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Amal Kumar Ghimeray,1 Un Sun Jung,1,2 Ha Youn Lee,1 Young Hoon Kim,1 Eun Kyung Ryu,1 Moon Sik Chang11R&D Center, Natural Solution Co., Ltd, Gojan-dong, Namdong-gu, Incheon, Republic of Korea; 2Department of Horticultural Biotechnology, Kyung Hee University, Yongin, Republic of KoreaBackground: In phytotherapy, the therapeutic potential is based on the combined action of different herbal drugs. Our objective was to evaluate the antioxidant, anti-collagenase (in vitro), and anti-wrinkle (in vivo) effect of combined formulation containing Ginkgo biloba, Punica granatum, Ficus carica, and Morus alba fruits extract.Methods: Antioxidant evaluation was based on the scavenging activity of free radicals (1,1-diphenyl-2-picrylhydrazyl, H2O2, and O2-) and the anti-collagenase activity was based on the reduction of collagenase enzyme in vitro. In an in vivo study, 21 female subjects were examined in a placebo-controlled trail. Facial wrinkle, especially the crow's feet region of eyes, was treated with topical formulated 2% cream for 56 days and compared with the placebo.Results: In the in vitro study, the combination of fruits extract showed a higher antioxidant activity which was comparable with the positive standard (ascorbic acid, butylated hydroxyanisole, and Trolox). The data also showed a dose-dependent inhibition of collagenase. In the in vivo study, treatment with 2% formulated cream for 56 days significantly reduced the percentage of wrinkle depth, length, and area with 11.5, 10.07, and 29.55, respectively.Conclusion: The combined formulation of fruit extracts showed excellent antioxidative and anti-collagenase activity as well as a significant effect on anti-wrinkle activity on human skin.Keywords: antioxidant, anti-collagenase, anti-wrinkle, fruits, topical formulation

Brain Machine Interfaces (BMIs) are promising technologies to rehabilitate the function of upper limbs in severely paralyzed patients. We succeeded in developing a BMI prosthetic arm for a monkey implanted with electrocorticogram (ECoG) electrodes and trained in a reaching task. It had stability in preventing the misclassification of ECoG patterns. However, the latency was about 200 ms as a trade-off for the stability. To improve the response of this BMI prosthetic arm, the generation of a trigger event by decoding muscle activity was adopted. It was performed to predict integrated electromyograms (iEMGs) from the ECoGs. Experiments were conducted to verify the availability of this method, and the results confirmed that the proposed method was superior to the conventional one. In addition, a performance test of the proposed method with actually achieved iEMGs instead of predicted iEMGs was performed, and we found that the motor intention is finely expressed through estimated muscle activity from brain activity rather than actual muscle activity.

Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.

Cancer has been identified as a leading cause of death worldwide, and the increasing number of cancer cases threatens to shorten the average life expectancy of people. Recently, we reported a 3-azido-3-deoxythymidine (AZT)-based amphipathic small molecule, ADG-2e that revealed a notable potency against tumor metastasis. To evaluate the anticancer potential of ADG-2e, we assessed its anticancer potency in vitro and in vivo. Anticancer screening of ADG-2e against cervical cancer cells, HeLa CCL2, and BT549 mammary gland ductal carcinoma showed significant inhibition of cancer cell proliferation. Furthermore, mechanistic investigations revealed that cancer cell death presumably proceeded through an oncosis mechanistic pathway because ADG-2e treated cells showed severe damage on the plasma membrane, a loss of membrane integrity, and leakage of α -tubulin and β -actin. Finally, evaluation of the antitumorigenic potential of ADG-2e in mouse xenograft models revealed that this compound potentially inhibits cancer cell proliferation. Collectively, these findings suggest that ADG-2e can evolve as an anticancer agent, which may represent a model for nucleoside-based small molecule anticancer drug discovery.

Motion estimation and compensation are necessary for improvement of tumor quantification analysis in positron emission tomography (PET) images. The aim of this study was to propose adaptive PET imaging with internal motion estimation and correction using regional artificial evaluation of tumors injected with low-dose and high-dose radiopharmaceuticals. In order to assess internal motion, molecular sieves imitating tumors were loaded with 18 F and inserted into the lung and liver regions in rats. All models were classified into two groups, based on the injected radiopharmaceutical activity, to compare the effect of tumor intensity. The PET study was performed with injection of F-18 fluorodeoxyglucose ( 18 F-FDG). Respiratory gating was carried out by external trigger device. Count, signal to noise ratio (SNR), contrast and full width at half maximum (FWHM) were measured in artificial tumors in gated images. Motion correction was executed by affine transformation with estimated internal motion data. Monitoring data were different from estimated motion. Contrast in the low-activity group was 3.57, 4.08 and 6.19, while in the high-activity group it was 10.01, 8.36 and 6.97 for static, 4 bin and 8 bin images, respectively. The results of the lung target in 4 bin and the liver target in 8 bin showed improvement in FWHM and contrast with sufficient SNR. After motion correction, FWHM was improved in both regions (lung: 24.56%, liver: 10.77%). Moreover, with the low dose of radiopharmaceuticals the PET image visualized specific accumulated radiopharmaceutical areas in the liver. Therefore, low activity in PET images should undergo motion correction before quantification analysis using PET data. We could improve quantitative tumor evaluation by considering organ region and tumor intensity.

Gold nanoparticles (AuNPs) have been widely used as nanocarriers in drug delivery to improve the efficiency of chemotherapy treatment and enhance early disease detection. The advantages of AuNPs include their excellent biocompatibility, easy modification and functionalization, facile synthesis, low toxicity, and controllable particle size. This study aimed to synthesize a conjugated citraconic anhydride link between morphologically homogeneous AuNPs and doxorubicin (DOX) (DOX-AuNP). The carrier was radiolabeled for tumor diagnosis using positron emission tomography (PET). The systemically designed DOX-AuNP was cleaved at the citraconic anhydride linker site under the mild acidic conditions of a cancer cell, thereby releasing DOX. Subsequently, the AuNPs aggregated via electrostatic attraction. HeLa cancer cells exhibited a high uptake of the radiolabeled DOX-AuNP. Moreover, PET tumor images were obtained using radiolabeled DOX-AuNP in cancer xenograft mouse models. Therefore, DOX-AuNP is expected to provide a valuable insight into the use of radioligands to detect tumors using PET., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance ( t 1/2 , 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4's capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation-dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli , demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 - unlike its N-terminal βTrCP degron motif - robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivo experiments showed that although STIL was not required for Plk4's initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4's ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4's autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.

89 Zr is an emerging radionuclide that plays an essential role in immuno-positron emission tomography (PET) imaging. The long half-life of 89 Zr (t 1/2 = 3.3 days) is favorable for evaluating the in vivo distribution of monoclonal antibodies. Thus, the use of 89 Zr is promising for monitoring antibody-based cancer therapies. Immuno-PET combines the sensitivity of PET with the specificity of antibodies. A number of studies have been conducted to investigate the feasibility of 89 Zr immuno-PET imaging for predicting the efficacy of radioimmunotherapy and antibody therapies, imaging target expression, detecting target-expressing tumors, and the monitoring of anti-cancer chemotherapies. In this review, we summarize the current status of PET imaging using 89 Zr in both preclinical and clinical studies by highlighting the use of immuno-PET for the targets of high clinical relevance. We also present 89 Zr-PET applications other than immuno-PET, such as nanoparticle imaging and cell tracking. Finally, we discuss the limitations and the ongoing research being performed to overcome the remaining hurdles.

To combat the escalating rise of antibacterial resistance, the development of antimicrobial peptides (AMPs) with a unique mode of action is considered an attractive strategy. However, proteolytic degradation of AMPs remains the greatest challenge in their transformation into therapeutics. Herein, we synthesized Fmoc-triazine amino acids that differ from each other by anchoring either cationic or hydrophobic residues. These unnatural amino acids were adopted for solid-phase peptide synthesis (SPPS) to synthesize a series of amphipathic antimicrobial peptidomimetics. From the antimicrobial screening, we found that the trimer, BJK-4 is the most potent short antimicrobial peptidomimetic without showing hemolytic activity and it displayed enhanced proteolytic stability. Moreover, the mechanism of action to kill bacteria was found to be an intracellular targeting., Competing Interests: The authors declare no conflict of interest.

Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex., (Copyright © 2020 American Society for Microbiology.)

Polo-like kinase 1 (Plk1) regulates cell cycle and cell proliferation, and is currently considered a potential biomarker in clinical trials for many cancers. A characteristic feature of Plks is their C-terminal polo-box domain (PBD). Pro-Leu-His-Ser-pThr (PLHS[pT])-the phosphopeptide inhibitor of the PBD of Plk1-induces apoptosis in cancer cells. However, because of the low cell membrane-penetration ability of PLHS[pT], new approaches are required to overcome these drawbacks. We therefore developed a vitamin E (VE) conjugate that is biodegradable by intracellular redox enzymes as an anticancer drug-delivery system. To ensure high efficiency of membrane penetration, we synthesized VE-S-S-PLHS[pT]KY ( 1 ) by conjugating PLHS[pT] to VE via a disulfide bond. We found that 1 penetrated cancer cell membranes, blocked cancer cell proliferation, and induced apoptosis in cancer cells through cell cycle arrest in the G2/M phase. We synthesized a radiolabeled peptide ( 124 I- 1 ), and the radioligand was evaluated in in vivo tumor uptake using positron emission tomography. This study shows that combination conjugates are an excellent strategy for specifically targeting Plk PBD. These conjugates have a dual function, with possible uses in anticancer therapy and tumor diagnosis.

Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.

Background and Purpose- Acceleration of longitudinal relaxation under hyperoxic challenge (ie, hyperoxia-induced ΔR 1 ) indicates oxygen accumulation and reflects baseline tissue oxygenation. We evaluated the feasibility of hyperoxia-induced ΔR 1 for evaluating cerebral oxygenation status and degree of ischemic damage in stroke. Methods- In 24-hour transient stroke rat models (n=13), hyperoxia-induced ΔR 1 , ischemic severity (apparent diffusion coefficient [ADC]), vasogenic edema (R 2 ), total and microvascular blood volume (superparamagnetic iron oxide-driven ΔR 2 * and ΔR 2 , respectively), and glucose metabolism activity ( 18 F-fluorodeoxyglucose uptake on positron emission tomography) were measured. The distribution of these parameters according to hyperoxia-induced ΔR 1 was analyzed. The partial pressure of tissue oxygen change during hyperoxic challenge was measured using fiberoptic tissue oximetry. In 4-hour stroke models (n=6), ADC and hyperoxia-induced ΔR 1 was analyzed with 2,3,5-triphenyltetrazolium chloride staining being a criterion of infarction. Results- Ischemic hemisphere showed significantly higher hyperoxia-induced ΔR 1 than nonischemic brain in a pattern depending on ADC. During hyperoxic challenge, ischemic hemisphere demonstrated uncontrolled increase of partial pressure of tissue oxygen, whereas contralateral hemisphere rapidly plateaued. Ischemic hemisphere also demonstrated significant correlation between hyperoxia-induced ΔR 1 and R 2 . Hyperoxia-induced ΔR 1 showed a significant negative correlation with 18 F-fluorodeoxyglucose uptake. The ADC, R 2 , ΔR 2 , and 18 F-fluorodeoxyglucose uptake showed a dichotomized distribution according to the hyperoxia-induced ΔR 1 as their slopes and values were higher at low hyperoxia-induced ΔR 1 (<50 ms -1 ) than at high ΔR 1 . In 4-hour stroke rats, the distribution of ADC according to the hyperoxia-induced ΔR 1 was similar with 24-hour stroke rats. The hyperoxia-induced ΔR 1 was greater in the infarct area (47±10 ms -1 ) than in peri-infarct area (16±4 ms -1 ; P<0.01). Conclusions- Hyperoxia-induced ΔR 1 adequately indicates cerebral oxygenation and can be a feasible biomarker to classify the degree of ischemia-induced damage in neurovascular function and metabolism in stroke brain.

This study retrospectively assessed whether time-lapse data relating to developmental timing and morphology were associated with clinical outcomes, with the eventual goal of using morphokinetic variables to select embryos prospectively for cryopreservation. In this study, we examined the clinical outcomes of single vitrified-warmed blastocyst transfer cycles that were cultured in a time-lapse incubation system. The morphokinetic variables included uneven pronuclei, an uneven blastomere, multinucleation, and direct, rapid, and irregular division. A total of 164 single vitrified-warmed blastocyst transfer cycles were analyzed (102 cycles of regularly developed blastocysts and 62 cycles of blastocysts with morphokinetic variables). No significant differences in the age of females or the standard blastocyst morphology were found between these two groups. The regularly developed blastocysts showed significantly higher implantation and clinical pregnancy rates than the blastocysts exhibiting morphokinetic variables (30.4% vs. 9.7% and 37.3% vs. 14.5%, respectively; p <0.01). The blastocysts that exhibited morphokinetic variables showed different mean development times compared with the regularly developed blastocysts. Although morphokinetic variables are known to have fatal impacts on embryonic development, a considerable number of embryos developed to the blastocyst stage. Morphokinetic variables had negative effects on the implantation and clinical pregnancy rates in vitrified-warmed blastocyst transfer cycles. These findings suggest that blastocysts cultured in a time-lapse incubation system should be considered for selective cryopreservation according to morphokinetic variables., Competing Interests: Conflict of interest: No potential conflict of interest relevant to this article was reported.

Amyloid-β (Aβ), a main pathogenic factor of Alzheimer's disease (AD), induces apoptosis accompanied by caspase activation. However, limited caspase activation and the suppression of the intrinsic apoptotic pathway (IAPW) are frequently observed upon Aβ treatment. In this study, we investigated whether these suppressive effects of Aβ can be overcome; we also examined the death-related pathways. Single treatments of cells with Aβ42 for up to 48 h barely induced caspase activation. In cells treated with Aβ42 twice for 2 h followed by 22 h (2+22 h) or for longer durations, the apoptotic protease activating factor-1 (Apaf-1) apoptosome was formed and caspases-3 and -9 were activated to a certain extent, suggesting the activation of the IAPW. However, the Aβ42-induced activation of the IAPW differed from that induced by treatment with other agents, such as staurosporine (STS) in that lower amounts of cytochrome c were released from the mitochondria, the majority of procaspase-9 in the Apaf-1 complex was not processed and caspase-3 was activated to a lesser extent in the peptide-treated cells. Thus, it seemed that the IAPW was not fully activated by Aβ42. The 30- and 41/43-kDa fragments derived from procaspase-8 were detected, which appear to be produced through the IAPW without death-inducing signaling-complex (DISC) formation, a key feature of the extrinsic apoptotic pathway (EAPW). Bid cleavage was observed only after caspase-3 activity reached its maximal levels, suggesting that the cleavage may contribute in a limited capacity to the amplification process of the IAPW in the Aβ-treated cells. Taken together, our data suggest that the IAPW, albeit functional only to a limited extent, plays a major role in Aβ42-induced apoptosis without the EAPW.

Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino acids. Through the systematic tuning of hydrophobicity, charge, and peptide length, we identified the shortest peptide Py11 with the most potent antimicrobial activity. Py11 displayed greater antimicrobial activity against antibiotic-resistant bacteria, including MRSA, MDRPA, and VREF, which was approximately 2-4 times higher than that of melittin. Besides its higher selectivity (therapeutic index) toward bacterial cells than LL-37, Py11 showed highly increased proteolytic stability against trypsin digestion and maintained its antimicrobial activity in the presence of physiological salts. Interestingly, Py11 exhibited higher anti-biofilm activity against MDRPA compared to LL-37. The results from fluorescence spectroscopy and transmission electron microscopy (TEM) suggested that Py11 kills bacterial cells possibly by integrity disruption damaging the cell membrane, leading to the cytosol leakage and eventual cell lysis. Furthermore, Py11 displayed significant anti-inflammatory (endotoxin-neutralizing) activity by inhibiting LPS-induced production of nitric oxide (NO) and TNF-α. Collectively, our results suggest that Py11 may serve as a model compound for the design of antimicrobial and antisepsis agents., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro . Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner . In an in vivo z ebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health.

Objective: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface., Methods: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development., Results: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups., Conclusion: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos., Competing Interests: No potential conflict of interest relevant to this article was reported.

Objective: To study the clinical outcomes of single frozen-thawed blastocyst transfer cycles according to the hatching status of frozen-thawed blastocysts., Methods: Frozen-thawed blastocysts were divided into three groups according to their hatching status as follows: less-than-expanded blastocyst (≤EdB), hatching blastocyst (HgB), and hatched blastocyst (HdB). The female age and infertility factors of each group were evaluated. The quality of the single frozen-thawed blastocyst was also graded as grade A, tightly packed inner cell mass (ICM) and many cells organized in the trophectoderm epithelium (TE); grade B, several and loose ICM and TE; and grade C, very few ICM and a few cells in the TE. The clinical pregnancy and implantation rate were compared between each group. The data were analyzed by either t-test or chi-square analysis., Results: There were no statistically significant differences in average female ages, infertility factors, or the distribution of blastocyst grades A, B, and C in each group. There was no significant difference in the clinical pregnancy and implantation rate of each group according to their blastocyst grade. However, there was a significant difference in the clinical pregnancy and implantation rate between each group. In the HdB group, the clinical pregnancy and implantation rate were similar regardless of the blastocyst quality., Conclusion: There was an effect on the clinical outcomes depending on whether the blastocyst hatched during single frozen-thawed blastocyst transfer. When performing single frozen-thawed blastocyst transfer, the hatching status of the frozen-thawed blastocyst may be a more important parameter for clinical outcomes than the quality of the frozen-thawed blastocyst.

Bronchial asthma is a chronic inflammatory disease of the airways characterized by a marked infiltration of eosinophils at the site of inflammation. Eotaxins are potent chemoattractants for eosinophils and play important roles in pathogenesis of asthma. In the course of screening for eotaxin-3 inhibitors, we found that wogonin showed potent inhibitory activity of interleukin-4 (IL-4)-induced eotaxin-3 expression in BEAS-2B cells. In this study, we examined the effects of wogonin on IL-4/STAT6 signaling pathway and biological implication in a mouse model of asthma. Wogonin inhibited IL-4-induced activation and nuclear translocation of STAT6 which plays a key role in either the transcription of STAT6-response genes or Th2 cytokine-mediated inflammation. Oral administration of wogonin significantly reduced activation of STAT6 in the lung and the expression of eotaxin and RANTES in bronchoalveolar lavage fluids. Histological examination of lung tissue demonstrated that wogonin significantly inhibited allergen-induced eosinophilic inflammation. Administration of wogonin reduced the total IgE and ovalbumin-specific IgE levels compared with the ovalbumin-challenged group. All of these data demonstrated that wogonin could alleviate airway inflammation through inhibition of STAT6 activation induced by Th2 cytokines. Our finding implicates a potential therapeutic value of wogonin in the treatment of asthma through regulation of IL-4/STAT6 signaling pathway.

Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.

Purpose: In the field of assisted reproductive technology (ART), medical accidents can result in serious legal and social consequences. This study was conducted to develop a security system (called IVF-guardian; IG) that could prevent mismatching or mix-ups in ART., Materials and Methods: A software program was developed in collaboration with outside computer programmers. A quick response (QR) code was used to identify the patients, gametes and embryos in a format that was printed on a label. There was a possibility that embryo development could be affected by volatile organic components (VOC) in the printing material and adhesive material in the label paper. Further, LED light was used as the light source to recognize the QR code. Using mouse embryos, the effects of the label paper and LED light were examined. The stability of IG was assessed when applied in clinical practice after developing the system. A total of 104 cycles formed the study group, and 82 cycles (from patients who did not want to use IG because of safety concerns and lack of confidence in the security system) to which IG was not applied comprised the control group., Results: Many of the label paper samples were toxic to mouse embryo development. We selected a particular label paper (P touch label) that did not affect mouse embryo development. The LED lights were non-toxic to the development of the mouse embryos under any experimental conditions. There were no differences in the clinical pregnancy rates between the IG-applied group and the control group (40/104 = 38.5 % and 30/82 = 36.6 %, respectively)., Conclusions: The application of IG in clinical practice did not affect human embryo development or clinical outcomes. The use of IG reduces the misspelling of patient names. Using IG, there was a disadvantage in that each treatment step became more complicated, but the medical staff improved and became sufficiently confident in ART to offset this disadvantage. Patients who received treatment using the IG system also went through a somewhat tedious process, but there were no complaints. These patients gained further confidence in the practitioners over the course of treatment.

In the last decade, drug delivery systems using biologically active molecules for cellular uptake of therapeutic targets have been studied for application and testing in clinical trials. For instance, the transactivator of transcription (TAT) peptide, or cell-penetrating peptide, was shown to deliver a variety of cargoes, including proteins, peptides, and nucleic acids. Polo-like kinase 1 (Plk1) plays key roles in the regulation of cell cycle events (e.g., mitotic progression). Plk1 was also shown to be activated and highly expressed in proliferating cells such as tumor cells. Amongst these phosphopeptides, Pro-Leu-His-Ser-p-Thr (PLHSpT), which is the minimal sequence for polo-box domain (PBD) binding, was shown to have an inhibitory effect and to induce apoptotic cell death. However, the phosphopeptide showed low cell membrane penetration. Thus, in our study, we synthesized Plk1 inhibitor TAT-PLHSpT to improve agent internalization into cells. TAT-PLHSpT was shown to internalize into the nucleus. The conjugation of TAT with PLHSpT inhibited cancer cell growth and survival. Moreover, it showed an increase in cellular uptake and inhibition of Plk1 kinase activity. Further studies are needed for biological evaluation of the new peptide in tumor-bearing animal models (in vivo). Our results prove that TAT-PLHSpT is a good candidate for specific PBD binding of Plk1 as a therapeutic agent for humans.

Oleanolic acid (OA) is a pentacyclic triterpenoid found in various plant species. Triterpenoid compounds have been shown to inhibit tumor proliferation and to induce apoptosis in cancer cells. We synthesized an OA derivative and evaluated its inhibitory effects on cell proliferation in human colon cancer. Radioisotope-labeled OA was prepared for noninvasive monitoring of tumor progression in vitro and in vivo. The OA derivative decreased cell survival in a concentration-dependent manner and increased apoptosis in HT-29 cells. Furthermore, it induced downregulation of cyclin D1, Cox-2, Bcl-2 and Bcl-xL mRNA expression and upregulation of the mRNA expression of the anti-apoptotic Bax protein in HT29 cells. NF-κB p65 and IκB expression also decreased, whereas expression of the apoptosis marker, the cleaved form of PARP-1, significantly increased in OA derivative-treated HT-29 cells. Radioisotope-labeled OA ( 68 Ga-NOTA-OA) showed significantly high tumor uptake, as assessed by biodistribution and positron emission tomography imaging analyses, at 1 h post-injection in the human colon cancer xenograft model. Our results demonstrate that the OA derivative has promising properties as an anticancer drug and as an imaging tool for tumor targeting.

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure-activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.

Peptides are ideal candidates for developing therapeutics. Polo-like kinase 1 is an important regulatory protein in the cell cycle and contains a C-terminal polo-box domain, which is the hallmark of this protein family. We developed a peptide inhibitor of polo-like kinase 1 that targets its polo-box domain. This new phosphopeptide, cRGDyK-S-S-CPLHSpT, preferentially penetrates the cancer cell membrane mediated by the integrin receptor, which is expressed at high levels by cancer cells. In the present study, using high performance liquid chromatography and mass spectroscopy, we determined the stability of cRGDyK-S-S-CPLHSpT and its cleavage by glutathione under typical conditions for cell culture. We further assessed the ability of the peptide to inhibit the proliferation of the U87MG glioma cell line. The phosphorylated peptide was stable, and the disulfide bond of cRGDyK-S-S-CPLHSpT was cleaved in 50 mM glutathione. This peptide inhibited the growth of cancer cells and changed their morphology. Therefore, we conclude that the phosphopeptide shows promise as a prodrug and has a high potential to act as an anticancer agent by inhibiting polo-like kinase 1 by binding its polo-box domain. These findings indicate the therapeutic potential of PLHSpT and peptides similarly targeted to surface receptors of cancer cells and to the functional domains of regulatory proteins.

Research into multifunctional nanoparticles is focused on creating an agent for use in an all-in-one multimodal imaging system that includes diagnostic imaging, drug delivery, and therapeutic monitoring. We designed a new dual-modality tumor-targeting agent with a new tumor-targeting molecule, oleanolic acid (OA), which is derived from a natural compound and coupled with a macrocyclic chelating agent such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), iron oxide nanoparticles (IONP), and radiolabeling components such as (68)Ga for dual-modality positron emission tomography (PET)/magnetic resonance imaging (MRI). We attempted to obtain fusion PET/MR images with the (68)Ga-NOTA-OA-IONP hybrid tumor-targeting imaging agent using colon cancer (HT-29) xenograft mice models. The HT-29 cancer cells showed high uptake of (68)Ga-NOTA-OA-IONP, which also had an inhibitory effect on the cells. Moreover, we obtained PET and MRI tumor images as well as fusion PET/MRI images of the tumors using (68)Ga-NOTA-OA-IONP. Therefore, the dual-modality cancer-targeting radiolabeled nanoparticle reported here is a potent imaging agent that is suitable for PET, MRI, and PET/MRI-based diagnosis of tumors; it also has the advantage of not only detecting tumor functionality, but also simultaneously aiding in tumor resolution., (Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.)

Micelles for mucosal immunity: A mucosal vaccine system based on γ-PGA nanomicelles and viral antigens was synthesized. The intranasal administration of the vaccine system induces a high immune response both in the humoral and cellular immunity (see picture)., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Micro-vibration culture system was examined to determine the effects on mouse and human embryo development and possible improvement of clinical outcomes in poor responders., Materials and Methods: The embryonic development rates and cell numbers of blastocysts were compared between a static culture group (n = 178) and a micro-vibration culture group (n = 181) in mice. The embryonic development rates and clinical results were compared between a static culture group (n = 159 cycles) and a micro-vibration culture group (n = 166 cycles) in poor responders. A micro-vibrator was set at a frequency of 42 Hz, 5 s/60 min duration for mouse and human embryo development., Results: The embryonic development rate was significantly improved in the micro-vibration culture group in mice (p < 0.05). The cell numbers of mouse blastocysts were significantly higher in the micro-vibration group than in the static culture group (p < 0.05). In the poor responders, the rate of high grade embryos was not significantly improved in the micro-vibration culture group on day 3. However, the optimal embryonic development rate on day 5 was improved in the micro-vibration group, and the total pregnancy rate and implantation rate were significantly higher in the micro-vibration group than in the static culture group (p < 0.05)., Conclusions: Micro-vibration culture methods have a beneficial effect on embryonic development in mouse embryos. In poor responders, the embryo development rate was improved to a limited extent under the micro-vibration culture conditions, but the clinical results were significantly improved.

Purpose: Vascular endothelial growth factor receptors (VEGFRs) are associated with tumor growth and induction of tumor angiogenesis and are known to be overexpressed in various human tumors. In the present study, we prepared and evaluated (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-benzyl (NOTA)-VEGF(121) as a positron emission tomography (PET) radioligand for the in vivo imaging of VEGFR expression., Methods: (68)Ga-NOTA-VEGF(121) was prepared by conjugation of VEGF(121) and p-SCN-NOTA, followed by radiolabeling with (68)GaCl(3) and then purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding of (68)Ga-NOTA-VEGF(121) was measured as a function of time. MicroPET and biodistribution studies of U87MG tumor xenografted mice were performed at 1, 2, and 4 h after injection of (68)Ga-NOTA-VEGF(121). The tumor tissues were then sectioned and subjected to immunostaining., Results: The decay-corrected radiochemical yield of (68)Ga-NOTA-VEGF(121) was 40 ± 4.5 % and specific activity was 243.1 ± 104.6 GBq/μmol (8.6 ± 3.7 GBq/mg). (68)Ga-NOTA-VEGF(121) was avidly taken up by HAECs in a time-dependent manner, and the uptake was blocked either by 32 % with VEGF(121) or by 49 % with VEGFR2 antibody at 4 h post-incubation. In microPET images of U87MG tumor xenografted mice, radioactivity was accumulated in tumors (2.73±0.32 %ID/g at 2 h), and the uptake was blocked by 40 % in the presence of VEGF(121). In biodistribution studies, tumor uptake (1.84±0.14 %ID/g at 2 h) was blocked with VEGF(121) at a similar level (52 %) to that of microPET images. Immunostaining analysis of U87MG tumor tissues obtained after the microPET imaging showed high levels of VEGFR2 expression., Conclusion: These results demonstrate that (68)Ga-NOTA-VEGF(121) has potential for the in vivo imaging of VEGFR expression. In addition, our results also suggest that the in vivo characteristics of radiolabeled VEGF depend on the properties of the radioisotope and the chelator used.

Objective: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates., Methods: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 µg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups., Results: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05)., Conclusion: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.

Polo-like kinase 1 (Plk1) is a regulator of cell cycle progression during mitosis; it is overexpressed in many different tumors and has been implicated as a potential antimitotic target. Plks are characterized by the presence of a highly conserved C-terminal polo-box domain (PBD) that is involved in regulating kinase activity. The phosphopeptide Pro-Leu-His-Ser-p-Thr (PLHSpT) is a potent selective inhibitor of the PBD of human plk1 that acts by inducing mitotic arrest and apoptotic cell death in cancer cells. We synthesized cRGDyK-S-S-CPLHSpT to exploit the drug delivery and molecular imaging using positron emission tomography (PET). The peptide was blocked dramatically proliferation of tumor in vitro and in vivo. It was attempted to develop and show a tumor PET image with the radiolabeled-peptide. Here we showed the peptide is promising not only as an anticancer drug, but also as a radioligand for tumor diagnosis with PET. We expect that our contribution will provide new insights into the design of Plk1 peptide inhibitors and have significant implications for anticancer therapy and tumor diagnosis., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

KR-31831 ((2R,3R,4S)-6-amino-4-[N-(4-chloropheyl)-N-(1H-imidazol-2ylmethyl)amino]-3-hydroxyl-2-methyl-2-dimethoxymethyl-3,4-dihydro-2H-1-benzopyran), an angiogenesis inhibitor, was evaluated in tumor-bearing mice using molecular imaging technology. Pre-treatment microPET images were acquired on SKOV-3 cell-implanted nude mice after injection with (64)Cu-DOTA-VEGF(121). KR-31831 (50 mg/kg) was then injected intraperitoneally into the treatment group (n=3), while injection vehicle was injected into the control (n=4) and blocking (n=3) groups. After injections occurred daily for 28 days, all groups of mice underwent post-treatment microPET imaging after injection with (64)Cu-DOTA-VEGF(121). The post-treatment images showed high tumor uptake in the control group and reduced tumor uptake in both the blocking and treatment groups. ROI analysis of the tumor images revealed 6.25%±1.18% ID/g at 1 h, 6.55%±0.69% ID/g at 2 h, and 4.68%±0.63% ID/g at 16 h in the control group; 3.87%±0.45% ID/g at 1 h, 4.50%±0.44% ID/g at 2 h, and 3.63%±0.25% ID/g at 16 h in the blocking group; and 4.03%±0.74% ID/g at 1 h, 4.37%±0.67% ID/g at 2 h, and 3.83%±0.90% ID/g at 16 h in the treatment group. Biodistribution obtained after the post-treatment microPET imaging also demonstrated high tumor uptake (3.74%±0.27% ID/g) in the control group and reduced uptakes in both the blocking group (2.69%±0.73% ID/g, P<.05) and the treatment group (3.11%±0.25% ID/g, P<.05), which correlated well with microPET imaging data. Immunofluorescence analysis showed higher levels of VEGFR2 and CD31 expressions in tumor tissues of the control and blocking groups than in tumor tissues of the treatment group. These results suggest that the antiangiogenic activity of KR-31831 is mediated through VEGFR2 and microPET serves as a useful molecular imaging tool for evaluation of a newly developed angiogenesis inhibitor, KR-31831., (Copyright © 2012 Elsevier Inc. All rights reserved.)

One of the obvious disadvantages of natural peptides is their liability to proteases. Among the several solutions for this issue, peptoids or oligomers of N-substituted glycine have emerged as a promising tool that may enhance the stability of proteolysis-susceptible natural peptides. We have synthesized the drosocin and its glyco-peptoid analogues linked O-GalNAc at the Thr(11) residue. One of our glyco-peptoid analogues showed an increased antibacterial activity by the modification of the Thr(11) residue with glyco-peptoid. Structure-activity relationship studies revealed that the antibacterial activity by glyco-peptoid drosocin requires three key elements: free hydroxyl group on the carbohydrate moiety, γ-methyl group of the Thr(11) residue derivative and (S)-configuration over (R)-configuration., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The human serum albumin tissue inhibitor of metalloproteinase 2 (HSA-TIMP2) is known to possess antitumor activity, which has been attributed to its ability to inhibit endothelial cell proliferation by binding to integrin receptors. In this study, a fusion protein, cyclic arginine-glycine-aspartate (RGD)-HSA-TIMP2, formed by conjugating HSA-TIMP2 with a RGD peptide, and its (123)I- and (68)Ga-labeled compounds, were synthesized and evaluated with in vivo tumor imaging using single photon emission computed tomography (SPECT) and positron emission tomography (PET). RGD-HSA-TIMP2 was synthesized by covalent bonding of the RGD peptide to the side chain amino groups of HSA-TIMP2 from a two-step reaction involving from activation with N-succinimidyl iodoacetate. This conjugation improved the anticancer effect of HSA-TIMP2 in cancer cells. The (123)I- and (68)Ga-labeled fusion proteins were prepared and subsequently injected into the tail veins of mice bearing human glioblastoma cancer U87MG xenografts for SPECT and PET imaging and biodistribution studies. Tumor uptake of radioligand was high in both the PET images and in the biodistribution studies at 3 h after injection. These studies demonstrated that the new fusion protein has potential not only as an anticancer agent but also as a radioligand for the diagnosis of tumors., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

We examined biogenic amine (BA) content and the diversity of lactic acid bacteria (LAB) population in Korean rice wine, Makgeolli, stored at 4 and 20°C. Among 8 BAs examined by HPLC, only putrescine was detected in low levels during 30days of storage at 4°C. However, at 20°C, several BAs were detected and total BA concentration increased during storage. The numbers of LAB in 4°C Makgeolli remained constant while those in 20°C Makgeolli were markedly changed. Denaturing gradient gel electrophoresis (DGGE) analysis shows the major LAB population in 4°C Makgeolli to be Pediococcus acidilactici while that in 20°C Makgeolli to be Lactobacillus plantarum, which has been known to produce BAs. The present study suggests that if Makgeolli is stored at an improper temperature, the diversity of LAB populations and BA formation can be markedly increased. Thus, low temperature storage and transport of the products are required to maintain a BA-free state., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to determine the safety and clinical effect of artificial shrinkage (AS) in terms of assisted hatching of fresh blastocysts. Also, we evaluated the correlation between patient age and the effect of AS on clinical outcome., Methods: Two AS methods, using a 29-gauge needle and laser pulse, were compared. Seventy-three blastocysts were shrunk using a 29-gauge needle and the same number of other blastocysts were shrunk by a laser pulse. We evaluated the shrunken blastocysts hourly and considered them viable if they re-expanded >70%. Blastocyst transfer cycles (n=134) were divided into two groups: a control group consisted of the cycles whose intact embryos were transferred (n=100), while the AS group consisted of the cycles whose embryos were replaced following AS (n=34). The implantation and pregnancy rates of the control group and AS group were compared (p<0.05)., Results: The re-expansion rates of the 29-gauge needle and laser pulse AS groups were similar (56 [76.7%] vs. 62 [84.9%], respectively). All of the remaining shrunken blastocysts were re-expanded within 2 hours. There was no degeneration of shrunken blastocysts. The total and clinical pregnancy rate of the AS group (23 [67.6%]; 20 [58.8%], respectively) was significantly higher than that of the control group (47 [47.0%]; 39 [39.0%], respectively). In the older patient group, there was no difference in the clinical outcomes between the AS and control groups., Conclusion: These results suggest that AS of blastocoele cavity, followed by the transfer, would be a useful approach to improve the clinical outcome in cycles in which fresh blastocyst stage embryos are transferred.

Background: β-catenin plays a pivotal role in hair follicle development and hair growth cycle., Objective: The aim of this study was to identify β-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells., Methods: Primary cultured ORS cells were transduced with recombinant adenovirus expressing N-terminal truncated β-catenin (constitutive active form), and β-catenin-regulated genes were identified., Results: Overexpression of the constitutively active form of β-catenin led to induction of Sox9 expression at both mRNA and protein levels. To investigate the potential role of Sox9, we made the recombinant adenovirus expressing green fluorescent protein-tagged Sox9, and then transduced into cultured ORS cells. Interestingly, Sox9 induced the expression of keratin 15, increased the proliferation of ORS cells in vitro, and enhanced colony-forming activity., Conclusion: Our results suggest that Sox9 is a β-catenin-regulated gene in ORS cells, and has potential importance in the regulation of hair follicle homeostasis.

Purpose: The development of a new bifunctional chelator, which holds radiometals strongly in living systems, is a prerequisite for the successful application of disease-specific biomolecules to medical diagnosis and therapy. Recently, TE2A was reported to make kinetically more stable Cu(II) complexes than TETA. Herein, we report a new synthetic route to TE2A and explore its potential as a bifunctional chelator., Methods: TE2A was synthesized using the regioselective alkylation of benzyl bromoacetate and successive deprotection of the methylene bridge and benzyl group. Salt-free TE2A was radiolabeled with (64)Cu and microPET imaging was performed to follow the clearance pattern of the (64)Cu-TE2A complex. TE2A was conjugated with cyclic RGD peptide and the TE2A-c(RGDyK) conjugate was radiolabeled with (64)Cu., Results: TE2A was prepared in salt-free form from cyclam in an overall yield of 74%. The microPET images showed that (64)Cu-TE2A is excreted rapidly from the body by the kidney and liver. TE2A was successfully conjugated with c(RGDyK) peptide through one carboxylate group and the TE2A-c(RGDyK) conjugate was radiolabeled with (64)Cu in 94% yield within 30 min., Conclusion: TE2A can be used by itself as a bifunctional chelator without any further structural modification.

Background: Plasminogen activator inhibitor-2 (PAI-2) is an enzyme inhibitor which is involved in various biological processes including cell differentiation, tissue regrowth and regeneration. Although PAI-2 has been originally isolated as an extracellular inhibitor of urokinase plasminogen activator (uPA), recent studies indicate that PAI-2 has other intracellular effects in keratinocyte, such as the component of cornified envelope., Objective: The aim of this study is to investigate the expression and functional role of PAI-2 during the keratinocyte differentiation., Methods: We transduced keratinocytes with adenovirus harboring the expression cassette for PAI-2, then examined the effect on keratinocytes differentiation., Results: When cultured epidermal keratinocytes were treated with 1.2 mM calcium, PAI-2 expression was increased time-dependently at both mRNA and protein levels. The calcium-induced PAI-2 expression was abolished by treatment with p38 MAPK inhibitor, while overexpression of MKK6 led to the increase of PAI-2 expression. When PAI-2 was overexpressed by adenoviral transduction, the expression of keratinocyte differentiation markers such as involucrin, keratin 10 and loricrin was markedly increased. Concomitantly, overexpression of PAI-2 resulted in the retardation of cell growth, with the increase of Rb and p53., Conclusion: These results suggest that PAI-2 has a role for promoting the differentiation of epidermal keratinocytes., (Copyright (c) 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

A number of phosphatidylcholine (PC) cations spanning a mass range of 400-1000 Da are investigated using electrospray ionization mass spectrometry coupled with traveling wave ion mobility spectrometry (TWIMS). A high correlation between mass and mobility is demonstrated with saturated phosphatidylcholine cations in N(2). A significant deviation from this mass-mobility correlation line is observed for the unsaturated PC cation. We found that the double bond in the acyl chain causes a 5% reduction in drift time. The drift time is reduced at a rate of approximately 1% for each additional double bond. Theoretical collision cross sections of PC cations exhibit good agreement with experimentally evaluated values. Collision cross sections are determined using the recently derived relationship between mobility and drift time in TWIMS stacked ring ion guide (SRIG) and compared to estimated collision cross sections using an empiric calibration method. Computational analysis was performed using the modified trajectory (TJ) method with nonspherical N(2) molecules as the drift gas. The difference between estimated collision cross sections and theoretical collision cross sections of PC cations is related to the sensitivity of the PC cation collision cross sections to the details of the ion-neutral interactions. The origin of the observed correlation and deviation between mass and mobility of PC cations is discussed in terms of the structural rigidity of these molecules using molecular dynamic simulations.

Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.

Unlabelled: Both bombesin (BBN) analogs and cyclic RGD peptides have been suitably radiolabeled for prostate cancer imaging. However, the limited expression of gastrin-releasing peptide receptor (GRPR) and integrin alpha(v)beta(3) as well as unfavorable in vivo kinetics limited further applications of these imaging agents. We hypothesize that a peptide ligand recognizing both GRPR and integrin will be advantageous because of its dual-receptor-targeting ability., Methods: A BBN-RGD heterodimer was synthesized from bombesin(7-14) and c(RGDyK) through a glutamate linker and then labeled with (18)F via the N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) prosthetic group. The receptor-binding characteristics and tumor-targeting efficacy of (18)F-FB-BBN-RGD were tested in vitro and in vivo., Results: FB-BBN-RGD had comparable integrin alpha(v)beta(3)-binding affinity with c(RGDyK) and comparable GRPR-binding affinity with BBN(7-14). (18)F-FB-BBN-RGD had significantly higher tumor uptake compared with monomeric RGD and monomeric BBN peptide tracer analogs at all time points examined. The PC-3 tumor uptake of (18)F-FB-BBN-RGD was inhibited only partially in the presence of an excess amount of unlabeled BBN(7-14) or c(RGDyK) but was blocked completely in the presence of both BBN(7-14) and c(RGDyK). Compared with (18)F-FB-BBN and (18)F-FB-RGD, (18)F-FB-BBN-RGD also had improved pharmacokinetics, resulting in a significantly higher imaging quality., Conclusion: Dual integrin alpha(v)beta(3) and GRPR recognition showed significantly improved tumor-targeting efficacy and pharmacokinetics compared with (18)F-labeled RGD and BBN analogs. The same heterodimeric ligand design may also be applicable to other receptor system combinations and other imaging modalities.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive loss of neurotransmitters, as well as acetylcholinesterase and nicotinic acetylcholine receptors in the central nervous system that leads to learning and memory deficits, among other problems. The disease is associated with increased production and accumulation extracellular amyloid plaques and neurofibrillary tangles in aging human brain, shown in postmortem exams. New methods for reliable in vivo measurement of brain therefore would be much more ideal. PET and SPECT imaging are sensitive methods for the quantitation of AD biomarkers. The development of molecular imaging agents for AD is critically important in the early diagnosis, neuropathogenesis studies and treatment of AD. A number of potential diagnostic PET and SPECT imaging agents targeting AD have been synthesized and evaluated. Although many agents showed excellent results for in vitro monitoring of the disease, there are only several radioligands with high selectivity and specificity to binding sites and appropriate pharmacokinetics, such as [11C]MP4A, [11C]PMP, [11C]nicotine, 2- or 6-[18F]fluoro-A-85380, [11C]SB-13, [11C]PIB, and [18F]FDDNP, that have been tested in AD patients. Here we review some recent progress and development of AD imaging agents using PET and SPECT in human clinical studies.

Objectives: Alzheimer's disease (AD) is characterized by reduced acetylcholinesterase (AChE) activity in the post-mortem tissues of AD patients. Therefore, AChE has been an attractive target for the diagnosis of AD. In the present study, 5,7-dihydro-3-[2-(1-(phenylmethyl)-4-piperidinyl)ethyl]-6H-pyrrolo[3,2-f]-1,2-benzisoxazol-6-one (CP-118,954), a potent AChE inhibitor, was labelled with radioiodine and evaluated as an AChE imaging agent for SPECT., Methods: Radioiodine-labelled CP-118,954 was prepared from CP-144,885 and [(125)I]iodobenzyl bromide, and anti-AChE activities of iodine-substituted CP-118,954 were measured. Metabolism studies were carried out in samples of blood and whole brain of mice injected with 2-[(123)I]iodo-CP-118,954 ((123)I-1). Tissue distribution studies were also performed in mice injected with I-1, and samples of blood, thyroid, stomach, and brain tissue (cerebellum, striatum and cortex) were removed, weighed and counted., Results: Of the ligands, 2-iodo-CP-118,954 exhibited higher binding affinity for AChE (IC50=24 nM) than the other positional isomers. 2-[(125)I]Iodo-CP-118,954 was found to have a lipophilicity (log P=2.1) favouring brain permeability and metabolic stability in mouse brain, but a marginal target (striatum) to non-target (cerebellum) uptake ratio (1.1) in mouse brain., Conclusion: This result demonstrates that 2-[(125)I]iodo-CP-118,954 may be unsuitable for AChE imaging. These findings suggest that radioligands suitable for AChE imaging should have not only a specific structure but also a sub-nanomolar to low nanomolar IC50.