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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.

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.

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: 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.

Transdermal drug delivery is a convenient route to transport pharmaceuticals. However, its application is limited to a few compounds. Microneedle (MN) patches have demonstrated a high efficiency for delivering poorly permeable drugs and gained significant attention due to the advantages of being painless and convenient for patients as well as providing efficient delivery. This study presents dissolvable polyvinylpyrrolidone (PVP)-based MNs that do not require needle removal while providing the rapid release of encapsulated insulin. PVP with two molecular weights, PVP10 and PVP360, were used to fabricate the needle portion of the MNs, and PVP360/sodium carboxymethyl cellulose (CMC) was used for preparation of the backing layer. The detailed characteristics and in vitro skin penetration capability of a series of PVP360/PVP10 MNs were analyzed. The penetration depths of PVP MNs were evaluated using optical coherence tomography. The PVP360/PVP10 = 1:3 MN patch was chosen for ex vivo and in vivo studies. Ex vivo drug release profiles show that the permeation efficiency of Lissamine green B (LGB)- and fluorescein isothiocyanate-labeled BSA (FITC-BSA)-loaded PVP MN patches was much higher than the groups of patches without MNs. Furthermore, to estimate the feasibility of PVP MNs for diabetes treatment, insulin-loaded PVP MN patches were administered to diabetic mice to evaluate glycemic control. The relative pharmacologic availability revealed that the MN patch has an immediate and effective effect on hypoglycemic administration. This study demonstrates that dissolvable PVP MNs may serve as a promising device to deliver macromolecules or protein drugs for efficient transdermal drug delivery.

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.

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.

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.

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCe) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCe before cerebral ischemia confers neuroprotection. The role of endogenous PKCe after cerebral ischemia remains elusive. Here we used male PKCe-null mice to assess the effects of PKCe 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 PKCe-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCe-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 PKCe-null mice after tGCI. Male PKCe-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 PKCe deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCe-null mice.

The key factors affecting the success of neural engineering using neural stem/progenitor cells (NSPCs) are the neuron quantity, the guidance of neurite outgrowth, and the induction of neurons to form functional synapses at synaptic junctions. Herein, a biomimetic material comprising a supported lipid bilayer (SLB) with adsorbed sequential polyelectrolyte multilayer (PEM) films was fabricated to induce NSPCs to form functional neurons without the need for serum and growth factors in a short-term culture. SLBs are suitable artificial substrates for neural engineering due to their structural similarity to synaptic membranes. In addition, PEM film adsorption provides protection for the SLB as well as the ability to vary the surface properties to evaluate the effects of physical and mechanical signals on NSPC differentiation. Our results revealed that NSPCs were inducible on SLB-PEM films consisting of up to eight alternating layers. In addition, the process outgrowth length, the percentage of differentiated neurons, and the synaptic function were regulated by the number of layers and the surface charge of the outermost layer. The average process outgrowth length was greater than 500 μm on SLB-PLL/PLGA (n = 7.5) after only 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were obviously enhanced on the SLB-PEM system compared with those on the PEM-only substrates. These results suggest that the PEM films can induce NSPC adhesion and differentiation and that an SLB base may enhance neuron differentiation and trigger the formation of functional synapses.

To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real-time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P

This study characterized the expression and subcellular localization of the IGF-1R in human corneal epithelial cells. Using a human telomerase-immortalized corneal epithelial cell line, IGF-1R expression and localization was assayed by immunofluorescence and subcellular fractionation followed by western blot. IGF-1R expression was confirmed in primary cultured human corneal epithelial cells. Nuclear localization was assessed under basal and IGF-1 stimulated culture conditions; phosphorylation status of the receptor in response to IGF-1 was demonstrated by western blot. IGF-1R:E-cadherin interactions were detected by immunofluorescence and co-immunoprecipitation of whole cell lysates. The results of this study demonstrated that IGF-1R localized predominantly to the nucleus and in a perinuclear cap pattern which co-localized with the Golgi complex in proliferating corneal epithelial cells. There was no difference in nuclear localization between primary or telomerized cell lines. Subcellular fractionation confirmed IGF-1Rα- and β-subunit localization in soluble and chromatin-bound nuclear fractions. Neither growth factor withdrawal nor IGF-1 stimulation altered nuclear IGF-1R. At points of cell-cell contact, IGF-1R co-localized with E-cadherin; co- immunoprecipitation assays confirmed the presence of an IGF-1R:E-cadherin complex. Importantly, this is the first report to identify IGF-1R in the nucleus and complexed with E-cadherin at points of cell-cell contact in corneal epithelial cells. Nuclear trafficking appeared to be independent of ligand-mediated events at the plasma membrane. The identification of IGF-1R in the nucleus and complexed with E-cadherin suggests novel regulatory functions outside the canonical ligand-induced endocytosis signaling pathway.

Neural stem/progenitor cells (NSPCs) are a possible candidate for advancing development and lineage control in neural engineering. Differentiated protocols have been developed in this field to generate neural progeny and to establish neural networks. However, continued refinement is required to enhance differentiation specificity and prevent the generation of unwanted cell types. In this study, we fabricated a niche-modulated system to investigate surface effects on NSPC differentiation by the formation of polyelectrolyte multilayer (PEM) films governed by electrostatic interactions of poly-l-glutamine acid as a polyanion and poly-l-lysine as a polycation. The serum- and chemical agent-free system provided a clean and clear platform to observe in isolation the interaction between surface niche and stem cell differentiation. We found that NSPCs were inducible on PEM films of up to eight alternating layers. In addition, neurite outgrowth, neuron percentage, and synaptic function were regulated by layer number and the surface charge of the terminal layer. The average process outgrowth length was over 500μm on PLL/PLGA(n=7.5) only after 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were enhanced as the number of layers increased, especially when the terminal layer was poly-l-lysine. Our results achieve important targets of neural engineering, including long processes, large neural network size, and large amounts of functional neurons. Our methodology for nanoscale control of material deposition can be successfully applied for surface modification, neural niche modulation, and neural engineering applications.

SUMOylation of viral proteins is widespread and serves to modify or regulate the properties of those proteins. Papillomaviruses are a large group of small DNA viruses that infect the skin, leading to benign lesions (warts) that in some cases can progress to malignancy. The papillomavirus life cycle is intimately connected with the differentiation process of stratified epithelium, and several viral early proteins function to modulate the host cell environment. One of the critical early proteins is the E2 protein, which functions in both viral replication and transcription. In the present paper, we demonstrate that E2 proteins are SUMOylated and that overexpression of SUMOylation results in a dramatic increase in intracellular levels of the E2 protein. We have shown previously that there is increased SUMOylation during keratinocyte differentiation, suggesting that the levels of E2 protein may be tied to changes in the cellular SUMOylation state during differentiation. In addition to itself being regulated by SUMOylation, E2 appears to influence the SUMOylation state of one of its binding partners, the cellular transcription factor, C/EBP (CCAAT/enhancer-binding protein). Overall, these observations indicate a complex interplay between this viral protein and the host SUMOylation system.

Purpose The aim of this study was to establish a relationship between hypoxic stress and the expression of ΔNp63α in an established rabbit contact lens model and in cultured corneal epithelial cells. Methods New Zealand white rabbits were fit in one eye with either a non-oxygen transmissible or hyper-oxygen-permeable rigid contact lens for 24 hrs of wear; the contralateral eye was used as a control. All the rabbits underwent a bilateral nictitating membranectomy to facilitate lens retention. ΔNp63α expression was analyzed by immunofluorescence and western blot. Telomerase-immortalized human corneal epithelial cells (hTCEpi) were grown in serum-free media and treated with the hypoxia-mimetic cobalt chloride to simulate hypoxia for 6 hrs (short term) or 24 hrs (prolonged). Transcriptional activity and protein levels were assessed using luciferase reporter assays, reverse transcription polymerase chain reaction, and western blot. Cell viability was assessed by live/dead assay. Results Compared with the non-lens wearing eye, 24 hrs of non-oxygen transmissible lens wear in vivo decreased ΔNp63α protein levels in both the limbal and central corneal epithelium; this decrease was not found in the hyper-oxygen transmissible lens group. In hTCEpi cells in vitro, hypoxia increased the activity of the ΔN promoter but reduced the levels of ΔNp63α mRNA after 24 hrs of prolonged culture. Similarly, ΔNp63α expression levels were unaffected from short-term exposure but decreased after 24 hrs. Live/dead assay confirmed the presence of viable cells after CoCl2 treatment at 6- and 24-hr time points. Cells treated for 24 hrs were viable but were smaller and rounded with signs of membrane blebbing, consistent with early stages of apoptosis. Conclusions Hypoxic stress induced by either prolonged wear of a nonoxygen transmissible lens in vivo or hypoxic-mimic conditions by cobalt chloride in vitro downregulates ΔNp63α in the corneal epithelium. The loss of ΔNp63α in response to hypoxic stress may contribute to the disruption of normal renewal mechanisms reported with low oxygen transmissible contact lens wear and prolonged eyelid closure.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329–338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125–136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.