Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

"Lighting up" fluoride: cellular imaging and zebrafish model interrogations using a simple ESIPT-based mycophenolic acid precursor-based probe.

The discovery and implementation of media that derive from bioinspired designs and bear optical readouts featuring large Stokes shifts are of continued interest to a wide variety of researchers and clinicians. Myco-F, a novel mycophenolic acid precursor-based probe features a cleavable tert-butyldimethylsiloxy group to allow for fluoride detection. Myco-F exhibits high selectivity and specificity towards F- (Stokes shift = 120 nm). All measurements were performed in complete aqueous media (LOD=0.38 µM). Myco-F enables detection of fluoride ions in living HEK293 cells and localizes in the eye region (among other regions) of the zebrafish. DFT calculations support the proposed ESIPT working photomechanism.

A Versatile Strategy for Screening Custom-Designed Warhead-Armed Cyclic Peptide Inhibitors.

Demand for peptide-based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide-based drug discovery, we developed a general scheme for cell-based screening of cyclic peptide inhibitors armed with a user-designed warhead. We combined unnatural amino acid incorporation and split intein-mediated peptide cyclization techniques and integrated a yeast-based colorimetric screening assay to generate a new scheme that we call the custom-designed warhead-armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5-6Q, that targets human histone deacetylase 8 (HDAC8) with a KD value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.

Synergistic effects of autocrine motility factor and methyl jasmonate on human breast cancer cells.

Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option.

Noncanonical Amino Acids in Synthetic Biosafety and Post-translational Modification Studies.

The incorporation of noncanonical amino acids (ncAAs) has been extensively studied because of its broad applicability. In the past decades, various in vitro and in vivo ncAA incorporation approaches have been developed to generate synthetic recombinant proteins. Herein, we discuss the methodologies for ncAA incorporation, and their use in diverse research areas, such as in synthetic biosafety and for studies of post-translational modifications.

The effect of autocrine motility factor alone and in combination with methyl jasmonate on liver cancer cell growth.

Neoplastic cells secrete autocrine motility factor (AMF) to stimulate the motility of cancer cells. In this study, AMF secreted from HT-29 colorectal cancer cells selectively suppressed liver cancer cells by downregulating pAKT and ß-catenin. In addition, HT-29 AMF significantly augmented the activity of methyl jasmonate against liver cancer cells and is a promising alternative for liver cancer therapy.

Predicting the Cochlear Dead Regions Using a Machine Learning-Based Approach with Oversampling Techniques.

Background and Objectives: Determining the presence or absence of cochlear dead regions (DRs) is essential in clinical practice. This study proposes a machine learning (ML)-based model that applies oversampling techniques for predicting DRs in patients. Materials and Methods: We used recursive partitioning and regression for classification tree (CT) and logistic regression (LR) as prediction models. To overcome the imbalanced nature of the dataset, oversampling techniques to duplicate examples in the minority class or to synthesize new examples from existing examples in the minority class were adopted, namely the synthetic minority oversampling technique (SMOTE). Results: The accuracy results of the 10-fold cross-validation of the LR and CT with the original data were 0.82 (±0.02) and 0.93 (±0.01), respectively. The accuracy results of the 10-fold cross-validation of the LR and CT with the oversampled data were 0.66 (±0.02) and 0.86 (±0.01), respectively. Conclusions: This study is the first to adopt the SMOTE method to assess the role of oversampling methods on audiological datasets and to develop an ML-based model. Considering that the SMOTE method did not improve the model's performance, a more flexible model or more clinical features may be needed.

Autocrine motility factor secreted by HeLa cells inhibits the growth of many cancer cells by regulating AKT/ERK signaling.

In cell competition, a secreted death signal can determine cell fate. However, the nature of such a signal remains unclear. In this study, conditioned medium from HeLa cells (HeLa CM) inhibited growth of A549 and MCF-7 cells. Through HeLa CM fractionation, glucose 6-phosphate isomerase/autocrine motility factor (GPI/AMF) was identified as the main growth inhibitor. Previously, AMF was known for its mitogenic, motogenic, and differentiation functions and was implicated in tumor progression and metastasis. HeLa CM lost its growth inhibitory property after treatment with erythrose-4-phosphate (E4P) or anti-GPI antibody. Purified HeLa recombinant AMF (rAMF) proteins inhibited the growth of A549, MDA-MB-232, MCF-7, AsPC-1, DU145, Hep-2, Hep G2, and HT-29 cells. However, growth of HL-60, SKOV3, U-87 MG, SNU-484, U-87 MG, and 3T3-L1 cells was little affected. In a Transwell assay, HeLa rAMF effectively reduced A549 cell migration and invasion. HeLa rAMF effectively induced apoptosis in A549 cells, apparently by reducing the levels of Bcl-2, GPI, and poly(ADP-ribose) polymerase (PARP)14 and activating caspase-3 and p53. HeLa rAMF antagonized HER2 and the AMF receptor (AMFR or GP78) in relation to the AKT/EKT signaling pathway. These results suggest that HeLa AMF could act as a diffusible death signal that could induce cancer cell-selective growth inhibition and apoptosis.

Efficacy of three proton-pump inhibitor therapeutic strategies on laryngopharyngeal reflux disease; a prospective randomized double-blind study.

OBJECTIVES: Proton-pump inhibitor (PPI) prescribing practices in laryngopharyngeal reflux disease (LPR) differ among physicians. We assessed the improvement in reflux symptom index (RSI) and reflux finding score (RFS) after treating LPR with three different regimens. DESIGN: A prospective, double-blind, randomized clinical trial. SETTING: Chungnam national university hospital in Korea. PARTICIPANTS: From July 2015 to July 2017, 100 patients with LPR included in the study. The patients were prescribed one of the following regimens for 3 months: group A, ilaprazole 10 mg, once a day (QD), n = 29; group B, ilaprazole 10 mg, twice a day (BID), n = 27; and group C, ilaprazole 10 mg BID plus mosapride citrate 5 mg three times a day (TID), n = 44. MAIN OUTCOME MEASURES: The total RSI and RFS scores and each subitems in RSI and FRS of the patients were evaluated. RESULTS: Total RFS and RSI scores improved significantly at the 3-month follow-up in all groups, and the improvements were of similar magnitudes. Regarding the RFS, the degrees of improvement in vocal cord oedema (P = 0.002) and diffuse laryngeal oedema (P = 0.003) scores differed significantly among the three groups. Moreover, overweight or obese patients in group C showed the greatest improvement in RFS. However, age had no effect on treatment efficacy. CONCLUSION: Three PPI therapeutic strategies showed similar efficacies against LPR according to total RFS and RSI scores. The addition of a prokinetic resulted in improvements in specific endoscopic findings, such as vocal cord oedema and diffuse laryngeal oedema. Furthermore, the addition of a prokinetic to PPI therapy was particularly beneficial for overweight or obese patients.

Chd1p recognizes H3K36Ac to maintain nucleosome positioning near the transcription start site.

In Saccharomyces cerevisiae, the ATP-dependent chromatin remodeler, Chd1p, globally affects nucleosome positioning at coding regions, where nucleosomes are specifically and directionally aligned with respect to the transcription start site (TSS). Various auxiliary domains of remodelers play critical roles by performing specialized functions that are unique to the type of remodeler. Here, we report that yeast Chd1p directly binds to acetylated histone H3K36 (H3K36Ac) via its chromodomain, and that H3K36Ac stimulates the nucleosome sliding activity of Chd1p in vitro. Furthermore, we use genome-wide analysis to demonstrate that H3K36Ac promotes the remodeling activity of Chd1p to maintain chromatin stability at the 5' ends of genes in vivo. Our work linking Chd1p with H3K36Ac provides novel insights into how the nucleosome remodeling activity of Chd1p is controlled near the TSS.

Chemical biology approaches for studying posttranslational modifications.

Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.

Radiological parameters related to success of the round window approach in cochlear implantation: A retrospective study.

OBJECTIVES: To evaluate the radiologic parameters related to success of round window (RW) approach for cochlear implantation (CI). DESIGN: A retrospective cohort study. SETTING: Academic-tertiary centre. PARTICIPANTS: Eighty-four consecutive patients without inner ear anomaly who underwent CI with the intent of the RW approach were included. The RW approach was performed through the facial recess after posterior tympanotomy (RW group). When the RW approach was not possible despite maximum effort to expose the RW, promontory cochleostomy (PC) was performed (PC group). MAIN OUTCOME MEASURES: The following radiologic parameters were compared between the two groups: (a) Width of the facial recess, (b) oblique distance between the cochlear basal turn (CBT) and facial nerve (FN), (c) anteroposterior distance between the posterior margin of the RW and FN and (d) angle between the EAC and CBT. RESULTS: Seventy patients (83.3%) were implanted using the RW approach, and 14 patients (16.7%) underwent the PC approach for CI. The anteroposterior distance between the posterior margin of the RW and FN and the angle between the EAC and CBT in the RW group were significantly longer and wider than those in the PC group (P < 0.001 and P = 0.001, respectively). Multivariate analysis revealed that these two parameters were independent parameters for success of the RW approach. CONCLUSIONS: The distance between the posterior margin of the RW and FN and the angle between the EAC and CBT are associated with success of RW approach. Therefore, preoperative radiologic analysis of the two parameters might help CI surgeons to select RW approach.

Genetic incorporation of Nε-acetyllysine reveals a novel acetylation-sumoylation switch in yeast.

The lysine acetylation of proteins plays a key role in regulating protein functions, thereby controlling a wide range of cellular processes. Despite the prevalence and significance of lysine acetylation in eukaryotes, however, its systematic study has been challenged by the technical limitations of conventional approaches for selective lysine acetylation in vivo. Here, we report the in vivo study of lysine acetylation via the genetic incorporation of Nε-acetyllysine in yeast. We demonstrate that a newly discovered acetylation-sumoylation switch precisely controls the localization and cellular function of the yeast septin protein, Cdc11, during the cell cycle. This approach should facilitate the comprehensive in vivo study of lysine acetylation across a wide range of proteins in eukaryotic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Sound Localization and Speech Perception in Noise of Pediatric Cochlear Implant Recipients: Bimodal Fitting Versus Bilateral Cochlear Implants.

OBJECTIVES: The aim of this study was to compare binaural performance of auditory localization task and speech perception in babble measure between children who use a cochlear implant (CI) in one ear and a hearing aid (HA) in the other (bimodal fitting) and those who use bilateral CIs. DESIGN: Thirteen children (mean age ± SD = 10 ± 2.9 years) with bilateral CIs and 19 children with bimodal fitting were recruited to participate. Sound localization was assessed using a 13-loudspeaker array in a quiet sound-treated booth. Speakers were placed in an arc from -90° azimuth to +90° azimuth (15° interval) in horizontal plane. To assess the accuracy of sound location identification, we calculated the absolute error in degrees between the target speaker and the response speaker during each trial. The mean absolute error was computed by dividing the sum of absolute errors by the total number of trials. We also calculated the hemifield identification score to reflect the accuracy of right/left discrimination. Speech-in-babble perception was also measured in the sound field using target speech presented from the front speaker. Eight-talker babble was presented in the following four different listening conditions: from the front speaker (0°), from one of the two side speakers (+90° or -90°), from both side speakers (±90°). Speech, spatial, and quality questionnaire was administered. RESULTS: When the two groups of children were directly compared with each other, there was no significant difference in localization accuracy ability or hemifield identification score under binaural condition. Performance in speech perception test was also similar to each other under most babble conditions. However, when the babble was from the first device side (CI side for children with bimodal stimulation or first CI side for children with bilateral CIs), speech understanding in babble by bilateral CI users was significantly better than that by bimodal listeners. Speech, spatial, and quality scores were comparable with each other between the two groups. CONCLUSIONS: Overall, the binaural performance was similar to each other between children who are fit with two CIs (CI + CI) and those who use bimodal stimulation (HA + CI) in most conditions. However, the bilateral CI group showed better speech perception than the bimodal CI group when babble was from the first device side (first CI side for bilateral CI users or CI side for bimodal listeners). Therefore, if bimodal performance is significantly below the mean bilateral CI performance on speech perception in babble, these results suggest that a child should be considered to transit from bimodal stimulation to bilateral CIs.

Truncated SRA RNA derivatives inhibit estrogen receptor-α-mediated transcription.

The steroid receptor RNA activator (SRA) is a long non-coding RNA (lncRNA) that acts as a putative coactivator for steroid receptor-mediated transcription. A recent study showed that SRA RNA can be structurally dissected into four domains comprising various secondary structures, but the contribution of each domain to the coactivation ability of SRA RNA was previously unknown. Here, we assessed the functional contributions of the various domains of SRA. We examined the effects of each domain on the coactivation of estrogen receptor-α (ERα)-mediated transcription of a luciferase reporter gene in HeLa cells. Then the detailed domain analysis was focused on domain III (D3) not only with the reporter gene in HeLa cells, but also with ERα-responsive genes in MCF7 breast cancer cells. Domain deletion analysis showed that the deletion of any domain decreased the luciferase activity, and that deletion of D3 caused the largest decrease. This D3 deletion effect was not recovered by co-expression of D3 alone; moreover, the expression of D3 fragments (particularly helices H15-H18, which are highly conserved across vertebrates) inhibited luciferase expression in HeLa cells. Moreover, a fragment containing helices H15-H18 reduced ERα-responsive gene expression in MCF7 breast cancer cells. Our findings indicate that D3 inhibited ERα-mediated transcription of a reporter gene in HeLa cells and that helices H15-H18, as a core element responsible for the D3-driven inhibition, reduced expression of ERα-responsive genes in breast cancer cells.

Effects of triamcinolone-impregnated nasal dressing on subjective and objective outcomes following endoscopic sinus surgery.

The aim of this study was to compare the effects of triamcinolone (TA)- and saline-soaked biodegradable nasal dressing on subjective symptoms, wound healing and improvement of olfactory dysfunction in patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) after undergoing endoscopic sinus surgery (ESS). The study was a prospective, randomized, double-blinded, placebo-controlled study. A total of 80 patients undergoing bilateral ESS for CRSwNP were enrolled and randomly assigned to two groups. Nasal dressing was impregnated with normal saline in the control group, while patients received triamcinolone-impregnated dressing in the TA group. Sino-Nasal Outcome Test 20 (SNOT-20) and Korean Version of the Sniffin' Stick (KVSS) II test were used to assess the patients' condition preoperatively and at postoperative 1 and 3 months. Lund-Kennedy (L-K) and perioperative sinus endoscopy (POSE) scores were assessed on postoperative months 1, 2, and 3. There were significant differences between the control group and the TA group in terms of postoperative L-K scores and POSE scores at 1 and 2 months. The postoperative endoscopic scores were significantly decreased in the TA group compared to the control at 1 month. Olfactory functions were significantly improved at postoperative 3 months (p = 0.0099) compared to the preoperative score in the TA group. Significant improvement in the olfactory functions among anosmic and hyposmic patients at postoperative 1 month (p = 0.0475) and 3 months (p = 0.0019) compared to their preoperative olfactory function score was observed only in the TA group. TA-impregnated dressing had a significant advantage over saline-soaked dressing with regard to postoperative wound healing and improvement of olfactory function.

Metformin increases survival in hormone receptor-positive, HER2-positive breast cancer patients with diabetes.

INTRODUCTION: Metformin use has recently been observed to decrease both the rate and mortality of breast cancer. Our study was aim to determine whether metformin use is associated with survival in diabetic breast cancer patients by breast cancer subtype and systemic treatment. METHODS: Data from the Asan Medical Center Breast Cancer Database from 1997 to 2007 were analyzed. The study cohort comprised 6,967 nondiabetic patients, 202 diabetic patients treated with metformin, and 184 diabetic patients that did not receive metformin. Patients who were divided into three groups by diabetes status and metformin use were also divided into four subgroups by hormone receptor and HER2-neu status. RESULTS: In Kaplan-Meier analysis, the metformin group had a significantly better overall and cancer specific survival outcome compared with non metformin diabetic group (P <0.005 for both). There was no difference in survival between the nondiabetic and metformin groups. In multivariate analysis, Compared with metformin group, patients who did not receive metformin tended to have a higher risk of metastasis with HR 5.37 (95 % CI, 1.88 to 15.28) and breast cancer death with HR 6.51 (95 % CI, 1.88 to 15.28) on the hormone receptor-positive and HER2-negative breast cancer. The significant survival benefit of metformin observed in diabetic patients who received chemotherapy and endocrine therapy (HR for disease free survival 2.14; 95 % CI 1.14 to 4.04) was not seen in diabetic patients who did not receive these treatments. CONCLUSION: Patients receiving metformin treatment when breast cancer diagnosis show a better prognosis only if they have hormone receptor-positive, HER2-positive tumors. Metformin treatment might provide a survival benefit when added to systemic therapy in diabetic patients.

Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg.

Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters.

Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.

Dopamine and Cu+/2+ can induce oligomerization of α-synuclein in the absence of oxygen: Two types of oligomerization mechanisms for α-synuclein and related cell toxicity studies.

α-Synuclein oligomers can induce neurotoxicity and are implicated in Parkinson's disease etiology and disease progression. Many studies have reported α-synuclein oligomerization by dopamine (DA) and transition metal ions, but few studies provide insight into joint influences of DA and Cu2+ . In this study, DA and Cu2+ were coadministered aerobically to measure α-synuclein oligomerization under these conditions. In the presence of oxygen, DA induced α-synuclein oligomerization in a dose-dependent manner. Cu+/2+ did not effect oligomerization in such a manner in the presence of DA. By electrophoresis, Cu2+ was found easily to induce oligomerization with DA. This implies that oligomerization invoked by DA is reversible in the presence of Cu2+, which appears to be mediated by noncovalent bond interactions. In the absence of oxygen, DA induced less oligomerization of α-synuclein, whereas DA/Cu2+ induced aerobic-level amounts of oligomers, suggesting that DA/Cu2+ induces oligomerization independent of oxygen concentration. Radical species were detected through electron paramagnetic resonance (EPR) spectroscopic analysis arising from coincubation of DA/Cu2+ with α-synuclein. Redox reactions induced by DA/Cu2+ were observed in multimer regions of α-synuclein oligomers through NBT assay. Cellular toxicity results confirm that, for normal and hypoxic conditions, copper or DA/Cu2+ can induce cell death, which may arise from copper redox chemistry. From these results, we propose that DA and DA/Cu2+ induce different mechanisms of α-synuclein oligomerization, cross-linking with noncovalent (or reversible covalent) bonding vs. likely radical-mediated covalent modification.