USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

Cisplatin is a chemotherapy drug that causes a plethora of DNA lesions and inhibits DNA transcription and replication, resulting in the induction of apoptosis in cancer cells. However, over time, patients develop resistance to cisplatin due to repeated treatment and thus the treatment efficacy is limited. Therefore, identifying an alternative therapeutic strategy combining cisplatin treatment along with targeting factors that drive cisplatin resistance is needed. CRISPR/Cas9 system-based genome-wide screening for the deubiquitinating enzyme (DUB) subfamily identified USP28 as a potential DUB that governs cisplatin resistance. USP28 regulates the protein level of microtubule-associated serine/threonine kinase 1 (MAST1), a common kinase whose expression is elevated in several cisplatin-resistant cancer cells. The expression level and protein turnover of MAST1 is a major factor driving cisplatin resistance in many cancer types. Here we report that the USP28 interacts and extends the half-life of MAST1 protein by its deubiquitinating activity. The expression pattern of USP28 and MAST1 showed a positive correlation across a panel of tested cancer cell lines and human clinical tissues. Additionally, CRISPR/Cas9-mediated gene knockout of USP28 in A549 and NCI-H1299 cells blocked MAST1-driven cisplatin resistance, resulting in suppressed cell proliferation, colony formation ability, migration and invasion in vitro. Finally, loss of USP28 destabilized MAST1 protein and attenuated tumor growth by sensitizing cells to cisplatin treatment in mouse xenograft model. We envision that targeting the USP28-MAST1 axis along with cisplatin treatment might be an alternative therapeutic strategy to overcome cisplatin resistance in cancer patients.

Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

ßTrCP1 promotes SLC35F2 protein ubiquitination and inhibits cancer progression in HeLa cells.

The solute carrier family 35 F2 (SLC35F2) belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, we identified ßTrCP1 interacting with and reducing the SLC35F2 protein level. ßTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between ßTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of ßTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, we demonstrate that ßTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of ßTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, we envision that ßTrCP1 may be a potential target for cancer therapeutics.

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.

Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP, and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. In addition, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.

Biportal endoscopic extraforaminal lumbar interbody fusion using a 3D-printed porous titanium cage with large footprints: technical note and preliminary results.

PURPOSE: The aim of this study was to introduce biportal endoscopic extraforaminal lumbar interbody fusion (BE-EFLIF), which involves insertion of a cage through a more lateral side as compared to the conventional corridor of transforaminal lumbar interbody fusion. We described the advantages and surgical steps of 3D-printed porous titanium cage with large footprints insertion through multi-portal approach, and preliminary results of this technique. METHODS: This retrospective study included 12 consecutive patients who underwent BE-EFLIF for symptomatic single-level lumbar degenerative disease. Clinical outcomes, including a visual analog scale (VAS) for back and leg pain and the Oswestry disability index (ODI), were collected at preoperative months 1 and 3, and 6 months postoperatively. In addition, perioperative data and radiographic parameters were analyzed. RESULTS: The mean patient age, follow-up period, operation time, and volume of surgical drainage were 68.3 ± 8.4 years, 7.6 ± 2.8 months, 188.3 ± 42.4 min, 92.5 ± 49.6 mL, respectively. There were no transfusion cases. All patients showed significant improvement in VAS and ODI postoperatively, and these were maintained for 6 months after surgery (P < 0.001). The anterior and posterior disc heights significantly increased after surgery (P < 0.001), and the cage was ideally positioned in all patients. There were no incidences of early cage subsidence or other complications. CONCLUSIONS: BE-EFLIF using a 3D-printed porous titanium cage with large footprints is a feasible option for minimally invasive lumbar interbody fusion. This technique is expected to reduce the risk of cage subsidence and improve the fusion rate.

PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury.

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1-0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

Structural basis for HOCl recognition and regulation mechanisms of HypT, a hypochlorite-specific transcriptional regulator.

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes.

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2-/- mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Structure and function of the hypochlorous acid-induced flavoprotein RclA from Escherichia coli.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+ We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

Perivascular Stem Cell-Derived Cyclophilin A Improves Uterine Environment with Asherman's Syndrome via HIF1α-Dependent Angiogenesis.

Asherman's syndrome (AS) is characterized by intrauterine adhesions or fibrosis resulting from scarring inside the endometrium. AS is associated with infertility, recurrent miscarriage, and placental abnormalities. Although mesenchymal stem cells show therapeutic promise for the treatment of AS, the molecular mechanisms underlying its pathophysiology remain unclear. We ascertained that mice with AS, like human patients with AS, suffer from extensive fibrosis, oligo/amenorrhea, and infertility. Human perivascular stem cells (hPVSCs) from umbilical cords repaired uterine damage in mice with AS, regardless of their delivery routes. In mice with AS, embryo implantation is aberrantly deferred, which leads to intrauterine growth restriction followed by no delivery at term. hPVSC administration significantly improved implantation defects and subsequent poor pregnancy outcomes via hypoxia inducible factor 1α (HIF1α)-dependent angiogenesis in a dose-dependent manner. Pharmacologic inhibition of HIF1α activity hindered hPVSC actions on pregnancy outcomes, whereas stabilization of HIF1α activity facilitated such actions. Furthermore, therapeutic effects of hPVSCs were not observed in uterine-specific HIF1α-knockout mice with AS. Secretome analyses of hPVSCs identified cyclophilin-A as the major paracrine factor for hPVSC therapy via HIF1α-dependent angiogenesis. Collectively, we demonstrate that hPVSCs-derived cyclophilin-A facilitates HIF1α-dependent angiogenesis to ameliorate compromised uterine environments in mice with AS, representing the major pathophysiologic features of humans with AS.

High-resolution structures of annexin A5 in a two-dimensional array.

Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 self-assembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca2+) and confirmed the Ca2+-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. High-resolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca2+ concentration and the phospholipid composition of the membrane.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice.

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.-Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

Importance of Deubiquitination in Macrophage-Mediated Viral Response and Inflammation.

Ubiquitination and deubiquitination play a fundamental role in the signaling pathways associated with innate and adaptive immune responses. Macrophages are key sentinels for the host defense, triggering antiviral and inflammatory responses against various invading pathogens. Macrophages recognize the genetic material of these pathogens as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) through the activation of its pattern recognition receptors (PRRs), initiating the cascade of immune signaling, which leads to the production of pro- and anti-inflammatory cytokines that initiates the appropriate immune response. Macrophage-mediated immune response is highly regulated and tightly controlled by the ubiquitin system since its abnormal activation or dysregulation may result in the severe pathogenesis of numerous inflammatory and autoimmune diseases. Deubiquitinating enzymes (DUBs) play a crucial role in reversing the ubiquitination and controlling the magnitude of the immune response. During infection, pathogens manipulate the host defense system by regulating DUBs to obtain nutrients and increase proliferation. Indeed, the regulation of DUBs by small molecule inhibitors has been proposed as an excellent way to control aberrant activation of immune signaling molecules. This review is focused on the complex role of DUBs in macrophage-mediated immune response, exploring the potential use of DUBs as therapeutic targets in autoimmune and inflammatory diseases by virtue of small molecule DUB inhibitors.

Reduced Fecal Calprotectin and Inflammation in a Murine Model of Atopic Dermatitis Following Probiotic Treatment.

Atopic dermatitis (AD) is one of the most common skin diseases with inflammation, chronic relapses, and intense pruritus. Its pathogenesis includes genetic susceptibility, an abnormal epidermal lipid barrier, and an increased production of IgE due to immune dysregulation. Recently, AD has been reported to be associated with intestinal inflammation and dysbiosis in human and murine models. Various probiotics are being used to control intestinal dysbiosis and inflammatory reactions. However, it is difficult to predict or determine the therapeutic effects of the probiotics, since it is rare for clinicians to use the probiotics alone to treat AD. It is also difficult to check whether the intestinal inflammation in patients with AD has improved since probiotic treatment. The aim of the present study was to determine whether mice with induced atopic dermatitis had any changes in fecal calprotectin, an indicator of intestinal inflammation, after probiotic administration. Our results showed that the fecal calprotectin levels in mice with induced dermatitis decreased significantly after the administration of probiotics. In addition, epidermal skin lesions were attenuated and inflammatory-related cytokines were downregulated after the administration of probiotics in mice with induced dermatitis. These results suggest that changes in fecal calprotectin levels could be used to assess the effectiveness of a probiotic strain as an adjuvant treatment for AD.

Dipeptidyl peptidase-4 inhibitor sitagliptin induces vasorelaxation via the activation of Kv channels and PKA.

The present study investigated the vasorelaxant effects of sitagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor in aortic rings pre-contracted with phenylephrine (Phe). Sitagliptin induced vasorelaxation in a concentration-dependent manner but the inhibition of voltage-dependent K+ (Kv) channels by pretreatment with 4-aminopyridine (4-AP) effectively reduced this effect. By contrast, the inhibition of inward rectifier K+ (Kir) channels by pretreatment with barium (Ba2+), large-conductance calcium (Ca2+)-activated K+ (BKCa) channels with paxilline, and adenosine triphosphate (ATP)-sensitive K+ (KATP) channels with glibenclamide did not change this effect. Although the application of SQ 22536, which is an adenylyl cyclase inhibitor, also did not change this effect, treatment with KT 5720, a protein kinase A (PKA) inhibitor, effectively reduced the vasorelaxant effects of sitagliptin. ODQ, which is a guanylyl cyclase inhibitor, and KT 5823, a protein kinase G (PKG) inhibitor, did not impact the effect. Furthermore, neither the inhibition of Ca2+ channels by pretreatment with nifedipine nor the inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps by pretreatment with thapsigargin changed the effect. Similarly, the effects of sitagliptin were not altered by eliminating the endothelium, by pretreatment with a nitric oxide (NO) synthase inhibitor (L-NAME), or by inhibition of small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) using apamin and TRAM-34. Taken together, these results suggest that sitagliptin induces vasorelaxation by inhibiting both membrane potential (Em)-dependent and -independent vasoconstriction and activating PKA and Kv channels independently of PKG signaling pathways, other K+ channels, SERCA pumps, and the endothelium.

The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

The anticholinergic drug oxybutynin inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

This study demonstrates the inhibitory effect of anticholinergic drug oxybutynin on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Oxybutynin inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 11.51 ± 0.38 µmol/L and a Hill coefficient (n) of 2.25 ± 0.12. Application of oxybutynin shifted the activation curve to the right and the inactivation curve to the left. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 and the Kv2.1 subtype inhibitor guangxitoxin suppressed the oxybutynin-induced inhibition of the Kv current. However, application of the Kv7 subtype inhibitor linopirdine did not affect the inhibition by oxybutynin of the Kv current. The anticholinergic drug atropine did not inhibit the Kv current nor influence oxybutynin-induced inhibition of the Kv current. From these results, we concluded that oxybutynin inhibited the vascular Kv current in a concentration-dependent manner by influencing the steady-state activation and inactivation curves independent of its anticholinergic effect.

Novel S-Bend Resonator Based on a Multi-Mode Waveguide with Mode Discrimination for a Refractive Index Sensor.

In this paper, a multi-mode waveguide-based optical resonator is proposed for an integrated optical refractive index sensor. Conventional optical resonators have been studied for single-mode waveguide-based resonators to enhance the performance, but mass production is limited owing to the high fabrication costs of nano-scale structures. To overcome this problem, we designed an S-bend resonator based on a micro-scale multi-mode waveguide. In general, multi-mode waveguides cannot be utilized as optical resonators, because of a performance degradation resulting from modal dispersion and an output transmission with multi-peaks. Therefore, we exploited the mode discrimination phenomenon using the bending loss, and the resulting S-bend resonator yielded an output transmission without multi-peaks. This phenomenon is utilized to remove higher-order modes efficiently using the difference in the effective refractive index between the higher-order and fundamental modes. As a result, the resonator achieved a Q-factor and sensitivity of 2.3 × 103 and 52 nm/RIU, respectively, using the variational finite-difference time-domain method. These results show that the multi-mode waveguide-based S-bend resonator with a wide line width can be utilized as a refractive index sensor.