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Enzalutamide (ENZ) is a second-generation androgen receptor (AR) antagonist used for the treatment of castration-resistant prostate cancer (CRPC) and reportedly prolongs survival time within a year of starting therapy. However, CRPC patients can develop ENZ resistance (ENZR), mainly driven by abnormal reactivation of AR signaling, involving increased expression of the full-length AR (ARfl) or dominantly active androgen receptor splice variant 7 (ARv7) and ARfl/ARv7 heterodimers. There is currently no efficient treatment for ENZR in CRPC. Herein, a small molecule LLU-206 was rationally designed based on the ENZ structure and exhibited potent inhibition of both ARfl and constitutively active ARv7 to inhibit PCa proliferation and suppress ENZR in CRPC. Mechanically, LLU-206 promoted ARfl/ARv7 protein degradation and decreased ARfl/ARv7 heterodimers through mouse double minute 2-mediated ubiquitination. Finally, LLU-206 exhibited favorable pharmacokinetic properties with poor permeability across the blood-brain barrier, leading to a lower prevalence of adverse effects, including seizure and neurotoxicity, than ENZ-based therapies. In a nutshell, our findings demonstrated that LLU-206 could effectively inhibit ARfl/ARv7-driven CRPC by dual-targeting of ARfl/ARv7 heterodimers and protein degradation, providing new insights for the design of new-generation AR inhibitors to overcome ARfl/ARv7-driven CRPC.

SummaryThe Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism remains poorly understood. Here, we show thatUnc-51-likekinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (Ci(R)/Gli(R)) into an activator form (Ci(A)/Gli(A)). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates Ci(R)/Gli(R), sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of Ci(A)/Gli(A). Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.

Objective To investigate the effect of protein tyrosine phosphatase receptor type O (PTPRO) on the phagocytic activity of alveolar epithelial cells in LPS-induced acute lung injury. Methods Mice were randomly divided into the normal control group and LPS stimulation group. The infiltration of inflammatory cells was detected by HE staining. The cytokine TNF-α level in lung was analyzed by ELISA. Western blotting was performed to detect the effect of LPS on PTPRO protein expression in lung. After the expression of PTPRO in MLE-12 cells was silenced by siRNA in vitro, flow cytometry was used to detect the effects of LPS and PTPRO siRNA on the phagocytic activity of MLE-12 cells, and the effects of LPS and PTPRO siRNA on the expression of PTPRO, AKT and phosphorylated AKT protein were measured by Western blotting. Results After the establishment of murine acute lung injury model by LPS injection(1 mg/kg), the infiltrated polymorphonuclear leukocytes were markedly increased. The level of TNF-α in lung tissue and the expression of PTPRO in MLE-12 cells were both significantly increased after LPS stimulation. However, the activity of MLE-12 cells to phagocytose fluorescent microbeads was evidently decreased after silencing PTPRO. Furthermore, silencing PTPRO induced a remarkable decrease in the phosphorylation of AKT in MLE-12 cells. Conclusion PTPRO can promote phagocytic activity of MLE-12 cells via activating AKT signaling pathway.

A theoretical model for studying the temperature properties of singlemode-multimode-singlemode (SMS) fiber structure fabricated by absorptive multimode fiber (MMF) cladding is established. Moreover, an SMS-based temperature sensor is fabricated and experimentally demonstrated. Experimental results show that the dip wavelength of the transmission spectrum changes linearly with temperature, which is in good agreement with the simulated results obtained by using the model. Further, a comprehensive study of temperature characteristics affected by the thermo-optic effect, thermal expansion effect, and thermal effect of absorption characteristics is performed for SMS fiber optic structures with different refractive indexes, thermo-optic coefficients, and absorption properties of MMF cladding, MMF core diameters, and thermal expansion coefficients of packaging shell. According to the obtained rules, investigations are carried out into the thermal response of an SMS fiber structure resulting from combined thermal effects for temperature performance optimization. Excellent temperature stability with a temperature sensitivity of 0 pm/°C or good temperature sensitivity of −441.58 pm/°C is achieved accordingly.

Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous Smo

The GPCR-family protein Smoothened (Smo) is an obligatory signal transducer of the Hedgehog (Hh) signaling pathway. Binding of Hh to its receptor Patched (Ptc) alleviates Ptc-mediated inhibition of Smo, allowing Smo to activate the Cubitus interruptus (Ci)/Gli family of zinc finger transcription factors. The activation of Smo is an early and crucial event in Hh signal transduction. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2/GRK2), and atypical PKC (aPKC). Here, we describe the assays used to examine the phosphorylation and activity of Smo, including in vitro kinase assay, phospho-specific antibodies, luciferase reporter assay, cell surface accumulation, and ciliary localization assays. These assays provide powerful tools to study Smo phosphorylation and activation, leading to mechanistic insight into Smo regulation.

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.

Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. Here, we show that graded Hh induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. We demonstrated that Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. We found that the C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. Our study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.

Carbon dioxide (CO2) was converted into formate (HCO2−) in a catalytic reduction system mainly composed of potassium borohydride (KBH4) and nanoscale bimetallic nickel–copper alloy (NBN–C), in which, the average CO2 conversion efficiency of 41.92% was obtained under the optimal experimental conditions, with a HCO2− selectivity of 53.42%. Various characterization methods were employed to investigate the physicochemical properties of NBN–C and the results indicated that NBN–C was a core-shell mesoporous catalyst with key active sites containing Ni0 and Cu0, at which the catalytic reduction of CO2 took place. The reaction mechanism was proposed based on these characterizations and relevant literatures.

The simultaneous removal of SO2 and NO from flue gas was firstly carried out by heterogeneous UV-Fenton-like process using Iron-containing polyoxometalates complex (denoted as FeIIIAspPW) as catalyst. The results showed that SO2 achieved complete removal under all experimental conditions. The simultaneous removal efficiencies of 100% for SO2 and 84.27% for NO were obtained under the established optimal experimental conditions. The main reaction products were identified as nitrate and sulfate based on the characterization of ion chromatography (IC) and material balance summary. Especially, the physicochemical properties of the fresh and spent catalyst were characterized systematically by a series of techniques, which manifested the stability of the catalyst and the existence of redox pair Fe3+/Fe2+ during the catalytic process. In combination with correlative references, the reaction mechanism was proposed, which stemmed from the chemisorption of H2O2 by hydrogen bonding and the synergetic effect between the Fenton-like catalysis of ferric iron and photo-catalysis of PW12O403− occurred on the surface of FeIIIAspPW.

Summary The Fused (Fu) kinase is a key transducer of Hedgehog signaling, but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites. For complete details on the use and execution of this protocol, please refer to Han et al. (2019)., Graphical Abstract, Highlights • Purification of constitutively active Fu from insect cells for in vitro kinase assay • Priming phosphorylation by Fu can allow secondary in vitro kinase assay • High-purity protein elution with Flag M2 affinity agarose and 3X Flag peptide • Sensitive phospho-protein detection via pIMAGO-biotin kit or specific antibodies, The Fused (Fu) kinase is a key transducer of Hedgehog signaling but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites.

Stem cell activity has to be regulated throughout adult life to maintain tissue homeostasis. Li et al. find that the membrane-associated kinase Gilgamesh (Gish/CK1γ) restricts JNK signaling and Drosophila intestinal stem cell proliferation by phosphorylating and destabilizing Rho1., Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. Here, we show that the membrane-associated kinase Gilgamesh (Gish)/CK1γ maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, we find that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, our study identifies Gish/CK1γ as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts.

Tourism is one of the largest and fastest developing industries in the world. As most protected areas have the purpose of both nature conservation and providing recreational opportunities, national parks and a variety of protected areas have become popular destinations for tourists worldwide. This paper critiques the appropriateness of current tourism developments of iconic National Parks in Australia and South Africa. The methods applied to this study include (a) analyzing case studies on different national parks to understand the management situation, (b) criticize existing tourism developments in Australia and South Africa with evidence, using conceptual model for situation analysis and suggestions for more rational management plans, and (c) comparing and contrasting Australia with South Africa. The theoretical method of ecological micro-landscapes is also presented to the study of protection and management of national parks and protected areas. The study could become a useful guide for the improvements on present protected area governance practices.

We experimentally study intracavity four-wave mixing (FWM) using Λ -type, three-level electromagnetically induced transparency (EIT) atoms confined in an optical ring cavity. The original dark-state peak disappears accompanying the generation of intracavity FWM when the coupling light is changed from traveling-wave to standing wave. Additionally, this FWM signal is on resonance with the optical cavity only when the coupling laser is detuned from the atomic resonance center, where the absorption is drastically reduced for both the probe and FWM fields. At last, we do theoretically analysis for the susceptibility of this composite atom–cavity system.

In order to solve the problem of modeling, deduction and measurement of operational concepts, a method of modeling and measurement of operational concepts based on Joint Mission Thread (JMT) and Activity-Based Method (ABM) is proposed. Combining the actual situation of our country and its own characteristics, according to the characteristics of ABM and JMT, the operational concepts are modeled. Through model deduction based on OPM (Object-Process Methodology), the function and logic of the operational concepts are tested. Combining with JMT, the operational concepts are decomposed into indicators to realize the measurability of the operational concepts. This method can provide reference for the normative description, modeling, testing and index decomposition of operational concepts.

Hippo (Hpo) signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie (Yki)/Yap via a cytoplasmic kinase cassette containing an upstream kinase Hpo/MST1/2 and a downstream kinase Warts (Wts)/Lats1/2. Here we show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus to prevent its nuclear accumulation and restrict Hpo pathway target gene expression. PRP4K inactivation accelerates whereas excessive PRP4K inhibits Yki-driven tissue overgrowth. PRP4K phosphorylates a subset of Wts/Lats1/2 sites on Yki/Yap to inhibit the binding of Yki/Yap to the Scalloped (Sd)/TEAD transcription factor and exclude Yki/Yap nuclear localization depending on nuclear export. Furthermore, PRP4K inhibits proliferation and invasiveness of cultured breast cancer cells and its high expression correlates with good prognosis in breast cancer patients. Our study unravels an unanticipated layer of Hpo pathway regulation and suggests that PRP4K-mediated Yki/Yap phosphorylation in the nucleus provides a fail-safe mechanism to restrict aberrant pathway activation., The Hippo signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie /Yap. Here the authors show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus, which prevents its nuclear accumulation and inhibits Hippo signaling.

In a Λ-type three-level atomic system coupled by an off-resonant standing-wave, the reflected four-wave mixing (FWM) spectrum is studied. It shows that the maximum reflection efficiency occurs when both of the coupling and probe fields are tuned off resonances from the atomic transitions. The essence of enhanced reflection is that the nonlinear efficiency of the FWM based on coherent atoms is improved due to the significant reduction of phase mismatch. The theoretical analysis shows good agreement with the experimental results. Furthermore, the influence of the atomic number density on the coupling frequency detuning of the optimum reflection efficiency and the linewidth are also investigated.

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.

Sparsity and reconstruction error are two main objectives to be optimized in sparse signal reconstruction. In this paper, sparse signals are reconstructed by optimizing these two objectives simultaneously. This reconstruction method mainly consists of three steps. First, a one-dimension-dominated method is used to find a uniformly distributed optimal compromise solution set between these two objectives. Second, the Iterative Half Thresholding method is employed to improve the sparsity. Third, a robust selection method is proposed to choose a final solution from the solution set. The proposed method is compared with eight sparse reconstruction algorithms on twelve sparse test instances. Experimental results show that the proposed algorithm is able to reconstruct both noisy and noiseless sparse signals. In addition, the effectiveness of the proposed algorithm is demonstrated in practical application instances.

Facial expression recognition is a challenging task, because it is difficult to recognize facial expressions of different persons if they are of diverse races and ages. Extracting distinctive feature from original facial image is a critical step for successful facial expression recognition. This paper proposes sparse representation feature for facial expression recognition. First of all, a dictionary is established using training images. Then sparse representation feature is extracted by sparse representation orthogonal matching pursuit method. Finally the extracted features of different expressions are classified by two-hidden-layer extreme learning machine. Facial expression images of both Cohn-Kanade and JAFFE databases are classified using sparse representation feature. Experimental results show that the sparse representation feature is suitable for facial expression recognition.

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli into its activator form (Ci(A)/Gli(A)), but the underlying mechanism remains poorly understood. Here we demonstrate that Hh stimulates the phosphorylation of Ci by the Ser/Thr kinase Fused (Fu) and that Fu-mediated phosphorylation of Ci promotes its activation. We find that Fu directly phosphorylates Ci on Ser218 and Ser1230, which primes its further phosphorylation by CK1 on adjacent sties. These phosphorylation events alter Ci binding to the pathway inhibitor Suppressor of fused (Sufu) and facilitate the recruitment of Transportin and the transcriptional coactivator CBP. Furthermore, we provide evidence that Sonic hedgehog (Shh) activates Gli2 by stimulating its phosphorylation on conserved sites through the Fu family kinases ULK3 and mFu/STK36 in a manner depending on Gli2 ciliary localization. Hence, Fu family kinase-mediated phosphorylation of Ci/Gli serves as a conserved mechanism that activates the Hh pathway transcription factor.

The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an "exposed" location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufu-binding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.

Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (GliA) in wild-type but not in Sufu mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers., Author summary The secreted Hedgehog (Hh) protein plays an evolutionarily conserved role in both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling activity contributes to a wide range of human diseases, including birth defects and cancer. Hh signaling in vertebrates critically depends on the primary cilium, a microtubule-based plasma membrane protrusion present on the surface of most mammalian cells. Upon ligand stimulation, Hh pathway components, including the seven-transmembrane protein Smoothened (Smo) and Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting are still poorly understood. Here, we discover that the PY-type nuclear localization signal (PY-NLS) and the nuclear import factor karyopherinβ2 (Kapβ2) regulate Gli ciliary localization and Hh pathway activity. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization without affecting Smo ciliary accumulation in response to Hh. Kapβ2 regulates the formation of the active form of Gli, which is required for proper Hh signaling in zebrafish embryos and cultured cerebellum granule neuron progenitors (CGNPs). Kapβ2 depletion impaired the growth of medulloblastoma cells driven by an oncogenic form of Smo. Finally, Kapβ2 is a transcriptional target of the Hh pathway, forming a positive feedback loop to promote Gli activation. Our study reveals the molecular mechanism underlying the regulation of Gli ciliary targeting and identifies Kapβ2 as a potential cancer drug target.

Sparse reconstruction is a technique to reconstruct sparse signal from a small number of samples. In sparse reconstruction problems, the sparsity and measurement error should be minimized simultaneously, therefore they can be solved by multi-objective optimization algorithms. Most multi-objective optimizers aim to obtain the complete Pareto front. However only solutions in knee region of Pareto front are preferred in sparse reconstruction problems. It is a waste of time to obtain the whole Pareto front. In this paper, a knee point driven multi-objective particle swarm optimization algorithm (KnMOPSO) is proposed to solve sparse reconstruction problems. KnMOPSO aims to find the local part of Pareto front so that it can solve the sparse reconstruction problems fast and accurately. In KnMOPSO personal best particles and global best particle are selected with knee point selection scheme. In addition, solutions which are more likely to be knee points are preferred to others.

Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo., A study of both fruit flies and vertebrate cells shows that the plasma membrane-associated kinase Gish/CK1γ promotes transduction of the Hedgehog signal by phosphorylating Smoothened., Author Summary The secreted glycoprotein Hedgehog (Hh) plays a conserved role in embryonic development and adult tissue homeostasis in species ranging from Drosophila to humans. Deregulation of Hh signal transduction contributes to a wide range of human disorders, including birth defects and cancer. The seven-transmembrane protein Smoothened (Smo) is an obligatory and conserved Hh signal transducer, but how Hh stimulates its activity remains unclear. Here we identify a plasma membrane associated kinase, Gilgamesh (Gish)/CK1γ, as a positive regulator of the Hh signaling activity. We find that Gish activates Hh signaling by phosphorylating a specific site in the Smo C-terminal intracellular tail. Phosphorylation of Smo by Gish is required for maximal activation of Smo and depends on membrane association of Gish and prior phosphorylation of Smo by protein kinase A (PKA). We also find that Hh stimulates the association of Smo with Gish after it travels to the plasma membrane, thus facilitating its phosphorylation by Gish. Finally, we provide evidence that CK1γ is found at the primary cilium in mammals and phosphorylates Smo to activate the Hh pathway. Our results uncover a conserved role of Gish/CK1γ in the regulation of Smo phosphorylation and provide new insight into the molecular underpinning of how Hh signal is transduced across the plasma membrane.

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.

Dexamethasone (DEX) has been routinely used as a pre-treatment in the clinical application of paclitaxel (PTX) to treat ovarian cancer. However, PTX-induced apoptosis might be inhibited by DEX. This study was undertaken to investigate the effects of DEX on the apoptosis induced by PTX. Both of SKOV-3 and HO-8910 human ovarian cancer cells were divided into four groups: (1) untreated (Con); (2) treated with DEX (0.1 μM) alone; (3) treated with PTX (50 nM); and (4) pre-treated with DEX (0.1 μM), and 24 h later, treated with PTX (DEX + PTX). Cell proliferation was determined by the 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di- phenytetrazoliumromide (MTT) dye uptake method, while cell apoptosis was analyzed by propidium iodide (PI) staining and flow cytometry. Then, reverse transcription polymerase chain reactions (RT-PCRs) were applied to semi-quantitative analysis, followed by western blot analysis. Statistical analysis was performed, with Fisher’s least significant difference test. Our results demonstrated that DEX can differentially inhibit SKOV-3 and HO-8910 cell proliferation induced by PTX and decrease the apoptosis rates in cancer cells. Pre-treatment with DEX could up-regulate the expressions of members of anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) and members of IAP family (survivin). The expression of cleaved caspase-3 was down-regulated by DEX, shown by semi-quantitative RT-PCRs and western blot analysis. Our data gained invaluable insights of the antagonistic mechanisms of DEX on PTX-induced cancer cell death and may provide new methods of using DEX as antineoplastic drugs or agents in the clinical treatment for ovarian cancer patients.

The cavity transmission spectrum is experimentally investigated in Λ-type three-level atoms coupled to a standing-wave cavity system. It is shown that the dark-state polariton peak is not generated at resonance but rather at off-resonance. The theoretical analysis reveals that the absence of an on-resonance dark-state polariton peak is mainly caused by the strong absorption of the intracavity medium to the probe cavity mode counterpropagating with the coupling field due to the Doppler shift in the hot atoms. Moreover, the optimal frequency position of the cavity mode for an efficient dark-state polariton peak is also demonstrated.

Background Dexamethasone (DEX) has been routinely used as a pre-treatment in the clinical application of paclitaxel (PTX) to treat ovarian cancer. However, PTX-induced apoptosis might be inhibited by DEX. This study was undertaken to investigate the effects of DEX on the apoptosis induced by PTX. Methods Both of SKOV-3 and HO-8910 human ovarian cancer cells were divided into four groups: (1) untreated (Con); (2) treated with DEX (0.1 μM) alone; (3) treated with PTX (50 nM); and (4) pre-treated with DEX (0.1 μM), and 24 h later, treated with PTX (DEX + PTX). Cell proliferation was determined by the 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di- phenytetrazoliumromide (MTT) dye uptake method, while cell apoptosis was analyzed by propidium iodide (PI) staining and flow cytometry. Then, reverse transcription polymerase chain reactions (RT-PCRs) were applied to semi-quantitative analysis, followed by western blot analysis. Statistical analysis was performed, with Fisher’s least significant difference test. Results Our results demonstrated that DEX can differentially inhibit SKOV-3 and HO-8910 cell proliferation induced by PTX and decrease the apoptosis rates in cancer cells. Pre-treatment with DEX could up-regulate the expressions of members of anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) and members of IAP family (survivin). The expression of cleaved caspase-3 was down-regulated by DEX, shown by semi-quantitative RT-PCRs and western blot analysis. Conclusions Our data gained invaluable insights of the antagonistic mechanisms of DEX on PTX-induced cancer cell death and may provide new methods of using DEX as antineoplastic drugs or agents in the clinical treatment for ovarian cancer patients.

The Hedgehog (Hh) family of secreted proteins governs a myriad of key developmental processes by regulating Ci/Gli transcription factors at multiple levels including nuclear/cytoplasmic shuttling. Here we investigate the mechanism underlying the regulation of Ci/Gli subcellular localization by identifying and characterizing a novel nuclear localization sequence (NLS) in the N-terminal conserved domain of Ci/Gli that matches the PY-NLS consensus. We demonstrate that the PY-NLS functions in parallel with a previously identified bipartite NLS to promote nuclear localization and activity of full-length Ci. We find that Transportin (Trn), a Drosophila homolog of Kapβ2, is responsible for PY-NLS-mediated nuclear localization of Ci. Furthermore, we show that the tumor suppressor and conserved Hh pathway component Suppressor of fused (Sufu) opposes Trn-mediated Ci nuclear import by masking its PY-NLS. Finally, we provide evidence that Gli proteins also contain a functional PY-NLS and that mammal Sufu employs a similar mechanism to regulate Gli nuclear translocation. Our study not only provides a mechanistic insight into how Sufu regulates Ci/Gli subcellular localization and Hh signaling but also reveals a role of Trn/Kapβ2 in developmental regulation.

As part of continuing studies of the identification of gene organization and cloning of novel alpha-conotoxins, the first alpha4/4-conotoxin identified in a vermivorous Conus species, designated Qc1.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds (I-III, II-IV connectivity) in a native globular configuration. The mature peptide of Qc1.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qc1.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qc1.2 was shown to selectively inhibit both rat neuronal alpha3beta2 and alpha3beta4 subtypes of nicotinic acetylcholine receptors with low potency. A block of about 63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized alpha-conotoxin members, the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.

Background: Dexamethasone (DEX) has been routinely used as a pre-treatment in the clinical application of paclitaxel (PTX) to treat ovarian cancer. However, PTX-induced apoptosis might be inhibited by DEX. This study was undertaken to investigate the effects of DEX on the apoptosis induced by PTX. Methods: Both of SKOV-3 and HO-8910 human ovarian cancer cells were divided into four groups: (1) untreated (Con); (2) treated with DEX (0.1 μM) alone; (3) treated with PTX (50 nM); and (4) pre-treated with DEX (0.1 μM), and 24 h later, treated with PTX (DEX + PTX). Cell proliferation was determined by the 3-(4,5)-dimethylthiahiazo (�z-y1)-3,5-di- phenytetrazoliumromide (MTT) dye uptake method, while cell apoptosis was analyzed by propidium iodide (PI) staining and flow cytometry. Then, reverse transcription polymerase chain reactions (RT-PCRs) were applied to semi-quantitative analysis, followed by western blot analysis. Statistical analysis was performed, with Fisher’s least significant difference test. Results: Our results demonstrated that DEX can differentially inhibit SKOV-3 and HO-8910 cell proliferation induced by PTX and decrease the apoptosis rates in cancer cells. Pre-treatment with DEX could up-regulate the expressions of members of anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) and members of IAP family (survivin). The expression of cleaved caspase-3 was down-regulated by DEX, shown by semi-quantitative RT-PCRs and western blot analysis. Conclusions: Our data gained invaluable insights of the antagonistic mechanisms of DEX on PTX-induced cancer cell death and may provide new methods of using DEX as antineoplastic drugs or agents in the clinical treatment for ovarian cancer patients.