Vascular Damage and Repair - Are Small-Diameter Vascular Grafts Still the "Holy Grail" of Tissue Engineering?

Cardiovascular diseases are the most important cause of morbidity and mortality in the civilized world. Stenosis or occlusion of blood vessels leads not only to events that are directly life-threatening, such as myocardial infarction or stroke, but also to a significant reduction in quality of life, for example in lower limb ischemia as a consequence of metabolic diseases. The first synthetic polymeric vascular replacements were used clinically in the early 1950s. However, they proved to be suitable only for larger-diameter vessels, where the blood flow prevents the attachment of platelets, pro-inflammatory cells and smooth muscle cells on their inner surface, whereas in smaller-diameter grafts (6 mm or less), these phenomena lead to stenosis and failure of the graft. Moreover, these polymeric vascular replacements, like biological grafts (decellularized or devitalized), are cell-free, i.e. there are no reconstructed physiological layers of the blood vessel wall, i.e. an inner layer of endothelial cells to prevent thrombosis, a middle layer of smooth muscle cells to perform the contractile function, and an outer layer to provide innervation and vascularization of the vessel wall. Vascular substitutes with these cellular components can be constructed by tissue engineering methods. However, it has to be admitted that even about 70 years after the first polymeric vascular prostheses were implanted into human patients, there are still no functional small-diameter vascular grafts on the market. The damage to small-diameter blood vessels has to be addressed by endovascular approaches or by autologous vascular substitutes, which leads to some skepticism about the potential of tissue engineering. However, new possibilities of this approach lie in the use of modern technologies such as 3D bioprinting and/or electrospinning in combination with stem cells and pre-vascularization of tissue-engineered vascular grafts. In this endeavor, sex-related differences in the removal of degradable biomaterials by the cells and in the behavior of stem cells and pre-differentiated vascular cells need to be taken into account. Key words: Blood vessel prosthesis, Regenerative medicine, Stem cells, Footprint-free iPSCs, sr-RNA, Dynamic bioreactor, Sex-related differences.

Synthesis and SAR investigation of biphenylaminoquinoline derivatives with benzyloxy substituents as promising anticancer agents.

The biphenyl scaffold represents a prominent privileged structure within the realms of organic chemistry and drug development. Biphenyl derivatives have demonstrated notable biological activities, including antimicrobial, anti-inflammatory, anti-HIV, and the treatment of neuropathic pain. Importantly, their anticancer abilities should not be underestimated. In this context, the present study involves the design and synthesis of a series of biphenyl derivatives featuring an additional privileged structure, namely the quinoline core. We have also diversified the substituents attached to the benzyloxy group at either the meta or para position of the biphenyl ring categorized into two distinct groups: [4,3']biphenylaminoquinoline-substituted and [3,3']biphenylaminoquinoline-substituted compounds. We embarked on an assessment of the cytotoxic activities of these derivatives in colorectal cancer cell line SW480 and prostate cancer cell line DU145 for exploring the structure-activity relationship. Furthermore, we determined the IC50 values of selected compounds that exhibited superior inhibitory effects on cell viability against SW480, DU145 cells, as well as MDA-MB-231 and MiaPaCa-2 cells. Notably, [3,3']biphenylaminoquinoline derivative 7j displayed the most potent cytotoxicity against these four cancer cell lines, SW480, DU145, MDA-MB-231, and MiaPaCa-2, with IC50 values of 1.05 µM, 0.98 µM, 0.38 µM, and 0.17 µM, respectively. This highly promising outcome underscores the potential of [3,3']biphenylaminoquinoline 7j for further investigation as a prospective anticancer agent in future research endeavors.

Enhancing precision in lung tumor ablation through innovations in CT-guided technique and angle control.

BACKGROUND: This retrospective study aimed to evaluate the precision and safety outcomes of image-guided lung percutaneous thermal ablation (LPTA) methods, focusing on radiofrequency ablation (RFA) and microwave ablation (MWA). The study utilized an innovative angle reference guide to facilitate these techniques in the treatment of lung tumors. METHODS: This study included individuals undergoing LPTA with the assistance of laser angle guide assembly (LAGA) at our hospital between April 2011 and March 2021. We analyzed patient demographics, tumor characteristics, procedure details, and complications. Logistic regressions were employed to assess risk factors associated with complications. RESULTS: A total of 202 patients underwent ablation for 375 lung tumors across 275 sessions involving 495 ablations. Most procedures used RFA, especially in the right upper lobe, and the majority of ablations were performed in the prone position (49.7%). Target lesions were at a median depth of 39.3 mm from the pleura surface, and remarkably, 91.9% required only a single puncture. Complications occurred in 31.0% of ablations, with pneumothorax being the most prevalent (18.3%), followed by pain (12.5%), sweating (6.5%), fever (5.0%), cough (4.8%), hemothorax (1.6%), hemoptysis (1.2%), pleural effusion (2.0%), skin burn (0.6%), and air emboli (0.2%). The median procedure time was 21 min. Notably, smoking/chronic obstructive pulmonary disease emerged as a significant risk factor for complications. CONCLUSION: The LAGA-assisted LPTA enhanced safety by improving accuracy and reducing risks. Overall, this investigation contributes to the ongoing efforts to refine and improve the clinical application of these thermal ablation techniques in the treatment of lung tumors.

Novel 4-aminoquinoline analogs targeting the HIF-1α signaling pathway.

Background: The aminoquinoline core exhibits versatile pharmacological properties, particularly in the area of anticancer activity. This study was designed to investigate the potential of the 4-aminoquinoline scaffold in the development of anticancer agents by targeting the HIF-1α signaling pathway. Methodology: The authors synthesized multiple derivatives of 4-aminoquinoline containing heterocyclic rings by a microwave reactor and assessed the cytotoxicity and inhibitory effects of these derivatives on the HIF-1α signaling pathway. Conclusion: Compound 3s was identified as the most promising HIF-1α inhibitor due to its exceptional antiproliferative effects, with IC50 values of 0.6 and 53.3 nM observed in MiaPaCa-2 and MDA-MB-231 cells, respectively. Furthermore, compound 3s was found to inhibit HIF-1α expression by decreasing the level of HIF-1α mRNA.

The Association between Cholesterol Levels and Severity of Normal Tension Glaucoma.

Purpose: To evaluate the serum lipid levels, including total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) of patients with normal tension glaucoma (NTG) and to investigate the relationship between serum HDL levels and the severity of NTG. Methods: In this cross-sectional, case-control study, 282 NTG subjects and 202 control subjects were enrolled from the outpatient clinic of the Department of Ophthalmology at Taichung Veterans General Hospital in central Taiwan from 2015 to 2021. Fasting cholesterol, HDL, and LDL levels were evaluated using a biochemical analyzer (ARCHITECT c16000). Glaucoma severity was classified by visual field test as mild (mean deviation [MD] ≥ -6.0dB), moderate (-12dB ≤ MD < -6 dB), and severe (MD < -12dB), based on the mean deviation. Results: HDL levels were significantly lower in the NTG group compared with the control subjects (47 ± 18mg/dl versus 53 ± 18mg/dl; p = 0.03). There were no statistically significant differences in total cholesterol or LDL levels between the NTG and control subjects (total cholesterol levels: 194 ± 39mg/dl versus 190 ± 32mg/dl; p > 0.05; LDL levels: 113 ± 30mg/dl versus 110 ± 29mg/dl; p > 0.05). The mean serum HDL levels were lowest in the severe group (41 ± 11mg/dl) followed by the moderate (45 ± 16mg/dl) and mild (50 ± 15mg/dl) groups, with significant differences among the three groups (p = 0.02). The multivariate regression analysis revealed a statistically significant negative correlation between HDL and vertical cup-to-disc ratio (VCDR; B =-0.16, p = 0.03) among all NTG patients and a positive correlation between HDL and retinal nerve fiber layer (RNFL; r = 0.34, p = 0.03) among all NTG patients. Conclusions: A significantly lower serum HDL concentration was found in the NTG patients, which was negatively associated with disease severity. The findings warrant further study to elucidate the role of these phenomena in the pathogenesis of glaucoma.

Effect of Muller's muscle-conjunctival resection on the upper eyelid crease position in Asian eyelids: a retrospective cohort study.

PURPOSE: Investigating the effect of Muller's muscle-conjunctival resection (MMCR) on the eyelid crease position. METHODS: This retrospective study included patients with unilateral acquired blepharoptosis who underwent MMCR during October 2018-December 2021. The following factors were recorded: preoperative, after phenylephrine, postoperative marginal reflex distance1 (MRD1) and tarsal platform show (TPS) of bilateral eyelids. The primary outcome was to measure the change in TPS and evaluate the factors associated with post-operative TPS. The secondary outcomes included exploring the rate of MRD1 and TPS symmetry after the operation. RESULTS: Forty patients were included in the final analysis. The mean MRD1 of the ptotic eye was 1.28 ± 0.78 mm, 2.79 ± 0.66 mm and 3.20 ± 0.67 mm before, after phenylephrine and after the operation, respectively. The mean TPS of the ptotic eye was 5.90 ± 1.86 mm, 3.96 ± 1.49 mm and 2.79 ± 1.63 mm before, after phenylephrine and after the operation, respectively. Changes in mean TPS after the phenylephrine test and post-operation were statistically significant (p < 0.001). The linear regression model revealed that the absolute change in TPS after phenylephrine drop and absolute change in MRD1 post-operation were significantly correlated with the absolute change in TPS post-operation. Besides, the ratio of symmetry in MRD1 and TPS was greatly improved post-operation (82.5% and 70.0% respectively). CONCLUSION: MMCR is an effective surgical method for ptosis correction as it can not only correct the eyelid crease position but also narrow the wide TPS. This method is particularly beneficial to patients with both mild to moderate ptosis and an asymmetric crease height.

Golgin Imh1 and GARP complex cooperate to restore the impaired SNARE recycling transport induced by ER stress.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which acts through various mechanisms to reduce ER stress. While the UPR has been well studied for its effects on the ER, its impact on the Golgi is less understood. The Golgi complex receives transport vesicles from the endosome through two types of tethering factors: long coiled-coil golgin and the multisubunit Golgi-associated retrograde protein (GARP) complex. Here, we report that ER stress increases the phosphorylation of golgin Imh1 to maintain the GARP-mediated recycling of the SNAREs Snc1 and Tlg1. We also identify a specific function of the Golgi affected by ER stress and elucidate a homeostatic response to restore this function, which involves both an Ire1-dependent and a MAP kinase Slt2/ERK2-dependent mechanism. Furthermore, our findings advance a general understanding of how two different types of tethers act cooperatively to mediate a transport pathway.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

Synthesis and evaluation of biarylquinoline derivatives as novel HIF-1α inhibitors.

Hypoxia-inducible factor (HIF)-1α is a key transcription factor that contributes to aggressive and drug-resistant phenotypes in tumor cells under hypoxic conditions. Therefore, targeting HIF-1α represents a promising therapeutic strategy for cancer drug development. In the present study, we designed, synthesized, and evaluated a new series of biarylquinoline derivatives as potential HIF-1α inhibitors based on structure-activity relationship. Among these derivatives, compound 7f represents the optimal agent with IC50 values of 28 nM and 15 nM in suppressing the viability of MiaPaCa-2 and MDA-MB-231 cells, respectively. Compound 7f also exhibited potent efficacy in inhibiting hypoxia-induced migration of MDA-MB-231 and MiaPaCa-2 cells. Mechanistically, compound 7f suppressed HIF-1α expression by blocking transcription and protein translation, in lieu of facilitating protein degradation. Moreover, this HIF-1α downregulation was associated with compound 7f's ability to concomitantly inhibit multiple signaling pathways governing HIF-1 α expression at different levels, including those mediated by STAT3, MEK/ERK MAPK, and mTOR/4E-BP1. Together, these findings underscore the translational potential of these biarylquinoline derivatives to be developed as novel HIF-1α inhibitors, which warrants further investigations.

Shifting from postauricular to transcanal microscopic tympanoplasty may have similar frequency-specific improvements with better air-bone-gap closure at low frequencies and a minimal learning-curve effect.

The shift from postauricular to transcanal microscopic tympanoplasty brings potential advantages of minimal morbidity, less postoperative pain, patient comfort, and surgical ease and speed, but also uncertainties of unfamiliar grafting material, an inadequate operation view, and an uncertain learning curve. These challenges might affect the successful repair rate and the frequency-specific hearing outcome, which is important for hearing perception. Rare studies reported frequency-specific hearing outcome with the learning curve for shifting from postauricular to transcanal microscopic tympanoplasty. Here, from Jul. 2013 to Nov. 2018, we compared patients in a shift from postauricular approach (35 ears) to transcanal approach (35 ears) of microscopic type-1 tympanoplasty. The results show that both of postauricular and transcanal microscopic tympanoplasties reduced the mean air-bone gap, 0.5k Hz gap, and 1k Hz gap after the surgery. The further analyses on gap change as a function of frequency (0.5, 1, 2, and 4k Hz) show that both of postauricular and transcanal tympanoplasties improved postoperative air-bone gap among the levels of frequency. The post hoc comparisons display a common gap reduction difference between 0.5k and 4k Hz. The successful repair rate did not differ between the 2 groups. There was no correlation between the postoperative mean gap change and the surgery date, suggesting a minimal learning-curve effect. The results of similar frequency-specific improvements and a minimal learning-curve effect may help to ease the concerns of those uncertainties before the shift.

Novel HIF-1α inhibitor CDMP-TQZ for cancer therapy.

Aim: Tumor cells adapt to hypoxic microenvironments by releasing the key transcription factor HIF-1α, which promotes angiogenesis, glycolytic phenotype, metastasis and erythropoiesis, allowing proliferation amid low oxygen levels. Therefore, therapeutic targeting of HIF-1α represents a viable strategy for cancer therapy. Methods & Results: The authors synthesized a series of novel tetrahydroquinazoline derivatives in six steps and demonstrated that their development had a unique ability to suppress HIF-1α expression through proteasomal degradation. Conclusion: Among these compounds, CDMP-TQZ (8bf) exhibited the highest antiproliferative potency in human cancer cells, in part through downregulation of HIF-1α.

Bone-conduction threshold and air-bone gap may predict frequency-specific air-conduction threshold after tympanoplasty.

Postoperative hearing improvement is one of the main expectations for patients receiving tympanoplasty. The capacity to predict postoperative hearing may help to counsel a patient properly and avoid untoward expectations. It is difficult to predict postoperative hearing without knowing the disease process in the middle ear, which can only be assessed intraoperatively. However, the duration and extent of the underlying pathologies may represent in bone-conduction threshold and air-bone gap. Here in patients undergoing tympanoplasty without ossiculoplasty, we sorted and separated the surgery dates into the first group to build the predicting models and the second group to test the predictions. There were 87 and 30 ears, respectively. No specific enrollment or exclusion criteria were based on underlying pathologies such as the perforation size of the tympanic membrane or the middle ear conditions. The results show that bone-conduction threshold and air-bone gap together predicted air-conduction threshold after the surgery, including each frequency of 0.5k, 1k, 2k, and 4k Hz. The discrepancies between the predictions and recordings did not differ among these four frequencies. Of the variance in mean postoperative air-conduction threshold, 56.7% was linearly accounted for by these two preoperative predictors in this sample. The results suggest a trend that, the higher the frequency, the larger the part was accounted for by these two preoperative predictors. These together may help a surgeon to estimate frequency-specific hearing outcome after the surgery, answer patients' questions with quantitative statistics, and counsel patients with proper expectations.

Development of Novel Rhodacyanine-Based Heat Shock Protein 70 Inhibitors.

BACKGROUND: A growing body of evidence suggests that Hsp70, which is overexpressed in human breast tumors, plays a role in tumorigenesis and tumor progression in breast cancer as well as in its aggressive phenotypes. Hsp70 constitutes a potential therapeutic target in the treatment of this disease. METHODS: We developed a new series of rhodacyanine-based Hsp70 inhibitors, represented by compounds 1 and 6, in which the cationic pyridin-1-ium or thiazol-3-ium ring of existing Hsp70 inhibitors (e.g., JG-40 and JG-98) was replaced by a corresponding benzo- fused N-heterocycle. RESULTS: Several lines of evidence suggest that these benzo-fused derivatives may exert their antitumor activities, in part, by targeting Hsp70. These putative inhibitors displayed differential antiproliferative efficacy against breast cancer cells (IC50 as low as 0.25 µM) versus nontumorigenic MCF-10A breast epithelial cells (IC50 ≥ 5 µM). This was correlated with the corresponding Hsp70 expression levels. Using a protein refolding assay, we confirmed that these agents effectively inhibited the chaperone activity of Hsp70. Moreover, these inhibitors effectively suppressed the expression of well-known oncogenic client proteins of Hsp70's, including FoxM1, HuR, and Akt, which paralleled their antiproliferative efficacy. Supporting the established role of Hsp70 in regulating protein refolding, these derivatives induced autophagy, as manifested by the conversion of LC3B-I to LC3B-II. Notably, these putative Hsp70 inhibitors did not cause a compensatory elevation in Hsp90 expression, contrasting with the previously reported effects of Hsp90 inhibitors on Hsp70 upregulation. CONCLUSION: Together with the finding that compounds 1 and 6 showed improved microsomal stability, these results suggest the translational potential of these putative Hsp70 inhibitors to foster new strategies for cancer therapy. However, whether these benzo-fused rhodacyanines act on kinases or other targets remains unclear. It is currently under investigation.

Synthesis and biological evaluation of thiophenylbenzofuran derivatives as potential P-glycoprotein inhibitors.

P-Glycoprotein (P-gp) overexpression is a major mechanism by which cancer cells acquire the multidrug resistance (MDR) phenotype, and is associated with poor clinical outcome in patients. In an effort to develop MDR-reversal agents, we synthesized and evaluated a series of thiophenylbenzofuran derivatives (4-31) as P-gp inhibitors, among which compounds 4, 10, and 14 represented the optimal agent in reversing the MDR phenotype. These P-gp inhibitors were dramatically effective than verapamil in sensitizing the human ABCB1-overexpressing ABCB1/Flp-In™-293 cells and MDR KBvin cells to a series of chemotherapeutic agents, including vincristine and paclitaxel, as manifested by multi-fold decreases in the respective IC50 values to therapeutically attainable levels.

Repeated intravitreal injections of antivascular endothelial growth factor in patients with neovascular age-related macular degeneration may increase the risk of ischemic optic neuropathy.

BACKGROUND: Previous case reports have demonstrated the occurrence of ischemic optic neuropathy (ION) following intravitreal injections of antivascular endothelial growth factor (anti-VEGF). However, no previous studies have investigated the impact of injection numbers on the risk of ION. The aim of our study was to investigate whether repeated intravitreal injections of anti-VEGF would increase the risk of subsequent ION in patients with neovascular age-related macular degeneration (AMD). METHODS: A population-based, retrospective cohort study using the Taiwan National Health Insurance Research Database was conducted from 2007 to 2013. Neovascular AMD patients receiving intravitreal injections of anti-VEGF during the study period were enrolled in the study cohort. Enrollees were divided into three groups according to the categorized levels of injection number (first level: < 10 times, second level: 10-15 times, and third level: > 15 times). Kaplan-Meier curves were generated to compare the cumulative hazard of subsequent ION among the three groups. Cox regression analyses were used to estimate crude and adjusted hazard ratios (HRs) for ION development with respect to the different levels of injection numbers. The confounders included for adjustment were age, sex, and comorbidities (diabetes, hypertension, hyperlipidemia, ischemic heart disease, and glaucoma). RESULTS: In total, the study cohort included 77,210 patients. Of these, 26,520, 38,010, and 12,680 were in the first-, second-, and third-level groups, respectively. The Kaplan-Meier method revealed that the cumulative hazards of ION were significantly higher in those who had a higher injection number. After adjusting for confounders, the adjusted HRs for ION in the second- and third-level groups were 1.91 (95% confidence interval [CI], 1.32-2.76) and 2.20 (95% CI, 1.42-3.43), respectively, compared with those in the first-level group. CONCLUSIONS: Among patients with neovascular AMD, those who receive a higher number of anti-VEGF injections have a significantly higher risk of developing ION compared with individuals who receive a lower number of injections.

Genetic inhibition of PKCε attenuates neurodegeneration after global cerebral ischemia in male mice.

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.

PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration.

BACKGROUND: Global cerebral ischemia triggers neurodegeneration in the hippocampal CA1 region, but the mechanism of neuronal death remains elusive. The epsilon isoform of protein kinase C (PKCε) has recently been identified as a master switch that controls the nucleocytoplasmic trafficking of ATF2 and the survival of melanoma cells. It is of interest to assess the role of PKCε-ATF2 signaling in neurodegeneration. RESULTS: Phosphorylation of ATF2 at Thr-52 was reduced in the hippocampus of PKCε null mice, suggesting that ATF2 is a phosphorylation substrate of PKCε. PKCε protein concentrations were significantly reduced 4, 24, 48 and 72 h after transient global cerebral ischemia, resulting in translocation of nuclear ATF2 to the mitochondria. Degenerating neurons staining positively with Fluoro-Jade C exhibited cytoplasmic ATF2. CONCLUSIONS: Our results support the hypothesis that PKCε regulates phosphorylation and nuclear sequestration of ATF2 in hippocampal neurons during ischemia-induced neurodegeneration.

Hyaluronic Acid-Based Multilayer Films Regulate Hypoxic Multicellular Aggregation of Pancreatic Cancer Cells with Distinct Cancer Stem-Cell-like Properties.

In vitro spherical cancer models have been widely used in cancer stem cell (CSC) research, and the ability of CSCs to form multicellular colonies is recognized as a morphological marker. However, although several spherical/colony models share a common three-dimensional (3D) conformation, each model displays its own intrinsic properties. Thus, the CSC phenotypes with distinct multicellular aggregate morphologies must be defined and clarified. Here, a novel 3D model was designed to regulate the type of pancreatic CSC colonies that form using niche mimetic hyaluronic acid (HA)-based multilayer nanofilms and hypoxia. The multicellular aggregate morphology, CSC phenotypes, CSC-related marker expression, cell cycle, invasion, and drug resistance were determined. On the basis of the results of a cell morphology analysis, colonies formed on multilayer nanofilms in response to both normoxia and hypoxia, but round and island-type colonies, were investigated. Immunostaining results revealed a significantly higher expression of stem cell markers, such as OCT4, CXCR4, and CD44v6, in colonies that formed on multilayer nanofilms. These colonies also expressed higher levels of E-cadherin, hypoxia-inducible factor-1α, and vimentin, particularly the round-type colonies that formed on HA-based multilayer nanofilms, [poly(allylamine) (PAH)/HA]3, indicating that these colonies exhibit hybrid and metastable epithelial/mesenchymal phenotypes. Moreover, the cell cycle and invasion tests revealed that most of the cells in colonies growing on multilayer nanofilms showed a quiescent, slow cycling phenotype but displayed higher invasion after induction. Furthermore, a hypoxic environment strongly influences the drug resistance. This study describes a useful tool to investigate the diverse phenotypes of pancreatic CSC colonies and to study their regulatory factors that may benefit CSC research.

Assessment of veterinary drugs in plants using pharmacokinetic approaches: The absorption, distribution and elimination of tetracycline and sulfamethoxazole in ephemeral vegetables.

The present study was carried out to demonstrate novel use of pharmacokinetic approaches to characterize drug behaviors/movements in the vegetables with implications to food safety. The absorption, distribution, metabolism and most importantly, the elimination of tetracycline (TC) and sulfamethoxazole (SMX) in edible plants Brassica rapa chinensis and Ipomoea aquatica grown hydroponically were demonstrated and studied using non-compartmental pharmacokinetic analysis. The results revealed drug-dependent and vegetable-dependent pharmacokinetic differences and indicated that ephemeral vegetables could have high capacity accumulating antibiotics (up to 160 µg g-1 for TC and 38 µg g-1 for SMX) within hours. TC concentration in the root (Cmax) could reach 11 times higher than that in the cultivation fluid and 3-28 times higher than the petioles/stems. Based on the volume of distribution (Vss), SMX was 3-6 times more extensively distributed than TC. Both antibiotics showed evident, albeit slow elimination phase with elimination half-lives ranging from 22 to 88 hours. For the first time drug elimination through the roots of a plant was demonstrated, and by viewing the root as a central compartment and continuous infusion without a loading dose as drug administration mode, it is possible to pharmacokinetically monitor the movement of antibiotics and their fate in the vegetables with more detailed information not previously available. Phyto-pharmacokinetic could be a new area worth developing new models for the assessment of veterinary drugs in edible plants.

Niche Mimicking for Selection and Enrichment of Liver Cancer Stem Cells by Hyaluronic Acid-Based Multilayer Films.

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that exhibit capacities for self-renewal, tumor initiation, disease relapse or metastasis, and resistance to chemotherapy and radiotherapy. However, the major obstacle associated with the use of CSCs is the difficulty in their isolation and enrichment. According to recent studies, CSCs share similar properties with normal stem cells, and it has been observed that hyaluronan (HA) plays a key factor in CSCs niches and that HA-mediated CD44 interaction promotes tumor progression. Therefore, HA-based multilayer films were used to fabricate sequential surface properties variation and to mimic CSC niches. A quartz crystal microbalance was used to investigate the layer-by-layer adsorption of PAH/HA multilayer films. Colony formation was observed on a series of poly(allylamine hydrochloride) PAH/HA multilayer films, and cytotoxicity and cell viability were evaluated by MTT, LDH and live/dead assay. It was observed that the cells isolated from (PAH/HA)3 displayed the best colony formation ability and that the expression of CD133/CD44 double positive cells was up-regulated to approximately 70% after 7 days of culture. Furthermore, the cells isolated from (PAH/HA)3 displayed higher chemo-resistance than the control group. The stem-cell-related genes expression of selected cells from (PAH/HA)3 after 7 days of culture was significantly different from that of the control group. In conclusion, this study provides a label-free selection and enrichment system that could serve as a new strategy for the future development of CSC selection and drug evaluation in cancer therapy.