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Yuyu Huang,1,2,&ast; Haoran Yu,3,&ast; Min Liang,1,2 Shule Hou,1,2 Junmin Chen,1,2 Fan Zhang,1,2 Xiayu Sun,1,2 Huan Jia,2,4,5 Jun Yang1,2,5 1Department of Otorhinolaryngology Head and Neck Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, People’s Republic of China; 2Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases (14DZ2260300), Shanghai, 200125, People’s Republic of China; 3ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai, 200031, People’s Republic of China; 4Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, People’s Republic of China; 5Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, 200031, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jun YangENT Ward of Xinhua Hospital, Floor 18, Building 8, 1665 Kongjiang Road, Yangpu District, Shanghai, 200092, People’s Republic of ChinaTel +86 13764981808Email yangjun@xinhuamed.com.cnHuan JiaENT Ward of Ninth People’s Hospital, Floor 2, Building 1, 639 Zhizaoju Road, Huangpu District, Shanghai, 200011, People’s Republic of ChinaTel +86 13601797481Email huan_jia_orl@163.comObjective: To investigate the hearing protection outcomes of different drug-eluting analog electrode arrays implanted into guinea pig cochleae.Methods: Sixty guinea pigs were randomly divided into a negative control group and five experimental groups implanted separately with blank (drug carrier), dexamethasone (DXM), aracytine (Ara-C), Ara-C+DXM, and nicotinamide adenine dinucleotide (NAD+) eluting analog electrode arrays. Micro CT was used to supervise the surgical procedure. Auditory brainstem response (ABR) thresholds of the guinea pigs were measured and analyzed.Results and Conclusions: Compared with the negative control, all other groups showed a significant increase in ABR threshold (p< 0.001) after surgery. Among them, there was no obvious difference between the blank (0 vs 90 days: 59.70± 10.57 vs 64.60± 9.47 dB SPL) and the NAD+ group (0 vs 90 days: 59.90± 9.87 vs 64.70± 8.65 dB SPL). On the other hand, the ABR thresholds in the DXM (0 days: 58.10± 10.73 dB SPL; 90 days: 51.70± 9.07 dB SPL) and the Ara-C group (0 days: 59.00± 10.05 dB SPL; 90 days: 51.60± 8.48 dB SPL) decreased significantly compared with the former two groups (p< 0.001). However, the Ara-C+DXM group showed no further benefit (p> 0.05). In addition, a significantly higher survival rate of spiral ganglion neurons in cochleae was observed in the Ara-C and/or DXM groups.Keywords: guinea pig, cochlear implant, drug-eluting electrode, hearing protection

The MAP kinase high osmolarity glycerol 1 (Hog1) plays a central role in responding to external oxidative stress in budding yeast Saccchromyces cerevisiae. However, the downstream responsive elements regulated by Hog1 remain poorly understood. In this study, we report that a Sporisorium scitamineum ortholog of Hog1, named as SsHog1, induced transcriptional expression of a putative cytochrome P450 oxidoreductase encoding gene SsCPR1, to antagonize oxidative stress. We found that upon exposure to hydrogen peroxide (H2 O2 ), SsHog1 underwent strikingly phosphorylation, which was proved to be critical for transcriptional induction of SsCPR1. Loss of SsCPR1 led to hypersensitive to oxidative stress similar as the sshog1Δ mutant did, but was resistant to osmotic stress, which is different from the sshog1Δ mutant. On the other hand, overexpression of SsCPR1 in the sshog1Δ mutant could partially restore its ability of oxidative stress tolerance, which indicated that the Hog1 MAP kinase regulates the oxidative stress response specifically through cytochrome P450 (SsCpr1) pathway. Overall, our findings highlight a novel MAPK signaling pathway mediated by Hog1 in regulation of the oxidative stress response via the cytochrome P450 system, which plays an important role in host-fungus interaction. This article is protected by copyright. All rights reserved.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP3R) activity, thus led to increased intracellular Ca2+ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP3R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca2+ accumulation, and pretreatment with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP3R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP3R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults1. No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities2,3. How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 A2 of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers—which are largely disordered in their free state—demonstrates an example of a ‘folding-upon-partner-binding’ mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs. A cryo-electron microscopy reconstruction shows the ring-shaped structure of the polymerase and capping domains of the L protein bound to a tetramer of phosphoprotein P of the human metapneumovirus.

Background POU3F4 is the causative gene for X-linked deafness-2 (DFNX2), characterized by incomplete partition type III (IP-III) malformation of the inner ear. The purpose of this study was to investigate the clinical characteristics and molecular findings in IP-III patients by Sanger or nanopore single-molecule sequencing. Methods Diagnosis of IP-III was mainly based on clinical characteristics including radiological and audiological findings. Sanger sequencing of POU3F4 was carried out for these IP-III patients. For those patients with negative results for POU3F4 Sanger sequencing, nanopore long-read single-molecule sequencing was used to identify the possible pathogenic variants. Hearing intervention outcomes of hearing aids (HAs) fitting and cochlear implantation (CI) were also analyzed. Aided pure tone average (PTA) was further compared between two groups of patients according to their different locations of POU3F4 variants: in the exon region or in the upstream region. Results In total, 18 male patients from 14 unrelated families were diagnosed with IP-III. 10 variants were identified in POU3F4 by Sanger sequencing and 6 of these were reported for the first time (p.Gln181*, p.Val215Gly, p.Arg282Gln, p.Gln316*, c.903_912 delins TGCCA and p.Arg205del). Four different deletions that varied from 80 to 486 kb were identified 876–1503 kb upstream of POU3F4 by nanopore long-read single-molecule sequencing. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutations. Among these 18 patients, 7 had bilateral HAs and 10 patients received unilateral CI. The mean aided PTA for HAs and CI users were 41.1 ± 5.18 and 40.3 ± 7.59 dB HL respectively. The mean PTAs for patients with the variants located in the exon and upstream regions were 39.6 ± 6.31 versus 43.0 ± 7.10 dB HL, which presented no significant difference (p = 0.342). Conclusions Among 14 unrelated IP-III patients, 28.6% (4/14) had no definite mutation in exon region of POU3F4. However, possible pathogenic deletions were identified in upstream region of this gene. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutation. There was no significant difference of hearing intervention outcomes between the IP-III patients with variants located in the exon region and in the upstream region.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases., Graphical Abstract

In the present study, the borate anions intercalated MgAl-layered double hydroxides (B-LDHs) microsphere and its calcinated product (B-LDOs) have been prepared by precursor method and characterized by the XRD, FT-IR, XPS, TG-DSC, SEM and TEM. This microsphere with hierarchical porous nanostructure was assembled by nanosheets. The B-LDHs and B-LDOs samples have high specific surface area of 138.6 m2 g −1 and 123.0 m2 g−1 measured by N2 adsorption−desorption, respectively. They were used as adsorbents to remove Congo red (CR) dye in an aqueous solution, exhibiting excellent adsorption performance with maximum adsorption capacities of 166 and 273 mg g −1 for CR respectively, which outperform most of the reported adsorbents in literatures. The adsorption processes nicely fitted with the kinetic pseudo-second-order rate equation and Langmuir adsorption model. The effects of B-LDOs sample dosage, pH and temperature of CR solution on adsorption were investigated. The possible adsorption mechanisms were also proposed. It can be expected that both samples, especially B-LDOs sample with higher adsorption efficiency and good reusability, could be considered as promising candidates for wastewater treatment.

Borate is a kind of green and environmental protective flame-retardant, which has been used in fiber, resin and wood and the like. To solve this bottleneck problem of prepared larger particles for borate flame-retardant which makes its poor flame retardant in the research and application, we prepared the calcium borate 2CaO•5B2O3•5H2O nanoplate and micro/nanosheet under hydrothermal conditions by adjusting pH of solution and reaction temperature, which were characterized by the XRD, FTIR, TGA, FESEM and TEM. The flame retardant properties of the prepared 2CaO•5B2O3•5H2O samples were investigated by using multiple thermochemical strategies (including TGA, DSC and non-isothermal decomposition kinetics), and further verified by limited oxygen index method. The nanoplate sample has the less TG mass loss and heat release for DSC, as well as the bigger LOI value, and apparent activation energy E a. These show that the flame retardant property of prepared 2CaO•5B2O3•5H2O nanoplate sample is stronger than that of micro/nanosheet sample. The possible flame retarding mechanism has been proposed. In addition, the horizontal comparison shows that the flame retardant property of prepared 2CaO•5B2O3•5H2O nanoplate is also better than that of 2CaO•B2O3•H2O nanosheet. This research indicated that 2CaO•5B2O3•5H2O nanoplate could be a potential utility-type flame retardant.

The Institute of Modern Physics (IMP) of the Chinese Academy of Sciences (CAS) is building a compact neutron source using a superconducting linac. The injector RFQ will operate in continuous wave (CW) mode, accelerating a 10-mA deuteron beam from 20 keV/u to 1.5 MeV/u. To minimize the possibility of beam loss in the downstream superconducting linac and maintain high acceleration efficiency, we take the 99.9% longitudinal beam emittance as the key optimization parameter for the beam dynamics design of the RFQ, and include an internal buncher to reduce the longitudinal beam emittance. This paper describes the design procedures, beam dynamics simulations and preliminary beam commissioning results for this RFQ. Simulation results show that 99.9% longitudinal beam emittance at the RFQ exit is optimized to 3.5 pi mm mrad, which is 0.13 times the longitudinal acceptance of the downstream superconducting linac. Beam commissioning results demonstrate that the RFQ can accelerate 7.8 mA H 2 + to 3.11 MeV. The transmission efficiency from a Faraday cup before the RFQ to the beam dump after the RFQ is 97.6%. The measured transmission efficiency agrees well with simulation results at different values for the inter-vane voltage.

Acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) are life-threatening conditions with high morbility and mortality, underscoring the urgent need for novel treatments. Leaves of the medicinal herb Microcos paniculata have been traditionally used for treating upper airway infections, by virtue of its content of flavonoids such as apigenin C-glycosides (ACGs). C-glycosides have been shown to exert strong anti-inflammatory properties, although their mechanism of action remains unknown. Herein, hypothesizing that ACGs from M. paniculata inhibit progression of ALI, we used the experimental model of lipopolysaccharide (LPS)-induced ALI in BALB/c mice to evaluate the therapeutic potential of purified ACGs. Our results showed that M. paniculata ACGs inhibited lung inflammation in animals undergoing ALI. The protective effects of ACGs were assessed by determination of cytokine levels and in situ analysis of lung inflammation. ACGs reduced the pulmonary edema and microvascular permeability, demonstrating a dose-dependent down-regulation of LPS-induced TNF-α, IL-6 and IL-1β expression in lung tissue and bronchoalveolar lavage fluid, along with reduced apoptosis. Moreover, metabolic profiling of mice serum and subsequent Ingenuity Pathway Analysis suggested that ACGs activated protective protein networks and pathways involving inflammatory regulators and apoptosis-related factors, such as JNK, ERK1/2 and caspase-3/7, suggesting that ACGs-dependent effects were related to MAPKs and mitochondrial apoptosis pathways. These results were further supported by evaluation of protein expression, showing that ACGs blocked LPS-activated phosphorylation of p38, ERK1/2 and JNK on the MAPKs signaling, and significantly upregulated the expression of Bcl-2 whilst down-regulated Bax and cleaved caspase-3. Remarkably, ACGs inhibited the LPS-dependent TLR4 and TRPC6 upregulation observed during ALI. Our study shows for the first time that ACGs inhibit acute inflammation and apoptosis by suppressing activation of TLR4/TRPC6 signaling pathway in a murine model of ALI. Our findings provide new evidence for better understanding the anti-inflammatory effects of ACGs. In this regard, ACGs could be exploited in the development of novel therapeutics for ALI and ARDS.

Cardiac hypertrophy is one of the major risk factors for chronic heart failure. The role of endophilinA2 (EndoA2) in clathrin-mediated endocytosis and clathrin-independent endocytosis is well documented. In the present study, we tested the hypothesis that EndoA2 protects against angiotensin II (Ang II)-induced cardiac hypertrophy by mediating intracellular angiotensin II type 1 receptor (AT1-R) trafficking in neonatal rat cardiomyocytes (NRCMs). Cardiac hypertrophy was evaluated by using cell surface area and quantitative RT-PCR (qPCR) analyses. For the first time, we found that EndoA2 attenuated cardiac hypertrophy and fibrosis induced by Ang II. Moreover, EndoA2 inhibited apoptosis induced by excessive endoplasmic reticulum stress (ERS), which accounted for the beneficial effects of EndoA2 on cardiac hypertrophy. We further revealed that there was an interaction between EndoA2 and AT1-R.The expression levels of EndoA2, which inhibits AT1-R transport from the cytoplasm to the membrane, and the interaction between EndoA2 and AT1-R were obviously decreased after Ang II treatment. Furthermore, Ang II inhibited the co-localization of AT1-R with GRP-78, which was reversed by EndoA2 overexpression. In conclusion, our results suggested that EndoA2 plays a role in protecting against cardiac hypertrophy induced by Ang II, possibly by inhibiting AT1-R transport from the cytoplasm to the membrane to suppress signal transduction.

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H2O2 in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H2O2-induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation, whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H2O2 treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.

Protein kinases are central mediators of signal-transduction cascades and attractive drug targets for therapeutic intervention. Since kinases are structurally and mechanistically related to each other, kinase inhibitor selectivity is often investigated by kinase profiling and considered as an important index for drug discovery. We here describe a versatile web server termed ProfKin for structure-based kinase profiling, which is based on a kinase-ligand focused database (KinLigDB). It provides all ready-to-use 3D structure coordinates of 4219 kinase-ligand complex structures covering 297 human kinases and the associated information, particularly including binding site type, binding ligand type, interaction fingerprints, downstream molecules and related human diseases. The web server works via predicting possible binding modes for the query molecule, prioritizing the binding modes guided by an interaction fingerprint analysis method, and giving a list of ranked kinases by a comprehensive index. Users can freely select entire or part of the KinLigDB database, e.g. via subfamily and binding site type, to customize the profiling contents. The superimpositions of the predicted binding poses of the query molecule with reference binding modes can be visually inspected on the website. The additional classification attributes and phylogenetic tree are also given for each top-ranked kinase.

p-Aminoazobenzene (pAAB) is a hazardous azo dye that causes considerable harm to human health and the environment. The development of novel and sensitive sensors for the rapid detection of pAAB is in high demand. In this study, a simple fluorescent sensor for pAAB detection is designed based on carbon dots (CDs) which are prepared using green carbon source of Momordica charantia L. via a facile hydrothermal approach. The fluorescence spectra of CDs exhibit considerable overlap with the absorption band of pAAB, and the fluorescence is specifically suppressed in the presence of pAAB ascribed to the inner filter effect. Good and wide linearity is observed in the pAAB concentration range of 0.01–12.5 μg mL−1 with a lower detection limit of 3.9 ng mL−1. The established method achieves good results with a rapid analysis of pAAB in different practical water and soil samples. The as-constructed fluorescent sensor provides a simple, rapid, economical and eco-friendly platform and possesses prospective applications for the effective, selective and sensitive detection of pAAB in the environmental field.

The construction of prodrugs has been a popular strategy to overcome the limitations of chemotherapeutic drugs. However, complicated synthesis procedures and laborious purification steps make the fabrication of amphiphilic prodrugs rather difficult. By harnessing the concept of host-guest interaction, we designed and prepared a supra-amphiphile consisting of a dendritic cyclodextrin host and an adamantane/naphthalimide-modified camptothecin guest through glutathione-responsive disulfide linkage. This host-guest complex could self-assemble in aqueous solution to give nanosized vesicles. When the disulfide bond in adamantane/naphthalimide-modified camptothecin was cleaved by glutathione, the fluorescence of the freed adamantane/naphthalimide unit showed a significant red shift with enhanced intensity. Such glutathione-responsive fluorescence change allows for intracellular imaging and simultaneous monitoring of drug release in real time. On account of abundant positively charged amine groups on the supramolecular vesicle surface, siRNA (siPlK1) could be efficiently loaded on the vesicle. The gel retardation and fluorescence experiments proved that the siPlK1 was successfully bonded to the supramolecular vesicle. The vesicle with dendritic cyclodextrin ring exhibited negligible cytotoxicity even at high concentrations, avoiding the shortcoming of cytotoxicity from commonly used gene vectors. In vitro studies demonstrated that the loaded siRNA was transported into cancer cells to improve cancer therapeutic efficacy. Thus, we developed a prodrug-based supramolecular amphiphile via the host-guest interaction with better therapeutic performance than free camptothecin. The assembled system was utilized as a drug/gene vector to achieve combinational gene therapy and chemotherapy with a synergistic effect, providing an alternative strategy to deliver both prodrug and therapeutic gene.

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (V(mem)) and quiescence. We found that V(mem) is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas V(mem) is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101–mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high V(mem) and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101–treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and V(mem) in the Artemia model.

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H 2 O 2 in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation,whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H 2 O 2 treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.

A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk −1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, −1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (∼2%). Accordingly, the bottom 2–4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (∼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes. MOE (Min. of Education, S’pore) Accepted version

Minus-one programmed ribosomal frameshifting (-1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon -1 RF, the ribosomal reading frame is shifted from the normal zero frame to the -1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson-Crick base pairs, respectively, through major-groove parallel PNA·RNA-RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate -1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs.

To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (−8.6 kcal/mol). The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

Background The aim of this study was to identify the incidence of endolymphatic hydrops using 3-dimensional fluid-attenuated inversion recovery (3D-FLAIR) magnetic resonance imaging (MRI) in the contralateral ear in patients with unilateral Ménière’s disease (MD). Material/Methods This was a prospective study. 3D-FLAIR MRI was performed with a 3 Tesla (3 T) unit 24 h after the intratympanic administration of gadolinium (Gd) in 30 unilateral MD patients with an asymptomatic contralateral ear. The incidence of contralateral involvement in unilateral MD patients and the potential correlations between the affected and contralateral ears were analyzed. Results Endolymphatic hydrops was observed in 7 of the 30 (23.3%) asymptomatic ears. The mean PTA of the asymptomatic ears in the contralateral hydrops patients (33.0±6.1 dB) was significantly higher compared with the non-hydrops patients (17.8±5.7 dB). The patients with observed contralateral hydrops exhibited a significantly longer duration of the disease compared with the non-hydrops patients (6.7±6.3 vs. 2.9±3.1 years, respectively). Furthermore, the patients with contralateral hydrops had a worse hearing level in the affected ears compared with the non-hydrops patients (70.3±7.4 vs. 52.5±3.8 dB, respectively). Conclusions Endolymphatic hydrops is closely related to hearing loss but does not necessarily result in Ménière’s symptoms. Patients with a long history of MD and severe hearing loss in the affected ear are more likely to exhibit endolymphatic hydrops in the asymptomatic contralateral ear. Adequate attention should focus on unilateral MD patients with contralateral ear hydrops because of the potential to develop bilateral MD.

Objectives: We describe the various molecular and cellular pathways that lead to early and delayed loss of residual hearing after cochlear implantation. Methods: We performed a systematic review using the Medline database with the key words cochlear implant, residual hearing, inflammation, apoptosis, and necrosis. Results: The mechanisms underlying the loss of residual hearing after cochlear implantation are multiple. Early hearing loss may be provoked by the surgical access to the inner ear spaces and by trauma caused by insertion of the electrode array. After the initial trauma, an acute inflammatory response promotes elevated levels of cytokines and reactive oxygen species, which in turn promote sensory cell loss by apoptosis, necrosis, and necrosis-like programmed cell death. Treatments that counteract such an inflammatory reaction, production of reactive oxygen species, and apoptosis are effective at preventing hair cell degeneration. However, delayed hearing loss appears to be a consequence of chronic inflammation with development of fibrotic tissue. The mechanisms that lead to fibrosis are poorly understood, and standard anti-inflammatory drugs are insufficient for preventing its development. Conclusions: Cochlear implantation is followed by an inflammatory response involving several pathways that lead to either short-term or long-term sensory hair cell degeneration. Future studies should focus on revealing the precise molecular mechanisms induced by cochlear implantation to allow the discovery of new targets for the effective prevention and treatment of loss of residual hearing.

Aptamers, that exist naturally in living cells as functional elements and can switch nonfluorescent natural targets to fluorophores, are very useful in developing highly sensitive and selective biosensors and screening functional agents. This work demonstrates that human telomeric G-quadruplex (HTG) can serve as a potential fluorophore-switching aptamer (FSA) to target a natural isoquinoline alkaloid. We found that, among the G-quadruplexes studied here and the various structurally similar alkaloids including epiberberine (EPI), berberine (BER), palmatine (PAL), jatrorrhizine (JAT), coptisine (COP), worenine (WOR), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT), only the HTG DNA, especially with a 5′-TA-3′ residue at the 5′ end of the G-quadruplex tetrad (5′-TAG3(TTAG3)3-3′, TA[Q]) as the minimal sequence, is the most efficient FSA to selectively light up the EPI fluorescence. Compared to the 5′ end flanking sequences, the 3′ end flanking sequences of the tetrad contribute significantly less to the recognition of EPI. The binding affinity of EPI to TA[Q] (Kd = 37 nM) is at least 20 times tighter than those of the other alkaloids. The steady-state absorption, steady-state/time-resolved fluorescence, and NMR studies demonstrate that EPI most likely interact with the 5′ end flanking sequence substructure beyond the core [Q] and the G-quadruplex tetrad in a much more specific manner than the other alkaloids. The highly selective and tight binding of EPI with the FSA and significantly enhanced fluorescence suggest the potential development of a selective EPI sensor (detection limit of 10 nM). More importantly, EPI, as the brightest FSA emitter among the alkaloids, can also serve as an efficient conformation probe for HTG DNA and discriminate the DNA G-quadruplex from the RNA counterpart. Furthermore, EPI can bind stoichiometrically to each G-quadruplex unit of long HTG DNA multimer with the most significant fluorescence enhancement, which has not been achieved by the previously reported probes. Our work suggests the potential use of EPI as a bioimaging probe and a therapeutic DNA binder. Accepted version

With the excellent electric performance, metal oxide arresters (MOA) have been widely used in the electric power system. In order to improve power supply reliability and reduce economic loss, it's necessary to conduct the health evaluation of electrical equipments. This paper takes metal oxide arrester as a research object. Firstly, condition parameters are selected, which can be gotten through preventive experiments, on-line monitoring and operation maintenance records, constructing the rational state evaluation index system. Secondly, the analytic hierarchy process (AHP) method is adopted to determine the weight for each condition parameter. Thirdly, the health evaluation model of metal oxide arresters is established based on the fuzzy grey clustering theory. Then, metal oxide arresters are taken for example shows that the comprehensive evaluation result is consistent with the actual state of equipment, which proves the proposed model and method's effectiveness and validity.

Salt stress limits plant growth and crop productivity and is an increasing threat to agriculture worldwide. In this study, proteomic and physiological responses of Brassica napus leaves under salt stress were investigated. Seedlings under salt treatment showed growth inhibition and photosynthesis reduction. A comparative proteomic analysis of seedling leaves exposed to 200 mM NaCl for 24 h, 48 h and 72 h was conducted. Forty-four protein spots were differentially accumulated upon NaCl treatment and 42 of them were identified, including several novel salt-responsive proteins. To determine the functional roles of these proteins in salt adaptation, their dynamic changes in abundance were analyzed. The results suggested that the up-accumulated proteins, which were associated with protein metabolism, damage repair and defense response, might contribute to the alleviation of the deleterious effect of salt stress on chlorophyll biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves. This study will lead to a better understanding of the molecular basis of salt stress adaptation in Brassica napus and provides a basis for genetic engineering of plants with improved salt tolerance in the future.