) were occasionally detected for several days after injection. The relative proportions of the individual plasmids of GX-12 remained relatively constant at the injection site until 90 min post-injection. The concentration of plasmid DNA in tissues other than the injection site peaked at 90 min post-injection and decreased to undetectable levels at 8 h post-injection. The rapid in vivo degradation of GX-12 and absence of persistence in non-target tissues suggest that the risk of potential gene-related toxicities by GX-12 administration, such as expression in non-target tissues, insertional mutagenesis and germline transmission, is minimal.

Inhibitory effects of the standardized extract (DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide synthase expression and nuclear transcription factor kappa B activation in mouse skin.

Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.